SIRT5 Sustains Stemness and Promotes Metastasis and Angiogenesis in Cervical Cancer via Activation of the TGF-β Signaling Pathway

SIRT5 Sustains Stemness and Promotes Metastasis and Angiogenesis in Cervical Cancer via Activation of the TGF-β Signaling Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article SIRT5 Sustains Stemness and Promotes Metastasis and Angiogenesis in Cervical Cancer via Activation of the TGF-β Signaling Pathway Feiting Xie, Hao huang, Xiaojing Chen, Lingfang Wang, Fenfen Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8614743/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: SIRT5, a mitochondrial NAD+‑dependent deacetylase, plays diverse roles in cancer. This study explored its expression, clinical relevance, and functions in cervical cancer. Methods: We systematically analyzed SIRT5 expression in cervical cancer tissues, assessed its correlation with patient survival, and investigated its functional roles through overexpression and knockdown experiments in vitro and in vivo. Results: SIRT5 was significantly overexpressed in cervical cancer tissues and correlated with poor patient survival. Functionally, SIRT5 overexpression enhanced cell migration, invasion, and stemness phenotypes (tube and sphere formation), while its knockdown reversed these effects. Mechanistically, SIRT5 upregulated stemness markers SOX2 and Nanog and activated Smad2/3 phosphorylation. Inhibition of the TGF-β signaling pathway reversed these SIRT5-induced effects. In vivo, SIRT5-overexpressing xenografts showed increased p-Smad2, Nanog, and CD31 expression, promoting metastasis and angiogenesis, but tumor growth remained unchanged. Conclusions: These findings suggest that SIRT5 facilitates cervical cancer metastasis and angiogenesis via TGF-β signaling, identifying it as a potential therapeutic target. cervical cancer SIRT5 TGF-β signaling pathway Tumor stemness angiogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Cervical cancer is the fourth most common malignancy in women worldwide and the second leading cause of cancer-related deaths among women in developing countries. 1 , 2 Annually, approximately 500,000 women are diagnosed with cervical cancer, resulting in over 300,000 deaths globally. 3 , 4 It is well-established that nearly all cervical carcinoma cases (99.7%) are associated with human papillomavirus (HPV) infection, with HPV16 and HPV18—the high-risk carcinogenic genotypes—accounting for approximately 55–60% and 10–15% of all cervical cancers, respectively. 5 Fortunately, the development of vaccines targeting the HPV types most commonly linked to cervical cancer has significantly reduced its global incidence. 6 , 7 Despite substantial advancements in diagnostic techniques and treatments that have improved outcomes for cervical cancer patients, the prognosis for those with regional or distant metastases remains poor. 8 , 9 However, the molecular mechanisms underlying cervical cancer metastasis are not yet fully understood. 10 Sirtuin (SIRT) are a family of class III histone deacetylases that regulate post-translational modifications of protein substrates involved in diverse metabolic pathways in an NAD-dependent manner. 11-13 Among seven members, SIRT1–7, 3 of which (SIRT3–5) are localized primarily in mitochondria. 14-16 SIRT5 functions as an NAD-dependent lysine demalonylase, desuccinylase and deglutarylase that specifically removes malonyl, succinyl and glutaryl groups from target proteins. 17 SIRT5 play complex roles in normal and diseased cells, with gain or loss of the sirt5 gene site occurs regularly across a series of cancers, though often under conditions with non-focal genomic events leading to alterations in plentiful flanking genes over and above SIRT5. 18 , 19 SIRT5 mRNA expression is discoverable across a wide number of cancers, such as colorectal cancer and gastric cancer, and so on. 20-22 A variety of previous studies have shown that SIRT5 expression is heightened in specific tumors relative to the corresponding adjacent or normal tissue type. 23 Recent studies have indicated that SIRT5 could promote the breast cancer tumorigenesis by stabilizing mitochondrial glutaminase, contribute to colorectal cancer growth by regulating T Cell activity, and influence the proliferation, invasion and migration of prostate cancer cells through acetyl-CoA acetyltransferase 1. 24-26 Meanwhile, contradictory findings also exist. Hypoxia-induced suppression of SIRT has been reported to promote the proliferation, migration and invasion of hepatocellular carcinoma cells. 27 , 28 These findings suggest that SIRT5 may act as either an oncogene or a tumor suppressor, depending on the cancer type. 29 Despite these insights, the role of SIRT5 in the malignant progression of cervical cancer remains largely unknown. In this study, SIRT5 protein expression levels were evaluated using tissue microarrays (TMA) containing cervical cancer tissues and adjacent normal tissues, and its clinical correlations were analyzed. 30 To further determine the role of SIRT5 in cervical cancer cell both in vitro and in vivo , stable SIRT5-overexpressing and knockdown cell lines were established using a lentiviral system. Our data indicated that the expression of SIRT5 was upregulated in cervical cancer tissues, and play an important role in regulating of malignant phenotypes such as cell migration, invasion potential, and angiogenesis. Furthermore, SIRT5 appeared to function as an oncogene in cervical cancer by promoting epithelial-mesenchymal transition (EMT) and enhancing the stem-like properties of cervical cancer cells through activation of the TGF-β signaling pathway. To the best of our knowledge, this is the first study to investigate the role of SIRT5 in cervical cancer, providing new insights into its function and offering a potential therapeutic target for the treatment of cervical cancer. Materials and Methods 2.1 Cell culture and stable cell lines generation Human cervical cancer cell lines, Hela and Siha, were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM (Gibco) supplemented with 10% FBS (BI), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained at 37°C in a humidified incubator with 5% CO₂. To generate stable cell lines, lentiviruses for SIRT5 overexpression, knockdown, and corresponding controls were purchased from GeneChem. After infection and GFP expression confirmation, cells were treated with 2 μg/mL puromycin for two weeks to select stable cell lines. Successful SIRT5 overexpression or knockdown was validated via western blotting. 2.2 Immunohistochemistry Cervical cancer tissues and adjacent normal tissues were collected from patients at the Women’s Hospital, School of Medicine, Zhejiang University, with written informed consent. The study was approved by the Institutional Ethics Committee. IHC staining was performed as described previously. 31 SIRT5 staining intensity and percentage of positive cells were scored, and the immunoreactivity score (IRS) was calculated using the formula: IRS = intensity score × quantity score . Staining was categorized as negative (≤3), weak positive (>3 but ≤6), or strong positive (>6). Primary antibodies used for IHC included anti-SIRT5 (1:100, Abcam), anti-Ki67 (1:100, CST), anti-p-Smad2 (1:150, Abcam), anti-Nanog (1:100, CST), and anti-CD31 (1:100, Proteintech). 2.3 Western blotting Cells were lysed in RIPA buffer containing 1% protease inhibitor cocktail (Sigma). Protein samples were separated on 12% or 15% SDS-PAGE gels and transferred onto PVDF membranes (Millipore). Membranes were blocked in 5% milk for 2 hours, incubated overnight with primary antibodies at 4°C, followed by HRP-conjugated secondary antibodies for 2 hours at room temperature. Protein bands were visualized using X-ray film (Fujifilm) after chemiluminescent detection. β-Actin served as the loading control. Primary antibodies included anti-SIRT5, EMT markers (E-cadherin, N-cadherin, Vimentin, β-catenin), stemness markers (SOX2, OCT4, Nanog), and TGF-β signaling proteins (Smad2, p-Smad2, Smad3, p-Smad3). 2.4 Cellular Assays 2.4.1 Cell Proliferation Assay Proliferation was assessed using the MTT assay. Stable cell lines were seeded in 96-well plates (5,000 cells/well) and incubated for 24 and 48 hours before measuring absorbance at 570 nm. 2.4.2 Colony formation assay Stable Hela and Siha cells (500 cells/well) were seeded in 6-well plates and cultured for 14 days. Colonies were fixed with methanol, stained with crystal violet, and counted under a microscope. 2.4.3 Migration and Invasion Assays Cell migration was analyzed using a Transwell system (8 μm pore size, Corning). For invasion assays, inserts were pre-coated with Matrigel (BD). Cells were seeded in serum-free DMEM in the upper chamber, while the lower chamber contained DMEM with 10% FBS. After 24 hours, migrated/invaded cells were fixed, stained with crystal violet, and counted under a microscope. 2.4.4 Tube formation assay Stable Siha cells were seeded on Matrigel-coated wells, and capillary-like structures were observed after 2 hours. Tube lengths were quantified using ImageJ software. 2.4.5 Sphere-formation assay Cells were seeded at 1,000 cells/well in a 24-well plate and cultured in DMEM/F12 medium supplemented with EGF, FGFb, and B27 for 8–10 days. Spheres were imaged under a microscope. 2.5 Immunofluorescence assay for smad2/3 cellular translocation Stable Siha cells overexpressing SIRT5 were seeded in 6-well plates and fixed with 4% formaldehyde. Cells were permeabilized with methanol, blocked with 3% BSA, and incubated with anti-Smad2/3 (1:150, CST) followed by Alexa Fluor 647-conjugated secondary antibody. Nuclei were stained with DAPI, and images were captured using a fluorescent microscope. 2.6 In vivo study Male BALB/c nude mice (4–6 weeks old, 20 g) were purchased from GemPharmatech, China, and housed under standard conditions. Xenografts were established by subcutaneously injecting 5 × 10⁶ Siha cells stably overexpressing SIRT5. Tumor volume was calculated using the formula: V = (a × b²)/2, where "a" is the tumor length and "b" is the width. Tumor tissues were fixed in formalin and embedded in paraffin for IHC analysis. Body weight was monitored to assess treatment toxicity. All animal experiments followed AAALAC and IACUC guidelines. 2.7 Statistical analysis Data are presented as mean ± S.D. from at least three independent experiments, except for animal experiments (mean ± SEM). Statistical analyses were performed using GraphPad Prism 5.0. Student’s t-test was used for comparisons, while survival curves were analyzed with the log-rank test. Correlations between SIRT5 expression and clinicopathological features were assessed using the chi-square test. A P-value < 0.05 (*) was considered statistically significant. Results 3.1 The express ions of SIRT5 protein level in cervical carcinoma samples w ere significantly higher than th ose of normal tissue samples To explore the clinical significance of SIRT5 in cervical cancer, we first analyzed its expression in cancer tissues (N=115) and adjacent normal tissues (N=35) using immunohistochemistry (IHC). As shown in Figure 1A, SIRT5 protein expression levels were notably higher in cervical cancer tissues compared to the adjacent ones. Furthermore, the staining score analysis confirmed that SIRT5 expression was significantly elevated in cancer tissues (Figure 1B, P < 0.001). Furthermore, a survival analysis using the Kaplan-Meier estimator was performed to evaluate the correlation between SIRT5 protein expression and the overall survival rate of cervical cancer patients. The results revealed that higher SIRT5 protein expression was significantly associated with a lower overall survival rate (Figure 1C, P = 0.018). Additionally, analysis of the correlation between SIRT5 expression and clinicopathological characteristics showed that SIRT5 expression was positively associated with tumor grade in cervical cancer patients (Table 1, P = 0.024). In summary, these results suggest that SIRT5 may act as a driver of cervical carcinogenesis and progression, highlighting its potential role as a biomarker and therapeutic target in cervical cancer 3.1 SIRT5 exerts slight effects on cell proliferation and colony formation activity in cervical cancer cells To investigate the function of SIRT5 in cervical cancer, stable Hela cell line overexpressing SIRT5 and stable Siha cell lines overexpressing or knock-down SIRT5 were constructed by infecting with lenti-sirt5 or lenti-shR-sirt5, respectively. Corresponding control cell lines were generated using control lentiviruses. Western blotting was then performed to confirm the expression levels of SIRT5 in these stable cell lines. As expected, a significant increase in protein level of SIRT5 was observed in Hela and Siha cells in the lenti-sirt5 group compared with those in the lenti-control group, while a remarkable decrease in protein level of SIRT5 in Siha cells was observed in the lenti-shR-sirt5 group compared with that in the lenti-shR-control group (Figure. 2A). While the ectopic expression of SIRT5 had inconsistent effects on cell proliferation in stable Hela or Siha cells, there were no significantly change in proliferation potential in all stable cell lines, regardless of whether SIRT5 was overexpressed or knocked down (Figure 2B). Additionally, as shown in Figure. 2C, the overexpression of SIRT5 apparently increased the efficiency of colony formation in stable Hela cells, while silence of SIRT5 significantly impaired the colony formation potential in stable Siha cells. However, overexpression of SIRT5 only slightly increased colony formation in stable Siha cells, and this effect was not statistically significant (Figure 2C). Therefore, these results demonstrated that overexpression or knock-down of SIRT5 has both mild influence on cell proliferation and colony formation in cervical cancer cells. 3.2 Overexpression of SIRT5 strengthens cell migration and invasion by inducing EMT in cervical cancer cells To investigate the effects of SIRT5 on cellular behavior in Hela and Siha cell lines, Transwell assays were performed to evaluate cell migration and invasion. As shown in Figure 3A, significantly accelerated cell migration and invasion potential were observed in the stable Hela and Siha cells in lenti-sirt5 groups compared to those in lenti-control groups. Conversely, in the lenti-shR-SIRT5 group, both migration and invasion of stable Siha cells were significantly suppressed compared to the lenti-shR-control group (Figure 3A, right panel). Therefore, these results demonstrated that SIRT5 positively regulated cell migration and invasion in cervical cancer. To further explore the underlying mechanisms of SIRT5 in regulating metastasis in cervical cancer cells, we examined the expression of epithelial and mesenchymal markers, as well as related transcription factors. Specifically, the epithelial marker E-cadherin, mesenchymal markers N-cadherin and vimentin, transcription factors including ZEB1, Slug, and Snail, and the tight junction protein β-catenin were analyzed in stable Hela and Siha cell lines overexpressing SIRT5. As shown in Figure 3B, ectopic expression of SIRT5 distinctly upregulated the mesenchymal markers N-cadherin, vimentin, slightly upregulated transcription factors ZEB1 and Slug, while downregulating the tight junction protein β-catenin. For the E-cadherin and Snail, there was no consistent results obtained in the stable Hela and Siha cell lines. Quantitative analysis of these protein levels was conducted and is presented in the accompanying histogram. Taken together, these results suggest that SIRT5 promotes cervical cancer cell metastasis by inducing epithelial-mesenchymal transition (EMT). 3.3 Overexpression SIRT5 promotes stemness phenotypes in cervical cancer cells Cancer stem cells (CSCs) are a small subpopulation of cells that have the ability to self-renew and are present in the majority of tumors, and contribute to the tumorigenesis, angiogenesis, and resistance to cytotoxic drugs and ionizing radiation of multiple cancer types including cervical cancer. In order to assess the role of SIRT5 in angiogenesis, in vitro tube formation assay was performed in stable Siha cell lines overexpressing or knock-down SIRT5. The results verified that significantly stronger tube formation activity was observed in the Sirt5-overexpressed Siha cells, while SIRT5-silence effectively weakened the tube formation potential (Figure. 4A). The quantity of tube well length was shown in the histogram (Figure. 4B). An in vitro spheroid formation assay has been diffusely documented as a common means to electively enrich cervical cancer cells with stem cell-like properties. Our data indicated that the number and size of spheres of stable Hela and Siha cell lines in lenti-sirt5 groups were more and larger those in lenti-control groups (Figure. 4C). Reverse effects of SIRT5 silence were observed in stable Siha cell line in lenti-shR-SIRT5 group compared with those in lenti-shR-control group (Figure. 4C). Moreover, several key stem cell-related transcriptional factors including SOX2, OCT4 and Nanog were examined in stable Hela and Siha cells with SIRT5 overexpressed. As shown in Figure. 4D, a remarkable increase in expression of SOX2 was observed in both Hela and Siha cell in lenti-sirt5 groups compared to those in lenti-control groups (Figure. 4D). In conclusion, SIRT5 play an evoked role in maintaining stemness phenotypes of cervical cancer cells. 3.4 SIRT5-induced cervical cancer cells motility dependents on the activation of TGF-β signaling pathway To uncover the underlying mechanisms in SIRT5 in regulating cervical cancer malignant properties, the activation of canonical TGF-β signaling pathway was determined in stable Hela and Siha cell lines overexpressing SIRT5. The results showed that SIRT5 overexpression significantly increased Smad2/3 phosphorylation in both Hela and Siha cells (Figure 5A & B). Additionally, in stable Siha cells, Smad2/3 was observed to translocate into the nucleus, as evidenced by a decrease in Smad2/3 levels in the cytoplasmic fraction and a corresponding increase in the nuclear fraction (Figure 5C). This nuclear localization of Smad2/3 was further confirmed through immunofluorescence microscopy, which showed the colocalization of red fluorescence-labeled Smad2/3 with DAPI-stained nuclei (Figure 5D). Furthermore, treatment with the TGF-β-specific inhibitor ITD-1 effectively reversed the enhanced migration and invasion abilities of stable Siha cells overexpressing SIRT5 (Figure 5E & F). In summary, these findings indicate that SIRT5-induced migration and invasion in cervical cancer cells are dependent on the activation of the TGF-β signaling pathway. 3.5 SIRT5 inhibits the growth of stable Siha cells-derived xenograft but promotes stemness and angiogenesis in vivo To subsequent investigate the function of sirt5 in vivo , stable Siha cells transduced with either lenti-control or lenti-SIRT5 were subcutaneously injected into nude mice. Tumor volume and weight were measured every 4 days, starting 10 days after injection. As shown in Figure. 6A and B, SIRT5 overexpression slightly inhibited tumor growth with no statistical significance which was consistent with the effect on cell proliferation in Siha cells in vitro . The weight variation of the mice during the experiment is presented in Figure 6C. After 34 days, the xenograft tumors were surgically removed, and H&E staining along with IHC assays were performed to assess cell necrosis and protein expression. As shown in Figure 6D, no significant differences in cell necrosis were observed between xenografts derived from lenti-control-Siha and lenti-SIRT5-Siha cells. Moreover, compared with lenti-control-siha-derived xenografts, those derived from lenti-SIRT5-Siha cells exhibited higher protein levels of SIRT5 (Figure 6E), Ki67 (Figure 6F), p-Smad2 (Figure 6G), Nanog (Figure 6H), and CD31 (Figure 6I). All these data suggested that SIRT5 played a critical role in maintaining stemness and promoting angiogenesis in vivo , at least in part through the activation of the TGF-β signaling pathway. Discussion The dual roles of SIRT5 as both an oncogene and tumor suppressor across different cancer types reflect its context-dependent functions, likely influenced by tumor type, metabolic status, and microenvironment. For instance, SIRT5 acts as an oncogene in breast cancer, colorectal cancer and ovarian cancer, while acted as tumor suppressor in liver cancer, and gastric cancer. 25 , 26 , 32-34 Recent studies have increasingly highlighted the involvement of SIRT5 in regulating nearly all malignant phenotypes of cancers, including tumorigenesis, cell proliferation, metastasis, drug resistance, immunoregulation. 35-38 In cervical cancer, our study establishes SIRT5 as a key regulator of metastasis and angiogenesis, primarily through the activation of the TGF-β signaling pathway. This pathway’s role in promoting EMT, stemness, and angiogenesis is well-documented in cervical cancer, and our findings position SIRT5 as an upstream activator of this axis. 39 , 40 As one of the three sirtuins (SIRT3–5) primarily localized in the mitochondria, SIRT5 plays a pivotal role in regulating mitochondrial metabolism, a central hub of cellular energy production and biosynthesis. 41 Notably, SIRT5 has been shown to stabilize mitochondrial glutaminase (GLS), an enzyme frequently upregulated during tumorigenesis, thereby promoting breast cancer progression via enhanced glutamine metabolism. 25 , 42 In colorectal cancer (CRC), SIRT5 supports the anaplerotic entry of glutamine into the TCA cycle by activating GLUD1 in a deglutarylation-dependent manner. 43-45 These studies underline the critical role of SIRT5 in reprogramming mitochondrial metabolism to support cancer cell growth and survival. While other sirtuin family members, such as SIRT1 and SIRT6, have been implicated in cervical cancer progression, the role of SIRT5 in cervical cancer remains unexplored. 46-48 Furthermore, the involvement of SIRT5 in stabilizing GLS and regulating glutaminolysis suggests a potential metabolic link to TGF-β activation, a hypothesis warranting further investigation. 49 Our study investigated the expression and biological roles of SIRT5 in cervical cancer and its underlying mechanisms. Using tissue microarrays and IHC assays, we found that SIRT5 protein levels were significantly higher in cervical cancer tissues compared to adjacent normal tissues. High SIRT5 expression was associated with poor overall survival and positively correlated with tumor grade, indicating its potential role in cervical carcinogenesis and progression (Figure 1, Table 1). To further explore its biological functions, we generated stable Hela and Siha cell lines overexpressing or knocking down SIRT5. While SIRT5 had minimal impact on cell proliferation and colony formation (Figure 2), it significantly promoted cell migration and invasion by enhancing epithelial-mesenchymal transition (EMT) and stemness, as evidenced by increased tube and spheroid formation and upregulation of the stemness-related transcription factor SOX2 (Figures 3 and 4). Mechanistically, we demonstrated that SIRT5 activated the TGF-β signaling pathway, a critical regulator of tumor proliferation, metastasis, and stemness in cervical cancer. Overexpression of SIRT5 increased Smad2/3 phosphorylation and facilitated their nuclear translocation, as confirmed by immunofluorescence analysis (Figure 5). Furthermore, treatment with ITD-1, a TGF-β pathway inhibitor, effectively suppressed SIRT5-induced migration and invasion, highlighting the indispensable role of this pathway in SIRT5-mediated tumor progression. To validate these findings in vivo, we used a xenograft model with stable Siha cells overexpressing SIRT5. Although SIRT5 slightly reduced tumor growth with lower Ki67 expression, it significantly enhanced the expression of p-Smad2, the stemness marker Nanog, and the angiogenesis marker CD31, indicating its role in promoting stemness and angiogenesis in vivo (Figure 6). Collectively, our results reveal that SIRT5 contributes to cervical cancer metastasis and angiogenesis by activating the TGF-β signaling pathway, providing new insights into the molecular mechanisms of cervical cancer progression and identifying SIRT5 as a potential therapeutic target. Small-molecule inhibitors of SIRT5 or TGF-β pathway inhibitors, like ITD-1, could potentially reduce metastatic burden and improve patient outcomes. Additionally, the use of SIRT5 as a biomarker for aggressive cervical cancer warrants further clinical validation. Conclusions In summary, our study provides the first evidence that SIRT5 plays a critical role in cervical cancer progression by promoting metastasis, stemness, and angiogenesis. Mechanistically, SIRT5 exerts its pro-tumorigenic effects primarily through the activation of the TGF-β signaling pathway, which drives EMT and enhances stem-like properties. These findings not only shed light on the molecular mechanisms underlying SIRT5’s role in cervical cancer but also suggest that SIRT5 could serve as a potential biomarker and therapeutic target for managing cervical cancer metastasis and progression. Further studies are warranted to explore the detailed metabolic and signaling interactions regulated by SIRT5, as well as its clinical potential in cervical cancer treatment. Declarations AUTHOR'S CONTRIBUTION: The authors confirm their contribution to the paper as follows: FX wrote the main manuscript text. HH prepared figures 1-3, XJ prepared figures 4-6. FW and LW contributed equally to this work as last authors.All authors reviewed the manuscript. Ethics approval and consent to participate The study were approved by the Ethics Committee of Women's Hospital, School of Medicine, Zhejiang University (Approval No. IRB-20230172-R). Human and animal rights All procedures performed in studies involving human participants were in accordance with the ethical standards of institutional and/or research committee and with the 1975 Declaration of Helsinki, as revised in 2013. Consent for publication Written informed consent was obtained from the patient for publication of this article and any accompanying images. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding This work was supported by National Natural Science Foundation of China, Grant No. 81672568; and the National Natural Science Foundation of China, Grant No. 32300460; and the Zhejiang Provincial Natural Science Foundation, Grant No. LQ23H160031 Conflict of interests The authors declare no conflict of interest financial or otherwise. Acknowledgment DECLARED NONE. References Xu, M., Cao, C., Wu, P., Huang, X. & Ma, D. Advances in cervical cancer: current insights and future directions. Cancer communications (London, England) 45 , 77-109, doi:10.1002/cac2.12629 (2025). Hyeon, D. Y. et al. Proteogenomic characterization of molecular and cellular targets for treatment-resistant subtypes in locally advanced cervical cancers. Molecular cancer 24 , 77, doi:10.1186/s12943-025-02256-3 (2025). Legut, M., Dolton, G., Mian, A. A., Ottmann, O. G. & Sewell, A. K. CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells. 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Wang, T. et al. SIRT5-mediated BCAT1 desuccinylation and stabilization leads to ferroptosis insensitivity and promotes cell proliferation in glioma. Cell death & disease 16 , 261, doi:10.1038/s41419-025-07626-9 (2025). Zhang, J. et al. SIRT5-modified human umbilical cord mesenchymal stem cells loaded with antioxidant polydopamine nanozyme enhance parpi resistance in ovarian cancer via fatty acid metabolism reprogramming. Journal of nanobiotechnology 23 , 485, doi:10.1186/s12951-025-03516-6 (2025). Wang, M., Meng, J., Wang, H., Hu, H. & Hong, Y. Atractylodes macrocephala III suppresses EMT in cervical cancer by regulating IGF2BP3 through ETV5. Journal of cellular and molecular medicine 28 , e18081, doi:10.1111/jcmm.18081 (2024). Wu, M., Chen, G., Liao, X., Xiao, L. & Zheng, J. YTHDF2 interference suppresses the EMT of cervical cancer cells and enhances cisplatin chemosensitivity by regulating AXIN1. Drug development research 83 , 1190-1200, doi:10.1002/ddr.21942 (2022). Yan, J. J. et al. SIRT5 modulates mitochondria function via mitophagy and antioxidant mechanisms to facilitate oocyte maturation in mice. International journal of biological macromolecules 306 , 141488, doi:10.1016/j.ijbiomac.2025.141488 (2025). Choi, H. et al. Disruption of redox balance in glutaminolytic triple negative breast cancer by inhibition of glutaminase and glutamate export. Neoplasia (New York, N.Y.) 61 , 101136, doi:10.1016/j.neo.2025.101136 (2025). Ke, Z. et al. Emerging roles of mitochondrial sirtuin SIRT5 in succinylation modification and cancer development. Frontiers in immunology 16 , 1531246, doi:10.3389/fimmu.2025.1531246 (2025). Shen, H. et al. Mitochondrial Sirtuins in Cancer: A Revisited Review from Molecular Mechanisms to Therapeutic Strategies. Theranostics 14 , 2993-3013, doi:10.7150/thno.97320 (2024). Wang, Y. Q. et al. Sirtuin5 contributes to colorectal carcinogenesis by enhancing glutaminolysis in a deglutarylation-dependent manner. Nature communications 9 , 545, doi:10.1038/s41467-018-02951-4 (2018). Van Meter, M., Gorbunova, V. & Seluanov, A. SIRT6: a promising target for cancer prevention and therapy. Advances in experimental medicine and biology 818 , 181-196, doi:10.1007/978-1-4471-6458-6_9 (2014). So, D. et al. Cervical cancer is addicted to SIRT1 disarming the AIM2 antiviral defense. Oncogene 37 , 5191-5204, doi:10.1038/s41388-018-0339-4 (2018). Jiang, W. et al. The PIK3CA E542K and E545K mutations promote glycolysis and proliferation via induction of the β-catenin/SIRT3 signaling pathway in cervical cancer. Journal of hematology & oncology 11 , 139, doi:10.1186/s13045-018-0674-5 (2018). Zhang, K. & Jagannath, C. Crosstalk between metabolism and epigenetics during macrophage polarization. Epigenetics & chromatin 18 , 16, doi:10.1186/s13072-025-00575-9 (2025). Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. 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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-8614743","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":591830942,"identity":"82baf971-32d8-48c2-8703-f0f0a61d2e37","order_by":0,"name":"Feiting Xie","email":"","orcid":"","institution":"Women's Hospital, School of Medicine, Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Feiting","middleName":"","lastName":"Xie","suffix":""},{"id":591830943,"identity":"4e729996-9432-4bbf-9b30-1666a80d9c67","order_by":1,"name":"Hao huang","email":"","orcid":"","institution":"Women's Hospital, School of Medicine, Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"huang","suffix":""},{"id":591830944,"identity":"869be784-6f3a-4ca2-9497-caa3a5b1c160","order_by":2,"name":"Xiaojing Chen","email":"","orcid":"","institution":"Women's Hospital, School of Medicine, Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Xiaojing","middleName":"","lastName":"Chen","suffix":""},{"id":591830945,"identity":"2d351bd9-e06b-4753-845f-d9113fb124c9","order_by":3,"name":"Lingfang Wang","email":"","orcid":"","institution":"Women's Hospital, School of Medicine, Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Lingfang","middleName":"","lastName":"Wang","suffix":""},{"id":591830946,"identity":"f4c11e22-6775-4839-a6c5-82444a391043","order_by":4,"name":"Fenfen Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIie3RMUvDQBTA8RcCmUKzviNgv8KVg1BB6Fc5KXS6SqfSQWpAyCS4RvBD5CNcODBLatcbOlQE6dAhkl08la6Xdit4/+k9eL/pAbhc59oCIALwJeDPJo8hNQBJIeAnk5D+bV2EViu1k4sNRvFD21xmcNHT3GtnNlLfTIay/kDyvCooyYARzf04t5BEioR9ZmpJ9bTghlwXmgd+aCPrfULLL4UjLbbSkLtuogXblqlCisJLDeG0i4z0PgH5ohD1hAG+4uCpfruPbYQ8CtbIW4VRPn5vcX7V71XjsrURU4CHycffZ3qpHZjD5jB5je3O5XK5/m3fQ4JSRKeMRQYAAAAASUVORK5CYII=","orcid":"","institution":"Women's Hospital, School of Medicine, Zhejiang University","correspondingAuthor":true,"prefix":"","firstName":"Fenfen","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-01-16 03:08:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8614743/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8614743/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102894864,"identity":"db0586a0-c3de-4d5a-aa58-e0e5a885db7f","added_by":"auto","created_at":"2026-02-18 06:14:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3743438,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of SIRT5 protein in cervical cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunohistochemistry (IHC) analysis of SIRT5 expression in adjacent normal tissues and cervical cancer tissues. SIRT5 protein levels are indicated by brown staining, with hematoxylin counterstaining in blue. Representative images are shown. (B) of SIRT5 IHC scores in human cervical cancer tissues and adjacent normal tissues. Scoring was performed using the MBP method as described in the Materials and Methods section. Statistical analysis was conducted using a Student’s t-test (***P \u0026lt; 0.001). \u0026nbsp;(C) Kaplan-Meier survival analysis showing the correlation between SIRT5 expression and overall survival in cervical cancer patients. Data were obtained from the tissue microarray and compared using a log-rank test (P = 0.018).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8614743/v1/0e6ba7381ed7d541f5c5e859.png"},{"id":102894860,"identity":"72a98cee-63bf-4fda-a0f4-b4766b8cdfbf","added_by":"auto","created_at":"2026-02-18 06:14:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1126607,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe role of SIRT5 in cell proliferation and colony formation in cervical cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot analysis confirming the efficacies of SIRT5 overexpression or knockdown in stable Hela and Siha cell lines. Quantitative analysis of SIRT5 protein levels is presented in the histogram. (B) Effects of SIRT5 overexpression or knockdown on cell proliferation in Hela and Siha cells were determined using the MTT assay. (C) Representative images of colony formation in Hela and Siha cells with SIRT5 overexpression or knockdown. Colony formation was quantified, and the surviving fraction is shown. Data represent one of three independent experiments (*P \u0026lt; 0.05; **P \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8614743/v1/54579607449183830d76e747.png"},{"id":102894863,"identity":"0703f1f3-4648-4739-883d-8b7addf9e04c","added_by":"auto","created_at":"2026-02-18 06:14:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1500327,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSIRT5 overexpression promotes cell migration, invasion, and EMT in cervical cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Transwell assays showing the effect of SIRT5 overexpression or knockdown on cell migration and invasion in Hela and Siha cells. Quantitative analysis of migrated and invaded cells is displayed in the histogram. (B) Western blot analysis of EMT markers in stable Hela and Siha cell lines. Quantitative analysis of protein levels is shown in the histogram. Data represent one of three independent experiments (*P \u0026lt; 0.05; **P \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8614743/v1/723ed9412edd8bfb7d9de647.png"},{"id":102964550,"identity":"c2b3670b-44e9-4fb3-b076-f8e529244f1a","added_by":"auto","created_at":"2026-02-19 04:22:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1760466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSIRT5 overexpression induces stemness phenotypes in cervical cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Effect of SIRT5 overexpression or knockdown on vascularization in Siha cells. (B) Quantitative analysis of tube formation length is presented in the histogram. (C) Sphere formation assays showing the effect of SIRT5 overexpression or knockdown in Hela and Siha cells. (D) Western blot analysis of stemness markers in stable Hela and Siha cell lines. Quantitative analysis of stemness marker protein levels is shown in the histogram. Data represent one of three independent experiments (*P \u0026lt; 0.05; **P \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8614743/v1/7e06c116102f723154fecb28.png"},{"id":102894861,"identity":"3c9bf196-1656-45e4-ba47-6ad70f09cba3","added_by":"auto","created_at":"2026-02-18 06:14:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1846744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSIRT5 promotes cervical cancer cell motility through activation of the TGF-β signaling pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot analysis of Smad2/3 phosphorylation in Hela and Siha cells overexpressing SIRT5. (B) Quantitative analysis of Smad2/3 phosphorylation levels is shown in the histogram. (C) Western blot analysis of cytoplasmic and nuclear Smad2/3 localization in Siha cells overexpressing SIRT5. Quantitative analysis of Smad2/3 levels in cytoplasmic and nuclear fractions is shown in the histogram. (D) Fluorescence microscopy images showing nuclear localization of Smad2/3 in stable Siha cells. (E) Transwell assays demonstrating the effect of SIRT5 overexpression on migration and invasion in stable Siha cells with or without treatment with the TGF-β inhibitor ITD-1. (F) Quantitative analysis of migrated and invaded cells from panel E is shown in the histogram. Data represent one of three independent experiments (*P \u0026lt; 0.05; **P \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8614743/v1/361e98420de08f2b324d4eb0.png"},{"id":102894865,"identity":"5393e6ef-771e-4cfb-881b-c101e7131e62","added_by":"auto","created_at":"2026-02-18 06:14:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3343401,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSIRT5 enhances stemness, activates the TGF-β signaling pathway, and promotes angiogenesis in vivo.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSIRT5-overexpressing Siha cells and control cells were subcutaneously injected into nude mice (5 × 10⁶ cells/mouse). (A) Representative images of mice bearing tumors. (B) Tumor volumes were measured using calipers and calculated with the formula: V = (a × b²)/2, where \"a\" is the length and \"b\" is the width in millimeters. Data are presented as mean ± S.E.M. (n = 5). (C) Body weight changes in the two groups are shown as mean ± S.E.M. (n = 5). (D) H\u0026amp;E staining of tumor sections from the lenti-control and lenti-SIRT5 groups. (E–I) IHC analysis of tumor tissues for SIRT5 (E), Ki67 (F), p-Smad2 (G), Nanog (H), and CD31 (I) expression. Representative images are shown at ×400 magnification.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8614743/v1/704a70ad0f9c0a840797ca16.png"},{"id":103298615,"identity":"ac66efb6-3ef7-4631-b09c-5ccb00ffe372","added_by":"auto","created_at":"2026-02-24 07:43:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16313393,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8614743/v1/33f6dc88-85e7-4ff4-9e57-8abffbb7bb1d.pdf"},{"id":102964388,"identity":"59b8fbf0-26c0-45bb-8e20-651918e791df","added_by":"auto","created_at":"2026-02-19 04:22:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19566,"visible":true,"origin":"","legend":"","description":"","filename":"table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8614743/v1/4d49be7bac5b5322d112acd5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"SIRT5 Sustains Stemness and Promotes Metastasis and Angiogenesis in Cervical Cancer via Activation of the TGF-β Signaling Pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCervical cancer is the fourth most common malignancy in women worldwide and the second leading cause of cancer-related deaths among women in developing countries.\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e2\u003c/sup\u003e Annually, approximately 500,000 women are diagnosed with cervical cancer, resulting in over 300,000 deaths globally.\u003csup\u003e3\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e4\u003c/sup\u003e It is well-established that nearly all cervical carcinoma cases (99.7%) are associated with human papillomavirus (HPV) infection, with HPV16 and HPV18\u0026mdash;the high-risk carcinogenic genotypes\u0026mdash;accounting for approximately 55\u0026ndash;60% and 10\u0026ndash;15% of all cervical cancers, respectively.\u003csup\u003e5\u003c/sup\u003e Fortunately, the development of vaccines targeting the HPV types most commonly linked to cervical cancer has significantly reduced its global incidence.\u003csup\u003e6\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e7\u003c/sup\u003e Despite substantial advancements in diagnostic techniques and treatments that have improved outcomes for cervical cancer patients, the prognosis for those with regional or distant metastases remains poor.\u003csup\u003e8\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e9\u003c/sup\u003e However, the molecular mechanisms underlying cervical cancer metastasis are not yet fully understood.\u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eSirtuin (SIRT) are a family of class III histone deacetylases that regulate post-translational modifications of protein substrates involved in diverse metabolic pathways in an NAD-dependent manner.\u003csup\u003e11-13\u003c/sup\u003e Among seven members, SIRT1\u0026ndash;7, 3 of which (SIRT3\u0026ndash;5) are localized primarily in mitochondria.\u003csup\u003e14-16\u003c/sup\u003e SIRT5 functions as an NAD-dependent lysine demalonylase, desuccinylase and deglutarylase that specifically removes malonyl, succinyl and glutaryl groups from target proteins.\u003csup\u003e17\u003c/sup\u003e SIRT5 play complex roles in normal and diseased cells, with gain or loss of the \u003cem\u003esirt5\u003c/em\u003e gene site occurs regularly across a series of cancers, though often under conditions with non-focal genomic events leading to alterations in plentiful flanking genes over and above SIRT5.\u003csup\u003e18\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e19\u003c/sup\u003e SIRT5 mRNA expression is discoverable across a wide number of cancers, such as colorectal cancer and gastric cancer, and so on.\u003csup\u003e20-22\u003c/sup\u003e A variety of previous studies have shown that SIRT5 expression is heightened in specific tumors relative to the corresponding adjacent or normal tissue type.\u003csup\u003e23\u003c/sup\u003e Recent studies have indicated that SIRT5 could promote the breast cancer tumorigenesis by stabilizing mitochondrial glutaminase, contribute to colorectal cancer growth by regulating T Cell activity, and influence the proliferation, invasion and migration of prostate cancer cells through acetyl-CoA acetyltransferase 1.\u003csup\u003e24-26\u003c/sup\u003e Meanwhile, contradictory findings also exist. Hypoxia-induced suppression of SIRT has been reported to promote the proliferation, migration and invasion of hepatocellular carcinoma cells.\u003csup\u003e27\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e28\u003c/sup\u003e These findings suggest that SIRT5 may act as either an oncogene or a tumor suppressor, depending on the cancer type.\u003csup\u003e29\u003c/sup\u003e Despite these insights, the role of SIRT5 in the malignant progression of cervical cancer remains largely unknown.\u003c/p\u003e\n\u003cp\u003eIn this study, SIRT5 protein expression levels were evaluated using tissue microarrays (TMA) containing cervical cancer tissues and adjacent normal tissues, and its clinical correlations were analyzed.\u003csup\u003e30\u003c/sup\u003e To further determine the role of SIRT5 in cervical cancer cell both\u003cem\u003e in vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, stable SIRT5-overexpressing and knockdown cell lines were established using a lentiviral system. Our data indicated that the expression of SIRT5 was upregulated in cervical cancer tissues, and play an important role in regulating of malignant phenotypes such as cell migration, invasion potential, and angiogenesis. Furthermore, SIRT5 appeared to function as an oncogene in cervical cancer by promoting epithelial-mesenchymal transition (EMT) and enhancing the stem-like properties of cervical cancer cells through activation of the TGF-\u0026beta; signaling pathway. To the best of our knowledge, this is the first study to investigate the role of SIRT5 in cervical cancer, providing new insights into its function and offering a potential therapeutic target for the treatment of cervical cancer.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Cell culture and stable cell lines generation \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman cervical cancer cell lines, Hela and Siha, were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM (Gibco) supplemented with 10% FBS (BI), 100 U/mL penicillin, and 100 \u0026mu;g/mL streptomycin. Cells were maintained at 37\u0026deg;C in a humidified incubator with 5% CO₂. To generate stable cell lines, lentiviruses for SIRT5 overexpression, knockdown, and corresponding controls were purchased from GeneChem. After infection and GFP expression confirmation, cells were treated with 2 \u0026mu;g/mL puromycin for two weeks to select stable cell lines. Successful SIRT5 overexpression or knockdown was validated via western blotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Immunohistochemistry \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCervical cancer tissues and adjacent normal tissues were collected from patients at the Women\u0026rsquo;s Hospital, School of Medicine, Zhejiang University, with written informed consent. The study was approved by the Institutional Ethics Committee. IHC staining was performed as described previously.\u003csup\u003e31\u003c/sup\u003e SIRT5 staining intensity and percentage of positive cells were scored, and the immunoreactivity score (IRS) was calculated using the formula: \u003cstrong\u003eIRS = intensity score \u0026times; quantity score\u003c/strong\u003e. Staining was categorized as negative (\u0026le;3), weak positive (\u0026gt;3 but \u0026le;6), or strong positive (\u0026gt;6). Primary antibodies used for IHC included anti-SIRT5 (1:100, Abcam), anti-Ki67 (1:100, CST), anti-p-Smad2 (1:150, Abcam), anti-Nanog (1:100, CST), and anti-CD31 (1:100, Proteintech).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Western blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were lysed in RIPA buffer containing 1% protease inhibitor cocktail (Sigma). Protein samples were separated on 12% or 15% SDS-PAGE gels and transferred onto PVDF membranes (Millipore). Membranes were blocked in 5% milk for 2 hours, incubated overnight with primary antibodies at 4\u0026deg;C, followed by HRP-conjugated secondary antibodies for 2 hours at room temperature. Protein bands were visualized using X-ray film (Fujifilm) after chemiluminescent detection. \u0026beta;-Actin served as the loading control. Primary antibodies included anti-SIRT5, EMT markers (E-cadherin, N-cadherin, Vimentin, \u0026beta;-catenin), stemness markers (SOX2, OCT4, Nanog), and TGF-\u0026beta; signaling proteins (Smad2, p-Smad2, Smad3, p-Smad3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Cellular Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.1 Cell Proliferation Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProliferation was assessed using the MTT assay. Stable cell lines were seeded in 96-well plates (5,000 cells/well) and incubated for 24 and 48 hours before measuring absorbance at 570 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.2 Colony formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStable Hela and Siha cells (500 cells/well) were seeded in 6-well plates and cultured for 14 days. Colonies were fixed with methanol, stained with crystal violet, and counted under a microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.3 Migration and Invasion Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell migration was analyzed using a Transwell system (8 \u0026mu;m pore size, Corning). For invasion assays, inserts were pre-coated with Matrigel (BD). Cells were seeded in serum-free DMEM in the upper chamber, while the lower chamber contained DMEM with 10% FBS. After 24 hours, migrated/invaded cells were fixed, stained with crystal violet, and counted under a microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.4 \u003c/strong\u003e\u003cstrong\u003eTube formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStable Siha cells were seeded on Matrigel-coated wells, and capillary-like structures were observed after 2 hours. Tube lengths were quantified using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.5 Sphere-formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded at 1,000 cells/well in a 24-well plate and cultured in DMEM/F12 medium supplemented with EGF, FGFb, and B27 for 8\u0026ndash;10 days. Spheres were imaged under a microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Immunofluorescence assay for smad2/3 cellular translocation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStable Siha cells overexpressing SIRT5 were seeded in 6-well plates and fixed with 4% formaldehyde. Cells were permeabilized with methanol, blocked with 3% BSA, and incubated with anti-Smad2/3 (1:150, CST) followed by Alexa Fluor 647-conjugated secondary antibody. Nuclei were stained with DAPI, and images were captured using a fluorescent microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 \u003cem\u003eIn vivo\u003c/em\u003e study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale BALB/c nude mice (4\u0026ndash;6 weeks old, 20 g) were purchased from GemPharmatech, China, and housed under standard conditions. Xenografts were established by subcutaneously injecting 5 \u0026times; 10⁶ Siha cells stably overexpressing SIRT5. Tumor volume was calculated using the formula: V = (a \u0026times; b\u0026sup2;)/2, where \u0026quot;a\u0026quot; is the tumor length and \u0026quot;b\u0026quot; is the width. Tumor tissues were fixed in formalin and embedded in paraffin for IHC analysis. Body weight was monitored to assess treatment toxicity. All animal experiments followed AAALAC and IACUC guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as mean \u0026plusmn; S.D. from at least three independent experiments, except for animal experiments (mean \u0026plusmn; SEM). Statistical analyses were performed using GraphPad Prism 5.0. Student\u0026rsquo;s t-test was used for comparisons, while survival curves were analyzed with the log-rank test. Correlations between SIRT5 expression and clinicopathological features were assessed using the chi-square test. A P-value \u0026lt; 0.05 (*) was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 The express\u003c/strong\u003e\u003cstrong\u003eions\u003c/strong\u003e\u003cstrong\u003e of SIRT5 protein level\u003c/strong\u003e\u003cstrong\u003ein cervical carcinoma samples w\u003c/strong\u003e\u003cstrong\u003eere\u003c/strong\u003e\u003cstrong\u003e significantly higher than th\u003c/strong\u003e\u003cstrong\u003eose\u003c/strong\u003e\u003cstrong\u003e of normal tissue \u003c/strong\u003e\u003cstrong\u003esamples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the clinical significance of SIRT5 in cervical cancer, we first analyzed its expression in cancer tissues (N=115) and adjacent normal tissues (N=35) using immunohistochemistry (IHC). As shown in Figure 1A, SIRT5 protein expression levels were notably higher in cervical cancer tissues compared to the adjacent ones. Furthermore, the staining score analysis confirmed that SIRT5 expression was significantly elevated in cancer tissues (Figure 1B, P \u0026lt; 0.001). Furthermore, a survival analysis using the Kaplan-Meier estimator was performed to evaluate the correlation between SIRT5 protein expression and the overall survival rate of cervical cancer patients. The results revealed that higher SIRT5 protein expression was significantly associated with a lower overall survival rate (Figure 1C, P = 0.018). Additionally, analysis of the correlation between SIRT5 expression and clinicopathological characteristics showed that SIRT5 expression was positively associated with tumor grade in cervical cancer patients (Table 1, P = 0.024). In summary, these results suggest that SIRT5 may act as a driver of cervical carcinogenesis and progression, highlighting its potential role as a biomarker and therapeutic target in cervical cancer\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e3.1 SIRT5 exerts slight effects on cell proliferation and colony formation activity in cervical cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the function of SIRT5 in cervical cancer, stable Hela cell line overexpressing SIRT5 and stable Siha cell lines overexpressing or knock-down SIRT5 were constructed by infecting with lenti-sirt5 or lenti-shR-sirt5, respectively. Corresponding control cell lines were generated using control lentiviruses. Western blotting was then performed to confirm the expression levels of SIRT5 in these stable cell lines. As expected, a significant increase in protein level of SIRT5 was observed in Hela and Siha cells in the lenti-sirt5 group compared with those in the lenti-control group, while a remarkable decrease in protein level of SIRT5 in Siha cells was observed in the lenti-shR-sirt5 group compared with that in the lenti-shR-control group (Figure. 2A). \u003c/p\u003e\n\u003cp\u003eWhile the ectopic expression of SIRT5 had inconsistent effects on cell proliferation in stable Hela or Siha cells, there were no significantly change in proliferation potential in all stable cell lines, regardless of whether SIRT5 was overexpressed or knocked down (Figure 2B). Additionally, as shown in Figure. 2C, the overexpression of SIRT5 apparently increased the efficiency of colony formation in stable Hela cells, while silence of SIRT5 significantly impaired the colony formation potential in stable Siha cells. However, overexpression of SIRT5 only slightly increased colony formation in stable Siha cells, and this effect was not statistically significant (Figure 2C). Therefore, these results demonstrated that overexpression or knock-down of SIRT5 has both mild influence on cell proliferation and colony formation in cervical cancer cells.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e3.2 Overexpression of SIRT5 strengthens cell migration and invasion by inducing EMT in cervical cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effects of SIRT5 on cellular behavior in Hela and Siha cell lines, Transwell assays were performed to evaluate cell migration and invasion. As shown in Figure 3A, significantly accelerated cell migration and invasion potential were observed in the stable Hela and Siha cells in lenti-sirt5 groups compared to those in lenti-control groups. Conversely, in the lenti-shR-SIRT5 group, both migration and invasion of stable Siha cells were significantly suppressed compared to the lenti-shR-control group (Figure 3A, right panel). Therefore, these results demonstrated that SIRT5 positively regulated cell migration and invasion in cervical cancer.\u003c/p\u003e\n\u003cp\u003eTo further explore the underlying mechanisms of SIRT5 in regulating metastasis in cervical cancer cells, we examined the expression of epithelial and mesenchymal markers, as well as related transcription factors. Specifically, the epithelial marker E-cadherin, mesenchymal markers N-cadherin and vimentin, transcription factors including ZEB1, Slug, and Snail, and the tight junction protein \u0026beta;-catenin were analyzed in stable Hela and Siha cell lines overexpressing SIRT5. As shown in Figure 3B, ectopic expression of SIRT5 distinctly upregulated the mesenchymal markers N-cadherin, vimentin, slightly upregulated transcription factors ZEB1 and Slug, while downregulating the tight junction protein \u0026beta;-catenin. For the E-cadherin and Snail, there was no consistent results obtained in the stable Hela and Siha cell lines. Quantitative analysis of these protein levels was conducted and is presented in the accompanying histogram. Taken together, these results suggest that SIRT5 promotes cervical cancer cell metastasis by inducing epithelial-mesenchymal transition (EMT).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Overexpression SIRT5 promotes stemness phenotypes in cervical cancer cells \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCancer stem cells (CSCs) are a small subpopulation of cells that have the ability to self-renew and are present in the majority of tumors, and contribute to the tumorigenesis, angiogenesis, and resistance to cytotoxic drugs and ionizing radiation of multiple cancer types including cervical cancer. In order to assess the role of SIRT5 in angiogenesis,\u003cem\u003e in vitro\u003c/em\u003e tube formation assay was performed in stable Siha cell lines overexpressing or knock-down SIRT5. The results verified that significantly stronger tube formation activity was observed in the Sirt5-overexpressed Siha cells, while SIRT5-silence effectively weakened the tube formation potential (Figure. 4A). The quantity of tube well length was shown in the histogram (Figure. 4B).\u003c/p\u003e\n\u003cp\u003eAn \u003cem\u003ein vitro\u003c/em\u003e spheroid formation assay has been diffusely documented as a common means to electively enrich cervical cancer cells with stem cell-like properties. Our data indicated that the number and size of spheres of stable Hela and Siha cell lines in lenti-sirt5 groups were more and larger those in lenti-control groups (Figure. 4C). Reverse effects of SIRT5 silence were observed in stable Siha cell line in lenti-shR-SIRT5 group compared with those in lenti-shR-control group (Figure. 4C). Moreover, several key stem cell-related transcriptional factors including SOX2, OCT4 and Nanog were examined in stable Hela and Siha cells with SIRT5 overexpressed. As shown in Figure. 4D, a remarkable increase in expression of SOX2 was observed in both Hela and Siha cell in lenti-sirt5 groups compared to those in lenti-control groups (Figure. 4D). In conclusion, SIRT5 play an evoked role in maintaining stemness phenotypes of cervical cancer cells.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e3.4 SIRT5-induced cervical cancer cells motility dependents on the activation of TGF-\u0026beta; signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo uncover the underlying mechanisms in SIRT5 in regulating cervical cancer malignant properties, the activation of canonical TGF-\u0026beta; signaling pathway was determined in stable Hela and Siha cell lines overexpressing SIRT5. The results showed that SIRT5 overexpression significantly increased Smad2/3 phosphorylation in both Hela and Siha cells (Figure 5A \u0026amp; B). Additionally, in stable Siha cells, Smad2/3 was observed to translocate into the nucleus, as evidenced by a decrease in Smad2/3 levels in the cytoplasmic fraction and a corresponding increase in the nuclear fraction (Figure 5C). This nuclear localization of Smad2/3 was further confirmed through immunofluorescence microscopy, which showed the colocalization of red fluorescence-labeled Smad2/3 with DAPI-stained nuclei (Figure 5D). Furthermore, treatment with the TGF-\u0026beta;-specific inhibitor ITD-1 effectively reversed the enhanced migration and invasion abilities of stable Siha cells overexpressing SIRT5 (Figure 5E \u0026amp; F). In summary, these findings indicate that SIRT5-induced migration and invasion in cervical cancer cells are dependent on the activation of the TGF-\u0026beta; signaling pathway.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e3.5 SIRT5 inhibits the growth of stable Siha cells-derived xenograft but promotes stemness and angiogenesis \u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo subsequent investigate the function of sirt5 \u003cem\u003ein vivo\u003c/em\u003e, stable Siha cells transduced with either lenti-control or lenti-SIRT5 were subcutaneously injected into nude mice. Tumor volume and weight were measured every 4 days, starting 10 days after injection. As shown in Figure. 6A and B, SIRT5 overexpression slightly inhibited tumor growth with no statistical significance which was consistent with the effect on cell proliferation in Siha cells\u003cem\u003e in vitro\u003c/em\u003e. The weight variation of the mice during the experiment is presented in Figure 6C. After 34 days, the xenograft tumors were surgically removed, and H\u0026amp;E staining along with IHC assays were performed to assess cell necrosis and protein expression. As shown in Figure 6D, no significant differences in cell necrosis were observed between xenografts derived from lenti-control-Siha and lenti-SIRT5-Siha cells. Moreover, compared with lenti-control-siha-derived xenografts, those derived from lenti-SIRT5-Siha cells exhibited higher protein levels of SIRT5 (Figure 6E), Ki67 (Figure 6F), p-Smad2 (Figure 6G), Nanog (Figure 6H), and CD31 (Figure 6I). All these data suggested that SIRT5 played a critical role in maintaining stemness and promoting angiogenesis \u003cem\u003ein vivo\u003c/em\u003e, at least in part through the activation of the TGF-\u0026beta; signaling pathway.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe dual roles of SIRT5 as both an oncogene and tumor suppressor across different cancer types reflect its context-dependent functions, likely influenced by tumor type, metabolic status, and microenvironment. For instance, SIRT5 acts as an oncogene in breast cancer, colorectal cancer and ovarian cancer, while acted as tumor suppressor in liver cancer, and gastric cancer.\u003csup\u003e25\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e26\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e32-34\u003c/sup\u003e Recent studies have increasingly highlighted the involvement of SIRT5 in regulating nearly all malignant phenotypes of cancers, including tumorigenesis, cell proliferation, metastasis, drug resistance, immunoregulation.\u003csup\u003e35-38\u003c/sup\u003e In cervical cancer, our study establishes SIRT5 as a key regulator of metastasis and angiogenesis, primarily through the activation of the TGF-\u0026beta; signaling pathway. This pathway\u0026rsquo;s role in promoting EMT, stemness, and angiogenesis is well-documented in cervical cancer, and our findings position SIRT5 as an upstream activator of this axis.\u003csup\u003e39\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e40\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAs one of the three sirtuins (SIRT3\u0026ndash;5) primarily localized in the mitochondria, SIRT5 plays a pivotal role in regulating mitochondrial metabolism, a central hub of cellular energy production and biosynthesis.\u003csup\u003e41\u003c/sup\u003e Notably, SIRT5 has been shown to stabilize mitochondrial glutaminase (GLS), an enzyme frequently upregulated during tumorigenesis, thereby promoting breast cancer progression via enhanced glutamine metabolism.\u003csup\u003e25\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e42\u003c/sup\u003e In colorectal cancer (CRC), SIRT5 supports the anaplerotic entry of glutamine into the TCA cycle by activating GLUD1 in a deglutarylation-dependent manner.\u003csup\u003e43-45\u003c/sup\u003e These studies underline the critical role of SIRT5 in reprogramming mitochondrial metabolism to support cancer cell growth and survival. While other sirtuin family members, such as SIRT1 and SIRT6, have been implicated in cervical cancer progression, the role of SIRT5 in cervical cancer remains unexplored.\u003csup\u003e46-48\u003c/sup\u003e Furthermore, the involvement of SIRT5 in stabilizing GLS and regulating glutaminolysis suggests a potential metabolic link to TGF-\u0026beta; activation, a hypothesis warranting further investigation.\u003csup\u003e49\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eOur study investigated the expression and biological roles of SIRT5 in cervical cancer and its underlying mechanisms. Using tissue microarrays and IHC assays, we found that SIRT5 protein levels were significantly higher in cervical cancer tissues compared to adjacent normal tissues. High SIRT5 expression was associated with poor overall survival and positively correlated with tumor grade, indicating its potential role in cervical carcinogenesis and progression (Figure 1, Table 1). To further explore its biological functions, we generated stable Hela and Siha cell lines overexpressing or knocking down SIRT5. While SIRT5 had minimal impact on cell proliferation and colony formation (Figure 2), it significantly promoted cell migration and invasion by enhancing epithelial-mesenchymal transition (EMT) and stemness, as evidenced by increased tube and spheroid formation and upregulation of the stemness-related transcription factor SOX2 (Figures 3 and 4).\u003c/p\u003e\n\u003cp\u003eMechanistically, we demonstrated that SIRT5 activated the TGF-\u0026beta; signaling pathway, a critical regulator of tumor proliferation, metastasis, and stemness in cervical cancer. Overexpression of SIRT5 increased Smad2/3 phosphorylation and facilitated their nuclear translocation, as confirmed by immunofluorescence analysis (Figure 5). Furthermore, treatment with ITD-1, a TGF-\u0026beta; pathway inhibitor, effectively suppressed SIRT5-induced migration and invasion, highlighting the indispensable role of this pathway in SIRT5-mediated tumor progression.\u003c/p\u003e\n\u003cp\u003eTo validate these findings in vivo, we used a xenograft model with stable Siha cells overexpressing SIRT5. Although SIRT5 slightly reduced tumor growth with lower Ki67 expression, it significantly enhanced the expression of p-Smad2, the stemness marker Nanog, and the angiogenesis marker CD31, indicating its role in promoting stemness and angiogenesis in vivo (Figure 6). Collectively, our results reveal that SIRT5 contributes to cervical cancer metastasis and angiogenesis by activating the TGF-\u0026beta; signaling pathway, providing new insights into the molecular mechanisms of cervical cancer progression and identifying SIRT5 as a potential therapeutic target. Small-molecule inhibitors of SIRT5 or TGF-\u0026beta; pathway inhibitors, like ITD-1, could potentially reduce metastatic burden and improve patient outcomes. Additionally, the use of SIRT5 as a biomarker for aggressive cervical cancer warrants further clinical validation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, our study provides the first evidence that SIRT5 plays a critical role in cervical cancer progression by promoting metastasis, stemness, and angiogenesis. Mechanistically, SIRT5 exerts its pro-tumorigenic effects primarily through the activation of the TGF-\u0026beta; signaling pathway, which drives EMT and enhances stem-like properties. These findings not only shed light on the molecular mechanisms underlying SIRT5\u0026rsquo;s role in cervical cancer but also suggest that SIRT5 could serve as a potential biomarker and therapeutic target for managing cervical cancer metastasis and progression. Further studies are warranted to explore the detailed metabolic and signaling interactions regulated by SIRT5, as well as its clinical potential in cervical cancer treatment.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR\u0026apos;S CONTRIBUTION:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm their contribution to the paper as follows: FX wrote the main manuscript text. HH prepared figures 1-3, XJ prepared figures 4-6. FW and LW contributed equally to this work as last authors.All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study were approved by the Ethics Committee of Women\u0026apos;s Hospital, School of Medicine, Zhejiang University (Approval No. IRB-20230172-R).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eHuman and animal rights\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving human participants were in accordance with the ethical standards of institutional and/or research committee and with the 1975 Declaration of Helsinki, as revised in 2013.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from the patient for publication of this article and any accompanying images.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China, Grant No. 81672568; and the National Natural Science Foundation of China, Grant No. 32300460; and the Zhejiang Provincial Natural Science Foundation, Grant No. LQ23H160031\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest financial or otherwise.\u003c/p\u003e\n\u003cp\u003eAcknowledgment\u003c/p\u003e\n\u003cp\u003eDECLARED NONE.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eXu, M., Cao, C., Wu, P., Huang, X. \u0026amp; Ma, D. 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This study explored its expression, clinical relevance, and functions in cervical cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eWe systematically analyzed SIRT5 expression in cervical cancer tissues, assessed its correlation with patient survival, and investigated its functional roles through overexpression and knockdown experiments in vitro and in vivo.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eSIRT5 was significantly overexpressed in cervical cancer tissues and correlated with poor patient survival. Functionally, SIRT5 overexpression enhanced cell migration, invasion, and stemness phenotypes (tube and sphere formation), while its knockdown reversed these effects. Mechanistically, SIRT5 upregulated stemness markers SOX2 and Nanog and activated Smad2/3 phosphorylation. Inhibition of the TGF-β signaling pathway reversed these SIRT5-induced effects. In vivo, SIRT5-overexpressing xenografts showed increased p-Smad2, Nanog, and CD31 expression, promoting metastasis and angiogenesis, but tumor growth remained unchanged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThese findings suggest that SIRT5 facilitates cervical cancer metastasis and angiogenesis via TGF-β signaling, identifying it as a potential therapeutic target.\u003c/p\u003e","manuscriptTitle":"SIRT5 Sustains Stemness and Promotes Metastasis and Angiogenesis in Cervical Cancer via Activation of the TGF-β Signaling Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-18 06:14:46","doi":"10.21203/rs.3.rs-8614743/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3320b831-61ea-460f-875a-a5f5c5d52083","owner":[],"postedDate":"February 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-12T04:38:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-18 06:14:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8614743","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8614743","identity":"rs-8614743","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}