Targeting glycolytic reprogramming by tsRNA-0032 for treating pathological lymphangiogenesis

Targeting glycolytic reprogramming by tsRNA-0032 for treating pathological lymphangiogenesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting glycolytic reprogramming by tsRNA-0032 for treating pathological lymphangiogenesis Biao Yan, Fan Ye, Ziran Zhang, Lianjun Shi, Wan Mu, Shuting Lu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4896824/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jan, 2025 Read the published version in Cell Death & Disease → Version 1 posted 9 You are reading this latest preprint version Abstract Lymphangiogenesis is vital for tissue fluid homeostasis, immune function, and lipid absorption. Disruption of this process is implicated in diseases such as cancer, inflammation, and autoimmune disorders. In this study, we elucidate the role of tsRNA-0032 in lymphangiogenesis and its molecular mechanisms. tsRNA-0032 expression is significantly diminished in corneal suture and LPS-induced human lymphatic endothelial cell (HLEC) models under inflammatory conditions. Overexpression of tsRNA-0032 suppresses lymphangiogenesis by inhibiting HLEC proliferation, migration, and tube formation. Moreover, overexpression of tsRNA-0032 inhibits suture-induced mouse corneal lymphangiogenesis in vivo . tsRNA-0032 is mainly found in the cytoplasm and interacts with Ago2 protein. Overexpression of tsRNA-0032 leads to a reduction in ATP production and lowers the levels of pyruvate and lactate by targeting PKM2, which is crucial for the final step of glycolysis. This regulation of glycolysis impacts the cellular energy and metabolic balance in HLECs, contributing to the inhibition of lymphangiogenesis. Clinical data show that tsRNA-0032 levels are markedly lower in corneal tissues from transplant recipients compared to donors, whereas PKM2 expression is elevated, underscoring the clinical significance of the tsRNA-0032/PKM2 axis in corneal lymphangiogenesis. This study provides novel insights into lymphangiogenesis regulation and offers potential therapeutic targets for lymphatic-related diseases. tsRNA-0032 lymphangiogenesis glycolysis PKM2 corneal suture Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION The lymphatic vessel system is an important component of circulatory system, working in concert with vascular system to maintain fluid balance, support immune function, and facilitate lipid absorption 1 . Lymphangiogenesis, the process of generating new lymphatic vessels, is primarily controlled by lymphatic endothelial cells (LECs). LECs typically maintain a quiescent state except during embryonic development 2 . However, under pathological conditions such as tumor metastasis 3 , cardiovascular diseases 4 , and various inflammatory diseases, LECs are activated, stimulating the proliferation, migration, and subsequent formation of new lymphatic vasculature. Within the ocular environment, dysfunctional lymphatic vessels can contribute to keratitis 5 , dry eye 6 , and transplantation rejection 7 . Despite extensive research, our comprehension of lymphangiogenesis remains incomplete, and clinical interventions targeting neoplastic lymphatic vessels are severely limited. Current therapeutic approaches primarily focus on inhibiting VEGFR3 8 or targeting LEC-autonomous activation of mutant pathways 9 through the use of rapamycin, gene therapy, or surgical resection. The mTOR inhibitor, rapamycin, has been a cornerstone in organ transplantation for many years, primarily because of its potent immunosuppressive properties 10 . However, long-term efficacy and adverse effects remain poorly characterized, and the complex interplay of lymphatic biology necessitates a nuanced approach when considering lymphangiogenesis as a therapeutic target for inflammatory and immune-related diseases. Thus, a deeper understanding of lymphangiogenesis is imperative for developing innovative and comprehensive treatment modalities for lymphangiogenesis-associated diseases. The occurrence and progression of pathological lymphangiogenesis require a complex interplay of various cytokines and growth factors. Existing studies have shown that small extracellular vesicle (sEVs) 11 , pro-inflammatory cytokines 12 , ROS 13 and various non-coding RNAs can regulate lymphangiogenesis. sEVs play a crucial role in the transport and distribution within lymphatic system. When sEVs are injected subcutaneously into the tail base and tumor tissues, they preferentially accumulate in lymph nodes. Pro-inflammatory cytokines and reactive oxygen species (ROS) promote lymphangiogenesis by regulating the proliferation and migration of lymphatic endothelial cells. Non-coding RNAs can regulate the function of lymphatic endothelial cells and lymphangiogenesis process by regulating gene expression. tRNA-derived small RNAs (tsRNAs) have emerged as a novel class of small non-coding RNAs, arising from specific cleavage of precursor or mature tRNAs. These cleavage events are predominantly triggered by stress conditions, such as amino acid deficiency, phosphate starvation, UV radiation, heat shock, hypoxia, oxidative damage, and viral infection. The production of tsRNAs is notably limited under normal growth conditions, indicating their role as tRNA-derived stress-induced RNAs. This suggests that tsRNAs may play crucial roles in the cellular response to stress, potentially regulating gene expression and maintaining cellular homeostasis during adverse conditions 14 . Emerging evidence underscores the capacity of tsRNAs to modify RNAs and interact with proteins, thereby exerting significant influence over diverse biological processes including gene silencing, ribosome biogenesis, and epigenetic regulation 15 . Increasing studies have revealed the involvement of tsRNAs in the progression of various diseases, such as tumor lymphatic metastasis, systemic inflammation, and lipid absorption disorders 16 , 17 , 18 . Given the established link between tsRNAs and angiogenesis 19 , 20 , it is imperative to investigate their potential regulatory function in lymphangiogenesis. A comprehensive understanding of this relationship may unveil novel insights into disease pathogenesis and inform the development of innovative therapeutic strategies. In this study, we investigated the role of tsRNA-0032, a specific cleavage product of tRNA-GTG-His, in lymphangiogenesis. Our findings demonstrate that tsRNA-0032 expression is downregulated during lymphangiogenesis, while its upregulation attenuates lymphangiogenesis and suppresses LEC hyperactivation under inflammatory conditions. Mechanistically, tsRNA-0032 regulates lymphangiogenesis by modulating glycolysis. These findings position tsRNA-0032 as a potential regulator of LEC dysfunction, warranting further investigation into its therapeutic potential for dysregulated lymphangiogenesis. MATERIALS AND METHODS Animal experiment Animals were obtained from Nanjing Junke Bioengineering Corporation and treated in accordance with the Association for Research in Vision and Ophthalmology’s guidelines for the use of animals in ophthalmic and vision research. All procedures were approved by the Animal Experiment Management Committee of the author’s institute. Corneal suture model A corneal lymphangiogenesis model was established to induce inflammation and stimulate lymphatic vessel growth towards the corneal center. Male C57BL/6J mice (6–8 weeks old) underwent anesthesia with xylazine (10 mg/kg) and ketamine (100 mg/kg) administered intraperitoneally. Pupil dilation was achieved using phenylephrine and tropicamide eye drops. Three interrupted 11 − 0 nylon sutures were placed in the corneal stroma near the limbus, spaced 120 degrees apart. Gatifloxacin ointment was applied topically to prevent infection. Corneas were harvested seven days post-surgery for subsequent analysis. Cell culture and transfection Primary human lymphatic endothelial cells (HLECs; Promocell, Germany, C-12216) were cultured in Endothelial Cell Basal Medium MV (Promocell, Germany, C-22220) supplemented with 12% fetal bovine serum (ScienCell, USA) and penicillin/streptomycin (100 U/mL, 100 µg/mL; Gibco, USA) in a humidified incubator at 37°C with 5% CO 2 . tsRNA-0032 mimics, inhibitors, and their respective negative controls (NCs) were designed and synthesized by GENERAY (China). HLECs were transfected with mimics or inhibitors using Lipofectamine 3000 (Invitrogen, USA, L3000015) according to the manufacturer’s protocol when cells reached approximately 80% confluence. Fluorescence in situ hybridization (FISH) The cellular localization of tsRNA-0032 in HLECs was determined by FISH assay. Cy3-labeled probes, 5’-CGAACCGAGGTTGCTGCGGCC-3’, specific to tsRNA-0032 were designed and synthesized by Servicebio (Wuhan, China). The signals of the probes were detected by a Fluorescent In Situ Hybridization Kit (Servicebio, China) according to the manufacturer’s instructions. Images were captured by a fluorescence microscope (Olympus, Japan) Nucleoplasmic separation assay Nuclear and cytoplasmic RNAs in HLECs were isolated using the Cytoplasmic and Nuclear RNA Purification Kit (NORGEN BIOTEK, Canada, 21000) according to the manufactures’ instruction. The collected RNAs were reverse transcribed into cDNAs, followed with qRT-PCR analysis. 2 −ΔΔCt method was used to analyze the relative expression levels of genes in the nuclear and cytoplasmic fractions. RNA immunoprecipitation (RIP) RIP was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA, 17–701) according to the manufacturer's protocol. Antibodies targeting Ago, PKM2, or IgG were incubated with magnetic beads overnight at 4°C. Following immunoprecipitation, co-precipitated RNA was extracted and subjected to qRT-PCR analysis to detect the expression of tsRNA-0032 and PKM2, thereby confirming the enrichment of target RNAs. Luciferase reporter assay HLECs were seeded into 96-well plates and cultured in complete medium until reaching 60–70% confluence. Cells were co-transfected with either Luc-PKM2 WT, Luc-PKM2 Mut, or an empty vector, along with tsRNA-0032 mimic or a negative control mimic using Lipofectamine 3000 (Invitrogen, USA, L3000015). After a 24-hour incubation, firefly and Renilla luciferase activities were measured in each group using a luciferase reporter assay kit (Promega, USA, E1910). Measurement of extracellular acidification rate (ECAR) 1 × 10 4 of HLECs were seeded onto Seahorse XFe96/XF Pro cell culture microplate (Agilent Technologies, 103794-100). They were cultured in XF base medium (pH 7.4) in a non-CO2 incubator at 37℃ for 1 h. Glucose (10 mM), glutamine (1 mM), 2-DG (50 mM), and oligomycin (1 µM) were sequentially added into the plates at specific time points following the manufacturer’s guidelines. ECAR was collected using Seahorse XFe96 Analyzer (Agilent Technologies) and analyzed using the Seahorse XFe96 software. Quantification of pyruvate and lactate 1 × 10 6 of HLECs were seeded onto 6-well plate and received different treatment respectively. Pyruvate and lactate levels were quantified using the pyruvate assay kit (Jiancheng Bioengineering Institute, China, A081–1–1) and lactate assay kit (Jiancheng Bioengineering Institute, China, A019-2-1). Absorbance was measured using the Multickan Skyhigh Microplate Reader (ThermoFisher Scientific, USA, A51119700DPC). Statistical analysis Unpaired Student’s t-tests were used to compare differences between two groups, while one-way analysis of variance (ANOVA) was employed for comparisons among multiple groups. Normal distribution of data was verified prior to ANOVA analysis. Data are presented as mean ± standard deviation. Statistical significance was set at P < 0.05. All statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, USA). RESULTS tsRNA-0032 expression is down-regulated during inflammatory stress Normal lymphatic vessels are confined to the conjunctiva and corneal limbus. However, pathological conditions such as inflammation or trauma can induce lymphatic vessel growth into the corneal center 21 . To investigate the relationship between tsRNA-0032 expression and lymphangiogenesis, we employed corneal suture and LPS-induced HLEC models. In a corneal suture mouse model, qRT-PCR analysis revealed a significant downregulation of tsRNA-0032 expression in sutured corneas compared to the controls (Fig. 1 A). Similarly, LPS-induced HLECs exhibited decreased tsRNA-0032 levels (Fig. 1 B). Given that tsRNA is generated from tRNA by ANG and Dicer 22 , we explored the underlying mechanism of tsRNA-0032 down-regulation. We silenced ANG or Dicer in HLECs under normal condition and LPS-treated condition. The results showed that transfection of ANG siRNA2/3 or Dicer siRNA2/3 led to reduced levels of ANG or Dicer expression (Fig. 1 C and 1 D). ANG knockdown significantly reduced tsRNA-0032 expression, while Dicer silencing had no effect both under normal condition and LPS-treated condition (Fig. 1 E-H). Taken together, these results suggest that tsRNA-0032 may act as a potential regulator of lymphangiogenesis, and its downregulation in pathological conditions is primarily mediated by ANG. tsRNA-0032 inhibits lymphatic endothelial cell function in vitro To investigate the role of tsRNA-0032 in lymphangiogenesis, HLECs were transfected with tsRNA-0032 mimics or inhibitors. Compared with the control group, transfection of tsRNA-0032 mimics significantly increased tsRNA-0032 expression, while transfection of tsRNA-0032 inhibitor decreased it (Fig. 2 A). Cell viability was assessed using CCK8 assays, which showed that tsRNA-0032 overexpression reduced cell viability, whereas tsRNA-0032 silencing increased it (Fig. 2 B). 5-Ethynyl-2’-deoxyuridine (EdU) assays demonstrated that tsRNA-0032 overexpression decreased HLEC proliferation, while silencing tsRNA-0032 promoted proliferation (Fig. 2 C). Transwell assays revealed that tsRNA-0032 mimic transfection inhibited HLEC migration, whereas its inhibitor promoted migration (Fig. 2 D). Matrigel tube formation and spheroid sprouting assays were conducted to evaluate tube formation and sprouting abilities. tsRNA-0032 mimic transfection reduced tube-like structure formation and sprouting in HLECs, while the inhibitor enhanced these processes (Fig. 2 E and 2 F). These results indicate that tsRNA-0032 regulates the biological function of HLECs in vitro. tsRNA-0032 inhibits pathological lymphangiogenesis in vivo To investigate the role of tsRNA-0032 in lymphangiogenesis in vivo , a suture-induced mouse corneal lymphangiogenesis model was employed. tsRNA-0032 agomir or antagomir was injected into the conjunctival sac to regulate tsRNA-0032 expression, as confirmed by qRT-PCR (Fig. 3 A). Up-regulation of tsRNA-0032 via agomir injection reduced corneal lymphangiogenesis, while down-regulation via antagomir injection increased it, as assessed by LYVE-1 immunofluorescence staining (Fig. 3 B and 3 C). tsRNA-0032 regulates HLEC function via targeting PKM2 To elucidate the mechanism underlying tsRNA-0032-mediated lymphangiogenesis, we investigated the subcellular localization of tsRNA-0032 in HLECs using fluorescence in situ hybridization (FISH). Results demonstrated that tsRNA-0032 is predominantly localized to the cytoplasm (Fig. 4 A). Nucleo-cytoplasmic fractionation assays further confirmed the predominantly cytoplasmic localization of tsRNA-0032 (Fig. 4 B). Given that Ago2 is a key protein involved in post-transcriptional gene regulation mediated by tsRNAs 23 , 24 , we conducted RIP assays to determine whether tsRNA-0032 exerts its biological role by binding to Ago2. RIP results demonstrated that Ago2, but not the negative control IgG, immunoprecipitated tsRNA-0032 (Fig. 4 C). Utilizing the tRFTar database ( http://www.rnanut.net/tRFTar/ ), we then analyzed potential downstream pathways and target genes of the tsRNA-0032/Ago2 complex. The database predicted that tsRNA-0032 regulates cellular glycolysis by targeting PKM2 and regulates cell fatty acid synthesis by targeting fatty acid synthase (FASN). To further identify the target gene of tsRNA-0032 in HLECs, qRT-PCR and Western blot analyses were performed. tsRNA-0032 overexpression significantly decreased PKM2 mRNA and protein levels, while its knockdown exhibited the opposite effect. In contrast, FASN mRNA and protein levels remained unaffected by tsRNA-0032 modulation (Fig. 4 D-F). Base pairing complementarity between tsRNA-0032 and PKM2 was confirmed (Fig. 4 G). Luciferase reporter assays demonstrated that tsRNA-0032 overexpression significantly reduced the luciferase activity of the wild-type PKM2 3'-UTR, but not its mutant form, indicating a direct interaction between tsRNA-0032 and the PKM2 3'-UTR (Fig. 4 H). Furthermore, RNA pull-down assays revealed that PKM2 was specifically enriched in the tsRNA-0032-bound fraction compared to the miR-484 control (Fig. 4 I). Collectively, these findings establish that tsRNA-0032 regulates HLEC function by directly targeting PKM2. tsRNA-0032/PKM2 signaling axis alters glycolysis in HLECs PKM2 is the rate-limiting enzyme in the final step of glycolysis, catalyzing the conversion of phosphoenolpyruvate (PEP) to pyruvate 25 . tsRNA-0032/PKM2 signaling axis regulates lymphatic endothelial cell function by modulating cellular glycolysis. To assess glycolytic activity, extracellular acidification rate (ECAR) was measured in HLECs using a Seahorse XF extracellular flux analyzer. tsRNA-0032 overexpression suppressed glycolysis, glycolytic capacity, and glycolytic reserve, while PKM2 overexpression partially rescued this inhibitory effect (Fig. 5 A, 5 B). Conversely, tsRNA-0032 knockdown enhanced glycolytic activity, which was attenuated by PKM2 silencing (Fig. S1 A, S1B). To further validate the impact of tsRNA-0032 on glycolysis, pyruvate and lactate production were quantified. tsRNA-0032 overexpression decreased pyruvate and lactate levels, effects partially reversed by PKM2 overexpression (Fig. 5 C, 5 D). Conversely, tsRNA-0032 knockdown increased pyruvate and lactate production, an effect reversed by PKM2 silencing (Fig. S1 C, S1D). Given the central role of glycolysis in ATP production, ATP levels were measured. tsRNA-0032 overexpression decreased ATP production, rescued by PKM2 overexpression, while tsRNA-0032 knockdown increased ATP production, attenuated by PKM2 silencing (Fig. 5 E, Fig. S1 E). To assess the specificity of tsRNA-0032's effect on glycolysis, the expression of other key glycolytic enzymes, HK2 and PFKFB3, was evaluated; no significant changes were observed (Fig. S1 F). Collectively, these findings demonstrate that the tsRNA-0032/PKM2 signaling axis specifically regulates glycolysis in HLECs. tsRNA-0032/PKM2 signaling axis is involved in lymphangiogenesis in vitro To confirm whether tsRNA-0032/PKM2 signaling axis is involved in lymphangiogenesis, we explored whether PKM2 alone could affect lymphangiogenesis in vitro . PKM2 pcDNA3.1 vector and PKM2 siRNA were used to modulate the expression level of PKM2, and the efficiency of interventions was demonstrated by qRT-PCR assays and western blots (Fig. S2 A and S2B). PKM2 overexpression promoted HLECs proliferation, migration, tube formation and sprouting activity, while PKM2 silencing reduced these cellular functions (Fig. S2 C-S2F). To investigate whether modulating PKM2 expression could reverse lymphatic system dysfunction induced by tsRNA-0032, we employed shikonin, a specific inhibitor of PKM2 activity with no reported effects on PKM1 or PKL 26 , 27 . CCK-8 assay determined that 1 µM shikonin was non-toxic to HLECs (Fig. S3A). To investigate the functional rescue of tsRNA-0032-mediated HLEC dysfunction, PKM2 was overexpressed. PKM2 overexpression effectively reversed the inhibitory effects of tsRNA-0032 on HLEC proliferation, migration, tube formation, and sprouting (Fig. 6 A-D). These findings collectively indicate that the tsRNA-0032/PKM2 signaling axis plays a critical role in regulating lymphatic endothelial cell function. tsRNA-0032/PKM2 signaling axis is involved in regulating lymphangiogenesis in vivo To elucidate the in vivo functional significance of tsRNA-0032/PKM2 signaling axis in lymphangiogenesis, a corneal suture model was established. Injection of tsRNA-0032 agomir into the conjunctival sac led to a reduction in corneal lymphangiogenic area, an effect phenocopied by treatment with shikonin, a specific PKM2 inhibitor. Conversely, overexpression of PKM2 through vector-mediated gene delivery effectively counteracted the anti-lymphangiogenic effects induced by tsRNA-0032 agomir, as evidenced by the increased formation and length of new lymphatic vessels (Fig. 7 A, 7 B). These findings collectively underscore the critical role of tsRNA-0032/PKM2 signaling axis in regulating corneal lymphangiogenesis and suggest its potential as a therapeutic target for this pathological condition. Clinical relevance of tsRNA-0032-mediated signaling in corneal lymphangiogenesis Keratitis, an inflammation of the cornea, is frequently accompanied by the abnormal growth of lymphatic vessels, a condition known as corneal lymphangiogenesis 28 , 29 . To investigate the potential clinical implications of tsRNA-0032/PKM2 signaling axis in this context, corneal tissue samples were collected from both keratitis patients (recipients) and healthy individuals (donors). qRT-PCR analysis revealed a marked downregulation of tsRNA-0032 and a concomitant upregulation of PKM2 in the corneal tissue of keratitis patients compared to healthy controls (Fig. 8 A, 8 B). These findings strongly suggest a correlation between the dysregulation of the tsRNA-0032/PKM2 signaling axis and the development of corneal lymphangiogenesis in keratitis patients. DISCUSSION Although physiological lymphangiogenesis is rare in healthy adults, pathological lymphangiogenesis plays a significant role in various disease processes, including inflammation, lymphedema, organ transplant rejection, tumor metastasis, and cardiovascular disease 30 . In inflammatory diseases such as rheumatoid arthritis, inflammation-induced lymphangiogenesis regulates fluid drainage, immune cell migration, and the removal of inflammatory mediators, ultimately accelerating inflammation resolution 31 . Conversely, following organ transplantation, lymphangiogenesis can trigger immune system reactivation in draining lymph nodes, potentially leading to organ rejection 32 . In cancer, tumor cells spread to lymph nodes via the lymphatic system, and inhibiting tumor lymphangiogenesis and lymph node metastasis in animal models effectively impedes tumor progression 33 , 34 . A common feature among these lymphangiogenesis-related diseases is the activation of the VEGF-C/VEGFR3 pathway 35 , 36 . While therapies targeting the primary regulatory pathways of lymphangiogenesis, similar to antiangiogenic drugs targeting VEGF, appear promising for treating these diseases, they have not fully met expectations. Therefore, it is crucial to elucidate the mechanisms of lymphangiogenesis in detail and explore alternative treatment strategies. As a novel class of non-coding RNAs, tsRNAs have emerged as critical regulators of diverse cellular processes and are increasingly recognized for their involvement in a wide range of physiological and pathological conditions 37 , 38 . For example, tsRNA-04002 alleviates intervertebral disc degeneration by targeting PRKCA to inhibit apoptosis of nucleus pulposus cells 39 . Sunyang Ying et al. demonstrated tRF-Gln-CTG-026 ameliorates liver injury by alleviating global protein synthesis. Angiogenin-mediated tsRNAs control inflammation and metabolic disorder by regulating NLRP3 inflammasome 40 . We previously found that tRNA-Cys-5-0007 plays a dual role of anti-angiogenesis and anti-inflammatory in ocular vascular disease 20 . This study focused on the functional characterization of tsRNA-0032, a tRNA-derived small RNA, in the context of lymphangiogenesis. Our findings revealed a consistent downregulation of tsRNA-0032 expression in both suture-induced murine corneal and LPS-stimulated HLEC models. Gain- and loss-of-function experiments demonstrated that tsRNA-0032 overexpression inhibits, while knockdown enhances, lymphangiogenesis, suggesting a suppressive role for tsRNA-0032 in this process. Lymphangiogenesis, the formation of new lymphatic vessels from pre-existing lymphatic networks, is a critical process mediated by LECs. These specialized endothelial cells, residing within the lymphatic vasculature, undergo proliferation, migration, and tube formation in response to specific stimuli to drive lymphatic vessel growth. Characterized by a unique metabolic profile, LECs exhibit a strong reliance on aerobic glycolysis to fuel their energy-intensive functions 12 . This metabolic phenomenon, known as the Warburg effect, is characterized by an increased reliance on glycolysis for ATP production even in the presence of oxygen. While seemingly less efficient than oxidative phosphorylation in terms of ATP yield, this metabolic shift is advantageous for rapidly proliferating cells. By channeling glucose towards glycolysis, cells can efficiently allocate resources for macromolecular biosynthesis while simultaneously mitigating oxidative stress through reduced ROS production 13 . Upon activation and transition from a quiescent to a proliferative and migratory state, HLECs exhibit a marked increase in glycolytic flu 41 . Impaired glycolysis profoundly compromises cellular function, as evidenced by various pathological conditions. For instance, podocyte-specific PKM2 deletion exacerbates angiotensin II-induced glomerular and podocyte injury, characterized by foot process effacement and proteinuria 42 . Similarly, glycolytic dysfunction in natural killer cells accelerates lung cancer progression 43 . As the pivotal enzyme catalyzing phosphoenolpyruvate conversion to pyruvate, PKM2 plays a crucial role in glycolysis 26 . Our study demonstrates that tsRNA-0032 regulates glycolysis by targeting PKM2. tsRNA-0032 overexpression inhibits glycolysis, leading to reduced pyruvate and lactate production, essential metabolites for cellular energy metabolism 44 , 45 . These metabolic alterations ultimately impair lymphatic endothelial cell function and suppress lymphangiogenesis. Notably, dysregulated pyruvate and lactate levels are implicated in various diseases, including Alzheimer's disease and tumor-draining lymph nodes 46 , 47 . Pyruvate and lactate kit demonstrated that tsRNA-0032 upregulation decreased pyruvate and lactate production, while tsRNA-0032 downregulation increased their production. Therefore, tsRNA-0032 influenced energy supply and the balance between metabolite production and consumption via targeting at PKM2, thus regulating lymphangiogenesis. To further corroborate the clinical relevance of the tsRNA-0032/PKM2 axis in lymphangiogenesis, we examined tsRNA-0032 and PKM2 expression levels in corneal tissues obtained from keratitis patients who had undergone corneal transplantation. Compared to corneal samples from corresponding healthy donors, those from keratitis patients exhibited significantly reduced tsRNA-0032 levels and concomitantly elevated PKM2 levels. These findings provide compelling evidence for the involvement of the tsRNA-0032/PKM2 signaling pathway in the pathogenesis of corneal lymphangiogenesis and suggest that targeting this pathway may offer a promising therapeutic strategy for the management of lymphangiogenesis-related ocular diseases. In conclusion, this study unveiled a critical role for tsRNA-0032 in regulating lymphatic endothelial cell function and subsequent lymphangiogenesis. Our findings demonstrate that tsRNA-0032 inhibits lymphatic endothelial cell proliferation, migration, tube formation, and sprouting in vitro. Furthermore, in vivo models of corneal suture-induced and subcutaneous Matrigel plug-induced lymphangiogenesis confirmed the inhibitory effects of tsRNA-0032 on lymphatic vessel formation. Mechanistically, we revealed that tsRNA-0032 interacts with Ago2 and directly targets PKM2, a key glycolytic enzyme, to suppress glycolytic reprogramming in lymphatic endothelial cells. These results collectively highlight the therapeutic potential of targeting the tsRNA-0032/PKM2 axis for the treatment of lymphangiogenesis-related diseases. Declarations DATA AVAILABILITY The data from this study are available from the authors upon request. ACKNOWLEDGEMENTS This work was generously supported by the grants from the National Natural Science Foundation of China (no.81770945 to Dr Yan; no. 81570859 and 82070983 to Dr Jiang). DECLARATION OF INTERESTS The authors declare that they have no conflict of interest. AUTHOR CONTRIBUTIONS B.Y. and Q.J. designed research; F.Y., Z.R.Z., L.J.S., S.T.L., and W.M. performed research; F.Y. and Z.R.Z. analyzed data; F.Y. and B.Y. wrote the article. References Hu Z, Zhao X, Wu Z, Qu B, Yuan M, Xing Y , et al. Lymphatic vessel: origin, heterogeneity, biological functions, and therapeutic targets. Signal Transduct Target Ther 2024, 9 (1) : 9. Tammela T, Alitalo K. Lymphangiogenesis: Molecular mechanisms and future promise. Cell 2010, 140 (4) : 460-476. Stacker SA, Williams SP, Karnezis T, Shayan R, Fox SB, Achen MG. Lymphangiogenesis and lymphatic vessel remodelling in cancer. Nat Rev Cancer 2014, 14 (3) : 159-172. Oliver G, Kipnis J, Randolph GJ, Harvey NL. The Lymphatic Vasculature in the 21(st) Century: Novel Functional Roles in Homeostasis and Disease. Cell 2020, 182 (2) : 270-296. Lee HK, Lee SM, Lee DI. 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Inflammation and Lymphatic Function. Front Immunol 2019, 10: 308. Wong BW. Lymphatic vessels in solid organ transplantation and immunobiology. Am J Transplant 2020, 20 (8) : 1992-2000. Dadras SS, Lange-Asschenfeldt B, Velasco P, Nguyen L, Vora A, Muzikansky A , et al. Tumor lymphangiogenesis predicts melanoma metastasis to sentinel lymph nodes. Mod Pathol 2005, 18 (9) : 1232-1242. Dieterich LC, Tacconi C, Ducoli L, Detmar M. Lymphatic vessels in cancer. Physiol Rev 2022, 102 (4) : 1837-1879. Lim L, Bui H, Farrelly O, Yang J, Li L, Enis D , et al. Hemostasis stimulates lymphangiogenesis through release and activation of VEGFC. Blood 2019, 134 (20) : 1764-1775. Lin QY, Zhang YL, Bai J, Liu JQ, Li HH. VEGF-C/VEGFR-3 axis protects against pressure-overload induced cardiac dysfunction through regulation of lymphangiogenesis. Clin Transl Med 2021, 11 (3) : e374. Zhang L, Liu J, Hou Y. Classification, function, and advances in tsRNA in non-neoplastic diseases. Cell Death Dis 2023, 14 (11) : 748. Zhao Y, Li X, Ye C, Huang C, Lv X, Li J. The biogenesis, mechanism and function of the tRNA-derived small RNA (tsRNA): a review compared with microRNA. Am J Cancer Res 2023, 13 (5) : 1656-1666. Pan J, Liu Z, Shen B, Xu J, Dai G, Xu W , et al. tsRNA-04002 alleviates intervertebral disk degeneration by targeting PRKCA to inhibit apoptosis of nucleus pulposus cells. J Orthop Surg Res 2023, 18 (1) : 413. Cai J, Li C, Liu S, Tan M, Sun Y, Sun X , et al. Angiogenin-mediated tsRNAs control inflammation and metabolic disorder by regulating NLRP3 inflammasome. Cell Death Differ 2024. Caruso P, Dunmore BJ, Schlosser K, Schoors S, Dos Santos C, Perez-Iratxeta C , et al. Identification of MicroRNA-124 as a Major Regulator of Enhanced Endothelial Cell Glycolysis in Pulmonary Arterial Hypertension via PTBP1 (Polypyrimidine Tract Binding Protein) and Pyruvate Kinase M2. Circulation 2017, 136 (25) : 2451-2467. Chen Z, Zhu Z, Liang W, Luo Z, Hu J, Feng J , et al. Reduction of anaerobic glycolysis contributes to angiotensin II-induced podocyte injury with foot process effacement. Kidney Int 2023, 103 (4) : 735-748. Cong J, Wang X, Zheng X, Wang D, Fu B, Sun R , et al. Dysfunction of Natural Killer Cells by FBP1-Induced Inhibition of Glycolysis during Lung Cancer Progression. Cell Metab 2018, 28 (2) : 243-255 e245. Vegran F, Boidot R, Michiels C, Sonveaux P, Feron O. Lactate influx through the endothelial cell monocarboxylate transporter MCT1 supports an NF-kappaB/IL-8 pathway that drives tumor angiogenesis. Cancer Res 2011, 71 (7) : 2550-2560. Galaz A, Cortes-Molina F, Arce-Molina R, Romero-Gomez I, Mardones GA, Felipe Barros L , et al. Imaging of the Lactate/Pyruvate Ratio Using a Genetically Encoded Forster Resonance Energy Transfer Indicator. Anal Chem 2020, 92 (15) : 10643-10650. Rossi A, Rigotto G, Valente G, Giorgio V, Basso E, Filadi R , et al. Defective Mitochondrial Pyruvate Flux Affects Cell Bioenergetics in Alzheimer's Disease-Related Models. Cell Rep 2020, 30 (7) : 2332-2348 e2310. Riedel A, Helal M, Pedro L, Swietlik JJ, Shorthouse D, Schmitz W , et al. Tumor-Derived Lactic Acid Modulates Activation and Metabolic Status of Draining Lymph Node Stroma. Cancer Immunol Res 2022, 10 (4) : 482-497. Additional Declarations (Not answered) Supplementary Files Supplementaldata1.docx Supplementaldata2.pdf Cite Share Download PDF Status: Published Journal Publication published 28 Jan, 2025 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 20 Sep, 2024 Review # 2 received at journal 14 Sep, 2024 Review # 1 received at journal 29 Aug, 2024 Reviewer # 2 agreed at journal 26 Aug, 2024 Reviewer # 1 agreed at journal 18 Aug, 2024 Reviewers invited by journal 18 Aug, 2024 Submission checks completed at journal 12 Aug, 2024 Editor assigned by journal 11 Aug, 2024 First submitted to journal 11 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-4896824","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":341626405,"identity":"91017ed1-f231-4bea-a42a-4173de21858d","order_by":0,"name":"Biao Yan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIie3RMQ6CMBSA4WdMYCG61kHxCBgSNIHDtAtd0NmBoS6Murp5BY5Q0wQXCCuj7g5OxolYoow2uJnYf3pN+qVtCqDT/WYGYIAJAG8W/e7EBRDfEBlhr90diHMqsvM5DuhhU2YI1j5hZsHVJF9RB2fhMuXCQJBTwqwVVhMeeQgbYpmCJL1EEIYsR03KqyS1oDZrSN2FVPIUkggMzcV6rAMZVde5Q7bhTL7FXeCMuokVqcmgjLzL4x7Y9v54qW6xP96ZuZpMeTshOWF4f5Mqm7XTkH3epdPpdP/dE9BQSGuFVM7mAAAAAElFTkSuQmCC","orcid":"","institution":"Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Biao","middleName":"","lastName":"Yan","suffix":""},{"id":341626406,"identity":"9a47594e-b79e-4452-885a-eac3bf1615e1","order_by":1,"name":"Fan Ye","email":"","orcid":"","institution":"Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Fan","middleName":"","lastName":"Ye","suffix":""},{"id":341626407,"identity":"915df2d0-d50b-4623-8dcd-749d1f29ad1e","order_by":2,"name":"Ziran Zhang","email":"","orcid":"","institution":"The Affiliated Eye Hospital, Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ziran","middleName":"","lastName":"Zhang","suffix":""},{"id":341626408,"identity":"3ca8b6ea-5b12-4c72-a402-31132cc94d81","order_by":3,"name":"Lianjun Shi","email":"","orcid":"","institution":"The Affiliated Eye Hospital, Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lianjun","middleName":"","lastName":"Shi","suffix":""},{"id":341626409,"identity":"eb87cdf2-b1ca-4356-9160-3ef217e8178e","order_by":4,"name":"Wan Mu","email":"","orcid":"","institution":"Eye \u0026 ENT Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Wan","middleName":"","lastName":"Mu","suffix":""},{"id":341626410,"identity":"155392aa-80eb-4b46-bbfb-7fffbef9a133","order_by":5,"name":"Shuting Lu","email":"","orcid":"","institution":"The Affiliated Eye Hospital, Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuting","middleName":"","lastName":"Lu","suffix":""},{"id":341626411,"identity":"81ef6f62-0d94-4d0c-b371-21f5a3504655","order_by":6,"name":"Xiumiao Li","email":"","orcid":"","institution":"The Affiliated Eye Hospital, Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiumiao","middleName":"","lastName":"Li","suffix":""},{"id":341626412,"identity":"70b1b6b7-5d67-4f24-9d0e-ae3e4764ec1a","order_by":7,"name":"qin jiang","email":"","orcid":"","institution":"nanjing medical school","correspondingAuthor":false,"prefix":"","firstName":"qin","middleName":"","lastName":"jiang","suffix":""}],"badges":[],"createdAt":"2024-08-12 00:10:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4896824/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4896824/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-025-07366-w","type":"published","date":"2025-01-28T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":64452545,"identity":"6829f050-1ce3-4932-a809-84cbd17c8aac","added_by":"auto","created_at":"2024-09-13 10:46:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":369867,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003etsRNA-0032 expression is down-regulated during inflammatory stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) qRT-PCR assays were performed to measure tsRNA-0032 expression levels in the corneas of C57BL/6 mice from the untreated group (Ctrl) and 3-day, 1-week, and 2-week suture group (n = 6). (B) qRT-PCR assays were used to compare tsRNA-0032 expression levels in HLECs exposed to inflammatory stress (LPS, 100 ng/ml) for 6, 12, and 24 h, or left untreated (Ctrl) (n = 4). (C and D) HLECs were transfected with ANG siRNA 1-3, Dicer siRNA 1-3, Scr siRNA as a negative control, or left untreated (Ctrl) for 24 h. qRT-PCR assays were conducted to detect ANG and Dicer expression levels (n = 4). (E-H) HLECs were transfected with ANG siRNA2/3, Dicer siRNA2/3, Scr siRNA as a negative control, or left untreated (Ctrl), and incubated without or without LPS (100 ng/mL) for 24 h. qRT-PCR assays were conducted to measure tsRNA-0032 expression levels (n = 4). Significant differences were evaluated by one-way ANOVA followed by a post hoc Bonferroni test. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 versus the Ctrl group.\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4896824/v1/85f4e5197bd2cfe60e32ac22.jpg"},{"id":64452550,"identity":"8b8ebbac-74b9-44cf-bdd2-c335b10776d1","added_by":"auto","created_at":"2024-09-13 10:46:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":797290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003etsRNA-0032 inhibits lymphatic endothelial cell function\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vitro\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A-F) HLECs were transfected with negative control (NC) mimic, tsRNA-0032 mimic, NC inhibitor, tsRNA-0032 inhibitor or left untreated (Ctrl) for 6 h and then exposed to LPS (100 ng/ml) for 12 h. The expression levels of tsRNA-0032 were determined by qRT-PCRs (A, n = 4). Cell viability was detected by CCK-8 assay (B, n = 4). Cell proliferation was detected by EdU staining. EdU, red; DAPI, blue. Scale bar, 20 μm (C, n = 4). Cell migration was detected by transwell assays. Scale bar, 20 μm (D, n = 4). Tube formation ability was detected by Martigel assays. Scale bar, 100 μm (E, n = 4). Cell sprouting ability was detected by spheroid sprouting assays. Scale bar, 100 μm (F, n = 4). The significant difference was determined by one-way ANOVA followed by Bonferroni’s post hoc test. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 versus Ctrl group.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4896824/v1/210be280003be92b57b807da.jpg"},{"id":64453223,"identity":"a032a7a2-31c5-42b3-9034-49ede8f291d8","added_by":"auto","created_at":"2024-09-13 10:54:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":430838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003etsRNA-0032 inhibits pathological lymphangiogenesis\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Six-to-eight-week-old C57BL/6 mice were transfected with a negative control agomir (NC agomir), tsRNA-0032 agomir, NC antagomir, or tsRNA-0032 antagomir using subconjunctival injection, or left untreated (Ctrl). qRT-PCR was performed to determine tsRNA-0032 expression levels (n = 5, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, one-way ANOVA followed by post hoc Bonferroni test). (B) C57BL/6 mice received corneal sutures, which were left in place for 7 days. Neolymphatic vessels in the flat-mounted cornea were visualized by the fluorescence signal of LYVE-1 (n = 5). Scale bar: 200 μm. (C) Quantification of neolymphatic vessel coverage area and total length was conducted (n = 5, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, one-way ANOVA followed by post hoc Bonferroni test, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 versus Ctrl group).\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4896824/v1/8780b0bb08432cd8c4af9303.jpg"},{"id":64453738,"identity":"89a93fb1-19f4-4fde-b2f3-270899ed6f64","added_by":"auto","created_at":"2024-09-13 11:02:30","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":555878,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003etsRNA-0032 regulates HLEC function by targeting PKM2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization (FISH) assays were conducted to determine the intracellular distribution of tsRNA-0032. Scale bar: 10 μm. (B) Nucleoplasmic separation assays were performed to quantify the intracellular distribution of tsRNA-0032, with 18S rRNA and U6 serving as cytoplasmic and nuclear controls, respectively. (C) Cellular fractions were isolated from HLECs and immunoprecipitated using Ago2 or IgG antibodies. The amount of tsRNA-0032 in the immunoprecipitates was determined by qRT-PCRs (n = 4, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Student’s \u003cem\u003et\u003c/em\u003e-test). Western blot assays were used to detect the specific interaction between tsRNA-0032 and Ago2 (n = 4). (D) qRT-PCR assays were conducted to detect mRNA expression levels of PKM2 and FASN in HLECs transfected with a negative control (NC) mimic, tsRNA-0032 mimic, NC inhibitor, tsRNA-0032 inhibitor, or left untreated (Ctrl) (n = 4, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, one-way ANOVA followed by Bonferroni test). (E and F) Western blots were performed to detect protein expression levels of PKM2 and FASN in HLECs transfected with NC mimic, tsRNA-0032 mimic, NC inhibitor, tsRNA-0032 inhibitor, or left untreated (Ctrl) (n = 4, one-way ANOVA followed by Bonferroni’s post hoc test, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 versus Ctrl group). (G) Nucleobase complementary alignment between tsRNA-0032 and PKM2 is shown. (H) The luciferase activity of WT-Luc-PKM2 or mutant Luc-PKM2 was determined after transfection with tsRNA-0032 mimic or NC mimic in HLECs (n = 4, Student’s \u003cem\u003et\u003c/em\u003e-test, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 versus Ctrl group). (I) The 3ʹ-end biotinylated tRF-0032 or biotinylated miR-484 was transfected into HLECs. After streptavidin capture, the levels of PKM2 and GAPDH in the input and bound fractions were detected by qRT-PCRs (n = 4, Student’s \u003cem\u003et\u003c/em\u003e-test, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 versus Ctrl group).\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4896824/v1/2f4a0534f69303de7f703b25.jpg"},{"id":64452548,"identity":"3362e88b-9b37-44ef-bc62-bbb26431d3aa","added_by":"auto","created_at":"2024-09-13 10:46:30","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":556045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003etsRNA-0032/PKM2 signaling axis alters glycolysis in HLECs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHLECs were transfected with a negative control (NC) mimic, tsRNA-0032 mimic, tsRNA-0032 plus PKM2 null vector, or tsRNA-0032 plus PKM2 overexpression vector. The Seahorse XFe96 Analyzer was used to measure the extracellular acidification rate (ECAR) of HLECs (A). Glycolysis, glycolytic capacity, and glycolytic reserve were calculated from the ECAR results (B). Pyruvate, lactate, and ATP production in HLECs were detected using pyruvate quantification, lactate quantification, and ATP quantification kits, respectively (C - E, n = 4). Significant differences were evaluated by one-way ANOVA followed by the Bonferroni test. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 between the marked groups.\u003c/p\u003e","description":"","filename":"Figure5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4896824/v1/cd42b75de0227d123657cd7a.jpg"},{"id":64453224,"identity":"05af0a4b-c06a-4a16-8e0f-d683c44be45a","added_by":"auto","created_at":"2024-09-13 10:54:30","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":733099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003etsRNA-0032/PKM2 signaling axis is involved in lymphangiogenesis \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHLECs were transfected with a negative control (NC) mimic, tsRNA-0032 mimic, tsRNA-0032 plus PKM2 null vector, tsRNA-0032 plus PKM2 overexpression vector, or exposed to shikonin for 6 h and then exposed to LPS (100 ng/mL) for 12 h. Cell proliferation was detected by EdU staining. EdU, red; DAPI, blue. Scale bar, 20 μm (A, n = 4). Cell migration was detected by Transwell assays. Scale bar, 20 μm (B, n = 4). Tube formation ability was detected by Matrigel assays. Scale bar, 100 μm (C, n = 4). Sprouting ability was detected by spheroid sprouting assays. Scale bar, 100 μm (D, n = 4). The significant difference was evaluated by one-way ANOVA followed by Bonferroni test. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 versus NC mimic group, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 between the marked groups.\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4896824/v1/6f5620e8be585556b51cd1a4.jpg"},{"id":64452546,"identity":"bf539f00-5384-4ea7-a23a-453555591cec","added_by":"auto","created_at":"2024-09-13 10:46:30","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":391728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003etsRNA-0032/PKM2 signaling axis is involved in lymphangiogenesis \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Six-to-eight-week-old C57BL/6 mice were transfected with a negative control (NC) agomir, tsRNA-0032 agomir, tsRNA-0032 plus PKM2 null vector, tsRNA-0032 plus PKM2 overexpression vector, or exposed to shikonin and then received corneal sutures. Neolymphatic vessels in the flat-mounted cornea were visualized by the fluorescence signal of LYVE-1 (n = 5). Scale bar, 200 μm. (B) Quantification of neolymphatic vessel coverage area and total length was performed (n = 5, one-way ANOVA followed by post hoc Bonferroni test, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 versus Ctrl group).\u003c/p\u003e","description":"","filename":"Figure7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4896824/v1/c4192ff81859f0ff8bf3bb7e.jpg"},{"id":64452552,"identity":"f668a670-7fb5-473b-b3e2-33a8a49c4e49","added_by":"auto","created_at":"2024-09-13 10:46:30","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":142106,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical relevance of tsRNA-0032-mediated signaling in corneal lymphangiogenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) qRT-PCR assays were performed to detect the expression of tsRNA-0032 in donors and recipients of corneal allograft transplantation (n = 15). (B) qRT-PCR assays were conducted to compare the expression of tsRNA-0032 between donors and recipients of corneal allograft transplantation (n = 15). Significant differences were evaluated using Student’s \u003cem\u003et\u003c/em\u003e-test. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 versus the donor group.\u003c/p\u003e","description":"","filename":"Figure8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4896824/v1/5d9250e13be7e765386a844f.jpg"},{"id":74905059,"identity":"8354b2c3-fa96-4182-8010-d4ee87906453","added_by":"auto","created_at":"2025-01-28 08:07:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5153357,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4896824/v1/a9fa58e1-d4cf-4492-a5ad-07f340f5046a.pdf"},{"id":64452554,"identity":"ccf145ad-3432-43f1-b75c-d5d09a69f94d","added_by":"auto","created_at":"2024-09-13 10:46:30","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1232013,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaldata1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4896824/v1/43e7644397f2a1499f04ca6b.docx"},{"id":64452555,"identity":"0cf71c23-fa0a-41bb-9f93-78892af103b0","added_by":"auto","created_at":"2024-09-13 10:46:30","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":11365417,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaldata2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4896824/v1/e65776ba0de0a6fa15a91cb1.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Targeting glycolytic reprogramming by tsRNA-0032 for treating pathological lymphangiogenesis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe lymphatic vessel system is an important component of circulatory system, working in concert with vascular system to maintain fluid balance, support immune function, and facilitate lipid absorption \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Lymphangiogenesis, the process of generating new lymphatic vessels, is primarily controlled by lymphatic endothelial cells (LECs). LECs typically maintain a quiescent state except during embryonic development \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, under pathological conditions such as tumor metastasis \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, cardiovascular diseases \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, and various inflammatory diseases, LECs are activated, stimulating the proliferation, migration, and subsequent formation of new lymphatic vasculature. Within the ocular environment, dysfunctional lymphatic vessels can contribute to keratitis \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, dry eye \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, and transplantation rejection \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite extensive research, our comprehension of lymphangiogenesis remains incomplete, and clinical interventions targeting neoplastic lymphatic vessels are severely limited. Current therapeutic approaches primarily focus on inhibiting VEGFR3 \u003csup\u003e8\u003c/sup\u003e or targeting LEC-autonomous activation of mutant pathways \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e through the use of rapamycin, gene therapy, or surgical resection. The mTOR inhibitor, rapamycin, has been a cornerstone in organ transplantation for many years, primarily because of its potent immunosuppressive properties \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, long-term efficacy and adverse effects remain poorly characterized, and the complex interplay of lymphatic biology necessitates a nuanced approach when considering lymphangiogenesis as a therapeutic target for inflammatory and immune-related diseases. Thus, a deeper understanding of lymphangiogenesis is imperative for developing innovative and comprehensive treatment modalities for lymphangiogenesis-associated diseases.\u003c/p\u003e \u003cp\u003eThe occurrence and progression of pathological lymphangiogenesis require a complex interplay of various cytokines and growth factors. Existing studies have shown that small extracellular vesicle (sEVs) \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, pro-inflammatory cytokines \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, ROS \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and various non-coding RNAs can regulate lymphangiogenesis. sEVs play a crucial role in the transport and distribution within lymphatic system. When sEVs are injected subcutaneously into the tail base and tumor tissues, they preferentially accumulate in lymph nodes. Pro-inflammatory cytokines and reactive oxygen species (ROS) promote lymphangiogenesis by regulating the proliferation and migration of lymphatic endothelial cells. Non-coding RNAs can regulate the function of lymphatic endothelial cells and lymphangiogenesis process by regulating gene expression.\u003c/p\u003e \u003cp\u003etRNA-derived small RNAs (tsRNAs) have emerged as a novel class of small non-coding RNAs, arising from specific cleavage of precursor or mature tRNAs. These cleavage events are predominantly triggered by stress conditions, such as amino acid deficiency, phosphate starvation, UV radiation, heat shock, hypoxia, oxidative damage, and viral infection. The production of tsRNAs is notably limited under normal growth conditions, indicating their role as tRNA-derived stress-induced RNAs. This suggests that tsRNAs may play crucial roles in the cellular response to stress, potentially regulating gene expression and maintaining cellular homeostasis during adverse conditions \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Emerging evidence underscores the capacity of tsRNAs to modify RNAs and interact with proteins, thereby exerting significant influence over diverse biological processes including gene silencing, ribosome biogenesis, and epigenetic regulation \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Increasing studies have revealed the involvement of tsRNAs in the progression of various diseases, such as tumor lymphatic metastasis, systemic inflammation, and lipid absorption disorders \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Given the established link between tsRNAs and angiogenesis \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, it is imperative to investigate their potential regulatory function in lymphangiogenesis. A comprehensive understanding of this relationship may unveil novel insights into disease pathogenesis and inform the development of innovative therapeutic strategies.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the role of tsRNA-0032, a specific cleavage product of tRNA-GTG-His, in lymphangiogenesis. Our findings demonstrate that tsRNA-0032 expression is downregulated during lymphangiogenesis, while its upregulation attenuates lymphangiogenesis and suppresses LEC hyperactivation under inflammatory conditions. Mechanistically, tsRNA-0032 regulates lymphangiogenesis by modulating glycolysis. These findings position tsRNA-0032 as a potential regulator of LEC dysfunction, warranting further investigation into its therapeutic potential for dysregulated lymphangiogenesis.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiment\u003c/h2\u003e \u003cp\u003eAnimals were obtained from Nanjing Junke Bioengineering Corporation and treated in accordance with the Association for Research in Vision and Ophthalmology\u0026rsquo;s guidelines for the use of animals in ophthalmic and vision research. All procedures were approved by the Animal Experiment Management Committee of the author\u0026rsquo;s institute.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCorneal suture model\u003c/h2\u003e \u003cp\u003eA corneal lymphangiogenesis model was established to induce inflammation and stimulate lymphatic vessel growth towards the corneal center. Male C57BL/6J mice (6\u0026ndash;8 weeks old) underwent anesthesia with xylazine (10 mg/kg) and ketamine (100 mg/kg) administered intraperitoneally. Pupil dilation was achieved using phenylephrine and tropicamide eye drops. Three interrupted 11\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon sutures were placed in the corneal stroma near the limbus, spaced 120 degrees apart. Gatifloxacin ointment was applied topically to prevent infection. Corneas were harvested seven days post-surgery for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and transfection\u003c/h2\u003e \u003cp\u003ePrimary human lymphatic endothelial cells (HLECs; Promocell, Germany, C-12216) were cultured in Endothelial Cell Basal Medium MV (Promocell, Germany, C-22220) supplemented with 12% fetal bovine serum (ScienCell, USA) and penicillin/streptomycin (100 U/mL, 100 \u0026micro;g/mL; Gibco, USA) in a humidified incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. tsRNA-0032 mimics, inhibitors, and their respective negative controls (NCs) were designed and synthesized by GENERAY (China). HLECs were transfected with mimics or inhibitors using Lipofectamine 3000 (Invitrogen, USA, L3000015) according to the manufacturer\u0026rsquo;s protocol when cells reached approximately 80% confluence.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFluorescence\u003c/b\u003e \u003cb\u003ein situ\u003c/b\u003e \u003cb\u003ehybridization (FISH)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe cellular localization of tsRNA-0032 in HLECs was determined by FISH assay. Cy3-labeled probes, 5\u0026rsquo;-CGAACCGAGGTTGCTGCGGCC-3\u0026rsquo;, specific to tsRNA-0032 were designed and synthesized by Servicebio (Wuhan, China). The signals of the probes were detected by a Fluorescent \u003cem\u003eIn Situ\u003c/em\u003e Hybridization Kit (Servicebio, China) according to the manufacturer\u0026rsquo;s instructions. Images were captured by a fluorescence microscope (Olympus, Japan)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eNucleoplasmic separation assay\u003c/h2\u003e \u003cp\u003eNuclear and cytoplasmic RNAs in HLECs were isolated using the Cytoplasmic and Nuclear RNA Purification Kit (NORGEN BIOTEK, Canada, 21000) according to the manufactures\u0026rsquo; instruction. The collected RNAs were reverse transcribed into cDNAs, followed with qRT-PCR analysis. 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method was used to analyze the relative expression levels of genes in the nuclear and cytoplasmic fractions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRNA immunoprecipitation (RIP)\u003c/h2\u003e \u003cp\u003eRIP was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA, 17\u0026ndash;701) according to the manufacturer's protocol. Antibodies targeting Ago, PKM2, or IgG were incubated with magnetic beads overnight at 4\u0026deg;C. Following immunoprecipitation, co-precipitated RNA was extracted and subjected to qRT-PCR analysis to detect the expression of tsRNA-0032 and PKM2, thereby confirming the enrichment of target RNAs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase reporter assay\u003c/h2\u003e \u003cp\u003eHLECs were seeded into 96-well plates and cultured in complete medium until reaching 60\u0026ndash;70% confluence. Cells were co-transfected with either Luc-PKM2 WT, Luc-PKM2 Mut, or an empty vector, along with tsRNA-0032 mimic or a negative control mimic using Lipofectamine 3000 (Invitrogen, USA, L3000015). After a 24-hour incubation, firefly and Renilla luciferase activities were measured in each group using a luciferase reporter assay kit (Promega, USA, E1910).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of extracellular acidification rate (ECAR)\u003c/h2\u003e \u003cp\u003e1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e of HLECs were seeded onto Seahorse XFe96/XF Pro cell culture microplate (Agilent Technologies, 103794-100). They were cultured in XF base medium (pH 7.4) in a non-CO2 incubator at 37℃ for 1 h. Glucose (10 mM), glutamine (1 mM), 2-DG (50 mM), and oligomycin (1 \u0026micro;M) were sequentially added into the plates at specific time points following the manufacturer\u0026rsquo;s guidelines. ECAR was collected using Seahorse XFe96 Analyzer (Agilent Technologies) and analyzed using the Seahorse XFe96 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of pyruvate and lactate\u003c/h2\u003e \u003cp\u003e1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e of HLECs were seeded onto 6-well plate and received different treatment respectively. Pyruvate and lactate levels were quantified using the pyruvate assay kit (Jiancheng Bioengineering Institute, China, A081\u0026ndash;1\u0026ndash;1) and lactate assay kit (Jiancheng Bioengineering Institute, China, A019-2-1). Absorbance was measured using the Multickan Skyhigh Microplate Reader (ThermoFisher Scientific, USA, A51119700DPC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eUnpaired Student\u0026rsquo;s t-tests were used to compare differences between two groups, while one-way analysis of variance (ANOVA) was employed for comparisons among multiple groups. Normal distribution of data was verified prior to ANOVA analysis. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003etsRNA-0032 expression is down-regulated during inflammatory stress\u003c/h2\u003e \u003cp\u003eNormal lymphatic vessels are confined to the conjunctiva and corneal limbus. However, pathological conditions such as inflammation or trauma can induce lymphatic vessel growth into the corneal center \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. To investigate the relationship between tsRNA-0032 expression and lymphangiogenesis, we employed corneal suture and LPS-induced HLEC models. In a corneal suture mouse model, qRT-PCR analysis revealed a significant downregulation of tsRNA-0032 expression in sutured corneas compared to the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Similarly, LPS-induced HLECs exhibited decreased tsRNA-0032 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Given that tsRNA is generated from tRNA by ANG and Dicer \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, we explored the underlying mechanism of tsRNA-0032 down-regulation. We silenced ANG or Dicer in HLECs under normal condition and LPS-treated condition. The results showed that transfection of ANG siRNA2/3 or Dicer siRNA2/3 led to reduced levels of ANG or Dicer expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). ANG knockdown significantly reduced tsRNA-0032 expression, while Dicer silencing had no effect both under normal condition and LPS-treated condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-H). Taken together, these results suggest that tsRNA-0032 may act as a potential regulator of lymphangiogenesis, and its downregulation in pathological conditions is primarily mediated by ANG.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003etsRNA-0032 inhibits lymphatic endothelial cell function\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the role of tsRNA-0032 in lymphangiogenesis, HLECs were transfected with tsRNA-0032 mimics or inhibitors. Compared with the control group, transfection of tsRNA-0032 mimics significantly increased tsRNA-0032 expression, while transfection of tsRNA-0032 inhibitor decreased it (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Cell viability was assessed using CCK8 assays, which showed that tsRNA-0032 overexpression reduced cell viability, whereas tsRNA-0032 silencing increased it (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). 5-Ethynyl-2\u0026rsquo;-deoxyuridine (EdU) assays demonstrated that tsRNA-0032 overexpression decreased HLEC proliferation, while silencing tsRNA-0032 promoted proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Transwell assays revealed that tsRNA-0032 mimic transfection inhibited HLEC migration, whereas its inhibitor promoted migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Matrigel tube formation and spheroid sprouting assays were conducted to evaluate tube formation and sprouting abilities. tsRNA-0032 mimic transfection reduced tube-like structure formation and sprouting in HLECs, while the inhibitor enhanced these processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results indicate that tsRNA-0032 regulates the biological function of HLECs \u003cem\u003ein vitro.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003etsRNA-0032 inhibits pathological lymphangiogenesis\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the role of tsRNA-0032 in lymphangiogenesis \u003cem\u003ein vivo\u003c/em\u003e, a suture-induced mouse corneal lymphangiogenesis model was employed. tsRNA-0032 agomir or antagomir was injected into the conjunctival sac to regulate tsRNA-0032 expression, as confirmed by qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Up-regulation of tsRNA-0032 via agomir injection reduced corneal lymphangiogenesis, while down-regulation via antagomir injection increased it, as assessed by LYVE-1 immunofluorescence staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003etsRNA-0032 regulates HLEC function via targeting PKM2\u003c/h2\u003e \u003cp\u003eTo elucidate the mechanism underlying tsRNA-0032-mediated lymphangiogenesis, we investigated the subcellular localization of tsRNA-0032 in HLECs using fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization (FISH). Results demonstrated that tsRNA-0032 is predominantly localized to the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNucleo-cytoplasmic fractionation assays further confirmed the predominantly cytoplasmic localization of tsRNA-0032 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Given that Ago2 is a key protein involved in post-transcriptional gene regulation mediated by tsRNAs \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, we conducted RIP assays to determine whether tsRNA-0032 exerts its biological role by binding to Ago2. RIP results demonstrated that Ago2, but not the negative control IgG, immunoprecipitated tsRNA-0032 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Utilizing the tRFTar database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rnanut.net/tRFTar/\u003c/span\u003e\u003cspan address=\"http://www.rnanut.net/tRFTar/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), we then analyzed potential downstream pathways and target genes of the tsRNA-0032/Ago2 complex. The database predicted that tsRNA-0032 regulates cellular glycolysis by targeting PKM2 and regulates cell fatty acid synthesis by targeting fatty acid synthase (FASN).\u003c/p\u003e \u003cp\u003eTo further identify the target gene of tsRNA-0032 in HLECs, qRT-PCR and Western blot analyses were performed. tsRNA-0032 overexpression significantly decreased PKM2 mRNA and protein levels, while its knockdown exhibited the opposite effect. In contrast, FASN mRNA and protein levels remained unaffected by tsRNA-0032 modulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F).\u003c/p\u003e \u003cp\u003eBase pairing complementarity between tsRNA-0032 and PKM2 was confirmed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Luciferase reporter assays demonstrated that tsRNA-0032 overexpression significantly reduced the luciferase activity of the wild-type PKM2 3'-UTR, but not its mutant form, indicating a direct interaction between tsRNA-0032 and the PKM2 3'-UTR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Furthermore, RNA pull-down assays revealed that PKM2 was specifically enriched in the tsRNA-0032-bound fraction compared to the miR-484 control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Collectively, these findings establish that tsRNA-0032 regulates HLEC function by directly targeting PKM2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003etsRNA-0032/PKM2 signaling axis alters glycolysis in HLECs\u003c/h2\u003e \u003cp\u003ePKM2 is the rate-limiting enzyme in the final step of glycolysis, catalyzing the conversion of phosphoenolpyruvate (PEP) to pyruvate \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. tsRNA-0032/PKM2 signaling axis regulates lymphatic endothelial cell function by modulating cellular glycolysis. To assess glycolytic activity, extracellular acidification rate (ECAR) was measured in HLECs using a Seahorse XF extracellular flux analyzer. tsRNA-0032 overexpression suppressed glycolysis, glycolytic capacity, and glycolytic reserve, while PKM2 overexpression partially rescued this inhibitory effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Conversely, tsRNA-0032 knockdown enhanced glycolytic activity, which was attenuated by PKM2 silencing (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, S1B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further validate the impact of tsRNA-0032 on glycolysis, pyruvate and lactate production were quantified. tsRNA-0032 overexpression decreased pyruvate and lactate levels, effects partially reversed by PKM2 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Conversely, tsRNA-0032 knockdown increased pyruvate and lactate production, an effect reversed by PKM2 silencing (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC, S1D). Given the central role of glycolysis in ATP production, ATP levels were measured. tsRNA-0032 overexpression decreased ATP production, rescued by PKM2 overexpression, while tsRNA-0032 knockdown increased ATP production, attenuated by PKM2 silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eE). To assess the specificity of tsRNA-0032's effect on glycolysis, the expression of other key glycolytic enzymes, HK2 and PFKFB3, was evaluated; no significant changes were observed (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eF). Collectively, these findings demonstrate that the tsRNA-0032/PKM2 signaling axis specifically regulates glycolysis in HLECs.\u003c/p\u003e \u003cp\u003e \u003cb\u003etsRNA-0032/PKM2 signaling axis is involved in lymphangiogenesis\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo confirm whether tsRNA-0032/PKM2 signaling axis is involved in lymphangiogenesis, we explored whether PKM2 alone could affect lymphangiogenesis \u003cem\u003ein vitro\u003c/em\u003e. PKM2 pcDNA3.1 vector and PKM2 siRNA were used to modulate the expression level of PKM2, and the efficiency of interventions was demonstrated by qRT-PCR assays and western blots (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA and S2B). PKM2 overexpression promoted HLECs proliferation, migration, tube formation and sprouting activity, while PKM2 silencing reduced these cellular functions (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC-S2F).\u003c/p\u003e \u003cp\u003eTo investigate whether modulating PKM2 expression could reverse lymphatic system dysfunction induced by tsRNA-0032, we employed shikonin, a specific inhibitor of PKM2 activity with no reported effects on PKM1 or PKL \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. CCK-8 assay determined that 1 \u0026micro;M shikonin was non-toxic to HLECs (Fig. S3A). To investigate the functional rescue of tsRNA-0032-mediated HLEC dysfunction, PKM2 was overexpressed. PKM2 overexpression effectively reversed the inhibitory effects of tsRNA-0032 on HLEC proliferation, migration, tube formation, and sprouting (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D). These findings collectively indicate that the tsRNA-0032/PKM2 signaling axis plays a critical role in regulating lymphatic endothelial cell function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003etsRNA-0032/PKM2 signaling axis is involved in regulating lymphangiogenesis\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the \u003cem\u003ein vivo\u003c/em\u003e functional significance of tsRNA-0032/PKM2 signaling axis in lymphangiogenesis, a corneal suture model was established. Injection of tsRNA-0032 agomir into the conjunctival sac led to a reduction in corneal lymphangiogenic area, an effect phenocopied by treatment with shikonin, a specific PKM2 inhibitor. Conversely, overexpression of PKM2 through vector-mediated gene delivery effectively counteracted the anti-lymphangiogenic effects induced by tsRNA-0032 agomir, as evidenced by the increased formation and length of new lymphatic vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). These findings collectively underscore the critical role of tsRNA-0032/PKM2 signaling axis in regulating corneal lymphangiogenesis and suggest its potential as a therapeutic target for this pathological condition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eClinical relevance of tsRNA-0032-mediated signaling in corneal lymphangiogenesis\u003c/h2\u003e \u003cp\u003eKeratitis, an inflammation of the cornea, is frequently accompanied by the abnormal growth of lymphatic vessels, a condition known as corneal lymphangiogenesis \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. To investigate the potential clinical implications of tsRNA-0032/PKM2 signaling axis in this context, corneal tissue samples were collected from both keratitis patients (recipients) and healthy individuals (donors). qRT-PCR analysis revealed a marked downregulation of tsRNA-0032 and a concomitant upregulation of PKM2 in the corneal tissue of keratitis patients compared to healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). These findings strongly suggest a correlation between the dysregulation of the tsRNA-0032/PKM2 signaling axis and the development of corneal lymphangiogenesis in keratitis patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAlthough physiological lymphangiogenesis is rare in healthy adults, pathological lymphangiogenesis plays a significant role in various disease processes, including inflammation, lymphedema, organ transplant rejection, tumor metastasis, and cardiovascular disease \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In inflammatory diseases such as rheumatoid arthritis, inflammation-induced lymphangiogenesis regulates fluid drainage, immune cell migration, and the removal of inflammatory mediators, ultimately accelerating inflammation resolution \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Conversely, following organ transplantation, lymphangiogenesis can trigger immune system reactivation in draining lymph nodes, potentially leading to organ rejection \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In cancer, tumor cells spread to lymph nodes via the lymphatic system, and inhibiting tumor lymphangiogenesis and lymph node metastasis in animal models effectively impedes tumor progression \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. A common feature among these lymphangiogenesis-related diseases is the activation of the VEGF-C/VEGFR3 pathway \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. While therapies targeting the primary regulatory pathways of lymphangiogenesis, similar to antiangiogenic drugs targeting VEGF, appear promising for treating these diseases, they have not fully met expectations. Therefore, it is crucial to elucidate the mechanisms of lymphangiogenesis in detail and explore alternative treatment strategies.\u003c/p\u003e \u003cp\u003eAs a novel class of non-coding RNAs, tsRNAs have emerged as critical regulators of diverse cellular processes and are increasingly recognized for their involvement in a wide range of physiological and pathological conditions \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. For example, tsRNA-04002 alleviates intervertebral disc degeneration by targeting PRKCA to inhibit apoptosis of nucleus pulposus cells \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Sunyang Ying et al. demonstrated tRF-Gln-CTG-026 ameliorates liver injury by alleviating global protein synthesis. Angiogenin-mediated tsRNAs control inflammation and metabolic disorder by regulating NLRP3 inflammasome \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. We previously found that tRNA-Cys-5-0007 plays a dual role of anti-angiogenesis and anti-inflammatory in ocular vascular disease \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. This study focused on the functional characterization of tsRNA-0032, a tRNA-derived small RNA, in the context of lymphangiogenesis. Our findings revealed a consistent downregulation of tsRNA-0032 expression in both suture-induced murine corneal and LPS-stimulated HLEC models. Gain- and loss-of-function experiments demonstrated that tsRNA-0032 overexpression inhibits, while knockdown enhances, lymphangiogenesis, suggesting a suppressive role for tsRNA-0032 in this process.\u003c/p\u003e \u003cp\u003eLymphangiogenesis, the formation of new lymphatic vessels from pre-existing lymphatic networks, is a critical process mediated by LECs. These specialized endothelial cells, residing within the lymphatic vasculature, undergo proliferation, migration, and tube formation in response to specific stimuli to drive lymphatic vessel growth. Characterized by a unique metabolic profile, LECs exhibit a strong reliance on aerobic glycolysis to fuel their energy-intensive functions \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This metabolic phenomenon, known as the Warburg effect, is characterized by an increased reliance on glycolysis for ATP production even in the presence of oxygen. While seemingly less efficient than oxidative phosphorylation in terms of ATP yield, this metabolic shift is advantageous for rapidly proliferating cells. By channeling glucose towards glycolysis, cells can efficiently allocate resources for macromolecular biosynthesis while simultaneously mitigating oxidative stress through reduced ROS production \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Upon activation and transition from a quiescent to a proliferative and migratory state, HLECs exhibit a marked increase in glycolytic flu \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Impaired glycolysis profoundly compromises cellular function, as evidenced by various pathological conditions. For instance, podocyte-specific PKM2 deletion exacerbates angiotensin II-induced glomerular and podocyte injury, characterized by foot process effacement and proteinuria \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Similarly, glycolytic dysfunction in natural killer cells accelerates lung cancer progression \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. As the pivotal enzyme catalyzing phosphoenolpyruvate conversion to pyruvate, PKM2 plays a crucial role in glycolysis \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Our study demonstrates that tsRNA-0032 regulates glycolysis by targeting PKM2. tsRNA-0032 overexpression inhibits glycolysis, leading to reduced pyruvate and lactate production, essential metabolites for cellular energy metabolism \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. These metabolic alterations ultimately impair lymphatic endothelial cell function and suppress lymphangiogenesis. Notably, dysregulated pyruvate and lactate levels are implicated in various diseases, including Alzheimer's disease and tumor-draining lymph nodes \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Pyruvate and lactate kit demonstrated that tsRNA-0032 upregulation decreased pyruvate and lactate production, while tsRNA-0032 downregulation increased their production. Therefore, tsRNA-0032 influenced energy supply and the balance between metabolite production and consumption via targeting at PKM2, thus regulating lymphangiogenesis.\u003c/p\u003e \u003cp\u003eTo further corroborate the clinical relevance of the tsRNA-0032/PKM2 axis in lymphangiogenesis, we examined tsRNA-0032 and PKM2 expression levels in corneal tissues obtained from keratitis patients who had undergone corneal transplantation. Compared to corneal samples from corresponding healthy donors, those from keratitis patients exhibited significantly reduced tsRNA-0032 levels and concomitantly elevated PKM2 levels. These findings provide compelling evidence for the involvement of the tsRNA-0032/PKM2 signaling pathway in the pathogenesis of corneal lymphangiogenesis and suggest that targeting this pathway may offer a promising therapeutic strategy for the management of lymphangiogenesis-related ocular diseases.\u003c/p\u003e \u003cp\u003eIn conclusion, this study unveiled a critical role for tsRNA-0032 in regulating lymphatic endothelial cell function and subsequent lymphangiogenesis. Our findings demonstrate that tsRNA-0032 inhibits lymphatic endothelial cell proliferation, migration, tube formation, and sprouting in vitro. Furthermore, in vivo models of corneal suture-induced and subcutaneous Matrigel plug-induced lymphangiogenesis confirmed the inhibitory effects of tsRNA-0032 on lymphatic vessel formation. Mechanistically, we revealed that tsRNA-0032 interacts with Ago2 and directly targets PKM2, a key glycolytic enzyme, to suppress glycolytic reprogramming in lymphatic endothelial cells. These results collectively highlight the therapeutic potential of targeting the tsRNA-0032/PKM2 axis for the treatment of lymphangiogenesis-related diseases.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data from this study are available from the authors upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was generously supported by the grants from the National Natural Science Foundation of China (no.81770945 to Dr Yan; no. 81570859 and 82070983 to Dr Jiang).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATION OF INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB.Y. and Q.J. designed research; F.Y., Z.R.Z., L.J.S., S.T.L., and W.M. performed research; F.Y. and Z.R.Z. analyzed data; F.Y. and B.Y. wrote the article.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHu Z, Zhao X, Wu Z, Qu B, Yuan M, Xing Y\u003cem\u003e, et al.\u003c/em\u003e Lymphatic vessel: origin, heterogeneity, biological functions, and therapeutic targets. \u003cem\u003eSignal Transduct Target Ther\u003c/em\u003e 2024, \u003cstrong\u003e9\u003c/strong\u003e(1)\u003cstrong\u003e:\u003c/strong\u003e 9.\u003c/li\u003e\n\u003cli\u003eTammela T, Alitalo K. 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Disruption of this process is implicated in diseases such as cancer, inflammation, and autoimmune disorders. In this study, we elucidate the role of tsRNA-0032 in lymphangiogenesis and its molecular mechanisms. tsRNA-0032 expression is significantly diminished in corneal suture and LPS-induced human lymphatic endothelial cell (HLEC) models under inflammatory conditions. Overexpression of tsRNA-0032 suppresses lymphangiogenesis by inhibiting HLEC proliferation, migration, and tube formation. Moreover, overexpression of tsRNA-0032 inhibits suture-induced mouse corneal lymphangiogenesis \u003cem\u003ein vivo\u003c/em\u003e. tsRNA-0032 is mainly found in the cytoplasm and interacts with Ago2 protein. Overexpression of tsRNA-0032 leads to a reduction in ATP production and lowers the levels of pyruvate and lactate by targeting PKM2, which is crucial for the final step of glycolysis. This regulation of glycolysis impacts the cellular energy and metabolic balance in HLECs, contributing to the inhibition of lymphangiogenesis. Clinical data show that tsRNA-0032 levels are markedly lower in corneal tissues from transplant recipients compared to donors, whereas PKM2 expression is elevated, underscoring the clinical significance of the tsRNA-0032/PKM2 axis in corneal lymphangiogenesis. This study provides novel insights into lymphangiogenesis regulation and offers potential therapeutic targets for lymphatic-related diseases.\u003c/p\u003e","manuscriptTitle":"Targeting glycolytic reprogramming by tsRNA-0032 for treating pathological lymphangiogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-13 10:46:25","doi":"10.21203/rs.3.rs-4896824/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-09-20T14:17:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-09-15T02:58:05+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-08-30T01:12:44+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-08-27T02:03:24+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-08-18T11:59:20+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-08-18T06:25:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-12T11:24:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-12T00:06:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2024-08-12T00:06:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"65ea017d-82cb-48c0-b58c-8f24d66885b8","owner":[],"postedDate":"September 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-28T08:07:51+00:00","versionOfRecord":{"articleIdentity":"rs-4896824","link":"https://doi.org/10.1038/s41419-025-07366-w","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2025-01-28 05:00:00","publishedOnDateReadable":"January 28th, 2025"},"versionCreatedAt":"2024-09-13 10:46:25","video":"","vorDoi":"10.1038/s41419-025-07366-w","vorDoiUrl":"https://doi.org/10.1038/s41419-025-07366-w","workflowStages":[]},"version":"v1","identity":"rs-4896824","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4896824","identity":"rs-4896824","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}