MiR-30c-5p targets SIRT1 to promote apoptosis in ovarian granulosa cells

MiR-30c-5p targets SIRT1 to promote apoptosis in ovarian granulosa cells | 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 MiR-30c-5p targets SIRT1 to promote apoptosis in ovarian granulosa cells Lifei Zhou, Bo Zheng, Yan Luo, Pingping Zhang, Fangfang Dai, mingming Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5752476/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 : Polycystic ovary syndrome (PCOS) is an endocrine and metabolic disorder characterized by abnormal follicular development and is the leading cause of infertility among women of reproductive age. miR-30c-5p, a newly discovered microRNA (miRNA), plays a crucial role in numerous pathological conditions, such as malignancies, reproductive dysfunctions, and metabolic irregularities. However, the role of miR-30c-5p in PCOS and the apoptosis of granulosa cells (GCs) is not fully understood. Research methods : GCs were isolated from the follicular fluid of 23 patients with PCOS and 21 normal controls. The expression of miR-30c-5p and Sirtuin 1 (SIRT1) was detected using quantitative real-time polymerase chain reaction (qRT-PCR). A Spearman correlation analysis was conducted to evaluate the relationship between the expression levels of these markers. Proliferation and apoptosis in human granulosa tumor cell lines (KGN) were analyzed following the overexpression or underexpression of miR-30c-5p, utilizing the Cell Counting Kit-8, flow cytometry, and western blot techniques. TargetScan was used to identify potential targets of miR-30c-5p, which were subsequently validated through qRT-PCR, western blot, and a dual luciferase reporter gene assay. Results : The results of our study suggest that the levels of miR-30c-5p aresignificantly elevated in individuals with PCOS compared with the control group. Conversely, we observed a significant decrease in the expression of SIRT1 in patients with PCOS compared with the control group. Moreover, miR-30c-5p expression was negatively correlated with SIRT1. The upregulation of miR-30c-5p suppressed the expression of SIRT1 and Bcl-2, inhibited GC proliferation, and increased the expression of Bax. Conclusion: Our findings suggest that miR-30c-5p inhibits the proliferation of KGN cells by targeting SIRT1. Thus, this study enhances the current understanding of the mechanisms underlying apoptosis in PCOS and GCs. miR-30c-5p SIRT1 PCOS Ovarian granulosa cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Polycystic ovary syndrome (PCOS) is the most prevalent endocrine and metabolic disorder, affecting 5–10% of women of reproductive age [1] . This syndrome is characterized by high androgen levels, ovulation disorders, polycystic ovaries, and insulin resistance (IR). In addition to clinical manifestations such as irregular menstruation, infertility, obesity, and hirsutism, women with PCOS have a higher susceptibility to metabolic diseases such as cardiovascular disease, hypertension, and diabetes [2–4] . Although lifestyle modifications, hormone therapy, and technologies such as in vitro fertilization and embryo transfer (IVF-ET) have notably improved pregnancy rates, outcomes still do not meet expectations. PCOS is influenced by genetic, environmental, and hormonal factors that significantly contribute to its development. Recent studies have highlighted the critical role of microRNAs (miRNAs) in the pathogenesis of PCOS, although the exact mechanisms are yet to be fully understood [5] . In the study of PCOS, granulosa cells (GCs) surrounding oocytes play a role not only in regulating follicular growth through the secretion of hormones and cytokines but also in other aspects [6] . These cells not only provide nutrients and oxygen to support the normal maturation of developing oocytes but also produce a series of signaling molecules that promote or inhibit follicular growth while interacting with other cells involved in follicle development [7–9] . However, abnormal function of GCs can cause various issues. For example, GC dysfunction in patients with PCOS may lead to impaired follicular development, ovulatory disorders, and infertility [10] . This is because the dysfunctional GCs do not provide adequate support and regulation to the developing oocyte. In addition, recent studies have confirmed these findings, observing that GC dysfunction in PCOS is associated with decreased cellular proliferation and increased apoptosis, which contribute to follicular growth atresia [11] . MiRNAs are a class of short-stranded non-coding RNAs that regulate the expression of transcribed genes during tissue differentiation and development, thereby impacting cellular function and metabolic status. Research has indicated that the abnormal expression of miRNAs is linked to the pathological progression of various ailments, including tumors, reproductive disorders, and metabolic disorders [12] . miR-30c-5p acts as a tumor suppressor in several malignancies, such as ovarian, endometrial, and papillary thyroid cancers, influencing cancer cell proliferation, migration, apoptosis, and angiogenesis [13–15] . Furthermore, some researchers have found that miR-30c-5p levels are significantly elevated in the blood, follicular fluid, and GCs of patients with PCOS, identified through sequencing [16–18] . However, the exact mechanism remains unclear. Sirtuin 1 (SIRT1), a deacetylase predominantly located in the nucleus, alters the function and localization of cellular proteins. Initially studied extensively as a longevity gene, its association with fertility defects was first discovered in 2003. SIRT1 regulates mitochondrial biogenesis, defense against oxidative stress, and energy homeostasis, thereby enhancing GC proliferation and spermatogenesis [19–20] . Some researchers have found that overexpression of miR-23a inhibits SIRT1 expression and increases apoptosis in human GCs [21] . Bioinformatics analysis has revealed complementary pairing between miR-30c-5p and the 3ʹ-untranslated region (UTR) of SIRT1 mRNA. However, the role of miR-30c-5p in regulating SIRT1 and its impact on the development of PCOS remain unclear. Therefore, this study aimed to explore the differential expression of miR-30c-5p and SIRT1 in GCs obtained from individuals with PCOS, as well as their role and significance in apoptosis. The findings from this research contribute to our understanding of the underlying mechanisms underlying abnormal proliferation and apoptosis in GCs associated with PCOS, offering valuable insights for both diagnosis and treatment approaches for PCOS. Materials and methods Patients and GC extraction This study included 23 individuals diagnosed with PCOS and 21 individuals without PCOS who underwent intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) at the Xingtai Fertility Hospital's Center for Reproductive Medicine. The Institutional Review Board of the Center for Reproductive Medicine, Xingtai Fertility Hospital, approved this study(202401). The controls consisted of 23 individuals diagnosed with PCOS based on the 2003 Rotterdam criteria [22] , alongside individuals who, despite their regular menstrual cycles and normal ovarian function, experienced infertility owing to tubal or male factors. Patients over the age of 35, those with abnormalities in the reproductive or endocrine systems, and those with chromosomal abnormalities were excluded. GCs collected from the enrolled patients were immediately stored at -80°C for subsequent quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis. Cell culture and transfection The human ovarian tumor GC line (KGN) was generously provided by the Hebei Maternal and Child Health Institute, China. These cell lines were cultured in DMEM/F12 (Gibco, USA) supplemented with 9% fetal bovine serum (Viva Cell, Shanghai, China) and 1% penicillin/streptomycin (Solarbio, Beijing, China). Cultivation was performed at 37°C in an incubator with a 5% CO 2 atmosphere. KGN cells were divided into four groups: miR-30c-5p mimic, miR-30c-5 mimic negative control (NC) miR-30c-5 inhibitor, and miR-30c-5p inhibitor NC (RUIBO, China). The miR-30c-5p mimic, miR-30c-5 mimic NC, miR-30c-5 inhibitor, and miR-30c-5p inhibitor NC were transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). After replacing the complete medium with DMEM (Gibco, USA), the cells were incubated at 37°C in a 5% CO 2 atmosphere for 6 h, after which the complete medium was replaced. qRTPCR analysis The miRNA and mRNA from the patients were extracted using kits from TianGen (China), and the RNA concentration and purity were assessed and found satisfactory. Subsequently, the RNA was converted to cDNA using the corresponding kits (TianGen, China), respectively. qRT-PCR (Life Technologies, Singapore) was performed under the following reaction conditions: an initial pre-denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 32 s. The relative expression levels of miRNA and mRNA were quantified using the 2 −ΔΔCT method, with GAPDH serving as the internal reference. Primers for miR-30c-5p and SIRT1 were designed and synthesized by RUIBO, while the forward primer for GAPDH was 5’-GGAGCGAGATCCCTCCAAAAT-3’, and the reverse primer was 5’-GGCTGTTGTCATACTTCTCATGGG-3’, both synthesized by TongYong (China). Western blotting The cells were treated with RIPA lysate containing protease inhibitors (Solarbio, Beijing, China) to extract total protein. After centrifugation, the resulting supernatant was utilized for the separation of proteins via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by their transfer onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% skim milk powder for 120 min and then incubated overnight at 4°C with primary antibodies SIRT1 (San Ying, 1:1500 dilution, Wuhan, China), GAPDH (San Ying, 1:15000 dilution, Wuhan, China), Bax (GB15690, Servicebio, China, 1:1000 dilution), Bcl-2 (GB154380, Servicebio, China, 1:1000 dilution), and β-actin (GB15003, Servicebio, China, 1:5000 dilution). This was followed by incubation with a horseradish peroxidase-labeled sheep anti-rabbit secondary antibody (ZhengNeng, 1:10000 dilution, China) at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence detection reagent and quantified densitometrically with ImageJ software(v.1.46, National Institute of Health of Bethesda, Bethesda, MD, USA). Dual luciferase assay SIRT1 was predicted to be a target of miR-30c-5p by TargetScan ( https://www.targetscan.org/vert ) and miRDB ( https://mirdb.org/ ). Wild-type (WT) and mutant (MUT) sequences of the SIRT1 3′-UTR target site were synthesized and then cloned into the pmirGLO vector. Log-phase 293T cells were seeded into 96-well plates and cultured overnight at 37°C in an atmosphere containing 5% CO 2 until transfection for 48 h. The following experimental groups were established: a blank group, pmirGLO + NC, pmirGLO + hsa-miR-30c-5p, SIRT1-WT + NC, SIRT1-WT + hsa-miR-30c-5p, SIRT1-MUT + NC, and SIRT1-MUT + hsa-miR-30c-5p. Each group consisted of three replicate wells, with 200 ng of plasmid per well and a final miRNA concentration of 100 nM per well. After 36 h of transfection, luciferase activity was measured using the Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA). Cell counting Kit-8 (CCK-8) assay Transfected granulocytes were seeded into 96-well plates at a density of 1500 cells/well. Four replicate plates were prepared to evaluate cell proliferation at 0, 24, 48, and 72 h, respectively. Each group within the plates consisted of five parallel wells. To each well, 100 µL of DMEM/F12 basal medium was added before placing the plates in a cell culture incubator for continued cultivation. After incubation, 10 µL of CCK-8 reagent (Shanghai, China) was added to each well, and the OD at 450 nm was measured using an enzyme marker. Cell apoptosis assay Forty-eight hours post-transfection, KGN cells were harvested using trypsin (Solarbio, Beijing, China), washed twice with 2 mL of pre-cooled sterile phosphate-buffered saline (PBS), and resuspended in the same buffer to a concentration of 1 × 10 6 cells/mL. The 5× Binding Buffer (MULTI SCIENCES, China) was diluted to a 1× working solution using double-distilled water, and 500 µL of this solution was used to resuspend the cells. Next, 5 µL of Annexin V-FITC and 10 µL of propidium iodide (PI) (both from MULTI SCIENCES, China) were added to the cell suspension. The mixture was incubated for 5 min in the dark at room temperature. Apoptotic cells were then detected using a flow cytometer with FITC and PI detection channels at excitation/emission wavelengths of 488 nm/530 nm and 535 nm/615 nm, respectively. The apoptosis rate was calculated as the sum of early and late apoptosis rates. Statistical analysis Statistical analysis was performed using SPSS software (version 22), with data expressed as mean ± standard deviation (SD). The Kolmogorov–Smirnov test was used to assess the normality of data distribution. Statistical significance was determined using the Student's t-test for normally distributed variables and the Mann–Whitney U test for non-parametric tests. A P-value < 0.05 was considered an indicator of statistical significance. Results Clinical and endocrine indices in patients with PCOS and controls Clinical and endocrine indices of patients with PCOS and controls are shown in Table 1 . No statistically significant differences were observed in age, estrogen (E2), and progesterone (P) between patients with PCOS and controls (P > 0.05 for all). Body mass index (BMI), basal luteinizing hormone (LH) levels, and testosterone (T) levels were significantly higher, while follicle-stimulating hormone and prolactin levels were significantly lower (P < 0.05 for all). Table 1 Clinical and endocrine parameters of patients with PCOS compared with controls. Basic parameters Control(n = 21) PCOS(n = 23) P- Value Age/(years) 28.86 ± 3.38 27.91 ± 3.69 0.405 BMI/(kg/m 2 ) 25.13 ± 4.05 26.85 ± 2.13 0.003* FSH(IU/L) 5.87 ± 1.02 5.79 ± 1.70 0.040* E2(pg/ml) 32.50(24.15,46.55) 35.10(26.42,46.60) 0.503 P(ng/ml) 0.37(0.24,0.48) 0.48(0.35,0.58) 0.095 PRL(ng/ml) 14.20(12.30,22.00) 11.00(7.71,16.10) 0.024* LH(IU/L) 3.81(3.07,5.60) 7.01(4.68,10.50) 0.007* T(ng/ml) 20.00(20.00,30.20) 34.00(25.70,46.00) 0.001* The data are represented as mean ± SD for normally distributed variables and as median with interquartile range for non-normally distributed variables. Statistical significance was defined as P < 0.05. miR-30c-5p expression is elevated and SIRT1 expression is decreased in GCs of patients with PCOS The qRT-PCR results indicated an increased expression of miR-30c-5p in patients with PCOS (Fig. 1 A), while the expression level of SIRT1 mRNA was reduced in the GCs of patients with PCOS compared with controls (Fig. 1 B). These results were consistent with those observed at the protein level (Figs. 1 C, 1 D). A Spearman correlation analysis was conducted to explore the relationship between miR-30c-5p and SIRT1 mRNA. The results showed that SIRT1 mRNA was negatively correlated with the expression of miR-30c-5p in the GCs of patients with PCOS (Fig. 1 E). miR-30c-5p inhibits cell proliferation and promotes apoptosis To investigate the functional role of miR-30c-5p in GCs from patients with PCOS, we established KGN cell lines stably expressing high levels of miR-30c-5p through transfection with a miR-30c-5p mimic, as confirmed in the graph (Fig. 2 A). CCK-8 experiments showed that cell viability in the miR-30c-5p mimic group decreased at 72 h post-transfection compared with cells in the miR-30c-5p mimic NC. In contrast, cells transfected with the miR-30c-5p inhibitor exhibited higher viability than those in the miR-30c-5p inhibitor NC group at 24, 48, and 72 h after transfection (Fig. 2 B, 2 C). Flow cytometry assays showed that miR-30c-5P increased the apoptosis rate in GCs compared with the miR-30c-5p mimic NC group, whereas the miR-30c-5p inhibitor group showed the opposite effect (Fig. 3 A). Furthermore, the miR-30c-5p mimic group exhibited upregulation of Bax expression and downregulation of Bcl-2 expression. The reverse pattern was observed in the miR-30c-5p inhibitor group (Figs. 4 A, 4 B). The results indicate that miR-30c-5p inhibits the proliferation of KGN cells and promotes apoptosis. SIRT1 is a predicted target of miR-30c-5p To elucidate the potential mechanism of miR-30c-5p in PCOS, we initially utilized TargetScan, miRDB, mirtarbase, and miRWalk websites to predict the target genes of miR-30c-5p. Both TargetScan and miRDB websites predicted that miR-30c-5P exhibits a complementary pairing sequence with the SIRT1 3ʹ-UTR region with complementary paired sequences (5A). SIRT1-WT + hsa-miR-30c-5p exhibited a significant decrease in luciferase activity compared with the SIRT1-WT + NC group, suggesting that hsa-miR-30c-5p may target SIRT1. Conversely, SIRT1-MUT + hsa-miR-30c-5p did not display significant changes in luciferase activity compared with the SIRT1-MUT + NC group, suggesting that the mutation site is critical for miR-30c-5p targeting SIRT1 (Figs. 5 B). Furthermore, to investigate the regulatory role of miR-30c-5p on SIRT1 expression, we performed both qRT-PCR and western blotting. Our results suggest that miR-30c-5p mimics downregulated the mRNA and protein levels of SIRT1, whereas inhibitors of miR-30c-5p upregulated the mRNA and protein levels of SIRT1 (Fig. 6 A, 6 B). These results suggest that miR-30c-5p may exert its effects through SIRT1 expression. Discussion The objective of this research was to explore the potential role of miR-30c-5p in the atypical proliferation of GCs in PCOS and to elucidate its underlying mechanisms. Our results showed that miR-30c-5p expression was significantly elevated in the GCs of infertile women with PCOS, while SIRT1 expression was significantly decreased and negatively correlated with miR-30c-5p levels in these patients. These findings validate our initial hypothesis. To verify the relationship between miR-30c-5p and SIRT1, we transfected KGN cells with miR-30c-5p mimics, miR-30c-5p inhibitors, and their respective NCs. The results showed that transfection with miR-30c-5p mimics resulted in decreased mRNA and protein expression of SIRT1 in KGN cells, whereas the miR-30c-5p inhibitor elevated both mRNA and protein levels of SIRT1 in KGN cells. Furthermore, miR-30c-5p directly targeted the 3ʹ-UTR of SIRT1 mRNA. Additionally, we observed decreased cell proliferation and increased apoptosis, including elevated Bax expression and decreased Bcl-2 expression, in the miR-30c-5p mimics group. On the contrary, the miR-30c-5p inhibitor group showed increased cell proliferation, decreased apoptosis rate, decreased Bax expression, and elevated Bcl-2 expression. Our findings provide evidence that miR-30c-5p can regulate the proliferation of KGN cells and promote apoptosis through the SIRT1 pathway in GCs. In recent years, an increasing number of studies have shown that the dysregulated role of miRNAs is a significant etiological factor in the pathogenesis of PCOS [23] . Liu et al. found that miR-96-5p is downregulated in the serum and GCs of patients with PCOS and polycystic mice. This downregulation inhibits the synthesis of estrogen and suppresses GC proliferation by targeting FOXO1 [24] . Du et al. found that miR-424 inhibits proliferation and promotes apoptosis in human ovarian GCs by directly targeting and inhibiting the expression of Apelin and APJ [25] . Wei et al. found that miR-874-3p expression is upregulated in PCOS and promotes testosterone-induced apoptosis of GCs through inhibition of HDAC1-mediated P53 deacetylation [26] . Therefore, miRNAs can influence the occurrence and development of PCOS through various pathways. Regarding miR-30c-5p, which is located on the short arm of chromosome 1 and the long arm of chromosome 6, most studies have focused on tumors and vascular smooth muscle, with relatively few investigations in PCOS [27] . Hu et al. found that miR-30c-5p inhibits oxidized LDL-induced inflammatory responses and apoptosis in human umbilical cord by downregulating the PTEN axis [28]. Liu et al. found that lidocaine inhibits oxidized LDL-induced venous endothelial cell inflammatory responses and apoptosis by oxidized low-density lipoprotein [28] . Liu et al. found that lidocaine inhibits proliferation and cisplatin resistance in cutaneous squamous cell carcinoma by regulating the miR-30c targeting of the SIRT1 pathway [29] . Yuan et al. found that upregulation of miR-30c-5p inhibits the growth, apoptosis, and migration of glioma cells by targeting Bcl-2 [30] . Zheng et al. reported that decreased miR-30c-5p expression promotes hepatocellular carcinoma progression by targeting RAB32 gene expression [31] . In addition, some researchers suggest that miR-30c-5p may be involved in the pathogenesis of PCOS based on sequencing and bioconfidence predictions. Another study reported that the level of miR-30c-5p is increased in the ovaries of polycystic mice, aligning with our findings in patients with polycystic ovaries [32–33] . However, the specific mechanism of its action has not yet been clarified. SIRT1 plays a crucial role in cell proliferation, autophagy, IR, and oxidative stress and serves as an important reproductive signaling pathway and regulator within the hypothalamic-pituitary-ovarian (HPO) axis [34] . Han et al. found that SIRT1 inhibits GC apoptosis by activating both the extracellular signal-regulated kinase1/2(ERK1/2 )and NF-kappa B(NF-κB)signaling pathways [35] . Wu et al. found that naringin supplementation improves IR and hormone levels, attenuates pathological changes in ovarian tissues, and increases SIRT1 expression in polycystic rats [36] . Huo et al. observed that resveratrol promotes follicular development in PCOS rats by upregulating SIRT1 expression via the glycolytic pathway [37] . Contrarily, another study found that while the expression of SIRT1 is significantly increased in patients over 40 years of age, it is reduced in patients with PCOS [38] . Consistent with these studies, our present study revealed a reduction in SIRT1 expression in GC tissues of patients with PCOS. Moreover, we found that the overexpression of miR-30c-5p inhibits SIRT1 expression. The aim of this study was to elucidate the functions of miR-30c-5p and SIRT1, as well as their relationship in the GCs of patients with PCOS. However, our study has some limitations. Firstly, the cells used were derived from cell lines, which cannot fully represent the cellular status of patients with PCOS. Secondly, the experimental conclusions were not validated in animal models. Therefore, future experimental designs should include the construction of animal models to more thoroughly investigate the correlation between miR-30c-5p, SIRT1, and PCOS. Summary We observed that miR-30c-5p expression was significantly elevated in patients with PCOS and negatively correlated with reduced SIRT1 levels, potentially impacting follicle development and oocyte quality in patients. In addition, cellular experiments demonstrated that overexpression of miR-30c-5p promoted apoptosis in KGN cells and inhibited the proliferation of GCs by targeting SIRT1. These findings suggest that miR-30c-5p may play a role in the pathophysiological mechanisms and progression of PCOS. These results provide a novel target for the treatment of PCOS. Declarations Acknowledgements The authors thank all individuals who participated in this study and supported this research. Author contributions LFZ: Analysis of data and manuscript writing, YL and MMZ : Data analysis and manuscript revision. PPZ : Validation, Supervision, Resources, Data curation. FFD and BZ: Sample collection. YLL: Project administration, Funding acquisition. Funding This work was supported by the 2025 Government-funded Training Program for Excellence in Clinical Medicine (ZF2025019) vailability of data and materials The data is available in reasonable circumstances Ethical approval All experiments and the ovarian granulosa cells used were approved by the Ethics Committee of Hebei Xingtai Fertility Hospital 2024 (Approval No. 202401). Informed consent Follicular fluid and clinical data were collected with the informed consent of patients. Competing interests The authors declare no competing interests. References Yu Z, Li Y, Zhao S, Liu F, Zhao H, Chen ZJ. Evidence of positive selection of genetic variants associated with PCOS. Hum Reprod. 2023 Nov 20;38(Supplement_2):ii57-ii68. He T, Sun Y, Zhang Y, Zhao S, Zheng Y, Hao G, Shi Y. MicroRNA-200b and microRNA-200c are up-regulated in PCOS granulosa cell and inhibit KGN cell proliferation via targeting PTEN. Reprod Biol Endocrinol. 2019 Aug 17;17(1):68. Li Y, Wu X, Miao S, Cao Q. 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Yuan, LQ, Zhang, T, Xu, L, et al. miR-30c-5p inhibits glioma proliferation and invasion via targeting Bcl2. TRANSL CANCER RES. 2021; 10 (1): 337-348. He, Z, Tian, M, Fu, X. Reduced expression of miR-30c-5p promotes hepatocellular carcinoma progression by targeting RAB32. Mol Ther Nucleic Acids. 2021; 26 603-612. De Nardo Maffazioli, G, Baracat, EC, Soares, JM, et al. Evaluation of circulating microRNA profiles in Brazilian women with polycystic ovary syndrome: A preliminary study. PLoS One. 2022; 17 (10): e0275031. Hu, J, Lin, F, Yin, Y, et al. Adipocyte-derived exosomal miR-30c-5p promotes ovarian angiogenesis in polycystic ovary syndrome via the SOCS3/STAT3/VEGFA pathway. J STEROID BIOCHEM. 2023; 230 106278. Li, X, He, Y, Wu, S, et al. Regulation of SIRT1 in Ovarian Function: PCOS Treatment. CURR ISSUES MOL BIOL. 2023; 45 (3): 2073-2089. Han, Y.; Luo, H.; Wang, H.; Cai, J.; Zhang, Y. SIRT1 induces resistance to apoptosis in human granulosa cells by activating the ERK pathway and inhibiting NF-κB signaling with anti-inflammatory functions. Apoptosis 2017, 22, 1260–1272 Wu, YX, Yang, XY, Han, BS, et al. Naringenin regulates gut microbiota and SIRT1/ PGC-1ɑ signaling pathway in rats with letrozole-induced polycystic ovary syndrome. BIOMED PHARMACOTHER. 2022; 153 113286. Huo, P, Li, M, Le, J, et al. Resveratrol improves follicular development of PCOS rats via regulating glycolysis pathway and targeting SIRT1. SYST BIOL REPROD MED. 2022; 69 (2): 153-165. González-Fernández, R, Martín-Ramírez, R, Rotoli, D, et al. Granulosa-Lutein Cell Sirtuin Gene Expression Profiles Differ between Normal Donors and Infertile Women. Int J Mol Sci. 2019; 21 (1): Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5752476","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":397347880,"identity":"a70bcec9-27d1-44a9-b66f-f3ce2f294573","order_by":0,"name":"Lifei Zhou","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lifei","middleName":"","lastName":"Zhou","suffix":""},{"id":397347881,"identity":"2369608c-a8d3-41cf-8a74-91b61db2ca78","order_by":1,"name":"Bo Zheng","email":"","orcid":"","institution":"Xingtai infertility Specialist Hospital","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Zheng","suffix":""},{"id":397347882,"identity":"f96c5e77-c366-4479-9bd3-c2459dc559bb","order_by":2,"name":"Yan Luo","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Luo","suffix":""},{"id":397347883,"identity":"d124b8f1-9a30-490c-a6ac-078d80d5c04a","order_by":3,"name":"Pingping Zhang","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pingping","middleName":"","lastName":"Zhang","suffix":""},{"id":397347884,"identity":"7f30a2a5-9cbd-4354-b94a-378273d837af","order_by":4,"name":"Fangfang Dai","email":"","orcid":"","institution":"Xingtai infertility Specialist Hospital","correspondingAuthor":false,"prefix":"","firstName":"Fangfang","middleName":"","lastName":"Dai","suffix":""},{"id":397347885,"identity":"bae1226a-618b-44a7-9a54-60e2b0e0ec51","order_by":5,"name":"mingming Zhang","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"prefix":"","firstName":"mingming","middleName":"","lastName":"Zhang","suffix":""},{"id":397347886,"identity":"8750edb6-522e-47a7-8ac4-ab0c6dd38252","order_by":6,"name":"Yali Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApUlEQVRIiWNgGAWjYBACPmYQWSAhx8BMrBY2sEoDCWMStIBJA4bEBqIdxsbOwCbBYGCRPr+d9+AHhhqbaGIcBtIikbvhMF+yBMOxtFyC1iG0MPMYSDA2HCZeS7p8M4/xD5K0JDAc5jEj2hZmC6AWww1ALRYJxPiFn/8A4w2Gijp5+f4zxjc+1NgQ1gLU9EX6D4ydQFg5GDB/IFLhKBgFo2AUjFQAAFD3KcLHFVVhAAAAAElFTkSuQmCC","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yali","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-01-02 14:23:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5752476/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5752476/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73086951,"identity":"c3b2a662-106b-4827-9b32-24f6db0396d2","added_by":"auto","created_at":"2025-01-06 14:57:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":839407,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of miR-30c-5p and SIRT1 in GCs of PCOS patients. A miR-30c-5p expression was elevated in GCs of PCOS patients. B SIRT1 mRNA expression was decreased in GCs of PCOS patients. C-D Protein expression of SIRT1 was decreased in PCOS patients. E Evaluated by using Spearman's correlation analysis. The correlation between miR-30c-5p and SIRT1. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared with the non-PCOS group\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5752476/v1/96549994b12b3e06d9cac736.png"},{"id":73086962,"identity":"4ae6a120-8e94-48b1-8cc8-f6fdbe4b400d","added_by":"auto","created_at":"2025-01-06 14:57:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1178468,"visible":true,"origin":"","legend":"\u003cp\u003emiR-30c-5p inhibits cell proliferation and promotes apoptosis. A levels of miR-30c-5p in each group after transfection, detected by qRT-PCR. B-C cell proliferation was performed using CCK-8 assay at 24, 48, and 72 hours after cell transfection. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5752476/v1/978a1cd1fd91d7aaeae54f8a.png"},{"id":73087436,"identity":"786200b8-7737-4b06-840a-438c8831deb3","added_by":"auto","created_at":"2025-01-06 15:05:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":781394,"visible":true,"origin":"","legend":"\u003cp\u003eA apoptosis rates of KGN cells in each group were detected by flow cytometry. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5752476/v1/081f6a3419213d63c48bb6dd.png"},{"id":73086950,"identity":"f50f7da8-abc5-4a32-81cb-2ca6dcccaa03","added_by":"auto","created_at":"2025-01-06 14:57:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":516738,"visible":true,"origin":"","legend":"\u003cp\u003eA-B Bax and Bcl- 2 protein expression. Compared with the corresponding negative control, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5752476/v1/ed6117df5f114863021ba59e.png"},{"id":73086984,"identity":"09d9fea4-d1e5-4acf-b188-b1f183e40502","added_by":"auto","created_at":"2025-01-06 14:57:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":419057,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eSIRT1 is a regulatory target of miR-30c-5p B A dual luciferase reporter assay was used to confirm the relationship between miR-30c- 5 p and SIRT1. Data are expressed as mean ±SD; Statistical analysis was carried out using Student's t-tests. ***P=0.0009; NS, not significant.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5752476/v1/b15a243600313d7122fc1407.png"},{"id":73086961,"identity":"0fbd8378-1031-43bf-a64f-81dfca0b898b","added_by":"auto","created_at":"2025-01-06 14:57:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2420978,"visible":true,"origin":"","legend":"\u003cp\u003eSIRT1 is a direct target of miR-30c-5p. A SIRT1 mRNA expression was assessed in cells transfected with miR-30c-5p overexpression or knockdown using qRT-PCR analysis. B SIRT1 protein expression was assessed in cells transfected withmiR-30c-5p overexpression or knockdown using protein blotting analysis. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 compared with the corresponding negative control.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5752476/v1/351e02ea946a7a034ae13746.png"},{"id":74945925,"identity":"79fed5c0-9709-4f29-987d-ce2e03a74987","added_by":"auto","created_at":"2025-01-28 15:32:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7438989,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5752476/v1/a4ef8155-bbbf-4170-bba8-5e524521a09d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"MiR-30c-5p targets SIRT1 to promote apoptosis in ovarian granulosa cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePolycystic ovary syndrome (PCOS) is the most prevalent endocrine and metabolic disorder, affecting 5\u0026ndash;10% of women of reproductive age \u003csup\u003e[1]\u003c/sup\u003e. This syndrome is characterized by high androgen levels, ovulation disorders, polycystic ovaries, and insulin resistance (IR). In addition to clinical manifestations such as irregular menstruation, infertility, obesity, and hirsutism, women with PCOS have a higher susceptibility to metabolic diseases such as cardiovascular disease, hypertension, and diabetes \u003csup\u003e[2\u0026ndash;4]\u003c/sup\u003e. Although lifestyle modifications, hormone therapy, and technologies such as in vitro fertilization and embryo transfer (IVF-ET) have notably improved pregnancy rates, outcomes still do not meet expectations. PCOS is influenced by genetic, environmental, and hormonal factors that significantly contribute to its development. Recent studies have highlighted the critical role of microRNAs (miRNAs) in the pathogenesis of PCOS, although the exact mechanisms are yet to be fully understood \u003csup\u003e[5]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the study of PCOS, granulosa cells (GCs) surrounding oocytes play a role not only in regulating follicular growth through the secretion of hormones and cytokines but also in other aspects \u003csup\u003e[6]\u003c/sup\u003e. These cells not only provide nutrients and oxygen to support the normal maturation of developing oocytes but also produce a series of signaling molecules that promote or inhibit follicular growth while interacting with other cells involved in follicle development \u003csup\u003e[7\u0026ndash;9]\u003c/sup\u003e. However, abnormal function of GCs can cause various issues. For example, GC dysfunction in patients with PCOS may lead to impaired follicular development, ovulatory disorders, and infertility \u003csup\u003e[10]\u003c/sup\u003e. This is because the dysfunctional GCs do not provide adequate support and regulation to the developing oocyte. In addition, recent studies have confirmed these findings, observing that GC dysfunction in PCOS is associated with decreased cellular proliferation and increased apoptosis, which contribute to follicular growth atresia \u003csup\u003e[11]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMiRNAs are a class of short-stranded non-coding RNAs that regulate the expression of transcribed genes during tissue differentiation and development, thereby impacting cellular function and metabolic status. Research has indicated that the abnormal expression of miRNAs is linked to the pathological progression of various ailments, including tumors, reproductive disorders, and metabolic disorders\u003csup\u003e[12]\u003c/sup\u003e. miR-30c-5p acts as a tumor suppressor in several malignancies, such as ovarian, endometrial, and papillary thyroid cancers, influencing cancer cell proliferation, migration, apoptosis, and angiogenesis \u003csup\u003e[13\u0026ndash;15]\u003c/sup\u003e. Furthermore, some researchers have found that miR-30c-5p levels are significantly elevated in the blood, follicular fluid, and GCs of patients with PCOS, identified through sequencing \u003csup\u003e[16\u0026ndash;18]\u003c/sup\u003e. However, the exact mechanism remains unclear.\u003c/p\u003e \u003cp\u003eSirtuin 1 (SIRT1), a deacetylase predominantly located in the nucleus, alters the function and localization of cellular proteins. Initially studied extensively as a longevity gene, its association with fertility defects was first discovered in 2003. SIRT1 regulates mitochondrial biogenesis, defense against oxidative stress, and energy homeostasis, thereby enhancing GC proliferation and spermatogenesis \u003csup\u003e[19\u0026ndash;20]\u003c/sup\u003e. Some researchers have found that overexpression of miR-23a inhibits SIRT1 expression and increases apoptosis in human GCs \u003csup\u003e[21]\u003c/sup\u003e. Bioinformatics analysis has revealed complementary pairing between miR-30c-5p and the 3ʹ-untranslated region (UTR) of SIRT1 mRNA. However, the role of miR-30c-5p in regulating SIRT1 and its impact on the development of PCOS remain unclear. Therefore, this study aimed to explore the differential expression of miR-30c-5p and SIRT1 in GCs obtained from individuals with PCOS, as well as their role and significance in apoptosis. The findings from this research contribute to our understanding of the underlying mechanisms underlying abnormal proliferation and apoptosis in GCs associated with PCOS, offering valuable insights for both diagnosis and treatment approaches for PCOS.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients and GC extraction\u003c/h2\u003e \u003cp\u003eThis study included 23 individuals diagnosed with PCOS and 21 individuals without PCOS who underwent intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) at the Xingtai Fertility Hospital's Center for Reproductive Medicine. The Institutional Review Board of the Center for Reproductive Medicine, Xingtai Fertility Hospital, approved this study(202401). The controls consisted of 23 individuals diagnosed with PCOS based on the 2003 Rotterdam criteria \u003csup\u003e[22]\u003c/sup\u003e, alongside individuals who, despite their regular menstrual cycles and normal ovarian function, experienced infertility owing to tubal or male factors. Patients over the age of 35, those with abnormalities in the reproductive or endocrine systems, and those with chromosomal abnormalities were excluded. GCs collected from the enrolled patients were immediately stored at -80\u0026deg;C for subsequent quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture and transfection\u003c/h3\u003e\n\u003cp\u003eThe human ovarian tumor GC line (KGN) was generously provided by the Hebei Maternal and Child Health Institute, China. These cell lines were cultured in DMEM/F12 (Gibco, USA) supplemented with 9% fetal bovine serum (Viva Cell, Shanghai, China) and 1% penicillin/streptomycin (Solarbio, Beijing, China). Cultivation was performed at 37\u0026deg;C in an incubator with a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere.\u003c/p\u003e \u003cp\u003eKGN cells were divided into four groups: miR-30c-5p mimic, miR-30c-5 mimic negative control (NC) miR-30c-5 inhibitor, and miR-30c-5p inhibitor NC (RUIBO, China). The miR-30c-5p mimic, miR-30c-5 mimic NC, miR-30c-5 inhibitor, and miR-30c-5p inhibitor NC were transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). After replacing the complete medium with DMEM (Gibco, USA), the cells were incubated at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere for 6 h, after which the complete medium was replaced.\u003c/p\u003e\n\u003ch3\u003eqRTPCR analysis\u003c/h3\u003e\n\u003cp\u003eThe miRNA and mRNA from the patients were extracted using kits from TianGen (China), and the RNA concentration and purity were assessed and found satisfactory. Subsequently, the RNA was converted to cDNA using the corresponding kits (TianGen, China), respectively. qRT-PCR (Life Technologies, Singapore) was performed under the following reaction conditions: an initial pre-denaturation at 95\u0026deg;C for 15 min, followed by 40 cycles of denaturation at 95\u0026deg;C for 10 s, annealing at 60\u0026deg;C for 20 s, and extension at 72\u0026deg;C for 32 s. The relative expression levels of miRNA and mRNA were quantified using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method, with GAPDH serving as the internal reference. Primers for miR-30c-5p and SIRT1 were designed and synthesized by RUIBO, while the forward primer for GAPDH was 5\u0026rsquo;-GGAGCGAGATCCCTCCAAAAT-3\u0026rsquo;, and the reverse primer was 5\u0026rsquo;-GGCTGTTGTCATACTTCTCATGGG-3\u0026rsquo;, both synthesized by TongYong (China).\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eThe cells were treated with RIPA lysate containing protease inhibitors (Solarbio, Beijing, China) to extract total protein. After centrifugation, the resulting supernatant was utilized for the separation of proteins via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by their transfer onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% skim milk powder for 120 min and then incubated overnight at 4\u0026deg;C with primary antibodies SIRT1 (San Ying, 1:1500 dilution, Wuhan, China), GAPDH (San Ying, 1:15000 dilution, Wuhan, China), Bax (GB15690, Servicebio, China, 1:1000 dilution), Bcl-2 (GB154380, Servicebio, China, 1:1000 dilution), and β-actin (GB15003, Servicebio, China, 1:5000 dilution). This was followed by incubation with a horseradish peroxidase-labeled sheep anti-rabbit secondary antibody (ZhengNeng, 1:10000 dilution, China) at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence detection reagent and quantified densitometrically with ImageJ software(v.1.46, National Institute of Health of Bethesda, Bethesda, MD, USA).\u003c/p\u003e\n\u003ch3\u003eDual luciferase assay\u003c/h3\u003e\n\u003cp\u003eSIRT1 was predicted to be a target of miR-30c-5p by TargetScan (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.targetscan.org/vert\u003c/span\u003e\u003cspan address=\"https://www.targetscan.org/vert\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and miRDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mirdb.org/\u003c/span\u003e\u003cspan address=\"https://mirdb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Wild-type (WT) and mutant (MUT) sequences of the SIRT1 3\u0026prime;-UTR target site were synthesized and then cloned into the pmirGLO vector. Log-phase 293T cells were seeded into 96-well plates and cultured overnight at 37\u0026deg;C in an atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e until transfection for 48 h. The following experimental groups were established: a blank group, pmirGLO\u0026thinsp;+\u0026thinsp;NC, pmirGLO\u0026thinsp;+\u0026thinsp;hsa-miR-30c-5p, SIRT1-WT\u0026thinsp;+\u0026thinsp;NC, SIRT1-WT\u0026thinsp;+\u0026thinsp;hsa-miR-30c-5p, SIRT1-MUT\u0026thinsp;+\u0026thinsp;NC, and SIRT1-MUT\u0026thinsp;+\u0026thinsp;hsa-miR-30c-5p. Each group consisted of three replicate wells, with 200 ng of plasmid per well and a final miRNA concentration of 100 nM per well. After 36 h of transfection, luciferase activity was measured using the Dual-Luciferase\u0026reg; Reporter Assay System (Promega, Madison, WI, USA).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell counting Kit-8 (CCK-8) assay\u003c/h2\u003e \u003cp\u003eTransfected granulocytes were seeded into 96-well plates at a density of 1500 cells/well. Four replicate plates were prepared to evaluate cell proliferation at 0, 24, 48, and 72 h, respectively. Each group within the plates consisted of five parallel wells. To each well, 100 \u0026micro;L of DMEM/F12 basal medium was added before placing the plates in a cell culture incubator for continued cultivation. After incubation, 10 \u0026micro;L of CCK-8 reagent (Shanghai, China) was added to each well, and the OD at 450 nm was measured using an enzyme marker.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell apoptosis assay\u003c/h3\u003e\n\u003cp\u003eForty-eight hours post-transfection, KGN cells were harvested using trypsin (Solarbio, Beijing, China), washed twice with 2 mL of pre-cooled sterile phosphate-buffered saline (PBS), and resuspended in the same buffer to a concentration of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL. The 5\u0026times; Binding Buffer (MULTI SCIENCES, China) was diluted to a 1\u0026times; working solution using double-distilled water, and 500 \u0026micro;L of this solution was used to resuspend the cells. Next, 5 \u0026micro;L of Annexin V-FITC and 10 \u0026micro;L of propidium iodide (PI) (both from MULTI SCIENCES, China) were added to the cell suspension. The mixture was incubated for 5 min in the dark at room temperature. Apoptotic cells were then detected using a flow cytometer with FITC and PI detection channels at excitation/emission wavelengths of 488 nm/530 nm and 535 nm/615 nm, respectively. The apoptosis rate was calculated as the sum of early and late apoptosis rates.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using SPSS software (version 22), with data expressed as mean \u0026plusmn; standard deviation (SD). The Kolmogorov\u0026ndash;Smirnov test was used to assess the normality of data distribution. Statistical significance was determined using the Student's t-test for normally distributed variables and the Mann\u0026ndash;Whitney U test for non-parametric tests. A P-value \u0026lt; 0.05 was considered an indicator of statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eClinical and endocrine indices in patients with PCOS and controls\u003c/h2\u003e \u003cp\u003eClinical and endocrine indices of patients with PCOS and controls are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. No statistically significant differences were observed in age, estrogen (E2), and progesterone (P) between patients with PCOS and controls (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05 for all). Body mass index (BMI), basal luteinizing hormone (LH) levels, and testosterone (T) levels were significantly higher, while follicle-stimulating hormone and prolactin levels were significantly lower (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical and endocrine parameters of patients with PCOS compared with controls.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasic parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl(n\u0026thinsp;=\u0026thinsp;21)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePCOS(n\u0026thinsp;=\u0026thinsp;23)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP-\u003c/em\u003eValue\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge/(years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.86\u0026thinsp;\u0026plusmn;\u0026thinsp;3.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.91\u0026thinsp;\u0026plusmn;\u0026thinsp;3.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.405\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI/(kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.13\u0026thinsp;\u0026plusmn;\u0026thinsp;4.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.003*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFSH(IU/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.040*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE2(pg/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32.50(24.15,46.55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.10(26.42,46.60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.503\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP(ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.37(0.24,0.48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.48(0.35,0.58)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.095\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePRL(ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.20(12.30,22.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.00(7.71,16.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.024*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLH(IU/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.81(3.07,5.60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.01(4.68,10.50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.007*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT(ng/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.00(20.00,30.20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.00(25.70,46.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe data are represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for normally distributed variables and as median with interquartile range for non-normally distributed variables. Statistical significance was defined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003emiR-30c-5p expression is elevated and SIRT1 expression is decreased in GCs of patients with PCOS\u003c/h2\u003e \u003cp\u003eThe qRT-PCR results indicated an increased expression of miR-30c-5p in patients with PCOS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), while the expression level of SIRT1 mRNA was reduced in the GCs of patients with PCOS compared with controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These results were consistent with those observed at the protein level (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC,\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). A Spearman correlation analysis was conducted to explore the relationship between miR-30c-5p and SIRT1 mRNA. The results showed that SIRT1 mRNA was negatively correlated with the expression of miR-30c-5p in the GCs of patients with PCOS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003emiR-30c-5p inhibits cell proliferation and promotes apoptosis\u003c/h2\u003e \u003cp\u003eTo investigate the functional role of miR-30c-5p in GCs from patients with PCOS, we established KGN cell lines stably expressing high levels of miR-30c-5p through transfection with a miR-30c-5p mimic, as confirmed in the graph (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). CCK-8 experiments showed that cell viability in the miR-30c-5p mimic group decreased at 72 h post-transfection compared with cells in the miR-30c-5p mimic NC. In contrast, cells transfected with the miR-30c-5p inhibitor exhibited higher viability than those in the miR-30c-5p inhibitor NC group at 24, 48, and 72 h after transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Flow cytometry assays showed that miR-30c-5P increased the apoptosis rate in GCs compared with the miR-30c-5p mimic NC group, whereas the miR-30c-5p inhibitor group showed the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, the miR-30c-5p mimic group exhibited upregulation of Bax expression and downregulation of Bcl-2 expression. The reverse pattern was observed in the miR-30c-5p inhibitor group (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The results indicate that miR-30c-5p inhibits the proliferation of KGN cells and promotes apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSIRT1 is a predicted target of miR-30c-5p\u003c/h2\u003e \u003cp\u003eTo elucidate the potential mechanism of miR-30c-5p in PCOS, we initially utilized TargetScan, miRDB, mirtarbase, and miRWalk websites to predict the target genes of miR-30c-5p. Both TargetScan and miRDB websites predicted that miR-30c-5P exhibits a complementary pairing sequence with the SIRT1 3ʹ-UTR region with complementary paired sequences (5A). SIRT1-WT\u0026thinsp;+\u0026thinsp;hsa-miR-30c-5p exhibited a significant decrease in luciferase activity compared with the SIRT1-WT\u0026thinsp;+\u0026thinsp;NC group, suggesting that hsa-miR-30c-5p may target SIRT1. Conversely, SIRT1-MUT\u0026thinsp;+\u0026thinsp;hsa-miR-30c-5p did not display significant changes in luciferase activity compared with the SIRT1-MUT\u0026thinsp;+\u0026thinsp;NC group, suggesting that the mutation site is critical for miR-30c-5p targeting SIRT1 (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Furthermore, to investigate the regulatory role of miR-30c-5p on SIRT1 expression, we performed both qRT-PCR and western blotting. Our results suggest that miR-30c-5p mimics downregulated the mRNA and protein levels of SIRT1, whereas inhibitors of miR-30c-5p upregulated the mRNA and protein levels of SIRT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These results suggest that miR-30c-5p may exert its effects through SIRT1 expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe objective of this research was to explore the potential role of miR-30c-5p in the atypical proliferation of GCs in PCOS and to elucidate its underlying mechanisms. Our results showed that miR-30c-5p expression was significantly elevated in the GCs of infertile women with PCOS, while SIRT1 expression was significantly decreased and negatively correlated with miR-30c-5p levels in these patients. These findings validate our initial hypothesis. To verify the relationship between miR-30c-5p and SIRT1, we transfected KGN cells with miR-30c-5p mimics, miR-30c-5p inhibitors, and their respective NCs. The results showed that transfection with miR-30c-5p mimics resulted in decreased mRNA and protein expression of SIRT1 in KGN cells, whereas the miR-30c-5p inhibitor elevated both mRNA and protein levels of SIRT1 in KGN cells. Furthermore, miR-30c-5p directly targeted the 3ʹ-UTR of SIRT1 mRNA. Additionally, we observed decreased cell proliferation and increased apoptosis, including elevated Bax expression and decreased Bcl-2 expression, in the miR-30c-5p mimics group. On the contrary, the miR-30c-5p inhibitor group showed increased cell proliferation, decreased apoptosis rate, decreased Bax expression, and elevated Bcl-2 expression. Our findings provide evidence that miR-30c-5p can regulate the proliferation of KGN cells and promote apoptosis through the SIRT1 pathway in GCs.\u003c/p\u003e \u003cp\u003eIn recent years, an increasing number of studies have shown that the dysregulated role of miRNAs is a significant etiological factor in the pathogenesis of PCOS \u003csup\u003e[23]\u003c/sup\u003e. Liu et al. found that miR-96-5p is downregulated in the serum and GCs of patients with PCOS and polycystic mice. This downregulation inhibits the synthesis of estrogen and suppresses GC proliferation by targeting FOXO1 \u003csup\u003e[24]\u003c/sup\u003e. Du et al. found that miR-424 inhibits proliferation and promotes apoptosis in human ovarian GCs by directly targeting and inhibiting the expression of Apelin and APJ \u003csup\u003e[25]\u003c/sup\u003e. Wei et al. found that miR-874-3p expression is upregulated in PCOS and promotes testosterone-induced apoptosis of GCs through inhibition of HDAC1-mediated P53 deacetylation \u003csup\u003e[26]\u003c/sup\u003e. Therefore, miRNAs can influence the occurrence and development of PCOS through various pathways. Regarding miR-30c-5p, which is located on the short arm of chromosome 1 and the long arm of chromosome 6, most studies have focused on tumors and vascular smooth muscle, with relatively few investigations in PCOS \u003csup\u003e[27]\u003c/sup\u003e. Hu et al. found that miR-30c-5p inhibits oxidized LDL-induced inflammatory responses and apoptosis in human umbilical cord by downregulating the PTEN axis [28]. Liu et al. found that lidocaine inhibits oxidized LDL-induced venous endothelial cell inflammatory responses and apoptosis by oxidized low-density lipoprotein \u003csup\u003e[28]\u003c/sup\u003e. Liu et al. found that lidocaine inhibits proliferation and cisplatin resistance in cutaneous squamous cell carcinoma by regulating the miR-30c targeting of the SIRT1 pathway \u003csup\u003e[29]\u003c/sup\u003e. Yuan et al. found that upregulation of miR-30c-5p inhibits the growth, apoptosis, and migration of glioma cells by targeting Bcl-2 \u003csup\u003e[30]\u003c/sup\u003e. Zheng et al. reported that decreased miR-30c-5p expression promotes hepatocellular carcinoma progression by targeting RAB32 gene expression \u003csup\u003e[31]\u003c/sup\u003e. In addition, some researchers suggest that miR-30c-5p may be involved in the pathogenesis of PCOS based on sequencing and bioconfidence predictions. Another study reported that the level of miR-30c-5p is increased in the ovaries of polycystic mice, aligning with our findings in patients with polycystic ovaries \u003csup\u003e[32\u0026ndash;33]\u003c/sup\u003e. However, the specific mechanism of its action has not yet been clarified.\u003c/p\u003e \u003cp\u003eSIRT1 plays a crucial role in cell proliferation, autophagy, IR, and oxidative stress and serves as an important reproductive signaling pathway and regulator within the hypothalamic-pituitary-ovarian (HPO) axis \u003csup\u003e[34]\u003c/sup\u003e. Han et al. found that SIRT1 inhibits GC apoptosis by activating both the extracellular signal-regulated kinase1/2(ERK1/2 )and NF-kappa B(NF-κB)signaling pathways \u003csup\u003e[35]\u003c/sup\u003e. Wu et al. found that naringin supplementation improves IR and hormone levels, attenuates pathological changes in ovarian tissues, and increases SIRT1 expression in polycystic rats \u003csup\u003e[36]\u003c/sup\u003e. Huo et al. observed that resveratrol promotes follicular development in PCOS rats by upregulating SIRT1 expression via the glycolytic pathway \u003csup\u003e[37]\u003c/sup\u003e. Contrarily, another study found that while the expression of SIRT1 is significantly increased in patients over 40 years of age, it is reduced in patients with PCOS \u003csup\u003e[38]\u003c/sup\u003e. Consistent with these studies, our present study revealed a reduction in SIRT1 expression in GC tissues of patients with PCOS. Moreover, we found that the overexpression of miR-30c-5p inhibits SIRT1 expression.\u003c/p\u003e \u003cp\u003eThe aim of this study was to elucidate the functions of miR-30c-5p and SIRT1, as well as their relationship in the GCs of patients with PCOS. However, our study has some limitations. Firstly, the cells used were derived from cell lines, which cannot fully represent the cellular status of patients with PCOS. Secondly, the experimental conclusions were not validated in animal models. Therefore, future experimental designs should include the construction of animal models to more thoroughly investigate the correlation between miR-30c-5p, SIRT1, and PCOS.\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSummary\u003c/h2\u003e \u003cp\u003eWe observed that miR-30c-5p expression was significantly elevated in patients with PCOS and negatively correlated with reduced SIRT1 levels, potentially impacting follicle development and oocyte quality in patients. In addition, cellular experiments demonstrated that overexpression of miR-30c-5p promoted apoptosis in KGN cells and inhibited the proliferation of GCs by targeting SIRT1. These findings suggest that miR-30c-5p may play a role in the pathophysiological mechanisms and progression of PCOS. These results provide a novel target for the treatment of PCOS.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors thank all individuals who participated in this study and supported this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLFZ: Analysis of data and manuscript writing, YL and MMZ : Data analysis and manuscript revision. PPZ : Validation, Supervision, Resources, Data curation. FFD and BZ: Sample collection. YLL: Project administration, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the 2025 Government-funded Training Program for Excellence in Clinical Medicine (ZF2025019)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003evailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data is available in reasonable circumstances\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All experiments and the ovarian granulosa cells used were approved by the Ethics Committee of Hebei Xingtai Fertility Hospital 2024 (Approval No. 202401).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollicular fluid and clinical data were collected with the informed consent of patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYu Z, Li Y, Zhao S, Liu F, Zhao H, Chen ZJ. 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BIOMED PHARMACOTHER. 2022; 153 113286.\u003c/li\u003e\n\u003cli\u003eHuo, P, Li, M, Le, J, et al. Resveratrol improves follicular development of PCOS rats via regulating glycolysis pathway and targeting SIRT1. SYST BIOL REPROD MED. 2022; 69 (2): 153-165. \u003c/li\u003e\n\u003cli\u003eGonz\u0026aacute;lez-Fern\u0026aacute;ndez, R, Mart\u0026iacute;n-Ram\u0026iacute;rez, R, Rotoli, D, et al. Granulosa-Lutein Cell Sirtuin Gene Expression Profiles Differ between Normal Donors and Infertile Women. Int J Mol Sci. 2019; 21 (1): \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"miR-30c-5p, SIRT1, PCOS, Ovarian granulosa cells","lastPublishedDoi":"10.21203/rs.3.rs-5752476/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5752476/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Polycystic ovary syndrome (PCOS) is an endocrine and metabolic disorder characterized by abnormal follicular development and is the leading cause of infertility among women of reproductive age. miR-30c-5p, a newly discovered microRNA (miRNA), plays a crucial role in numerous pathological conditions, such as malignancies, reproductive dysfunctions, and metabolic irregularities. However, the role of miR-30c-5p in PCOS and the apoptosis of granulosa cells (GCs) is not fully understood.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch methods\u003c/strong\u003e: GCs were isolated from the follicular fluid of 23 patients with PCOS and 21 normal controls. The expression of miR-30c-5p and Sirtuin 1 (SIRT1) was detected using quantitative real-time polymerase chain reaction (qRT-PCR). A Spearman correlation analysis was conducted to evaluate the relationship between the expression levels of these markers. Proliferation and apoptosis in human granulosa tumor cell lines (KGN) were analyzed following the overexpression or underexpression of miR-30c-5p, utilizing the Cell Counting Kit-8, flow cytometry, and western blot techniques. TargetScan was used to identify potential targets of miR-30c-5p, which were subsequently validated through qRT-PCR, western blot, and a dual luciferase reporter gene assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The results of our study suggest that the levels of miR-30c-5p aresignificantly elevated in individuals with PCOS compared with the control group. Conversely, we observed a significant decrease in the expression of SIRT1 in patients with PCOS compared with the control group. Moreover, miR-30c-5p expression was negatively correlated with SIRT1. The upregulation of miR-30c-5p suppressed the expression of SIRT1 and Bcl-2, inhibited GC proliferation, and increased the expression of Bax.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Our findings suggest that miR-30c-5p inhibits the proliferation of KGN cells by targeting SIRT1. Thus, this study enhances the current understanding of the mechanisms underlying apoptosis in PCOS and GCs.\u003c/p\u003e","manuscriptTitle":"MiR-30c-5p targets SIRT1 to promote apoptosis in ovarian granulosa cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-06 14:57:13","doi":"10.21203/rs.3.rs-5752476/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":"d1f12976-8f8c-4350-a2ac-e179dead750c","owner":[],"postedDate":"January 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-28T15:24:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-06 14:57:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5752476","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5752476","identity":"rs-5752476","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}