BAP1 serves as a clear-cell renal cell carcinoma suppressor and is inversely regulated by miR-200c-3p

BAP1 serves as a clear-cell renal cell carcinoma suppressor and is inversely regulated by miR-200c-3p | 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 BAP1 serves as a clear-cell renal cell carcinoma suppressor and is inversely regulated by miR-200c-3p Du Wei, Ge Wenyu, Yu Ling, Chen Hongzhe, Wang Dongmei, Xu Xinglu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4161288/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 The initiation and development of malignant tumor is always accompanied by a series of complex gene expression alterations inside the cells. As a tumor suppressor, the deubiquitinating enzyme BAP1 has been identified as an important regulator on the outcomes and biological properties of clear-cell renal cell carcinoma (ccRCC). However, BAP1-involved intracellular regulatory cascades in clear-cell renal cell carcinoma are still not fully understood. In this study, we provided evidence that the protein levels of BAP1 were dramatically diminished in clear-cell renal cell carcinoma in vitro and in vivo. Notably, the relatively low expression of BAP1 is significantly associated with worse prognosis in ccRCC patients. Besides, through the prediction of bioinformatics methods and verification of biological experiments, we confirmed that miR-200c-3p was the direct upstream regulator of BAP1. Taken together, our study presents an important role of miR-200c-3p/BAP1 in the development of ccRCC, which provided an alternative strategy for treating ccRCC in clinical practice. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Renal cell carcinoma (RCC) is one of the most lethal of human disease, accounting for 2–3% of adult malignancies in the world, while clear-cell renal cell carcinoma accounts for approximately 80% of RCC [ 1 , 2 ] . The early diagnosis and complete surgical excision are the main factors contributing to a definitive treatment of ccRCC. The type of treatment depends on multiple factors including the stage of clear-cell renal cell carcinoma, the type of clear-cell renal cell carcinoma, pre-existing or comorbid conditions and overall health and age of the patient. Although early-stage ccRCC is curable by surgery resection, the prognosis for ccRCC patients is still poor for the resistant to chemotherapy and radiotherapy in most cases [ 3 , 4 ] . Therefore, the improvement of new diagnostic and treatment strategies for ccRCC is urgently required. MicroRNAs (miRNAs) are endogenous, small noncoding RNAs with 19–22 nucleotides in length that modulate biological processes at the post-transcriptional levels [ 5 , 6 ] . Accumulating evidence has demonstrated that miRNAs are differently expressed in various human cancers including ccRCC [ 7 ] . Different expression profile of miRNAs is strongly related to numerous biological processes such as cell proliferation, migration, invasion, metastasis and so forth [ 8 – 10 ] . Up to now, more than 1000 human miRNAs have been identified and reported as molecular biomarkers for diagnosis, prognosis and treatment of diseases. Thus, it is extremely necessary to explore the specific miRNA in ccRCC as tumor markers with enough specificity and sensitivity. Epigenetic is a rapidly growing field that studies gene expression modifications not involving changes in the DNA sequence [ 11 , 12 ] . Ubiquitination is one of the main areas of epigenetic research [ 13 ] . Among the de-ubiquitinating protein family, BRCA1 associated protein-1 (BAP1) is a deubiquitinating enzyme with an ubiquitin carboxy-terminal hydrolase (UCH) domain. Plenty of researches have illustrated that BAP1 was involved in numerous cellular processes, such as cell fate determination, stem cell pluripotency and other biological processes [ 14 – 16 ] . In cancers, BAP1 was first shown to act as a tumor suppressor, where its deubiquitinase (UCH) domain and nuclear localization sequences were required for BAP1 to suppress cell growth [ 17 ] . Furthermore, BAP1 mutations have been identified in malignant neoplasms [ 18 ] . However, the roles of BAP1 in the initiation and progression of human renal cell carcinoma remains controversial. Thus, elucidating the BAP1-involved intricate regulative network and epigenetic could shed light on how their deregulation contributes to the development of ccRCC. Since cancers may have multiple pathologic factors, we tried to explain the pathogenesis of renal cell carcinoma from the perspective of epigenetic, especially the upstream of BAP1 in our research. In the present study, our date showed that BAP1 was a tumor suppressor gene in ccRCC and was closely associated with the prognosis of ccRCC. Furthermore, BAP1 was a direct target gene of miR-200c-3p and that miR-200c-3p could inhibit the expression of BAP1, which contributed to the progression of clear-cell renal cell carcinoma. Consequently, enhanced expression of miR-200c-3p promoted the proliferation and suppressed the apoptosis of clear-cell renal cell carcinoma cells in vitro. Therefore, in the present study, we provided evidence that targeting miR-200c-3p/BAP1 may be an alternative strategy for treating clear-cell renal cell carcinoma in clinical practice. Materials and Methods Human tissues and cells The clear-cell renal cell carcinoma and matched normal adjacent tissues were derived from patients undergoing surgical procedure from April 2015 to December 2019. All the patients have provided the written consent, and the Ethics Committee from Heilongjiang Provincial Hospital approved all aspects of this study. Tissue fragments were immediately frozen in liquid nitrogen at the time of surgery. The human renal carcinoma cell lines were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI 1640medium (Gibco, Grand Island, NY, USA) supplemented with 12% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA). All the cells were maintained at 37°C in humidified 5% CO 2 atmosphere. Cell transfection The cells were inoculated into complete medium in a 6-well cell culture plate at the appropriate cell density for transfection (40 − 50%). Over-expression of miR-200c-3p was achieved by transfecting renal cancer cells with a miR-200c-3p mimic, while knockdown of miR-200c-3p was achieved by transfecting a miRNA inhibitor. The mimic and inhibitor were purchased from GenePharma (Shanghai, China). The siRNA sequences of the human BAP1 cDNA (si-BAP1) and shRNA-BAP1 were designed and synthesized by GenePharma (Shanghai, China). A scrambled siRNA that did not bind the human BAP1 cDNA was designed and synthesized as a negative control. For over-expression of BAP1, a plasmid encoding the full-length human BAP1 was purchased from Invitrogen (Carlsbad, CA, USA).While an empty plasmid was served as the negative control. Quantitative reverse-transcription polymerase chain reaction. Total RNA was harvested and extracted from tissues or celllines using Trizol (Invitrogen, Life, USA), according to the manufacturer’s instructions. For miR-200c-3p, cDNA was synthesized from total RNA (0.5 mg) using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, USA). For BAP1, cDNA was synthesized from total RNA (0.5 mg) using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA), with random primer. FormiR-200c-3p, the reverse transcription primer sequence was 5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCCATCAT-3’. Real-time quantitative PCR was performed using SYBR Master Mix (Roche, Life, USA).All the primers were designed and synthesized by the Sangon Biotech (Shanghai, China). The primers for miR-200c-3p were as follows: 5’-ACACTCCAGCTGGGTAATACTGCCGGGTAAT-3’(forward) and 5’-TGGTGTCGTGGAGTCG-3’ (reverse). The primers for BAP1 were as follows: 5’-GACCCAGGCCTCTTCACC-3’(forward) and 5’-AGTCCTTCATGCGACTCAGG-3’ (reverse). The primers for U6 were as follows: 5’-CTCGCTTCGGCAGCACA-3’(forward) and 5’-AACGCTTCACGAATTTGCGT-3’ (reverse). These assays were performed on the 7500 Fast Real-Time PCR System (Applied Biosystem, USA). The results were calculated using the ΔΔ threshold cycle (Ct) method and then normalized to the endogenous reference control gene U6 for miR-200c-3p and GAPDH for BAP1. Protein extraction and western blotting The protein contents were determined with a BCA Protein Assay Kit (Beyotime, Shanghai, China).The samples were mixed with loading buffer (Beyotime, Shanghai, China) and boiled for 10 min for denaturation. Then the samples were subjected to electrophoresis using 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), and were later transferred onto a polyvinylidene fluoride (PVDF) membrane, which was afterwards incubated with 5% nonfat milk (Beyotime, Shanghai, China) for 2 h at room temperature, and then probed with antibodies against Ki67 (Santa Cruz, USA), Cyclin D (Santa Cruz, USA), P53 (Abcam, Cambridge, United Kingdom), and AKT (Abcam, Cambridge, United Kingdom) overnight at 4°C on a shaker. The membranes were then washed thrice with 0.05% TBST (TBS mixed with 0.05% tween) and incubated with Alexa Fluor labeled secondary antibodies (Beyotime, A0460 and A0468, Shanghai, China) for 1 h in the dark at room temperature. Finally, the bands in the membranes were detected with the Odyssey instrument (Li-COR, American Gene Corp. USA), and the Odyssey v1.2 software was used to analyze the density of the bands. Cell proliferation assay After 24 hours post-transfection, the proliferation of cells in each group was detected using the Cell Counting Kit-8 (Beyotime, C0037, Shanghai, China) according to the manufacturer’s instructions. Briefly, 10 µLCCK-8 liquid was added to the test well and incubated with cells for3 hours at 37°C. The absorbance of each well was measured at a wave length of 450 nm. Transwell migration and invasion assay. To detect the migration ability of cells, chamber of a Transwell plate with 8 µM pore size (Costar, USA) were used. Firstly, cells (1×10 4 ) were seeded in the upper chamber with serum-free medium, while complete medium was added to the bottom wells of the chambers. All Transwell chambers were incubated at 37°C and 5% CO 2 in culture medium. The cells that had not migrated to the other side of the membrane were removed from the upper face of the filters, then the remaining cells were fixed with 95% ethanol for 15min and washed in tap water. Crystal violet solution (4g / L) was added to each chamber for 10 min, after which the chambers were washed again in tap water. Cells were then counted in five randomly chosen visual fields (200×) and the average value was calculated.Similar chambers coated with Matrigel (BD, USA) were used to determine invasive ability in the cell invasion assay. Tumor xenograft assay in vivo. The research in nude mice was conducted in accordance with the ethical standards and national guidelines, which was approved by the Ethics Committee of Heilongjiang Provincial Hospital. The 4 weeks old nude mice (BALB/c-nu/nu) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Caki-1 cells were first transfected with miR-200c-3p inhibitor or co-transfected with miR-200c-3p inhibitor and BAP-1 shRNA, designed and synthesized by GenePharma (Shanghai, China). Then the mice were subcutaneously injected with 0.3 ml tumor cell suspension. After 4 weeks, the mice were sacrificed and the tumors were then removed for measuring the size and weight, and the following immunohistochemistry assay. Statistical analysis All statistical analyses were performed with Graphpad prism 5 software. All data were expressed as mean ± standard deviation (SD). Statistical comparisons between two groups were conducted with the Student’s t-test. Univariate analysis of variance (ANOVA) was used for comparisons among multiple groups. A p value < 0.05 was considered statistically significant. Result Down regulation of BAP1 in vivo and in vitro. Date from The Cancer Genome Atlas (TCGA) dataset showed that the expression level of BAP1 was obviously derceased in clear-cell renal cell carcinoma (ccRCC), compared with the matched paracancerous tissues (Fig. 1 A). To further assess the expression of BAP1 in ccRCC, we performed immunohistochemistry to detect the expression of BAP1 in the tissues of ccRCC. As shown in Fig. 1 B, the protein expression levels of BAP1 in ccRCC tissues were significantly lower than that of normal tissues, which were consistent with the results from the TCGA database. Meanwhile, analysis by RT-PCR revealed that the mRNA expression levels of BAP1 in ccRCC tissues were also significantly reduced than that of normal tissues (Fig. 1 C). Then, to determine the prognostic significance of BAP1 expression in ccRCC, we performed Kaplan-Meier survival analysis to assess the correlation between BAP1 expression and ccRCC prognosis. As depicted in Fig. 1 D, the overall survival time of patients with low expression level of BAP1 were obviously shorter than that of patients with relatively high expression level of BAP1. Besides, the mRNA and protein levels of BAP1 were also dramatically down-regulated both in ccRCC cell lines (786-O and Caki-1 cell lines (Fig. 1 E-F), compared with normal human renal proximal tubular cells (HK-2 cells). Collectively, the above evidence suggested that BAP1 might be involved in the progression of ccRCC and affect the prognosis of patients. Detection of an inverse correlation between miR-200c-3p and BAP1 levels in ccRCC. Emerging evidence have suggested that miRNAs were aberrantly expressed in multiple cancers, which raised the question that miRNAs might play a pivotal role in ccRCC and our interest was directed to explore whether the expression of BAP1 was associated with abnormal expression of miRNAs. Thus, to identify the potential upstream miRNAs which may regulate the expression level of BAP1, we measured the expression profile of miRNAs in a corhot of ccRCC tissues and their matched normal renal tissues using the gene chip analysis. The analysis showed that 159 miRNAs were up-regulated and 124 miRNAs were down-regulated in ccRCC tissues, compared with their matched normal renal tissues. The expression profile of miRNAs in ccRCC tissues and their matched normal renal tissues was presented as the volcano map in Fig. 2 A. Since the expression levels of potential miRNAs were negatively correlated with BAP1, we selected miRNAs with abnormally elevated expression in the microarray results; meanwhile, two computational bioinformatics database (TargetScan and miRanda) were used in combination to identify potential miRNAs that have potential binding sites with the 3’UTR site of BAP1. Among the candidate miRNAs, miR-223-3p, miR-200c-3p, miR-185-5p and miR-543 were predicted to be BAP1 regulators and were up-regulated in these ccRCC tissues compared with matched normal renal tissues (Fig. 2 B). In vitro assay also confirmed that miR-223-3p, miR-200c-3p, miR-185-5p and miR-543 were up-regulated in both in 786-O and Caki-1 ccRCC cell lines, compared to HK-2 cells. Considering that the role of miR-200c-3p in ccRCC has not been elucidated, our studies focused on the expression of miR-200c-3p and its potential association with BAP1 in ccRCC. Subsequently, Pearson correlation analysis indicated that the expression of BAP1 was negatively correlated with miR-200c-3p (Fig. 2 D), which strongly suggested that miR-200c-3p was likely to regulate the expression of BAP1 in ccRCC. In the differential gene expression analysis (Heatmap) from gene chip analysis (Fig. 2 E), it was found that BAP1 is significantly decreased in ccRCC. Furthermore, KEGG analysis (Fig. 2 F) revealed that pathways such as the cell cycle are significantly activated in ccRCC. These abnormally activated pathways may be associated with the reported role of BAP1 as a tumor suppressor gene, affecting the proliferation and apoptosis of tumor cells. Validation of BAP1 as a direct target of miR-200c-3p. To determine the direct biding relationship between miR-200c-3p and the 3’UTR site of BAP1 mRNA, dual-luciferase reporter assay using the HEK293T cells co-transfected with BAP1 3’UTR WT/MUT and miR-200c-3pminic/inhibitor (Fig. 4 ) was performed. The results showed that the miR-200c-3p minic group exhibited inhibited luciferase activity of wild (WT) plasmids but has no influence on the luciferase activity of mutant (MUT) plasmids, indicating that miR-200c-3p suppresses the expression of BAP1 by directly binding to BAP1 3’UTR region. MiR-200c-3p promotes the proliferation in ccRCC cells by inhibiting BAP1 . Subsequently, we evaluated the biological effects of the miR-200c-3p-driven repression of BAP1 expression in ccRCC cells. First, we knocked down the expression of miR-200c-3p by transfection of miR-200c-3p inhibitor in Caki-1 cells. The knockdown efficiency was confirmed by RT-PCR analysis (Fig. 4 A). As illustrated in Fig. 4 B-C, knockdown of miR-200c-3p could dramatically increase the mRNA (Fig. 4 B) and protein (Fig. 4 C) expression of BAP1 in Caki-1 ccRCC cells. Moreover, we also knocked down the expression of BAP1 by transfection of cells with BAP1 siRNA. The results showed that transfection of BAP1 siRNA significantly inhibited both the mRNA (Fig. 4 D) and protein (Fig. 4 E) expression levels of BAP1. To determine whether the expression pattern of miR-200c-3p/BAP1 has influence in the proliferation of Caki-1 ccRCC cells, CCK-8 assay was performed. As expected, the results showed that knockdown of miR-200c-3p markedly inhibited the proliferation of Caki-1 ccRCC cells, compared with control group. However, this phenomenon could be largely neutralized by co-transfection of BAP1 siRNA, compared with negative control group. The effect of miR-200c-3p/BAP1 on the growth of tumor xenografts. To confirm the effect of miR-200c-3p/BAP1 on the growth of tumor xenografts, we seeded nude mice with normal Caki-1 cells, or Caki-1 cells transfected with miR-200c-3p inhibitor, or miR-200c-3p inhibitor with BAP-1 shRNA cells, or negative control. The results in vivo were consistent with in vitro results. As shown in Fig. 5 B-C, the tumor weight and volume were significantly decreased in miR-200c-3p inhibitor group, while this phenomenon was largely neutralized in the co-transfection of BAP1 shRNA group, compared with negative control group. Besides, the results of immunohistochemistry in Fig. 5 D also showed that miR-200c-3p inhibitor significantly inhibited the expression of Ki-67, which was an important marker of tumor proliferation. Co-transfection of BAP1 shRNA with miR-200c-3p inhibitor reversed the above anti-tumor effect by miR-200c-3p inhibitor alone. As a tumor suppressor gene, the expression of BAP1 determined by immunohistochemistry was opposite to that of Ki-67. These above evidences strongly indicated that a typical miR-200c-3p/BAP1 mediated post-transcriptional regulation mechanism was involved in the progression of ccRCC. Discussion Currently, with the improvement in the diagnosis and treatment, the diagnostic and therapy rate of ccRCC has been greatly improved. However, there are also a large number of patients who are not optimally treated due to lack of early clinical symptoms and sensitive biomarkers [ 19 , 20 ] . Therefore, it is urgent to find new and effective targets to improve the level of precise diagnosis and clinical treatment. From the TCGA database we found that the expression of BAP1 is significantly decreased in ccRCC tissues compared to that of normal paracancerous tissue. Subsequent prognostic analysis also showed that, as a tumor suppressor gene, the expression of BAP1 was closely related to the survival of ccRCC patients. What’s more, through miRNA chip assay and bioinformatics analysis, we screened out the possible upstream regulatory molecules of BAP1, miR-200c-3p. Correspondingly, the expression level of BAP1 showed an opposite trend to that of miR-200c-3p in ccRCC tissues. The subsequent experiments further confirmed that miR-200c-3p directly targeted and regulated the expression of BAP1. Targeting miR-200c-3p/BAP1 can regulate the proliferation of ccRCC cells. The above evidence strongly indicated that miR-200c-3p/BAP1 was involved in the regulation of ccRCC progression. There are still some shortcomings in the current research. We have not done in vivo and in vitro experiments to further determine the over-expression effect of miR-200c-3p/BAP1 in the progression of ccRCC, and we do not fully determine whether there are other upstream regulatory molecules of BAP1. In addition, how miR-200c-3p itself is differentially expressed in ccRCC also needs further research to elucidate. In summary, the present research demonstrated that BAP1 level was reduced in tissues from ccRCC patients; patients with low BAP1 expression have a worse prognosis than patients with high BAP1 expression. Notably, we further investigated the upstream of BAP1 and demonstrated that miR-200c-3p directly bided to 3’UTR site of BAP1 mRNA and suppressed BAP1 expression. Targeting miR-200c-3p/BAP1 has significant influence on the proliferation of ccRCC cells. Therefore, our present study showed that miR-200c-3p/BAP1 might be a potential therapeutic target for future ccRCC diagnosis and clinical treatments. Declarations Ethics approval and consent to participate All the patients have provided the written consent, and the Ethics Committee from Heilongjiang Provincial Hospital approved all aspects of this study Availability of Data and Materials The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Consent for Publication Written informed consent for publication was obtained from all participants. Competing Interests The authors declare that there is no conflict of interest in the manuscript. Funding Research project of Heilongjiang Provincial Health Commission: 20211111000145. Author Contribution Du Wei was responsible for the design of the study and writing of the manuscript. Ge Wenyu,Yu Ling and Chen Hongzhe performed the experiments, including cell culture , PCR and western blotting. Wang Dongmei participated in the animal experiments. Xu Xinglu designed the study and conducted the statistical analysis. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work was appropriately investigated and resolved. References Chaffer, C.L. and Weinberg, R.A.A perspective on cancer cell metastasis. Science , 2011 331, 1559-1564. 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Cancer Res , 2021 81, 2807-2814. Ventii, K.H., Devi, N.S., Friedrich, K.L., Chernova, T.A., Tighiouart, M., Van Meir, E.G. and Wilkinson, K.D.BRCA1-associated protein-1 is a tumor suppressor that requires deubiquitinating activity and nuclear localization. Cancer Res , 2008 68, 6953-6962. Kury, S., Ebstein, F., Molle, A., Besnard, T., Lee, M.K., Vignard, V., Hery, T., Nizon, M., Mancini, G.M.S., Giltay, J.C. et al. Rare germline heterozygous missense variants in BRCA1-associated protein 1, BAP1, cause a syndromic neurodevelopmental disorder. Am J Hum Genet , 2022 109, 361-372. Cochetti, G., Cari, L., Maula, V., Cagnani, R., Paladini, A., Del Zingaro, M., Nocentini, G. and Mearini, E.Validation in an Independent Cohort of MiR-122, MiR-1271, and MiR-15b as Urinary Biomarkers for the Potential Early Diagnosis of Clear Cell Renal Cell Carcinoma. Cancers (Basel) , 2022 14. Sequeira, J.P., Constancio, V., Salta, S., Lobo, J., Barros-Silva, D., Carvalho-Maia, C., Rodrigues, J., Braga, I., Henrique, R. and Jeronimo, C.LiKidMiRs: A ddPCR-Based Panel of 4 Circulating miRNAs for Detection of Renal Cell Carcinoma. Cancers (Basel) , 2022 14. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4161288","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":291228494,"identity":"bc413173-b6b1-4fea-82a6-1ad643e62c45","order_by":0,"name":"Du Wei","email":"","orcid":"","institution":"Heilongjiang Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Du","middleName":"","lastName":"Wei","suffix":""},{"id":291228495,"identity":"14cb7bda-5764-4cd7-94c1-56621e1ce2fe","order_by":1,"name":"Ge Wenyu","email":"","orcid":"","institution":"Heilongjiang Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ge","middleName":"","lastName":"Wenyu","suffix":""},{"id":291228496,"identity":"4f42b79d-35d5-44e2-a5f3-a7b32174237f","order_by":2,"name":"Yu Ling","email":"","orcid":"","institution":"Heilongjiang Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Ling","suffix":""},{"id":291228497,"identity":"5dba27af-d2fe-4b9d-a769-7fe462ebf610","order_by":3,"name":"Chen Hongzhe","email":"","orcid":"","institution":"Heilongjiang Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Hongzhe","suffix":""},{"id":291228498,"identity":"6aae634c-3a26-4bff-922f-e84a5749e8e7","order_by":4,"name":"Wang Dongmei","email":"","orcid":"","institution":"Heilongjiang Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wang","middleName":"","lastName":"Dongmei","suffix":""},{"id":291228499,"identity":"8cc368c3-462c-4caf-86c4-e2f3f1e9270c","order_by":5,"name":"Xu Xinglu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYDACZiB+AMT8zMwHHxCvJQGIJdvZkg2ItwmkxeA8j5kAUaoNjvMefpHYdjhx82EGMwaGGptoglokm/nSLBLb0hK3HWZIe8BwLC23gZAWfmYeM4PENhuQluMGjA2HCWthg2iRSNzczNgmQZQWoC3GD0C2bGBmZiNOi2QzjxlDwrk04xmH2ZgNEojxi8H5M8YfPpQdlu3vP//xwYcaG8JaQN6RgDMTiFAOAswfiFQ4CkbBKBgFIxUAAGcSOxx/suPQAAAAAElFTkSuQmCC","orcid":"","institution":"Heilongjiang Provincial Hospital","correspondingAuthor":true,"prefix":"","firstName":"Xu","middleName":"","lastName":"Xinglu","suffix":""}],"badges":[],"createdAt":"2024-03-25 07:07:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4161288/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4161288/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55112076,"identity":"eaa01de3-b1ec-4567-b30e-f685c10ed9d2","added_by":"auto","created_at":"2024-04-22 19:06:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":570791,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The expression of BAP1 from\u003cstrong\u003e \u003c/strong\u003eTCGA. (B) The expression of BAP1 in ccRCC tissues and paracancerous tissues, detected by immunohistochemistry (Magnification×200). (C) The mRNA expression of BAP1 in ccRCC tissues and paracancerous tissues, detected by RT-PCR. n=24. (D) The survivorship curve of ccRCC patient by Kaplan-Meier survival analysis. (E) The mRNA expression of BAP1 in ccRCC cell lines (Caki-1, 786-O) and normal cells (HK-2), detected by RT-PCR. n=6. (F) The protein expression of BAP1 in ccRCC cell lines (Caki-1, 786-O) and normal cells (HK-2), detected by Western blot. n=6. *P≤0.05; **P≤0.01.\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4161288/v1/040b471c37d8f2bad22fb770.jpg"},{"id":55112075,"identity":"18b6b1e9-932f-43eb-8dac-43b71382993b","added_by":"auto","created_at":"2024-04-22 19:06:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2320382,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The expression profile of miRNAs in ccRCC tissues and paracancerous tissues, detected by miRNA chip assay. (B) The expression of four miRNAs in ccRCC tissues and paracancerous tissues, detected by RT-PCR. n=24. (C) The expression of four miRNAs in ccRCC cell lines and normal cell line. (D) Pearson correlation analysis indicated that the expression of BAP1 was negatively correlated with miR-200c-3p. (E) Heatmap represents the differentially expressed genes in ccRCC. (F) Aberrant pathways identified by KEGG analysis in ccRCC, compared to normal tissues.*P≤0.05; **P≤0.01.\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4161288/v1/123a6fde4c0a6863967e5604.jpg"},{"id":55112077,"identity":"52eaa004-8cb6-4c2f-81ca-108a66201958","added_by":"auto","created_at":"2024-04-22 19:06:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":212407,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The predicted binding site between miR-200c-3p and 3’UTR region of BAP1 and the artificially constructed mutant sequences. (B) The results of dual-luciferase reporter gene assay to determine the direct biding state between miR-200c-3p and 3’UTR region of BAP1. MiR-200c-3p significantly inhibited the fluorescence of the constructed plasmids, but not in the mutant BAP1 constructed plasmids. #P≤0.05; **P≤0.01.\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4161288/v1/d8050a1d0f8c3cb9491dcda9.jpg"},{"id":55113705,"identity":"b4565cae-6943-4272-9d6e-92143071b215","added_by":"auto","created_at":"2024-04-22 19:14:42","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":267231,"visible":true,"origin":"","legend":"\u003cp\u003e(A) MiR-200c-3p inhibitor significantly inhibited the expression of miR-200c-3p, compared with the negative control. n=6. (B) MiR-200c-3p inhibitor significantly promoted the mRNA expression of BAP1, compared with the negative control. n=6. (C) MiR-200c-3p inhibitor significantly promoted the protein expression of BAP1, compared with the negative control. n=6. (D) The BAP1 siRNA significantly inhibited the mRNA expression of BAP1, compared with the negative control. n=6. (E) The BAP1 siRNA significantly inhibited the protein expression of BAP1, compared with the negative control. n=6. (F) The proliferation of Caki-1 cells was significantly inhibited by miR-200c-3p inhibitor, which largely reversed by BAP-1 siRNA. n=6. *P≤0.05; #P≤0.05; **P≤0.01.\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4161288/v1/03d7e819876770182bc93a26.jpg"},{"id":55112079,"identity":"a146ac59-ad26-4480-8653-f63c08098f2f","added_by":"auto","created_at":"2024-04-22 19:06:42","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3223911,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The representative images of tumor xenografts. (B) The statistical graph of tumor weight. n=6. (C) The statistical graph of tumor volume. n=6. (D) The expression of Ki-67 and BAP-1, determined by immunohistochemistry. *P≤0.05; #P≤0.05.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4161288/v1/75b8c38a7fde1d0d68496564.jpg"},{"id":64211437,"identity":"d340bcd1-5b47-4a25-aaf6-4be8056b81f8","added_by":"auto","created_at":"2024-09-10 07:35:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9621710,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4161288/v1/1cb6b296-7abe-40bb-9f73-12b8ccb2c740.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"BAP1 serves as a clear-cell renal cell carcinoma suppressor and is inversely regulated by miR-200c-3p","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRenal cell carcinoma (RCC) is one of the most lethal of human disease, accounting for 2\u0026ndash;3% of adult malignancies in the world, while clear-cell renal cell carcinoma accounts for approximately 80% of RCC\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The early diagnosis and complete surgical excision are the main factors contributing to a definitive treatment of ccRCC. The type of treatment depends on multiple factors including the stage of clear-cell renal cell carcinoma, the type of clear-cell renal cell carcinoma, pre-existing or comorbid conditions and overall health and age of the patient. Although early-stage ccRCC is curable by surgery resection, the prognosis for ccRCC patients is still poor for the resistant to chemotherapy and radiotherapy in most cases \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Therefore, the improvement of new diagnostic and treatment strategies for ccRCC is urgently required.\u003c/p\u003e \u003cp\u003eMicroRNAs (miRNAs) are endogenous, small noncoding RNAs with 19\u0026ndash;22 nucleotides in length that modulate biological processes at the post-transcriptional levels \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Accumulating evidence has demonstrated that miRNAs are differently expressed in various human cancers including ccRCC\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Different expression profile of miRNAs is strongly related to numerous biological processes such as cell proliferation, migration, invasion, metastasis and so forth \u003csup\u003e[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Up to now, more than 1000 human miRNAs have been identified and reported as molecular biomarkers for diagnosis, prognosis and treatment of diseases. Thus, it is extremely necessary to explore the specific miRNA in ccRCC as tumor markers with enough specificity and sensitivity.\u003c/p\u003e \u003cp\u003eEpigenetic is a rapidly growing field that studies gene expression modifications not involving changes in the DNA sequence \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Ubiquitination is one of the main areas of epigenetic research\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Among the de-ubiquitinating protein family, BRCA1 associated protein-1 (BAP1) is a deubiquitinating enzyme with an ubiquitin carboxy-terminal hydrolase (UCH) domain. Plenty of researches have illustrated that BAP1 was involved in numerous cellular processes, such as cell fate determination, stem cell pluripotency and other biological processes\u003csup\u003e[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. In cancers, BAP1 was first shown to act as a tumor suppressor, where its deubiquitinase (UCH) domain and nuclear localization sequences were required for BAP1 to suppress cell growth \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Furthermore, BAP1 mutations have been identified in malignant neoplasms\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. However, the roles of BAP1 in the initiation and progression of human renal cell carcinoma remains controversial. Thus, elucidating the BAP1-involved intricate regulative network and epigenetic could shed light on how their deregulation contributes to the development of ccRCC. Since cancers may have multiple pathologic factors, we tried to explain the pathogenesis of renal cell carcinoma from the perspective of epigenetic, especially the upstream of BAP1 in our research.\u003c/p\u003e \u003cp\u003eIn the present study, our date showed that BAP1 was a tumor suppressor gene in ccRCC and was closely associated with the prognosis of ccRCC. Furthermore, BAP1 was a direct target gene of miR-200c-3p and that miR-200c-3p could inhibit the expression of BAP1, which contributed to the progression of clear-cell renal cell carcinoma. Consequently, enhanced expression of miR-200c-3p promoted the proliferation and suppressed the apoptosis of clear-cell renal cell carcinoma cells in vitro. Therefore, in the present study, we provided evidence that targeting miR-200c-3p/BAP1 may be an alternative strategy for treating clear-cell renal cell carcinoma in clinical practice.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHuman tissues and cells\u003c/h2\u003e \u003cp\u003eThe clear-cell renal cell carcinoma and matched normal adjacent tissues were derived from patients undergoing surgical procedure from April 2015 to December 2019. All the patients have provided the written consent, and the Ethics Committee from Heilongjiang Provincial Hospital approved all aspects of this study. Tissue fragments were immediately frozen in liquid nitrogen at the time of surgery. The human renal carcinoma cell lines were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI 1640medium (Gibco, Grand Island, NY, USA) supplemented with 12% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA). All the cells were maintained at 37\u0026deg;C in humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell transfection\u003c/h2\u003e \u003cp\u003eThe cells were inoculated into complete medium in a 6-well cell culture plate at the appropriate cell density for transfection (40\u0026thinsp;\u0026minus;\u0026thinsp;50%). Over-expression of miR-200c-3p was achieved by transfecting renal cancer cells with a miR-200c-3p mimic, while knockdown of miR-200c-3p was achieved by transfecting a miRNA inhibitor. The mimic and inhibitor were purchased from GenePharma (Shanghai, China). The siRNA sequences of the human BAP1 cDNA (si-BAP1) and shRNA-BAP1 were designed and synthesized by GenePharma (Shanghai, China). A scrambled siRNA that did not bind the human BAP1 cDNA was designed and synthesized as a negative control. For over-expression of BAP1, a plasmid encoding the full-length human BAP1 was purchased from Invitrogen (Carlsbad, CA, USA).While an empty plasmid was served as the negative control.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantitative reverse-transcription polymerase chain reaction.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTotal RNA was harvested and extracted from tissues or celllines using Trizol (Invitrogen, Life, USA), according to the manufacturer\u0026rsquo;s instructions. For miR-200c-3p, cDNA was synthesized from total RNA (0.5 mg) using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, USA). For BAP1, cDNA was synthesized from total RNA (0.5 mg) using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA), with random primer. FormiR-200c-3p, the reverse transcription primer sequence was 5\u0026rsquo;-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCCATCAT-3\u0026rsquo;. Real-time quantitative PCR was performed using SYBR Master Mix (Roche, Life, USA).All the primers were designed and synthesized by the Sangon Biotech (Shanghai, China). The primers for miR-200c-3p were as follows: 5\u0026rsquo;-ACACTCCAGCTGGGTAATACTGCCGGGTAAT-3\u0026rsquo;(forward) and 5\u0026rsquo;-TGGTGTCGTGGAGTCG-3\u0026rsquo; (reverse). The primers for BAP1 were as follows: 5\u0026rsquo;-GACCCAGGCCTCTTCACC-3\u0026rsquo;(forward) and 5\u0026rsquo;-AGTCCTTCATGCGACTCAGG-3\u0026rsquo; (reverse). The primers for U6 were as follows: 5\u0026rsquo;-CTCGCTTCGGCAGCACA-3\u0026rsquo;(forward) and 5\u0026rsquo;-AACGCTTCACGAATTTGCGT-3\u0026rsquo; (reverse). These assays were performed on the 7500 Fast Real-Time PCR System (Applied Biosystem, USA). The results were calculated using the ΔΔ threshold cycle (Ct) method and then normalized to the endogenous reference control gene U6 for miR-200c-3p and GAPDH for BAP1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and western blotting\u003c/h2\u003e \u003cp\u003eThe protein contents were determined with a BCA Protein Assay Kit (Beyotime, Shanghai, China).The samples were mixed with loading buffer (Beyotime, Shanghai, China) and boiled for 10 min for denaturation. Then the samples were subjected to electrophoresis using 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), and were later transferred onto a polyvinylidene fluoride (PVDF) membrane, which was afterwards incubated with 5% nonfat milk (Beyotime, Shanghai, China) for 2 h at room temperature, and then probed with antibodies against Ki67 (Santa Cruz, USA), Cyclin D (Santa Cruz, USA), P53 (Abcam, Cambridge, United Kingdom), and AKT (Abcam, Cambridge, United Kingdom) overnight at 4\u0026deg;C on a shaker. The membranes were then washed thrice with 0.05% TBST (TBS mixed with 0.05% tween) and incubated with Alexa Fluor labeled secondary antibodies (Beyotime, A0460 and A0468, Shanghai, China) for 1 h in the dark at room temperature. Finally, the bands in the membranes were detected with the Odyssey instrument (Li-COR, American Gene Corp. USA), and the Odyssey v1.2 software was used to analyze the density of the bands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation assay\u003c/h2\u003e \u003cp\u003eAfter 24 hours post-transfection, the proliferation of cells in each group was detected using the Cell Counting Kit-8 (Beyotime, C0037, Shanghai, China) according to the manufacturer\u0026rsquo;s instructions. Briefly, 10 \u0026micro;LCCK-8 liquid was added to the test well and incubated with cells for3 hours at 37\u0026deg;C. The absorbance of each well was measured at a wave length of 450 nm.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranswell migration and invasion assay.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo detect the migration ability of cells, chamber of a Transwell plate with 8 \u0026micro;M pore size (Costar, USA) were used. Firstly, cells (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e) were seeded in the upper chamber with serum-free medium, while complete medium was added to the bottom wells of the chambers. All Transwell chambers were incubated at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e in culture medium. The cells that had not migrated to the other side of the membrane were removed from the upper face of the filters, then the remaining cells were fixed with 95% ethanol for 15min and washed in tap water. Crystal violet solution (4g / L) was added to each chamber for 10 min, after which the chambers were washed again in tap water. Cells were then counted in five randomly chosen visual fields (200\u0026times;) and the average value was calculated.Similar chambers coated with Matrigel (BD, USA) were used to determine invasive ability in the cell invasion assay.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTumor xenograft assay in vivo.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe research in nude mice was conducted in accordance with the ethical standards and national guidelines, which was approved by the Ethics Committee of Heilongjiang Provincial Hospital. The 4 weeks old nude mice (BALB/c-nu/nu) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Caki-1 cells were first transfected with miR-200c-3p inhibitor or co-transfected with miR-200c-3p inhibitor and BAP-1 shRNA, designed and synthesized by GenePharma (Shanghai, China). Then the mice were subcutaneously injected with 0.3 ml tumor cell suspension. After 4 weeks, the mice were sacrificed and the tumors were then removed for measuring the size and weight, and the following immunohistochemistry assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed with Graphpad prism 5 software. All data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical comparisons between two groups were conducted with the Student\u0026rsquo;s t-test. Univariate analysis of variance (ANOVA) was used for comparisons among multiple groups. A p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cstrong\u003eDown regulation of BAP1 in vivo and in vitro.\u003c/strong\u003e Date from The Cancer Genome Atlas (TCGA) dataset showed that the expression level of BAP1 was obviously derceased in clear-cell renal cell carcinoma (ccRCC), compared with the matched paracancerous tissues (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). To further assess the expression of BAP1 in ccRCC, we performed immunohistochemistry to detect the expression of BAP1 in the tissues of ccRCC. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, the protein expression levels of BAP1 in ccRCC tissues were significantly lower than that of normal tissues, which were consistent with the results from the TCGA database. Meanwhile, analysis by RT-PCR revealed that the mRNA expression levels of BAP1 in ccRCC tissues were also significantly reduced than that of normal tissues (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Then, to determine the prognostic significance of BAP1 expression in ccRCC, we performed Kaplan-Meier survival analysis to assess the correlation between BAP1 expression and ccRCC prognosis. As depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD, the overall survival time of patients with low expression level of BAP1 were obviously shorter than that of patients with relatively high expression level of BAP1. Besides, the mRNA and protein levels of BAP1 were also dramatically down-regulated both in ccRCC cell lines (786-O and Caki-1 cell lines (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE-F), compared with normal human renal proximal tubular cells (HK-2 cells). Collectively, the above evidence suggested that BAP1 might be involved in the progression of ccRCC and affect the prognosis of patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of an inverse correlation between miR-200c-3p and BAP1 levels in ccRCC.\u003c/strong\u003e Emerging evidence have suggested that miRNAs were aberrantly expressed in multiple cancers, which raised the question that miRNAs might play a pivotal role in ccRCC and our interest was directed to explore whether the expression of BAP1 was associated with abnormal expression of miRNAs. Thus, to identify the potential upstream miRNAs which may regulate the expression level of BAP1, we measured the expression profile of miRNAs in a corhot of ccRCC tissues and their matched normal renal tissues using the gene chip analysis. The analysis showed that 159 miRNAs were up-regulated and 124 miRNAs were down-regulated in ccRCC tissues, compared with their matched normal renal tissues. The expression profile of miRNAs in ccRCC tissues and their matched normal renal tissues was presented as the volcano map in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA. Since the expression levels of potential miRNAs were negatively correlated with BAP1, we selected miRNAs with abnormally elevated expression in the microarray results; meanwhile, two computational bioinformatics database (TargetScan and miRanda) were used in combination to identify potential miRNAs that have potential binding sites with the 3\u0026rsquo;UTR site of BAP1. Among the candidate miRNAs, miR-223-3p, miR-200c-3p, miR-185-5p and miR-543 were predicted to be BAP1 regulators and were up-regulated in these ccRCC tissues compared with matched normal renal tissues (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). In vitro assay also confirmed that miR-223-3p, miR-200c-3p, miR-185-5p and miR-543 were up-regulated in both in 786-O and Caki-1 ccRCC cell lines, compared to HK-2 cells. Considering that the role of miR-200c-3p in ccRCC has not been elucidated, our studies focused on the expression of miR-200c-3p and its potential association with BAP1 in ccRCC. Subsequently, Pearson correlation analysis indicated that the expression of BAP1 was negatively correlated with miR-200c-3p (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD), which strongly suggested that miR-200c-3p was likely to regulate the expression of BAP1 in ccRCC. In the differential gene expression analysis (Heatmap) from gene chip analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE), it was found that BAP1 is significantly decreased in ccRCC. Furthermore, KEGG analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF) revealed that pathways such as the cell cycle are significantly activated in ccRCC. These abnormally activated pathways may be associated with the reported role of BAP1 as a tumor suppressor gene, affecting the proliferation and apoptosis of tumor cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation of BAP1 as a direct target of miR-200c-3p.\u003c/strong\u003eTo determine the direct biding relationship between miR-200c-3p and the 3\u0026rsquo;UTR site of BAP1 mRNA, dual-luciferase reporter assay using the HEK293T cells co-transfected with BAP1 3\u0026rsquo;UTR WT/MUT and miR-200c-3pminic/inhibitor (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) was performed. The results showed that the miR-200c-3p minic group exhibited inhibited luciferase activity of wild (WT) plasmids but has no influence on the luciferase activity of mutant (MUT) plasmids, indicating that miR-200c-3p suppresses the expression of BAP1 by directly binding to BAP1 3\u0026rsquo;UTR region.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMiR-200c-3p promotes the proliferation in ccRCC cells by inhibiting BAP1\u003c/strong\u003e. Subsequently, we evaluated the biological effects of the miR-200c-3p-driven repression of BAP1 expression in ccRCC cells. First, we knocked down the expression of miR-200c-3p by transfection of miR-200c-3p inhibitor in Caki-1 cells. The knockdown efficiency was confirmed by RT-PCR analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). As illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB-C, knockdown of miR-200c-3p could dramatically increase the mRNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB) and protein (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC) expression of BAP1 in Caki-1 ccRCC cells. Moreover, we also knocked down the expression of BAP1 by transfection of cells with BAP1 siRNA. The results showed that transfection of BAP1 siRNA significantly inhibited both the mRNA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD) and protein (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE) expression levels of BAP1. To determine whether the expression pattern of miR-200c-3p/BAP1 has influence in the proliferation of Caki-1 ccRCC cells, CCK-8 assay was performed. As expected, the results showed that knockdown of miR-200c-3p markedly inhibited the proliferation of Caki-1 ccRCC cells, compared with control group. However, this phenomenon could be largely neutralized by co-transfection of BAP1 siRNA, compared with negative control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe effect of miR-200c-3p/BAP1 on the growth of tumor xenografts.\u003c/strong\u003e To confirm the effect of miR-200c-3p/BAP1 on the growth of tumor xenografts, we seeded nude mice with normal Caki-1 cells, or Caki-1 cells transfected with miR-200c-3p inhibitor, or miR-200c-3p inhibitor with BAP-1 shRNA cells, or negative control. The results in vivo were consistent with in vitro results. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB-C, the tumor weight and volume were significantly decreased in miR-200c-3p inhibitor group, while this phenomenon was largely neutralized in the co-transfection of BAP1 shRNA group, compared with negative control group. Besides, the results of immunohistochemistry in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD also showed that miR-200c-3p inhibitor significantly inhibited the expression of Ki-67, which was an important marker of tumor proliferation. Co-transfection of BAP1 shRNA with miR-200c-3p inhibitor reversed the above anti-tumor effect by miR-200c-3p inhibitor alone. As a tumor suppressor gene, the expression of BAP1 determined by immunohistochemistry was opposite to that of Ki-67. These above evidences strongly indicated that a typical miR-200c-3p/BAP1 mediated post-transcriptional regulation mechanism was involved in the progression of ccRCC.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCurrently, with the improvement in the diagnosis and treatment, the diagnostic and therapy rate of ccRCC has been greatly improved. However, there are also a large number of patients who are not optimally treated due to lack of early clinical symptoms and sensitive biomarkers \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is urgent to find new and effective targets to improve the level of precise diagnosis and clinical treatment.\u003c/p\u003e \u003cp\u003eFrom the TCGA database we found that the expression of BAP1 is significantly decreased in ccRCC tissues compared to that of normal paracancerous tissue. Subsequent prognostic analysis also showed that, as a tumor suppressor gene, the expression of BAP1 was closely related to the survival of ccRCC patients. What\u0026rsquo;s more, through miRNA chip assay and bioinformatics analysis, we screened out the possible upstream regulatory molecules of BAP1, miR-200c-3p. Correspondingly, the expression level of BAP1 showed an opposite trend to that of miR-200c-3p in ccRCC tissues. The subsequent experiments further confirmed that miR-200c-3p directly targeted and regulated the expression of BAP1. Targeting miR-200c-3p/BAP1 can regulate the proliferation of ccRCC cells. The above evidence strongly indicated that miR-200c-3p/BAP1 was involved in the regulation of ccRCC progression.\u003c/p\u003e \u003cp\u003eThere are still some shortcomings in the current research. We have not done in vivo and in vitro experiments to further determine the over-expression effect of miR-200c-3p/BAP1 in the progression of ccRCC, and we do not fully determine whether there are other upstream regulatory molecules of BAP1. In addition, how miR-200c-3p itself is differentially expressed in ccRCC also needs further research to elucidate.\u003c/p\u003e \u003cp\u003eIn summary, the present research demonstrated that BAP1 level was reduced in tissues from ccRCC patients; patients with low BAP1 expression have a worse prognosis than patients with high BAP1 expression. Notably, we further investigated the upstream of BAP1 and demonstrated that miR-200c-3p directly bided to 3\u0026rsquo;UTR site of BAP1 mRNA and suppressed BAP1 expression. Targeting miR-200c-3p/BAP1 has significant influence on the proliferation of ccRCC cells. Therefore, our present study showed that miR-200c-3p/BAP1 might be a potential therapeutic target for future ccRCC diagnosis and clinical treatments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the patients have provided the written consent, and the Ethics Committee from Heilongjiang Provincial Hospital approved all aspects of this study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent for publication was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearch project of Heilongjiang Provincial Health Commission: 20211111000145.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDu Wei was responsible for the design of the study and writing of the manuscript. Ge Wenyu,Yu Ling and Chen Hongzhe performed the experiments, including cell culture , PCR and western blotting. Wang Dongmei participated in the animal experiments. Xu Xinglu designed the study and conducted the statistical analysis. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work was appropriately investigated and resolved.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChaffer, C.L. and Weinberg, R.A.A perspective on cancer cell metastasis. \u003cstrong\u003e\u003cem\u003eScience\u003c/em\u003e\u003c/strong\u003e, \u003cstrong\u003e2011\u003c/strong\u003e 331, 1559-1564.\u003c/li\u003e\n\u003cli\u003eZhou, Y., Li, C., Wang, Z., Tan, S., Liu, Y., Zhang, H. and Li, X.CircRNAs as Novel Biomarkers and Therapeutic Targets in Renal Cell Carcinoma. \u003cstrong\u003e\u003cem\u003eFront Mol Biosci\u003c/em\u003e\u003c/strong\u003e, \u003cstrong\u003e2022\u003c/strong\u003e 9, 833079.\u003c/li\u003e\n\u003cli\u003eBallesteros, P.A., Chamorro, J., Roman-Gil, M.S., Pozas, J., Gomez Dos Santos, V., Granados, A.R., Grande, E., Alonso-Gordoa, T. and Molina-Cerrillo, 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Detection of Renal Cell Carcinoma. \u003cstrong\u003e\u003cem\u003eCancers (Basel)\u003c/em\u003e\u003c/strong\u003e, \u003cstrong\u003e2022\u003c/strong\u003e 14.\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":"","lastPublishedDoi":"10.21203/rs.3.rs-4161288/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4161288/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe initiation and development of malignant tumor is always accompanied by a series of complex gene expression alterations inside the cells. As a tumor suppressor, the deubiquitinating enzyme BAP1 has been identified as an important regulator on the outcomes and biological properties of clear-cell renal cell carcinoma (ccRCC). However, BAP1-involved intracellular regulatory cascades in clear-cell renal cell carcinoma are still not fully understood. In this study, we provided evidence that the protein levels of BAP1 were dramatically diminished in clear-cell renal cell carcinoma in vitro and in vivo. Notably, the relatively low expression of BAP1 is significantly associated with worse prognosis in ccRCC patients. Besides, through the prediction of bioinformatics methods and verification of biological experiments, we confirmed that miR-200c-3p was the direct upstream regulator of BAP1. Taken together, our study presents an important role of miR-200c-3p/BAP1 in the development of ccRCC, which provided an alternative strategy for treating ccRCC in clinical practice.\u003c/p\u003e","manuscriptTitle":"BAP1 serves as a clear-cell renal cell carcinoma suppressor and is inversely regulated by miR-200c-3p","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-22 19:06:24","doi":"10.21203/rs.3.rs-4161288/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":"c4a86be8-c5f2-4292-be25-185dd323fbe3","owner":[],"postedDate":"April 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-10T07:27:36+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-22 19:06:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4161288","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4161288","identity":"rs-4161288","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}