miR-203a通过靶向抑制CDK6调控膀胱癌细胞增殖及放射敏感性  

miR-203a regulates bladder cancer cell proliferation and radiosensitivity by targeting CDK6

作　　者：王磊[1] 乔庆东[1] 黄海航[2] 李浩然 何云峰[4] Wang Lei;Qiao Qingdong;Huang Haihang;Li Haoran;He Yunfeng(Department of Urology I,Xinxiang Central Hospital,Xinxiang 453000,China;Department of Radiotherapy,Xinxiang Central Hospital,Xinxiang 453000,China;School of Basic Medicine,Xinxiang Medical College,Xinxiang 453000,China;Department of Urology,First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]新乡市中心医院泌尿外一科,453000 [2]新乡市中心医院放疗科,453000 [3]新乡医学院基础医学院,453000 [4]重庆医科大学附属第一医院泌尿外科,400016

出　　处：《中华放射肿瘤学杂志》2021年第2期191-197,共7页Chinese Journal of Radiation Oncology

基　　金：河南省科技发展计划项目(172102310371);河南省医学科技攻关计划联合共建项目(2018020928)。

摘　　要：目的探讨miR-203a在膀胱癌(BC)细胞系(RT-112、T24、5637、UM-UC-3细胞)中的表达及对细胞增殖及放射敏感性的影响。方法将miR-203a mimics、miR-203a inhibitor、CDK6siRNA、CDK6表达质粒及相应阴性对照(NC)转染入BC细胞中。实时荧光定量PCR检测miR-203a在RT-112、T24、5637、UM-UC-3细胞和人膀胱上皮永生化细胞系(SV-HUC-1)中的表达。CCK8实验研究miR-203a和CDK6对BC细胞系增殖的调控。克隆形成实验研究miR-203a和CDK6对BC细胞系放射敏感性的影响。荧光素酶报告基因实验验证miR-203a的靶基因。蛋白印迹法检测miR-203a对CDK6蛋白表达的影响。采用单因素方差分析进行多组间比较、t检验进行两组间比较。结果与SV-HUC-1细胞相比,miR-203a在RT-112、T24、5637、UM-UC-3细胞中的表达明显降低(P<0.05)。与NC相比,过表达miR-203a抑制BC细胞系增殖(P<0.05),敲低miR-203a促进BC细胞系增殖(P<0.05)。与NC相比,过表达miR-203a增加BC细胞系的放射敏感性(P<0.05),敲低miR-203a减弱BC细胞系的放射敏感性(P<0.05)。CDK6是miR-203a的作用靶点。与NC组相比,过表达miR-203a显著降低了CDK6蛋白水平(P<0.05),敲低miR-203a显著上调了CDK6蛋白水平(P<0.05)。在转染miR-203a mimics的T24和UM-UC-3细胞中过表达CDK6后,与miR-203a mimics组相比,细胞增殖能力升高、放射敏感性降低(P<0.05);在转染miR-203a inhibitor的RT-112和5637细胞中沉默CDK6后,与miR-203a inhibitor组相比,细胞增殖能力降低、放射敏感性升高(P<0.05)。结论miR-203a在BC细胞系中低表达,可作为抑癌基因抑制BC细胞系增殖和增强放射敏感性。Objective To investigate the expression of miR-203a in bladder cancer(BC)cell lines(RT-112,T24,5637,UM-UC-3)and evaluate the effects on BC cell proliferation and radiosensitivity.Methods Mir-203a mimics,mir-203a inhibitor,CDK6siRNA,CDK6 expression plasmid and corresponding negative controls were transfected into BC cells.Quantitative real-time PCR was used to detect the expression of miR-203a in BC cell lines and human bladder epithelial immortalized cell line(SV-HUC-1).CCK8 assay was used to investigate the regulation of miR-203a and cyclin-dependent kinases 6(CDK6)on the proliferation of BC cells.Colony formation assay was performed to assess the effect of miR-203a and CDK6 on the radiosensitivity of BC cells.The target gene of miR-203a was confirmed by luciferase reporter assay.The effect of miR-203a on CDK6 protein expression was detected by Western blot.Multi-group comparison was performed by one-way ANOVA and two-group comparison was conducted by t-test.Results Compared with the SV-HUC-1 cells,the expression levels of miR-203a in RT-112,T24,5637 and UM-UC-3 cells were significantly down-regulated(all P<0.05).Compared with NC group,overexpression of miR-203a significantly inhibited the proliferation of BC cells,whereas knockdown of miR-203a significantly promoted the proliferation of BC cells(both P<0.05).Compared with NC group,overexpression of miR-203a significantly increased the sensitivity of BC cells to radiotherapy,whereas knockdown of miR-203a significantly weakened the sensitivity of BC cells to radiotherapy(both P<0.05).CDK6 was the target of miR-203a.Compared with NC group,overexpression of miR-203a significantly down-regulated the expression level of CDK6 protein,whereas knockdown of miR-203a significantly up-regulated the expression level of CDK6 protein(both P<0.05).After overexpression of CDK6 in T24 and UM-UC-3 cells transfected with miR-203a mimics,the cell proliferation ability was significantly increased,whereas the sensitivity to radiotherapy was significantly decreased compared with mir-2

关 键 词：miR-203a 细胞周期蛋白依赖性激酶 放射敏感性 膀胱癌细胞系 

分 类 号：R737.14[医药卫生—肿瘤]