CircNR3C1 Inhibits Proliferation Of Bladder Cancer Cells Through MiR-27a-3p/cyclin D1 Axis

As one of the most frequently occurring tumor of urinary system,bladder cancer ranks ninth in prevalent cancer worldwide.Each year,approximately 430000 patients are diagnosed with bladder cancer worldwide.However,the therapeutic efficacy still displayed confined according to most clinical trials of chemotherapy for advanced bladder cancer.Thus,discovering new molecular mechanisms and searching for potential therapeutic targets will contribute to improving the benefits of treatment on bladder cancer.As a new type of RNAs,covalently closed circular RNA molecules(circRNAs)were originally discovered and considered as by-products of aberrant splicing with little functional potential 40 years ago.Next-generation RNA sequencing(RNA-Seq)from nonpolyadenylated RNA transcripts and bioinformatic analyses recently exhibited that circRNAs were widely expressed in diverse mammalian cell lines and across various species.Studies have shown that circRNAs displayed some conservation,high abundance,cell type-or tissue-and developmental stage-specific expression.Recently,dozens of circRNAs have been shown to play important role in human cancers.In previous study,we firstly confirmed that circular RNA NR3C1(circNR3C1)was downregulated in bladder cancer tissues according to High-Throughput Sequencing,and further demonstrated the same expression characteristics in bladder cancer cell lines compared to normal urothelial cell line.For clarify the biofunction of circNR3C1 in bladder cancer cells,we carried out hands of experiments in vitro and vivo.To further uncover the molecular mechanism,we performed some experiments in vitro using bladder cancer celllines.In adition,we would analyze the correlation between differential expression of circNR3C1 and clinical indicators.Here,our study will be elaborated predominantly in three parts: Part Ⅰ Expression of circNR3C1 in human bladder cancer and biological effects of circNR3C1 on bladder cancer cellsObjective: Investigeate the expression of circNR3C1 in human bladder tissues and celllines.To further expore the biological behviors of circNR3C1 on bladder cancer cells.Methods: 1.Polymerase chain reaction(PCR),Norther blot and Rnase R treatment were used to idenfy circN3C1 in bladder cancer cells.2.Real-time PCR(qRT-PCR)was carried out to detect circNR3C1 expression levels in 42 pairs of bladder cancer tissues and adjacent normal bladder tissues and bladder cell lines.Statistical analysis was used to analyze the correlation between circNR3C1 and clinical indicators.Fluorescence in situ hybridization(FISH)was performed to explore the distribution of circNR3C1 in bladder cancer cells.3.The circNR3C1 overexpression plasmid was constructed and transfected into EJ and T24 T cells,and colony formation assay,cell counting kit-8(CCK-8)assay,5-ethynyl-2’-deoxyuridine(EdU)assay,cell cycle assay,transwell invasion and migration assay were performed to explore the biological effects of circNR3C1 in bladder cancer cells.4.Tumor xenografts experiments was carried out to detected the role of circNR3C1 on tumor growth of xenografts.Results: 1.CircNR3C1 was derived from exon-2 of gene NR3C1 and conservatively existent in bladder cancer cells.2.Comparing with normal bladder tissues and normal urothelial cells(SV-HUC-1),circNR3C1 was downregulated in human bladder cancer tissues and bladder cancer celllines(EJ,T24 T and UMUC3).The differential expression was not correlative with clinical indicators.CircNR3C1 was manly localized in cytoplasm rather than in nucleus.3.Colony-forming ability of circNR3C1 over-expression cells were significantly lower than control in both cell lines.Over-expression of circNR3C1 induced cell cycle arrest at G0/G1 phase in EJ and T24 T cells compared with controls.There was no obviously differentiation between over-expression groups and controls on transwell migration and matrigel invasion assays.4.The growth of xenograft tumor growth was obviously inhibited in circNR3C1 overpression group compared with control group.Conclusion: circNR3C1 was down-regulated in bladder cancer cells and bladder cancer celllines,and mainly located in the cytoplasm of bladder cancer cells.Overexpressing circNR3C1 could inhibits cell proliferation and tumor growth of bladder cancer.Part Ⅱ Circ NR3C1 down-regulates the cyclin D1 expression and inhibits cell proliferation of bladder cancerObjective: To find out the downstream target gene of circ NR3C1 and clarify the molecular mechanisms of circ NR3C1 that inhibited cell proliferation and cell cycle,and further investigate the biological efficts of bladder cancer cells that circ NR3C1 regulates the expression of it’s target gene.Methods: 1.Circ NR3C1 plasmid was transfected into EJ and T24 T cells,and the cycle related proteins including CDK2,CDK4,CDK6,cyclin D1,cyclin E,P21 and P27 were chose to perform western blot assays in circ NR3C1/vector stably transfected EJ and T24 T cells as well as xenografts from mice.2.Cell cycle assay and western blot reversing experiments were performed to further confirm the effects on cell cycle and protein expression caused by the regulation of circ NR3C1 on target gene.3.Cell clone assay and Ed U assay reversing experiments were carried out to confirm the effects on cell proliferation of bladder cancer cells induced by the regulation of circ NR3C1 on target gene.4.Immunohistochemistry(IHC)analysis were used to detect the expression of target gene in human bladder cancer tissues and mice xenograft tumor tissues.Result: 1.The protein expression levels of cyclin D1 were dramatically decreased in circ NR3C1 overexpressed bladder cancer cells and tissues from xenografted tumor compared with vector groups,while the others shown no change between two sides.2.Cyclin D1 could moderately reversed cell cycle arrest at G0/G1 phase and down-regulated expression of cyclin D1 induced by circ NR3C1.3.The decreased proliferation caused by circ NR3C1 could partly be reversed by cyclin D1 in colony formation assay and Ed U assay.4.The expression of cyclin D1 was inhibited by overexpressing circ NR3C1 and was up-regulated in human bladder cancer tissues comparing with normal tissues.Conclusion: Circ NR3C1 induced G0/G1 arrest of bladder cancer cells through suppressing the expression of cyclin D1,and subsequently inhibited the proliferation of bladder cancer cells.Part Ⅲ Circ NR3C1 inhibits cell proliferation of bladder cancer through sponging mi R-27a-3p to downregulate expression of cyclin D1Objective: To further explore the molecular mechanism that circ NR3C1 downregulates the expression of cyclin D1 and inhibits cell proliferation.Methods: 1.Design the circ NR3C1 pull-down pribe.PCR and pull-down assay was performed to verify effectiveness and specificity of the probe.2.Using database(starbase)to predict the candidate mi RNA,which could interact with circ NR3C1.Pull-down assay and FISH colocalization was carried out to confirm the most appropriate candidates which not only could be sponged by circ NR3C1 but also play roles on cell proliferation.3.Western blot,cell clone assay,Ed U,cell cycle assay reverse experiments were carried out to further confirm the inhibitory effect caused by the regulation of circ NR3C1 on target mi RNA.4.Design the dual luciferase reporter plasmid of cyclin D1 5’UTR including wild type and mutational type and clarify the mechanism that target mi RNA regulates cyclin D1 expression.Results: 1.Circ NR3C1 could be specificly and substantially pulled down using circ NR3C1 probe.2.mi R-23a-3p and mi R-27a-3p could interacte with circ NR3C1 and silence mi R-27-3p could downregulate cyclin D1 expression.3.overexpressing circ NR3C1 suppressed cyclin D1 expression by sponging mi R-27a-3p to induce cell cycle arrest at G0/G1,and subsequently inhibited proliferation of bladder cancer cells.4.mi R-27a-3p could directly targets 5’UTR of cyclin D1 and promote cyclin D1 expression.Conclusion: Circ NR3C1 suppressed cyclin D1 expression via sponging mi R-27a-3p to decrease interactions between mi R-27a-3p and 5’UTR of cyclin D1 and inhibits cell proliferation of bladder cancer cells.