微小RNA191-5p通过靶向CDK6抑制胃癌细胞的生长  

miRNA-191-5p represses cell growth by targeting CDK6 in gastric cancer

作　　者：曹楠婧 侯会会 李峰[2] 郭素堂[2] 汪毅 Cao Nanjing;Hou Huihui;Li Feng;Guo Sutang;Wang Yi(Department of Biochemistry and Molecular Biology,Basic Medical College,Shanxi Medical University,Taiyuan 030001,China;Shanxi Cancer Hospital,Taiyuan 030013,China;Department of VIP Medical Services,National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100021,China)

机构地区：[1]山西医科大学生物化学与分子生物学教研室,太原030001 [2]山西省肿瘤医院,太原030013 [3]国家癌症中心,国家肿瘤临床医学研究中心,中国医学科学院北京协和医学院肿瘤医院特需医疗部,北京100021

出　　处：《中华医学杂志》2020年第46期3689-3693,共5页National Medical Journal of China

基　　金：山西省重点研发计划项目(201803D31166);山西省科技厅基础面上项目(201801D121303)。

摘　　要：目的:探讨微小RNA191-5p(miR-191-5p)对胃癌细胞迁移、克隆形成和增殖的影响。方法:采用实时定量-聚合酶链反应(Real-time PCR)检测分析了来自山西省肿瘤医院的60例胃癌患者的胃癌组织(C组)及其癌旁正常组织(N组)中miR-191-5p的表达水平;应用pcDNA3.1载体构建过表达miR-191-5p的重组质粒(pcDNA-mRNA-191-5p),实现miR-191-5p在胃癌细胞中的过表达,用miRNA-191-5p inhibitor实现miR-191-5p在胃癌细胞中的低表达;分别用划痕愈合实验、克隆形成实验和CCK-8法检测细胞迁移、克隆形成和增殖能力;Targetscan预测miR-191-5p与周期素依赖性激酶6(CDK6)的结合位点,并通过双荧光报告基因验证;Western印迹检测miR-191-5p对p21和CDK6蛋白表达的影响。结果:与N组相比,C组中有53例(88%)胃癌组织中出现miR-191-5p的表达下调;C组miR-191-5p的表达水平为0.43±0.13,显著低于N组的0.88±0.12, P<0.001,过表达miR-191-5p能显著抑制胃癌细胞的迁移、克隆形成和增殖能力,差异有统计学意义( P<0.05);双荧光报告基因证实miR-191-5p与CDK6的3′UTR结合;Western印迹显示胃癌细胞中pcDNA-miR-191-5p下调了CDK6表达而上调了p21表达。 结论:miR-191-5p表达下调可能参与胃癌的发生发展,过表达miR-191-5p能下调CDK6并抑制胃癌细胞生长。Objective To investigate the effects of miR-191-5p on cell migration,clone formation and proliferation of gastric cancer(GC)cells.Methods The level of miR-191-5p expression was detected by real-time reverse transcriptase-polymerase chain reaction(RT-PCR)in 60 paired GC tissues and their adjacent normal tissues.miR-191-5p overexpression was achieved by transfection of construct pcDNA-miR-191-5p into GC cells.The migration,clone formation and proliferation of GC cells were detected by the scratch wound assay,clone formation assay and cell counting kit-8(CCK-8),respectively.Low expression of miR-191-5p was achieved with miRNA-191-5p inhibitor.The binding sites of cyclin-dependent kinase 6(CDK6)and miR-191-5p were analyzed using TargetScan software,and the interaction of CDK6 and miR-191-5p was verified using dual-fluorescence reporter gene expression.Western blot(WB)was used to detect the effect of miR-191-5p on the expression of p21 and CDK6 proteins.Results miR-191-5p decreased in 53 cases(88%)of GC tissues compared to their controls.Furthermore,overexpression of miR-191-5p effectively inhibited the migration,clone formation and proliferation of GC cells(P<0.05).Dual-fluorescence reporter confirmed that miR-191-5p bound to 3′UTR of CDK6.WB showed that pcDNA-miR-191-5p inhibited the CDK6 expression but promoted the p21.Conclusion Down-regulation of miR-191-5p has a correlation with the progression of GC.Overexpression of miR-191-5p can decrease the expression of CDK6 and inhibit the growth of GC cells.

关 键 词：胃肿瘤 微小RNA191-5p 周期素依赖性激酶6 迁移 增殖 

分 类 号：R735.2[医药卫生—肿瘤]