Targeting angiogenin as a therapeutic strategy for age-related osteoporosis

Targeting angiogenin as a therapeutic strategy for age-related osteoporosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting angiogenin as a therapeutic strategy for age-related osteoporosis Ye Xiao, Weihong Kuang, Zhuolin Peng, Mengzhu Yuan, Mingsheng Ye, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7830644/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract With aging, the accumulation of cellular stress and cellular senescence drives the progression of osteoporosis. As a conserved cellular stress response, angiogenin (ANG) induces the cleavage of cytoplasmic transfer RNAs (tRNAs) to generate tRNA-derived stress-induced RNAs (tiRNAs) with diverse functional roles in various diseases. However, their biological functions in regulating osteoporosis remain poorly understood. In our study, we observed that angiogenin levels increase in senescent Bone Marrow Stromal Cells (BMSCs), and that angiogenin promotes age-dependent accumulation of 5'-tiRNA-Glu -CTC by cleaving tRNA-Glu under oxidative stress. The 5'-tiRNA-Glu -CTC disrupts the stability of anti-senescence and pro-osteogenic mRNAs, leading to bone-fat imbalance and thereby accelerating bone loss. Blocking 5'-tiRNA-Glu -CTC wiht antisense oligonucleotide (ASO) or inhibiting angiogenin with NCI-65828 alleviates BMSCs senescence and age-related bone loss. Clinical sample detection and analysis revealed that elevated serum levels of 5'-tiRNA-Glu -CTC in patients with osteoporosis exhibit a positive correlation with bone resorption markers and a negative correlation with bone formation markers. These findings suggest that 5'-tiRNA-Glu -CTC may serve as a potential biomarker for diagnosing age-related osteoporosis. Collectively, our study sheds new light on the role of ANG-induced 5'-tiRNAs in regulating BMSCs senescence and highlights angiogenin as a promising therapeutic target for age-related osteoporosis. Health sciences/Endocrinology/Endocrine system and metabolic diseases/Metabolic bone disease/Osteoporosis Biological sciences/Drug discovery/Biomarkers/Diagnostic markers Osteoporosis Angiogenin Aging Bone Marrow Stromal Cells tRNA-derived stress-induced RNAs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Osteoporosis is defined as a systemic chronic metabolic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, and constitutes a major risk factor for fracture 1 . Evidence demonstrates that chronic oxidative stress- induced DNA damage, cellular apoptosis, and cellular senescence of bone marrow stromal cells (BMSCs) is an important factor that contributes to the disease 2 , 3 . Additionally, oxidative stress triggers angiogenin (ANG)-mediated production of transfer RNA-derived small RNAs (tsRNAs) by altering tRNA modification profiles 4 . As cellular stress response products, dysregulated tsRNAs participate in various cellular processes; however, their biological functions in regulating osteoporosis remain unclear. Oxidative stress is defined as a dynamic imbalance between the generation of reactive oxygen species (ROS) and the elimination capacity of antioxidant systems, inducing the disruption of physiological redox homeostasis and subsequently triggers an adaptive or pathological cellular stress response 5 . This imbalance can lead to oxidative damage to biological macromolecules (such as DNA, proteins, lipids), which in turn causes defects in cell differentiation, apoptosis, mitochondrial dysfunction and inflammation. There is increasing evidence that oxidative stress disrupts bone homeostasis and is involved in the development of osteoporosis 6 . Specifically, high levels of ROS inhibit the expression of osteogenic differentiation markers such as ALP, OCN, COL1, and Runx2 by activating the MAPKs pathway 7 . ROS also activates the FOXO transcription factor, reduces the availability of β-catenin, and downregulates the Wnt/β-catenin signaling pathway, thereby inhibiting osteoblastogenesis 8 , 9 . It cannot be ignored that high reactive oxygen species (ROS) levels in the senescent microenvironment (SME) induce bone marrow derived stromal cells (BMSCs) senescence, which is considered another important factor contributing to bone loss 10 . Oxidative stress also promote osteoclast differentiation through NF-κB, which increase the expression of transcriptional factors c-Fos and NFATc1 to regulate the genes tartrate-resistant acid phosphatase and cathepsin K involved in osteoclastogenesis and bone resorption 11 , 12 . Moreover, RANKL-induced osteoclastogenesis is enhanced via activation of MAPKs and NF-κB 13 . However, oxidative stress also suppresses Nrf2 pathway, leading to enhancement of bone resorption 14 . Moreover, ROS induce osteocyte apoptosis and alter the RANKL/OPG ratio, which triggers signals that promote the local recruitment of osteoclasts and excessive osteoclastogenesis 15 , 16 . It is evident that the progression of osteoporosis is closely correlated with ROS, which induces BMSCs senescence and prevents osteoblast differentiation, induces osteoclast differentiation and activity, and enhances apoptotic osteocytes, thereby altering the RANKL/OPG ratio. Recently, the tRNA cleavage activity of angiogenin (ANG) triggered by oxidative stress- a critical component of eukaryotic cellular responses to and mitigation of environmental stress- has caught our attention, which often involves a decrease in general translation and an increase in preferential translation of stress-response genes 17 . Angiogenin (ANG) is a stress-induced, secreted ribonuclease and exhibits various biological activities, extending from inducing angiogenesis to stimulating cell proliferation and, more recently, to promoting cell survival 18 . The biological functions of ANG depend on its subcellular localization. In the nucleus, angiogenin promotes ribosomal RNA (rRNA) transcription, thus facilitating cell growth and proliferation. Under stress conditions, angiogenin translocates to the cytosol where it cleaves mature tRNA into transfer RNA-derived small RNAs (tsRNAs) 19 . The dysregulation of angiogenin-induced tsRNAs has been proposed to have multiple cellular functions, including inhibition of ribosome biogenesis, inhibition of protein translation, and inhibition of apoptosis, and participation in various diease processes 17 , 20 . However, whether angiogenin-induced tsRNAs are involved in the progression of osteoporosis pathogenesis remains unexplored. tRNA-derived small RNAs (tsRNAs), generated through cleavage of mature or precursor tRNAs (pre-tRNAs) at distinct sites, are now recognized as a novel class of regulatory non-coding RNAs 21 , 22 . So far, the nomenclature for tsRNA has not been established and its different names are accorded to the length and cleavage position on tRNA or pre-tRNA. tsRNAs of 18 ~ 30 nt in length are categorized into two types: Type I and Type II. Type I tsRNAs (tRF-5 and tRF-3) are produced via Dicer-mediated cleavage of the D-loop or T-loop of mature tRNAs, while Type II tsRNAs (tRF-1) are generated through cleavage of the 3' trailer sequences of pre-tRNAs by Endonuclease Z (RNase Z)/cytoplasmic ribonuclease Z2 (ELAC2). Additionally, tsRNAs of 30 ~ 40 nt in length are termed "tRNA halves," often referred to as "tiRNA (tRNA-derived stress-induced RNAs)" due to their stress-induced characteristics and generated through cleavage tRNAs at the anticodon loop by nuclease angiogenin 23 . Recently, tsRNAs have been recognized as novel therapeutic targets and regulatory molecules, exerting pivotal roles through multiple mechanisms. First, they promote mRNA degradation via DICER-dependent biogenesis by binding to AGO proteins, thereby mimicking miRNA behavior to downregulate gene expression 24 . These tsRNAs also mediate translational regulation in an AGO proteins-independent manner by exerting structural effects on mRNAs or rRNAs 25 . Second, they displace RNA-binding proteins (RBPs) from pro-oncogenic or metabolic mRNAs, suppressing cancer metastasis, or alternatively bind RBPs to either protect mRNAs from decay or enhance the stability of metabolic mRNAs, promoting tumorigenesis 26 – 29 . Third, they repress translation- a function that remains among the most extensively studied roles of tsRNAs in molecular biology. Under stress, the nuclease angiogenin cleaves tRNAs and generates 3'-tiRNA and 5'-tiRNA. Notably, 5'-tiRNA, but not 3'-tiRNA, inhibits global protein synthesis by forming intermolecular RNA G-quadruplexes (RG4) or binding to polyadenylate-binding protein 1 (PABPC1), thereby displacing PABPC1 and eIF4A/G/E from m⁷G-capped mRNAs and resulting in the sequestration of initiation factors 30 – 33 . Interestingly, 5'-tiRNA may decrease global translation of mRNAs containing weak internal ribosomal entry sites (IRESs) while selectively upregulating the translation of mRNAs containing strong IRESs that are involved in pro-survival and anti-apoptotic pathways, thereby promoting cell survival under adverse conditions 34 . Researchers also have proposed that ANG-induced tsRNAs may contribute to the formation of SGs in response to cellular stress, which indicated that ANG plays an active role in stress response of the cells and may promote cell survival via this mechanism 35 . In summary, the diverse regulatory mechanisms of tsRNAs render them closely linked to the progression of diverse human diseases, encompassing cancer, neurodegenerative disorders, metabolic syndromes, viral infections, and inflammasome-related diseases 36 . Considerable evidence shows that tRFs are detectable in model organisms of different ages and are associated with age-related diseases, such as neurodegenerative diseases 37 , 38 . It remains largely unknown whether increased oxidative stress triggers angiogenin-induced tRNA cleavage, a mechanism potentially involved in the progression of osteoporosis-a systemic chronic metabolic and age-associated skeletal disease. In this study, we observed a translocation of angiogenin (ANG) from the nucleus to the cytosol in BMSCs during aging and further found that the ANG cleavage product 5'-tiRNA-Glu − CTC in BMSCs increased with aging in mice and humans. 5'-tiRNA-Glu − CTC disrupts the stability of anti-senescence and pro-osteogenic mRNAs by binding to their 3'UTR regions, including Capn1, Cdk1, Ccnd1, Foxm1 and Sp7. Moreover, we discovered that treatment with the ANG inhibitor NCI-65828 ameliorates age-related bone loss, indicating that inhibition of ANG activity may represent a potential therapeutic strategy for osteoporosis. Furthermore, we detected that the 5'-tiRNA-Glu − CTC levels in human serum were negatively correlated with BMD, Z-scores and osteogenic markers (PINP and BGP), and were positively correlated with osteoclast markers (CTX and TRAP), suggesting that 5'-tiRNA-Glu − CTC may serve as a promising serum biomarker for diagnosing osteoporosis. Materials and Methods Mice C57BL/6J mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. All mice were housed in specific pathogen-free animal facilities (22–24°C) at the Experimental Animal Research Center of Central South University under a 12-hour dark/light cycle, with free access to water and a standard diet. All animal care protocols and experiments were approved by the Medical Ethics Committee of Xiangya Hospital of Central South University. Human sample Serum samples from 40 osteoporosis patients were obtained from the Endocrinology Laboratory of Xiangya Hospital.​​ Osteoporosis diagnosis was confirmed by two or more experienced physicians at Xiangya Hospital using dual-energy X-ray absorptiometry (DXA) to measure bone mineral density (BMD), particularly at the lumbar spine. ​​The study was approved by the Clinical Ethics Committee of Xiangya Hospital, Central South University.​​ Throughout the communication process, the nature of the study and its potential implications were explained to all participants, and each provided written informed consent. Cell culture The BMSC cell line (MUBMX-01001, Cyagen) were cultured in MEM-αmedium (Procell, PM150421) supplemented with 10% Fetal bovine serum (Gibco, 10091-148) and 1% penicillin-streptomycin (Procell, PB180120), and maintained at 37°C in a humidified 5% CO₂ incubator. For primary BMSCs isolation and culture, Mouse bone marrow was flushed from mouse femurs and tibias using pre-chilled MEM-α complete medium via a 1 mL syringe, and the harvested cells were resuspended and then was incubated with antibodies targeting mouse Sca1 (108108; BioLegend), CD29 (102206;BioLegend), CD45 (103132༛BioLegend), and CD11b (101226༛BioLegend) at 4°C for 20 min, followed by FACS (BD Biosciences) sorting to isolate Sca1 + CD29 + CD45 − CD11b − cells as BMSCs. The sorted cells were seeded into 6 cm culture dishes and cultured to reach 90% confluency. The cells were then digested with 0.25% trypsin for about 2 min, and the detached cells were subsequently passaged into new 6 cm dishes to enrich the cell population. tsRNA-sequencing Total RNA was extracted from the serum of osteoporosis patients using the miRNeasy Serum Advanced Kit (217204, Qiagen) and quantified with the NanoDrop ND-1000 instrument. RNA modifications were removed from the RNA samples, followed by ligation with 3'-adapter and 5'-adapter to construct a sequencing library via PCR amplification. Recovery of 134 ~ 160bp PCR amplified fragment libraries via DNA gel electrophoresis. The libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ as sequencing clusters and sequenced on Illumina sequencer according to the manufacturers instructions. Raw sequencing data are used to the following analysis. Sequencing quality is examined by FastQC. Trimmed 5', 3'-adaptor bases by cutadapt. Trimmed reads are aligned allowing for 1 mismatch only to the mature transfer RNA (tRNA) sequences, then the rest of the unmapped reads are aligned allowing for 1 mismatch only to precursor tRNA sequences with bowtie software. The rest of the unmapped reads are aligned allowing for 1 mismatch only to microRNA (miRNA) reference sequences with miRDeep2. The expressions of tsRNA are evaluated using their sequencing counts and are normalized as counts per million of total aligned reads (CPM). The differentially expressed are screened based on the count value with R package edgeR. The scatter is ploted with differentially expressed results. RNA-sequencing Total RNA was isolated from BMSCs transfected with 5'-tsRNA-Lys − CTT , 5'-tiRNA-Glu − CTC , and negative RNA using the AG RNAex Pro Reagent (AG21102, Accurate Biology). RNA quality was examined by gel electrophoresis and quantitatived with Qubit (Thermo, Waltham, MA, USA). The sequencing libraries were prepared using VAHTSTM Stranded mRNA-seq Library Prep Kit for Illumina® and sequenced in collaboration with Genergy Biotechnology Co. Ltd. (Shanghai, China). Differentially expressed genes (DEGs) exhibiting two-fold changes and Benjamini and Hochberg-adjusted P values ≤ 0.05 were selected. Then DEGs were chosen for function and signaling pathway enrichment analysis using GO and KEGG databases. The significantly enriched pathways were determined when P < 0.05 and at least two affiliated genes were included. Biotinylated RNA pull down: BMSCs cell line was lysed on ice for 10 minutes using cell lysis buffer containing protease inhibitors, followed by homogenization at 60 Hz for 10 s, pausing for 10 s, and repeating 3 times. The lysate was then centrifuged at 14,000 rpm, 4°C for 10 min. The supernatant was collected into a new centrifuge tube, and was incubated with 20 µg biotin-labeled 5'-tiRNA-Glu − CTC (pre-denatured at 65°C for 10 min and cooled to 4°C) at 4°C with rotation for 2 h. Then, streptavidin beads were added and incubated at 4°C with rotation for 2 h. The RNA captured by the magnetic beads was eluted and subjected to commercial RIP-seq (GUANGZHOU RIBOBIO CO., LTD). Cell transfection The 5'-tiRNA-Glu − CTC , 5'-tsRNA-Lys − CTT , TRF-5004b or 5'-tiRNA-Glu − CTC antisense oligonucleotide (ASO) fragments were synthesized by Sangon Biotech (Shanghai, China). These tsRNA fragments weretransfected into the BMSCs using Lipofectamine 2000 (Invitrogen). After 6 h, the medium wasreplaced with appropriate medium for further experiments. Osteogenic differentiation assay and Alizarin Red S staining For osteogenic differentiation in vitro, primary BMSCs transfected with 5'-tiRNA-Glu − CTC , 5'-tsRNA-Lys − CTT , TRF-5004b, 5'-tiRNA-Glu − CTC antisense oligonucleotide or treated with Angiogenin inhibitor were cultured in 24-well plates for 21 days using α-MEM supplemented with 10% fetal bovine serum, 0.1 mM dexamethasone, 10 mM β-glycerophosphate, and 50 µM ascorbic acid-2-phosphate. The medium was replaced every 3 days. Then the cells were stained with 2% Alizarin Red S (Sigma-Aldrich, A5533, pH 4.2) to assess extracellular matrix mineralization. Alizarin Red S released from the extracellular matrix into cetylpyridinium chloride solution was quantified spectrophotometrically at 540 nm. Adipogenic differentiation assay and Oil Red O Staining For adipogenic differentiation in vitro, primary BMSCs transfected with 5'-tiRNA-Glu − CTC , 5'-tsRNA-Lys − CTT , TRF-5004b, 5'-tiRNA-Glu − CTC antisense oligonucleotide or treated with Angiogenin inhibitor were cultured in 24-well plates for 10 days using α-MEM supplemented with 10% fetal bovine serum, 0.5 mM 3-isobutyl-1-methylxanthine, 5 µg/ml insulin, and 1 µM dexamethasone. The medium was replaced every 3 days. Oil Red O (Sigma-Aldrich) staining was performed to detect lipids in mature adipocytes. The Oil Red O was extracted from the matrix and quantified spectrophotometrically at 492 nm. SA-β-gal staining For SA-β-gal staining in vitro, primary BMSCs transfected with 5'-tiRNA-Glu − CTC , 5'-tsRNA-Lys − CTT , TRF-5004b, 5'-tiRNA-Glu − CTC antisense oligonucleotide or treated with Angiogenin inhibitor were cultured in 24-well plates for 3 days using α-MEM medium. For senescence assessment, cellular senescence was detected using the Senescence-Associated β-Galactosidase Staining Kit (Solarbio, G1580) following the manufacturer's instructions. Fluorescence in situ hybridization (FISH) FISH for cells according to the manufacturer’s instructions using the RiboTM Fluorescent in Situ Hybridization kit (C10910). Briefly, young and aging primary BMSCs were seeded onto coverslips in 24-well plates. On the following day, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min and permeabilized for 5 min. Then, pre-hybridization was carried out at 37°C for 30 min, followed by hybridization with FITC-labeled-5'-tiRNA-Glu − CTC probe-containing hybridization buffer overnight at 37°C. The next day, the cells were washed with PBS and mounted with DAPI-containing medium for imaging under fluorescence microscope. FISH for bone tissue sections was performed as follows: 200 ng of FITC-labeled 5'-tiRNA-Glu − CTC , 5'-tsRNA-Lys − CTT and TRF-5004b probes were added to 10 mL hybridization buffer containing 2 × SSC, 10% dextran sulfate, 25 mM sodium phosphate, 100 mg salmon sperm DNA, and RNase inhibitor. The hybridization buffer was pre-warmed to 37°C, applied to the sections, and incubated in hybridization oven at 42°C for 24 h, followed by an additional 12 ~ 24 h of hybridization at 37°C. Then, the sections were washed with 2 × SSC buffer at 42°C for 10 min, followed by 3 times wash with 1×PBS. Finally, the sections were mounted with glycerol containing DAPI and immediately imaged using fluorescence microscope. Northern blotting assay Northern blot was performed using the Biotin Northern Blot Kit (for Small RNA) (R0220, Beyotime) according to the manufacturer’s protocol. Briefly, RNA samples were separated on a 15% urea-PAGE gel and the gel was transferred to a nylon membrane using semi-dry fast blotter (Guangzhou Dao One, FTB95) at 1.3A for 10 min. Then the membrane was crosslinked by UV irradiation for 5 min. The membrane was pre-hybridized at 37°C for 2 h, followed by hybridization with a biotin-labeled 5'-tiRNA-Glu − CTC probe (10 pmol/mL) at 37°C for 2 h. Subsequently, the membrane was incubated with HRP-streptavidin for 15 min. Signal detection was performed using a chemiluminescent substrate and imaging was visualized by ChemiDoc XRS Imaging System (Bio-Rad). tRNA-Glu cleavage in vitro The tRNA cleavage reaction was performed in a 10 µL mixture containing 0.5 µg tRNA-Glu, 40 mM Tris–HCl (pH 8.0), 20 mM KCl, 4 mM MgCl₂, and 2 mM DTT at 37°C for 40 min. Then the buffer was diluted with 2 × RNA loading buffer. The RNA samples were then denatured at 65°C for 10 min, immediately chilled on ice, and analyzed by Northern blotting to evaluate cleavage efficiency. RNA decay assay Cells transfected with 5'-tiRNA-Glu − CTC or negative control RNA were treated with actinomycin D (MCE, HY-17559) at a final concentration of 10 µg/mL. Total RNA was collected using TRIzol at 0, 1, 2, 8, and 16 h, followed by RT-qPCR analysis with GAPDH as the internal reference. The relative expression of the target gene at each time point (compared to 0 h) was calculated using the 2^(-ΔΔCT) method. The decay rate of mRNA concentration over time (dc/dt) is proportional to the degradation rate constant (k decay ) and the cytoplasmic mRNA concentration (c), as described by the equation: dc/dt = -k decay ·c. Thus, the mRNA half-life is calculated as: t (1/2) = ln(2)/k decay . Dual-Luciferase Reporter Assay Hek293T were transfected with either the pmirGLO-Foxm1, pmirGLO-Foxm1-mut, pmirGLO-Cdk1, pmirGLO-Cdk1-mut, pmirGLO-Capn1, pmirGLO-Capn1-mut, pmirGLO-Ccnd1, pmirGLO-Ccnd1-mut, pmirGLO-Sp7 and pmirGLO-Sp7-mut plasmids along with 5'-tiRNA-Glu − CTC or negative control RNA. After 48 hourstransfection, the cells were harvested and transferred to 96-well plates. The activities of firefly luciferase and Renilla luciferase were measured according to the manufacturer's instructions of the Dual-Glo® Luciferase Assay System (E2920, Promega). Flow cytometry BMSCs were transfected with 5'-tiRNA-Glu − CTC or negative control RNA, the cells were digested with pre-cooled 0.25% trypsin and washed with 1 × PBS to obtain cell pellet. The pellet was resuspended in BD fixation/permeabilization solution and fixed for 30 min at 4°C. Then cells were incubated with KI67 antibody (1:200, Proteintech, 27309-1-AP) at room temperature for 60 min and futher incubated with anti-Rabbit AF647 (1:200, proteintech, RGAR005) for 30 min. Cells were stained with DAPI (3 µg/mL) on ice for 10 min before analyzing. The proportions of cells in S-G2-M phase were calculated using FlowJo V10 (BD Biosciences). Intramedullary injection of adeno-associated virus The 5'-tiRNA-Glu − CTC and 5'-tiRNA-Glu − CTC − ASO sequence was cloned into pHBAAV-LEPR-Zs -Green shuttle plasmid (Hanbio Biotechnology Co.) and transformed into DH5α competent cells for large-scale amplification. The pHBAAV-LEPR-5'-tiRNA-Glu − CTC− -ZsGreen, pHBAAV-LEPR- 5'-tiRNA-Glu − CTC− ASO-ZsGreen and pHBAAV-LEPR-ZsGreen shuttle plasmids were cotransfected into HEK293T cells with pAAV-RC and pHelper plasmids. After transfection for 72 hours, HEK293T cells were collected and lysed, and the virus is purified by Adeno-Associated Virus (AAV) Purification Maxi Kit (V1469, Biomiga) and then concentrated by ultrafiltration tubes (Millipore, UFC905008).​​ A total of 1*10^11 vg of rAAV2/9-LEPR-5'-tiRNA-Glu − CTC or rAAV2/9-LEPR-5'-tiRNA-Glu − CTC -ASO and rAAV2/9-LEPR-Null was delivered via periosteal injection into the femoral bone marrow cavity and intervened for 1 month.​​ Preparation of bone-targeted liposomes Bone-targeted liposomes were prepared according to the following steps. Briefly, 100 mg of (AspSerSer, DSS)₆ peptide and 100 mg of DSPE-PEG2000-MAL were added to 5 mL of DMF and reacted via a maleimide-mediated reaction to form DSPE-PEG2000-(DSS)₆. The solution was purified through dialysis for 48h and freeze-dried to obtain the DSPE-PEG2000-(DSS)6. Then, 50mg ANG inhibitor NCI-65828, 150mg DSPE-PEG2000-(DSS)6, and 25mg egg yolk lecithin were co-dissolved in 6mL mixture of chloroform/methanol (1:1, V/V) solution. The mixture was formed into a thin film via a rotary evaporator, then hydrated under vacuum and sonicated at room temperature. The mixture was then sterilized via a 0.22-µm filter prior to subsequent experiments 39 . Calcein double-labeling assay Mice were intraperitoneally injected with calcein (25 mg/kg) at eight days and two days before euthanasia. Femurs were isolated, fixed in 70% ethanol, and embedded in methyl methacrylate. Sections (5 µm thick) were cut using a microtome and imaged under a fluorescence microscope. The mineral apposition rate (MAR) was measured to assess bone formation activity. Immunofluorescence and immunohistochemical staining and Histochemistry analysis For immunofluorescence staining, the bones were dissected and fixed in 4% paraformaldehyde for 24h, followed by decalcification in 0.5 M EDTA at 4°C for 21 days. The bone tissues were then embedded in cryo-embedding medium and sectioned into 6 µm slices. The sections were blocked with 3% BSA for 1 hour, incubated with primary antibodies SP7 (Abcam, AB209484) or Leptin R (AF497, NOVUS) at 4°C overnight, and then treated with Alexa Fluor 555 (1:200, Invitrogen, A31572) at room temperature for 1h the following day. The sections were mounted with DAPI-containing glycerol, and fluorescence signals were captured using Zeiss microscope (Apotome 3). For immunohistochemical staining, mouse bones were fixed in paraformaldehyde for 24 h after collection, decalcified at 4°C for 3 w, and then embedded in paraffin and sectioned at 6 µm. After deparaffinization and rehydration, the sections were stained with an OCN primary antibody and counterstained with hematoxylin. Stained images were quantified using ImageJ software. For histochemical staining, paraffin sections were processed for TRAP and H&E staining according to the manufacturer’s instructions. ELISA assay The concentration of human osteocalcin, Beta Crosslaps, Alkaline Phosphatase (ALP) of human serum were measured by using ELISA kit (Elabscience, E-EL-H1343, E-EL-H0960, E-BC-K009-M). The level of P I NP and TRAP-5b in human sreum were determined by using ELISA kit from JONLNBIO (JL14248, JL13353). The concentration of P I NP and CTX1 in mouse serum were detected by using ELISA kit from Elabscience (E-EL-M0233, E-EL-M3023). All measurements were performed according to the manufacturer’s instructions. RT-qPCR Total RNA was extracted with AG RNAex Pro Reagent (AG21102, Accurate Biology). RNA purity and concentration was determined by BioTek Epoch2 microplate reader (BioTek, USA) and 1 ug RNA was reverse transcribed into cDNA using the 5× Evo M-MLV RT Premix (AG11706, Accurate Biology). Real time PCR reaction was performed on ABI QuantStudio 3 system using 2X SYBR Green Pro Taq HS Premix (AG11718, Accurate Biology) in 20 uL reaction volumes containing 2 uL cDNA. The relative mRNA expression was normalized to GAPDH and calculated by 2 −ΔΔCT method. Western blotting assay Cells were lysed with lysis buffer containing protease inhibitor cocktail for 10 minutes on ice. The lysates were centrifuged at 12,000g for 10 minutes and the supernatant was transferred into a new tube. Supernatant was heated at 100°C for 10 minutes mixed with 6 × SDS loading buffer, then separated by SDS-PAGE and electro-transferred to PVDF membranes. After blocking with 5% skim milk for 2 hours, membranes were incubated with primary antibodies at 4°C overnight. Primary antibodies were FOXM1 (Proteintech, 13147-1-AP), CDK1 (Promab, 30181), CAPN1 (Promab, P07140), CCND1 (Promab, 31985), SP7 (Abcam, AB209484), LPL (Promab, P33464), P21 (Abcam, AB109199), P16 (Promab, P21210), ACTIN (Boster, BM0627). Secondary anti-mouse (Ap124p, MilliporeSigma) and anti-rabbit (Ap132p, MilliporeSigma) HRP-conjugated antibodies were applied the next day. Then the bands were visualized by ChemiDoc XRS Imaging System (Bio-Rad) with chemiluminescence reagent (Thermo Fisher Scientific, 32106). Micro-CT The femora were dissected from mice and fixed overnight in 4% paraformaldehyde. Then the bones were wrapped with sealing film and scanned using a high-resolution micro-computed tomography (µCT) system (Skyscan 1172, Bruker MicroCT, Kontich, Belgium). Image reconstruction was performed using NRecon software (version 1.6, Bruker MicroCT), followed by analysis with CTAn (version 1.9) for trabecular and cortical bone parameters (BV/TV, Tb.Th, Tb.N and Tb.Sp ) and 3D visualization with CTVol (version 2.0) with the region of 5% of the femoral length belowe the growth plate. Quantification and statistical analysis Data is presented as mean ± SEM and exhibits continuous normal distribution. Statistical analyses were performed using Excel or GraphPad Prism 8 software. Comparisons between two groups were conducted using two-tailed Student’s t-tests, and one-way or two-way ANOVA were used for comparisons among multiple groups. All experiments were repeated three times, and representative experiments are shown. For consistency in comparisons, significance across all figures is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. Sample sizes for in vivo and in vitro experiments were determined based on prior experience. All samples were randomly assigned, and no animals were excluded from the experiments. Results 1. Aging induces the production of 5 ' -tsRNA-Glu − CTC in Bone Marrow Mesenchymal Stromal Cells (BMSCs) Previous studies have demonstrated that the osteogenic-adipogenic differentiation bias of BMSCs during aging constitutes a significant cause of age-related bone loss 40 . To test whether transfer RNA-derived small RNAs (tsRNA) is involved in this progression, we first performed small RNA sequencing on BMSCs derived from 3-month-old (young) and 20-month-old (aged) mice to identify the expression pattern of tsRNAs during BMSCs aging. We identified two novel tsRNAs, TRF-mmu-053 and TRF-mmu-007, and a known tsRNA, TRF-5004b, which exhibited a pronounced increase in aged BMSCs compared to young BMSCs, with counts > 500, logFC > 2, and p < 0.05 (Fig. 1 A). By analyzing the secondary structures of TRF-mmu-053 and TRF-mmu-007 using R2DT 41 . We found that TRF-mmu-053 partly matched of tRNA-Glu − CTC and was characterized as 5'-tiRNA-Glu − CTC , while TRF-mmu-007 partly matched of tRNA-Lys − CTT and was defined as 5'-tsRNA-Lys − CTT (Fig. 1 I). To validate the increased expression of the three tsRNAs, we performed fluorescence in situ hybridization (FISH) on aged (20-month-old) bone tissues and young (3-month-old) samples, utilizing specific DNA probes complementary to 5'-tiRNA-Glu − CTC , 5'-tsRNA-Lys − CTT and TRF-5004b. Indeed, the three tsRNAs exhibited a significant increase in expression within aged bone tissues compared to young samples (Fig. 1 B and C). The colocalization assay combining Leptin Receptor (LEPR) immunofluorescence staining and FISH staining revealed that 5'-tiRNA-Glu − CTC and 5'-tsRNA-Lys − CTT were primarily expressed in BMSCs. Specifically, 5'-tiRNA-Glu − CTC exhibited a higher colocalization rate with LEPR (Fig. 1 B and D). We subsequently assessed the regulatory effects of the three tsRNAs on BMSCs differentiation in vitro. Primary BMSCs were transfected with the three tsRNAs and incubated with osteogenic or adipogenic differentiation medium. Alizarin Red S and Oil Red O staining revealed that treatment with the three tsRNAs significantly inhibited calcium nodule formation while promoting lipid droplet formation. Among the tested conditions, 5'-tiRNA-Glu − CTC and 5'-tsRNA-Lys − CTT exhibited a stronger inhibitory effect on calcium nodule formation and a greater promoting effect on lipid droplet formation compared to TRF-5004b (Fig. 1 F–H). SA beta-gal staining showed that over-expressing the three tsRNAs exhibited a higher proportion of β-Gal-positive senescent cells compared to the control group (Fig. 1 E and H). Similarly, relative to TRF-5004b, 5'-tiRNA-Glu − CTC and 5'-tsRNA-Lys − CTT exhibited a stronger pro-senescence effect (Fig. 1 E and H). These data suggested that 5'-tiRNA-Glu − CTC and 5'-tsRNA-Lys − CTT and TRF-5004b disrupt the lineage fate of BMSCs and induce their senescence. Based on the results of in situ hybridization and cellular staining, we selected 5'-tiRNA-Glu − CTC and 5'-tsRNA-Lys − CTT for the follow-up research. To identify the mRNA regulatory patterns governed by 5'-tiRNA-Glu − CTC and 5'-tsRNA-Lys − CTT , we collected BMSCs transfected with 5'-tiRNA-Glu − CTC and 5'-tsRNA-Lys − CTT and performed RNA sequencing. The data showed that over-expression of 5'-tiRNA-Glu − CTC resulted in 477 upregulated and 674 downregulated genes, while over-expression 5'-tsRNA-Lys − CTT led to 482 upregulated and 481 downregulated genes (Fig. 1 J). KEGG pathway enrichment analysis revealed that these differentially expressed genes from 5'-tiRNA-Glu − CTC over-expression group were significant enrichmented in cell adhesion molecules, cellular senescence and cell cycle pathways (Fig. 1 K). Meanwhile, these differentially expressed genes from 5'-tsRNA-Lys − CTT over-expression group were significant enrichmented in cell adhesion molecules, cell cycle pathways and calcium signaling pathway (Fig. 1 K). Examination of differentially expressed genes related to senescence, osteogenesis, and adipogenesis showed downregulation of anti-aging genes (e.g., Foxm1 , Cdk1 , Ccnd1 ) and osteogenic genes (e.g., Sp7 , Col1a1 , Dmp4 ), and upregulation of adipogenic (e.g., Lpl , Pparg , Fabp4 ) and pro-inflammatory genes (e.g., IL-6 , IL-10 , Ccl3 ) (Fig. 1 L). These results were validated by real-time quantitative PCR (RT-qPCR) (Fig. 1 M). Given that the results of the colocalization assay and RNA sequencing, 5'-tiRNA-Glu − CTC may be act as a more important regulator driving BMSCs differentiation shift and cellular senescence. It is consistent with a previous result showing a regulatory role of 5'-tiRNA-Glu − CTC in brain aging and age-related memory decline 38 . Therefore, we selected 5'-tiRNA-Glu − CTC for further study. ​ ​​​2. 5 ' -tiRNA-Glu − CTC accelerates bone loss in both male and female Mice​​ To explore the role of tRNA-derived small RNAs on bone metabolism in vivo, we utilized rAAV serotype 2/9 with LEPR promoter for delivery of 5'-tiRNA-Glu − CTC (rAAV2/9-LEPR-5'-tiRNA-Glu − CTC ) to BMSCs. Young (3-month-old) and aged (12-month-old) male and female mice received intra-bone marrow injections of rAAV-delivered 5'-tiRNA-Glu − CTC or rAAV-2/9 vector (Fig. 2 A). All mice received two intraperitoneal calcein injections within the last six days of the one-month period before serum, bone, and bone marrow collection. To explore the overexpression efficiency of 5'-tiRNA-Glu − CTC in BMSCs, we isolated and cultured BMSCs from the 5'-tiRNA-Glu − CTC group and the AAV-2/9 vector group. RT-qPCR analysis showed that 5'-tiRNA-Glu − CTC levels were significantly increased in the 5'-tiRNA-Glu − CTC group compared to the control group, indicating that rAAV2/9 successfully targeted BMSCs and boosted 5'-tiRNA-Glu − CTC expression in BMSCs (Fig. 2 B and Fig. 3 A). ELISA analysis revealed significantly reduced serum PINP levels and elevated CTX levels in both young and aged treated mice compared to control groups (Fig. 2 C and D). These findings suggest that 5'-tiRNA-Glu − CTC exerts an inhibitory effect on bone formation. Micro-computed tomography (micro-CT) analysis of the distal femoral metaphysis revealed significantly reduced trabecular bone mass, trabecular thickness and trabecular number and increased trabecular separation in AAV-5'-tiRNA-Glu − CTC treated male mice compared to control groups (Fig. 2 E-I). Hematoxylin and eosin (H&E) staining confirmed a reduction in trabecular area in mice injected with rAAV-delivered 5'-tiRNA-Glu − CTC compared to the negative control group (Fig. 2 J and K). Immunohistochemical and immunofluorescence analyses revealed decreased numbers of osteoblasts, SP7-positive cells, and LEPR-positive cells, along with increased osteoclast numbers, in both young and aged male mice treated with rAAV-delivered 5'-tiRNA-Glu − CTC compared to placebo-treated groups (Fig. 2 L–S). Similarly, calcein double-labeling indicated a reduced mineral apposition rate (MAR) in the 5'-tiRNA-Glu − CTC -treated group relative to controls (Fig. 2 T and U). Consistently, AAV-5'-tiRNA-Glu − CTC -treated also exhibited significant reductions in bone mass and trabecular number in female mice (Fig. 3 B-F). Osteoblast numbers, SP7-positive cells, LEPR-positive cells, and MAR were decreased compared to control groups (Fig. 3 G-L and O-P). Interestingly, unlike in males, 5'-tiRNA-Glu − CTC overexpression did not alter osteoclast numbers in female mice, and its effects on trabecular thickness and separation were inconsistent across age groups (Fig. 3 M-N). These results demonstrated that 5'-tiRNA-Glu − CTC overexpression impairs bone formation. ​ ​​​3. Inhibition of 5 ' -tiRNA-Glu − CTC via ASO ameliorates BMSCs senescence and bone loss in aged mice These lines of evidence support 5'-tiRNA-Glu − CTC as a potential target for age-related bone less. Based on the biological mechanism by which antisense oligonucleotides bind to target RNA and induce RNase H-mediated RNA degradation. We next designed an antisense oligonucleotide (ASO) targeting 5'-tiRNA-Glu − CTC , and transfected it into primary bone marrow mesenchymal stromal cells (BMSCs) isolated from aged mice. Subsequently, we performed Alizarin Red S, Oil Red O staining and SA β-galactosidase staining assays to comprehensively evaluate the effect of ASO on BMSCs differentiation and senescence in osteogenic or adipogenic differentiation medium. The results showed that 5'-tiRNA-Glu − CTC -ASO treatment significantly reduced the number of senescent cells in aged BMSCs, enhanced mineralization (as indicated by increased calcium nodule formation), while decreased lipid droplet formation (as demonstrated by decreased Oil Red O staining), compared to the control group (Fig. 4 A–C). The data suggested that the inhibitor of 5'-tiRNA-Glu − CTC would ameliorate BMSC senescence and differentiation shift. Based on the ameliorative effects of 5'-tiRNA-Glu − CTC -ASO on aged BMSCs, we are eager to know whether the ASO-based inhibitor of 5'-tiRNA-Glu − CTC ameliorates age-associated bone loss in vivo. The rAAV2/9-LEPR-5'-tiRNA-Glu − CTC -ASO was constructed and delivered via intra-bone marrow injection into aged (18 month) male and female mice at 1×10¹¹ vg per mouse for a one-month intervention. The control group received injection of an equivalent volume of viral vector. Compared to control groups, mice treated with rAAV2/9-LEPR-5'-tiRNA-Glu − CTC -ASO exhibited increased bone mass, greater trabecular thickness and number, and reduced trabecular separation (Fig. 4 D–H). Beyond that​​, rAAV2/9-LEPR-5'-tiRNA-Glu − CTC -ASO-treated aged female mice had fewer marrow adipocytes, while males showed increased trabecular bone mass (Fig. 4 I and J). Immunostaining for osteocalcin, tartrate-resistant acid phosphatase (TRAP), SP7, and LEPR revealed that 5'-tiRNA-Glu − CTC -ASO-treated aged mice exhibited increased osteoblasts, SP7-positive cells, and LEPR-positive cells, decreased osteoclasts, and an improved mineral apposition rate (MAR) in both aged male and female mice compared with the control group (Fig. 4 K–T). These results demonstrated that the ASO-based inhibitor of 5'-tiRNA-Glu − CTC alleviates age-associated bone loss. ​ ​4. 5 ' -tiRNA-Glu − CTC induces BMSCs senescence by disrupting mRNA stability​ ​ It is well established that tiRNAs inhibits global translation or selectively suppress of specific gene expression by disrupting ribosomal assembly, interacting with initiation factors such as eIF4F, and promoting the formation of stress granules (SGs) 19 , 33 , 42 . Besides, certain 5'-tsRNAs can recognize the 3' UTR of mRNAs and repress their expression 23 .​ To explore the regulatory mechanism by which 5'-tiRNA-Glu − CTC modulates BMSC senescence and differentiation. Firstly, to identify specific mRNA targets that bind to 5'-tiRNA-Glu − CTC , we performed high-throughput sequencing of mRNA fragments pulled down by biotin-labeled 5'-tiRNA-Glu − CTC using streptavidin beads (Fig. 5 A). The distribution of sequencing reads across the genome was analyzed, and the results revealed sequencing reads were mapped to exonic (85.46%), intronic (9.91%) and intergenic (4.63%) (Fig. 5 B). The data indicated that the sequencing reads were mainly distributed in the exon region. To further identify target genes directly regulated by 5'-tiRNA-Glu − CTC , we performed an overlap analysis between the result of 5'-tiRNA-Glu − CTC pulled down assay followed by high-throughput sequencing and mRNA expression profiling via mRNA-seq (Fig. 1 L), which identified 312 overlapping genes (Fig. 5 C). We next sought to elucidate the biological pathways and functions commonly associated with these 312 overlapping genes across multiple conditions. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the 312 overlapping genes were significantly enriched in phagosome pathway, cellular senescence, antigen processing and presentation and the cell cycle pathways (Fig. 5 D and E). Based on these KEGG results, we focused subsequent analyses on genes related to cellular senescence and cell cycle regulation, including Foxm1, Cdk1, Capn1 and Ccnd1 . Through the analysis of peak reads distributed across genes from high-throughput sequencing results, we identified 5'-tiRNA-Glu − CTC mainly binds to the 3′UTRs of Foxm1, Cdk1, Capn1, Ccnd1 , and Sp7 (Fig. 5 F). Based on the complementary sequences of Foxm1, Cdk1, Capn1, Ccnd1 , and Sp7 retrieved from National Center for Biotechnology Information (NCBI) database, we identified seed binding sites for 5'-tiRNA-Glu − CTC (Fig. 5 G). Previous studies have supported that small RNAs influence mRNA stability through binding to their 3'UTR regions and promoting degradation through AU-rich elements or by facilitating microRNA binding 43 . To further validate whether 5'-tiRNA-Glu − CTC impairs mRNA stability by binding to the 3′UTRs of these mRNA, we transfected BMSCs cell lines with 5'-tiRNA-Glu − CTC or negative RNA for 48h, followed by actinomycin D treatment and collected RNA samples at multiple time points over 16 h. We found that that the relative Foxm1 mRNA half-life was 30.42 h of BMSCs treated with negative RNA through RT-qPCR. Notably, in 5'-tiRNA-Glu − CTC -overexpressing BMSCs, Foxm1 mRNA levels were decreased faster upon actinomycin D treatment and the resulting relative Foxm1 mRNA half-life (10.36 h) was reduced by 65.94%. Meanwhile, other senescence-related mRNA half-life of Cdk1 , Capn1 and Ccnd1 were reduced by 45.4%, 23.96% and 59.64% respectively, and the osteogenic mRNA level of Sp7 was decreased by 61.2% (Fig. 5 H). The results demonstrated that 5'-tiRNA-Glu − CTC influences target genes' mRNA stability by binding to their 3′UTRs. To further verify whether 5'-tiRNA-Glu − CTC mediates mRNA degradation through the 3'UTR, we cloned the 3' UTR or mutated seed region of target genes into Dual-Luciferase Reporter Vector (pmirGLO) and constructed pmirGLO-WT or pmirGLO-Mut plasmids. The pmirGLO-WT or pmirGLO-Mut plasmids were co-transfected into HEK293T cells along with either 5'-tiRNA-Glu − CTC or negative control RNA. As expected, ectopic expression of 5'-tiRNA-Glu − CTC induced a significant decrease in Fluc activity of the WT senescence-related (e.g., Foxm1 , Cdk1 , Capn1 , Ccnd1 ) and osteogenic (e.g., Sp7 ) genes reporters. Conversely, this decrease was rescued by mutations in the seed regions of 3' UTRs (Fig. 5 I). The degradation of mRNAs will disrupt translation and lead to a decrease in protein translation. Western blotting confirmed that 5'-tiRNA-Glu − CTC overexpression in young BMSCs downregulated senescence-related (e.g., P16, P21, FOXM1, CDK1, CAPN1, CCND1) and osteogenic (e.g., SP7) proteins and upregulated adipogenic (e.g., LPL) proteins, while 5'-tiRNA-Glu − CTC -ASO treatment in aged BMSCs reversed these effects (Fig. 5 O). These findings suggest that 5'-tiRNA-Glu − CTC decreases target genes expression by binding to 3'UTR of mRNAs, thereby disrupting mRNA stability. Since the target genes like Foxm1, Cdk1, and Ccnd1 are closely associated with the cell cycle, we further performed flow cytometry to analyze the cell cycle distribution. 48 h after transfecting young primary BMSCs with 5'-tiRNA-Glu − CTC or negative RNA, we stained cells with Ki67 antibody and DAPI dye to categorize the cells into three stages: G 0 phase, G 1 phase, and G 2 /S/M phase. We found that the proportion of BMSCs transfected with 5'-tiRNA-Glu − CTC in the G 2 /S/M phase was significantly decreased compared to the control BMSCs (Fig. 5 J and k), indicating that 5'-tiRNA-Glu − CTC impairs the proliferative activity of BMSCs. Subsequently, we performed Ki67 immunofluorescence staining in primary BMSCs, which further validates that 5'-tiRNA-Glu − CTC inhibits cell proliferation (Fig. 5 L and M). To investigate whether ASO-based inhibitor of 5'-tiRNA-Glu − CTC will rescue aged primary BMSCs' senescence, we transfected 5'-tiRNA-Glu − CTC -ASO into aged BMSCs. The result indicated that 5'-tiRNA-Glu − CTC -ASO-expressed aged BMSCs increased expression of senescence-related (e.g., Foxm1 , Capn1 ) and osteogenic (e.g., Sp7 ) genes and decreased adipogenic gene (e.g., Lpl ) expression by RT-qPCR (Fig. 5 N) and western blotting assessment (Fig. 5 O). These results demonstrated that 5'-tiRNA-Glu − CTC regulates BMSC senescence and differentiation by impairing the mRNA stability of anti-senescence-related genes (e.g., Foxm1, Cdk1, and Ccnd1 ) and pro-osteogenic genes (e.g., Sp7 ) (Fig. 7 R). ​ ​5. Cellular stress triggers ANG-cleaving activity and induces 5 ' -tiRNA-Glu − CTC production Previous studies have demonstrated that tiRNA, a product of the endoribonuclease ANG (Angiogenin), is generated through the cleavage of specific mature tRNA under diverse stress conditions- including oxidative stress, heat shock, hypothermia, hypoxia, and cold shock 44 , 45 . As an important component of cellular stress responses, tiRNA plays a critical role in mitigating environmental stress and maintaining cellular homeostasis 44 . Aging is considered a chronic stress process characterized by increased oxidative stress; mounting evidences show that tsRNAs are differentially expressed in model organisms of different ages. Subsequently, we examined the changes in endoribonuclease expression during bone marrow mesenchymal stromal cells (BMSCs) senescence. The expression pattern of endoribonuclease in 2-month-old (young) and 24-month-old BMSCs was analyzed using RNA-sequencing 46 . As expected, we observed upregulation of multiple tRNA-cleaving endoribonucleases including ANG in aged BMSCs (Fig. 6 A). To further confirm the upregulation of ANG expression in aged BMSCs, we performed immunofluorescence co-staining of ANG and LEPR in bone tissues from 2- and 24-month-old mice. The result showed that ANG was specifically upregulated in aged BMSCs and exhibited a significant colocalization rate with LEPR (Fig. 6 B and C). Correspondingly, relative quantitative analysis of ANG levels via RT-qPCR in young and aged female and male mice also confirmed this result (Fig. 6 D). Next, we sought to determine whether ANG serves as the pivotal enzyme responsible for the formation of 5'-tiRNA-Glu − CTC in aged BMSCs. Firstly, we assessed ANG-mediated cleavage of tRNA-Glu using Northern blotting. The results revealed that ANG specifically cleaves full-length tRNA-Glu into a unique 5'-tiRNA-Glu − CTC in an in vitro reaction system. Notably, the abundance of this tiRNA exhibited a significant dose-dependent increase with ANG concentration (Fig. 6 L). Subsequently, we conducted in situ hybridization using a FITC-labeled 5'-tiRNA-Glu − CTC probe, combined with co-localization staining for ANG in both young and aged BMSCs. As expected, compared to young BMSCs, the levels of 5'-tiRNA-Glu − CTC and ANG expression were significantly increased in aged BMSCs (Fig. 6 K). Researchers have proposed that ANG-induced tiRNAs may contribute to the formation of SGs (stress granules) in response to cellular stress. The formation of stress granules (SGs) promotes cell survival under various stress conditions, including heat shock, oxidative stress, ischemia, and viral infection. We further investigated stress granule (SG) formation in aged BMSCs. We first treated mouse BMSCs with 500 µM sodium arsenite (NaAsO 2 ) for one hour—a well-established inducer of cellular oxidative stress. After treatment, we examined the expression of G3BP1, a canonical marker for stress granules (SGs). Notably, NaAsO 2 treatment significantly increased the proportion of G3BP1-positive cells compared to the control group (Fig. 6 E and F). This indicates that the stress induced by NaAsO 2 leads to the formation of stress granules, as evidenced by the upregulation of G3BP1. Furthermore, we performed immunofluorescence staining for G3BP1 to visualize the abundance of stress granules in aged BMSCs. The results revealed a significant increase in SG abundance in aged BMSCs compared to young BMSCs (Fig. 6 G and H). This suggests that aged BMSCs have a higher propensity to form stress granules under stress conditions. This observation suggests that enhanced SG formation may be beneficial to the survival of aged cells under physiological stress 47 . It has been reported that under stress, ANG translocates from the nucleus to the cytoplasm, where it cleaves specific tRNAs into tiRNAs, subsequently disrupting cellular physiological functions and inducing age-related diseases 38 . Similarly, in our experiments, treatment of BMSCs with NaAsO 2 —compared to the negative control group—not only increased cytoplasmic ANG levels but also led to elevated levels of 5'-tiRNA-Glu − CTC . Specifically, under normal conditions, ANG is predominantly localized in the nucleus of BMSCs, whereas under oxidative stress, it translocates to the cytoplasm and cleaves tRNA-Glu to generate 5'-tiRNA-Glu − CTC (Fig. 6 I–J). Consistent with this mechanism, compared to young BMSCs, aged BMSCs also exhibited pronounced translocation of ANG from the nucleus to the cytoplasm, along with higher levels of 5'-tiRNA-Glu − CTC , as further confirmed by in sit hybridization at the cellular level (Fig. 6 K). The results were further validated by western blotting, confirming the subcellular redistribution of ANG during the aging process of BMSCs (Fig. 6 M). These findings suggest that the distribution of ANG in the aged BMSCs undergoes a significant shift from the nucleus to the cytoplasm. Our results demonstrate that ANG shifts from the nucleus to the cytoplasm, triggering ANG-mediated tRNA cleavage activity as a critical contributing factor to cellular senescence. This finding suggests that targeting the inhibition of ANG activity may serve as a therapeutic strategy for age-related osteoporosis. A study screening small-molecule inhibitors targeting the ribonucleolytic activity of human angiogenin (ANG) demonstrated that NCI compound 65828 (NCI-65828) significantly inhibits ANG enzymatic activity- with Ki values < 100 µM. Subsequent studies revealed that ANG inhibition ameliorated cell stress induced by 5′tRNA-derived fragments (tiRNAs) and rescued the deleterious effects of NSun2 deficiency during neurodevelopment 48 , 49 . In a subsequent study, we aim to investigate whether angiogenin (ANG) inhibition, using the NCI compound 65828, can alleviate BMSCs senescence and age-associated bone loss. The molecular docking model of ANG with NCI-65828 was generated using AUTODOCK 3.0. In this model (Fig. 6 N), the azo group is positioned in the catalytic center alongside Glu140. The main-chain oxygen atom is bound by His37, His137, and the Lys64 side chain. Additionally, the side chains form hydrogen bonds with the residues Asp65 and Val66.This binding model differs from the reported binding pattern of the human ANG protein 48 . To determine the optimal concentration of ANG for cell treatment, we treated aged primary BMSCs with ANG inhibitor in a dose-response study. The CCK-8 assay showed that the ANG inhibitor-NCI-65828 promoted cell viability at lower concentrations (10 µM), whereas it inhibited cell proliferation at higher concentrations (> 50 µM) (Fig. 6 O). Western blotting analysis demonstrated that treatment with the ANG inhibitor NCI-65828 at a concentration of 10 µM significantly increases the expression of the protein targeted by 5'-tiRNA-Glu − CTC (Fig. 6 P). The results indicate that 10 µM NCI-65828 represents the optimal concentration for cellular treatment. We next treated aged BMSCs with NCI-65828 (10 µM) and found that it significantly reduced the area of SA-β-gal staining (Fig. 6 Q), enhanced calcium nodule formation, and decreased lipid droplet formation (Fig. 6 R and S). Collectively, these findings demonstrate that NCI-65828 effectively attenuates senescence in aged BMSCs, enhances osteogenic differentiation while inhibiting adipogenic differentiation, thereby modulating the lineage commitment balance. ​ ​​​6. Inhibitor of ANG-cleaving activity ameliorates age-related bone loss in aged mice​​ To investigate whether NCI-65828, as a targeted ANG inhibitor, ameliorates age-related bone loss, the NCI-65828 was encapsulated into bone-targeted liposomes and delivered via intra-bone marrrow injection into aged (18 month) male and female mice for a one-month intervention. Control group received injection of an equivalent volume of empty liposomes. Compared to control groups, mice treated with NCI-65828 exhibited increased bone mass, greater trabecular thickness and number, and reduced trabecular separation (Fig. 7 A–E). Furthermore, ANG inhibitor-treated mice exhibited higher levels of Procollagen type I N-terminal propeptide (PINP) and lower levels of C-terminal telopeptide of type I collagen (CTX) compared to the control group, as determined by ELISA analysis (Fig. 7 F and G). Histological analysis further demonstrated a lower proportion of adipocytes in female treatment groups and a larger trabecular area in male treatment groups (Fig. 7 H and I). In addition, we performed immunohistochemical (IHC) staining on paraffin sections of the femur and immunofluorescence (IF) staining on frozen sections of the tibia. Compared to control group, mice treated with the ANG inhibitor exhibited a higher proportion of osteoblasts, Sp7-positive cells, and LEPR-positive cells, along with a lower proportion of osteoclasts in bone tissue (Fig. 7 J–Q). These findings suggest that NCI-65828 treatment exerts a ameliorative effect on age-related bone loss, and inhibition of ANG activity is recognized as a potential therapeutic strategy for osteoporosis (Fig. 7 R). ​​7. 5 ' -tiRNA-Glu − CTC serves as a novel serum biomarker for assisting in the diagnosis of osteoporosis​ As we know, transfer RNA-derived fragments as (tsRNAs) have also been suggested as potential biomarkers for various diseases and health conditions. Studies have indicated that certain tsRNAs are specifically changed in the serum of osteoporosis patients 50 . In our study, we collected peripheral blood samples from 40 osteoporosis patients (aged 70 ~ 90 years) and serum samples from 40 age- and gender-matched healthy individuals. The serum samples from osteoporosis patients and those from healthy controls were each pooled separately into individual tubes and subsequently subjected to small RNA sequencing analysis. The subtype distribution demonstrated that differentially expressed (DE) tsRNAs derived from tRNA-Glu − CTC , tRNA-Glu − TTC , tRNA-Gly − GCC , tRNA-Phe − GAA and tRNA-Pro − TGG were the most abundant (Fig. 8 C). In our analysis of differentially expressed small RNAs, we found that small RNAs displayed a cholinergic-associated shift, from miRNAs to tsRNAs. Specifically, following filtration with a count threshold greater than 10, differential expression analysis of small RNAs revealed that 79% of the 323 DE tsRNAs including 5'-tiRNA-Glu − CTC were upregulated, whereas 55% of the 110 DE miRNAs were downregulated (Fig. 8 A and B). The observed cholinergic shift is consistent with the phenomenon seen in post-stroke patients when compared to healthy controls 51 . Notably, the 55% DE miRs included several miRs known to be perturbed in osteoporosis: has-miR-100-5p, has-miR-125-5p, and has-miR-17-5p 52,53 (Fig. 8 B). Our findings point toward tsRNAs/miRNAs as potential biomarkers for increased osteoporosis risk in these patients. In this study, we investigate whether serum levels of 5'-tiRNA-Glu − CTC could serve as a candidate diagnostic biomarker for osteoporosis, focusing on its expression patterns and clinical correlation with bone metabolism markers. Therefore, we quantified the serum levels of 5'-tiRNA-Glu − CTC , bone formation-related markers (including PINP, BGP/Osteocalcin), and bone resorption-related markers (including CTX and TRAP) in 40 osteoporosis patients using ELISA analysis. Although 5'-tiRNA-Glu − CTC did not show a strict correlation with age (Fig. 8 D), its levels were negatively correlated with BMD and Z-scores in osteoporosis patients (Fig. 8 E–F). Moreover, serum 5'-tiRNA-Glu − CTC levels correlated negatively with osteogenic markers (PINP and BGP; Fig. 8 G–H) and positively with osteoclast markers (CTX and TRAP; Fig. 8 J–K). A positive correlation was also observed between serum ANG and 5'-tiRNA-Glu − CTC levels (Fig. 8 L), which supports the proposed mechanism that ANG-mediated cleavage contributes to the biogenesis of this tRNA fragment. In contrast, correlations with ALP, serum calcium, and 25-hydroxyvitamin D3 remained weak and require further validation (Fig. 8 I, M–N). Together, these findings indicate that 5'-tiRNA-Glu − CTC may serve as a promising serum biomarker for diagnosing osteoporosis. Discussion Emerging evidence has implicated the accumulation of reactive oxygen species (ROS) and increased oxidative stress as causative factors in the pathogenesis of age-related osteoporosis 54 , 55 . As an important component of stress responses, angiogenin-induced tRNA cleavage neutralizes stress stimuli by decreasing general translation while reprogramming pro-survival and anti-apoptotic protein translation, conserving anabolic energy, and promoting cell survival 17 , 30 , 37 , 38 , 56 . However, whether increased oxidative stress triggers angiogenin-induced tRNA cleavage involved in the progression of age-related osteoporosis remains largely unknown. In this study, we demonstrated that oxidative stress induces angiogenin translocation from the nucleus to the cytosol, where it cleaves tRNA-Glu, leading to abundant accumulation of the 5'-tiRNA-Glu − CTC fragment in senescent BMSCs. The age-induced 5'-tiRNA-Glu − CTC alters mRNA metabolism through a regulatory mechanism highly similar to that of miRNAs, leading to bone-fat imbalance and thereby accelerating bone loss. This study elucidates a novel regulatory axis in age-related osteoporosis, wherein oxidative stress-induced tRNA fragmentation operates as a central regulatory mechanism. Observations demonstrate that tRNA fragments are present across diverse organisms and show stress-induced upregulation. In this study, we mainly focus on 5'-tiRNA-Glu − CTC , which is significantly accumulated in senescent BMSCs and was also detected in aging medial prefrontal cortices (mPFCs) 38 . As reported in a previous study, 5'-tiRNA-Glu − CTC serves as a major factor triggering age-related defects in cell function. Our data indicates that the identification of 5'-tiRNA-Glu − CTC as a critical factor contributes to the disruption of bone-fat differentiation balance in BMSCs. Research indicates that tiRNA suppresses protein translation through multiple mechanisms: (1) displacing the cap-binding complex eIF4F from capped mRNA, (2) competitively binding to LARS2 (a mitochondrial leucyl-tRNA synthetase) to disrupt mitochondria-encoded protein translation, or (3) reducing mRNA transcript stability 38 , 57 , 58 . In our study, we found that tiRNA, similar to miRNAs, suppresses its targets through a mechanism that may depend on its incorporation into Argonaute (Ago) protein complexes. Apart from this regulatory mechanism, tiRNAs may also interact with candidate proteins involved in BMSCs differentiation - a hypothesis that merits further investigation. It is worth noting that some tRNA-derived fragments (tRFs), which represent another type of fragments derived from tRNAs and are cleaved by Endonuclease Z (RNaseZ), cytoplasmic homologous ribonuclease Z2 (ELAC2), and Dicer, also exhibited a pronounced increase in senescent BMSCs. Unlike tiRNA, tRFs are processed by specific endonucleases and play diverse functions in various diseases 59 . It is suggested that tRFs may be involved in regulating BMSCs senescence and differentiation, which will be further investigated in our future research. Recently research reported that some novel endonucleases, such as schlafen family member 12 (SLFN12) selectively digests tRNALeu(TAA) producing more complex tRNA fragments, which is not similar to the traditional tRFs 42 . This implies that previously unidentified tRNA-cleaving endonucleases may exist, and that the structural complexity of tRFs could be greater than our current understanding. We believe that investigating the mechanisms underlying tRF-mediated regulation is significant importance while presenting significant challenges. Angiogenin (ANG), a member of the secreted ribonuclease (RNASE) superfamily, exerts diverse physiological functions, ranging from promoting angiogenesis to enabling cell growth, proliferation, and survival under adverse conditions 20 . Recently a study reported that angiogenin, secreted by osteoclasts, protects neighboring vascular cells against senescence 60 . In our study, we demonstrated that angiogenin-mediated cleavage of specific tRNAs generates tiRNA products, such as 5'-tiRNA-Glu − CTC , which induces BMSCs senescence and accelerates bone loss via alterations in mRNA metabolism. Our data indicate that angiogenin functions as a detrimental regulator of BMSCs senescence and differentiation. Notably, other previous studies have also identified angiogenin as a detrimental regulator, aligning with its established role in modulating hippocampal neurogenesis and age-related memory decline through angiogenin-mediated tRNA cleavage 38 , 49 . These controversial findings may be explained by the tightly regulated substrate recognition of angiogenin and its subcellular localization in response to cellular stress conditions 20 . Under growth conditions, angiogenin is translocated to nucleus and stimulates ribosomal RNA (rRNA) transcription, thus facilitating cell growth and proliferation. This may represent a biological process that promotes the proliferation of young BMSCs. Under stress conditions, angiogenin accumulates in cytoplasmic compartments, where it modulates the production of tiRNA and triggers stress granule (SG) formation to protect cells from cellular stress 61 – 63 . The physiological significance of this process lies in its promotion of senescent cell survival under cellular stress conditions 34 , 62 . In our study, the process by which angiogenin translocates to the cytosol appears to promote senescent BMSCs survival by triggering stress granule (SG) formation under oxidative stress. Meanwhile, angiogenin accumulates in the cytosol, where it cleaves specific tRNAs to generate tiRNA, thereby suppressing global protein translation. As demonstrated in our study, angiogenin-induced 5'-tiRNA repress the expression of anti-senescence and pro-osteogenic genes, including Foxm1 , Cdk1 , Capn1 , Ccnd1 , and Sp7 . Regarding the differential physiological functions, a proposed mechanism suggests that angiogenin-mediated tiRNA selectively inhibit the translation of mRNAs containing weak internal ribosome entry sites (IRES), while simultaneously enhancing the translation of mRNAs with strong IRES elements that promote cell survival under stress conditions 20 , 64 . As a result, physiological progression contributes to the survival of senescent BMSCs with impaired osteogenic differentiation potential under oxidative stress conditions. Another alternative hypothesis to explain the challenge role of angiogenin is proposed: Only a subset of senescent BMSCs exhibit high angiogenin expression, whereas young BMSCs show either no expression or significantly lower expression levels. Consequently, only these angiogenin-strongly positive senescent BMSCs are susceptible to angiogenin-induced tRNA cleavage under oxidative stress. This observation not only corroborates the well-documented heterogeneity of senescent cells but also positions angiogenin as a potential novel senescence marker 65 . Furthermore, these findings provide a mechanistic explanation for how pharmacological inhibition of angiogenin activation via NCI-65828 ameliorates BMSCs senescence and age-related bone loss. Previous studies have supported the notion that eliminating senescent cells enhances bone formation in age-related osteoporosis. Our findings also indicate that targeting the elimination of senescent BMSCs with elevated angiogenin expression considers as a viable therapeutic strategy for age-related osteoporosis 39 , 66 . Of note, there are four aspects of this field that are interesting and worth studying in the future. The first aspect pertains to the mechanism underlying angiogenin translocation from the nucleus to the cytosol under oxidative stress conditions, with a focus on identifying regulatory factors and signaling pathways involved in this process. The second aspect focuses on the factors influencing the activation of angiogenin under oxidative stress. By using immunoprecipitation with a specific anti-angiogenin antibody followed by LC-MS analysis, we aim to identify angiogenin-interacting proteins and further investigate which candidate binding proteins, such as RNH1, activate angiogenin under oxidative stress 67 – 69 . The third aspect concerns the reason why tRNA is cleaved by angiogenin. Studies have reported that Dnmt2 - and NSun2 -mediated cytosine-5 methylation (m5C) promotes tRNA stability. A lack of m5C modification increases stress-induced cleavage of tRNAs and sensitizes flies to oxidative stress 49 , 70 , 71 . This suggests that angiogenin tends to cleave tRNAs lacking m5C modification within the anti-codon loop, although this phenomenon is limited to specific tRNAs. Based on these findings, we tentatively propose that downregulation of the m5C-modifying enzymes Dnmt2 or NSun2 during aging reduces tRNA m5C modification. This reduction may subsequently enhance angiogenin-mediated tRNA cleavage under oxidative stress, although this effect appears confined to specific tRNA. The four aspect investigates strategies for the precise delivery of NCI compound 65828 (an angiogenin inhibitor) to senescent BMSCs, aiming to mitigate stress-induced tRNA cleavage. As angiogenin is highly expressed in liver tissue, it functions as an angiogenic factor and plays a critical role in angiogenesis 72 . If the BMSCs-targeted NCI-65828 drug delivery vector leaks into liver tissue, it is highly likely to impair liver vessel function. In the present study, we constructed a BMSCs-targeted drug delivery liposomes incorporating a bone affinity peptide (DSS)6, which exhibit high delivery efficiency in bone marrow and minimize systemic side effects 39 . It is suggested that the use of a bone-targeted delivery system efficiently eliminates senescent cells by delivering senolytics, this approach will open new avenues for addressing age-related bone disease 66 . We believe that addressing these four challenges in the future will enhance our understanding of how angiogenin-mediated tRNA cleavage influences bone disease. The increased or dysregulated occurrence of tsRNAs in biofluids such as serum, or sperm in various cancer or disease conditions makes them attractive candidates for biomarker development 73 – 76 . In our study, we observed that serum levels of 5ʹ-tiRNA-Glu − CTC in osteoporosis patients exhibited a negative correlation with bone mineral density (BMD), procollagen type I N-terminal propeptide (PINP), and osteocalcin (BGP), while demonstrating a positive correlation with C-terminal telopeptide of type I collagen (CTX) and tartrate-resistant acid phosphatase (TRAP). These findings suggest that 5ʹ-tiRNA-Glu − CTC may serve as a potential biomarker for diagnosing age-related osteoporosis. This observation provides the first clinical insight into the contribution of 5ʹ-tiRNA-Glu − CTC to the pathophysiological regulation of bone formation.​ In summary, this study demonstrates that angiogenin-mediated tRNA cleavage under oxidative stress serves as a critical trigger for age-related osteoporosis. We identified the first mechanistic link between 5ʹ-tRNA fragments producted by angiogenin and the age-related osteoporosis. Furthermore, targeting the inhibition of angiogenin with NCI-65828 represents a promising therapeutic strategy for this disease. Declarations Acknowledgements This work was supported by grants from China National Health Development Research Center (2025ZD0550400), National Natural Science Foundation of China (Grant No. 82471619), Natural Science Foundation of Hunan Province (Grant No. 2024JJ5459), and Scientific Research Program of FuRong Laboratory (No. 2024PT5104). Author contribution Y.X. and G.-Q.X. conceived the project and designed the experiments. W.-H.K. and Z.-L.P. performed the experiments. M.-Z.Y. and M.-S.Y. assisted with data acquisition. Y.L. and Y.X. performed the data analysis. Y.X. secured funding and provided project administration and supervision. W.-H.K. and Z.-L.P. integrated the data and drafted the manuscript together with Y.X. All authors reviewed and revised the manuscript. Declaration of interests The authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7830644","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":546662961,"identity":"e00f0cc7-cb27-4bb1-b1d6-7c0b9dda82c8","order_by":0,"name":"Ye 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1","display":"","copyAsset":false,"role":"figure","size":2121890,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAging induces the production of 5'-tsRNA-Glu-CTC in Bone Marrow Mesenchymal Stromal Cells (BMSCs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. A scatterplot of tsRNA sequencing from primary BMSCs of 3-, and 20-month-old mice (counts\u0026gt;1).\u003c/p\u003e\n\u003cp\u003eB. Representative images of LEPR (red), tsRNA(5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e/5'-tsRNA-Lys\u003csup\u003e-CTT\u003c/sup\u003e/TRF-5004b) FISH (green), and DAPI (blue) from\u003cstrong\u003e \u003c/strong\u003eprimary BMSCs of 3-, and 20-month-old mice (Scale bars, 50μm).\u003c/p\u003e\n\u003cp\u003eC. Quantitation of tsRNA(5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e/5'-tsRNA-Lys\u003csup\u003e-CTT\u003c/sup\u003e/TRF-5004b) -FISH positive area (n = 8).\u003c/p\u003e\n\u003cp\u003eD. Ratio of tsRNA(5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e/5'-tsRNA-Lys\u003csup\u003e-CTT\u003c/sup\u003e/TRF-5004b)-positive area in LEPR+ region (n = 8).\u003c/p\u003e\n\u003cp\u003eE-G. Representative images of β-Gal staining (E), Alizarin Red staining (F) and Oil Red O staining (G) of BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e/5'-tsRNA-Lys\u003csup\u003e-CTT\u003c/sup\u003e/TRF-5004b/negative control RNA (Scale bars, 200μm).\u003c/p\u003e\n\u003cp\u003eH. Quantitation of β-Gal staining, calcium mineralization and Oil Red O of tsRNA (5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e/5'-tsRNA-Lys\u003csup\u003e-CTT\u003c/sup\u003e/TRF-5004b/negative control RNA) (n=6).\u003c/p\u003e\n\u003cp\u003eI. Predicted secondary structure of 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e and 5'-tsRNA-Lys\u003csup\u003e-CTT\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eJ. Volcano plot of BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e or 5'-tsRNA-Lys\u003csup\u003e-CTT\u003c/sup\u003e compared to control group (fold change ≥2 , p value ＜ 0.05).\u003c/p\u003e\n\u003cp\u003eK. KEGG pathway enrichment of BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e or 5'-tsRNA-Lys\u003csup\u003e-CTT \u003c/sup\u003ecompared to control group.\u003c/p\u003e\n\u003cp\u003eL. Heatmap of differential expression mRNAs from BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e, 5'-tsRNA-Lys\u003csup\u003e-CTT \u003c/sup\u003eor negative control RNA (n = 3).\u003c/p\u003e\n\u003cp\u003eM. RT-qPCR analysis of differential expression mRNAs in BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e, 5'-tsRNA-Lys\u003csup\u003e-CTT \u003c/sup\u003eor negative control RNA\u003csup\u003e \u003c/sup\u003e(n=6).\u003c/p\u003e\n\u003cp\u003eData are shown as the mean ± SEM. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 by Student's t-test (C, D, H, M).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7830644/v1/31ad59fbc9e9972b1c9dd391.png"},{"id":96968394,"identity":"63c9f805-a718-4124-a226-fd5eb93fe239","added_by":"auto","created_at":"2025-11-28 07:00:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2006856,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e5'-tiRNA-Glu\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-CTC\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e accelerates bone loss in male Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Schematic diagram of experimental workflow. Young (3 mo) and aged (12 mo) male mice received intravenous injection of rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e or rAAV2/9-vehicle. Calcein was injected intraperitoneally at weeks 3 and 4. Femurs and tibias were collected at weeks 4 for analysis.\u003c/p\u003e\n\u003cp\u003eB. RT-qPCR analysis of the expression level of 5'-tiRNA-Glu\u003csup\u003e-CTC \u003c/sup\u003ein BMSCs from young and aged male mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eC. ELISA analysis the serum PINP levels in young and aged male mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eD. ELISA analysis the serum CTX levels in young and aged male mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eE. Representative micro-CT images of trabecular bone in young and aged male mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eF-I. Quantification of trabecular bone volume (Tb.BV/TV, F), trabecular thickness (Tb.Th, G), trabecular bone number (Tb.N, H), and trabecular separation (Tb.Sp, I) of young and aged male mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e (n=8).\u003c/p\u003e\n\u003cp\u003eJ-K. Representative images of H\u0026amp;E staining (J) and quantification of trabecular area (K) in femur sections of young and aged male mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC \u003c/sup\u003e(Scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003eL-M. Representative images of osteocalcin (OCN) staining (L) and quantification of number of OCN⁺ cells (M) of young and aged male mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e (Scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003eN-O. Representative images of SP7 staining (N) and quantification of number of SP7⁺ cells (O) of young and aged male mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC \u003c/sup\u003e(Scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003eP-Q. Representative images of LEPR staining (P) and quantification of LEPR⁺ area (Q) of young and aged male mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC \u003c/sup\u003e(Scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003eR-S. Representative images of TRAP staining (R) and quantification of number of TRAP⁺ cells (S) of young and aged male mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC \u003c/sup\u003e(Scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003eT-U. Representative images of calcein double-labeling (T) and analysis of mineral apposition rate (MAR, U) of young and aged male mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e (Scale bar, 20 μm).\u003c/p\u003e\n\u003cp\u003eData are shown as the mean ± SEM; n = 8. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 by Student's t-test (B–D, F–I, K M, O, Q, S, U).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7830644/v1/3244b9cdf7863bdfb40a4455.png"},{"id":96968409,"identity":"6696ec59-94e6-4a5e-8b43-c0495ddb3904","added_by":"auto","created_at":"2025-11-28 07:00:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1148075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e5'-tiRNA-Glu-CTC accelerates bone loss in female Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. RT-qPCR analysis of the expression level of 5'-tiRNA-Glu\u003csup\u003e-CTC \u003c/sup\u003ein BMSCs from young (3 mo) and aged (12 mo) female mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eB. Representative micro-CT images of trabecular bone of young and aged female mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eC-F. Quantification of trabecular bone volume (Tb.BV/TV, C), trabecular thickness (Tb.Th, D), trabecular bone number (Tb.N, E), and trabecular separation (Tb.Sp, F) of young and aged female mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e (n=8).\u003c/p\u003e\n\u003cp\u003eG-H. Representative images of osteocalcin (OCN) staining (G) and quantification of number of OCN⁺ cells (H) of young and aged female mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC \u003c/sup\u003e(Scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003eI-J. Representative images of SP7 staining (I) and quantification of number of SP7⁺ cells (J) of young and aged female mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC \u003c/sup\u003e(Scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003eK-L. Representative images of LEPR staining (K) and quantification of LEPR⁺ area (L) of young and aged female mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e (Scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003eM-N. Representative images of TRAP staining (M) and quantification of number of TRAP⁺ cells (N) of young and aged female mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e (Scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003eO-P. Representative images of calcein double-labeling (O) and analysis of mineral apposition rate (MAR, P) of young and aged female mice treated with rAAV2/9-vehicle or rAAV2/9-5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e (Scale bar, 20 μm)\u003c/p\u003e\n\u003cp\u003eData are shown as the mean ± SEM; n = 8. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 by Student's t-test (A, C–F, H, J, L, N, P).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7830644/v1/51d889a3ad7f163d5ac24ab4.png"},{"id":97137792,"identity":"7e9c14bb-94e6-4467-8cad-4cf2b7f94e49","added_by":"auto","created_at":"2025-12-01 09:58:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2145768,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of 5'-tiRNA-Glu\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-CTC\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e via ASO ameliorates BMSCs senescence and bone loss in aged mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Representative images of β-Gal staining and quantitation of β-Gal positive area of aged primary BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -ASO or negative control RNA (Scale bars, 200μm, n=6).\u003c/p\u003e\n\u003cp\u003eB. Representative images of Alizarin Red staining and quantitation of calcium mineralization of aged primary BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -ASO or negative control RNA (Scale bars, 200μm, n=6).\u003c/p\u003e\n\u003cp\u003eC. Representative images of Oil Red O staining and quantitation of Oil Red O of aged primary BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -ASO or negative control RNA (Scale bars, 200μm, n=6).\u003c/p\u003e\n\u003cp\u003eD. Representative micro-CT images of trabecular bone in aged (18 mo) mice treated with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -ASO or vehicle of both sexes.\u003c/p\u003e\n\u003cp\u003eE-H. Quantification of trabecular bone volume (Tb.BV/TV, E), trabecular thickness (Tb.Th, F), trabecular bone number (Tb.N, G), and trabecular separation (Tb.Sp, H) in aged mice treated with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -ASO or vehicle of both sexes (n=8).\u003c/p\u003e\n\u003cp\u003eI-J. Representative images of H\u0026amp;E staining (I) and quantification of adipocytes or trabecular area (J) in femur sections of aged mice treated with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -ASO or vehicle of both sexes (Scale bar, 50 μm, n=8).\u003c/p\u003e\n\u003cp\u003eK-L. Representative images of osteocalcin (OCN) staining (K) and quantification of number of OCN⁺ cells (L) in aged mice treated with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -ASO or vehicle of both sexes (Scale bar, 50 μm, n=8).\u003c/p\u003e\n\u003cp\u003eM-N. Representative images of SP7 staining (M) and quantification of number of SP7⁺ cells (N) in aged mice treated with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -ASO or vehicle of both sexes (Scale bar, 50 μm, n=8).\u003c/p\u003e\n\u003cp\u003eO-P. Representative images of LEPR staining (O) and quantification of LEPR⁺ area (P) in aged mice treated with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -ASO or vehicle of both sexes (Scale bar, 50 μm, n=8).\u003c/p\u003e\n\u003cp\u003eQ-R. Representative images of TRAP staining (Q) and quantification of number of TRAP⁺ cells (R) in aged mice treated with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -ASO or vehicle of both sexes (Scale bar, 50 μm, n=8).\u003c/p\u003e\n\u003cp\u003eS-T. Representative images of calcein double-labeling (S) and analysis of mineral apposition rate (MAR, T) in aged mice treated with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -ASO or vehicle of both sexes (Scale bar, 20 μm, n=8).\u003c/p\u003e\n\u003cp\u003eData are shown as the mean ± SEM. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 by Student's t-test (A-C, E-H, J, L, N, P, R, T).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7830644/v1/93eec7245f5547ddb431f9b2.png"},{"id":97136547,"identity":"b8ed7a4b-1005-461a-ab3f-33a64dfcee25","added_by":"auto","created_at":"2025-12-01 09:56:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1735271,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e5'-tiRNA-Glu\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-CTC\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e induces BMSCs senescence by disrupting mRNA stability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Young primary BMSCs of mice transfected with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e or negative control RNA were eluted for high-throughput sequencing and RNA sequencing.\u003c/p\u003e\n\u003cp\u003eB. Genomic distribution of 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e RIP-seq peaks.\u003c/p\u003e\n\u003cp\u003eC. Venn diagram representing the overlap genes between 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e RIP-seq targets and 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -overexpressing RNA-Seq targets.\u003c/p\u003e\n\u003cp\u003eD. Bubble plot of the top 20 enriched KEGG pathways.\u003c/p\u003e\n\u003cp\u003eE. Chord diagram of enriched pathways for the intersection genes of Figure5 C.\u003c/p\u003e\n\u003cp\u003eF. RIP-seq read coverage of \u003cem\u003eFoxm1, Cdk1, Capn1, Sp7,\u003c/em\u003e and \u003cem\u003eCcnd1\u003c/em\u003e from BMSCs. The dashed regions indicate the binding locations within the 3' UTR.\u003c/p\u003e\n\u003cp\u003eG. Sequence of 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e binding to the 3' UTR of targeted genes.\u003c/p\u003e\n\u003cp\u003eH. Reducing mRNA half-life of the representative targets (\u003cem\u003eFoxm1, Cdk1, Capn1, Sp7 \u003c/em\u003eand\u003cem\u003e Ccnd1\u003c/em\u003e) by transfecting with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e or negative control RNA in BMSCs.\u003c/p\u003e\n\u003cp\u003eI. Relative luciferase activity of wild-type or mutated of the representative targets (\u003cem\u003eFoxm1, Cdk1, Capn1, Sp7 \u003c/em\u003eand\u003cem\u003e Ccnd1\u003c/em\u003e) in HEK293T cells transfected with 5'-tiRNA-Glu\u003csup\u003e-CTC \u003c/sup\u003eor negative control RNA.\u003c/p\u003e\n\u003cp\u003eJ-K. Representative plots (J) and quantification (K) of the cell cycle in BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e or negative control RNA, as analyzed by flow cytometry (n=3).\u003c/p\u003e\n\u003cp\u003eL-M. Representative images of KI67 staining (L) and quantification of KI67⁺ cells (M) in BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e or negative control RNA (Scale bar, 50 μm, n=6).\u003c/p\u003e\n\u003cp\u003eN. RT-qPCR analysis of related mRNA in BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -ASO or negative control RNA (n=6).\u003c/p\u003e\n\u003cp\u003eO. Representative immunoblots of related proteins in BMSCs transfected with eithir 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e or negative control RNA (left) and either 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -ASO or negative control RNA (right).\u003c/p\u003e\n\u003cp\u003eData are shown as the mean ± SEM. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 by Student's t-test (H, I, K, M, N).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7830644/v1/746a9b186b5f50dc6dd9a7c2.png"},{"id":96968400,"identity":"602c49c8-d5b5-4a63-bc94-81a94573459b","added_by":"auto","created_at":"2025-11-28 07:00:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2410263,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular stress triggers ANG-cleaving activity and induces 5'-tiRNA-Glu\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-CTC\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Heatmap of differentially expressed of tRNA-cleaving enzymes mRNA in BMSCs of 2-month, and 24-month mice.\u003c/p\u003e\n\u003cp\u003eB. Representative images of LEPR (red), ANG (green), and DAPI (blue) in BMSCs of 2-month, and 24-month-old mice (Scale bars, 10μm).\u003c/p\u003e\n\u003cp\u003eC. Ratio of ANG-positive LEPR+ region (n = 8).\u003c/p\u003e\n\u003cp\u003eD. RT-qPCR analysis of ANG mRNA level in BMSCs of 2-, and 24-month-old mice of both sexes. (n=6).\u003c/p\u003e\n\u003cp\u003eE-F. Representative images (E) of G3BP1 staining and quantification of number of G3BP1⁺ cells (F) in BMSCs treated with NaAsO\u003csub\u003e2 \u003c/sub\u003eor vehicle\u003csub\u003e \u003c/sub\u003e(Scale bar, 20 μm, n=8).\u003c/p\u003e\n\u003cp\u003eG-H. Representative images (G) of G3BP1 staining and quantification of number of G3BP1⁺ cells (H) in BMSCs of 2-, and 24-month-old mice\u003csub\u003e \u003c/sub\u003e(Scale bar, 10 μm, n=8).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eI. Representative images of ANG (red), 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -FISH (green), and DAPI (blue) in BMSCs treated with Na₃AsO₃\u003csub\u003e \u003c/sub\u003eor vehicle (Scale bars, 10μm).\u003c/p\u003e\n\u003cp\u003eJ. Representative density plot showing co-localization of ANG (red), 5'-tiRNA-Glu-CTC–FISH (green), and DAPI (blue) by triple immunofluorescence in NaAsO\u003csub\u003e2\u003c/sub\u003e-treated group.\u003c/p\u003e\n\u003cp\u003eK. Representative images of ANG (red), 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e -FISH (green), and DAPI (blue) in BMSCs\u0026nbsp;\u0026nbsp; of 2-, and 24-month-old mice (Scale bars, 10μm).\u003c/p\u003e\n\u003cp\u003eL. Northern blotting analysis of tRNA cleavage efficiency in vitro under gradient concentrations of ANG.\u003c/p\u003e\n\u003cp\u003eM. Representative immunoblots of ANG levels in total homogenates and nuclear fractions of BMSCs in 2- and 24-month-old mice.\u003c/p\u003e\n\u003cp\u003eN. Molecular docking model of NCI-65828.\u003c/p\u003e\n\u003cp\u003eO. Cell viability measured by CCK-8 assay after treatment with gradient concentrations of NCI-65828.\u003c/p\u003e\n\u003cp\u003eP. Representative immunoblots of related proteins in aged primary BMSCs treated with different concentration of NCI-65828.\u003c/p\u003e\n\u003cp\u003eQ. Representative images of β-Gal staining and quantitation of β-Gal positive area of aged primary BMSCs treated with NCI-65828 or vehicle (Scale bars, 200μm, n=6).\u003c/p\u003e\n\u003cp\u003eR. Representative images of Alizarin Red staining and quantitation of calcium mineralization of aged primary BMSCs treated with NCI-65828 or vehicle (Scale bars, 200μm, n=6).\u003c/p\u003e\n\u003cp\u003eS. Representative images of Oil Red O staining and quantitation of Oil Red O of aged primary BMSCs treated with NCI-65828 or vehicle (Scale bars, 200μm, n=6).\u003c/p\u003e\n\u003cp\u003eData are shown as the mean ± SEM. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 by Student's t-test (C, D, F, H, N, P-R).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7830644/v1/a6ff086de558455def87f8c5.png"},{"id":96968395,"identity":"e80fdfb0-9b3e-46cb-80ea-2517e4901a11","added_by":"auto","created_at":"2025-11-28 07:00:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2151627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibitor of ANG-cleaving activity ameliorates age-related bone loss in aged mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Representative micro-CT images of trabecular bone in aged (18 mo) mice treated with NCI-65828 or vehicle of both sexes.\u003c/p\u003e\n\u003cp\u003eB-E. Quantification of trabecular bone volume (Tb.BV/TV, B), trabecular thickness (Tb.Th, C), trabecular bone number (Tb.N, D), and trabecular separation (Tb.Sp, E) in aged mice treated with NCI-65828 or vehicle of both sexes.\u003c/p\u003e\n\u003cp\u003eF. ELISA analysis the serum PINP levels in aged mice treated with NCI-65828 or vehicle of both sexes.\u003c/p\u003e\n\u003cp\u003eG. ELISA analysis the serum CTX levels in aged mice treated with NCI-65828 or vehicle of both sexes.\u003c/p\u003e\n\u003cp\u003eH-I. Representative images of H\u0026amp;E staining (H) and quantification of adipocytes or trabecular area (I) in femur sections in aged mice treated with NCI-65828 or vehicle of both sexes (Scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003eJ-K. Representative images of osteocalcin (OCN) staining (J) and quantification of number of OCN⁺ cells (K) in aged mice treated with NCI-65828 or vehicle of both sexes (Scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003eL-M. Representative images of SP7 staining (L) and quantification of number of SP7⁺ cells (M) in aged mice treated with NCI-65828 or vehicle of both sexes (Scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003eN-O. Representative images of LEPR staining (N) and quantification of LEPR⁺ area (O) in aged mice treated with NCI-65828 or vehicle of both sexes (Scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003eP-Q. Representative images of TRAP staining (P) and quantification of number of TRAP⁺ cells (Q) in aged mice treated with NCI-65828 or vehicle of both sexes (Scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003eR. Schematic model of mRNA destabilization by 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e deriving from ANG-mediated cleavage of tRNA.\u003c/p\u003e\n\u003cp\u003eData are shown as the mean ± SEM; n = 8. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 by Student's t-test (B-E, F-G, I, K, M, O, Q).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7830644/v1/ac4638e4681c118c59464965.png"},{"id":96968422,"identity":"46a88802-8826-4b1b-8012-27177868977b","added_by":"auto","created_at":"2025-11-28 07:00:43","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":727086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e5'-tiRNA-Glu\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-CTC\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e serves as a novel serum biomarker for assisting in the diagnosis of osteoporosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Scatterplot of tsRNA sequencing from serum of individuals with osteoporosis (fold change \u0026gt; 1.5).\u003c/p\u003e\n\u003cp\u003eB. Scatterplot of miRNA sequencing from serum of individuals with osteoporosis (fold change \u0026gt; 1.5).\u003c/p\u003e\n\u003cp\u003eC. Stacked bar chart showing the proportional distribution of various tsRNA types within corresponding tRNA.\u003c/p\u003e\n\u003cp\u003eD. Scatter plot of the correlation between 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e and age of individuals with osteoporosis(n=40).\u003c/p\u003e\n\u003cp\u003eE. Scatter plot of the correlation between 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e and Bone Mineral Density (BMD) of individuals with osteoporosis(n=40).\u003c/p\u003e\n\u003cp\u003eF.Scatter plot of the correlation between 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e and T-score of BMD of individuals with osteoporosis(n=40).\u003c/p\u003e\n\u003cp\u003eG.Scatter plot of the correlation between 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e and PINP of individuals with osteoporosis(n=40).\u003c/p\u003e\n\u003cp\u003eH.Scatter plot of the correlation between 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e and BGP of individuals with osteoporosis(n=40).\u003c/p\u003e\n\u003cp\u003eI.Scatter plot of the correlation between 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e and ALP of individuals with osteoporosis(n=40).\u003c/p\u003e\n\u003cp\u003eJ.Scatter plot of the correlation between 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e and CTX of individuals with osteoporosis(n=40).\u003c/p\u003e\n\u003cp\u003eK.Scatter plot of the correlation between 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e and TRAP of individuals with osteoporosis(n=40).\u003c/p\u003e\n\u003cp\u003eL.Scatter plot of the correlation between 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e and ANG of individuals with osteoporosis(n=40).\u003c/p\u003e\n\u003cp\u003eM.Scatter plot of the correlation between 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e and Calcium of individuals with osteoporosis(n=40).\u003c/p\u003e\n\u003cp\u003eN.Scatter plot of the correlation between 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e and 25-OH-D3 of individuals with osteoporosis(n=40).\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7830644/v1/46ce440edf25e3d844ffa63f.png"},{"id":97898813,"identity":"f0101d2c-8334-4891-bfa9-f54bdc7c02cf","added_by":"auto","created_at":"2025-12-10 15:39:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15717031,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7830644/v1/4547d0f2-e902-4be3-a353-13f95bb3e725.pdf"}],"financialInterests":"There is a conflict of interest\nThe authors declare no competing interests.","formattedTitle":"Targeting angiogenin as a therapeutic strategy for age-related osteoporosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoporosis is defined as a systemic chronic metabolic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, and constitutes a major risk factor for fracture\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Evidence demonstrates that chronic oxidative stress- induced DNA damage, cellular apoptosis, and cellular senescence of bone marrow stromal cells (BMSCs) is an important factor that contributes to the disease\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Additionally, oxidative stress triggers angiogenin (ANG)-mediated production of transfer RNA-derived small RNAs (tsRNAs) by altering tRNA modification profiles\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. As cellular stress response products, dysregulated tsRNAs participate in various cellular processes; however, their biological functions in regulating osteoporosis remain unclear.\u003c/p\u003e\u003cp\u003eOxidative stress is defined as a dynamic imbalance between the generation of reactive oxygen species (ROS) and the elimination capacity of antioxidant systems, inducing the disruption of physiological redox homeostasis and subsequently triggers an adaptive or pathological cellular stress response\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This imbalance can lead to oxidative damage to biological macromolecules (such as DNA, proteins, lipids), which in turn causes defects in cell differentiation, apoptosis, mitochondrial dysfunction and inflammation. There is increasing evidence that oxidative stress disrupts bone homeostasis and is involved in the development of osteoporosis\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Specifically, high levels of ROS inhibit the expression of osteogenic differentiation markers such as ALP, OCN, COL1, and Runx2 by activating the MAPKs pathway\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. ROS also activates the FOXO transcription factor, reduces the availability of β-catenin, and downregulates the Wnt/β-catenin signaling pathway, thereby inhibiting osteoblastogenesis\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. It cannot be ignored that high reactive oxygen species (ROS) levels in the senescent microenvironment (SME) induce bone marrow derived stromal cells (BMSCs) senescence, which is considered another important factor contributing to bone loss\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Oxidative stress also promote osteoclast differentiation through NF-κB, which increase the expression of transcriptional factors c-Fos and NFATc1 to regulate the genes tartrate-resistant acid phosphatase and cathepsin K involved in osteoclastogenesis and bone resorption\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Moreover, RANKL-induced osteoclastogenesis is enhanced via activation of MAPKs and NF-κB\u003csup\u003e13\u003c/sup\u003e. However, oxidative stress also suppresses Nrf2 pathway, leading to enhancement of bone resorption\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Moreover, ROS induce osteocyte apoptosis and alter the RANKL/OPG ratio, which triggers signals that promote the local recruitment of osteoclasts and excessive osteoclastogenesis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. It is evident that the progression of osteoporosis is closely correlated with ROS, which induces BMSCs senescence and prevents osteoblast differentiation, induces osteoclast differentiation and activity, and enhances apoptotic osteocytes, thereby altering the RANKL/OPG ratio. Recently, the tRNA cleavage activity of angiogenin (ANG) triggered by oxidative stress- a critical component of eukaryotic cellular responses to and mitigation of environmental stress- has caught our attention, which often involves a decrease in general translation and an increase in preferential translation of stress-response genes\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Angiogenin (ANG) is a stress-induced, secreted ribonuclease and exhibits various biological activities, extending from inducing angiogenesis to stimulating cell proliferation and, more recently, to promoting cell survival\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The biological functions of ANG depend on its subcellular localization. In the nucleus, angiogenin promotes ribosomal RNA (rRNA) transcription, thus facilitating cell growth and proliferation. Under stress conditions, angiogenin translocates to the cytosol where it cleaves mature tRNA into transfer RNA-derived small RNAs (tsRNAs)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The dysregulation of angiogenin-induced tsRNAs has been proposed to have multiple cellular functions, including inhibition of ribosome biogenesis, inhibition of protein translation, and inhibition of apoptosis, and participation in various diease processes\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. However, whether angiogenin-induced tsRNAs are involved in the progression of osteoporosis pathogenesis remains unexplored.\u003c/p\u003e\u003cp\u003etRNA-derived small RNAs (tsRNAs), generated through cleavage of mature or precursor tRNAs (pre-tRNAs) at distinct sites, are now recognized as a novel class of regulatory non-coding RNAs\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. So far, the nomenclature for tsRNA has not been established and its different names are accorded to the length and cleavage position on tRNA or pre-tRNA. tsRNAs of 18\u0026thinsp;~\u0026thinsp;30 nt in length are categorized into two types: Type I and Type II. Type I tsRNAs (tRF-5 and tRF-3) are produced via Dicer-mediated cleavage of the D-loop or T-loop of mature tRNAs, while Type II tsRNAs (tRF-1) are generated through cleavage of the 3' trailer sequences of pre-tRNAs by Endonuclease Z (RNase Z)/cytoplasmic ribonuclease Z2 (ELAC2). Additionally, tsRNAs of 30\u0026thinsp;~\u0026thinsp;40 nt in length are termed \"tRNA halves,\" often referred to as \"tiRNA (tRNA-derived stress-induced RNAs)\" due to their stress-induced characteristics and generated through cleavage tRNAs at the anticodon loop by nuclease angiogenin\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Recently, tsRNAs have been recognized as novel therapeutic targets and regulatory molecules, exerting pivotal roles through multiple mechanisms. First, they promote mRNA degradation via DICER-dependent biogenesis by binding to AGO proteins, thereby mimicking miRNA behavior to downregulate gene expression\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. These tsRNAs also mediate translational regulation in an AGO proteins-independent manner by exerting structural effects on mRNAs or rRNAs\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Second, they displace RNA-binding proteins (RBPs) from pro-oncogenic or metabolic mRNAs, suppressing cancer metastasis, or alternatively bind RBPs to either protect mRNAs from decay or enhance the stability of metabolic mRNAs, promoting tumorigenesis\u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Third, they repress translation- a function that remains among the most extensively studied roles of tsRNAs in molecular biology. Under stress, the nuclease angiogenin cleaves tRNAs and generates 3'-tiRNA and 5'-tiRNA. Notably, 5'-tiRNA, but not 3'-tiRNA, inhibits global protein synthesis by forming intermolecular RNA G-quadruplexes (RG4) or binding to polyadenylate-binding protein 1 (PABPC1), thereby displacing PABPC1 and eIF4A/G/E from m⁷G-capped mRNAs and resulting in the sequestration of initiation factors\u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Interestingly, 5'-tiRNA may decrease global translation of mRNAs containing weak internal ribosomal entry sites (IRESs) while selectively upregulating the translation of mRNAs containing strong IRESs that are involved in pro-survival and anti-apoptotic pathways, thereby promoting cell survival under adverse conditions\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Researchers also have proposed that ANG-induced tsRNAs may contribute to the formation of SGs in response to cellular stress, which indicated that ANG plays an active role in stress response of the cells and may promote cell survival via this mechanism\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In summary, the diverse regulatory mechanisms of tsRNAs render them closely linked to the progression of diverse human diseases, encompassing cancer, neurodegenerative disorders, metabolic syndromes, viral infections, and inflammasome-related diseases\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Considerable evidence shows that tRFs are detectable in model organisms of different ages and are associated with age-related diseases, such as neurodegenerative diseases\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. It remains largely unknown whether increased oxidative stress triggers angiogenin-induced tRNA cleavage, a mechanism potentially involved in the progression of osteoporosis-a systemic chronic metabolic and age-associated skeletal disease.\u003c/p\u003e\u003cp\u003eIn this study, we observed a translocation of angiogenin (ANG) from the nucleus to the cytosol in BMSCs during aging and further found that the ANG cleavage product 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e in BMSCs increased with aging in mice and humans. 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e disrupts the stability of anti-senescence and pro-osteogenic mRNAs by binding to their 3'UTR regions, including Capn1, Cdk1, Ccnd1, Foxm1 and Sp7. Moreover, we discovered that treatment with the ANG inhibitor NCI-65828 ameliorates age-related bone loss, indicating that inhibition of ANG activity may represent a potential therapeutic strategy for osteoporosis. Furthermore, we detected that the 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e levels in human serum were negatively correlated with BMD, Z-scores and osteogenic markers (PINP and BGP), and were positively correlated with osteoclast markers (CTX and TRAP), suggesting that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e may serve as a promising serum biomarker for diagnosing osteoporosis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMice\u003c/h2\u003e\u003cp\u003eC57BL/6J mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. All mice were housed in specific pathogen-free animal facilities (22\u0026ndash;24\u0026deg;C) at the Experimental Animal Research Center of Central South University under a 12-hour dark/light cycle, with free access to water and a standard diet. All animal care protocols and experiments were approved by the Medical Ethics Committee of Xiangya Hospital of Central South University.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eHuman sample\u003c/h3\u003e\n\u003cp\u003eSerum samples from 40 osteoporosis patients were obtained from the Endocrinology Laboratory of Xiangya Hospital.​​ Osteoporosis diagnosis was confirmed by two or more experienced physicians at Xiangya Hospital using dual-energy X-ray absorptiometry (DXA) to measure bone mineral density (BMD), particularly at the lumbar spine. ​​The study was approved by the Clinical Ethics Committee of Xiangya Hospital, Central South University.​​ Throughout the communication process, the nature of the study and its potential implications were explained to all participants, and each provided written informed consent.\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eThe BMSC cell line (MUBMX-01001, Cyagen) were cultured in MEM-αmedium (Procell, PM150421) supplemented with 10% Fetal bovine serum (Gibco, 10091-148) and 1% penicillin-streptomycin (Procell, PB180120), and maintained at 37\u0026deg;C in a humidified 5% CO₂ incubator. For primary BMSCs isolation and culture, Mouse bone marrow was flushed from mouse femurs and tibias using pre-chilled MEM-α complete medium via a 1 mL syringe, and the harvested cells were resuspended and then was incubated with antibodies targeting mouse Sca1 (108108; BioLegend), CD29 (102206;BioLegend), CD45 (103132༛BioLegend), and CD11b (101226༛BioLegend) at 4\u0026deg;C for 20 min, followed by FACS (BD Biosciences) sorting to isolate Sca1\u0026thinsp;+\u0026thinsp;CD29\u0026thinsp;+\u0026thinsp;CD45\u0026thinsp;\u0026minus;\u0026thinsp;CD11b\u0026thinsp;\u0026minus;\u0026thinsp;cells as BMSCs. The sorted cells were seeded into 6 cm culture dishes and cultured to reach 90% confluency. The cells were then digested with 0.25% trypsin for about 2 min, and the detached cells were subsequently passaged into new 6 cm dishes to enrich the cell population.\u003c/p\u003e\n\u003ch3\u003etsRNA-sequencing\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from the serum of osteoporosis patients using the miRNeasy Serum Advanced Kit (217204, Qiagen) and quantified with the NanoDrop ND-1000 instrument. RNA modifications were removed from the RNA samples, followed by ligation with 3'-adapter and 5'-adapter to construct a sequencing library via PCR amplification. Recovery of 134\u0026thinsp;~\u0026thinsp;160bp PCR amplified fragment libraries via DNA gel electrophoresis. The libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ as sequencing clusters and sequenced on Illumina sequencer according to the manufacturers instructions. Raw sequencing data are used to the following analysis. Sequencing quality is examined by FastQC. Trimmed 5', 3'-adaptor bases by cutadapt. Trimmed reads are aligned allowing for 1 mismatch only to the mature transfer RNA (tRNA) sequences, then the rest of the unmapped reads are aligned allowing for 1 mismatch only to precursor tRNA sequences with bowtie software. The rest of the unmapped reads are aligned allowing for 1 mismatch only to microRNA (miRNA) reference sequences with miRDeep2. The expressions of tsRNA are evaluated using their sequencing counts and are normalized as counts per million of total aligned reads (CPM). The differentially expressed are screened based on the count value with R package edgeR. The scatter is ploted with differentially expressed results.\u003c/p\u003e\n\u003ch3\u003eRNA-sequencing\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from BMSCs transfected with 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e, 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, and negative RNA using the AG RNAex Pro Reagent (AG21102, Accurate Biology). RNA quality was examined by gel electrophoresis and quantitatived with Qubit (Thermo, Waltham, MA, USA). The sequencing libraries were prepared using VAHTSTM Stranded mRNA-seq Library Prep Kit for Illumina\u0026reg; and sequenced in collaboration with Genergy Biotechnology Co. Ltd. (Shanghai, China). Differentially expressed genes (DEGs) exhibiting two-fold changes and Benjamini and Hochberg-adjusted P values\u0026thinsp;\u0026le;\u0026thinsp;0.05 were selected. Then DEGs were chosen for function and signaling pathway enrichment analysis using GO and KEGG databases. The significantly enriched pathways were determined when P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and at least two affiliated genes were included.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eBiotinylated RNA pull down:\u003c/h2\u003e\u003cp\u003eBMSCs cell line was lysed on ice for 10 minutes using cell lysis buffer containing protease inhibitors, followed by homogenization at 60 Hz for 10 s, pausing for 10 s, and repeating 3 times. The lysate was then centrifuged at 14,000 rpm, 4\u0026deg;C for 10 min. The supernatant was collected into a new centrifuge tube, and was incubated with 20 \u0026micro;g biotin-labeled 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e (pre-denatured at 65\u0026deg;C for 10 min and cooled to 4\u0026deg;C) at 4\u0026deg;C with rotation for 2 h. Then, streptavidin beads were added and incubated at 4\u0026deg;C with rotation for 2 h. The RNA captured by the magnetic beads was eluted and subjected to commercial RIP-seq (GUANGZHOU RIBOBIO CO., LTD).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell transfection\u003c/h3\u003e\n\u003cp\u003eThe 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e, TRF-5004b or 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e antisense oligonucleotide (ASO) fragments were synthesized by Sangon Biotech (Shanghai, China). These tsRNA fragments weretransfected into the BMSCs using Lipofectamine 2000 (Invitrogen). After 6 h, the medium wasreplaced with appropriate medium for further experiments.\u003c/p\u003e\n\u003ch3\u003eOsteogenic differentiation assay and Alizarin Red S staining\u003c/h3\u003e\n\u003cp\u003eFor osteogenic differentiation in vitro, primary BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e, TRF-5004b, 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e antisense oligonucleotide or treated with Angiogenin inhibitor were cultured in 24-well plates for 21 days using α-MEM supplemented with 10% fetal bovine serum, 0.1 mM dexamethasone, 10 mM β-glycerophosphate, and 50 \u0026micro;M ascorbic acid-2-phosphate. The medium was replaced every 3 days. Then the cells were stained with 2% Alizarin Red S (Sigma-Aldrich, A5533, pH 4.2) to assess extracellular matrix mineralization. Alizarin Red S released from the extracellular matrix into cetylpyridinium chloride solution was quantified spectrophotometrically at 540 nm.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAdipogenic differentiation assay and Oil Red O Staining\u003c/h2\u003e\u003cp\u003eFor adipogenic differentiation in vitro, primary BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e, TRF-5004b, 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e antisense oligonucleotide or treated with Angiogenin inhibitor were cultured in 24-well plates for 10 days using α-MEM supplemented with 10% fetal bovine serum, 0.5 mM 3-isobutyl-1-methylxanthine, 5 \u0026micro;g/ml insulin, and 1 \u0026micro;M dexamethasone. The medium was replaced every 3 days. Oil Red O (Sigma-Aldrich) staining was performed to detect lipids in mature adipocytes. The Oil Red O was extracted from the matrix and quantified spectrophotometrically at 492 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eSA-β-gal staining\u003c/h2\u003e\u003cp\u003eFor SA-β-gal staining in vitro, primary BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e, TRF-5004b, 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e antisense oligonucleotide or treated with Angiogenin inhibitor were cultured in 24-well plates for 3 days using α-MEM medium. For senescence assessment, cellular senescence was detected using the Senescence-Associated β-Galactosidase Staining Kit (Solarbio, G1580) following the manufacturer's instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eFluorescence in situ hybridization (FISH)\u003c/h2\u003e\u003cp\u003eFISH for cells according to the manufacturer\u0026rsquo;s instructions using the RiboTM Fluorescent in Situ Hybridization kit (C10910). Briefly, young and aging primary BMSCs were seeded onto coverslips in 24-well plates. On the following day, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min and permeabilized for 5 min. Then, pre-hybridization was carried out at 37\u0026deg;C for 30 min, followed by hybridization with FITC-labeled-5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e probe-containing hybridization buffer overnight at 37\u0026deg;C. The next day, the cells were washed with PBS and mounted with DAPI-containing medium for imaging under fluorescence microscope. FISH for bone tissue sections was performed as follows: 200 ng of FITC-labeled 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e and TRF-5004b probes were added to 10 mL hybridization buffer containing 2 \u0026times; SSC, 10% dextran sulfate, 25 mM sodium phosphate, 100 mg salmon sperm DNA, and RNase inhibitor. The hybridization buffer was pre-warmed to 37\u0026deg;C, applied to the sections, and incubated in hybridization oven at 42\u0026deg;C for 24 h, followed by an additional 12\u0026thinsp;~\u0026thinsp;24 h of hybridization at 37\u0026deg;C. Then, the sections were washed with 2 \u0026times; SSC buffer at 42\u0026deg;C for 10 min, followed by 3 times wash with 1\u0026times;PBS. Finally, the sections were mounted with glycerol containing DAPI and immediately imaged using fluorescence microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eNorthern blotting assay\u003c/h2\u003e\u003cp\u003eNorthern blot was performed using the Biotin Northern Blot Kit (for Small RNA) (R0220, Beyotime) according to the manufacturer\u0026rsquo;s protocol. Briefly, RNA samples were separated on a 15% urea-PAGE gel and the gel was transferred to a nylon membrane using semi-dry fast blotter (Guangzhou Dao One, FTB95) at 1.3A for 10 min. Then the membrane was crosslinked by UV irradiation for 5 min. The membrane was pre-hybridized at 37\u0026deg;C for 2 h, followed by hybridization with a biotin-labeled 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e probe (10 pmol/mL) at 37\u0026deg;C for 2 h. Subsequently, the membrane was incubated with HRP-streptavidin for 15 min. Signal detection was performed using a chemiluminescent substrate and imaging was visualized by ChemiDoc XRS Imaging System (Bio-Rad).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003etRNA-Glu cleavage in vitro\u003c/h2\u003e\u003cp\u003eThe tRNA cleavage reaction was performed in a 10 \u0026micro;L mixture containing 0.5 \u0026micro;g tRNA-Glu, 40 mM Tris\u0026ndash;HCl (pH 8.0), 20 mM KCl, 4 mM MgCl₂, and 2 mM DTT at 37\u0026deg;C for 40 min. Then the buffer was diluted with 2 \u0026times; RNA loading buffer. The RNA samples were then denatured at 65\u0026deg;C for 10 min, immediately chilled on ice, and analyzed by Northern blotting to evaluate cleavage efficiency.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eRNA decay assay\u003c/h2\u003e\u003cp\u003eCells transfected with 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e or negative control RNA were treated with actinomycin D (MCE, HY-17559) at a final concentration of 10 \u0026micro;g/mL. Total RNA was collected using TRIzol at 0, 1, 2, 8, and 16 h, followed by RT-qPCR analysis with GAPDH as the internal reference. The relative expression of the target gene at each time point (compared to 0 h) was calculated using the 2^(-ΔΔCT) method. The decay rate of mRNA concentration over time (dc/dt) is proportional to the degradation rate constant (k\u003csub\u003edecay\u003c/sub\u003e) and the cytoplasmic mRNA concentration (c), as described by the equation: dc/dt = -k\u003csub\u003edecay\u003c/sub\u003e\u0026middot;c. Thus, the mRNA half-life is calculated as: t\u003csub\u003e(1/2)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;ln(2)/k\u003csub\u003edecay\u003c/sub\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eDual-Luciferase Reporter Assay\u003c/h2\u003e\u003cp\u003eHek293T were transfected with either the pmirGLO-Foxm1, pmirGLO-Foxm1-mut, pmirGLO-Cdk1, pmirGLO-Cdk1-mut, pmirGLO-Capn1, pmirGLO-Capn1-mut, pmirGLO-Ccnd1, pmirGLO-Ccnd1-mut, pmirGLO-Sp7 and pmirGLO-Sp7-mut plasmids along with 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e or negative control RNA. After 48 hourstransfection, the cells were harvested and transferred to 96-well plates. The activities of firefly luciferase and Renilla luciferase were measured according to the manufacturer's instructions of the Dual-Glo\u0026reg; Luciferase Assay System (E2920, Promega).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometry\u003c/h2\u003e\u003cp\u003eBMSCs were transfected with 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e or negative control RNA, the cells were digested with pre-cooled 0.25% trypsin and washed with 1 \u0026times; PBS to obtain cell pellet. The pellet was resuspended in BD fixation/permeabilization solution and fixed for 30 min at 4\u0026deg;C. Then cells were incubated with KI67 antibody (1:200, Proteintech, 27309-1-AP) at room temperature for 60 min and futher incubated with anti-Rabbit AF647 (1:200, proteintech, RGAR005) for 30 min. Cells were stained with DAPI (3 \u0026micro;g/mL) on ice for 10 min before analyzing. The proportions of cells in S-G2-M phase were calculated using FlowJo V10 (BD Biosciences).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eIntramedullary injection of adeno-associated virus\u003c/h2\u003e\u003cp\u003eThe 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e and 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC \u0026minus;\u003c/sup\u003eASO sequence was cloned into pHBAAV-LEPR-Zs\u003c/p\u003e\u003cp\u003e-Green shuttle plasmid (Hanbio Biotechnology Co.) and transformed into DH5α competent cells for large-scale amplification. The pHBAAV-LEPR-5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u0026minus;\u003c/sup\u003e-ZsGreen, pHBAAV-LEPR-\u003c/p\u003e\u003cp\u003e5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u0026minus;\u003c/sup\u003eASO-ZsGreen and pHBAAV-LEPR-ZsGreen shuttle plasmids were cotransfected into HEK293T cells with pAAV-RC and pHelper plasmids. After transfection for 72 hours, HEK293T cells were collected and lysed, and the virus is purified by Adeno-Associated Virus (AAV) Purification Maxi Kit (V1469, Biomiga) and then concentrated by ultrafiltration tubes (Millipore, UFC905008).​​ A total of 1*10^11 vg of rAAV2/9-LEPR-5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e or rAAV2/9-LEPR-5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e-ASO and rAAV2/9-LEPR-Null was delivered via periosteal injection into the femoral bone marrow cavity and intervened for 1 month.​​\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of bone-targeted liposomes\u003c/h2\u003e\u003cp\u003eBone-targeted liposomes were prepared according to the following steps. Briefly, 100 mg of (AspSerSer, DSS)₆ peptide and 100 mg of DSPE-PEG2000-MAL were added to 5 mL of DMF and reacted via a maleimide-mediated reaction to form DSPE-PEG2000-(DSS)₆. The solution was purified through dialysis for 48h and freeze-dried to obtain the DSPE-PEG2000-(DSS)6. Then, 50mg ANG inhibitor NCI-65828, 150mg DSPE-PEG2000-(DSS)6, and 25mg egg yolk lecithin were co-dissolved in 6mL mixture of chloroform/methanol (1:1, V/V) solution. The mixture was formed into a thin film via a rotary evaporator, then hydrated under vacuum and sonicated at room temperature. The mixture was then sterilized via a 0.22-\u0026micro;m filter prior to subsequent experiments\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eCalcein double-labeling assay\u003c/h2\u003e\u003cp\u003eMice were intraperitoneally injected with calcein (25 mg/kg) at eight days and two days before euthanasia. Femurs were isolated, fixed in 70% ethanol, and embedded in methyl methacrylate. Sections (5 \u0026micro;m thick) were cut using a microtome and imaged under a fluorescence microscope. The mineral apposition rate (MAR) was measured to assess bone formation activity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence and immunohistochemical staining and Histochemistry analysis\u003c/h2\u003e\u003cp\u003eFor immunofluorescence staining, the bones were dissected and fixed in 4% paraformaldehyde for 24h, followed by decalcification in 0.5 M EDTA at 4\u0026deg;C for 21 days. The bone tissues were then embedded in cryo-embedding medium and sectioned into 6 \u0026micro;m slices. The sections were blocked with 3% BSA for 1 hour, incubated with primary antibodies SP7 (Abcam, AB209484) or Leptin R (AF497, NOVUS) at 4\u0026deg;C overnight, and then treated with Alexa Fluor 555 (1:200, Invitrogen, A31572) at room temperature for 1h the following day. The sections were mounted with DAPI-containing glycerol, and fluorescence signals were captured using Zeiss microscope (Apotome 3). For immunohistochemical staining, mouse bones were fixed in paraformaldehyde for 24 h after collection, decalcified at 4\u0026deg;C for 3 w, and then embedded in paraffin and sectioned at 6 \u0026micro;m. After deparaffinization and rehydration, the sections were stained with an OCN primary antibody and counterstained with hematoxylin. Stained images were quantified using ImageJ software. For histochemical staining, paraffin sections were processed for TRAP and H\u0026amp;E staining according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eELISA assay\u003c/h2\u003e\u003cp\u003eThe concentration of human osteocalcin, Beta Crosslaps, Alkaline Phosphatase (ALP) of human serum were measured by using ELISA kit (Elabscience, E-EL-H1343, E-EL-H0960, E-BC-K009-M). The level of P I NP and TRAP-5b in human sreum were determined by using ELISA kit from JONLNBIO (JL14248, JL13353). The concentration of P I NP and CTX1 in mouse serum were detected by using ELISA kit from Elabscience (E-EL-M0233, E-EL-M3023). All measurements were performed according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eRT-qPCR\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted with AG RNAex Pro Reagent (AG21102, Accurate Biology). RNA purity and concentration was determined by BioTek Epoch2 microplate reader (BioTek, USA) and 1 ug RNA was reverse transcribed into cDNA using the 5\u0026times; Evo M-MLV RT Premix (AG11706, Accurate Biology). Real time PCR reaction was performed on ABI QuantStudio 3 system using 2X SYBR Green Pro Taq HS Premix (AG11718, Accurate Biology) in 20 uL reaction volumes containing 2 uL cDNA. The relative mRNA expression was normalized to GAPDH and calculated by 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eWestern blotting assay\u003c/h2\u003e\u003cp\u003eCells were lysed with lysis buffer containing protease inhibitor cocktail for 10 minutes on ice. The lysates were centrifuged at 12,000g for 10 minutes and the supernatant was transferred into a new tube. Supernatant was heated at 100\u0026deg;C for 10 minutes mixed with 6 \u0026times; SDS loading buffer, then separated by SDS-PAGE and electro-transferred to PVDF membranes. After blocking with 5% skim milk for 2 hours, membranes were incubated with primary antibodies at 4\u0026deg;C overnight. Primary antibodies were FOXM1 (Proteintech, 13147-1-AP), CDK1 (Promab, 30181), CAPN1 (Promab, P07140), CCND1 (Promab, 31985), SP7 (Abcam, AB209484), LPL (Promab, P33464), P21 (Abcam, AB109199), P16 (Promab, P21210), ACTIN (Boster, BM0627). Secondary anti-mouse (Ap124p, MilliporeSigma) and anti-rabbit (Ap132p, MilliporeSigma) HRP-conjugated antibodies were applied the next day. Then the bands were visualized by ChemiDoc XRS Imaging System (Bio-Rad) with chemiluminescence reagent (Thermo Fisher Scientific, 32106).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eMicro-CT\u003c/h2\u003e\u003cp\u003eThe femora were dissected from mice and fixed overnight in 4% paraformaldehyde. Then the bones were wrapped with sealing film and scanned using a high-resolution micro-computed tomography (\u0026micro;CT) system (Skyscan 1172, Bruker MicroCT, Kontich, Belgium). Image reconstruction was performed using NRecon software (version 1.6, Bruker MicroCT), followed by analysis with CTAn (version 1.9) for trabecular and cortical bone parameters (BV/TV, Tb.Th, Tb.N and Tb.Sp ) and 3D visualization with CTVol (version 2.0) with the region of 5% of the femoral length belowe the growth plate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eQuantification and statistical analysis\u003c/h2\u003e\u003cp\u003eData is presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and exhibits continuous normal distribution. Statistical analyses were performed using Excel or GraphPad Prism 8 software. Comparisons between two groups were conducted using two-tailed Student\u0026rsquo;s t-tests, and one-way or two-way ANOVA were used for comparisons among multiple groups. All experiments were repeated three times, and representative experiments are shown. For consistency in comparisons, significance across all figures is indicated as follows: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Sample sizes for in vivo and in vitro experiments were determined based on prior experience. All samples were randomly assigned, and no animals were excluded from the experiments.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003e1. Aging induces the production of 5\u003c/b\u003e'\u003cb\u003e-tsRNA-Glu\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;CTC\u003c/b\u003e\u003c/sup\u003e \u003cb\u003ein Bone Marrow Mesenchymal Stromal Cells (BMSCs)\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePrevious studies have demonstrated that the osteogenic-adipogenic differentiation bias of BMSCs during aging constitutes a significant cause of age-related bone loss\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. To test whether transfer RNA-derived small RNAs (tsRNA) is involved in this progression, we first performed small RNA sequencing on BMSCs derived from 3-month-old (young) and 20-month-old (aged) mice to identify the expression pattern of tsRNAs during BMSCs aging. We identified two novel tsRNAs, TRF-mmu-053 and TRF-mmu-007, and a known tsRNA, TRF-5004b, which exhibited a pronounced increase in aged BMSCs compared to young BMSCs, with counts\u0026thinsp;\u0026gt;\u0026thinsp;500, logFC\u0026thinsp;\u0026gt;\u0026thinsp;2, and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). By analyzing the secondary structures of TRF-mmu-053 and TRF-mmu-007 using R2DT\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. We found that TRF-mmu-053 partly matched of tRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e and was characterized as 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, while TRF-mmu-007 partly matched of tRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e and was defined as 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). To validate the increased expression of the three tsRNAs, we performed fluorescence in situ hybridization (FISH) on aged (20-month-old) bone tissues and young (3-month-old) samples, utilizing specific DNA probes complementary to 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e and TRF-5004b. Indeed, the three tsRNAs exhibited a significant increase in expression within aged bone tissues compared to young samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and C). The colocalization assay combining Leptin Receptor (LEPR) immunofluorescence staining and FISH staining revealed that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e and 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e were primarily expressed in BMSCs. Specifically, 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e exhibited a higher colocalization rate with LEPR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and D). We subsequently assessed the regulatory effects of the three tsRNAs on BMSCs differentiation in vitro. Primary BMSCs were transfected with the three tsRNAs and incubated with osteogenic or adipogenic differentiation medium. Alizarin Red S and Oil Red O staining revealed that treatment with the three tsRNAs significantly inhibited calcium nodule formation while promoting lipid droplet formation. Among the tested conditions, 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e and 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e exhibited a stronger inhibitory effect on calcium nodule formation and a greater promoting effect on lipid droplet formation compared to TRF-5004b (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u0026ndash;H). SA beta-gal staining showed that over-expressing the three tsRNAs exhibited a higher proportion of β-Gal-positive senescent cells compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and H). Similarly, relative to TRF-5004b, 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e and 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e exhibited a stronger pro-senescence effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and H). These data suggested that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e and 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e and TRF-5004b disrupt the lineage fate of BMSCs and induce their senescence. Based on the results of in situ hybridization and cellular staining, we selected 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e and 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e for the follow-up research. To identify the mRNA regulatory patterns governed by 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e and 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e, we collected BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e and 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e and performed RNA sequencing. The data showed that over-expression of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e resulted in 477 upregulated and 674 downregulated genes, while over-expression 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e led to 482 upregulated and 481 downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). KEGG pathway enrichment analysis revealed that these differentially expressed genes from 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e over-expression group were significant enrichmented in cell adhesion molecules, cellular senescence and cell cycle pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). Meanwhile, these differentially expressed genes from 5'-tsRNA-Lys\u003csup\u003e\u0026minus;\u0026thinsp;CTT\u003c/sup\u003e over-expression group were significant enrichmented in cell adhesion molecules, cell cycle pathways and calcium signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). Examination of differentially expressed genes related to senescence, osteogenesis, and adipogenesis showed downregulation of anti-aging genes (e.g., \u003cem\u003eFoxm1\u003c/em\u003e, \u003cem\u003eCdk1\u003c/em\u003e, \u003cem\u003eCcnd1\u003c/em\u003e) and osteogenic genes (e.g., \u003cem\u003eSp7\u003c/em\u003e, \u003cem\u003eCol1a1\u003c/em\u003e, \u003cem\u003eDmp4\u003c/em\u003e), and upregulation of adipogenic (e.g., \u003cem\u003eLpl\u003c/em\u003e, \u003cem\u003ePparg\u003c/em\u003e, \u003cem\u003eFabp4\u003c/em\u003e) and pro-inflammatory genes (e.g., \u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eIL-10\u003c/em\u003e, \u003cem\u003eCcl3\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). These results were validated by real-time quantitative PCR (RT-qPCR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM). Given that the results of the colocalization assay and RNA sequencing, 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e may be act as a more important regulator driving BMSCs differentiation shift and cellular senescence. It is consistent with a previous result showing a regulatory role of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e in brain aging and age-related memory decline\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Therefore, we selected 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e for further study.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e​\u003cb\u003e​​​2. 5\u003c/b\u003e'\u003cb\u003e-tiRNA-Glu\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;CTC\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eaccelerates bone loss in both male and female Mice​​\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo explore the role of tRNA-derived small RNAs on bone metabolism in vivo, we utilized rAAV serotype 2/9 with LEPR promoter for delivery of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e (rAAV2/9-LEPR-5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e) to BMSCs. Young (3-month-old) and aged (12-month-old) male and female mice received intra-bone marrow injections of rAAV-delivered 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e or rAAV-2/9 vector (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). All mice received two intraperitoneal calcein injections within the last six days of the one-month period before serum, bone, and bone marrow collection. To explore the overexpression efficiency of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e in BMSCs, we isolated and cultured BMSCs from the 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e group and the AAV-2/9 vector group. RT-qPCR analysis showed that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e levels were significantly increased in the 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e group compared to the control group, indicating that rAAV2/9 successfully targeted BMSCs and boosted 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e expression in BMSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). ELISA analysis revealed significantly reduced serum PINP levels and elevated CTX levels in both young and aged treated mice compared to control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). These findings suggest that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e exerts an inhibitory effect on bone formation. Micro-computed tomography (micro-CT) analysis of the distal femoral metaphysis revealed significantly reduced trabecular bone mass, trabecular thickness and trabecular number and increased trabecular separation in AAV-5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e treated male mice compared to control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-I). Hematoxylin and eosin (H\u0026amp;E) staining confirmed a reduction in trabecular area in mice injected with rAAV-delivered 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e compared to the negative control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ and K). Immunohistochemical and immunofluorescence analyses revealed decreased numbers of osteoblasts, SP7-positive cells, and LEPR-positive cells, along with increased osteoclast numbers, in both young and aged male mice treated with rAAV-delivered 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e compared to placebo-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL\u0026ndash;S). Similarly, calcein double-labeling indicated a reduced mineral apposition rate (MAR) in the 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e-treated group relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eT and U). Consistently, AAV-5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e-treated also exhibited significant reductions in bone mass and trabecular number in female mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-F). Osteoblast numbers, SP7-positive cells, LEPR-positive cells, and MAR were decreased compared to control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-L and O-P). Interestingly, unlike in males, 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e overexpression did not alter osteoclast numbers in female mice, and its effects on trabecular thickness and separation were inconsistent across age groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM-N). These results demonstrated that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e overexpression impairs bone formation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e​\u003cb\u003e​​​3. Inhibition of 5\u003c/b\u003e'\u003cb\u003e-tiRNA-Glu\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;CTC\u003c/b\u003e\u003c/sup\u003e \u003cb\u003evia ASO ameliorates BMSCs senescence and bone loss in aged mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThese lines of evidence support 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e as a potential target for age-related bone less. Based on the biological mechanism by which antisense oligonucleotides bind to target RNA and induce RNase H-mediated RNA degradation. We next designed an antisense oligonucleotide (ASO) targeting 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, and transfected it into primary bone marrow mesenchymal stromal cells (BMSCs) isolated from aged mice. Subsequently, we performed Alizarin Red S, Oil Red O staining and SA β-galactosidase staining assays to comprehensively evaluate the effect of ASO on BMSCs differentiation and senescence in osteogenic or adipogenic differentiation medium. The results showed that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e-ASO treatment significantly reduced the number of senescent cells in aged BMSCs, enhanced mineralization (as indicated by increased calcium nodule formation), while decreased lipid droplet formation (as demonstrated by decreased Oil Red O staining), compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;C). The data suggested that the inhibitor of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e would ameliorate BMSC senescence and differentiation shift. Based on the ameliorative effects of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e-ASO on aged BMSCs, we are eager to know whether the ASO-based inhibitor of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e ameliorates age-associated bone loss in vivo. The rAAV2/9-LEPR-5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e-ASO was constructed and delivered via intra-bone marrow injection into aged (18 month) male and female mice at 1\u0026times;10\u0026sup1;\u0026sup1; vg per mouse for a one-month intervention. The control group received injection of an equivalent volume of viral vector. Compared to control groups, mice treated with rAAV2/9-LEPR-5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e-ASO exhibited increased bone mass, greater trabecular thickness and number, and reduced trabecular separation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026ndash;H). Beyond that​​, rAAV2/9-LEPR-5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e-ASO-treated aged female mice had fewer marrow adipocytes, while males showed increased trabecular bone mass (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI and J). Immunostaining for osteocalcin, tartrate-resistant acid phosphatase (TRAP), SP7, and LEPR revealed that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e-ASO-treated aged mice exhibited increased osteoblasts, SP7-positive cells, and LEPR-positive cells, decreased osteoclasts, and an improved mineral apposition rate (MAR) in both aged male and female mice compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK\u0026ndash;T). These results demonstrated that the ASO-based inhibitor of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e alleviates age-associated bone loss.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e​\u003cb\u003e​4. 5\u003c/b\u003e'\u003cb\u003e-tiRNA-Glu\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;CTC\u003c/b\u003e\u003c/sup\u003e \u003cb\u003einduces BMSCs senescence by disrupting mRNA stability​\u003c/b\u003e​\u003c/p\u003e\u003cp\u003eIt is well established that tiRNAs inhibits global translation or selectively suppress of specific gene expression by disrupting ribosomal assembly, interacting with initiation factors such as eIF4F, and promoting the formation of stress granules (SGs)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Besides, certain 5'-tsRNAs can recognize the 3' UTR of mRNAs and repress their expression\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.​ To explore the regulatory mechanism by which 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e modulates BMSC senescence and differentiation. Firstly, to identify specific mRNA targets that bind to 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, we performed high-throughput sequencing of mRNA fragments pulled down by biotin-labeled 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e using streptavidin beads (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The distribution of sequencing reads across the genome was analyzed, and the results revealed sequencing reads were mapped to exonic (85.46%), intronic (9.91%) and intergenic (4.63%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The data indicated that the sequencing reads were mainly distributed in the exon region. To further identify target genes directly regulated by 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, we performed an overlap analysis between the result of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e pulled down assay followed by high-throughput sequencing and mRNA expression profiling via mRNA-seq (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL), which identified 312 overlapping genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). We next sought to elucidate the biological pathways and functions commonly associated with these 312 overlapping genes across multiple conditions. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the 312 overlapping genes were significantly enriched in phagosome pathway, cellular senescence, antigen processing and presentation and the cell cycle pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and E). Based on these KEGG results, we focused subsequent analyses on genes related to cellular senescence and cell cycle regulation, including \u003cem\u003eFoxm1, Cdk1, Capn1\u003c/em\u003e and \u003cem\u003eCcnd1\u003c/em\u003e. Through the analysis of peak reads distributed across genes from high-throughput sequencing results, we identified 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e mainly binds to the 3\u0026prime;UTRs of \u003cem\u003eFoxm1, Cdk1, Capn1, Ccnd1\u003c/em\u003e, and \u003cem\u003eSp7\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Based on the complementary sequences of \u003cem\u003eFoxm1, Cdk1, Capn1, Ccnd1\u003c/em\u003e, and \u003cem\u003eSp7\u003c/em\u003e retrieved from National Center for Biotechnology Information (NCBI) database, we identified seed binding sites for 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Previous studies have supported that small RNAs influence mRNA stability through binding to their 3'UTR regions and promoting degradation through AU-rich elements or by facilitating microRNA binding\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. To further validate whether 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e impairs mRNA stability by binding to the 3\u0026prime;UTRs of these mRNA, we transfected BMSCs cell lines with 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e or negative RNA for 48h, followed by actinomycin D treatment and collected RNA samples at multiple time points over 16 h. We found that that the relative \u003cem\u003eFoxm1\u003c/em\u003e mRNA half-life was 30.42 h of BMSCs treated with negative RNA through RT-qPCR. Notably, in 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e-overexpressing BMSCs, \u003cem\u003eFoxm1\u003c/em\u003e mRNA levels were decreased faster upon actinomycin D treatment and the resulting relative \u003cem\u003eFoxm1\u003c/em\u003e mRNA half-life (10.36 h) was reduced by 65.94%. Meanwhile, other senescence-related mRNA half-life of \u003cem\u003eCdk1\u003c/em\u003e, \u003cem\u003eCapn1\u003c/em\u003e and \u003cem\u003eCcnd1\u003c/em\u003e were reduced by 45.4%, 23.96% and 59.64% respectively, and the osteogenic mRNA level of \u003cem\u003eSp7\u003c/em\u003e was decreased by 61.2% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). The results demonstrated that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e influences target genes' mRNA stability by binding to their 3\u0026prime;UTRs. To further verify whether 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e mediates mRNA degradation through the 3'UTR, we cloned the 3' UTR or mutated seed region of target genes into Dual-Luciferase Reporter Vector (pmirGLO) and constructed pmirGLO-WT or pmirGLO-Mut plasmids. The pmirGLO-WT or pmirGLO-Mut plasmids were co-transfected into HEK293T cells along with either 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e or negative control RNA. As expected, ectopic expression of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e induced a significant decrease in Fluc activity of the WT senescence-related (e.g., \u003cem\u003eFoxm1\u003c/em\u003e, \u003cem\u003eCdk1\u003c/em\u003e, \u003cem\u003eCapn1\u003c/em\u003e, \u003cem\u003eCcnd1\u003c/em\u003e) and osteogenic (e.g., \u003cem\u003eSp7\u003c/em\u003e) genes reporters. Conversely, this decrease was rescued by mutations in the seed regions of 3' UTRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). The degradation of mRNAs will disrupt translation and lead to a decrease in protein translation. Western blotting confirmed that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e overexpression in young BMSCs downregulated senescence-related (e.g., P16, P21, FOXM1, CDK1, CAPN1, CCND1) and osteogenic (e.g., SP7) proteins and upregulated adipogenic (e.g., LPL) proteins, while 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e-ASO treatment in aged BMSCs reversed these effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eO). These findings suggest that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e decreases target genes expression by binding to 3'UTR of mRNAs, thereby disrupting mRNA stability. Since the target genes like \u003cem\u003eFoxm1, Cdk1, and Ccnd1\u003c/em\u003e are closely associated with the cell cycle, we further performed flow cytometry to analyze the cell cycle distribution. 48 h after transfecting young primary BMSCs with 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e or negative RNA, we stained cells with Ki67 antibody and DAPI dye to categorize the cells into three stages: G\u003csub\u003e0\u003c/sub\u003e phase, G\u003csub\u003e1\u003c/sub\u003e phase, and G\u003csub\u003e2\u003c/sub\u003e/S/M phase. We found that the proportion of BMSCs transfected with 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e in the G\u003csub\u003e2\u003c/sub\u003e/S/M phase was significantly decreased compared to the control BMSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ and k), indicating that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e impairs the proliferative activity of BMSCs. Subsequently, we performed Ki67 immunofluorescence staining in primary BMSCs, which further validates that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e inhibits cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL and M).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo investigate whether ASO-based inhibitor of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e will rescue aged primary BMSCs' senescence, we transfected 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e-ASO into aged BMSCs. The result indicated that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e-ASO-expressed aged BMSCs increased expression of senescence-related (e.g., \u003cem\u003eFoxm1\u003c/em\u003e, \u003cem\u003eCapn1\u003c/em\u003e) and osteogenic (e.g., \u003cem\u003eSp7\u003c/em\u003e) genes and decreased adipogenic gene (e.g., \u003cem\u003eLpl\u003c/em\u003e) expression by RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN) and western blotting assessment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eO). These results demonstrated that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e regulates BMSC senescence and differentiation by impairing the mRNA stability of anti-senescence-related genes (e.g., \u003cem\u003eFoxm1, Cdk1, and Ccnd1\u003c/em\u003e) and pro-osteogenic genes (e.g., \u003cem\u003eSp7\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eR).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e​\u003cb\u003e​5. Cellular stress triggers ANG-cleaving activity and induces 5\u003c/b\u003e'\u003cb\u003e-tiRNA-Glu\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;CTC\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eproduction\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePrevious studies have demonstrated that tiRNA, a product of the endoribonuclease ANG (Angiogenin), is generated through the cleavage of specific mature tRNA under diverse stress conditions- including oxidative stress, heat shock, hypothermia, hypoxia, and cold shock\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. As an important component of cellular stress responses, tiRNA plays a critical role in mitigating environmental stress and maintaining cellular homeostasis\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Aging is considered a chronic stress process characterized by increased oxidative stress; mounting evidences show that tsRNAs are differentially expressed in model organisms of different ages. Subsequently, we examined the changes in endoribonuclease expression during bone marrow mesenchymal stromal cells (BMSCs) senescence. The expression pattern of endoribonuclease in 2-month-old (young) and 24-month-old BMSCs was analyzed using RNA-sequencing\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. As expected, we observed upregulation of multiple tRNA-cleaving endoribonucleases including ANG in aged BMSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To further confirm the upregulation of ANG expression in aged BMSCs, we performed immunofluorescence co-staining of ANG and LEPR in bone tissues from 2- and 24-month-old mice. The result showed that ANG was specifically upregulated in aged BMSCs and exhibited a significant colocalization rate with LEPR (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and C). Correspondingly, relative quantitative analysis of ANG levels via RT-qPCR in young and aged female and male mice also confirmed this result (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Next, we sought to determine whether ANG serves as the pivotal enzyme responsible for the formation of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e in aged BMSCs. Firstly, we assessed ANG-mediated cleavage of tRNA-Glu using Northern blotting. The results revealed that ANG specifically cleaves full-length tRNA-Glu into a unique 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e in an in vitro reaction system. Notably, the abundance of this tiRNA exhibited a significant dose-dependent increase with ANG concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). Subsequently, we conducted in situ hybridization using a FITC-labeled 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e probe, combined with co-localization staining for ANG in both young and aged BMSCs. As expected, compared to young BMSCs, the levels of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e and ANG expression were significantly increased in aged BMSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). Researchers have proposed that ANG-induced tiRNAs may contribute to the formation of SGs (stress granules) in response to cellular stress. The formation of stress granules (SGs) promotes cell survival under various stress conditions, including heat shock, oxidative stress, ischemia, and viral infection. We further investigated stress granule (SG) formation in aged BMSCs. We first treated mouse BMSCs with 500 \u0026micro;M sodium arsenite (NaAsO\u003csub\u003e2\u003c/sub\u003e) for one hour\u0026mdash;a well-established inducer of cellular oxidative stress. After treatment, we examined the expression of G3BP1, a canonical marker for stress granules (SGs). Notably, NaAsO\u003csub\u003e2\u003c/sub\u003e treatment significantly increased the proportion of G3BP1-positive cells compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and F). This indicates that the stress induced by NaAsO\u003csub\u003e2\u003c/sub\u003e leads to the formation of stress granules, as evidenced by the upregulation of G3BP1. Furthermore, we performed immunofluorescence staining for G3BP1 to visualize the abundance of stress granules in aged BMSCs. The results revealed a significant increase in SG abundance in aged BMSCs compared to young BMSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and H). This suggests that aged BMSCs have a higher propensity to form stress granules under stress conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis observation suggests that enhanced SG formation may be beneficial to the survival of aged cells under physiological stress\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. It has been reported that under stress, ANG translocates from the nucleus to the cytoplasm, where it cleaves specific tRNAs into tiRNAs, subsequently disrupting cellular physiological functions and inducing age-related diseases\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Similarly, in our experiments, treatment of BMSCs with NaAsO\u003csub\u003e2\u003c/sub\u003e\u0026mdash;compared to the negative control group\u0026mdash;not only increased cytoplasmic ANG levels but also led to elevated levels of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e. Specifically, under normal conditions, ANG is predominantly localized in the nucleus of BMSCs, whereas under oxidative stress, it translocates to the cytoplasm and cleaves tRNA-Glu to generate 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eI\u0026ndash;J). Consistent with this mechanism, compared to young BMSCs, aged BMSCs also exhibited pronounced translocation of ANG from the nucleus to the cytoplasm, along with higher levels of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, as further confirmed by in sit hybridization at the cellular level (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). The results were further validated by western blotting, confirming the subcellular redistribution of ANG during the aging process of BMSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eM). These findings suggest that the distribution of ANG in the aged BMSCs undergoes a significant shift from the nucleus to the cytoplasm.\u003c/p\u003e\u003cp\u003eOur results demonstrate that ANG shifts from the nucleus to the cytoplasm, triggering ANG-mediated tRNA cleavage activity as a critical contributing factor to cellular senescence. This finding suggests that targeting the inhibition of ANG activity may serve as a therapeutic strategy for age-related osteoporosis. A study screening small-molecule inhibitors targeting the ribonucleolytic activity of human angiogenin (ANG) demonstrated that NCI compound 65828 (NCI-65828) significantly inhibits ANG enzymatic activity- with Ki values\u0026thinsp;\u0026lt;\u0026thinsp;100 \u0026micro;M. Subsequent studies revealed that ANG inhibition ameliorated cell stress induced by 5\u0026prime;tRNA-derived fragments (tiRNAs) and rescued the deleterious effects of NSun2 deficiency during neurodevelopment\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In a subsequent study, we aim to investigate whether angiogenin (ANG) inhibition, using the NCI compound 65828, can alleviate BMSCs senescence and age-associated bone loss. The molecular docking model of ANG with NCI-65828 was generated using AUTODOCK 3.0. In this model (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eN), the azo group is positioned in the catalytic center alongside Glu140. The main-chain oxygen atom is bound by His37, His137, and the Lys64 side chain. Additionally, the side chains form hydrogen bonds with the residues Asp65 and Val66.This binding model differs from the reported binding pattern of the human ANG protein\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. To determine the optimal concentration of ANG for cell treatment, we treated aged primary BMSCs with ANG inhibitor in a dose-response study. The CCK-8 assay showed that the ANG inhibitor-NCI-65828 promoted cell viability at lower concentrations (10 \u0026micro;M), whereas it inhibited cell proliferation at higher concentrations (\u0026gt;\u0026thinsp;50 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eO). Western blotting analysis demonstrated that treatment with the ANG inhibitor NCI-65828 at a concentration of 10 \u0026micro;M significantly increases the expression of the protein targeted by 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eP). The results indicate that 10 \u0026micro;M NCI-65828 represents the optimal concentration for cellular treatment. We next treated aged BMSCs with NCI-65828 (10 \u0026micro;M) and found that it significantly reduced the area of SA-β-gal staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eQ), enhanced calcium nodule formation, and decreased lipid droplet formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eR and S). Collectively, these findings demonstrate that NCI-65828 effectively attenuates senescence in aged BMSCs, enhances osteogenic differentiation while inhibiting adipogenic differentiation, thereby modulating the lineage commitment balance.\u003c/p\u003e\u003cp\u003e​\u003cb\u003e​​​6. Inhibitor of ANG-cleaving activity ameliorates age-related bone loss in aged mice​​\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate whether NCI-65828, as a targeted ANG inhibitor, ameliorates age-related bone loss, the NCI-65828 was encapsulated into bone-targeted liposomes and delivered via intra-bone marrrow injection into aged (18 month) male and female mice for a one-month intervention. Control group received injection of an equivalent volume of empty liposomes. Compared to control groups, mice treated with NCI-65828 exhibited increased bone mass, greater trabecular thickness and number, and reduced trabecular separation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;E). Furthermore, ANG inhibitor-treated mice exhibited higher levels of Procollagen type I N-terminal propeptide (PINP) and lower levels of C-terminal telopeptide of type I collagen (CTX) compared to the control group, as determined by ELISA analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eF and G). Histological analysis further demonstrated a lower proportion of adipocytes in female treatment groups and a larger trabecular area in male treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eH and I). In addition, we performed immunohistochemical (IHC) staining on paraffin sections of the femur and immunofluorescence (IF) staining on frozen sections of the tibia. Compared to control group, mice treated with the ANG inhibitor exhibited a higher proportion of osteoblasts, Sp7-positive cells, and LEPR-positive cells, along with a lower proportion of osteoclasts in bone tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ\u0026ndash;Q). These findings suggest that NCI-65828 treatment exerts a ameliorative effect on age-related bone loss, and inhibition of ANG activity is recognized as a potential therapeutic strategy for osteoporosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003eR).\u003c/p\u003e\u003cp\u003e\u003cb\u003e​​7. 5\u003c/b\u003e'\u003cb\u003e-tiRNA-Glu\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;CTC\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eserves as a novel serum biomarker for assisting in the diagnosis of osteoporosis​\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs we know, transfer RNA-derived fragments as (tsRNAs) have also been suggested as potential biomarkers for various diseases and health conditions. Studies have indicated that certain tsRNAs are specifically changed in the serum of osteoporosis patients\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In our study, we collected peripheral blood samples from 40 osteoporosis patients (aged 70\u0026thinsp;~\u0026thinsp;90 years) and serum samples from 40 age- and gender-matched healthy individuals. The serum samples from osteoporosis patients and those from healthy controls were each pooled separately into individual tubes and subsequently subjected to small RNA sequencing analysis. The subtype distribution demonstrated that differentially expressed (DE) tsRNAs derived from tRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, tRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;TTC\u003c/sup\u003e, tRNA-Gly\u003csup\u003e\u0026minus;\u0026thinsp;GCC\u003c/sup\u003e, tRNA-Phe\u003csup\u003e\u0026minus;\u0026thinsp;GAA\u003c/sup\u003e and tRNA-Pro\u003csup\u003e\u0026minus;\u0026thinsp;TGG\u003c/sup\u003e were the most abundant (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). In our analysis of differentially expressed small RNAs, we found that small RNAs displayed a cholinergic-associated shift, from miRNAs to tsRNAs. Specifically, following filtration with a count threshold greater than 10, differential expression analysis of small RNAs revealed that 79% of the 323 DE tsRNAs including 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e were upregulated, whereas 55% of the 110 DE miRNAs were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and B). The observed cholinergic shift is consistent with the phenomenon seen in post-stroke patients when compared to healthy controls\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Notably, the 55% DE miRs included several miRs known to be perturbed in osteoporosis: has-miR-100-5p, has-miR-125-5p, and has-miR-17-5p \u003csup\u003e52,53\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Our findings point toward tsRNAs/miRNAs as potential biomarkers for increased osteoporosis risk in these patients. In this study, we investigate whether serum levels of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e could serve as a candidate diagnostic biomarker for osteoporosis, focusing on its expression patterns and clinical correlation with bone metabolism markers. Therefore, we quantified the serum levels of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, bone formation-related markers (including PINP, BGP/Osteocalcin), and bone resorption-related markers (including CTX and TRAP) in 40 osteoporosis patients using ELISA analysis. Although 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e did not show a strict correlation with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), its levels were negatively correlated with BMD and Z-scores in osteoporosis patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE\u0026ndash;F). Moreover, serum 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e levels correlated negatively with osteogenic markers (PINP and BGP; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG\u0026ndash;H) and positively with osteoclast markers (CTX and TRAP; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ\u0026ndash;K). A positive correlation was also observed between serum ANG and 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eL), which supports the proposed mechanism that ANG-mediated cleavage contributes to the biogenesis of this tRNA fragment. In contrast, correlations with ALP, serum calcium, and 25-hydroxyvitamin D3 remained weak and require further validation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI, M\u0026ndash;N). Together, these findings indicate that 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e may serve as a promising serum biomarker for diagnosing osteoporosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEmerging evidence has implicated the accumulation of reactive oxygen species (ROS) and increased oxidative stress as causative factors in the pathogenesis of age-related osteoporosis\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. As an important component of stress responses, angiogenin-induced tRNA cleavage neutralizes stress stimuli by decreasing general translation while reprogramming pro-survival and anti-apoptotic protein translation, conserving anabolic energy, and promoting cell survival\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. However, whether increased oxidative stress triggers angiogenin-induced tRNA cleavage involved in the progression of age-related osteoporosis remains largely unknown. In this study, we demonstrated that oxidative stress induces angiogenin translocation from the nucleus to the cytosol, where it cleaves tRNA-Glu, leading to abundant accumulation of the 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e fragment in senescent BMSCs. The age-induced 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e alters mRNA metabolism through a regulatory mechanism highly similar to that of miRNAs, leading to bone-fat imbalance and thereby accelerating bone loss. This study elucidates a novel regulatory axis in age-related osteoporosis, wherein oxidative stress-induced tRNA fragmentation operates as a central regulatory mechanism.\u003c/p\u003e\u003cp\u003eObservations demonstrate that tRNA fragments are present across diverse organisms and show stress-induced upregulation. In this study, we mainly focus on 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, which is significantly accumulated in senescent BMSCs and was also detected in aging medial prefrontal cortices (mPFCs)\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. As reported in a previous study, 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e serves as a major factor triggering age-related defects in cell function. Our data indicates that the identification of 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e as a critical factor contributes to the disruption of bone-fat differentiation balance in BMSCs. Research indicates that tiRNA suppresses protein translation through multiple mechanisms: (1) displacing the cap-binding complex eIF4F from capped mRNA, (2) competitively binding to LARS2 (a mitochondrial leucyl-tRNA synthetase) to disrupt mitochondria-encoded protein translation, or (3) reducing mRNA transcript stability\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. In our study, we found that tiRNA, similar to miRNAs, suppresses its targets through a mechanism that may depend on its incorporation into Argonaute (Ago) protein complexes. Apart from this regulatory mechanism, tiRNAs may also interact with candidate proteins involved in BMSCs differentiation - a hypothesis that merits further investigation. It is worth noting that some tRNA-derived fragments (tRFs), which represent another type of fragments derived from tRNAs and are cleaved by Endonuclease Z (RNaseZ), cytoplasmic homologous ribonuclease Z2 (ELAC2), and Dicer, also exhibited a pronounced increase in senescent BMSCs. Unlike tiRNA, tRFs are processed by specific endonucleases and play diverse functions in various diseases\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. It is suggested that tRFs may be involved in regulating BMSCs senescence and differentiation, which will be further investigated in our future research. Recently research reported that some novel endonucleases, such as schlafen family member 12 (SLFN12) selectively digests tRNALeu(TAA) producing more complex tRNA fragments, which is not similar to the traditional tRFs\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. This implies that previously unidentified tRNA-cleaving endonucleases may exist, and that the structural complexity of tRFs could be greater than our current understanding. We believe that investigating the mechanisms underlying tRF-mediated regulation is significant importance while presenting significant challenges.\u003c/p\u003e\u003cp\u003eAngiogenin (ANG), a member of the secreted ribonuclease (RNASE) superfamily, exerts diverse physiological functions, ranging from promoting angiogenesis to enabling cell growth, proliferation, and survival under adverse conditions\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Recently a study reported that angiogenin, secreted by osteoclasts, protects neighboring vascular cells against senescence\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. In our study, we demonstrated that angiogenin-mediated cleavage of specific tRNAs generates tiRNA products, such as 5'-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e, which induces BMSCs senescence and accelerates bone loss via alterations in mRNA metabolism. Our data indicate that angiogenin functions as a detrimental regulator of BMSCs senescence and differentiation. Notably, other previous studies have also identified angiogenin as a detrimental regulator, aligning with its established role in modulating hippocampal neurogenesis and age-related memory decline through angiogenin-mediated tRNA cleavage\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. These controversial findings may be explained by the tightly regulated substrate recognition of angiogenin and its subcellular localization in response to cellular stress conditions\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Under growth conditions, angiogenin is translocated to nucleus and stimulates ribosomal RNA (rRNA) transcription, thus facilitating cell growth and proliferation. This may represent a biological process that promotes the proliferation of young BMSCs. Under stress conditions, angiogenin accumulates in cytoplasmic compartments, where it modulates the production of tiRNA and triggers stress granule (SG) formation to protect cells from cellular stress\u003csup\u003e\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. The physiological significance of this process lies in its promotion of senescent cell survival under cellular stress conditions\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. In our study, the process by which angiogenin translocates to the cytosol appears to promote senescent BMSCs survival by triggering stress granule (SG) formation under oxidative stress. Meanwhile, angiogenin accumulates in the cytosol, where it cleaves specific tRNAs to generate tiRNA, thereby suppressing global protein translation. As demonstrated in our study, angiogenin-induced 5'-tiRNA repress the expression of anti-senescence and pro-osteogenic genes, including \u003cem\u003eFoxm1\u003c/em\u003e, \u003cem\u003eCdk1\u003c/em\u003e, \u003cem\u003eCapn1\u003c/em\u003e, \u003cem\u003eCcnd1\u003c/em\u003e, and \u003cem\u003eSp7\u003c/em\u003e. Regarding the differential physiological functions, a proposed mechanism suggests that angiogenin-mediated tiRNA selectively inhibit the translation of mRNAs containing weak internal ribosome entry sites (IRES), while simultaneously enhancing the translation of mRNAs with strong IRES elements that promote cell survival under stress conditions\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. As a result, physiological progression contributes to the survival of senescent BMSCs with impaired osteogenic differentiation potential under oxidative stress conditions. Another alternative hypothesis to explain the challenge role of angiogenin is proposed: Only a subset of senescent BMSCs exhibit high angiogenin expression, whereas young BMSCs show either no expression or significantly lower expression levels. Consequently, only these angiogenin-strongly positive senescent BMSCs are susceptible to angiogenin-induced tRNA cleavage under oxidative stress. This observation not only corroborates the well-documented heterogeneity of senescent cells but also positions angiogenin as a potential novel senescence marker\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Furthermore, these findings provide a mechanistic explanation for how pharmacological inhibition of angiogenin activation via NCI-65828 ameliorates BMSCs senescence and age-related bone loss. Previous studies have supported the notion that eliminating senescent cells enhances bone formation in age-related osteoporosis. Our findings also indicate that targeting the elimination of senescent BMSCs with elevated angiogenin expression considers as a viable therapeutic strategy for age-related osteoporosis\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOf note, there are four aspects of this field that are interesting and worth studying in the future. The first aspect pertains to the mechanism underlying angiogenin translocation from the nucleus to the cytosol under oxidative stress conditions, with a focus on identifying regulatory factors and signaling pathways involved in this process. The second aspect focuses on the factors influencing the activation of angiogenin under oxidative stress. By using immunoprecipitation with a specific anti-angiogenin antibody followed by LC-MS analysis, we aim to identify angiogenin-interacting proteins and further investigate which candidate binding proteins, such as RNH1, activate angiogenin under oxidative stress\u003csup\u003e\u003cspan additionalcitationids=\"CR68\" citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. The third aspect concerns the reason why tRNA is cleaved by angiogenin. Studies have reported that \u003cem\u003eDnmt2\u003c/em\u003e- and \u003cem\u003eNSun2\u003c/em\u003e-mediated cytosine-5 methylation (m5C) promotes tRNA stability. A lack of m5C modification increases stress-induced cleavage of tRNAs and sensitizes flies to oxidative stress\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. This suggests that angiogenin tends to cleave tRNAs lacking m5C modification within the anti-codon loop, although this phenomenon is limited to specific tRNAs. Based on these findings, we tentatively propose that downregulation of the m5C-modifying enzymes \u003cem\u003eDnmt2\u003c/em\u003e or \u003cem\u003eNSun2\u003c/em\u003e during aging reduces tRNA m5C modification. This reduction may subsequently enhance angiogenin-mediated tRNA cleavage under oxidative stress, although this effect appears confined to specific tRNA. The four aspect investigates strategies for the precise delivery of NCI compound 65828 (an angiogenin inhibitor) to senescent BMSCs, aiming to mitigate stress-induced tRNA cleavage. As angiogenin is highly expressed in liver tissue, it functions as an angiogenic factor and plays a critical role in angiogenesis\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. If the BMSCs-targeted NCI-65828 drug delivery vector leaks into liver tissue, it is highly likely to impair liver vessel function. In the present study, we constructed a BMSCs-targeted drug delivery liposomes incorporating a bone affinity peptide (DSS)6, which exhibit high delivery efficiency in bone marrow and minimize systemic side effects\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. It is suggested that the use of a bone-targeted delivery system efficiently eliminates senescent cells by delivering senolytics, this approach will open new avenues for addressing age-related bone disease\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. We believe that addressing these four challenges in the future will enhance our understanding of how angiogenin-mediated tRNA cleavage influences bone disease.\u003c/p\u003e\u003cp\u003eThe increased or dysregulated occurrence of tsRNAs in biofluids such as serum, or sperm in various cancer or disease conditions makes them attractive candidates for biomarker development\u003csup\u003e\u003cspan additionalcitationids=\"CR74 CR75\" citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. In our study, we observed that serum levels of 5ʹ-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e in osteoporosis patients exhibited a negative correlation with bone mineral density (BMD), procollagen type I N-terminal propeptide (PINP), and osteocalcin (BGP), while demonstrating a positive correlation with C-terminal telopeptide of type I collagen (CTX) and tartrate-resistant acid phosphatase (TRAP). These findings suggest that 5ʹ-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e may serve as a potential biomarker for diagnosing age-related osteoporosis. This observation provides the first clinical insight into the contribution of 5ʹ-tiRNA-Glu\u003csup\u003e\u0026minus;\u0026thinsp;CTC\u003c/sup\u003e to the pathophysiological regulation of bone formation.​\u003c/p\u003e\u003cp\u003eIn summary, this study demonstrates that angiogenin-mediated tRNA cleavage under oxidative stress serves as a critical trigger for age-related osteoporosis. We identified the first mechanistic link between 5ʹ-tRNA fragments producted by angiogenin and the age-related osteoporosis. Furthermore, targeting the inhibition of angiogenin with NCI-65828 represents a promising therapeutic strategy for this disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from China National Health Development Research Center (2025ZD0550400), National Natural Science Foundation of China (Grant No. 82471619), Natural Science Foundation of Hunan Province (Grant No. 2024JJ5459), and Scientific Research Program of FuRong Laboratory (No. 2024PT5104). \u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.X. and G.-Q.X. conceived the project and designed the experiments. W.-H.K. and Z.-L.P. performed the experiments. M.-Z.Y. and M.-S.Y. assisted with data acquisition. Y.L. and Y.X. performed the data analysis. Y.X. secured funding and provided project administration and supervision. W.-H.K. and Z.-L.P. integrated the data and drafted the manuscript together with Y.X. All authors reviewed and revised the manuscript.\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSies, H., Berndt, C., and Jones, D.P. (2017). Oxidative Stress. 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Science \u003cem\u003e389\u003c/em\u003e, eadp5384. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.adp5384.http://doi.org/10.1126/science.adp5384\u003c/span\u003e\u003cspan address=\"10.1126/science.adp5384.10.1126/science.adp5384\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Osteoporosis, Angiogenin, Aging, Bone Marrow Stromal Cells, tRNA-derived stress-induced RNAs","lastPublishedDoi":"10.21203/rs.3.rs-7830644/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7830644/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWith aging, the accumulation of cellular stress and cellular senescence drives the progression of osteoporosis. As a conserved cellular stress response, angiogenin (ANG) induces the cleavage of cytoplasmic transfer RNAs (tRNAs) to generate tRNA-derived stress-induced RNAs (tiRNAs) with diverse functional roles in various diseases. However, their biological functions in regulating osteoporosis remain poorly understood. In our study, we observed that angiogenin levels increase in senescent Bone Marrow Stromal Cells (BMSCs), and that angiogenin promotes age-dependent accumulation of 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e by cleaving tRNA-Glu under oxidative stress. The 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e disrupts the stability of anti-senescence and pro-osteogenic mRNAs, leading to bone-fat imbalance and thereby accelerating bone loss. Blocking 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e wiht antisense oligonucleotide (ASO) or inhibiting angiogenin with NCI-65828 alleviates BMSCs senescence and age-related bone loss. Clinical sample detection and analysis revealed that elevated serum levels of 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e in patients with osteoporosis exhibit a positive correlation with bone resorption markers and a negative correlation with bone formation markers. These findings suggest that 5'-tiRNA-Glu\u003csup\u003e-CTC\u003c/sup\u003e may serve as a potential biomarker for diagnosing age-related osteoporosis. Collectively, our study sheds new light on the role of ANG-induced 5'-tiRNAs in regulating BMSCs senescence and highlights angiogenin as a promising therapeutic target for age-related osteoporosis.\u003c/p\u003e","manuscriptTitle":"Targeting angiogenin as a therapeutic strategy for age-related osteoporosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-28 07:00:35","doi":"10.21203/rs.3.rs-7830644/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2103712b-08ae-445e-84d5-73d387be55ce","owner":[],"postedDate":"November 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58154338,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases/Metabolic bone disease/Osteoporosis"},{"id":58154339,"name":"Biological sciences/Drug discovery/Biomarkers/Diagnostic markers"}],"tags":[],"updatedAt":"2025-12-09T23:35:49+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-28 07:00:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7830644","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7830644","identity":"rs-7830644","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}