Overexpression of MIR-302C-3P Leads to Apoptosis in Human Renal Mesangial Cells

Overexpression of MIR-302C-3P Leads to Apoptosis in Human Renal Mesangial 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 Overexpression of MIR-302C-3P Leads to Apoptosis in Human Renal Mesangial Cells Wentao Huang, Baojun Hao, Yongqiang Chen, Jinyan Lin, Ting-Ting Wu, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5675630/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 Objective: The apoptosis of glomerular mesangial cells is closely related to the occurrence and development of diabetic nephropathy (DN). However, the mechanism remains unclear. In the present study, we found that the high-glucose cultures induced the upregulation of the miRNA-302c-3p. This study aimed to investigate the effects of over expression of miR-302c-3p on human glomerular mesangial cells (HRMCs). Materials and Methods: HRMCs were cultured in vitro and divided into 2 groups: ①: siRNA-NC group, ② siRNA-TIMP3 group. The miR-302c-3p expression was up-regulated in HRMCs by using miR-302c-3p mimic. We measured the expression of MDA, SOD, CAT, and ROS to examine the oxidative stress of cells. The apoptosis rate of cells was determined by flow cytometry, and the mitochondrial membrane potential (MMP) was examined by the JC-1 detection kit. Real-time polymerase chain reaction (PCR) was used to detect the relative expression of p53 and Survivin. Protein expression of Bcl-2, BAX, caspase-3, p27, Apaf-1, Cytochrome C, and β-actin was analyzed by Western blot. Results: Compared with the control group, we found that the overexpression of miR-302c-3p significantly increased the oxidative stress and apoptosis on HRMCs. The level of p53 was significantly increased ( p <0.01), and the level of Survivin was reduced significantly ( p <0.05) in the miR-302c-3p mimic group. Western blot results showed that the expression of BAX, p27, Apaf-1 and Cytochrome C was significantly up-regulated; the expression of Bcl-2 was significantly down-regulated in the HRMCs in miR-302c-3p mimic group. Conclusions: These findings indicated that overexpression of miR-302c-3p induced the apoptosis of HRMCs, and might be associated with the oxidative stress. miR-302c-3p oxidative stress cell apoptosis diabetic nephropathy human renal mesangial cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Diabetes mellitus (DM) is a widely prevalent chronic metabolic disease characterized by a persistent hyperglycemic state, usually caused by insufficient insulin secretion or insulin resistance. As the course of DM progresses, patients face the risk of multiple complications, among which diabetic nephropathy (DN) is one of the most serious and common microvascular complications, with an incidence rate of about 20–40%. The pathological characteristics of DN are proliferation, extracellular matrix accumulation and fibrosis.Various states caused by diabetes, such as inflammation, hyperglycemia, long-term cellular stress and mitochondrial damage, are involved in the apoptotic process of different cell types in DN ( Alicic et al., 2017 ). Human renal mesangial cells (HRMCs) are glomerular native cells located between the glomerular capillary rings, responsible for the secretory cell matrix, cytokine production, support of the glomerular capillary plexus, phagocytosis, and the removal of macromolecular substances (Vasavada et al., 2005). Increasing evidence suggests that the apoptosis of HRMCs is important in the structural changes of the nephron and in the loss of renal function ( Tung et al., 2018 ). However, due to the complex pathogenesis of DN, including inflammation, epithelial-mesenchymal transition (EMT), oxidative stress, mitochondrial damage, cell apoptosis, and genetic / epigenetic factors, the underlying mechanisms have not been fully elucidated. Therefore, a thorough understanding of the molecular mechanisms regulating the apoptosis of HRMCs is important for the prevention and treatment of kidney diseases. MicroRNAs (miRNAs) is a short non-coding RNA that regulates gene expression by binding to the 3 ′ untranslated region (3 ′ UTR) of the target mRNA, leading to the degradation or translation repression of the mRNA. Among these miRNAs, miR-302c-3p, which is one of the key members of the miR-302/367 family. Recent studies further suggest that the miR-302/367 cluster plays an important role in regulating cell proliferation, differentiation, and reprogramming processes. As a member of this family, miR-302c-3p plays a key role in a variety of biological processes, including embryonic development, tumorigenesis and development, and intervertebral disc degeneration. Moreover, there is increasing evidence that miRNAs is involved in the development of diabetic nephropathy (DN) . In this study, we examined the expression of miR-302c-3p in a high glucose environment and the levels of its downstream regulated target genes. These target genes are inextricably linked with oxidative stress, apoptosis, and inflammatory factors in the glomerular cells. They lead to glomerular dysfunction and ultimately to the development of diabetic nephropathy. The regulation mechanism is shown in Fig. 7 (Mechanism diagram). Materials and Methods Cell Culture and Treatment HRMCs were purchased from American Type Culture Collections (Manassas, VA). Cells were maintained in MCM medium (ScienCell, CA) with 2% FBS, 1% penicillin-streptomycin and 1% growth factor, cultured in an incubator set to 37 ◦C, 5% CO2. Since proliferative activity vanished, the cells were passaged every 3 days until the 9th passage. Cells were divided into 2 groups: ①: siRNA-NC group: empty vector control group, ② siRNA-TIMP 3 group: siRNA-TIMP3 transfection concentration was 20 nM, and the transfection time was 72 hours. MiRNA Transfection MiR-NC, miR-302c-3p mimic were obtained from Biomics (Nantong, China). For preparation, HRMCs were incubated for 24 hours in antibiotic-free MCM medium, then miRNA was transfected into HRMCs with Lipofectamine RNAimax Transfection Reagent (Life Technologies, Invitrogen) according to the manufacturer’s instructions for 72 hours for further research. Oxidative Stress Analysis According to the instructions of superoxide dismutase (SOD) assay kit (Jiancheng, Nanjing, China), catalase (CAT) assay kit (Jiancheng, Nanjing, China), malondialdehyde (MDA) assay kit (Jiancheng, Nanjing, China) and reactive oxygen species (ROS) assay kit (Jiancheng, Nanjing, China), the oxidative stress conditions of the cells were determined. The absorbance was measured respectively at 550 nm (SOD), 405 nm (CAT) and 532 nm (MDA) by using a 96-well microplate reader (SpectraMax M5, Molecular Devices, CA), and the ROS level was measured by detecting the fluorescence intensity of DCF probes by Flow cytometry (BD Biosciences, San Jose, CA). Each of the above-mentioned experiments was repeated for ≥ 3 times. Apoptosis assay Cells were collected and washed with PBS, and then suspended in the 500 µL binding buffer. Finally, cells were stained with 5 µL Annexin V-EGFP and 5 µL Propidium Iodide (PI) (Keygen, Nantong, China) for 20 minutes in the dark at room temperature, the apoptosis rate was measured by flow cytometry (BD Bioscience Company, San Jose, California) and cell exploration software (BD Bioscience). Mitochondrial Membrane Potential Analysis The mitochondrial membrane potential (MMP) was detected by a fluorescence microscope (Nikon ECLIPSE TE2000-E, Japan) following the instruction of JC-1 assay kit (Beyotime, Shanghai China), and the red/green fluorescence intensity represented the distribution of JC-1 monomer and aggregates in mitochondria. Each of the above-mentioned experiments was repeated for ≥ 3 times. Cytosol was separated from cells with the Mitochondrial/cytosol Fractionation Kit (Beyotime). The release of cytochrome c from mitochondria was evaluated by Western blot analysis of cytosolic protein samples as described previously. Quantitative RT-PCR Analysis Total RNA was extracted using the RNA isolator Total RNA Extraction Reagent (Vazyme, Nanjing China) according to the manufacturer's instructions. The cDNA was transcribed from template RNA using PrimeScript™ RT Reagent Kit (Takara, Dalian, China), then analyzed the expressions of p53, Survivin, FN by SYBR Green assays (Bio-Rad, California, USA) with the help of Roche Light Cycler 480 system. According to the method of 2Δ Δ CT, the expression of every gene was standardized to β -actin and expressed relative to the normal group. Western Blot Analysis The whole cell or cytosol lysates were prepared by IP cell lysis buffer (Beyotime, Shanghai China), and quantified. Proteins (20 µg) were fractionated on 12% SDS-PAGE, blotted onto a PVDF (polyvinylidene difluoride) membrane (Millipore, Billerica, MA). Western blotting was carried out as described above (Zhang et al., 2023 ). Primary antibodies included Bcl-2 (Proteintech), BAX (Proteintech), caspase-3 (Proteintech), p27 (Abclonal), Apaf-1 (Proteintech), Cytochrome C (Beyotim), and β-actin (Abclonal), which were chosen as loading controls. The membrane was incubated with HRP labeled anti-rabbit (Abclonal) or anti-mouse (Abclonal) secondary antibody, visualized by chemiluminescence, and analyzed by gray-scale scanning with Image J software (National Institute of Health Bethesda MD, USA). Statistical Analysis All data are presented as the mean ± SD from at least three independent experiments. Student's test or One-way ANOVA and Tukey test were used to compare the differences between two or more groups, and *P<0.05 or **P<0.01 showed statistical significance. Results 3.1 High glucose-induced the upregulation of miR-302c-3p in HRMCs We found that miR-302c-3p expression was 3.4-fold higher than the control group after high-glucose treatment (**p < 0.01 vs. NG), which also provided the experimental basis for our follow-up studies. (Fig. 6 ) 3.2 Effect of miR-302c-3p on oxidative stress in HRMCs To reveal the redox status in HRMCs after transfected with miR-302c-3p mimic, we detected the intracellular ROS, the levels of MDA, the activities of SOD and CAT, which were critical detection indexs in the enzyme antioxidant system. The results indicated that miR-302c-3p significantly resulted in the overproduction of ROS (Fig. 1 A), increased SOD levels (Fig. 1 B), and decreased the MDA levels (Fig. 1 C) and CAT activities (Fig. 1 D) compared with the control group. The administration of antioxidant NAC reversed the above performance. 3.3 Effect of miR-302c-3p on apoptosis in HRMCs Compared with the control, transfection with miR-302c-3p mimics increased the rate of apoptosis (Fig. 2 A, 2 B). 3.4 Effect of MiR-302c-3p Mimic on Mitochondrial Membrane Potential in HRMCs JC-1 fluorescent probe labeled cells and the changes of mitochondrial membrane potential in cells were observed by fluorescence microscope, and the results were shown in Fig. 3 . The overall relative fluorescence intensity of the miR-302c-3p mimic group was markedly lower than that in the control group, indicating that transfection of miR-302c-3p mimic could reduce the mitochondrial membrane potential in HRMCs. 3.5 Effect of MiR-302c-3p on the Expression of P53 and Survivin in HRMCs The qRT-PCR results showed miR-302c-3p mimics upregulated the expression of p53, but compared with the control group, the expression of Survivin was down-regulated (Fig. 4 ). 3.6 Effect of MiR-302c-3p on the Expression of BAX, APAF-1, p27 et al in HRMCs Western blot results showed that miR-302c-3p mimics upregulated the expression of BAX, APAF-1, p27 and Cyt C, downregulated Bcl-2 in cytosol, compared with the control (Fig. 5 A, 5 B). Discussion DM is a metabolic disorder, characterized by the absolute or relative lack of insulin secretion and insulin utilization disorders, mainly marked by hyperglycemia (ADA, 2023). Among them, DN as a serious complication of diabetes, is mainly caused by the damage to renal blood vessels and glomeruli caused by long-term hyperglycemia (KDOQI, 2012).With the gradual progression of the disease, symptoms such as proteinuria, hypertension and edema will gradually appear, and in severe cases may even lead to the occurrence of renal insufficiency and uremia (Vasanth et al., 2018). Therefore,it is particularly critical and urgent to explore its new therapeutic targets. As important functional cells of the glomeruli, mesangial cells play a crucial role in maintaining the normal physiological function of the glomeruli. When the internal environment changes, such as during an inflammatory reaction, mesangial cells may proliferate or undergo apoptosis, which may lead to nephropathy(Sun et al., 2018 ).The typical renal histopathological features of diabetic nephropathy include diffuse or segmental expansion of mesangial cells, apoptosis of mesangial cells, and proliferation of the intercellular matrix (Zhang et al., 2023 ).Among them, the apoptosis of mesangial cells is one of the key factors leading to renal failure. In diabetes, persistent hyperglycemia poses a serious threat to the kidney and other organs. Previous studies have revealed that the expression of microRNA-302c-3p (miR-302c-3p) in human renal mesangial cells (HRMCs) increases significantly in high-glucose conditions, suggesting its pivotal role in kidney injury. Furthermore, we observed that this altered expression of miR-302c-3p in HRMCs is closely associated with oxidative stress status, mitochondrial function, and the expression of genes involved in apoptosis. As a non-coding RNA, miR-302c-3p regulates gene expression at the post-transcriptional level by silencing or degrading target mRNAs, making it an important biomarker for human diseases.Aberrant expression of miRNAs has been shown to be closely associated with the development of diabetic nephropathy (DN). For example, Zhang et al. found that microRNA-22 promoted renal tubulointerstitial fibrosis in DN by targeting PTEN and inhibiting autophagy (Zhang et al., 2018 ).Similarly, Li et al. showed that miR-218 regulates DN by regulating IKK-beta and inhibiting NF- κ B-mediated inflammatory responses (Li et al., 2020). In addition, Zhang et al. found that non-coding RNA NEAT1 affects cell pyroptosis in the DN by modulating the miR-34c / NLRP 3 axis (Zhan et al., 2020 ). MiR-302c-3p, as a member of the miR-302/367 cluster, plays a role in maintaining stem cell pluripotency and cancer formation (Gu et al., 2017 ; Suh MR et al., 2004 ).Mounting evidence suggests that this cluster plays a crucial role in cell proliferation, differentiation, and reprogramming (Gao et al., 2015 ;Kuo et al., 2012 ;Kang et al., 2012 ).Ma et al. found that downregulation of miR-302c expression was associated with poor prognosis and low overall survival in gastric cancer tissues (Ma G et al., 2017 ). Li et al. found that MiR-302c-3p inhibited epithelial stromal transformation (EMT) in human endometrial cancer cells by targeting ZEB 1 and acted as a suppressor of NF- κ B signaling in human lung epithelial cells A549, promoting anti-pathogen invasion and inflammatory response (Ma et al., 2017 ).These studies highlight the important role of miR-302c in the apoptotic process. Our study found that the expression of miR-302c-3p was significantly upregulated in HRMCs cultured with high glucose. This study further investigated the effect of overexpression of miR-302c-3p on apoptosis in human mesangial cells. We found that overexpression of miR-302c-3p caused oxidative stress and apoptosis in HRMCs cultured with high glucose. Oxidative stress plays a pivotal role in the progression of diabetic nephropathy. In the diabetic state, persistent hyperglycemia leads to an excessive mitochondrial load, generating abundant reactive oxygen species (ROS). These ROS damage normal proteins, lipids, and nucleic acids, exacerbating renal injury by activating signaling molecules and inducing mediators of damage. In addition, oxidative stress can also affect many signaling molecules and systems, such as transforming growth factor- β (TGF- β) and nuclear factor kappa-B (NF- κ B), which can further exacerbate kidney injury. Apoptosis, a programmed cell death essential for maintaining cellular homeostasis, is another crucial pathological process in diabetic nephropathy.Factors like hyperglycemia and oxidative stress can trigger the apoptotic pathway, leading to kidney cell death. Notably, the apoptosis of podocytes, epithelial cells in the glomerulus, is particularly critical in diabetic nephropathy, as their loss impairs the glomerular filtration barrier, resulting in albuminuria and decreased renal function.Diabetic nephropathy is the result of oxidative stress and apoptosis. In the hyperglycemic environment, the kidney suffers from continuous oxidative stress damage, while the increase in apoptosis also accelerates the decline of kidney function. Brown lee et al. found that there are a series of adaptive mechanisms in the body to protect cells from damage, and a variety of harmful stimuli can break the balance of oxidative stress and promote cell apoptosis (Brown lee et al., 2001). Kaneto et al. and Ha et al. found that oxidative stress mediates apoptosis through mitochondria, death receptors, and endoplasmic reticulum stress, and may also induce apoptosis via activation of the mitogen-activated protein kinase pathway, nuclear transcription factor KB, caspases, and other mechanisms (Kaneto H et al., 1999 ; Ha et al., 2015).Feng's studies point to the role of podocyte apoptosis in diabetic nephropathy, and especially how oxidative stress affects this process. Podocytes are an important component of the glomerulus, and their damage and apoptosis play a key role in the development of diabetic nephropathy. This study illustrates how hyperglycemia can cause podocyte damage and apoptosis by increasing oxidative stress, which then affects renal function (Naderi et al., 2003 ).Liu's review article explored the overall role of oxidative stress in diabetic nephropathy, discussing how it leads to kidney damage through various mechanisms, including affecting the function of glomeruli and renal tubules (Liu et al., 2019). Papachristoforou et al. extensively explored the mechanisms of diabetes complications, including the roles of oxidative stress and apoptosis, elucidating how these processes are linked to various diabetes complications, particularly diabetic nephropathy. (Papachristoforou et al., 2013).These studies show that oxidative stress and apoptosis are intertwined and interact, jointly driving the development and progression of diabetic nephropathy. However, we found that HRMCs transfected with miR-302c-3p analog showed a significant oxidative stress response, manifested by excessive production of ROS and altered activity of antioxidant enzymes. We also observed an increased rate of apoptosis, decreased mitochondrial membrane potential, after transfection of HRMCs with miR-302c-3p mimics, and altered expression of related apoptotic genes. Mitochondria are the energy factory and one of the main executors of apoptosis. These results suggest that miR-302c-3p may participate in the progression of DN by regulating oxidative stress, apoptosis, and mitochondrial function. This study also investigated the effect of the transfection of miR-302c-3p mimics on the expression of specific genes in HRMCs. The results demonstrated that the mimics significantly upregulated p53 expression while downregulating Survivin, providing important insights into its role in apoptosis regulation.Glomerular mesangial cells, as the most dynamic intrinsic cellular component of the kidney, are sensitive to various stimuli such as high glucose, angiotensin, and aldosterone. These stimuli manifest as enhanced oxidative stress, ultimately promoting the development of glomerulosclerosis.Among these stimuli, p53, a key stress response factor, is activated under conditions of DNA damage, oxidative stress, and others (Hernandez-Borrero et al., 2021). Activated p53 induces cell cycle arrest or apoptosis by regulating the expression of its downstream genes (Engeland et al., 2022).Specifically, p53 increases the expression level of Bax and downregulates Bcl-2 expression, thereby promoting apoptosis.Under oxidative stress conditions, especially when cellular DNA is damaged, p53 gene expression is up-regulated, as shown by accelerated mRNA translation and post-translational modifications.In this study, we observed a close association between the upregulation of p53 and the increase in apoptosis, further confirming its crucial role in the progression of diabetic nephropathy.On the other hand, Survivin, as a member of the apoptosis inhibitor protein family, typically exhibits downregulated expression during apoptosis(Bernardo et al., 2020 ).The reduction of Survivin expression in this study further supports the notion that miR-302c-3p promotes the apoptosis of HRMCs. Moreover, our study also found that the expression of a series of apoptosis-related proteins was significantly changed when miR-302c-3p was overexpressed in HRMCs. Specifically, the expression levels of pro-apoptotic proteins such as BAX, p27, Apaf-1, and cytochrome C were significantly upregulated, while the anti-apoptotic protein Bcl-2 was significantly downregulated. This pattern of change further confirmed the critical role of miR-302c-3p in promoting apoptosis of HRMCs.To gain a deeper understanding of this mechanism, we performed immunoblot analysis. The results showed that the expression of BAX, APAF-1, p27 and cytochrome C were significantly increased in HRMCs transfected with miR-302c-3p mimics compared with control cells. These proteins play pivotal roles in cell apoptosis. For instance, BAX and cytochrome C are key molecules in the mitochondria-mediated apoptotic pathway, while APAF-1 is involved in the formation of apoptotic bodies, further driving the transmission of apoptotic signals. Additionally, as a cell cycle inhibitor, the upregulation of p27 may further promote the progression of apoptosis (Guiley et al., 2019 ; Zhan et al., 2020 ).Notably, we also observed that Bcl-2 expression in the cytoplasm was significantly downregulated upon transfection of miR-302c-3p mimics. Bcl-2 is an important anti-apoptotic protein, and its downregulation may promote the development of apoptosis (Wang et al., 2009).These findings are consistent with previous findings and further highlighting the important role of miR-302c-3p in the regulation of apoptosis. In summary, our results indicate that miR-302c-3p expression rises significantly in mesangial cells in a high glucose setting, which is consistent with its key role in kidney injury. Furthermore, we observed significant changes in the expression of apoptosis-related genes and proteins in mesangial cells after the transfection of miR-302c-3p mimics, particularly in p53 and Survivin, further confirming its crucial role in the regulation of apoptosis.Additionally, we identified upregulation of a series of apoptosis-related proteins such as BAX, p27, Apaf-1 and cytochrome C, as well as downregulation of the antiapoptotic protein Bcl-2, and these changes together promoted apoptosis in mesangial cells. These findings not only deepen our understanding of the mechanism of mesangial apoptosis in diabetic nephropathy, explore the role of miR-302c-3p and its regulatory mechanism on cell apoptosis in diabetic nephropathy, but also provide potential targets for developing novel therapeutic strategies. In the future, we will further explore the specific mechanism of action of miR-302c-3p in diabetic nephropathy, with a view to providing better treatment options for patients. Declarations Author Contribution [Peng Wang and Shu-Lan Qin]contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Wei-Fen Zhuang, Zhu-Yuan Chen and Wen-Tao Huang], [Shi-Wen Liu, Ze-Ming Liu and Dan Xu, ] and [Bao-Jun Hao, Ying Lin and Ying-Zhen Huang]. The first draft of the manuscript was written by [Jin-Yan Lin] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Alicic,R.Z., Michele T, Rooney., Katherine R, Tuttle.(2017). Diabetic Kidney Disease: Challenges, Progress, and Possibilities. Clin J Am Soc Nephrol, 12(12), 0. doi:10.2215/CJN.11491116 Vasavada, Nina., & Agarwal, Rajiv.. (2005). Role of oxidative stress in diabetic nephropathy. Advances in chronic kidney disease, 12(2), 146-54.doi:10.1053/j.ackd.2005.01.001 Tung, CW., Yung-Chien, Hsu., Ya-Hsueh, Shih., Pey-Jium, Chang., Chun-Liang, Lin.(2018). 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Unraveling survivin expression in chronic myeloid leukemia: Molecular interactions and clinical implications. Blood Rev (2020) 43:100671. doi: 10.1016/j.blre.2020.100671. PubMed PMID: 32107072. Guiley KZ, Stevenson JW, Lou K, Barkovich KJ, Kumarasamy V, Wijeratne TU, Bunch KL, Tripathi S, Knudsen ES, Witkiewicz AK, Shokat KM, Rubin SM. p27 allosterically activates cyclin-dependent kinase 4 and antagonizes palbociclib inhibition. Science. 2019 Dec 13;366(6471):eaaw2106. doi: 10.1126/science.aaw2106. PMID: 31831640; PMCID: PMC7592119. Wang C, Youle R J. The role of mitochondria in apoptosis[J]. Annual review of genetics, 2009, 43(1): 95-118. 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. 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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-5675630","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":392404284,"identity":"ad997b00-1aed-4e7b-86bf-13a46c8cc095","order_by":0,"name":"Wentao Huang","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wentao","middleName":"","lastName":"Huang","suffix":""},{"id":392404285,"identity":"cc892028-0be2-4758-8991-33d162706b13","order_by":1,"name":"Baojun Hao","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Baojun","middleName":"","lastName":"Hao","suffix":""},{"id":392404286,"identity":"4f37e56e-8b8b-4bfe-8587-35d7e348e0a3","order_by":2,"name":"Yongqiang Chen","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yongqiang","middleName":"","lastName":"Chen","suffix":""},{"id":392404287,"identity":"3f79b32e-39a7-47c8-b37c-6580676f6fee","order_by":3,"name":"Jinyan Lin","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinyan","middleName":"","lastName":"Lin","suffix":""},{"id":392404288,"identity":"5e450d05-cf34-4692-a4a9-1e7a08b35318","order_by":4,"name":"Ting-Ting Wu","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ting-Ting","middleName":"","lastName":"Wu","suffix":""},{"id":392404289,"identity":"c1c5f4eb-2488-43dc-8a93-a6e4b2be5ee1","order_by":5,"name":"Shiwen Liu","email":"","orcid":"","institution":"Dalian Maritime University","correspondingAuthor":false,"prefix":"","firstName":"Shiwen","middleName":"","lastName":"Liu","suffix":""},{"id":392404290,"identity":"c800a294-c8bf-4117-aa31-3be373e0f99a","order_by":6,"name":"Dan Xu","email":"","orcid":"","institution":"Dalian Maritime University","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Xu","suffix":""},{"id":392404291,"identity":"806871d9-afba-49be-b2ae-03539e102613","order_by":7,"name":"Huixin Liang","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Huixin","middleName":"","lastName":"Liang","suffix":""},{"id":392404292,"identity":"79e19f85-cff8-4ddd-8da8-ef3ca668bf57","order_by":8,"name":"Ying Lin","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Lin","suffix":""},{"id":392404293,"identity":"8a387d08-cb9e-48e3-a778-d2f166c30062","order_by":9,"name":"Yingzhen Huang","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yingzhen","middleName":"","lastName":"Huang","suffix":""},{"id":392404294,"identity":"9d0af557-b790-4343-a3bd-cb58c2bf65f4","order_by":10,"name":"Qun Wang","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qun","middleName":"","lastName":"Wang","suffix":""},{"id":392404295,"identity":"509a5c9f-1d09-4e7b-bca7-823063f9eb1e","order_by":11,"name":"Jiong Le","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiong","middleName":"","lastName":"Le","suffix":""},{"id":392404296,"identity":"59f3d2df-b6ff-44ee-b273-f5c550154360","order_by":12,"name":"Yi Qian","email":"","orcid":"","institution":"Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Qian","suffix":""},{"id":392404297,"identity":"a997c8b4-9cc4-4a29-8957-89411687b108","order_by":13,"name":"Shulan Qin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACNmbmAwYfKiTk+NmbDxCnhY+9LaFwxhkLY8meYwnEaZHjOWPwmbetInHDDR8DIh0mkWO4cQabhDHDDZ6PN94w2MnpNhDUklZs8IFHQo5xdu9myzkMycZmBwhqSd5mOENCwphZ5uw2aR6GA4nbCGtJMP/NYyCR2CaR84xILTxHDIx5EiQSeyRy2IjUAgxkwxkHJIwleI4ZW84xIMIv8s3AqPz4r07O/njzwxtvKuzkCGpBARI8REYNshZSdYyCUTAKRsGIAADIy0DdDROEIgAAAABJRU5ErkJggg==","orcid":"","institution":"Southern Medical University","correspondingAuthor":true,"prefix":"","firstName":"Shulan","middleName":"","lastName":"Qin","suffix":""}],"badges":[],"createdAt":"2024-12-19 09:53:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5675630/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5675630/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72156577,"identity":"9ccd1b53-207b-4eff-b9bf-72982c8f07d8","added_by":"auto","created_at":"2024-12-23 08:56:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":323363,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of miR-302c-3p mimic on oxidative stress of HRMCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHRMCs were transfected with miR-302c-3p mimic or miR-NC at a concentration of 20 nM for 72 h in the absence or presence of antioxidant NAC.\u003c/p\u003e\n\u003cp\u003eA. ROS levels. B. SOD level. C. MDA levels. D. CAT activity. NAC: antioxidant.\u0026nbsp; *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 vs miR-NC. （\u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 vs miR-302c-3p mimic).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5675630/v1/f28b9b7636863dbfa0d046c9.png"},{"id":72156576,"identity":"f4b05d1f-96d7-48e5-a4cd-d3c39d26f956","added_by":"auto","created_at":"2024-12-23 08:56:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":112806,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of miR-302c-3p mimic on the apoptosis rate of HRMCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHRMCs were transfected with miR-302c-3p mimic or miR-NC at a concentration of 20 nM for 72 h. A. Flow cytometry assay chart; B. Statistical analysis of apoptotic cell death rates (**\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs miR-NC).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5675630/v1/0a35393b1899356aa3ab05ea.png"},{"id":72156582,"identity":"63c006c4-2d78-4184-bfae-76b761dc6402","added_by":"auto","created_at":"2024-12-23 08:56:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":525326,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of miR-302c-3p mimic on mitochondrial membrane potential in HRMCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Fluorescence detection of mitochondrial membrane potential (Representative graph of mitochondrial membrane potential fluorescence results, red: JC-1 polymer; green: JC-1 monomer; Scale bar: 100 µm). B. Analysis statistical results. (**\u003cem\u003eP\u003c/em\u003e＜0.01 vs miR-NC).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5675630/v1/b2256e26c2235308a0fe4395.png"},{"id":72156889,"identity":"9a5de40c-5621-43c6-9595-413539a5e39b","added_by":"auto","created_at":"2024-12-23 09:04:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":24651,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of miR-302c-3p mimic on p53 and Survivin mRNA expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHRMCs were transfected with miR-302c-3p mimic or miR-NC at a concentration of 20 nM for 72 h.（*\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01 vs miR-NC).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5675630/v1/a19f4727f00e2ea4d3655dec.png"},{"id":72156587,"identity":"5e7bf12a-4ae4-4304-bace-3569783989af","added_by":"auto","created_at":"2024-12-23 08:56:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":109030,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of miR-302c-3p mimic on apoptosis-associated proteins in HRMCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHRMCs were transfected with miR-302c-3p mimic or miR-NC at a concentration of 20 nM for 72 h. A. Representative graph of Westen Blot results; B. Grayscale analysis statistical results.（**\u003cem\u003eP\u003c/em\u003e＜0.01 vs miR-NC).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5675630/v1/2c1b0ec43f4a2ad091a4710b.png"},{"id":72156584,"identity":"9df319e4-9ab4-4c01-9cf2-6d7705962902","added_by":"auto","created_at":"2024-12-23 08:56:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantitative fluorescence PCR by probe method showed that miR-302c-3p was expressed 3.4 times higher than normal sugars after treatment with high glucose.( **P \u0026lt;0.01 vs NG ).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5675630/v1/c85b58cd270300caa1cc62bc.png"},{"id":72156578,"identity":"3495327d-7da8-428f-9151-461eb11569dd","added_by":"auto","created_at":"2024-12-23 08:56:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":135166,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanic diagram\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5675630/v1/03d8c47e64f29839846675a6.png"},{"id":73013221,"identity":"f1d8e658-29be-4816-a526-6b4964e6fbef","added_by":"auto","created_at":"2025-01-06 02:01:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1915085,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5675630/v1/b9a75491-25d8-4262-8253-8010e5b7b0a7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eOverexpression of MIR-302C-3P Leads to Apoptosis in Human Renal Mesangial Cells\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eDiabetes mellitus (DM) is a widely prevalent chronic metabolic disease characterized by a persistent hyperglycemic state, usually caused by insufficient insulin secretion or insulin resistance. As the course of DM progresses, patients face the risk of multiple complications, among which diabetic nephropathy (DN) is one of the most serious and common microvascular complications, with an incidence rate of about 20\u0026ndash;40%. The pathological characteristics of DN are proliferation, extracellular matrix accumulation and fibrosis.Various states caused by diabetes, such as inflammation, hyperglycemia, long-term cellular stress and mitochondrial damage, are involved in the apoptotic process of different cell types in DN (\u003c/span\u003eAlicic et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eHuman renal mesangial cells (HRMCs) are glomerular native cells located between the glomerular capillary rings, responsible for the secretory cell matrix, cytokine production, support of the glomerular capillary plexus, phagocytosis, and the removal of macromolecular substances (Vasavada et al., 2005). Increasing evidence suggests that the apoptosis of HRMCs is important in the structural changes of the nephron and in the loss of renal function (\u003c/span\u003eTung et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eHowever, due to the complex pathogenesis of DN, including inflammation, epithelial-mesenchymal transition (EMT), oxidative stress, mitochondrial damage, cell apoptosis, and\u003c/span\u003e genetic / epigenetic factors, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ethe underlying mechanisms have not been fully elucidated. Therefore, a thorough understanding of the molecular mechanisms regulating the apoptosis of HRMCs is important for the prevention and treatment of kidney diseases.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMicroRNAs (miRNAs) is a short non-coding RNA that regulates gene expression by binding to the 3 \u0026prime; untranslated region (3 \u0026prime; UTR) of the target mRNA, leading to the degradation or translation repression of the mRNA. Among these miRNAs, miR-302c-3p, which is one of the key members of the miR-302/367 family. Recent studies further suggest that the miR-302/367 cluster plays an important role in regulating cell proliferation, differentiation, and reprogramming processes. As a member of this family, miR-302c-3p plays a key role in a variety of biological processes, including embryonic development, tumorigenesis and development, and intervertebral disc degeneration. Moreover, there is increasing evidence that miRNAs is involved in the development of diabetic nephropathy (DN) .\u003c/span\u003e \u003c/p\u003e \u003cp\u003eIn this study, we examined the expression of miR-302c-3p in a high glucose environment and the levels of its downstream regulated target genes. These target genes are inextricably linked with oxidative stress, apoptosis, and inflammatory factors in the glomerular cells. They lead to glomerular dysfunction and ultimately to the development of diabetic nephropathy. The regulation mechanism is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003e (Mechanism diagram).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture and Treatment\u003c/h2\u003e \u003cp\u003eHRMCs were purchased from American Type Culture Collections (Manassas, VA). Cells were maintained in MCM medium (ScienCell, CA) with 2% FBS, 1% penicillin-streptomycin and 1% growth factor, cultured in an incubator set to 37 ◦C, 5% CO2. Since proliferative activity vanished, the cells were passaged every 3 days until the 9th passage. Cells were divided into 2 groups: ①: siRNA-NC group: empty vector control group, ② siRNA-TIMP 3 group: siRNA-TIMP3 transfection concentration was 20 nM, and the transfection time was 72 hours.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMiRNA Transfection\u003c/h3\u003e\n\u003cp\u003eMiR-NC, miR-302c-3p mimic were obtained from Biomics (Nantong, China). For preparation, HRMCs were incubated for 24 hours in antibiotic-free MCM medium, then miRNA was transfected into HRMCs with Lipofectamine RNAimax Transfection Reagent (Life Technologies, Invitrogen) according to the manufacturer\u0026rsquo;s instructions for 72 hours for further research.\u003c/p\u003e\n\u003ch3\u003eOxidative Stress Analysis\u003c/h3\u003e\n\u003cp\u003eAccording to the instructions of superoxide dismutase (SOD) assay kit (Jiancheng, Nanjing, China), catalase (CAT) assay kit (Jiancheng, Nanjing, China), malondialdehyde (MDA) assay kit (Jiancheng, Nanjing, China) and reactive oxygen species (ROS) assay kit (Jiancheng, Nanjing, China), the oxidative stress conditions of the cells were determined. The absorbance was measured respectively at 550 nm (SOD), 405 nm (CAT) and 532 nm (MDA) by using a 96-well microplate reader (SpectraMax M5, Molecular Devices, CA), and the ROS level was measured by detecting the fluorescence intensity of DCF probes by Flow cytometry (BD Biosciences, San Jose, CA). Each of the above-mentioned experiments was repeated for \u0026ge;\u0026thinsp;3 times.\u003c/p\u003e\n\u003ch3\u003eApoptosis assay\u003c/h3\u003e\n\u003cp\u003eCells were collected and washed with PBS, and then suspended in the 500 \u0026micro;L binding buffer. Finally, cells were stained with 5 \u0026micro;L Annexin V-EGFP and 5 \u0026micro;L Propidium Iodide (PI) (Keygen, Nantong, China) for 20 minutes in the dark at room temperature, the apoptosis rate was measured by flow cytometry (BD Bioscience Company, San Jose, California) and cell exploration software (BD Bioscience).\u003c/p\u003e\n\u003ch3\u003eMitochondrial Membrane Potential Analysis\u003c/h3\u003e\n\u003cp\u003eThe mitochondrial membrane potential (MMP) was detected by a fluorescence microscope (Nikon ECLIPSE TE2000-E, Japan) following the instruction of JC-1 assay kit (Beyotime, Shanghai China), and the red/green fluorescence intensity represented the distribution of JC-1 monomer and aggregates in mitochondria. Each of the above-mentioned experiments was repeated for \u0026ge;\u0026thinsp;3 times.\u003c/p\u003e \u003cp\u003eCytosol was separated from cells with the Mitochondrial/cytosol Fractionation Kit (Beyotime). The release of cytochrome c from mitochondria was evaluated by Western blot analysis of cytosolic protein samples as described previously.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative RT-PCR Analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using the RNA isolator Total RNA Extraction Reagent (Vazyme, Nanjing China) according to the manufacturer's instructions. The cDNA was transcribed from template RNA using PrimeScript\u0026trade; RT Reagent Kit (Takara, Dalian, China), then analyzed the expressions of p53, Survivin, FN by SYBR Green assays (Bio-Rad, California, USA) with the help of Roche Light Cycler 480 system. According to the method of 2Δ Δ CT, the expression of every gene was standardized to β -actin and expressed relative to the normal group.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern Blot Analysis\u003c/h3\u003e\n\u003cp\u003eThe whole cell or cytosol lysates were prepared by IP cell lysis buffer (Beyotime, Shanghai China), and quantified. Proteins (20 \u0026micro;g) were fractionated on 12% SDS-PAGE, blotted onto a PVDF (polyvinylidene difluoride) membrane (Millipore, Billerica, MA). Western blotting was carried out as described above (Zhang et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Primary antibodies included Bcl-2 (Proteintech), BAX (Proteintech), caspase-3 (Proteintech), p27 (Abclonal), Apaf-1 (Proteintech), Cytochrome C (Beyotim), and β-actin (Abclonal), which were chosen as loading controls. The membrane was incubated with HRP labeled anti-rabbit (Abclonal) or anti-mouse (Abclonal) secondary antibody, visualized by chemiluminescence, and analyzed by gray-scale scanning with Image J software (National Institute of Health Bethesda MD, USA).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD from at least three independent experiments. Student's test or One-way ANOVA and Tukey test were used to compare the differences between two or more groups, and *P\u0026lt;0.05 or **P\u0026lt;0.01 showed statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 High glucose-induced the upregulation of miR-302c-3p in HRMCs\u003c/h2\u003e \u003cp\u003eWe found that miR-302c-3p expression was 3.4-fold higher than the control group after high-glucose treatment (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. NG), which also provided the experimental basis for our follow-up studies. (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e3.2 Effect of miR-302c-3p on oxidative stress in\u003c/span\u003e \u003cb\u003eHRMCs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo reveal the redox status in HRMCs after transfected with miR-302c-3p mimic, we detected the intracellular ROS, the levels of MDA, the activities of SOD and CAT, which were critical detection indexs in the enzyme antioxidant system. The results indicated that miR-302c-3p significantly resulted in the overproduction of ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), increased SOD levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and decreased the MDA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and CAT activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) compared with the control group. The administration of antioxidant NAC reversed the above performance.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e3.3\u003c/span\u003e \u003cb\u003eEffect of miR-302c-3p on apoptosis in HRMCs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCompared with the control, transfection with miR-302c-3p mimics increased the rate of apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effect of MiR-302c-3p Mimic on Mitochondrial Membrane Potential in HRMCs\u003c/h2\u003e \u003cp\u003eJC-1 fluorescent probe labeled cells and the changes of mitochondrial membrane potential in cells were observed by fluorescence microscope, and the results were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The overall relative fluorescence intensity of the miR-302c-3p mimic group was markedly lower than that in the control group, indicating that transfection of miR-302c-3p mimic could reduce the mitochondrial membrane potential in HRMCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Effect of MiR-302c-3p on the Expression of P53 and Survivin in HRMCs\u003c/h2\u003e \u003cp\u003eThe qRT-PCR results showed miR-302c-3p mimics upregulated the expression of p53, but compared with the control group, the expression of Survivin was down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Effect of MiR-302c-3p on the Expression of BAX, APAF-1, p27 et al in HRMCs\u003c/h2\u003e \u003cp\u003eWestern blot results showed that miR-302c-3p mimics upregulated the expression of BAX, APAF-1, p27 and Cyt C, downregulated Bcl-2 in cytosol, compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDM is a metabolic disorder, characterized by the absolute or relative lack of insulin secretion and insulin utilization disorders, mainly marked by hyperglycemia (ADA, 2023). Among them, DN as a serious complication of diabetes, is mainly caused by the damage to renal blood vessels and glomeruli caused by long-term hyperglycemia (KDOQI, 2012).With the gradual progression of the disease, symptoms such as proteinuria, hypertension and edema will gradually appear, and in severe cases may even lead to the occurrence of renal insufficiency and uremia (Vasanth et al., 2018). Therefore,it is particularly critical and urgent to explore its new therapeutic targets. As important functional cells of the glomeruli, mesangial cells play a crucial role in maintaining the normal physiological function of the glomeruli. When the internal environment changes, such as during an inflammatory reaction, mesangial cells may proliferate or undergo apoptosis, which may lead to nephropathy(Sun et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).The typical renal histopathological features of diabetic nephropathy include diffuse or segmental expansion of mesangial cells, apoptosis of mesangial cells, and proliferation of the intercellular matrix (Zhang et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).Among them, the apoptosis of mesangial cells is one of the key factors leading to renal failure.\u003c/p\u003e \u003cp\u003eIn diabetes, persistent hyperglycemia poses a serious threat to the kidney and other organs. Previous studies have revealed that the expression of microRNA-302c-3p (miR-302c-3p) in human renal mesangial cells (HRMCs) increases significantly in high-glucose conditions, suggesting its pivotal role in kidney injury. Furthermore, we observed that this altered expression of miR-302c-3p in HRMCs is closely associated with oxidative stress status, mitochondrial function, and the expression of genes involved in apoptosis. As a non-coding RNA, miR-302c-3p regulates gene expression at the post-transcriptional level by silencing or degrading target mRNAs, making it an important biomarker for human diseases.Aberrant expression of miRNAs has been shown to be closely associated with the development of diabetic nephropathy (DN). For example, Zhang et al. found that microRNA-22 promoted renal tubulointerstitial fibrosis in DN by targeting PTEN and inhibiting autophagy (Zhang et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).Similarly, Li et al. showed that miR-218 regulates DN by regulating IKK-beta and inhibiting NF- κ B-mediated inflammatory responses (Li et al., 2020). In addition, Zhang et al. found that non-coding RNA NEAT1 affects cell pyroptosis in the DN by modulating the miR-34c / NLRP 3 axis (Zhan et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). MiR-302c-3p, as a member of the miR-302/367 cluster, plays a role in maintaining stem cell pluripotency and cancer formation (Gu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Suh MR et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).Mounting evidence suggests that this cluster plays a crucial role in cell proliferation, differentiation, and reprogramming (Gao et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e;Kuo et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e;Kang et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).Ma et al. found that downregulation of miR-302c expression was associated with poor prognosis and low overall survival in gastric cancer tissues (Ma G et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Li et al. found that MiR-302c-3p inhibited epithelial stromal transformation (EMT) in human endometrial cancer cells by targeting ZEB 1 and acted as a suppressor of NF- κ B signaling in human lung epithelial cells A549, promoting anti-pathogen invasion and inflammatory response (Ma et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).These studies highlight the important role of miR-302c in the apoptotic process. Our study found that the expression of miR-302c-3p was significantly upregulated in HRMCs cultured with high glucose. This study further investigated the effect of overexpression of miR-302c-3p on apoptosis in human mesangial cells. We found that overexpression of miR-302c-3p caused oxidative stress and apoptosis in HRMCs cultured with high glucose.\u003c/p\u003e \u003cp\u003eOxidative stress plays a pivotal role in the progression of diabetic nephropathy. In the diabetic state, persistent hyperglycemia leads to an excessive mitochondrial load, generating abundant reactive oxygen species (ROS). These ROS damage normal proteins, lipids, and nucleic acids, exacerbating renal injury by activating signaling molecules and inducing mediators of damage. In addition, oxidative stress can also affect many signaling molecules and systems, such as transforming growth factor- β (TGF- β) and nuclear factor kappa-B (NF- κ B), which can further exacerbate kidney injury. Apoptosis, a programmed cell death essential for maintaining cellular homeostasis, is another crucial pathological process in diabetic nephropathy.Factors like hyperglycemia and oxidative stress can trigger the apoptotic pathway, leading to kidney cell death. Notably, the apoptosis of podocytes, epithelial cells in the glomerulus, is particularly critical in diabetic nephropathy, as their loss impairs the glomerular filtration barrier, resulting in albuminuria and decreased renal function.Diabetic nephropathy is the result of oxidative stress and apoptosis. In the hyperglycemic environment, the kidney suffers from continuous oxidative stress damage, while the increase in apoptosis also accelerates the decline of kidney function. Brown lee et al. found that there are a series of adaptive mechanisms in the body to protect cells from damage, and a variety of harmful stimuli can break the balance of oxidative stress and promote cell apoptosis (Brown lee et al., 2001). Kaneto et al. and Ha et al. found that oxidative stress mediates apoptosis through mitochondria, death receptors, and endoplasmic reticulum stress, and may also induce apoptosis via activation of the mitogen-activated protein kinase pathway, nuclear transcription factor KB, caspases, and other mechanisms (Kaneto H et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Ha et al., 2015).Feng's studies point to the role of podocyte apoptosis in diabetic nephropathy, and especially how oxidative stress affects this process. Podocytes are an important component of the glomerulus, and their damage and apoptosis play a key role in the development of diabetic nephropathy. This study illustrates how hyperglycemia can cause podocyte damage and apoptosis by increasing oxidative stress, which then affects renal function (Naderi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).Liu's review article explored the overall role of oxidative stress in diabetic nephropathy, discussing how it leads to kidney damage through various mechanisms, including affecting the function of glomeruli and renal tubules (Liu et al., 2019). Papachristoforou et al. extensively explored the mechanisms of diabetes complications, including the roles of oxidative stress and apoptosis, elucidating how these processes are linked to various diabetes complications, particularly diabetic nephropathy. (Papachristoforou et al., 2013).These studies show that oxidative stress and apoptosis are intertwined and interact, jointly driving the development and progression of diabetic nephropathy. However, we found that HRMCs transfected with miR-302c-3p analog showed a significant oxidative stress response, manifested by excessive production of ROS and altered activity of antioxidant enzymes. We also observed an increased rate of apoptosis, decreased mitochondrial membrane potential, after transfection of HRMCs with miR-302c-3p mimics, and altered expression of related apoptotic genes. Mitochondria are the energy factory and one of the main executors of apoptosis. These results suggest that miR-302c-3p may participate in the progression of DN by regulating oxidative stress, apoptosis, and mitochondrial function.\u003c/p\u003e \u003cp\u003eThis study also investigated the effect of the transfection of miR-302c-3p mimics on the expression of specific genes in HRMCs. The results demonstrated that the mimics significantly upregulated p53 expression while downregulating Survivin, providing important insights into its role in apoptosis regulation.Glomerular mesangial cells, as the most dynamic intrinsic cellular component of the kidney, are sensitive to various stimuli such as high glucose, angiotensin, and aldosterone. These stimuli manifest as enhanced oxidative stress, ultimately promoting the development of glomerulosclerosis.Among these stimuli, p53, a key stress response factor, is activated under conditions of DNA damage, oxidative stress, and others (Hernandez-Borrero et al., 2021). Activated p53 induces cell cycle arrest or apoptosis by regulating the expression of its downstream genes (Engeland et al., 2022).Specifically, p53 increases the expression level of Bax and downregulates Bcl-2 expression, thereby promoting apoptosis.Under oxidative stress conditions, especially when cellular DNA is damaged, p53 gene expression is up-regulated, as shown by accelerated mRNA translation and post-translational modifications.In this study, we observed a close association between the upregulation of p53 and the increase in apoptosis, further confirming its crucial role in the progression of diabetic nephropathy.On the other hand, Survivin, as a member of the apoptosis inhibitor protein family, typically exhibits downregulated expression during apoptosis(Bernardo et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).The reduction of Survivin expression in this study further supports the notion that miR-302c-3p promotes the apoptosis of HRMCs.\u003c/p\u003e \u003cp\u003eMoreover, our study also found that the expression of a series of apoptosis-related proteins was significantly changed when miR-302c-3p was overexpressed in HRMCs. Specifically, the expression levels of pro-apoptotic proteins such as BAX, p27, Apaf-1, and cytochrome C were significantly upregulated, while the anti-apoptotic protein Bcl-2 was significantly downregulated. This pattern of change further confirmed the critical role of miR-302c-3p in promoting apoptosis of HRMCs.To gain a deeper understanding of this mechanism, we performed immunoblot analysis. The results showed that the expression of BAX, APAF-1, p27 and cytochrome C were significantly increased in HRMCs transfected with miR-302c-3p mimics compared with control cells. These proteins play pivotal roles in cell apoptosis. For instance, BAX and cytochrome C are key molecules in the mitochondria-mediated apoptotic pathway, while APAF-1 is involved in the formation of apoptotic bodies, further driving the transmission of apoptotic signals. Additionally, as a cell cycle inhibitor, the upregulation of p27 may further promote the progression of apoptosis (Guiley et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhan et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).Notably, we also observed that Bcl-2 expression in the cytoplasm was significantly downregulated upon transfection of miR-302c-3p mimics. Bcl-2 is an important anti-apoptotic protein, and its downregulation may promote the development of apoptosis (Wang et al., 2009).These findings are consistent with previous findings and further highlighting the important role of miR-302c-3p in the regulation of apoptosis.\u003c/p\u003e \u003cp\u003eIn summary, our results indicate that miR-302c-3p expression rises significantly in mesangial cells in a high glucose setting, which is consistent with its key role in kidney injury. Furthermore, we observed significant changes in the expression of apoptosis-related genes and proteins in mesangial cells after the transfection of miR-302c-3p mimics, particularly in p53 and Survivin, further confirming its crucial role in the regulation of apoptosis.Additionally, we identified upregulation of a series of apoptosis-related proteins such as BAX, p27, Apaf-1 and cytochrome C, as well as downregulation of the antiapoptotic protein Bcl-2, and these changes together promoted apoptosis in mesangial cells. These findings not only deepen our understanding of the mechanism of mesangial apoptosis in diabetic nephropathy, explore the role of miR-302c-3p and its regulatory mechanism on cell apoptosis in diabetic nephropathy, but also provide potential targets for developing novel therapeutic strategies. In the future, we will further explore the specific mechanism of action of miR-302c-3p in diabetic nephropathy, with a view to providing better treatment options for patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003e[Peng Wang and Shu-Lan Qin]contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Wei-Fen Zhuang, Zhu-Yuan Chen and Wen-Tao Huang], [Shi-Wen Liu, Ze-Ming Liu and Dan Xu, ] and [Bao-Jun Hao, Ying Lin and Ying-Zhen Huang]. The first draft of the manuscript was written by [Jin-Yan Lin] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlicic,R.Z., Michele T, Rooney., Katherine R, Tuttle.(2017). Diabetic Kidney Disease: Challenges, Progress, and Possibilities. 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N Engl J Med. 1988 May 19;318(20):1315-21. doi: 10.1056/NEJM198805193182007. PMID: 3283558.\u003c/li\u003e\n\u003cli\u003eKaneto H, Kajimoto Y, Miyagawa J, Matsuoka T, Fujitani Y, Umayahara Y, Hanafusa T, Matsuzawa Y, Yamasaki Y, Hori M. Beneficial effects of antioxidants in diabetes: possible protection of pancreatic beta-cells against glucose toxicity. Diabetes. 1999 Dec;48(12):2398-406. doi: 10.2337/diabetes.48.12.2398. PMID: 10580429.\u003c/li\u003e\n\u003cli\u003eHa H, Hwang IA, Park JH, Lee HB. Role of reactive oxygen species in the pathogenesis of diabetic nephropathy. Diabetes Res Clin Pract. 2008 Nov 13;82 Suppl 1:S42-5. doi: 10.1016/j.diabres.2008.09.017. Epub 2008 Oct 8. PMID: 18845352.\u003c/li\u003e\n\u003cli\u003eNaderi J, Hung M, Pandey S. Oxidative stress-induced apoptosis in dividing fibroblasts involves activation of p38 MAP kinase and over-expression of Bax: resistance of quiescent cells to oxidative stress. 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Cell cycle regulation: p53-p21-RB signaling. Cell Death Differ (2022) 29(5):946-60. doi: 10.1038/s41418-022-00988-z. PubMed PMID: 35361964.\u003c/li\u003e\n\u003cli\u003eBernardo PS, Lemos L, de Moraes GN, Maia RC. Unraveling survivin expression in chronic myeloid leukemia: Molecular interactions and clinical implications. Blood Rev (2020) 43:100671. doi: 10.1016/j.blre.2020.100671. PubMed PMID: 32107072.\u003c/li\u003e\n\u003cli\u003eGuiley KZ, Stevenson JW, Lou K, Barkovich KJ, Kumarasamy V, Wijeratne TU, Bunch KL, Tripathi S, Knudsen ES, Witkiewicz AK, Shokat KM, Rubin SM. p27 allosterically activates cyclin-dependent kinase 4 and antagonizes palbociclib inhibition. Science. 2019 Dec 13;366(6471):eaaw2106. doi: 10.1126/science.aaw2106. PMID: 31831640; PMCID: PMC7592119.\u003c/li\u003e\n\u003cli\u003eWang C, Youle R J. The role of mitochondria in apoptosis[J]. Annual review of genetics, 2009, 43(1): 95-118.\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-302c-3p, oxidative stress, cell apoptosis, diabetic nephropathy, human renal mesangial cells ","lastPublishedDoi":"10.21203/rs.3.rs-5675630/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5675630/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e The apoptosis of glomerular mesangial cells is closely related to the occurrence and development of diabetic nephropathy (DN). However, the mechanism remains unclear. In the present study, we found that the high-glucose cultures induced the upregulation of the miRNA-302c-3p. This study aimed to investigate the effects of over expression of miR-302c-3p on human glomerular mesangial cells (HRMCs).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods: \u003c/strong\u003eHRMCs were cultured in vitro and divided into 2 groups: ①: siRNA-NC group, ② siRNA-TIMP3 group. The miR-302c-3p expression was up-regulated in HRMCs by using miR-302c-3p mimic. We measured the expression of MDA, SOD, CAT, and ROS to examine the oxidative stress of cells. The apoptosis rate of cells was determined by flow cytometry, and the mitochondrial membrane potential (MMP) was examined by the JC-1 detection kit. Real-time polymerase chain reaction (PCR) was used to detect the relative expression of p53 and Survivin. Protein expression of Bcl-2, BAX, caspase-3, p27, Apaf-1, Cytochrome C, and β-actin was analyzed by Western blot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Compared with the control group, we found that the overexpression of miR-302c-3p significantly increased the oxidative stress and apoptosis on HRMCs. The level of p53 was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), and the level of Survivin was reduced significantly (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) in the miR-302c-3p mimic group. Western blot results showed that the expression of BAX, p27, Apaf-1 and Cytochrome C was significantly up-regulated; the expression of Bcl-2 was significantly down-regulated in the HRMCs in miR-302c-3p mimic group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThese findings indicated that overexpression of miR-302c-3p induced the apoptosis of HRMCs, and might be associated with the oxidative stress.\u003c/p\u003e","manuscriptTitle":"Overexpression of MIR-302C-3P Leads to Apoptosis in Human Renal Mesangial Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-23 08:56:03","doi":"10.21203/rs.3.rs-5675630/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":"6d4176e9-9835-4f34-a54c-0bfd5f19750a","owner":[],"postedDate":"December 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-15T17:38:24+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-23 08:56:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5675630","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5675630","identity":"rs-5675630","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}