微小RNA-143通过调控Kras表达影响前列腺癌PC-3细胞的增殖与迁移  

Effect of microRNA-143 on proliferation and migration of prostate cancer PC-3 cells by regulating Kras expression

作　　者：刘民[1] 李昕[2] 宋苗苗[1] Liu Min;Li Xin;Song Miaomiao(Department of Urology,Cangzhou Central Hospital,Cangzhou 061001,China;Department of Urology,the First Hospital,Peking University,Beijing 100034,China)

机构地区：[1]河北省沧州市中心医院泌尿外二科,061001 [2]北京大学第一医院泌尿外科,100034

出　　处：《中华实验外科杂志》2020年第2期283-285,共3页Chinese Journal of Experimental Surgery

摘　　要：目的探究微小RNA(miRNA,miR)-143通过调控Kras的表达影响前列腺癌PC-3细胞的增殖与迁移的机制。方法选用购自美国典型培养物保藏中心细胞株PC-3,保持在补充有10%胎牛血清(Hyclone)的F-12培养基(Hyclone)中。细胞在5%CO2和37℃的湿润环境下生长。根据实验需求对细胞进行实验分组,随机分为PC-3转染组、PC-3对照组。通过细胞计数试剂盒(CCK-8)试剂盒对细胞活力进行测定。通过实时定量反转录聚合酶链反应(RT-qPCR)检测各组细胞中miR-143与Kras mRNA表达;通过蛋白质印迹反应检测各组细胞因子CD133、Oct4、Klf4的蛋白表达情况。组间比较采用单因素方差分析或者重复测量的方差分析,组间两两比较采用LSD-t检验。结果RT-qPCR分析各组miR-143 mRA和Kras mRNA表达为:miR-143 mRNA PC-3对照组(1.06±0.02)较PC-3转染组(3.17±0.23)降低;Kras mRNA PC-3对照组(2.75±0.11)较PC-3转染组(1.23±0.03)升高,差异有统计学意义(t=5.167,P<0.05)。蛋白印迹检测发现CD133、Oct4、Klf4的蛋白表达PC-3对照组(2.67±0.11、3.18±0.23、2.84±0.26)较PC-3转染组(1.15±0.03、1.26±0.14、1.18±0.15)升高,差异有统计学意义(t=4.862,P<0.05)。细胞活力检测发现PC-3对照组(1.62±0.14)较PC-3转染组(0.58±0.02)细胞活力值升高,差异有统计学意义(t=4.154,P<0.05)。结论miR-143可通过抑制Kras的基因表达来控制前列腺PC-3的增殖及迁移状况,miR-143的高表达可有效抑制前列腺癌细胞的增殖及侵袭。Objective To investigate the mechanism of miRNA(miRNA,miR)-143 influencing the proliferation and migration of prostate cancer PC-3 cells by regulating the expression of Kras.Methods The bone metastatic PCa cell line PC-3 was purchased from the American Type Culture Collection and maintained in F-12 medium supplemented with 10%fetal bovine serum.Stably transfected cells were maintained in culture medium in the presence of puromycin.The cells were grown under a humidified atmosphere of 5%CO2 and 37℃.The cells were subgrouped according to the experimental requirements and randomly divided into PC-3 transfection group(the sequence of pri-mir-143 was cloned into pmscv-purine plasmid by restriction enzyme BglⅡ,and the constructed plasmid was combined with PIK vector by lentivirus method),and PC-3 control group(pMSCV empty vector combination was used as a control group).Cell viability was determined by cell counting kit-8(CCK-8)assay.The expression levels of miR-143 and Kras mRNA in each group were detected by RT-qPCR.The expression levels of cytokines CD133,Oct4 and Klf4 were detected by Western blotting.Results The miR-143 mRNA expression in PC-3 control group(1.06±0.02)was lower than in the PC-3 transfection group(3.17±0.23).The Kras mRNA expression in the PC-3 control group(2.75±0.11)was significantly increased as compared with the PC-3 transfection group[(1.23±0.03),t=5.167,P<0.05].Western blotting analysis showed that the protein expression of CD133,Oct4,and Klf4 was significantly higher in the PC-3 control group(2.67±0.11,3.18±0.23,2.84±0.26)than in the PC-3 transfection group(1.15±0.03,1.26±0.14,1.18±0.15;t=4.862,P<0.05).Cell viability assay showed that the PC-3 control group(1.62±0.14)had significantly higher cell viability than the PC-3 transfection group(0.58±0.02,t=4.154,P<0.05).Conclusion MiR-143 can control the proliferation and migration of prostate PC-3 by inhibiting the expression of Kras gene.The high expression of miR-143 can effectively inhibit the reproduction and invasion of prostat

关 键 词：前列腺细胞 微小RNA-143 KRAS 细胞增殖 细胞转染 

分 类 号：R737[医药卫生—肿瘤]