MicroRNA-3619-5p Suppresses Bladder Carcinoma Progression By Directly Targeting β-catenin And CDK2 And Activating P21^WAF1/CIP1

Part I Inhibitory Effect of miR-3619-5p on Proliferation and Metastasis of Bladder Cancer CellsOBJECTIVES: To investigate the effect of miR-3619-5p on the bladder cancer cells proliferation,senescence,apoptosis,colony formation,migration and invasion.METHODS: The expression of miR-3619-5p was detected by quantitative real-time PCR(q RT-PCR)in bladder cancer tissues and bladder cancer cells.ds Control,miR-3619-5p,anti-Control and anti-miR-3619-5p mimics were transfected into bladder cancer cell lines 5637 and T24 cells with a final concentration at 50 n M by liposome.Cells were harvested 72 hours after transfection and bladder cancer cells proliferation was evaluated by Cell Titer96? AQueous One Solution Cell Proliferation Assay(MTS)at 4 different time points following transfection(24 hours,48 hours,72 hours,and 96 hours).0.5% Crystal violet staining was used to detect the changes of colony forming ability of bladder tumor cells after transfection.Ed U assay was performed to evaluate the proliferation of bladder cancer cells.Cells senescence was assessed by β-galactosidase staining.Flow cytometry was conducted to evaluate the bladder cancer cells cycle distribution and apoptosis of 5637 and T24 cells.To construct a subcutaneous xenograft tumor model in nude mice to observe the changes of proliferation of bladder cancer cells in vitro.Scratch assay was performed to detect the migration of T24 and 5637 cells after transfection.was used to analyze the invasion and migration abilities of bladder cancer cells were tested by Transwell assay mothed.RESULTS: In bladder cancer tissues and bladder cancer cells(EJ,T24,5637 and J82),the expression of miR-3619-5p was significantly lower than those in para-cancer tissues and bladder epithelial cells SV-HUC-1 respectively.In bladder cancer cells T24 and 5637,MTS assay results showed that compared with the Mock and ds Control group,miR-3619-5p group inhibited bladder cancer cells proliferation(P<0.05).Colony formation assay showed that compared with Mock and ds Control groups,miR-3619-5p mimic transfected cells formed colonies significantly fewer in number and smaller in size(P<0.05).Ed U assay revealed that,compared with the ds Control group,miR-3619-5p significantly suppressed the proliferation of bladder tumor cells.Transfection of miR-3619-5p mimic into 5637 and T24 cells for 72 hours remarkably induced cell cycle G0/G1 arrest.Further statistical analysis showed that compared to ds Control groups,the proportion of cells in G0/G1 phase was increased and the proportion of cells in S phase and G2/M phase was decreased after transfection of miR-3619-5p mimics(P<0.05).In addition,transfection of miR-3619-5p mimic into 5637 and T24 cells led to more bladder cancer cells apoptosis in early and late phase than ds Control groups(P< 0.05)and also led to more cells senescent.Scratch assay,Migration chamber assay and Matrigel invasion chamber assay indicated that artificial expression of miR-3619-5p mimic potently inhibited T24 and 5637 cells migration and invasion at 72 hours post transfection(P < 0.05)compared with ds Control group.The anti-Control and anti-miR-3619-5p mimics were transfected into T24 and 5637 cells,respectively,we found that the elimination of miR-3619-5p could promote the cell proliferation and colony formation,inhibit cell apoptosis and promote cancer cell migration and invasion.CONCLUSIONS: miR-3619-5p was low expressed in bladder cancer tissues and cells,indicating that miR-3619-5p have an important tumor suppressor effect in bladder cancer.Overexpression of miR-3619-5p in T24 and 5637 cells can inhibit tumor cells colony formation and proliferation,promote apoptosis and senescence of tumor cells and induce tumor cell cycle G0/G1 arrest.In addition,elimination of endogenous miR-3619-5p in bladder cancer cells accelerates cell proliferation,migration and invasion,which reversely proves that miR-3619-5p plays a key role in inhibiting the proliferation,invasion and migration of bladder cancer cells.Part IIMechanism of miR-3619-5p in inhibiting bladder cancer cellsOBJECTIVES: It has been found that miR-3619-5p could bind to the p21 gene promoter and activate p21 gene expression in prostate cancer cells and inhibit the proliferation of prostate cancer cells.Whether does miR-3619-5p bind to p21 gene promoter and inhibit bladder cancer cells? Through software analysis,we found that miR-3619-5p can bind to 3’UTR of β-catenin and CDK2.So,what are the roles of β-catenin and CDK2 in miR-3619-5p inhibiting bladder cancer?METHODS: The expression of p21 m RNA was detected by quantitative real-time PCR(q RT-PCR)in 15 pairs of bladder cancer tissues and adjacent non-cancerous bladder tissues.The expressions of β-catenin,CDK2 and p21 protein in each group were detected by Immunohistochemistry.Western blot was used to detect the expression level of β-catenin,CDK2 and p21 protein in bladder cancer tissues and para-cancerous bladder mucosa respectively.Similarly,p21,β-catenin and CDK2 m RNA and protein levels in bladder cancer cells were detected by q RT-PCR and Western blot respectively.The biosynthesis negative control ds Control,miR-3619-5p mimics.ds Control and i R-3619-5p mimics were transfected into bladder cancer cell lines T24 and5637 by lipofection.The expression of p21 gene and its downstream genes’ m RNAs and proteins,β-catenin and CDK2 proteins and EMT related genes’ m RNAs and proteins were detected by q RT-PCR and Western blot 72 hours after transfection.Immunofluorescence staining was used to observe the subcellular expression of β-catenin protein in T24 and 5637 cells.In addition,chromatin immunoprecipitation(Ch IP)assay was used to detect whether biotin-labeled miR-3619-5p bind to the p21 promoter.Dual luciferase reporter assay detects whether miR-3619-5p binds to 3’UTR of β-catenin and CDK2.RESULTS: In bladder cancer tissues,m RNA and protein expression of p21 was significantly lower than those in para-cancer tissues,and the expression ofβ-catenin and CDK2 protein in bladder cancer tissues was significantly higher than that in para-cancerous tissues.The m RNA and protein expressions of p21 genes in bladder cancer cell lines(EJ,T24,5637 and J82)were significantly lower than those in bladder epithelial cells SV-HUC-1;β-catenin and CDK2 protein expression was significantly higher than in bladder epithelial cells.In bladder cancer cells 5637 and T24,the expression of p21 gene and its downstream genes’ m RNAs and proteins were significantly increased at 72 hours after transfection with miR-3619-5p mimics compared with that of ds Control transfection group and significantly inhibiting the expression ofβ-catenin and CDK2 protein expression.Transfection of miR-3619-5p significantly reduced the expression of β-catenin in the nuclear in 5637 and T24 cells.In addition,overexpression of miR-3619-5p induced up-regulation of epithelial markers and down-regulated expression of mesenchymal markers of EMT-related genes in tumor cells.Ch IP assay confirmed that miR-3619-5p activated p21 gene expression mainly through binding to specific sequence of p21 promoter in 5637 and T24 cells.Dual luciferase reporter assay showed that miR-3619-5p binds directly to β-catenin and CDK2 3’UTR in 5637 and T24.CONCLUSIONS: p21 m RNA was low expressed in human bladder cancer tissues and cancer cells;miR-3619-5p can activate its expression by targeted binding with specific sequence of promoter of p21 gene in 5637 and T24 cells.Moreover,while activating p21 gene,it also induced the change of its downstream gene expression and co-act with tumor cell proliferation and metastasis.miR-3619-5p can target-bind with specific sequences of β-catenin and CDK2 3’UTR to inhibit its expression in tumor cells.More importantly,miR-3619-5p inhibits the activation of Wnt/β-catenin signaling pathway,and inhibit the epithelial-mesenchymal transition(EMT)of tumor cells,which has a significant inhibitory effect on the occurrence and development of bladder cancer cells.