miR-222对膀胱癌增殖行为的影响及其机制研究

Effective and mechanism study of miR-222 on the proliferation of bladder cancer cell

目的 探讨miR-222表达水平变化对膀胱癌细胞增殖行为的调控作用及可能机制,阐明miR-222在膀胱癌中可能作用的靶向蛋白及其作用位点.方法 利用qRT-PCR法检测正常膀胱上皮细胞和膀胱癌细胞中的miR-222的表达水平;利用CCK8法和EdU染色法检测各组细胞的增殖能力;利用细胞周期试剂盒检测各组细胞周期的分布;利用Western blot法和qRT-PCR检测各组细胞内抑癌基因p27kip1和p57kip2的表达;利用TargetScan Human 7.1软件预测miR-222与p27kip1和p57kip2 mRNA 3 非编码区（UTR）的结合位点;利用双荧光素酶法验证miR-222与p27kip1和p57kip2的靶向结合位点.结果 miR-222在膀胱癌细胞中的表达水平显著高于正常膀胱上皮细胞;miR-222表达水平的上调会明显促进细胞的增殖并且抑制p27kip1和p57kip2的表达,下调则会抑制细胞的增殖,引起膀胱癌细胞的细胞周期GO/G1期滞留,并促进了p27kip1和p57kip2的表达;miR-222通过靶向结合目标蛋白p27kip1和p57kip2 mRNA的3＇-UTR抑制其转录.结论 miR-222在膀胱癌中特异性高表达,通过与p27kip1和p57kip2 mRNA的3＇-UTR特异性结合抑制其表达水平,并促进膀胱癌细胞周期由G1期向S期转变,从而促进了癌细胞的增殖.

 Objective To investigate the effects of miR-222 on the proliferation of bladder cancer cells and the potential mechanisms and elucidate the putative targets of miR-222 in bladder cancer cell lines and the binding sites of miR-222 on the targets.Methods The miR-222 expression of bladder cancer cells and normal bladder cell line were tested by qRT-PCR.The proliferartion levels and cell cycles of transfected cells were tested by CCK8 assay,EdU staining and cell cycle staining with propidium iodide.QRT-PCR and Western blot assays were conducted to investigate the expression of tumor suppressing genes p27kip1 and p57kip2.The binding sites of miR-222 with the 3＇-UTRs of the p27kip1 and p57kip2 mRNAs were predicted by the algorithm of TargetScan Human 7.1.Luciferase assay was employed to validate the binding sites of miR-222 on 3＇-UTRs of p27kip1 and p57kip2.Results In our study,we demonstrated that 4 bladder cancer cell lines have high expression levels of miR-222 when compare with normal bladder cells.Over-expression of miR-222 promoted T24 cell proliferation and reduced the expression of p27kip1 and p57kip2,while inhibition of miR-222 decreased the proliferation level of cells,induced cell cycle arresting in G0/G1 phase,and promoted the expression of p27kip1 and p57kip2.miR-222 inhibit p27kip1 and p57kip2 by binding to their 3＇-UTR regions.Conclusions The expresison levels of miR-222 are significantly up-regulated in bladder cancer,promoting cell proliferation by binding to the 3＇-UTRs of p27kipl and p57kip2 mRNAs,downregulating the expression levels of mRNAs and proteins of p27kip1 and p57kip2 and inducing cell cycle transition from G1 phase to S phase.