ciRS-7 Promotes Proliferation and Apoptosis in Insulinoma Cells via miR-7/MYRIP/Pax6 Signaling Pathway

ciRS-7 Promotes Proliferation and Apoptosis in Insulinoma Cells via miR-7/MYRIP/Pax6 Signaling Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ciRS-7 Promotes Proliferation and Apoptosis in Insulinoma Cells via miR-7/MYRIP/Pax6 Signaling Pathway Zhenlin Tan, Minli Hu, Dan Wu, Chen Liu, Zheng Feng, Zhimei Luo, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7700686/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: Pancreatic Neuroendocrine Tumors（PNETs）is a rare neuroendocrine tumor with a complex and not fully elucidated pathogenesis. In recent years, the role of non-coding RNAs in tumorigenesis has gained increasing attention. Circular RNA ciRS-7, known as a competitive endogenous RNA, acts as a sponge for tumor-suppressive miR-7, thereby modulating gene expression. Methods： The expression levels of ciRS-7 and miR-7 in NES2Y cells were detected by qRT-PCR. Subsequently, ciRS-7 was silenced in NES2Y cells, and the expression of apoptosis-related genes Bax and Caspase-3, as well as proliferation-related genes Myrip and Pax6, was evaluated by qRT-PCR and Western blot analysis. Cell proliferation was assessed using the CCK-8 assay and colony formation assay, while cell apoptosis was analyzed by flow cytometry Results: We detected significant upregulation of ciRS-7 in insulinoma cell lines NES2Y, whereas miR-7 expression was markedly downregulated. Knockdown of ciRS-7 significantly suppressed cellular proliferation while promoting apoptosis, concomitantly upregulating expression of apoptosis-related genes Bax and Caspase-3. Further study showed that ciRS-7 regulates Myrip and Pax6 expression through miR-7. Knockdown of Myrip suppresses cell proliferation, induces apoptosis, and reverses the functional expression patterns of ciRS-7 and miR-7. Conclusion: CiRS-7 functions as a miR-7 sponge, inducing insulinoma cell apoptosis by targeting miR-7 and modulating the Myrip /Pax6 signaling pathway. Pancreatic Neuroendocrine Tumors ciRS-7 miR-7 apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Pancreatic Neuroendocrine Tumors (PNETs) are a heterogeneous group of neoplasms arising from neuroendocrine cells within the pancreas, representing approximately 3% of all pancreatic tumors [ 1 ]. Although the incidence of PNETs remains relatively low, epidemiological studies have demonstrated a steady increase in the diagnosis of PNETs over the past two decades, likely due to advances in imaging techniques and increased clinical awareness [ 2 , 3 ]. The clinical presentation of PNETs varies widely, ranging from asymptomatic cases to severe hormone-related syndromes, and patient outcomes can differ significantly. Conventional treatment strategies include surgical resection, chemotherapy, and targeted therap [ 4 , 5 ]. However, therapeutic efficacy for advanced or metastatic disease remains suboptimal [ 6 ]. Therefore, a comprehensive understanding of the molecular mechanisms underlying PNET pathogenesis is essential, along with the identification of novel diagnostic biomarkers and potential therapeutic targets. Non-coding RNAs (ncRNAs) play critical regulatory roles in gene expression and have been widely associated with the development and progression of various cancers [ 7 ]. Among them, circular RNAs (circRNAs) represent a unique class of ncRNAs characterized by their covalently closed loop structures formed through back-splicing events, which render them resistant to degradation by exonucleases and thus highly stable and conserved across species [ 8 ]. Unlike linear RNAs, circRNAs are resistant to degradation by exonucleases due to their unique loop structure, making circRNAs remarkably stable and abundant in various tissues and body fluids, including blood and saliva [ 9 ]. CircRNAs participate in the regulation of gene expression through multiple mechanisms, including acting as microRNA (miRNA) sponges to modulate post-transcriptional gene regulation [ 10 ], interacting with RNA-binding proteins (RBPs) to influence RNA processing [ 11 ] and even regulating transcription and translation processes [ 12 ]. MicroRNA-7 (miR-7) is a widely expressed microRNA that has been shown to exert either tumor-suppressive or oncogenic functions depending on the cancer type and cellular context [ 13 ]. Importantly, downregulation of miR-7 has been observed in both type 2 diabetes and PNETs, suggesting its protective role against islet cell dysregulation [ 14 ]. In many cancers, miR-7 suppresses tumor cell proliferation, migration, and invasion by targeting key oncogenes such as epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) [ 15 ]. However, the expression and function of miR-7 are regulated by multiple factors, among which circRNAs serving as miRNA sponges represent a crucial regulatory mechanism [ 16 ]. CiRS-7, also known as CDR1as, was the first extensively studied circRNA capable of sequestering miR-7 through abundant binding sites, thereby relieving the inhibitory effects of miR-7 on its downstream target genes and promoting tumor progression [ 17 ]. Given the critical roles of ciRS-7 and miR-7 in tumor development and progression, as well as the essential functions of Myrip and Pax6 in insulin secretion and pancreatic development [ 18 ], the objective of this study is to elucidate the involvement of the ciRS-7/miR-7 regulatory axis in pancreatic islet cell tumorigenesis by modulating the expression of Myrip and Pax6. Our findings indicate that knockdown of the circular RNA ciRS-7 suppresses cell growth in pancreatic islet cell tumor cells by targeting miR-7 and modulating the Myrip and Pax6 signaling pathways, ultimately inducing apoptosis. These results suggest that ciRS-7 may serve as a potential therapeutic target for pancreatic islet cell tumors. Methods Quantitative reverse transcription polymerase chain reaction (qRT‒PCR) Quantitative reverse transcription polymerase chain reaction can reliably detect and quantitatively measure the products generated in each cycle of the PCR process [19]. Total RNA was extracted from HPDE6-C7 and NES2Y cells via TRIzol reagent (15596026, Thermo, USA) following the manufacturer’s instructions. MiRNA was extracted from HPDE6-C7 and NES2Y cells via TRIzol reagent (Life Technologies, Carlsbad, USA) following the manufacturer’s instructions. Total RNA was isolated, and an mRNA reverse transcription kit (CW2569, CWBIO, China) was used to reverse transcribe total RNA into cDNA. The expression levels of miR-7, ciRS-7, Myrip, Pax6, Bax and Caspase-3 were quantified via qRT‒PCR, and specific primers were used to perform qRT‒PCR. U6 or GAPDH was selected as the internal reference for analysis of target gene expression. The combination of Primer 8.0 software (Premier Biosoft, Palo Alto, USA) and Sangon Biotech (Shanghai, China) was used to design all the primers (Table 1). We used the 2 −ΔΔCT method to calculate the relative expression levels of each gene. Cell culture and transfection The HPDE6-C7 (NO.BNCC359453) cell line from BeNa Culture Cottection of Beijing and NES2Y (No.BFN607200613) cell lines were obtained from Biochannel Biotechnology of Nanjing. HPDE6-C7 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37°C in a humidified atmosphere containing 5% CO2. NES2Y cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Small interfering RNAs (siRNAs) (siRNAs, siciRS-7-NC, siciRS-7-1 and siciRS-7-2, siMyrip-NC, siMyrip-1 and siMyrip-2) were designed by Kidan Biotech of Guangzhou (Table 2). ciRS-7 and Myrip were knocked down by siRNA, and the empty vector was used as a negative control. All the cells were subsequently transfected with Lipofectamine RNA iMAX (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. Cell Counting Kit-8 The cells in the different groups were digested, counted, and seeded in 96-well plates (0030730119, Eppendorf, Germany) at 1x10 4 cells/well (100 μl per well). Each group was divided into three wells. After the cells had adhered to the wall, the medium was changed, and the intervention factors were added. After 48 hours of treatment, the culture medium was removed, and the cells were incubated with 110μl of DMEM containing 10μl of CCK-8 solution. After incubation at 37°C and 5% CO2 for 4 h, the absorbance value at 450 nm was analyzed with a Bio-Tek microplate (MB-530, Heales, China). Flow cytometry For the determination of cell apoptosis, cells were double-stained with Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI) to identify apoptotic populations. Following trypsin digestion without EDTA, the cells were collected, washed with phosphate-buffered saline (PBS), and resuspended in binding buffer. Annexin V-FITC and PI staining were performed according to the manufacturer's instructions (KGA108, KeyGEN Biotech Co., Ltd., Jiangsu, China). Cell apoptosis was then analyzed using an A00-1-11102 flow cytometer (Beckman Coulter, USA). Colony formation assay After transfection, the cells (6000/well) were seeded into six-well plates and cultured for 14 days in high-glucose DMEM supplemented with 10% FBS at 37°C and 5% CO2. The colonies were subsequently fixed in 4% paraformaldehyde and stained with 0.05% crystal violet. Western blot The total proteins were extracted with RIPA buffer (P0013B, Fude Biotech, China). The proteins were deposited onto a nitrocellulose filter membrane via polyacrylamide gel electrophoresis. The membranes were blocked with 5% (w/v) nonfat dry milk for 90 min before being incubated with primary antibodies overnight at 4°C. The primary antibodies used included those against GAPDH (CST 5174s, 1:1000), Myrip (Abcam ab10149, 1:1000), Pax6 (CST 60433, 1:1000), Bax (Proteintech 60267-1-Ig, 1:1000), and Caspase-3 (GeneTex GTX110543, 1:1000). The sections were then incubated with secondary anti-goat IgG (FDG007, 1:5000), anti-mouse IgG (FDM007, 1:5000) and anti-rabbit IgG (CST 7074s, 1:5000) antibodies for 1.5 h at room temperature. We used an ECL chromogenic substrate to visualize the protein bands. Statistical analysis Statistical analysis of functional outcomes was carried out using Student’s t-test for two-group comparisons and two-way ANOVA for multi-group comparisons. Data are presented as mean ± SEM. Significance levels are denoted as: NS, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Results Differential Expression of ciRS-7 and miR-7 in Pancreatic Neuroendocrine Tumors. Accumulating evidence has highlighted the involvement of non-coding RNAs, particularly circRNAs and miRNAs, in the initiation and progression of various cancers [20]. Given their emerging regulatory roles in tumorigenesis, we focused on the potential dysregulation of ciRS-7 and miR-7 in pancreatic neuroendocrine tumors (PNETs). To investigate the expression profiles of ciRS-7 and miR-7 in PNET cells, we performed RT-qPCR analysis in a normal human pancreatic ductal epithelial cell line (HPDE6-C7) and a pancreatic neuroendocrine tumor cell line (NES2Y). The results revealed that ciRS-7 was significantly upregulated in NES2Y cells compared to HPDE6-C7 cells (Figure 1A). Conversely, miR-7 expression was markedly downregulated in the tumor cell line relative to the normal control (Figure 1B). These findings suggest that ciRS-7 may exert oncogenic functions, whereas miR-7 may act as a tumor suppressor in the progression of pancreatic neuroendocrine tumors. Knockdown of ciRS-7 suppresses tumor cell proliferation and induces apoptosis CiRS-7 (also known as CDR1as) is a well-characterized circRNA containing multiple conserved binding sites for miR-7, allowing it to specifically sequester and inhibit this miRNA [21]. To validate the functional role of ciRS-7, we designed a small interfering RNA (siRNA) targeting ciRS-7 to silence its expression in pancreatic islet cell tumor cells. RT-qPCR analysis confirmed that si-ciRS-7 effectively reduced ciRS-7 expression levels (Figure 2A), indicating efficient transfection. Subsequently, CCK-8 and colony formation assays were performed to evaluate the impact of ciRS-7 silencing on cell proliferation. The CCK-8 assay revealed a significant decrease in cell viability following ciRS-7 knockdown (Figure 2B). Consistently, the colony formation assay showed that the clonogenic ability of tumor cells was markedly impaired after ciRS-7 suppression (Figure 2C). These results indicate that ciRS-7 promotes cell proliferation in pancreatic islet cell tumor cells. To further investigate the effect of ciRS-7 silencing on cell apoptosis, flow cytometric analysis was performed. Our results showed that knockdown of ciRS-7 significantly increased the apoptotic rate in NES2Y cells (Figure 2D). Consistently, RT-qPCR and Western blot analyses revealed that the expression levels of the apoptosis-related genes Bax and Caspase-3 were markedly upregulated following ciRS-7 suppression (Figure 2E). Collectively, these data indicate that Silencing of ciRS-7 suppresses the growth of NES2Y cells, suggesting that ciRS-7 may function as a pro-proliferative factor in promoting tumor cell proliferation. CiRS-7 regulates the expression of Myrip and Pax6 by modulating miR-7 Given the established role of ciRS-7 as a potent molecular sponge for miR-7 [22], we further investigated the functional consequences of ciRS-7 silencing on miR-7 bioavailability and its downstream targets. RT-qPCR analysis revealed that knockdown of ciRS-7 significantly increased miR-7 expression, while markedly decreasing the mRNA levels of Myrip and Pax6 (Figure 3A-3D). Consistent with transcriptional repression, Western blot analysis revealed a pronounced decrease in MYRIP and PAX6 protein expression (Figure 3E). These results suggest that ciRS-7 may regulate Myrip and Pax6 expression through modulating miR-7. Myrip is regulated by ciRS-7 and promotes cell proliferation while inhibiting apoptosis in NES2Y cells. To further elucidate the regulatory relationship between Myrip, ciRS-7, miR-7, and Pax6, we designed a small interfering RNA (siRNA) targeting Myrip to silence its expression in pancreatic islet cell tumor cells. Figures 4A and 4B validated the knockdown efficiency of si-Myrip using RT-qPCR and Western blot analysis, respectively. Myrip is an interacting partner of the small GTPase Rab27, playing a role in the trafficking and exocytosis of secretory granules [23]. To further investigate the regulatory effects of Myrip on its upstream and downstream genes in pancreatic neuroendocrine tumor cells, RT-qPCR was performed following Myrip knockdown. The results showed that silencing Myrip significantly reduced the mRNA expression levels of both its upstream regulator ciRS-7 and its downstream target Pax6, while markedly increasing miR-7 expression (Figures 4C-4G). CCK-8 and colony formation assays demonstrated that Myrip silencing led to a significant inhibition of cell proliferation (Figures 4H-4I). Flow cytometric analysis revealed a notable increase in apoptosis after Myrip knockdown (Figure 4J). Moreover, RT-qPCR and Western blot analyses revealed that silencing Myrip significantly upregulated the expression levels of Bax and Caspase-3 (Figure 4K-4N). Taken together, these results suggest that Myrip knockdown reverses the expression of ciRS-7 and its downstream targets (Myrip and Pax6), thereby inhibiting tumor cell proliferation and promoting apoptosis. Discussion The pathogenesis of pancreatic neuroendocrine tumor is complex and involves abnormalities in multiple genes and signaling pathways [ 24 ]. In this study, we investigated the role of ciRS-7 in islet cell tumorigenesis and uncovered a critical regulatory mechanism mediated by the ciRS-7/miR-7/Myrip/Pax6 signaling axis in modulating tumor cell proliferation and apoptosis. Our results demonstrate that ciRS-7 is highly expressed in pancreatic neuroendocrine tumor cells, whereas its target miR-7 is downregulated. This finding is consistent with the well-established function of ciRS-7 as a molecular sponge for miR-7 [ 25 ]. Specifically, ciRS-7 binds to and sequesters miR-7, thereby reducing its effective concentration and alleviating miR-7-mediated suppression of downstream target genes [ 26 ]. We further demonstrated that silencing ciRS-7 significantly inhibits the proliferation of pancreatic neuroendocrine tumor cells and promotes apoptosis, suggesting that ciRS-7 exerts oncogenic effects in this context. Notably, miR-7 expression was upregulated following ciRS-7 knockdown, which is consistent with its established function as a molecular sponge for miR-7. More importantly, we observed that the expression levels of Myrip and Pax6 were markedly reduced upon ciRS-7 silencing. Myrip, also known as Myosin Va and Rab-interacting protein, is involved in intracellular trafficking and cytoskeletal dynamics, processes that are essential for cell motility and functional polarization [ 27 ]. Pax6 has been widely recognized as a master regulator of pancreatic islet development and β-cell function, with critical implications in both normal physiology and pathological conditions such as tumorigenesis [ 28 , 29 ]. Our findings suggest that ciRS-7 may regulate the expression of Myrip and Pax6 through modulating miR-7, thereby influencing the biological behavior of pancreatic neuroendocrine tumor cells. Functionally, Myrip depletion recapitulated the tumor-suppressive effects of ciRS-7 knockdown, including reduced cell viability and increased apoptotic activity, highlighting its pro-tumorigenic role in pancreatic neuroendocrine tumors. Furthermore, we observed that silencing Myrip led to a significant downregulation of ciRS-7 and Pax6 expression, while miR-7 expression was upregulated. Our findings suggest that Myrip is not only regulated by the ciRS-7/miR-7 axis, but may also participate in this complex regulatory network and influence the expression of both ciRS-7 and Pax6. These results provide a foundation for further investigation into the clinical utility of the ciRS-7/miR-7/Myrip/Pax6 axis as a diagnostic marker or therapeutic target in pancreatic neuroendocrine tumors. Conclusion Through in vitro investigations, this study elucidates the mechanistic role of ciRS-7 in pancreatic islet cell tumors. We demonstrate that ciRS-7 is markedly upregulated in pancreatic neuroendocrine tumor cells and functions as a molecular sponge for miR-7. This interaction alleviates miR-7-mediated suppression of Myrip and Pax6, consequently promoting tumor cell proliferation while inhibiting apoptosis. The identification of this ciRS-7/miR-7/Myrip/Pax6 signaling axis provides novel insights into the molecular pathogenesis of pancreatic neuroendocrine tumors and reveals potential targets for future diagnostic and therapeutic development. Abbreviations RT-qPCR: quantitative reverse transcription PCR, siRNA: small interfering RNA, miRNA: microRNA, ciRS-7: circRNA-7, PNETs: pancreatic neuroendocrine tumors, RBPs: RNA-binding proteins, PI3K: phosphatidylinositol 3-kinase, EGFR: epidermal growth factor rreceptor, MAPK: mitogen-activated protein kinase, CCK-8: cell counting kit-8. Declarations Acknowledgments Not applicable. Author Contributions D.L. supported and supervised the study. C.L. and Z.F. responded to the study design. Z.T. conducted most experiments in vitro. Z.T., M.H., D.W. and Z.L. contributed to data collection. Z.T. and M.H. prepared the manuscript. D.L. reviewed the manuscript. Funding This study was supported by Shenzhen Key Medical Discipline Construction Fund (No.SZXK010), Shenzhen Science and Technology Project (JCYJ20220531093411025), Sanming Project of Medicine in Shenzhen(No.SZSM202411024). Data availability The datasets used in this paper are available online. Ethics approval and consent to participate This study does not involve animal experiments or clinical research involving human subjects; therefore, no animal or clinical ethics approval was required. Consent for publication Not applicable. Competing interests The authors report no potential conflicts of interest, including financial or personal relationships that could influence the findings presented in this study. Author details a Peking University Shenzhen Hospital, Shenzhen, 518000, China. b Department of Gastroenterology, Zhongshan City People’s Hospital, Zhongshan, 528403, China. c Shenzhen Hospital of Guangzhou University of Chinese Medicine, Shenzhen, 518034, China References Cives M, Strosberg JR. Gastroenteropancreatic Neuroendocrine Tumors. CA: A Cancer Journal for Clinicians. 2018;68:471–87. Cloyd JM. Non-functional neuroendocrine tumors of the pancreas: Advances in diagnosis and management. World Journal of Gastroenterology. 2015;21:9512. Dasari A, Shen C, Halperin D, Zhao B, Zhou S, Xu Y, et al. Trends in the Incidence, Prevalence, and Survival Outcomes in Patients With Neuroendocrine Tumors in the United States. JAMA Oncol. 2017;3:1335. Tacelli M, Gentiluomo M, Biamonte P, Castano JP, Berković MC, Cives M, et al. Pancreatic neuroendocrine neoplasms (pNENs): Genetic and environmental biomarkers for risk of occurrence and prognosis. Seminars in Cancer Biology. 2025;112:112–25. 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07:38:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7700686/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7700686/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93035587,"identity":"453f7edb-a8e9-4751-a676-da8b769f0ad9","added_by":"auto","created_at":"2025-10-08 11:05:22","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15646930,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-7700686/v1/31557a27b7e4e66b3ad522d3.docx"},{"id":93035575,"identity":"19fc29df-c680-468d-8f91-057ff63fe1f6","added_by":"auto","created_at":"2025-10-08 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11:05:24","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":79272,"visible":true,"origin":"","legend":"","description":"","filename":"122c9cfc183f4530a10b93f973739bff1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7700686/v1/8186a9c9fce11cfa08bb7842.xml"},{"id":93035616,"identity":"c86f0b48-c87e-4edb-8b41-070aa58b21f3","added_by":"auto","created_at":"2025-10-08 11:05:23","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":88810,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7700686/v1/bafedfb4f8fb82c2b7a52c8b.html"},{"id":93035576,"identity":"d56738f6-10d9-49cf-a751-8dbc4871d4a5","added_by":"auto","created_at":"2025-10-08 11:05:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":51672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression levels of ciRS-7 and miR-7 in pancreatic neuroendocrine tumor cell lines. \u003c/strong\u003e(A) ciRS-7 expression in HPDE6-C7 and NES2Y cells. (B) miR-7 expression in HPDE6-C7 and NES2Y cells. Data are presented as mean ± SEM from three independent experiments. Statistical significance was determined by Student’s t-test; \u003cem\u003e*P \u0026lt; 0.05, ****P \u0026lt; 0.0001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7700686/v1/f630ff4a750fb05b1c57aa42.png"},{"id":93035583,"identity":"a578cb99-9111-4cfe-907a-967255c03160","added_by":"auto","created_at":"2025-10-08 11:05:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":269909,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSilencing of ciRS-7 inhibits cell proliferation and induces apoptosis. \u003c/strong\u003e(A) RT-qPCR confirms effective knockdown of ciRS-7 by si-ciRS-7.\u003cstrong\u003e \u003c/strong\u003e(B) CCK-8 assay shows reduced cell viability after ciRS-7 silencing.\u003cstrong\u003e \u003c/strong\u003e(C) Colony formation assay demonstrates decreased clonogenic capacity upon ciRS-7 downregulation. (D) Flow cytometry analysis reveals increased apoptosis following ciRS-7 knockdown. (E-H) RT-qPCR and Western blot show upregulation of Bax and Caspase-3 expression after ciRS-7 suppression. \u003cem\u003e*P \u0026lt; 0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, ****P \u0026lt; 0.0001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7700686/v1/9b58796f959855ae56aba2f2.png"},{"id":93035590,"identity":"ac46cead-d968-4836-a179-58ee063f57d5","added_by":"auto","created_at":"2025-10-08 11:05:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":136700,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCiRS-7 regulates Myrip and Pax6 expression via modulation of miR-7.\u003c/strong\u003e (A-D) RT-qPCR shows that silencing ciRS-7 increases miR-7 expression and decreases Myrip and Pax6 mRNA levels. (E) Western blot confirms reduced protein expression of Myrip and Pax6 after ciRS-7 knockdown. \u003cem\u003e*P \u0026lt; 0.05, **P\u0026lt;0.01.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7700686/v1/3c71ffa68cacdec9fbbdba5a.png"},{"id":93035621,"identity":"ea0577b7-c785-4118-af70-679ef42bf9ad","added_by":"auto","created_at":"2025-10-08 11:05:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":346620,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional characterization of Myrip\u003c/strong\u003e \u003cstrong\u003ein NES2Y cells.\u003c/strong\u003e (A, B) RT-qPCR and Western blot confirm efficient knockdown of Myrip.(C-F) mRNA Expression of Myrip-Related Genes in NES2Y Cells After Myrip Silencing. (G) Protein levels of upstream and downstream genes following Myrip silencing. (H, I) CCK-8 and colony formation assays show that Myrip knockdown inhibits cell proliferation. (J) Flow cytometry reveals increased apoptosis after Myrip silencing. (K-M) RT-qPCR and Western blot demonstrate upregulation of Bax and Caspase-3 expression upon Myrip knockdown.\u003cem\u003e *P \u0026lt; 0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7700686/v1/cda9a9f499c5be52a9e51c3d.png"},{"id":95314043,"identity":"e578d9d7-d6db-48ee-8bb5-3e8e56674962","added_by":"auto","created_at":"2025-11-06 15:52:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1531565,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7700686/v1/cabdfd9c-5dc5-4be9-aebc-e3b4e121d351.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"ciRS-7 Promotes Proliferation and Apoptosis in Insulinoma Cells via miR-7/MYRIP/Pax6 Signaling Pathway","fulltext":[{"header":"Background","content":"\u003cp\u003ePancreatic Neuroendocrine Tumors (PNETs) are a heterogeneous group of neoplasms arising from neuroendocrine cells within the pancreas, representing approximately 3% of all pancreatic tumors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although the incidence of PNETs remains relatively low, epidemiological studies have demonstrated a steady increase in the diagnosis of PNETs over the past two decades, likely due to advances in imaging techniques and increased clinical awareness [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The clinical presentation of PNETs varies widely, ranging from asymptomatic cases to severe hormone-related syndromes, and patient outcomes can differ significantly. Conventional treatment strategies include surgical resection, chemotherapy, and targeted therap [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, therapeutic efficacy for advanced or metastatic disease remains suboptimal [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, a comprehensive understanding of the molecular mechanisms underlying PNET pathogenesis is essential, along with the identification of novel diagnostic biomarkers and potential therapeutic targets.\u003c/p\u003e\u003cp\u003eNon-coding RNAs (ncRNAs) play critical regulatory roles in gene expression and have been widely associated with the development and progression of various cancers [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Among them, circular RNAs (circRNAs) represent a unique class of ncRNAs characterized by their covalently closed loop structures formed through back-splicing events, which render them resistant to degradation by exonucleases and thus highly stable and conserved across species [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Unlike linear RNAs, circRNAs are resistant to degradation by exonucleases due to their unique loop structure, making circRNAs remarkably stable and abundant in various tissues and body fluids, including blood and saliva [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. CircRNAs participate in the regulation of gene expression through multiple mechanisms, including acting as microRNA (miRNA) sponges to modulate post-transcriptional gene regulation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], interacting with RNA-binding proteins (RBPs) to influence RNA processing [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and even regulating transcription and translation processes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. MicroRNA-7 (miR-7) is a widely expressed microRNA that has been shown to exert either tumor-suppressive or oncogenic functions depending on the cancer type and cellular context [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Importantly, downregulation of miR-7 has been observed in both type 2 diabetes and PNETs, suggesting its protective role against islet cell dysregulation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In many cancers, miR-7 suppresses tumor cell proliferation, migration, and invasion by targeting key oncogenes such as epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, the expression and function of miR-7 are regulated by multiple factors, among which circRNAs serving as miRNA sponges represent a crucial regulatory mechanism [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. CiRS-7, also known as CDR1as, was the first extensively studied circRNA capable of sequestering miR-7 through abundant binding sites, thereby relieving the inhibitory effects of miR-7 on its downstream target genes and promoting tumor progression [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Given the critical roles of ciRS-7 and miR-7 in tumor development and progression, as well as the essential functions of Myrip and Pax6 in insulin secretion and pancreatic development [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], the objective of this study is to elucidate the involvement of the ciRS-7/miR-7 regulatory axis in pancreatic islet cell tumorigenesis by modulating the expression of Myrip and Pax6. Our findings indicate that knockdown of the circular RNA ciRS-7 suppresses cell growth in pancreatic islet cell tumor cells by targeting miR-7 and modulating the Myrip and Pax6 signaling pathways, ultimately inducing apoptosis. These results suggest that ciRS-7 may serve as a potential therapeutic target for pancreatic islet cell tumors.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eQuantitative reverse transcription polymerase chain reaction (qRT‒PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative reverse transcription polymerase chain reaction can reliably detect and quantitatively measure the products generated in each cycle of the PCR process [19]. Total RNA was extracted from HPDE6-C7 and NES2Y cells via TRIzol reagent (15596026, Thermo, USA) following the manufacturer\u0026rsquo;s instructions. MiRNA was extracted from HPDE6-C7 and NES2Y cells via TRIzol reagent (Life Technologies, Carlsbad, USA) following the manufacturer\u0026rsquo;s instructions. Total RNA was isolated, and an mRNA reverse transcription kit (CW2569, CWBIO, China) was used to reverse transcribe total RNA into cDNA. The expression levels of miR-7, ciRS-7, Myrip, Pax6, Bax and Caspase-3 were quantified via qRT‒PCR, and specific primers were used to perform qRT‒PCR. U6 or GAPDH was selected as the internal reference for analysis of target gene expression. The combination of Primer 8.0 software (Premier Biosoft, Palo Alto, USA) and Sangon Biotech (Shanghai, China) was used to design all the primers (Table 1). We used the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method to calculate the relative expression levels of each gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe HPDE6-C7 (NO.BNCC359453) cell line from BeNa Culture Cottection of Beijing and NES2Y (No.BFN607200613) cell lines were obtained from Biochannel Biotechnology of Nanjing. HPDE6-C7 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin\u0026ndash;streptomycin at 37\u0026deg;C in a humidified atmosphere containing 5% CO2. NES2Y cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin\u0026ndash;streptomycin at 37\u0026deg;C in a humidified atmosphere containing 5% CO2. Small interfering RNAs (siRNAs) (siRNAs, siciRS-7-NC, siciRS-7-1 and siciRS-7-2, siMyrip-NC, siMyrip-1 and siMyrip-2) were designed by Kidan Biotech of Guangzhou (Table 2). ciRS-7 and Myrip were knocked down by siRNA, and the empty vector was used as a negative control. All the cells were subsequently transfected with Lipofectamine RNA iMAX (Invitrogen, Carlsbad, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Counting Kit-8\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells in the different groups were digested, counted, and seeded in 96-well plates (0030730119, Eppendorf, Germany) at 1x10\u003csup\u003e4\u003c/sup\u003e cells/well (100 \u0026mu;l per well). Each group was divided into three wells. After the cells had adhered to the wall, the medium was changed, and the intervention factors were added. After 48 hours of treatment, the culture medium was removed, and the cells were incubated with 110\u0026mu;l of DMEM containing 10\u0026mu;l of CCK-8 solution. After incubation at 37\u0026deg;C and 5% CO2 for 4 h, the absorbance value at 450 nm was analyzed with a Bio-Tek microplate (MB-530, Heales, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the determination of cell apoptosis, cells were double-stained with Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI) to identify apoptotic populations. Following trypsin digestion without EDTA, the cells were collected, washed with phosphate-buffered saline (PBS), and resuspended in binding buffer. Annexin V-FITC and PI staining were performed according to the manufacturer\u0026apos;s instructions (KGA108, KeyGEN Biotech Co., Ltd., Jiangsu, China). Cell apoptosis was then analyzed using an A00-1-11102 flow cytometer (Beckman Coulter, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter transfection, the cells (6000/well) were seeded into six-well plates and cultured for 14 days in high-glucose DMEM supplemented with 10% FBS at 37\u0026deg;C and 5% CO2. The colonies were subsequently fixed in 4% paraformaldehyde and stained with 0.05% crystal violet.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe total proteins were extracted with RIPA buffer (P0013B, Fude Biotech, China). The proteins were deposited onto a nitrocellulose filter membrane via polyacrylamide gel electrophoresis. The membranes were blocked with 5% (w/v) nonfat dry milk for 90 min before being incubated with primary antibodies overnight at 4\u0026deg;C. The primary antibodies used included those against GAPDH (CST 5174s, 1:1000), Myrip (Abcam ab10149, 1:1000), Pax6 (CST 60433, 1:1000), Bax (Proteintech 60267-1-Ig, 1:1000), and Caspase-3 (GeneTex GTX110543, 1:1000). The sections were then incubated with secondary anti-goat IgG (FDG007, 1:5000), anti-mouse IgG (FDM007, 1:5000) and anti-rabbit IgG (CST 7074s, 1:5000) antibodies for 1.5 h at room temperature. We used an ECL chromogenic substrate to visualize the protein bands.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis of functional outcomes was carried out using Student\u0026rsquo;s t-test for two-group comparisons and two-way ANOVA for multi-group comparisons. Data are presented as mean \u0026plusmn; SEM. Significance levels are denoted as: NS, non-significant; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDifferential Expression of ciRS-7 and miR-7 in Pancreatic Neuroendocrine Tumors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccumulating evidence has highlighted the involvement of non-coding RNAs, particularly circRNAs and miRNAs, in the initiation and progression of various cancers\u0026nbsp;[20]. Given their emerging regulatory roles in tumorigenesis, we focused on the potential dysregulation of ciRS-7 and miR-7 in pancreatic neuroendocrine tumors (PNETs). To investigate the expression profiles of ciRS-7 and miR-7 in PNET cells, we performed RT-qPCR analysis in a normal human pancreatic ductal epithelial cell line (HPDE6-C7) and a pancreatic neuroendocrine tumor cell line (NES2Y). The results revealed that ciRS-7 was significantly upregulated in NES2Y cells compared to HPDE6-C7 cells (Figure 1A). Conversely, miR-7 expression was markedly downregulated in the tumor cell line relative to the normal control (Figure 1B). These findings suggest that ciRS-7 may exert oncogenic functions, whereas miR-7 may act as a tumor suppressor in the progression of pancreatic neuroendocrine tumors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnockdown of ciRS-7 suppresses tumor cell proliferation and induces apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCiRS-7 (also known as CDR1as) is a well-characterized circRNA containing multiple conserved binding sites for miR-7, allowing it to specifically sequester and inhibit this miRNA\u0026nbsp;[21]. To validate the functional role of ciRS-7, we designed a small interfering RNA (siRNA) targeting ciRS-7 to silence its expression in pancreatic islet cell tumor cells. RT-qPCR analysis confirmed that si-ciRS-7 effectively reduced ciRS-7 expression levels (Figure 2A), indicating efficient transfection. Subsequently, CCK-8 and colony formation assays were performed to evaluate the impact of ciRS-7 silencing on cell proliferation. The CCK-8 assay revealed a significant decrease in cell viability following ciRS-7 knockdown (Figure 2B). Consistently, the colony formation assay showed that the clonogenic ability of tumor cells was markedly impaired after ciRS-7 suppression (Figure 2C). These results indicate that ciRS-7 promotes cell proliferation in pancreatic islet cell tumor cells.\u003c/p\u003e\n\u003cp\u003eTo further investigate the effect of ciRS-7 silencing on cell apoptosis, flow cytometric analysis was performed. Our results showed that knockdown of ciRS-7 significantly increased the apoptotic rate in NES2Y cells (Figure 2D). Consistently, RT-qPCR and Western blot analyses revealed that the expression levels of the apoptosis-related genes Bax and Caspase-3 were markedly upregulated following ciRS-7 suppression (Figure 2E). Collectively, these data indicate that Silencing of ciRS-7 suppresses the growth of NES2Y cells, suggesting that ciRS-7 may function as a pro-proliferative factor in promoting tumor cell proliferation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCiRS-7 regulates the expression of Myrip and Pax6 by modulating miR-7\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the established role of ciRS-7 as a potent molecular sponge for miR-7\u0026nbsp;[22], we further investigated the functional consequences of ciRS-7 silencing on miR-7 bioavailability and its downstream targets. RT-qPCR analysis revealed that knockdown of ciRS-7 significantly increased miR-7 expression, while markedly decreasing the mRNA levels of Myrip and Pax6 (Figure 3A-3D). Consistent with transcriptional repression, Western blot analysis revealed a pronounced decrease in MYRIP and PAX6 protein expression (Figure 3E). These results suggest that ciRS-7 may regulate Myrip and Pax6 expression through modulating miR-7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMyrip is regulated by ciRS-7 and promotes cell proliferation while inhibiting apoptosis in NES2Y cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further elucidate the regulatory relationship between Myrip, ciRS-7, miR-7, and Pax6, we designed a small interfering RNA (siRNA) targeting Myrip to silence its expression in pancreatic islet cell tumor cells. Figures 4A and 4B validated the knockdown efficiency of si-Myrip using RT-qPCR and Western blot analysis, respectively. Myrip is an interacting partner of the small GTPase Rab27, playing a role in the trafficking and exocytosis of secretory granules [23]. To further investigate the regulatory effects of Myrip on its upstream and downstream genes in pancreatic neuroendocrine tumor cells, RT-qPCR was performed following Myrip knockdown. The results showed that silencing Myrip significantly reduced the mRNA expression levels of both its upstream regulator ciRS-7 and its downstream target Pax6, while markedly increasing miR-7 expression (Figures 4C-4G). CCK-8 and colony formation assays demonstrated that Myrip silencing led to a significant inhibition of cell proliferation (Figures 4H-4I). Flow cytometric analysis revealed a notable increase in apoptosis after Myrip knockdown (Figure 4J). Moreover, RT-qPCR and Western blot analyses revealed that silencing Myrip significantly upregulated the expression levels of Bax and Caspase-3 (Figure 4K-4N). Taken together, these results suggest that Myrip knockdown reverses the expression of ciRS-7 and its downstream targets (Myrip and Pax6), thereby inhibiting tumor cell proliferation and promoting apoptosis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe pathogenesis of pancreatic neuroendocrine tumor is complex and involves abnormalities in multiple genes and signaling pathways [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this study, we investigated the role of ciRS-7 in islet cell tumorigenesis and uncovered a critical regulatory mechanism mediated by the ciRS-7/miR-7/Myrip/Pax6 signaling axis in modulating tumor cell proliferation and apoptosis. Our results demonstrate that ciRS-7 is highly expressed in pancreatic neuroendocrine tumor cells, whereas its target miR-7 is downregulated. This finding is consistent with the well-established function of ciRS-7 as a molecular sponge for miR-7 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Specifically, ciRS-7 binds to and sequesters miR-7, thereby reducing its effective concentration and alleviating miR-7-mediated suppression of downstream target genes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. We further demonstrated that silencing ciRS-7 significantly inhibits the proliferation of pancreatic neuroendocrine tumor cells and promotes apoptosis, suggesting that ciRS-7 exerts oncogenic effects in this context. Notably, miR-7 expression was upregulated following ciRS-7 knockdown, which is consistent with its established function as a molecular sponge for miR-7. More importantly, we observed that the expression levels of Myrip and Pax6 were markedly reduced upon ciRS-7 silencing. Myrip, also known as Myosin Va and Rab-interacting protein, is involved in intracellular trafficking and cytoskeletal dynamics, processes that are essential for cell motility and functional polarization [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Pax6 has been widely recognized as a master regulator of pancreatic islet development and β-cell function, with critical implications in both normal physiology and pathological conditions such as tumorigenesis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Our findings suggest that ciRS-7 may regulate the expression of Myrip and Pax6 through modulating miR-7, thereby influencing the biological behavior of pancreatic neuroendocrine tumor cells. Functionally, Myrip depletion recapitulated the tumor-suppressive effects of ciRS-7 knockdown, including reduced cell viability and increased apoptotic activity, highlighting its pro-tumorigenic role in pancreatic neuroendocrine tumors. Furthermore, we observed that silencing Myrip led to a significant downregulation of ciRS-7 and Pax6 expression, while miR-7 expression was upregulated. Our findings suggest that Myrip is not only regulated by the ciRS-7/miR-7 axis, but may also participate in this complex regulatory network and influence the expression of both ciRS-7 and Pax6. These results provide a foundation for further investigation into the clinical utility of the ciRS-7/miR-7/Myrip/Pax6 axis as a diagnostic marker or therapeutic target in pancreatic neuroendocrine tumors.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThrough in vitro investigations, this study elucidates the mechanistic role of ciRS-7 in pancreatic islet cell tumors. We demonstrate that ciRS-7 is markedly upregulated in pancreatic neuroendocrine tumor cells and functions as a molecular sponge for miR-7. This interaction alleviates miR-7-mediated suppression of Myrip and Pax6, consequently promoting tumor cell proliferation while inhibiting apoptosis. The identification of this ciRS-7/miR-7/Myrip/Pax6 signaling axis provides novel insights into the molecular pathogenesis of pancreatic neuroendocrine tumors and reveals potential targets for future diagnostic and therapeutic development.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eRT-qPCR: quantitative reverse transcription PCR, siRNA: small interfering RNA, miRNA: microRNA, ciRS-7: circRNA-7, PNETs: pancreatic neuroendocrine tumors, RBPs: RNA-binding proteins, PI3K: phosphatidylinositol 3-kinase, EGFR: epidermal growth factor rreceptor, MAPK: mitogen-activated protein kinase, CCK-8: cell counting kit-8.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.L. supported and supervised the study. C.L. and Z.F. responded to the study design. Z.T. conducted most experiments in vitro. Z.T., M.H., D.W. and Z.L. contributed to data collection. Z.T. and M.H. prepared the manuscript. D.L. reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Shenzhen Key Medical Discipline Construction Fund (No.SZXK010),\u0026nbsp;Shenzhen Science and Technology Project (JCYJ20220531093411025), Sanming Project of Medicine in Shenzhen(No.SZSM202411024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used in this paper are available online.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study does not involve animal experiments or clinical research involving human subjects; therefore, no animal or clinical ethics approval was required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no potential conflicts of interest, including financial or personal relationships that could influence the findings presented in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003ePeking University Shenzhen Hospital, Shenzhen, 518000, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003eDepartment of Gastroenterology, Zhongshan City People\u0026rsquo;s Hospital, Zhongshan, 528403, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u0026nbsp;\u003c/sup\u003eShenzhen Hospital of Guangzhou University of Chinese Medicine, Shenzhen, 518034, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCives M, Strosberg JR. 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Biomedicine \u0026amp; Pharmacotherapy. 2023;162:114672. \u003c/li\u003e\n\u003cli\u003eLiu L, Liu F-B, Huang M, Xie K, Xie Q-S, Liu C-H, et al. Circular RNA ciRS-7 promotes the proliferation and metastasis of pancreatic cancer by regulating miR-7-mediated EGFR/STAT3 signaling pathway. Hepatobiliary \u0026amp; Pancreatic Diseases International. 2019;18:580\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eKudo A, Akahoshi K, Ito S, Akashi T, Shimada S, Ogura T, et al. Downregulated Pancreatic Beta Cell Genes Indicate Poor Prognosis in Patients With Pancreatic Neuroendocrine Neoplasms. Annals of Surgery. 2020;271:732\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003ePanneerselvam A, Kannan A, Mariajoseph-Antony LF, Prahalathan C. PAX proteins and their role in pancreas. Diabetes Research and Clinical Practice. 2019;155:107792. \u003c/li\u003e\n\u003cli\u003eYongblah K, Alford SC, Ryan BC, Chow RL, Howard PL. Protecting Pax6 3\u0026prime; UTR from MicroRNA-7 Partially Restores PAX6 in Islets from an Aniridia Mouse Model. Molecular Therapy Nucleic Acids. 2018;13:144\u0026ndash;53. \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":"Pancreatic Neuroendocrine Tumors, ciRS-7, miR-7, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-7700686/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7700686/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Pancreatic Neuroendocrine Tumors（PNETs）is a rare neuroendocrine tumor with a complex and not fully elucidated pathogenesis. In recent years, the role of non-coding RNAs in tumorigenesis has gained increasing attention. Circular RNA ciRS-7, known as a competitive endogenous RNA, acts as a sponge for tumor-suppressive miR-7, thereby modulating gene expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods：\u003c/strong\u003eThe expression levels of ciRS-7 and miR-7 in NES2Y cells were detected by qRT-PCR. Subsequently, ciRS-7 was silenced in NES2Y cells, and the expression of apoptosis-related genes Bax and Caspase-3, as well as proliferation-related genes Myrip and Pax6, was evaluated by qRT-PCR and Western blot analysis. Cell proliferation was assessed using the CCK-8 assay and colony formation assay, while cell apoptosis was analyzed by flow cytometry\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe detected significant upregulation of ciRS-7 in insulinoma cell lines NES2Y, whereas miR-7 expression was markedly downregulated. Knockdown of ciRS-7 significantly suppressed cellular proliferation while promoting apoptosis, concomitantly upregulating expression of apoptosis-related genes Bax and Caspase-3. Further study showed that ciRS-7 regulates Myrip and Pax6 expression through miR-7. Knockdown of Myrip suppresses cell proliferation, induces apoptosis, and reverses the functional expression patterns of ciRS-7 and miR-7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eCiRS-7 functions as a miR-7 sponge, inducing insulinoma cell apoptosis by targeting miR-7 and modulating the Myrip /Pax6 signaling pathway.\u003c/p\u003e","manuscriptTitle":"ciRS-7 Promotes Proliferation and Apoptosis in Insulinoma Cells via miR-7/MYRIP/Pax6 Signaling Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 11:05:17","doi":"10.21203/rs.3.rs-7700686/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":"74b05a32-980d-452c-96b3-7a6f0060b383","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-06T09:09:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-08 11:05:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7700686","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7700686","identity":"rs-7700686","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}