LINC00323 induced by hypoxia promote cartilage callus by interacting with FUS to regulate PDGFB expression

LINC00323 induced by hypoxia promote cartilage callus by interacting with FUS to regulate PDGFB expression | 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 Research Article LINC00323 induced by hypoxia promote cartilage callus by interacting with FUS to regulate PDGFB expression Jiang 黄, Ju yong Wang, Xiang Yao Sun, Shuai An, Guang Lei Cao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3966058/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 Intermittent hypoxia has been reported to contribute beneficial effects on fracture healing depending on various factors like duration, frequency, and severity. Yet, little is known about the underlying molecular mechanism. Our previous study found that LINC00323 was up-regulated under hypoxic conditions, suggesting that it might play a final role in hypoxia-induced fracture repair. The present study is to investigate the osteogenic effect of LINC00323 in vitro and in vivo . Upregulation of LINC00323 enhanced the mineralization and activity ALP and increased the expression of osteogenic markers. Further analysis revealed that LINC00323 promoted PDGFB expression by binding FUS to regulate the growth and osteogenic differentiation of MC3T3-E1. Lentivirus mediated LINC00323 particles were injected into the fracture site of the tibia of mice, and fracture healing was evaluated by X-rays, micro-CT examination, biomechanical test and histological staining. Local injection of Lentivirus-LINC00323 increased bone mass, biomechanical strength and cartilage callus formation. These findings indicated that LINC00323 induced the differentiation of osteoblast-like cells via regulation of the expression of PDGFB, represents a theoretical basis to accelerate fracture healing. Bone QCT/µCT Matrix mineralization Molecular pathways - development Osteoblasts Injury/fracture healing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Fracture healing is a complex physiological process, crucial for the restoration of bone integrity and function after injury, which attend considerable interest in both clinical and research settings [ 1 , 2 ]. Advances in understanding the bone healing mechanisms have elucidated a series of complex cellular and molecular interactions, involving hematoma formation, inflammation, soft and hard callus formation, and extracellular matrix remodeling [ 3 – 5 ]. Emerging research is now focused on targeting specific pathways and molecules to accelerate and improve fracture healing, thus potentially reducing its financial impact [ 6 – 8 ]. While the traditional view of fracture healing emphasizes the role of mechanical factors and local biology, recent evidence suggests that systemic factors like oxygen tension also play a pivotal role [ 9 , 10 ]. Intermittent hypoxia, characterized by periodic exposure to low oxygen levels, has been increasingly recognized for its potential therapeutic benefits in enhancing fracture repair [ 10 ]. This paradoxical phenomenon, where transient low oxygen states can exert beneficial effects on bone healing, depends on various factors such as duration, frequency, and severity of hypoxic episodes [ 11 , 12 ]. However, the molecular mechanisms underlying these beneficial effects remain largely elusive. Previous studies have hinted at the involvement of non-coding RNAs (ncRNAs) in the cellular response to hypoxic conditions [ 13 – 15 ]. Among these, long non-coding RNAs (lncRNAs) have emerged as key regulators of gene expression, capable of modulating diverse biological processes including osteogenesis [ 13 ]. Recent studies suggest that LINC00323 regulate the expression of specific genes by interacting with chromatin or other RNA molecules and involved in the progression of cancer by affecting tumor cell proliferation, migration, and invasion [ 16 – 18 ]. Our previous work demonstrated that the LINC00323 is significantly up-regulated under hypoxic conditions, suggesting a possible functional role in hypoxia-induced osteogenic pathways and fracture healing processes [ 19 ]. But the molecular mechanisms of remains largely unknown. The present study aims to unravel the osteogenic effects of LINC00323 both in vitro and in vivo . We hypothesize that LINC00323, through its upregulation under hypoxic conditions, may enhance fracture healing by modulating key osteogenic pathways. We focus on its potential to influence the differentiation of osteoblast-like cells and its regulation of pivotal osteogenic markers. Notably, our investigation extends to the role of LINC00323 in modulating PDGFB expression, a growth factor known for its significant involvement in bone biology. We employ adenovirus-mediated LINC00323 particles, delivered locally to fracture sites in mice, to assess the consequent effects on bone mass, biomechanical strength, and cartilage callus formation, which might provide a deeper understanding of the molecular mechanisms through which intermittent hypoxia influences fracture healing. Materials and Methods GEO bioinformatics analysis GSE226245 was downloaded from the GEO database ( https://www.ncbi.nlm.nih.gov/geo ) and applied the DESeq2 software to obtain differentially expressed genes (DEGs) in sunitinib-resistant RCC cells 786-O(Sun-7R) with control or FUS knockdown. The threshold for screening DEGs was: log2 Fold Change |log2FC |> 1 and an adjusted p-value < 0.05. Cell culture and osteogenic differentiation MC3T3-E1 cells, a well-characterized mouse pre-osteoblast cell line, are obtained from the American Type Culture Collection (ATCC CRL-2594) and typically maintained in an α-MEM culture medium (Gibco BRL Life Technologies, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (Gibco BRL Life Technologies), and incubated in 5% CO 2 humidified incubator at 37°C. MC3T3-E1 cells were authenticated by STR DNA profiling analysis and tested for mycoplasma contamination. For osteoblast differentiation, MC3T3-E1 cells are exposed to a medium containing with 50 µg/mL l-ascorbic acid and 10 mM β-glycerophosphate. For hypoxia treatment, cells were cultured with serum-free medium under hypoxic conditions (5% CO 2 , 1% O 2 ) for 6 h in Heracell™ VIOS 160i Tri-Gas CO 2 Incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and then cultured with complete medium for reperfusion (5% CO 2 , 21% O 2 ) for 18 h in a normal incubator. Plasmids construction To overexpress LINC00323 in MC3T3-E1 cells, the full length of cDNA was cloned into pCDH-CMV-MCS-EF1α-Puro Cloning and Expression Lentivector (pCDH-CMV-MCS-EF1α-Puro (System Biosciences, USA). Lentiviruses particles were produced using the ViaFect™ transfection reagent (Promega, Madison, WI, USA) by co-transfection HEK293T cells with the packaging plasmids psPAX2 and pMD2.G. Lentiviruses particles were collected and infected MC3T3-E1 cells 48 h after transfection. 1 mg/ml puromycin (Sigma) were used to select. The efficiency of infection was assessed by qRT-PCR analysis. For gene knockdown, the small interfering RNA (siRNAs) targeting FUS (siFUS), PDGFB (siPDGFB), and negative control shRNA (siNC) were designed and provided by GenePharma (Shanghai, China). MC3T3-E1 cells were transfected with control or specific siRNAs 50 nM using the ViaFect™ transfection reagent (Promega). The efficiency of gene knockdown was evaluated using western blot 48 h after transfection. Reverse Transcription-PCR Total RNA was isolated from the cells with TRIzol Reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. A total of 500 ng RNA was subsequently transcribed into cDNA using PrimeScript™ RT reagent Kit (Takara Biotechnology co., LTD., Dalin, China). Real time PCRs were performed with TB Green® Fast qPCR Mix (Takara Biotechnology co.). Amplification by PCR was performed using the ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, USA). The primers used as follows: β-actin (ACTB), 5′-GGCTGTATTCCCCTCCATCG-3′ and 5′-CCAGTTGGTAACAATGCCATGT-3′; Runt-related transcription factor 2 (Runx2), 5′-TTCAACGATCTGAGATTTGTGGG-3′ and 5′-GGATGAGGAATGCGCCCTA-3′; collagen type I alpha 1 chain (Col1A1), 5′-GCTCCTCTTAGGGGCCACT-3′ and 5′-ATTGGGGACCCTTAGGCCAT-3′; osteocalcin (OCN), 5′-CAGGAGGGCAATAAGGTAGT-3′ and 5′-TCTGCTACAGGGAAAACAGT-3′; osteopontin (OPN), 5′-AGTTTCGCAGACCTGACATCC-3′ and 5′-TTCCTGACTATCAATCACATCGG-3′; The 2 −ΔΔCt method was employed to analyze gene expression. Cell Viability Assay For Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) assays, cells were seeded in a 96-well plate at a density of 8 × 10 3 cells per well. The absorbance was measured at 450 nm after 72h days with a microplate reader (Bio-Rad, CA, USA). ALP activity MC3T3-E1 cells were seeded into 6-well plate followed by differentiation for 21 days. For the ALP activity assay, the cells were washed with ice-cold PBS. After lysing, the supernatant was collected for measurement of the ALP activity and protein concentration. The ALP activity assay was assessed using Alkaline Phosphatase Assay Kit (Abcam, Cambridge, MA, USA) following the manufacturer’s protocol. ARS Staining For ARS staining, cells were washed with PBS three times and fixed in 70% ice-cold ethanol for 1 h at RT. The mineralized matrix was stained with 40 mM Alizarin red staining (Sigma Aldrich, St. Louis, MO, USA) under gentle agitation for 15 min at RT. After staining, the cells were washed with PBS 5 times. The red stain was destained with 10% (w/v) cetylpyridinium chloride (Sigma Aldrich) for 1 h, the absorbance at OD 570 nm was collected to assess the degree of mineralization. RNA pull-down assay The RNA-pulldown analysis was conducted as previously described using Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fischer Scientific, Waltham, MA, USA) according to the manufacturer’s instructions [ 20 ]. In brief, LINC00323 or PDGFB was labeled using Biotin RNA Labeling Mix (Roche, Basel, Switzerland), then digested with DNase I, protease inhibitor and RNase inhibitor. The supernatant of cell lysate was incubated with an equal amount of streptavidin magnetic beads at 37°C for 1 h. The protein level of FUS in the LINC00323-protein complexes was analyzed by western blot. RNA immunoprecipitation RNA immunoprecipitation (RIP) was conducted with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore Corp, Billerica, MA, USA) following the manufacturer’s protocol. In brief, cells were lysate and incubated with RIP immunoprecipitation buffer supplemented with magnetic beads conjugated with negative IgG or anti-FUS antibody. After digesting with Proteinase K, Immunoprecipitated RNAs were reversely transcribed into cDNA and following quantitative real-time PCR analysis. RNA stability assay For detection of mRNA stability of PDGFB, cells were treated with 5 µg/mL actinomycin D (Sigma-Aldrich) for 0, 20, 40, and 60 minutes, respectively. Then RNA was extracted and analyzed by RT-qPCR analysis. Hematoxylin and eosin (H&E) stain and Masson trichrome stain The fractured femur and calluses were collected on day 28 days after the bone fracture. After undergoing decalcification in 10% formic acid for a duration of one week, the samples were subsequently encased in paraffin wax. They were then cut longitudinally into sections with a thickness of 5 micrometers and carefully placed onto glass microscope slides. For the H&E staining of bone tissue, sections are first dewaxed in xylene and rehydrated through a graded alcohol series before being stained with hematoxylin to label nuclei. After rinsing, eosin is applied to stain cytoplasmic elements. In Masson's Trichrome staining, similarly prepared sections undergo sequential staining with Weigert's hematoxylin for nuclear definition, followed by Biebrich scarlet-acid fuchsin solution, and then differentiated in phosphomolybdic/phosphotungstic acid solution. Collagen is stained blue with aniline blue, providing contrast to red muscle fibers. The stained sections are dehydrated, cleared, and mounted. The slides were examined and photographed under an BX53 microscope light microscope (Olympus, Tokyo, Japan). Western blot Total cell proteins were extracted from cells using RIPA lysis buffer (Cell Signaling Technology, Berkeley, CA, USA) containing the protease inhibitors (Roche). After the protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific), Western blotting was performed using standard protocols. The primary antibodies used in the study were as follows: anti-FUS Polyclonal Antibody (Thermo Fischer Scientific), anti-PDGFB Polyclonal antibody (Proteintech, Wuhan, China), anti-PDGFR beta Polyclonal antibody (Proteintech), and anti-β-actin (Proteintech). β-Actin was used as the loading control. Radiographic analysis and micro-computed tomography examination At 28 days after the bone fracture, fracture healing of mouse tibial was examined with radiographs (X-ray) with MX-20 Specimen Radiography System (Faxitron Bioptics, LLC, Tucson, AZ, USA). Micro-computed tomography (µCT) scanning was performed to aseess the microstructure of the bone callus as our previous described [ 19 ]. Meanwhile, the bone structural indices, including including the bone volume fraction (BV/TV, %), trabecular number (Tb.N, 1/mm), trabecular thickness (Tb.Th, mm) and trabecular separation (Tb.Sp, mm) were calculated. Tibial Fracture Model and treatment An in vivo mouse tibial fracture model was constructed using 10-weeks age male C57BL/6 mice as described previously [ 19 ]. The mice were housed in sterilized cages at the Experimental Animal Center of Capital Medical University (Beijing, China). All animal care and experimental procedures were performed in adherence to the National Institute of Health guidelines for Care and Use of Laboratory Animals and were approved by Institutional Animal Care and Use Committee of SHZY. (Permission No: SHZY-202106AF). In all, 3 groups of animals (n = 8 each group) were used, including model group (model), model group with lentiviruses particles with control vector (vector), and model group with lentiviruses particles with LINC00323 (LINC00323). Finally, callus tissue around the fracture site was harvested for subsequent detection. Statistical analysis All data was presented as mean ± standard deviation (SD) from at least three independent experiments. GraphPad Prism 5 Demo (GraphPad, Inc., La Jolla, CA, USA) was used to statistical analysis. Student's t test was used to analyze the differences between two groups. Ordinary one-way ANOVA combined with Tukey's multiple comparisons test was used to compare differences among three or more groups. A p value less than 0.05 was considered statistically significant. Results LINC00323 contributes to the osteogenesis in MC3T3-E1 cells To investigate the key roles of LINC00323 in osteogenic differentiation, the expression of LINC00323 was determined in MC3T3-E1 cells during osteoblast differentiation. We found the expression level of LINC00323 was increased after induction of osteogenic differentiation (Fig. 1 A), suggesting that LINC00323 might play a role in osteogenesis. To further investigate the effect of LINC00323 on osteogenic differentiation, MC3T3-E1 cells were transduced with Lentivirus constructs expressing LINC00323 (LINC00323) or its control vector (vector), and Lentivirus encoding shRNA against LINC00323 (shLINC00323) or shRNA negative control (SCR). RT-qPCR was performed to confirm that LINC00323 was effectively upregulated in LINC00323-infected cells (Fig. 1 B), whereas was downregulated in shLINC00323-infected cells (Fig. 1 B) 36 hours later. The results of CCK-8 assay showed that overexpression of LINC00323 resulted in an increased viability of MC3T3-E1 cells under hypoxia state (Fig. 1 C). In contrast, a decreased viability of MC3T3-E1 cells was observed after LINC00323 silencing under hypoxia state (Fig. 1 C). However, there was no difference on cell proliferation whether LINC00323 overexpressing or LINC00323 silencing under normoxic state (Fig. 1 C). Flow cytometry was conducted to examine apoptosis in MC3T3-E1 cells with the LINC00323. As expected, overexpression of LINC00323 could partly hypoxia-induced MC3T3-E1 cells apoptosis, whereas the lack of LINC00323 further exacerbated the hypoxia-induced apoptosis (Fig. 1 D). Since LINC00323 might involve in osteogenic differentiation, then we assess the expression of osteogenic differentiation marker. As shown in Fig. 1 E, ALP activity of MC3T3-E1 cells in growth medium (Blank) or osteogenic medium (Control) was measured at 7 days, the ALP activity was markedly increased after LINC00323 overexpression (Fig. 1 E). As expect, knockdown of LINC00323 reduced the ALP activity of MC3T3-E1 cells (Fig. 1 E). Thereafter, the mRNA levels of ALP, COL1A1, OCN, and Runx2 were examined by RT-qPCR. The expression levels of these four markers showed a remarkable increase in LINC00323 overexpressed MC3T3-E1 cells (Fig. 1 F), whereas a result opposite to LINC00323 knockdown was observed (Fig. 1 F). In addition, LINC00323 exhibited remarkable function to promote osteoblast differentiation and mineralization, as evidenced by numerous alizarin red dye-bound nodules in LINC00323 overexpressed MC3T3-E1 cells (Fig. 1 G). In contrast, deletion of LINC00323 decreased the mineralization and calcified nodule formation in MC3T3-E1 cells (Fig. 1 G). Overall, the findings suggest that LINC00323 might contribute to fracture healing by enhanced the differentiation of osteogenesis. LINC00323 interacted with FUS To understand the molecular mechanisms of LINC00323 in osteogenesis, NcPath database ( http://ncpath.pianlab.cn/#/Home ) was used to predict LINC00323 interaction genes [ 21 ]. The RNA-binding protein FUS was reported to interact with LINC00323 by high throughput sequencing [ 22 ]. To confirm the relation between LINC00323 and FUS, RNA pull-down assay was performed and verified the binding of LINC00323 to FUS (Fig. 2 A). Conformably, the results from RIP displayed that LINC00323 was apparently enriched by FUS immunoprecipitation (Fig. 2 B). To determine whether the expression of LINC00323 affected the expression of FUS, RT-qPCR and western blot were performed to determine the expression of FUS after alteration of LINC00323. Upon LINC00323 knockdown, the mRNA and protein levels of FUS were found to be decreased in MC3T3-E1 cells (Fig. 2 C), whereas overexpression of LINC00323 enhanced the mRNA and protein levels of FUS (Fig. 2 C). Furthermore, the increase mRNA and protein levels of FUS were observed in MC3T3-E1 cells with hypoxia treatment (Fig. 2 D). In addition, LINC00323 exhibited no change of FUS both at mRNA and protein level under hypoxia state (Fig. 2 E), whereas a similar result to LINC00323 knockdown was observed (Fig. 2 E). These results collectively suggested that LINC00323 interacted with FUS and did not influence the transcription of FUS in MC3T3-E1 cells. LINC00323 interacted with FUS to increase the stability of PDGFB mRNA FUS has been reported to regulate the mRNA stability of downstream target genes, but genes mediated by FUS remained largely unknown [ 20 , 23 ]. A GEO dataset, GSE226245, was downloaded from the database to analyze differentially expressed genes (DEGs). Compared to the control group, 3 genes (RGS5, IFI44 and IGS15) were significantly increased and 6 genes (FOXL1, PDGFRB, RASD2, TGFB2-AS1, AMH, and PDGFB) were significantly decreased in FUS knockdown group (Fig. 3 A). Previous studies have shown that PDGFB was known to be a potent mitogen and chemoattractant for cells involved in wound healing and tissue regeneration, including cells critical for bone healing processes [ 24 , 25 ]. Then, PDGFB was chosen for further investigation. To determine whether FUS-mediated alterations of PDGFB, siRNA targeting FUS lead to a significant decrease of PDGFB both at mRNA and protein level (Fig. 3 B and 3 G). The increased expression of PDGFB was observed in hypoxia-induced MC3T3-E1 cells (Fig. 3 C). Further RNA pull-down assays using a biotin-labeled probe against mRNA of PDGFB were performed in MC3T3-E1 cells. Western blot illustrated that FUS could be detected by the probe specifically targeting PDGFB, but not by control probe (Fig. 3 D). RIP experiments also showed that the interaction between FUS and mRNA of PDGFB (Fig. 3 E). Then, we want to test whether LINC00323 regulates FUS expression. higher expression of PDGFB were observed in LINC00323 overexpressed MC3T3-E1 cells (Fig. 3 F). Besides, we also noticed that knockdown of LINC00323 significantly downregulated the expression of FUS (Fig. 3 G). To explore the mechanisms by which FUS silencing down-regulated PDGFB in MC3T3-E1 cells, the stability of PDGFB was examined. MDA-MB-231 cells were transfected with siRNA against LINC00323 or FUS and simultaneously treated with actinomycin D, a transcription inhibitor, and then the mRNA expression levels of PDGFB were examined at 0, 20, 40, and 60 minutes, respectively. The data revealed that the mRNA expression levels of PDGFB were decreased significantly after LINC00323 or FUS silencing (Fig. 3 H). These findings suggested that the combination of LINC00323 and FUS enhanced the stability of PDGFB through direct binding. LINC00323 regulated hypoxia-mediated MC3T3-E1 cells survival and osteogenic differentiation via the FUS/PDGFB axis Based on data from above experiments, we hypothesized that LINC00323 promoted osteogenic differentiation via regulating FUS/PDGFB axis. To clarify the effect of FUS/PDGFB axis on LINC00323-mediated osteogenic differentiation in fracture healing, LINC00323 overexpression together with siRNAs targeting FUS or PDGFB was transfected into MC3T3-E1 cells following hypoxia treatment (Fig. 4 A). When LINC00323 was overexpressed in MC3T3-E1 cells, knockdown of FUS or PDGFB could reverse the protecting effect of LINC00323 on the cell viability under hypoxia state (Fig. 4 B). Besides, FUS or PDGFB silencing partially reversed the effect of LINC00323 on cell apoptosis by flow cytometry (Fig. 4 C). At day 7 postosteogenic induction, knockdown of FUS or PDGFB significantly inhibited LINC00323-midated ALP activity (Fig. 4 D). In addition, FUS or PDGFB silencing partially attenuated the expression of osteogenic differentiation marker induced by LINC00323 overexpression under hypoxia state (Fig. 4 E). Consistently, alizarin red staining also verified that FUS or PDGFB silencing decreased the mineralization and calcified nodule formation in LINC00323 overexpressed MC3T3-E1 cells (Fig. 4 F). These results indicated that LINC00323 promoted osteoblastic differentiation of MC3T3-E1 cells via regulating FUS/PDGFB axis. LINC00323 accelerate the fracture healing process To elaborate the impact of LINC00323 on bone regeneration in vivo , a mouse tibial fracture model was constructed. One day after the fracture, 1 x 10 8 IU (integration units) lentivirus was locally injected into the subcutaneous region of a local fracture. Especially at day 28, the fracture healing process of the tibial was accelerated in the LINC00323 group compared with the Vector and Blank group based on the weekly X-ray radiographic images (Fig. 5 A). Confirming the results of X-ray, Micro-CT scanning showed increased bone and tissue volume of the fracture callus of mice with 28 days after fracture (Fig. 5 B). This was confirmed by a decrease of gap distance of cortical defects (Fig. 5 B). Higher BV/TV, Tb.N, and Tb.Th values and a lower Tb.Sp value were observed in LINC00323 group compared with the Vector and Blank group (Fig. 5 C). Consistent with the radiographic results, H&E and Masson's trichrome staining demonstrated better cortical growth and more collagen fiber in the LINC00323 group compared with the other two groups (Fig. 5 D). Subsequent measurement of the expression of PDGFB in bone healing defect in vivo was measured by immunohistochemical staining. The expression of PDGFB in tibial fracture region was significantly higher than the other two groups (Fig. 5 E), which indicated that the impact of LINC00323 on the fracture healing might be mediated by increased PDGFB. Taken together, these data showed that LINC00323 facilitated accelerated fracture healing process by promoting osteogenic differentiation in vivo . Discussion Hypoxia has been found to play a multifaceted and crucial role in bone healing, influencing various cellular processes and molecular pathways [ 26 , 27 ]. The ongoing research in this field holds significant promise for developing advanced therapeutic strategies to enhance bone regeneration and treat complex bone injuries [ 9 , 28 , 29 ]. However, translating these findings into clinical practice requires a deeper understanding of the optimal use of hypoxia in bone healing and its integration with other therapeutic modalities. Long non-coding RNAs (lncRNAs), which are transcripts longer than 200 nucleotides without protein-coding potential, have been found to regulate gene expression at multiple levels, including chromatin modification, transcription, and post-transcriptional processing [ 30 , 31 ]. LncRNAs represent a rapidly evolving area of research with significant potential to improve our understanding and treatment of fracture healing [ 13 , 32 , 33 ]. For example, lncRNA KCNQ1OT1 as a competing endogenous RNA of miR-701-3p to promote osteoblast proliferation, migration, and inhibit apoptosis in vitro and in vivo [ 34 ]. LncRNA ENST00000563492 promoted osteogenic differentiation of BMSCs and improved the osteogenesis-angiogenesis coupling process through enhancing the expression of VEGF during osteogenic differentiation of BMSCs [ 35 ]. An increased trend of LINC00323 was detected in the MC3T3-E1 cells treated with hypoxia by our previous study [ 19 ]. In our present study, LINC00323 was significantly upregulated during osteoblast differentiation in MC3T3-E1 cells. The enhancement of osteogenic differentiation following LINC00323 overexpression, as evidenced by increased activity of ALP and elevated expression of key osteogenic markers like RUNX2, COL1A1, and OCN. Conversely, the inhibitory effect on osteogenesis observed upon LINC00323 knockdown further underscores its crucial role in bone formation. Furthermore, there is an active interaction of FUS with LINC00323 and PDGFB during hypoxic conditions promotes the survival of osteoblasts and stimulates their differentiation into bone-forming cells, which is critical for the osteogenesis process (Fig. 6 ). In addition, LINC00323 promoted osteoblastic differentiation and fracture healing in a mouse model. In certain types of cancer, including breast cancer and liver cancer, the expression levels of LINC00323 have been linked to disease severity, prognosis, and survival rates [ 16 , 18 ]. LINC00323 was also reported to a strong hypoxia-dependent activation of intergenic lncRNAs [ 36 ]. In the current study, we demonstrated that the LINC00323 were dramatically increased during osteogenic differentiation. Overexpression of LINC00323 maintained cell viability of MC3T3-E1 and hypoxia induced cell apoptosis, consistent with previous studies highlighting the function role of LINC00323 under hypoxic conditions [ 36 ]. Previous studies have shown that lncRNAs can interacted with RNA-binding proteins to regulate physiological functions and the pathogenesis of certain diseases, including chromatin regulation, transcription regulation, scaffolds, post-transcriptional modification [ 37 – 39 ]. Online bioinformatic tools was used to predict the interaction with LINC01133 [ 21 ], which was supported by results of high throughput sequencing [ 22 ]. FUS (Fused in Sarcoma) was chosen due to FUS was an RNA-binding protein that involved in the export of mRNA from the nucleus to the cytoplasm and plays a role in mRNA stability and turnover [ 40 ]. LncRNA XIST was reported to interact with FUS and increased the stability of SPHK1 to promote osteoclast differentiation through SPHK1/S1P/ERK signaling pathway [ 40 ]. LncRNA GAS6-AS1 regulated colorectal cancer (CRC) proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) via recruiting FUS to stable TRIM14 mRNA stability [ 41 ]. Recent study has shown that ANRIL recruited and interacted with FUS to stable HIFA to mediate transcription of VEGFA and ANRIL, which accelerated wound healing in diabetic foot ulcers [ 42 ]. In the present study, the interaction between LINC00323 and FUS was confirmed by verified by RNA pull-down and RIP assays. The increased viability and osteogenic potential of MC3T3-E1 cells overexpressing LINC00323 under hypoxic conditions are particularly noteworthy. This suggests a potential adaptive mechanism mediated by LINC00323 in response to hypoxic stress, a common feature of the fracture healing microenvironment. These observations contribute to the understanding of how lncRNAs can modulate cellular responses in varying oxygen conditions, which is crucial for tissue regeneration processes. Through analysis of GSE226245, PDGFB was chosen for further investigation. PDGFB was a key growth factor involved in various cellular processes, including cell proliferation, migration, and angiogenesis [ 43 , 44 ]. PDGFB was also reported to play a crucial role in tissue development and repair, and its dysregulation is implicated in various pathologies [ 24 , 25 , 45 ]. The relationship between LINC00323 and FUS was confirmed by verified by RNA pull-down and RIP assays. Our study reveals indicated that the interaction between LINC00323 and FUS increased the stability of PDGFB. And knockdown LINC00323 or FUS decreased the expression of PDGFB. Further in vivo experiments showed that LINC00323 accelerated fracture healing consisted with the in vitro findings and suggested a translational potential for LINC00323 in therapeutic strategies aimed at enhancing bone repair. The high expression of PDGFB was observed in a mouse tibial fracture model with LINC00323 treatment. While this study provides significant insights, it also acknowledges certain limitations. The specific mechanisms by which LINC00323 modulates FUS and PDGFB, and their broader implications in the complex network of bone healing, require further exploration. Additionally, understanding the role of LINC00323 in various stages of bone healing and its interactions with different cellular components remains a crucial area for future research. Conclusion In conclusion, our study indicated that LINC00323 interacted with FUS to modulate the stability of PDGFB mRNA under hypoxic state, which established a theoretical foundation for novel therapeutic approaches aimed at accelerating fracture repair, thereby addressing a critical need in clinical orthopedics and trauma medicine. Declarations Declaration of competing interest The authors declare no conflict of interest. Author Contribution GLC and JH were involved in the design of the study, basic analysis of data, drafting of manuscript, and revising it for critical knowledge content. SA performed the statistical analysis. JYW and XYS were involved in the acquisition of data, drafting of manuscript, and revising it critically for critical knowledge content. All authors have read and approved the final submitted manuscript. Acknowledgements This work was supported by grants from the Beijing Municipal Natural Science Foundation (No. 7232073) Data availability statement The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. References Marsell R, Einhorn TA. The biology of fracture healing. Injury 2011;42:551–5. Cunningham BP, Brazina S, Morshed S, Miclau TR. Fracture healing: A review of clinical, imaging and laboratory diagnostic options. Injury 2017;48 Suppl 1:S69-75. Axelrad TW, Einhorn TA. Use of clinical assessment tools in the evaluation of fracture healing. Injury 2011;42:301–5. Manescu PV, Antoniac I, Antoniac A, Laptoiu D, Paltanea G, Ciocoiu R, et al. 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Bone 2023;167:116645. Jo S, Lee SH, Park J, Nam B, Kim H, Youn J, et al. Platelet-Derived Growth Factor B Is a Key Element in the Pathological Bone Formation of Ankylosing Spondylitis. J Bone Miner Res 2023;38:300–12. Zhang T, Yan S, Song Y, Chen C, Xu D, Lu B, et al. Exosomes secreted by hypoxia-stimulated bone-marrow mesenchymal stem cells promote grafted tendon-bone tunnel healing in rat anterior cruciate ligament reconstruction model. J Orthop Translat 2022;36:152–63. Chen HS, Yau YC, Ko PT, Yen BL, Ho CT, Hung SC. Mesenchymal Stem Cells From a Hypoxic Culture Can Improve Rotator Cuff Tear Repair. Cell Transplant 2022;31:73819807. Becerikli M, Reinkemeier F, Dadras M, Wallner C, Wagner JM, Drysch M, et al. TGF-beta pathway inhibition as the therapeutic acceleration of diabetic bone regeneration. J Orthop Res 2022;40:1810–26. Zheng X, Zhang X, Wang Y, Liu Y, Pan Y, Li Y, et al. Hypoxia-mimicking 3D bioglass-nanoclay scaffolds promote endogenous bone regeneration. Bioact Mater 2021;6:3485–95. Quinn JJ, Chang HY. Unique features of long non-coding RNA biogenesis and function. Nat Rev Genet 2016;17:47–62. Kanduri C. Long noncoding RNAs: Lessons from genomic imprinting. Biochim Biophys Acta 2016;1859:102–11. Chen J, Yang Y. LncRNA HAGLR absorbing miR-214-3p promotes BMP2 expression and improves tibial fractures. Am J Transl Res 2021;13:11065–80. Zhang Y, Yuan Q, Wei Q, Li P, Zhuang Z, Li J, et al. Long noncoding RNA XIST modulates microRNA-135/CREB1 axis to influence osteogenic differentiation of osteoblast-like cells in mice with tibial fracture healing. Hum Cell 2022;35:133–49. Chen L, Xiong Y, Yan C, Zhou W, Endo Y, Xue H, et al. LncRNA KCNQ1OT1 accelerates fracture healing via modulating miR-701-3p/FGFR3 axis. FASEB J 2020;34:5208–22. Ouyang Z, Tan T, Zhang X, Wan J, Zhou Y, Jiang G, et al. LncRNA ENST00000563492 promoting the osteogenesis-angiogenesis coupling process in bone mesenchymal stem cells (BMSCs) by functions as a ceRNA for miR-205-5p. Cell Death Dis 2020;11:486. Fiedler J, Breckwoldt K, Remmele CW, Hartmann D, Dittrich M, Pfanne A, et al. Development of Long Noncoding RNA-Based Strategies to Modulate Tissue Vascularization. J Am Coll Cardiol 2015;66:2005–15. Statello L, Guo CJ, Chen LL, Huarte M. Gene regulation by long non-coding RNAs and its biological functions. Nat Rev Mol Cell Biol 2021;22:96–118. Winkle M, El-Daly SM, Fabbri M, Calin GA. Noncoding RNA therapeutics - challenges and potential solutions. Nat Rev Drug Discov 2021;20:629–51. Rafiee A, Riazi-Rad F, Havaskary M, Nuri F. Long noncoding RNAs: regulation, function and cancer. Biotechnol Genet Eng Rev 2018;34:153–80. Zhang DW, Wang HG, Zhang KB, Guo YQ, Yang LJ, Lv H. LncRNA XIST facilitates S1P-mediated osteoclast differentiation via interacting with FUS. J Bone Miner Metab 2022;40:240–50. Chen Q, Zhou L, Ma D, Hou J, Lin Y, Wu J, et al. LncRNA GAS6-AS1 facilitates tumorigenesis and metastasis of colorectal cancer by regulating TRIM14 through miR-370-3p/miR-1296-5p and FUS. J Transl Med 2022;20:356. Wan J, Bao Y, Hou LJ, Li GJ, Du LJ, Ma ZH, et al. lncRNA ANRIL accelerates wound healing in diabetic foot ulcers via modulating HIF1A/VEGFA signaling through interacting with FUS. J Gene Med 2023;25:e3462. Miller CR, Hjelmeland AB. Breaking the feed forward inflammatory cytokine loop in the tumor microenvironment of PDGFB-driven glioblastomas. J Clin Invest 2023;133. Zheng Y, Ji S, Li X, Wen L. Qijia rougan formula ameliorates ECM deposition in hepatic fibrosis by regulating the JAK1/STAT6-microRNA-23a feedback loop in macrophage M2 polarization. Biomed Pharmacother 2023;168:115794. Kalra K, Eberhard J, Farbehi N, Chong JJ, Xaymardan M. Role of PDGF-A/B Ligands in Cardiac Repair After Myocardial Infarction. Front Cell Dev Biol 2021;9:669188. 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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-3966058","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":273790703,"identity":"a594ffdc-c170-437e-a855-6d2afcdb0026","order_by":0,"name":"Jiang 黄","email":"","orcid":"","institution":"Xuan Wu Hospital of the Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiang","middleName":"","lastName":"黄","suffix":""},{"id":273790704,"identity":"af0668cd-22b5-4249-8889-daa617219ffc","order_by":1,"name":"Ju yong Wang","email":"","orcid":"","institution":"Xuan Wu Hospital of the Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ju","middleName":"yong","lastName":"Wang","suffix":""},{"id":273790705,"identity":"7b2557f2-167c-4eb3-93e7-2d21e61ef5be","order_by":2,"name":"Xiang Yao Sun","email":"","orcid":"","institution":"Xuan Wu Hospital of the Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"Yao","lastName":"Sun","suffix":""},{"id":273790706,"identity":"dfb5803c-71c3-4e8f-9831-17bd83e61b1a","order_by":3,"name":"Shuai An","email":"","orcid":"","institution":"Xuan Wu Hospital of the Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuai","middleName":"","lastName":"An","suffix":""},{"id":273790707,"identity":"0ea91b92-5058-487c-92bb-532a611e07a8","order_by":4,"name":"Guang Lei Cao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBADHjZm5gMHPvwgQYsMHztb4sGZPSRosZHj5zE+zMFGhFL56MOHX/zccxjoMJ4Phxl4GOT5xQ7g12J4Li3NsucZSAvvhsMFFgyGM2cnENDSw2NmwHMAqmUGD0OCwW2CWvi/Gf4Ba+F5ACSJ0CLPw8P8GGILDwNxWgx42MyYZQ6kA7WwGQADWYKwX+R7mB9/fHPA2l6+//DjDx9+2MjzSxOy5QADmwQDQzOML4FfOdiWBgbmDwwMdYRVjoJRMApGwcgFAHVgQHeD7OCbAAAAAElFTkSuQmCC","orcid":"","institution":"Xuan Wu Hospital of the Capital Medical University","correspondingAuthor":true,"prefix":"","firstName":"Guang","middleName":"Lei","lastName":"Cao","suffix":""}],"badges":[],"createdAt":"2024-02-18 06:14:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3966058/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3966058/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51443292,"identity":"0ec244c0-f679-48ad-890e-477ff44da13e","added_by":"auto","created_at":"2024-02-21 17:59:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2952108,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of LINC00323 on the osteogenic differentiation of MC3T3-E1 cells. (A) The expression of LINC00323 in MC3T3-E1 cells during osteogenesis induction was measured by qRT-PCR. (B, C) The expression of LINC00323 was examined in MC3T3-E1 cells after overexpression of LINC00323 or knockdown of LINC00323 by qRT-PCR. (C) The effect of LINC00323 on MC3T3-E1 cell viability was detected under normoxic or hypoxic states. (D) Cell apoptosis was determined by Annexin V-FITC/PI staining followed by flow cytometry. (E) The ALP activity was measured using an ALP assay kit at day 7 post-osteoinduction. (F) The expression levels of Runx2, OPN, OCN, and Col1A1 in MC3T3-E1 cells after LINC00323 overexpressed or LINC00323 knockdown by qRT-PCR at day 7 post-osteoinduction. (G) Alizarin red staining showed that the osteoblastic differentiation of LINC00323-knockdown or LINC00323-overexpression MC3T3-E1 cells at 21 days post-osteoinduction. ** p \u0026lt; 0.01 compared to the control group.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3966058/v1/544c53e7a6263e67506bb54d.png"},{"id":51443289,"identity":"bd4c11ae-5ecc-4d05-a457-2e4e8bd76a25","added_by":"auto","created_at":"2024-02-21 17:59:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":288446,"visible":true,"origin":"","legend":"\u003cp\u003eLINC00323 bind to FUS. (A) The binding of LINC00323 and FUS measured by RNA-pulldown assay. (B) A RIP assay was performed to assess the interaction between LINC00323 and FUS. (C) FUS expression was assessed in MC3T3-E1 cells after LINC00323 knockdown and LINC00323 overexpressed by qRT-PCR and western blot, respectively. (D) FUS expression was assessed in MC3T3-E1 cells under hypoxic state. (E) FUS expression was assessed in MC3T3-E1 cells after LINC00323 knockdown and overexpression by qRT-PCR and western blot, respectively.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3966058/v1/2682f0eb1d547da1c010fb73.png"},{"id":51443290,"identity":"4a6935e2-8785-420b-a1dd-d4e5b72eb1bd","added_by":"auto","created_at":"2024-02-21 17:59:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":337849,"visible":true,"origin":"","legend":"\u003cp\u003eLINC00323 interacted with FUS to support the stability of PDGFB mRNA. (A) The heatmap of GSE226245. (B) The mRNA level of PDGFB in FUS knockdown MC3T3-E1 cells. (C) PDGFB expression was assessed in MC3T3-E1 cells under hypoxia state. RNA pull-down assay (D) and RIP assay (E) were performed to evaluate the interactions between FUS and PDGFB. The expression of PDGFB was assessed in MC3T3-E1 cells after LINC00323 overexpressed (F) and LINC00323 knockdown (G) by qRT-PCR and western blot, respectively. (H) Reducing PDGFB mRNA half-life was observed by LINC00323 silencing or FUS silencing in MC3T3-E1 cells. ** p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3966058/v1/f24630c188029b70f5d5a20a.png"},{"id":51444764,"identity":"6074f5b2-45d4-4840-93fd-5d97e95ed90b","added_by":"auto","created_at":"2024-02-21 18:07:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":745296,"visible":true,"origin":"","legend":"\u003cp\u003eLINC00323-modulated MC3T3-E1 cell behavior is reversed by FUS silencing or PDGFB silencing. (A) The protein levels of FUS, PDGFB and PDGFRB in MC3T3-E1 cells exposed to hypoxia with the indicated treatments. Cell viability (B), cell apoptosis (C), ALP activity (D) in MC3T3-E1 cells exposed to hypoxia with the indicated treatments. (E) Osteogenic factor and osteoblastic differentiation of MC3T3-E1 cells exposed to hypoxia with the indicated treatments. ** p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3966058/v1/b91f212af52aba56b7e001e6.png"},{"id":51443291,"identity":"3e3b0630-f131-4701-8b54-f5f31802906d","added_by":"auto","created_at":"2024-02-21 17:59:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5505596,"visible":true,"origin":"","legend":"\u003cp\u003eLINC00323 promotes fracture healing in mice. (A) Representative X-ray images of the fracture model with LINC00323 treatment on 28 days following fracture. (B) Representative μCT images of the fractured femora were taken 28 days following fracture. (C) The statistical diagrams of BV/TV, Tb.Th, Tb.N and Tb.Sp are presented according to standard three-dimensional microstructural analysis. (D) H\u0026amp;E staining and Masson's trichrome staining were performed on the femur fractured zone after surgery 28 days. (E) Immunostaining for PDGFB in fracture callus.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3966058/v1/e19b116e3c0c03dc6e8cde4d.png"},{"id":51443294,"identity":"91f0727f-b0b4-483c-9a07-c3f951fc9d71","added_by":"auto","created_at":"2024-02-21 17:59:42","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2971337,"visible":true,"origin":"","legend":"\u003cp\u003eA schematic diagram of LINC00323 involved in osteoblast function under normal and hypoxic conditions. Under normal oxygen levels, LINC00323 interacted with FUS to regulate PDGFB to maintain physiological functions. Under hypoxic conditions, increased interaction between LINC00323 and FUS enhance the stability of PDGFB mRNA to promote osteoblast survival and osteogenic differentiation.\u003c/p\u003e","description":"","filename":"Figure6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3966058/v1/6feb7d32b8ea44c3c0e4340e.jpeg"},{"id":54869223,"identity":"c46608de-34f8-4d42-b84a-377b5690f314","added_by":"auto","created_at":"2024-04-17 23:52:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5296706,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3966058/v1/d52b1703-6a87-4df4-b1f2-12392211d7af.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"LINC00323 induced by hypoxia promote cartilage callus by interacting with FUS to regulate PDGFB expression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFracture healing is a complex physiological process, crucial for the restoration of bone integrity and function after injury, which attend considerable interest in both clinical and research settings [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Advances in understanding the bone healing mechanisms have elucidated a series of complex cellular and molecular interactions, involving hematoma formation, inflammation, soft and hard callus formation, and extracellular matrix remodeling [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Emerging research is now focused on targeting specific pathways and molecules to accelerate and improve fracture healing, thus potentially reducing its financial impact [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While the traditional view of fracture healing emphasizes the role of mechanical factors and local biology, recent evidence suggests that systemic factors like oxygen tension also play a pivotal role [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Intermittent hypoxia, characterized by periodic exposure to low oxygen levels, has been increasingly recognized for its potential therapeutic benefits in enhancing fracture repair [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This paradoxical phenomenon, where transient low oxygen states can exert beneficial effects on bone healing, depends on various factors such as duration, frequency, and severity of hypoxic episodes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the molecular mechanisms underlying these beneficial effects remain largely elusive.\u003c/p\u003e \u003cp\u003ePrevious studies have hinted at the involvement of non-coding RNAs (ncRNAs) in the cellular response to hypoxic conditions [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Among these, long non-coding RNAs (lncRNAs) have emerged as key regulators of gene expression, capable of modulating diverse biological processes including osteogenesis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Recent studies suggest that LINC00323 regulate the expression of specific genes by interacting with chromatin or other RNA molecules and involved in the progression of cancer by affecting tumor cell proliferation, migration, and invasion [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our previous work demonstrated that the LINC00323 is significantly up-regulated under hypoxic conditions, suggesting a possible functional role in hypoxia-induced osteogenic pathways and fracture healing processes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. But the molecular mechanisms of remains largely unknown.\u003c/p\u003e \u003cp\u003eThe present study aims to unravel the osteogenic effects of LINC00323 both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. We hypothesize that LINC00323, through its upregulation under hypoxic conditions, may enhance fracture healing by modulating key osteogenic pathways. We focus on its potential to influence the differentiation of osteoblast-like cells and its regulation of pivotal osteogenic markers. Notably, our investigation extends to the role of LINC00323 in modulating PDGFB expression, a growth factor known for its significant involvement in bone biology. We employ adenovirus-mediated LINC00323 particles, delivered locally to fracture sites in mice, to assess the consequent effects on bone mass, biomechanical strength, and cartilage callus formation, which might provide a deeper understanding of the molecular mechanisms through which intermittent hypoxia influences fracture healing.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eGEO bioinformatics analysis\u003c/p\u003e \u003cp\u003eGSE226245 was downloaded from the GEO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and applied the DESeq2 software to obtain differentially expressed genes (DEGs) in sunitinib-resistant RCC cells 786-O(Sun-7R) with control or FUS knockdown. The threshold for screening DEGs was: log2 Fold Change |log2FC |\u0026gt; 1 and an adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eCell culture and osteogenic differentiation\u003c/p\u003e \u003cp\u003eMC3T3-E1 cells, a well-characterized mouse pre-osteoblast cell line, are obtained from the American Type Culture Collection (ATCC CRL-2594) and typically maintained in an α-MEM culture medium (Gibco BRL Life Technologies, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (Gibco BRL Life Technologies), and incubated in 5% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator at 37\u0026deg;C. MC3T3-E1 cells were authenticated by STR DNA profiling analysis and tested for mycoplasma contamination. For osteoblast differentiation, MC3T3-E1 cells are exposed to a medium containing with 50 \u0026micro;g/mL l-ascorbic acid and 10 mM β-glycerophosphate. For hypoxia treatment, cells were cultured with serum-free medium under hypoxic conditions (5% CO\u003csub\u003e2\u003c/sub\u003e, 1% O\u003csub\u003e2\u003c/sub\u003e) for 6 h in Heracell\u0026trade; VIOS 160i Tri-Gas CO\u003csub\u003e2\u003c/sub\u003e Incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and then cultured with complete medium for reperfusion (5% CO\u003csub\u003e2\u003c/sub\u003e, 21% O\u003csub\u003e2\u003c/sub\u003e) for 18 h in a normal incubator.\u003c/p\u003e \u003cp\u003ePlasmids construction\u003c/p\u003e \u003cp\u003eTo overexpress LINC00323 in MC3T3-E1 cells, the full length of cDNA was cloned into pCDH-CMV-MCS-EF1α-Puro Cloning and Expression Lentivector (pCDH-CMV-MCS-EF1α-Puro (System Biosciences, USA). Lentiviruses particles were produced using the ViaFect\u0026trade; transfection reagent (Promega, Madison, WI, USA) by co-transfection HEK293T cells with the packaging plasmids psPAX2 and pMD2.G. Lentiviruses particles were collected and infected MC3T3-E1 cells 48 h after transfection. 1 mg/ml puromycin (Sigma) were used to select. The efficiency of infection was assessed by qRT-PCR analysis.\u003c/p\u003e \u003cp\u003eFor gene knockdown, the small interfering RNA (siRNAs) targeting FUS (siFUS), PDGFB (siPDGFB), and negative control shRNA (siNC) were designed and provided by GenePharma (Shanghai, China). MC3T3-E1 cells were transfected with control or specific siRNAs 50 nM using the ViaFect\u0026trade; transfection reagent (Promega). The efficiency of gene knockdown was evaluated using western blot 48 h after transfection.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReverse Transcription-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from the cells with TRIzol Reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer\u0026rsquo;s protocol. A total of 500 ng RNA was subsequently transcribed into cDNA using PrimeScript\u0026trade; RT reagent Kit (Takara Biotechnology co., LTD., Dalin, China). Real time PCRs were performed with TB Green\u0026reg; Fast qPCR Mix (Takara Biotechnology co.). Amplification by PCR was performed using the ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, USA). The primers used as follows: β-actin (ACTB), 5\u0026prime;-GGCTGTATTCCCCTCCATCG-3\u0026prime; and 5\u0026prime;-CCAGTTGGTAACAATGCCATGT-3\u0026prime;; Runt-related transcription factor 2 (Runx2), 5\u0026prime;-TTCAACGATCTGAGATTTGTGGG-3\u0026prime; and 5\u0026prime;-GGATGAGGAATGCGCCCTA-3\u0026prime;; collagen type I alpha 1 chain (Col1A1), 5\u0026prime;-GCTCCTCTTAGGGGCCACT-3\u0026prime; and 5\u0026prime;-ATTGGGGACCCTTAGGCCAT-3\u0026prime;; osteocalcin (OCN), 5\u0026prime;-CAGGAGGGCAATAAGGTAGT-3\u0026prime; and 5\u0026prime;-TCTGCTACAGGGAAAACAGT-3\u0026prime;; osteopontin (OPN), 5\u0026prime;-AGTTTCGCAGACCTGACATCC-3\u0026prime; and 5\u0026prime;-TTCCTGACTATCAATCACATCGG-3\u0026prime;; The 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method was employed to analyze gene expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell Viability Assay\u003c/h2\u003e \u003cp\u003eFor Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) assays, cells were seeded in a 96-well plate at a density of 8 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well. The absorbance was measured at 450 nm after 72h days with a microplate reader (Bio-Rad, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eALP activity\u003c/h2\u003e \u003cp\u003eMC3T3-E1 cells were seeded into 6-well plate followed by differentiation for 21 days. For the ALP activity assay, the cells were washed with ice-cold PBS. After lysing, the supernatant was collected for measurement of the ALP activity and protein concentration. The ALP activity assay was assessed using Alkaline Phosphatase Assay Kit (Abcam, Cambridge, MA, USA) following the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eARS Staining\u003c/h2\u003e \u003cp\u003eFor ARS staining, cells were washed with PBS three times and fixed in 70% ice-cold ethanol for 1 h at RT. The mineralized matrix was stained with 40 mM Alizarin red staining (Sigma Aldrich, St. Louis, MO, USA) under gentle agitation for 15 min at RT. After staining, the cells were washed with PBS 5 times. The red stain was destained with 10% (w/v) cetylpyridinium chloride (Sigma Aldrich) for 1 h, the absorbance at OD 570 nm was collected to assess the degree of mineralization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRNA pull-down assay\u003c/h2\u003e \u003cp\u003eThe RNA-pulldown analysis was conducted as previously described using Pierce\u0026trade; Magnetic RNA-Protein Pull-Down Kit (Thermo Fischer Scientific, Waltham, MA, USA) according to the manufacturer\u0026rsquo;s instructions [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In brief, LINC00323 or PDGFB was labeled using Biotin RNA Labeling Mix (Roche, Basel, Switzerland), then digested with DNase I, protease inhibitor and RNase inhibitor. The supernatant of cell lysate was incubated with an equal amount of streptavidin magnetic beads at 37\u0026deg;C for 1 h. The protein level of FUS in the LINC00323-protein complexes was analyzed by western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA immunoprecipitation\u003c/h2\u003e \u003cp\u003eRNA immunoprecipitation (RIP) was conducted with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore Corp, Billerica, MA, USA) following the manufacturer\u0026rsquo;s protocol. In brief, cells were lysate and incubated with RIP immunoprecipitation buffer supplemented with magnetic beads conjugated with negative IgG or anti-FUS antibody. After digesting with Proteinase K, Immunoprecipitated RNAs were reversely transcribed into cDNA and following quantitative real-time PCR analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRNA stability assay\u003c/h2\u003e \u003cp\u003eFor detection of mRNA stability of PDGFB, cells were treated with 5 \u0026micro;g/mL actinomycin D (Sigma-Aldrich) for 0, 20, 40, and 60 minutes, respectively. Then RNA was extracted and analyzed by RT-qPCR analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin and eosin (H\u0026amp;E) stain and Masson trichrome stain\u003c/h2\u003e \u003cp\u003eThe fractured femur and calluses were collected on day 28 days after the bone fracture. After undergoing decalcification in 10% formic acid for a duration of one week, the samples were subsequently encased in paraffin wax. They were then cut longitudinally into sections with a thickness of 5 micrometers and carefully placed onto glass microscope slides. For the H\u0026amp;E staining of bone tissue, sections are first dewaxed in xylene and rehydrated through a graded alcohol series before being stained with hematoxylin to label nuclei. After rinsing, eosin is applied to stain cytoplasmic elements. In Masson's Trichrome staining, similarly prepared sections undergo sequential staining with Weigert's hematoxylin for nuclear definition, followed by Biebrich scarlet-acid fuchsin solution, and then differentiated in phosphomolybdic/phosphotungstic acid solution. Collagen is stained blue with aniline blue, providing contrast to red muscle fibers. The stained sections are dehydrated, cleared, and mounted. The slides were examined and photographed under an BX53 microscope light microscope (Olympus, Tokyo, Japan).\u003c/p\u003e \u003cp\u003eWestern blot\u003c/p\u003e \u003cp\u003eTotal cell proteins were extracted from cells using RIPA lysis buffer (Cell Signaling Technology, Berkeley, CA, USA) containing the protease inhibitors (Roche). After the protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific), Western blotting was performed using standard protocols. The primary antibodies used in the study were as follows: anti-FUS Polyclonal Antibody (Thermo Fischer Scientific), anti-PDGFB Polyclonal antibody (Proteintech, Wuhan, China), anti-PDGFR beta Polyclonal antibody (Proteintech), and anti-β-actin (Proteintech). β-Actin was used as the loading control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRadiographic analysis and micro-computed tomography examination\u003c/h2\u003e \u003cp\u003eAt 28 days after the bone fracture, fracture healing of mouse tibial was examined with radiographs (X-ray) with MX-20 Specimen Radiography System (Faxitron Bioptics, LLC, Tucson, AZ, USA). Micro-computed tomography (\u0026micro;CT) scanning was performed to aseess the microstructure of the bone callus as our previous described [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Meanwhile, the bone structural indices, including including the bone volume fraction (BV/TV, %), trabecular number (Tb.N, 1/mm), trabecular thickness (Tb.Th, mm) and trabecular separation (Tb.Sp, mm) were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTibial Fracture Model and treatment\u003c/h2\u003e \u003cp\u003eAn \u003cem\u003ein vivo\u003c/em\u003e mouse tibial fracture model was constructed using 10-weeks age male C57BL/6 mice as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The mice were housed in sterilized cages at the Experimental Animal Center of Capital Medical University (Beijing, China). All animal care and experimental procedures were performed in adherence to the National Institute of Health guidelines for Care and Use of Laboratory Animals and were approved by Institutional Animal Care and Use Committee of SHZY. (Permission No: SHZY-202106AF). In all, 3 groups of animals (n = 8 each group) were used, including model group (model), model group with lentiviruses particles with control vector (vector), and model group with lentiviruses particles with LINC00323 (LINC00323). Finally, callus tissue around the fracture site was harvested for subsequent detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data was presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from at least three independent experiments. GraphPad Prism 5 Demo (GraphPad, Inc., La Jolla, CA, USA) was used to statistical analysis. Student's t test was used to analyze the differences between two groups. Ordinary one-way ANOVA combined with Tukey's multiple comparisons test was used to compare differences among three or more groups. A p value less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLINC00323 contributes to the osteogenesis in MC3T3-E1 cells\u003c/h2\u003e \u003cp\u003eTo investigate the key roles of LINC00323 in osteogenic differentiation, the expression of LINC00323 was determined in MC3T3-E1 cells during osteoblast differentiation. We found the expression level of LINC00323 was increased after induction of osteogenic differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), suggesting that LINC00323 might play a role in osteogenesis. To further investigate the effect of LINC00323 on osteogenic differentiation, MC3T3-E1 cells were transduced with Lentivirus constructs expressing LINC00323 (LINC00323) or its control vector (vector), and Lentivirus encoding shRNA against LINC00323 (shLINC00323) or shRNA negative control (SCR). RT-qPCR was performed to confirm that LINC00323 was effectively upregulated in LINC00323-infected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), whereas was downregulated in shLINC00323-infected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) 36 hours later. The results of CCK-8 assay showed that overexpression of LINC00323 resulted in an increased viability of MC3T3-E1 cells under hypoxia state (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In contrast, a decreased viability of MC3T3-E1 cells was observed after LINC00323 silencing under hypoxia state (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). However, there was no difference on cell proliferation whether LINC00323 overexpressing or LINC00323 silencing under normoxic state (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Flow cytometry was conducted to examine apoptosis in MC3T3-E1 cells with the LINC00323. As expected, overexpression of LINC00323 could partly hypoxia-induced MC3T3-E1 cells apoptosis, whereas the lack of LINC00323 further exacerbated the hypoxia-induced apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince LINC00323 might involve in osteogenic differentiation, then we assess the expression of osteogenic differentiation marker. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, ALP activity of MC3T3-E1 cells in growth medium (Blank) or osteogenic medium (Control) was measured at 7 days, the ALP activity was markedly increased after LINC00323 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). As expect, knockdown of LINC00323 reduced the ALP activity of MC3T3-E1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Thereafter, the mRNA levels of ALP, COL1A1, OCN, and Runx2 were examined by RT-qPCR. The expression levels of these four markers showed a remarkable increase in LINC00323 overexpressed MC3T3-E1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), whereas a result opposite to LINC00323 knockdown was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In addition, LINC00323 exhibited remarkable function to promote osteoblast differentiation and mineralization, as evidenced by numerous alizarin red dye-bound nodules in LINC00323 overexpressed MC3T3-E1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). In contrast, deletion of LINC00323 decreased the mineralization and calcified nodule formation in MC3T3-E1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Overall, the findings suggest that LINC00323 might contribute to fracture healing by enhanced the differentiation of osteogenesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLINC00323 interacted with FUS\u003c/h2\u003e \u003cp\u003eTo understand the molecular mechanisms of LINC00323 in osteogenesis, NcPath database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ncpath.pianlab.cn/#/Home\u003c/span\u003e\u003cspan address=\"http://ncpath.pianlab.cn/#/Home\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to predict LINC00323 interaction genes [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The RNA-binding protein FUS was reported to interact with LINC00323 by high throughput sequencing [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To confirm the relation between LINC00323 and FUS, RNA pull-down assay was performed and verified the binding of LINC00323 to FUS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Conformably, the results from RIP displayed that LINC00323 was apparently enriched by FUS immunoprecipitation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To determine whether the expression of LINC00323 affected the expression of FUS, RT-qPCR and western blot were performed to determine the expression of FUS after alteration of LINC00323. Upon LINC00323 knockdown, the mRNA and protein levels of FUS were found to be decreased in MC3T3-E1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), whereas overexpression of LINC00323 enhanced the mRNA and protein levels of FUS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Furthermore, the increase mRNA and protein levels of FUS were observed in MC3T3-E1 cells with hypoxia treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In addition, LINC00323 exhibited no change of FUS both at mRNA and protein level under hypoxia state (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), whereas a similar result to LINC00323 knockdown was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). These results collectively suggested that LINC00323 interacted with FUS and did not influence the transcription of FUS in MC3T3-E1 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eLINC00323 interacted with FUS to increase the stability of PDGFB mRNA\u003c/h2\u003e \u003cp\u003eFUS has been reported to regulate the mRNA stability of downstream target genes, but genes mediated by FUS remained largely unknown [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. A GEO dataset, GSE226245, was downloaded from the database to analyze differentially expressed genes (DEGs). Compared to the control group, 3 genes (RGS5, IFI44 and IGS15) were significantly increased and 6 genes (FOXL1, PDGFRB, RASD2, TGFB2-AS1, AMH, and PDGFB) were significantly decreased in FUS knockdown group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Previous studies have shown that PDGFB was known to be a potent mitogen and chemoattractant for cells involved in wound healing and tissue regeneration, including cells critical for bone healing processes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Then, PDGFB was chosen for further investigation. To determine whether FUS-mediated alterations of PDGFB, siRNA targeting FUS lead to a significant decrease of PDGFB both at mRNA and protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). The increased expression of PDGFB was observed in hypoxia-induced MC3T3-E1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Further RNA pull-down assays using a biotin-labeled probe against mRNA of PDGFB were performed in MC3T3-E1 cells. Western blot illustrated that FUS could be detected by the probe specifically targeting PDGFB, but not by control probe (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). RIP experiments also showed that the interaction between FUS and mRNA of PDGFB (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Then, we want to test whether LINC00323 regulates FUS expression. higher expression of PDGFB were observed in LINC00323 overexpressed MC3T3-E1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Besides, we also noticed that knockdown of LINC00323 significantly downregulated the expression of FUS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). To explore the mechanisms by which FUS silencing down-regulated PDGFB in MC3T3-E1 cells, the stability of PDGFB was examined. MDA-MB-231 cells were transfected with siRNA against LINC00323 or FUS and simultaneously treated with actinomycin D, a transcription inhibitor, and then the mRNA expression levels of PDGFB were examined at 0, 20, 40, and 60 minutes, respectively. The data revealed that the mRNA expression levels of PDGFB were decreased significantly after LINC00323 or FUS silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). These findings suggested that the combination of LINC00323 and FUS enhanced the stability of PDGFB through direct binding.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eLINC00323 regulated hypoxia-mediated MC3T3-E1 cells survival and osteogenic differentiation via the FUS/PDGFB axis\u003c/h2\u003e \u003cp\u003eBased on data from above experiments, we hypothesized that LINC00323 promoted osteogenic differentiation via regulating FUS/PDGFB axis. To clarify the effect of FUS/PDGFB axis on LINC00323-mediated osteogenic differentiation in fracture healing, LINC00323 overexpression together with siRNAs targeting FUS or PDGFB was transfected into MC3T3-E1 cells following hypoxia treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). When LINC00323 was overexpressed in MC3T3-E1 cells, knockdown of FUS or PDGFB could reverse the protecting effect of LINC00323 on the cell viability under hypoxia state (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Besides, FUS or PDGFB silencing partially reversed the effect of LINC00323 on cell apoptosis by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). At day 7 postosteogenic induction, knockdown of FUS or PDGFB significantly inhibited LINC00323-midated ALP activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In addition, FUS or PDGFB silencing partially attenuated the expression of osteogenic differentiation marker induced by LINC00323 overexpression under hypoxia state (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Consistently, alizarin red staining also verified that FUS or PDGFB silencing decreased the mineralization and calcified nodule formation in LINC00323 overexpressed MC3T3-E1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These results indicated that LINC00323 promoted osteoblastic differentiation of MC3T3-E1 cells via regulating FUS/PDGFB axis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eLINC00323 accelerate the fracture healing process\u003c/h2\u003e \u003cp\u003eTo elaborate the impact of LINC00323 on bone regeneration \u003cem\u003ein vivo\u003c/em\u003e, a mouse tibial fracture model was constructed. One day after the fracture, 1 x 10\u003csup\u003e8\u003c/sup\u003e IU (integration units) lentivirus was locally injected into the subcutaneous region of a local fracture. Especially at day 28, the fracture healing process of the tibial was accelerated in the LINC00323 group compared with the Vector and Blank group based on the weekly X-ray radiographic images (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Confirming the results of X-ray, Micro-CT scanning showed increased bone and tissue volume of the fracture callus of mice with 28 days after fracture (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This was confirmed by a decrease of gap distance of cortical defects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Higher BV/TV, Tb.N, and Tb.Th values and a lower Tb.Sp value were observed in LINC00323 group compared with the Vector and Blank group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Consistent with the radiographic results, H\u0026amp;E and Masson's trichrome staining demonstrated better cortical growth and more collagen fiber in the LINC00323 group compared with the other two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Subsequent measurement of the expression of PDGFB in bone healing defect \u003cem\u003ein vivo\u003c/em\u003e was measured by immunohistochemical staining. The expression of PDGFB in tibial fracture region was significantly higher than the other two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), which indicated that the impact of LINC00323 on the fracture healing might be mediated by increased PDGFB. Taken together, these data showed that LINC00323 facilitated accelerated fracture healing process by promoting osteogenic differentiation \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHypoxia has been found to play a multifaceted and crucial role in bone healing, influencing various cellular processes and molecular pathways [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The ongoing research in this field holds significant promise for developing advanced therapeutic strategies to enhance bone regeneration and treat complex bone injuries [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, translating these findings into clinical practice requires a deeper understanding of the optimal use of hypoxia in bone healing and its integration with other therapeutic modalities. Long non-coding RNAs (lncRNAs), which are transcripts longer than 200 nucleotides without protein-coding potential, have been found to regulate gene expression at multiple levels, including chromatin modification, transcription, and post-transcriptional processing [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. LncRNAs represent a rapidly evolving area of research with significant potential to improve our understanding and treatment of fracture healing [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. For example, lncRNA KCNQ1OT1 as a competing endogenous RNA of miR-701-3p to promote osteoblast proliferation, migration, and inhibit apoptosis \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. LncRNA ENST00000563492 promoted osteogenic differentiation of BMSCs and improved the osteogenesis-angiogenesis coupling process through enhancing the expression of VEGF during osteogenic differentiation of BMSCs [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. An increased trend of LINC00323 was detected in the MC3T3-E1 cells treated with hypoxia by our previous study [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In our present study, LINC00323 was significantly upregulated during osteoblast differentiation in MC3T3-E1 cells. The enhancement of osteogenic differentiation following LINC00323 overexpression, as evidenced by increased activity of ALP and elevated expression of key osteogenic markers like RUNX2, COL1A1, and OCN. Conversely, the inhibitory effect on osteogenesis observed upon LINC00323 knockdown further underscores its crucial role in bone formation. Furthermore, there is an active interaction of FUS with LINC00323 and PDGFB during hypoxic conditions promotes the survival of osteoblasts and stimulates their differentiation into bone-forming cells, which is critical for the osteogenesis process (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In addition, LINC00323 promoted osteoblastic differentiation and fracture healing in a mouse model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn certain types of cancer, including breast cancer and liver cancer, the expression levels of LINC00323 have been linked to disease severity, prognosis, and survival rates [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. LINC00323 was also reported to a strong hypoxia-dependent activation of intergenic lncRNAs [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In the current study, we demonstrated that the LINC00323 were dramatically increased during osteogenic differentiation. Overexpression of LINC00323 maintained cell viability of MC3T3-E1 and hypoxia induced cell apoptosis, consistent with previous studies highlighting the function role of LINC00323 under hypoxic conditions [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious studies have shown that lncRNAs can interacted with RNA-binding proteins to regulate physiological functions and the pathogenesis of certain diseases, including chromatin regulation, transcription regulation, scaffolds, post-transcriptional modification [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Online bioinformatic tools was used to predict the interaction with LINC01133 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], which was supported by results of high throughput sequencing [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. FUS (Fused in Sarcoma) was chosen due to FUS was an RNA-binding protein that involved in the export of mRNA from the nucleus to the cytoplasm and plays a role in mRNA stability and turnover [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. LncRNA XIST was reported to interact with FUS and increased the stability of SPHK1 to promote osteoclast differentiation through SPHK1/S1P/ERK signaling pathway [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. LncRNA GAS6-AS1 regulated colorectal cancer (CRC) proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) via recruiting FUS to stable TRIM14 mRNA stability [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Recent study has shown that ANRIL recruited and interacted with FUS to stable HIFA to mediate transcription of VEGFA and ANRIL, which accelerated wound healing in diabetic foot ulcers [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In the present study, the interaction between LINC00323 and FUS was confirmed by verified by RNA pull-down and RIP assays. The increased viability and osteogenic potential of MC3T3-E1 cells overexpressing LINC00323 under hypoxic conditions are particularly noteworthy. This suggests a potential adaptive mechanism mediated by LINC00323 in response to hypoxic stress, a common feature of the fracture healing microenvironment. These observations contribute to the understanding of how lncRNAs can modulate cellular responses in varying oxygen conditions, which is crucial for tissue regeneration processes.\u003c/p\u003e \u003cp\u003eThrough analysis of GSE226245, PDGFB was chosen for further investigation. PDGFB was a key growth factor involved in various cellular processes, including cell proliferation, migration, and angiogenesis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. PDGFB was also reported to play a crucial role in tissue development and repair, and its dysregulation is implicated in various pathologies [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The relationship between LINC00323 and FUS was confirmed by verified by RNA pull-down and RIP assays. Our study reveals indicated that the interaction between LINC00323 and FUS increased the stability of PDGFB. And knockdown LINC00323 or FUS decreased the expression of PDGFB. Further \u003cem\u003ein vivo\u003c/em\u003e experiments showed that LINC00323 accelerated fracture healing consisted with the \u003cem\u003ein vitro\u003c/em\u003e findings and suggested a translational potential for LINC00323 in therapeutic strategies aimed at enhancing bone repair. The high expression of PDGFB was observed in a mouse tibial fracture model with LINC00323 treatment.\u003c/p\u003e \u003cp\u003eWhile this study provides significant insights, it also acknowledges certain limitations. The specific mechanisms by which LINC00323 modulates FUS and PDGFB, and their broader implications in the complex network of bone healing, require further exploration. Additionally, understanding the role of LINC00323 in various stages of bone healing and its interactions with different cellular components remains a crucial area for future research.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study indicated that LINC00323 interacted with FUS to modulate the stability of PDGFB mRNA under hypoxic state, which established a theoretical foundation for novel therapeutic approaches aimed at accelerating fracture repair, thereby addressing a critical need in clinical orthopedics and trauma medicine.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eGLC and JH were involved in the design of the study, basic analysis of data, drafting of manuscript, and revising it for critical knowledge content. SA performed the statistical analysis. JYW and XYS were involved in the acquisition of data, drafting of manuscript, and revising it critically for critical knowledge content. All authors have read and approved the final submitted manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from the Beijing Municipal Natural Science Foundation (No. 7232073)\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMarsell R, Einhorn TA. The biology of fracture healing. Injury 2011;42:551\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCunningham BP, Brazina S, Morshed S, Miclau TR. 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Breaking the feed forward inflammatory cytokine loop in the tumor microenvironment of PDGFB-driven glioblastomas. J Clin Invest 2023;133.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng Y, Ji S, Li X, Wen L. Qijia rougan formula ameliorates ECM deposition in hepatic fibrosis by regulating the JAK1/STAT6-microRNA-23a feedback loop in macrophage M2 polarization. Biomed Pharmacother 2023;168:115794.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalra K, Eberhard J, Farbehi N, Chong JJ, Xaymardan M. Role of PDGF-A/B Ligands in Cardiac Repair After Myocardial Infarction. Front Cell Dev Biol 2021;9:669188.\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":"Bone QCT/µCT, Matrix mineralization, Molecular pathways - development, Osteoblasts, Injury/fracture healing","lastPublishedDoi":"10.21203/rs.3.rs-3966058/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3966058/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntermittent hypoxia has been reported to contribute beneficial effects on fracture healing depending on various factors like duration, frequency, and severity. Yet, little is known about the underlying molecular mechanism. Our previous study found that LINC00323 was up-regulated under hypoxic conditions, suggesting that it might play a final role in hypoxia-induced fracture repair. The present study is to investigate the osteogenic effect of LINC00323 \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Upregulation of LINC00323 enhanced the mineralization and activity ALP and increased the expression of osteogenic markers. Further analysis revealed that LINC00323 promoted PDGFB expression by binding FUS to regulate the growth and osteogenic differentiation of MC3T3-E1. Lentivirus mediated LINC00323 particles were injected into the fracture site of the tibia of mice, and fracture healing was evaluated by X-rays, micro-CT examination, biomechanical test and histological staining. Local injection of Lentivirus-LINC00323 increased bone mass, biomechanical strength and cartilage callus formation. These findings indicated that LINC00323 induced the differentiation of osteoblast-like cells via regulation of the expression of PDGFB, represents a theoretical basis to accelerate fracture healing.\u003c/p\u003e","manuscriptTitle":"LINC00323 induced by hypoxia promote cartilage callus by interacting with FUS to regulate PDGFB expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-21 17:59:36","doi":"10.21203/rs.3.rs-3966058/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":"fd1d1d31-70a1-46c4-b9a2-20f962017d65","owner":[],"postedDate":"February 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-17T23:44:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-21 17:59:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3966058","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3966058","identity":"rs-3966058","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}