miR-383-5p Inhibits Human Malignant Melanoma via Targeting CENPF

miR-383-5p Inhibits Human Malignant Melanoma via Targeting CENPF | 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 miR-383-5p Inhibits Human Malignant Melanoma via Targeting CENPF Wanwan Jin, Huazhen Liu, Qin Xu, Nan Lin, Haiting Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-154404/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: Human malignant melanoma (MM) is one of the skin cancers with the highest mortality. In this study, we investigated the role of miR-383-5p on human MM cells. Methods: The expression of miR-383-5p was measured by quantitative real-time PCR assay. The cell proliferation, invasion and migration were detected by CCK8, clone formation and transwell assays. Flow cytometry assay was used to detect apoptosis. The binding of miR-383-5p and 3’UTR of CENPF mRNA was indicated by dual-luciferase assays. Results: We found that miR-383-5p inhibited the cells proliferation, migration and invasion, and promoted apoptosis of M14 and A375 cells. Biochemical analysis revealed that the expression of miR-383-5p was negatively correlated with CENPF expression in human MM, and the doul-luciferase report showed that miR-383-5p could effectively bind to the 3’UTR of CENPF. CENPF expression was up-regulated and predicted the prognosis of MM. In addition, high expression of CENPF can effectively remedy the resistance of cell proliferation and vitality caused by miR-383-5p. Conclusion: In conclusion, miR-383-5p acts as a tumor suppressor in human MM by targeting CENPF, suggesting that CENPF may be a potential therapeutic target for human MM. Cancer Biology miR-383-5p CENPF Human MM proliferation apoptosis Figures Figure 1 Figure 2 Figure 3 Introduction Human malignant melanoma (MM) is a highly malignant tumor with approximately 20,0000 new cases each year in the world(1). And the incidence of MM is increasing(1). According to the stage and severity of the disease, treatment methods are different, including chemotherapy, radiotherapy, immunotherapy and biotherapy(2). However, the current treatment outcomes are not ideal(3). Studying the pathogenesis of MM is essential to improve the efficacy of MM. MicroRNAs (miRNAs) are a class of endogenous, single-strand, short noncoding RNAs. miRNAs can bind to the 3'-untranslated region (3'UTR) base of the target mRNA to mediate translation inhibition and mRNA degradation(4). It has been reported that miRNAs can regulate the expression of more than 50% of human protein coding genes and play an important role in the biological processes of cell differentiation, metabolism, proliferation, apoptosis, and tumorigenesis(5–7). CENPF (centromere protein F) can regulate the movement of centromere, its expression changes with the cell cycle(8). CENPF has a wealth of biological functions, including mitotic regulation, microtubule mechanics, gene regulation, myocyte differentiation, histone methylation and so on(9). It was reported that CENPF is highly expressed in a variety of tumors. In some tumors, it is also related to clinical indicators, such as stage and prognosis(10, 11). In this study, we found that CENPF is highly expressed in MM tissues, and the high expression of CENPF predicted a poor prognosis. In addition, we found that miR-383-5p could directly target CENPF to play the role of tumor suppressor gene in human MM. Materials And Methods Agents Primers were ordered from Genewiz Company (Beijing, China). Antibodies, including anti-CENPF (Ag29688), anti-GAPDH (10494–1-AP, 1:5,000), and HRP sheep anti-rabbit/mouse (1:5,000) were ordered from PTG Company (Bellevue, WA, USA); Active-Caspase3 (#ab32042, 1:1,000), Bcl-2 (ab32124, 1:1,000), Bax (ab32503, 1:1,000), Cyclin D1 (ab134175, 1:1,000), p-AKT (ab38449, 1:1,000), and p-mTOR (ab109268, 1:1,000) were purchased from Abcam (Cambridge, United Kingdom). Cell culture and transfection Human MM cell lines (A375 and M14) were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Science (Shanghai, China) and were cultured in 10% fetal bovine serum (FBS), streptomycin (100 μg/mL), and penicillin (100 U/mL) DMEM medium at 37℃ with 5% CO 2 . The cells were divided into three groups at the logarithmic growth stage, then transfected with miR-383-5p mimics (miR-383-5p), miR-NC (NC) and miR-383-5p mimics+pcDNA 3.1-CENPF (miR-383-5p+CENPF) by using Lipofectamine 2000 (TMO, Waltham, MA, USA) according to the manufacturer’s guidelines. miR-383-5p mimics sequence is 5'-AGAUCAGAAGGUGAUUGUGGCU-3'. Reverse transcription quantitative PCR (qPCR) Ultrapure RNA Kit (CwBio, Beijing, China) was performed to isolate the total RNA after 48 h of transfection and cDNA was synthesized using HiFiScript cDNA Synthesis Kit (CwBio, Beijing, China). The expression level of miR-383-5p was calculated by 2 − △ Ct method. Reverse transcription primer of miR-383-5p: 5'-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAGCCAC-3'. The PCR primers for miR-383-5p are listed as follows: Forward: 5'-GGGAGATCAGAAGGTGATTGTGGCT-3' Reverse: 5'-CAGTGCGTGTCGTGGAGT-3' The primer sequence of U6 as internal reference is as follows: Forward: 5'-CTCGCTTCGGCAGCACA-3' Reverse: 5'-AACGCTTCACGAATTTGCGT-3' Cell growth assays Cell Counting Kit-8 (CCK8) (Solarbio, Beijing, China) assay and colony formation assay were carried out to evaluate the proliferation and viability ability of A375 and M14 cells. About 3000 cells were seeded into each well of 96-well-plate. OD values were detected every 24 hours. 10 μl CCK8 solution was added to the well two hours before the detection. Then the growth carve was drawn according the OD values. For the colony formation assay, 500 cells were planted in each well of a 6-well plate, and were cultured for 10 days. Then colonies were fixed with 10% neutral formalin for 1 h and dyed with crystal violet (Beyotime, Haimen, China). The cells were photographed under a microscope (Olympus, Tokyo, Japan). Cell apoptosis assay Flow cytometer (BD FACSC anto II, BD Biosciences, San Jose, CA, USA) was used to detect the apoptosis of A375 and M14 cells. After 24 hours of transfection, cells were culture in serum-free medium for 24 hours and collected. The apoptosis rate was analyzed by annexin V/FITC (4A Biotech Company, Beijing, China) according to the instructions. Annexin V/FITC and propidium iodide were used to evaluate the percentage of apoptosis. The flow results were analyzed and processed by Flowjo software. Cell invasion and migration assays The invasion and migration ability of A375 and M14 cells were evaluated with transwell chamber (Millipore, Billerica, MA, USA). For invasion assay, the frozen matrigel (356234, BD, Franklin Lakes, NJ, USA) was diluted to 1:6 in serum-free medium, and then the 40 μl matrigel was applied to the upper chamber. The 150 μl serum-free medium and 1×10 4 cells were added to the upper chamber, 500 μl complete medium was added to the lower chamber. After 24 hours of incubation, the surface of each upper chamber was wiped gently with a cotton swab. The cells of lower chamber were washed with PBS and fixed with 4 % PFA for 30 min, then stained with 0.1 % crystal violet for 20 min. For migration assay, there are no matrigel coating and the rest was the same as the invasion assay. Luciferase reporter assay Based on bioinformatics, we predicted a complementary relationship between miR-383-5p and 3'UTR of CENPF mRNA. The mutation vector of CENPF 3'UTR was constructed by point mutation. miR-383-5p mimics and CENPF 3'UTR wild type (WT), miR-NC and CENPF 3'UTR wild type (WT), miR-383-5p mimics and mutant vector (Mut), miR-NC and mutant vector (Mut) were transfected into A375 and M14 respectively. 48 hours later, the cells were collected and the luciferase activity was detected with the doul-luciferase reporter gene assay kit (Beyotime, China) according to the instructions. Western blot assays The protein of cells was extracted with RIPA (protease inhibitor, CwBio, Beijing, China) buffer. 20 μg protein samples were added into each lane containing 10% SDS-PAGE gel. After transfer, the polyvinylidene fluoride (PVDF) membrane was sealed with 5 % skimmed milk for 1 hour and probed with primary and secondary antibodies. After washing the membrane, ECL developer (B500024, Proteintech, Chicago, IL, USA) was added and Quantity One Software was used to assess the results. Statistical analyses The data were analyzed using SPSS software. Each experiment was repeated at least three times. The results were presented as mean ± SD. Differences between two groups were tested by Student's t-test, differences among more than two groups were tested by one-way ANOVA. P<0.05 was considered significant difference. Results miR-383-5p inhibits human MM cell proliferation, migration and invasion Firstly, we analyzed the expression level of miR-383-5p between human normal melanocytes (HEM) and MM cell lines (uacc62, M14, M21, and A375) with qPCR. As the results of Fig. 1 A, the expression level of miR-383-5p was down-regulated in MM cell lines compared with HEM. Then, we chose A375 and M14 cell lines for further study. A375 and M14 cells were transfected with miR-383-5p mimic (miR-383-5p group) or miR-NC (NC group) to investigate the role of miR-383-5p in human MM. The transfection efficiency was detected with qPCR. After transfection of miR-383-5p mimic, the expression level of miR-383-5p was significantly up-regulated (Fig. 1 B, P < 0.05). CCK8 and colony analysis showed that miR-383-5p inhibited the proliferation and growth ability of A375 and M14 cells (Fig. 1 C and D). In order to further elucidate the mechanism of miR-383-5p resistance in MM cells, apoptosis was analyzed. As shown in Fig. 2 A, miR-383-5p promoted the cell apoptosis (P < 0.05). In addition, miR-383-5p inhibited the migration and invasion ability of A375 and M14 cells (Fig. 2 B, P < 0.05). miR-383-5p directly targets CENPF in MM cells Furthermore, Starbase v3.0 online database was used to analyzed the co-expression of CENPF and miR-383-5p in human MM. As shown in Fig. 3 A, the expression of miR-383-5p in skin cutaneous melanoma (SKCM) tissues was negatively correlated with CENPF expression. It has been reported that miRNAs can mediate translation inhibition and mRNA degradation by pairing with the 3’-untranslated region (3’UTR) base of the target mRNA. Therefore, miR-383-5p was predicted to bind to the 3’UTR of CENPE to reduce the expression of CENPF in SKCM through Starbase v3.0 database (Fig. 3 B). The results of dual-luciferase reporter showed that the fluorescence intensity of the co-transfection group of miR-383-5p mimics and CENPF-WT was significantly lower than that of other co-transfection groups (Fig. 3 B). Furthermore, the results of western blot showed that miR-383-5p inhibited the protein expression of CENBP (Fig. 3 C). These results suggested that miR-383-5p could promote the translation inhibition and mRNA degradation of CENPF through base complementary. CENPF expression is up-regulated and predicts the prognosis of MM The expression patten of CENPF in SKCM tissues was analyzed basing on gene expression profiling interactive analysis (GEPIA). As shown in Fig. 3 D, the expression of CENPF was significantly up-regulated in human SKCM tissues than that in normal tissues. Moreover, we analyzed the overall survival rate (OS) of SKCM patients with higher or lower expression level of CENPF. The results showed that the high expression level of CENPF predicted a poor prognosis (Fig. 3 E). Upregulation of CENPF reverses the effects of miR-383-5p on proliferation and migration To further study the interaction between miR-383-5p and CENPF on MM cells, the pcDNA 3.1-CENPF and miR-383-5p mimics (miR-383-5p + CENPF) were transfected into A375 and M14 cells. As shown in Fig. 4F and G, the recovery of CENPF expression reversed the inhibitory effect of miR-383-5p on the proliferation and migration of MM cells. Taken together, CENPF could remedy the proliferation and migration resistance of MM cells induced by miR-383-5p. Discussion As an oncogene or tumor suppressor, miRNA provides new regulatory mechanisms(12) based on the biological function of target genes, and can be used for the classification and prognosis prediction of cancer(13). According to reports, miR-383-5p acts as a tumor suppressor in a variety of human cancers by modulating multiple target genes(14–18). Many studies have shown that the expression of miR-383-5p is down-regulated in various human cancer, and the reduction of miR-383-5p is considered a potential biomarker for metastasis and poor prognosis(14–18). In addition, miR-383-5p inhibits cell proliferation and mobility of various cancer cells, and induces apoptosis(14–18). Furthermore, the over-expression of miR-383-5p enhances the chemosensitivity of ovarian cancer cells(19) and prompts a better response outcome in sorafenib targeting therapy for hepatocellular carcinoma cells(20). However, nothing is known about the role of miR-383-5p in MM. In this study, we found that the over-expression of miR-383-5p inhibited the proliferation, migration and invasion of M14 and A375 cells, and induced cell apoptosis. Our results are consistent with previous studies that miR-383-5p functions as a tumor suppressor. TargetScan 7.1 database was used to predict the target gene of miR-383-5p, and CENPF as one of the target genes attracted our attention. Recent studies have revealed that the high expression of CENPF promotes cancer cell mitosis and cell cycle(21). The dual-luciferase reporter was carried out to verify whether CENPF is the direct target of miR-383-5p in human MM cells. The results showed that miR-383-5p could effectively bind to the 3’UTR of CENPF, indicating that miR-383-5p has direct interaction with CENPF. CCK8 and transwell experiments proved that high expression of CENPF can effectively remedy the resistance of cell proliferation and vitality caused by miR-383-5p. Conclusion In summary, the expression of miR-383-5p was negatively correlated with the expression of CENPF in human MM, and miR-383-5p regulated the development of human MM by targeting CENPF, which provides a new strategy for the treatment of human MM. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials Not applicable. Competing interests The authors declare that they have no competing interests. Funding No funding was received. Authors' contributions FC analyzed and interpreted the patient data regarding the hematological disease and the transplant. RH performed the histological examination of the kidney, and was a major contributor in writing the manuscript. All authors read and approved the final manuscript. Acknowledgements Not applicable. References Guo J, Qin S, Liang J, Lin T, Si L, Chen X, et al. Chinese Guidelines on the Diagnosis and Treatment of Melanoma (2015 Edition). Targeting the MAPK and PI3K pathways in combination with PD1 blockade in melanoma. Peripheral blood Th9 cells are a possible pharmacodynamic biomarker of nivolumab treatment efficacy in metastatic melanoma patients. Wang K, Liang Q, Wei L, Zhang W, Zhu PJTB. 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Yongdong, Dai, and, Lulu, Liu, and, et al. Characterization of the oncogenic function of centromere protein F in hepatocellular carcinoma. Zhuo YJ, Xi M, Wan Y-P, Hua W, Liu Y-L, Wan S, et al. Enhanced expression of centromere protein F predicts clinical progression and prognosis in patients with prostate cancer.35(4):966---72. Yu T, Liu L, Li J, Yan M, Lin HJO. MiRNA-10a is upregulated in NSCLC and may promote cancer by targeting PTEN. 2015;6(30):30239-50. Wang S, Zhao X, Wang J, Wen Y, Zhang L, Wang D, et al. Upregulation of microRNA-203 is associated with advanced tumor progression and poor prognosis in epithelial ovarian cancer. 2013;30(3):1-7. Wei C, Gao JJ. Downregulated miR-383-5p contributes to the proliferation and migration of gastric cancer cells and is associated with poor prognosis. PeerJ. 2019;7:e7882. Xu G, Li N, Zhang Y, Zhang J, Xu R, Wu Y. MicroRNA-383-5p inhibits the progression of gastric carcinoma via targeting HDAC9 expression. Braz J Med Biol Res. 2019;52(8):e8341. Zhao S, Gao X, Zang S, Li Y, Feng X, Yuan X. MicroRNA-383-5p acts as a prognostic marker and inhibitor of cell proliferation in lung adenocarcinoma by cancerous inhibitor of protein phosphatase 2A. Oncol Lett. 2017;14(3):3573-9. Zhang Z, Xu L, He L, Wang J, Shi X, Li Z, et al. MiR-891a-5p as a prognostic marker and therapeutic target for hormone receptor-positive breast cancer. J Cancer. 2020;11(13):3771-82. Hu Y, Ma Y, Liu J, Cai Y, Zhang M, Fang X. LINC01128 expedites cervical cancer progression by regulating miR-383-5p/SFN axis. BMC Cancer. 2019;19(1):1157. Jiang J, Xie C, Liu Y, Shi Q, Chen Y. Up-regulation of miR-383-5p suppresses proliferation and enhances chemosensitivity in ovarian cancer cells by targeting TRIM27. Biomed Pharmacother. 2019;109:595-601. Shi Y, Huang A. Effects of sorafenib on lung metastasis in rats with hepatocellular carcinoma: the role of microRNAs. Tumour Biol. 2015;36(11):8455-63. Fritzler MJ, Rattner JB, Luft LM, Edworthy SM, Casiano CA, Peebles C, et al. Historical perspectives on the discovery and elucidation of autoantibodies to centromere proteins (CENP) and the emerging importance of antibodies to CENP-F.10(4):194-200. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-154404","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":8894288,"identity":"7357cbbd-847c-461d-add5-94c658a697d7","order_by":0,"name":"Wanwan Jin","email":"","orcid":"","institution":"Yuying Children's Hospital of Wenzhou Medical College: Wenzhou Medical University Second Affiliated Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wanwan","middleName":"","lastName":"Jin","suffix":""},{"id":8894289,"identity":"7ae91d15-11e1-4cfc-92bf-9730c155c447","order_by":1,"name":"Huazhen Liu","email":"","orcid":"","institution":"Changhai Hospital Affiliated Naval Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Huazhen","middleName":"","lastName":"Liu","suffix":""},{"id":8894290,"identity":"a2306765-2c89-4ea4-9892-11d906b74d8c","order_by":2,"name":"Qin Xu","email":"","orcid":"","institution":"Yuying Children's Hospital of Wenzhou Medical College: Wenzhou Medical University Second Affiliated Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Xu","suffix":""},{"id":8894291,"identity":"70caec3e-cb22-449d-9c72-cabd4ca4f4f9","order_by":3,"name":"Nan Lin","email":"","orcid":"","institution":"Yuying Children's Hospital of Wenzhou Medical College: Wenzhou Medical University Second Affiliated Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nan","middleName":"","lastName":"Lin","suffix":""},{"id":8894292,"identity":"f816df58-12d5-4594-b6d1-13b34273a985","order_by":4,"name":"Haiting Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApUlEQVRIiWNgGAWjYBACAwhlw8PP30C8Fkag2jQZyRkHSNNy2MagIYFILebszc8f87ad5zFgOMD44WMOEVose44ZNs5su81jztzALDlzGzEOu5Fg2PARqMWy4QAbMy9xWtI/NiS2neMxOJBAtJYckC0HSNBi2XOmcOaMc8k8kjMONhPnF3P29g2fecrs7Pn5mw9++EiMFiQAip9RMApGwSgYBdQBAJLQN8Xm1lHnAAAAAElFTkSuQmCC","orcid":"","institution":"Yuying Children's Hospital of Wenzhou Medical College: Wenzhou Medical University Second Affiliated Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Haiting","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2021-01-25 18:19:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-154404/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-154404/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":5353243,"identity":"4d105424-40fb-4f18-81fe-9f85db475093","added_by":"auto","created_at":"2021-01-28 21:46:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1965480,"visible":true,"origin":"","legend":"miR-383-5p inhibits human MM cell proliferation. \nNotes: (A) The expression of miR-383-5p in four human melanoma cell lines, uacc62, M14, M21, and A375, and in one human normal melanocyte cell line, HEM. (B) The expression level of miR-383-5p was evaluated with qPCR. CCK8 assay (C) and plate clonality assay (D) was performed to assess the cell proliferation of M14 and A375 cells. *P\u003c0.05.\n","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-154404/v1/6e34b373dd70a82a7e19d6d0.png"},{"id":5353299,"identity":"01762168-856a-44c7-85fe-6342d56d88ee","added_by":"auto","created_at":"2021-01-28 21:49:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6229957,"visible":true,"origin":"","legend":"miR-383-5p induces apoptosis and inhibits proliferation of human MM cell. \nNotes: (A) Flow cytometric analysis was performed to estimate the apoptosis of A375 and M14 cells. (B) The invasion and migration of A375 and M14 was detected by transwell assay. *P\u003c0.05.\n","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-154404/v1/a7613f629f428bee262a259b.png"},{"id":5353245,"identity":"64febd4a-6ca1-472e-a016-f59589aae6f1","added_by":"auto","created_at":"2021-01-28 21:46:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5227670,"visible":true,"origin":"","legend":"Upregulation of CENPF reverses the effects of miR-383-5p on proliferation and migration. \nNotes: (A) The co-expression of CENPF and miR-383-5p in skin cutaneous melanoma (SKCM) tissues was assessed by using the starbase v3.0 online database. (B) upper line: The binding site of miR-383-5p on 3'UTR of CENPF; lower line: Dual-luciferase reporter analysis was performed in M14 cells. (C) The expression level of CENPF was evaluated with western blotting. (D) CENPF expression was significantly increased in SKCM tumor tissues compared with normal skin tissues, which from GEPIA. (E) The survival curve showed that SKCM patients with low CENPF level had better OS than those with high CENPF level. (F) Cell proliferation ability was detected by CCK8 assay. (G) Cell migration ability was assessed by transwell assay. *P\u003c0.05. \n","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-154404/v1/86ed5f799b5a8e39acc6ff64.png"},{"id":13653920,"identity":"1ee57101-524b-44a3-ab9e-ff8b170f1ecd","added_by":"auto","created_at":"2021-09-17 09:55:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1314640,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-154404/v1/65ee301b-ce31-4f8e-b13b-f74641d13d04.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003emiR-383-5p Inhibits Human Malignant Melanoma via Targeting CENPF\u003c/p\u003e","fulltext":[{"header":"Introduction","content":" \u003cp\u003eHuman malignant melanoma (MM) is a highly malignant tumor with approximately 20,0000 new cases each year in the world(1). And the incidence of MM is increasing(1). According to the stage and severity of the disease, treatment methods are different, including chemotherapy, radiotherapy, immunotherapy and biotherapy(2). However, the current treatment outcomes are not ideal(3). Studying the pathogenesis of MM is essential to improve the efficacy of MM.\u003c/p\u003e \u003cp\u003eMicroRNAs (miRNAs) are a class of endogenous, single-strand, short noncoding RNAs. miRNAs can bind to the 3'-untranslated region (3'UTR) base of the target mRNA to mediate translation inhibition and mRNA degradation(4). It has been reported that miRNAs can regulate the expression of more than 50% of human protein coding genes and play an important role in the biological processes of cell differentiation, metabolism, proliferation, apoptosis, and tumorigenesis(5\u0026ndash;7).\u003c/p\u003e \u003cp\u003eCENPF (centromere protein F) can regulate the movement of centromere, its expression changes with the cell cycle(8). CENPF has a wealth of biological functions, including mitotic regulation, microtubule mechanics, gene regulation, myocyte differentiation, histone methylation and so on(9). It was reported that CENPF is highly expressed in a variety of tumors. In some tumors, it is also related to clinical indicators, such as stage and prognosis(10, 11).\u003c/p\u003e \u003cp\u003eIn this study, we found that CENPF is highly expressed in MM tissues, and the high expression of CENPF predicted a poor prognosis. In addition, we found that miR-383-5p could directly target CENPF to play the role of tumor suppressor gene in human MM.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003ch2\u003e\u003cem\u003eAgents\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003ePrimers were ordered from Genewiz Company (Beijing, China). Antibodies, including anti-CENPF (Ag29688), anti-GAPDH (10494\u0026ndash;1-AP, 1:5,000), and HRP sheep anti-rabbit/mouse (1:5,000) were ordered from PTG Company (Bellevue, WA, USA); Active-Caspase3 (#ab32042, 1:1,000), Bcl-2 (ab32124, 1:1,000), Bax (ab32503, 1:1,000), Cyclin D1 (ab134175, 1:1,000), p-AKT (ab38449, 1:1,000), and p-mTOR (ab109268, 1:1,000) were purchased from Abcam (Cambridge, United Kingdom).\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eCell culture and transfection\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eHuman MM cell lines (A375 and M14) were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Science (Shanghai, China) and were cultured in 10% fetal bovine serum (FBS), streptomycin (100 \u0026mu;g/mL), and penicillin (100 U/mL) DMEM medium at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e. The cells were divided into three groups at the logarithmic growth stage, then transfected with miR-383-5p mimics (miR-383-5p), miR-NC (NC) and miR-383-5p mimics+pcDNA 3.1-CENPF (miR-383-5p+CENPF) by using Lipofectamine 2000 (TMO, Waltham, MA, USA) according to the manufacturer\u0026rsquo;s guidelines. miR-383-5p mimics sequence is 5'-AGAUCAGAAGGUGAUUGUGGCU-3'.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eReverse transcription quantitative PCR (qPCR)\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eUltrapure RNA Kit (CwBio, Beijing, China) was performed to isolate the total RNA after 48 h of transfection and cDNA was synthesized using HiFiScript cDNA Synthesis Kit (CwBio, Beijing, China). The expression level of miR-383-5p was calculated by 2\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e△\u003c/sup\u003e\u003csup\u003eCt\u003c/sup\u003e method.\u003c/p\u003e\n\u003cp\u003eReverse transcription primer of miR-383-5p: 5'-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAGCCAC-3'.\u003c/p\u003e\n\u003cp\u003eThe PCR primers for miR-383-5p are listed as follows:\u003c/p\u003e\n\u003cp\u003eForward: 5'-GGGAGATCAGAAGGTGATTGTGGCT-3'\u003c/p\u003e\n\u003cp\u003eReverse: 5'-CAGTGCGTGTCGTGGAGT-3'\u003c/p\u003e\n\u003cp\u003eThe primer sequence of U6 as internal reference is as follows:\u003c/p\u003e\n\u003cp\u003eForward: 5'-CTCGCTTCGGCAGCACA-3'\u003c/p\u003e\n\u003cp\u003eReverse: 5'-AACGCTTCACGAATTTGCGT-3'\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eCell growth assays\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eCell Counting Kit-8 (CCK8) (Solarbio, Beijing, China) assay and colony formation assay were carried out to evaluate the proliferation and viability ability of A375 and M14 cells. About 3000 cells were seeded into each well of 96-well-plate. OD values were detected every 24 hours. 10 \u0026mu;l CCK8 solution was added to the well two hours before the detection. Then the growth carve was drawn according the OD values. For the colony formation assay, 500 cells were planted in each well of a 6-well plate, and were cultured for 10 days. Then colonies were fixed with 10% neutral formalin for 1 h and dyed with crystal violet (Beyotime, Haimen, China). The cells were photographed under a microscope (Olympus, Tokyo, Japan).\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eCell apoptosis assay\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eFlow cytometer (BD FACSC anto II, BD Biosciences, San Jose, CA, USA) was used to detect the apoptosis of A375 and M14 cells. After 24 hours of transfection, cells were culture in serum-free medium for 24 hours and collected. The apoptosis rate was analyzed by annexin V/FITC (4A Biotech Company, Beijing, China) according to the instructions. Annexin V/FITC and propidium iodide were used to evaluate the percentage of apoptosis. The flow results were analyzed and processed by Flowjo software.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eCell invasion and migration assays\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eThe invasion and migration ability of A375 and M14 cells were evaluated with transwell chamber (Millipore, Billerica, MA, USA). For invasion assay, the frozen matrigel (356234, BD, Franklin Lakes, NJ, USA) was diluted to 1:6 in serum-free medium, and then the 40 \u0026mu;l matrigel was applied to the upper chamber. The 150 \u0026mu;l serum-free medium and 1\u0026times;10\u003csup\u003e4 \u003c/sup\u003ecells were added to the upper chamber, 500 \u0026mu;l complete medium was added to the lower chamber. After 24 hours of incubation, the surface of each upper chamber was wiped gently with a cotton swab. The cells of lower chamber were washed with PBS and fixed with 4 % PFA for 30 min, then stained with 0.1 % crystal violet for 20 min. For migration assay, there are no matrigel coating and the rest was the same as the invasion assay.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eLuciferase reporter assay\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eBased on bioinformatics, we predicted a complementary relationship between miR-383-5p and 3'UTR of CENPF mRNA. The mutation vector of CENPF 3'UTR was constructed by point mutation. miR-383-5p mimics and CENPF 3'UTR wild type (WT), miR-NC and CENPF 3'UTR wild type (WT), miR-383-5p mimics and mutant vector (Mut), miR-NC and mutant vector (Mut) were transfected into A375 and M14 respectively. 48 hours later, the cells were collected and the luciferase activity was detected with the doul-luciferase reporter gene assay kit (Beyotime, China) according to the instructions.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eWestern blot assays\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eThe protein of cells was extracted with RIPA (protease inhibitor, CwBio, Beijing, China) buffer. 20 \u0026mu;g protein samples were added into each lane containing 10% SDS-PAGE gel. After transfer, the polyvinylidene fluoride (PVDF) membrane was sealed with 5 % skimmed milk for 1 hour and probed with primary and secondary antibodies. After washing the membrane, ECL developer (B500024, Proteintech, Chicago, IL, USA) was added and Quantity One Software was used to assess the results.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eStatistical analyses\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eThe data were analyzed using SPSS software. Each experiment was repeated at least three times. The results were presented as mean \u0026plusmn; SD. Differences between two groups were tested by Student's t-test, differences among more than two groups were tested by one-way ANOVA. P\u0026lt;0.05 was considered significant difference.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":" \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003emiR-383-5p inhibits human MM cell proliferation, migration and invasion\u003c/h2\u003e \u003cp\u003eFirstly, we analyzed the expression level of miR-383-5p between human normal melanocytes (HEM) and MM cell lines (uacc62, M14, M21, and A375) with qPCR. As the results of Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the expression level of miR-383-5p was down-regulated in MM cell lines compared with HEM. Then, we chose A375 and M14 cell lines for further study. A375 and M14 cells were transfected with miR-383-5p mimic (miR-383-5p group) or miR-NC (NC group) to investigate the role of miR-383-5p in human MM. The transfection efficiency was detected with qPCR. After transfection of miR-383-5p mimic, the expression level of miR-383-5p was significantly up-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). CCK8 and colony analysis showed that miR-383-5p inhibited the proliferation and growth ability of A375 and M14 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and D). In order to further elucidate the mechanism of miR-383-5p resistance in MM cells, apoptosis was analyzed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, miR-383-5p promoted the cell apoptosis (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, miR-383-5p inhibited the migration and invasion ability of A375 and M14 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003emiR-383-5p directly targets CENPF in MM cells\u003c/h2\u003e \u003cp\u003eFurthermore, Starbase v3.0 online database was used to analyzed the co-expression of CENPF and miR-383-5p in human MM. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the expression of miR-383-5p in skin cutaneous melanoma (SKCM) tissues was negatively correlated with CENPF expression. It has been reported that miRNAs can mediate translation inhibition and mRNA degradation by pairing with the 3\u0026rsquo;-untranslated region (3\u0026rsquo;UTR) base of the target mRNA. Therefore, miR-383-5p was predicted to bind to the 3\u0026rsquo;UTR of CENPE to reduce the expression of CENPF in SKCM through Starbase v3.0 database (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The results of dual-luciferase reporter showed that the fluorescence intensity of the co-transfection group of miR-383-5p mimics and CENPF-WT was significantly lower than that of other co-transfection groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Furthermore, the results of western blot showed that miR-383-5p inhibited the protein expression of CENBP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These results suggested that miR-383-5p could promote the translation inhibition and mRNA degradation of CENPF through base complementary.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCENPF expression is up-regulated and predicts the prognosis of MM\u003c/h2\u003e \u003cp\u003eThe expression patten of CENPF in SKCM tissues was analyzed basing on gene expression profiling interactive analysis (GEPIA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, the expression of CENPF was significantly up-regulated in human SKCM tissues than that in normal tissues. Moreover, we analyzed the overall survival rate (OS) of SKCM patients with higher or lower expression level of CENPF. The results showed that the high expression level of CENPF predicted a poor prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eUpregulation of CENPF reverses the effects of miR-383-5p on proliferation and migration\u003c/h2\u003e \u003cp\u003eTo further study the interaction between miR-383-5p and CENPF on MM cells, the pcDNA 3.1-CENPF and miR-383-5p mimics (miR-383-5p\u0026thinsp;+\u0026thinsp;CENPF) were transfected into A375 and M14 cells. As shown in Fig.\u0026nbsp;4F and G, the recovery of CENPF expression reversed the inhibitory effect of miR-383-5p on the proliferation and migration of MM cells. Taken together, CENPF could remedy the proliferation and migration resistance of MM cells induced by miR-383-5p.\u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eAs an oncogene or tumor suppressor, miRNA provides new regulatory mechanisms(12) based on the biological function of target genes, and can be used for the classification and prognosis prediction of cancer(13). According to reports, miR-383-5p acts as a tumor suppressor in a variety of human cancers by modulating multiple target genes(14\u0026ndash;18). Many studies have shown that the expression of miR-383-5p is down-regulated in various human cancer, and the reduction of miR-383-5p is considered a potential biomarker for metastasis and poor prognosis(14\u0026ndash;18). In addition, miR-383-5p inhibits cell proliferation and mobility of various cancer cells, and induces apoptosis(14\u0026ndash;18). Furthermore, the over-expression of miR-383-5p enhances the chemosensitivity of ovarian cancer cells(19) and prompts a better response outcome in sorafenib targeting therapy for hepatocellular carcinoma cells(20). However, nothing is known about the role of miR-383-5p in MM.\u003c/p\u003e \u003cp\u003eIn this study, we found that the over-expression of miR-383-5p inhibited the proliferation, migration and invasion of M14 and A375 cells, and induced cell apoptosis. Our results are consistent with previous studies that miR-383-5p functions as a tumor suppressor.\u003c/p\u003e \u003cp\u003eTargetScan 7.1 database was used to predict the target gene of miR-383-5p, and CENPF as one of the target genes attracted our attention. Recent studies have revealed that the high expression of CENPF promotes cancer cell mitosis and cell cycle(21). The dual-luciferase reporter was carried out to verify whether CENPF is the direct target of miR-383-5p in human MM cells. The results showed that miR-383-5p could effectively bind to the 3\u0026rsquo;UTR of CENPF, indicating that miR-383-5p has direct interaction with CENPF. CCK8 and transwell experiments proved that high expression of CENPF can effectively remedy the resistance of cell proliferation and vitality caused by miR-383-5p.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eIn summary, the expression of miR-383-5p was negatively correlated with the expression of CENPF in human MM, and miR-383-5p regulated the development of human MM by targeting CENPF, which provides a new strategy for the treatment of human MM.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eNo funding was received.\u003c/p\u003e\n\u003ch2\u003eAuthors' contributions\u003c/h2\u003e\n\u003cp\u003eFC analyzed and interpreted the patient data regarding the hematological disease and the transplant. RH performed the histological examination of the kidney, and was a major contributor in writing the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGuo J, Qin S, Liang J, Lin T, Si L, Chen X, et al. Chinese Guidelines on the Diagnosis and Treatment of Melanoma (2015 Edition).\u003c/li\u003e\n\u003cli\u003eTargeting the MAPK and PI3K pathways in combination with PD1 blockade in melanoma.\u003c/li\u003e\n\u003cli\u003ePeripheral blood Th9 cells are a possible pharmacodynamic biomarker of nivolumab treatment efficacy in metastatic melanoma patients.\u003c/li\u003e\n\u003cli\u003eWang K, Liang Q, Wei L, Zhang W, Zhu PJTB. MicroRNA-608 acts as a prognostic marker and inhibits the cell proliferation in hepatocellular carcinoma by macrophage migration inhibitory factor. 2016;37(3):3823-30.\u003c/li\u003e\n\u003cli\u003eHu J, Qiu M, Jiang F, Zhang S, Yang X, Wang J, et al. MiR-145 regulates cancer stem-like properties and epithelial-to-mesenchymal transition in lung adenocarcinoma-initiating cells.35(9):8953-61.\u003c/li\u003e\n\u003cli\u003eJohnnidis JB, Harris MH, Wheeler RT, Stehling-Sun S, Lam MH, Kirak O, et al. Regulation of progenitor cell proliferation and granulocyte function by microRNA-223.451(7182):1125-9.\u003c/li\u003e\n\u003cli\u003eXing F, Wu K, Watabe KJCPD. MicroRNAs in Cancer Stem Cells: New Regulators of Stemness. 2014;20(33):-.\u003c/li\u003e\n\u003cli\u003eZhu X, Mancini MA, Chang KH, Liu CY, Chen CF, Shan B, et al. Characterization of a novel 350-kilodalton nuclear phosphoprotein that is specifically involved in mitotic-phase progression.15(9):5017-29.\u003c/li\u003e\n\u003cli\u003eMa L, Zhao X, Science XZJJoB. Mitosin/CENP-F in mitosis, transcriptional control, and differentiation.13(2):205-13.\u003c/li\u003e\n\u003cli\u003eYongdong, Dai, and, Lulu, Liu, and, et al. Characterization of the oncogenic function of centromere protein F in hepatocellular carcinoma.\u003c/li\u003e\n\u003cli\u003eZhuo YJ, Xi M, Wan Y-P, Hua W, Liu Y-L, Wan S, et al. Enhanced expression of centromere protein F predicts clinical progression and prognosis in patients with prostate cancer.35(4):966---72.\u003c/li\u003e\n\u003cli\u003eYu T, Liu L, Li J, Yan M, Lin HJO. MiRNA-10a is upregulated in NSCLC and may promote cancer by targeting PTEN. 2015;6(30):30239-50.\u003c/li\u003e\n\u003cli\u003eWang S, Zhao X, Wang J, Wen Y, Zhang L, Wang D, et al. Upregulation of microRNA-203 is associated with advanced tumor progression and poor prognosis in epithelial ovarian cancer. 2013;30(3):1-7.\u003c/li\u003e\n\u003cli\u003eWei C, Gao JJ. Downregulated miR-383-5p contributes to the proliferation and migration of gastric cancer cells and is associated with poor prognosis. PeerJ. 2019;7:e7882.\u003c/li\u003e\n\u003cli\u003eXu G, Li N, Zhang Y, Zhang J, Xu R, Wu Y. MicroRNA-383-5p inhibits the progression of gastric carcinoma via targeting HDAC9 expression. Braz J Med Biol Res. 2019;52(8):e8341.\u003c/li\u003e\n\u003cli\u003eZhao S, Gao X, Zang S, Li Y, Feng X, Yuan X. MicroRNA-383-5p acts as a prognostic marker and inhibitor of cell proliferation in lung adenocarcinoma by cancerous inhibitor of protein phosphatase 2A. Oncol Lett. 2017;14(3):3573-9.\u003c/li\u003e\n\u003cli\u003eZhang Z, Xu L, He L, Wang J, Shi X, Li Z, et al. MiR-891a-5p as a prognostic marker and therapeutic target for hormone receptor-positive breast cancer. J Cancer. 2020;11(13):3771-82.\u003c/li\u003e\n\u003cli\u003eHu Y, Ma Y, Liu J, Cai Y, Zhang M, Fang X. LINC01128 expedites cervical cancer progression by regulating miR-383-5p/SFN axis. BMC Cancer. 2019;19(1):1157.\u003c/li\u003e\n\u003cli\u003eJiang J, Xie C, Liu Y, Shi Q, Chen Y. Up-regulation of miR-383-5p suppresses proliferation and enhances chemosensitivity in ovarian cancer cells by targeting TRIM27. Biomed Pharmacother. 2019;109:595-601.\u003c/li\u003e\n\u003cli\u003eShi Y, Huang A. Effects of sorafenib on lung metastasis in rats with hepatocellular carcinoma: the role of microRNAs. Tumour Biol. 2015;36(11):8455-63.\u003c/li\u003e\n\u003cli\u003eFritzler MJ, Rattner JB, Luft LM, Edworthy SM, Casiano CA, Peebles C, et al. Historical perspectives on the discovery and elucidation of autoantibodies to centromere proteins (CENP) and the emerging importance of antibodies to CENP-F.10(4):194-200.\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-383-5p, CENPF, Human MM, proliferation, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-154404/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-154404/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eHuman malignant melanoma (MM) is one of the skin cancers with the highest mortality. In this study, we investigated the role of miR-383-5p on human MM cells. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThe expression of miR-383-5p was measured by quantitative real-time PCR assay. The cell proliferation, invasion and migration were detected by CCK8, clone formation and transwell assays. Flow cytometry assay was used to detect apoptosis. The binding of miR-383-5p and 3’UTR of CENPF mRNA was indicated by dual-luciferase assays.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe found that miR-383-5p inhibited the cells proliferation, migration and invasion, and promoted apoptosis of M14 and A375 cells. Biochemical analysis revealed that the expression of miR-383-5p was negatively correlated with CENPF expression in human MM, and the doul-luciferase report showed that miR-383-5p could effectively bind to the 3’UTR of CENPF. CENPF expression was up-regulated and predicted the prognosis of MM. In addition, high expression of CENPF can effectively remedy the resistance of cell proliferation and vitality caused by miR-383-5p. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eIn conclusion, miR-383-5p acts as a tumor suppressor in human MM by targeting CENPF, suggesting that CENPF may be a potential therapeutic target for human MM.\u003c/p\u003e","manuscriptTitle":"miR-383-5p Inhibits Human Malignant Melanoma via Targeting CENPF","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-01-28 21:46:12","doi":"10.21203/rs.3.rs-154404/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":"5fb53fbf-4365-48ef-8cf7-1bcca5d341ce","owner":[],"postedDate":"January 28th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":2132531,"name":"Cancer Biology"}],"tags":[],"updatedAt":"2021-02-08T13:46:48+00:00","versionOfRecord":[],"versionCreatedAt":"2021-01-28 21:46:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-154404","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-154404","identity":"rs-154404","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}