小分子双链RNAs联合应用抑制膀胱癌细胞增殖的实验研究  

Combination of small double-stranded RNAs suppress the proliferation of bladder cancer cells

作　　者：张庆松[1] 李传昌[1] 陈忠[1] 李凡[1] 袁慧星[1] 杨为民[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院泌尿外科,武汉430030

出　　处：《现代泌尿生殖肿瘤杂志》2016年第4期225-230,共6页Journal of Contemporary Urologic and Reproductive Oncology

基　　金：国家自然科学基金(81372759)

摘　　要：目的在膀胱癌细胞系T24和EJ细胞中转染两种不同的人工合成小分子双链RNA(dsRNA),观察联合应用与分别单独应用两种dsRNA对膀胱癌细胞生长的影响。方法根据对膀胱癌细胞的处理不同分为:1阴性对照组(dsControl)、dsP21-322组、dsP21-555组和dsP21-322+dsP21-555组;2dsControl组、dsP21-322组、dsP53-285组和dsP21-322+dsP53-285组。采用实时荧光定量聚合酶链反应(qPCR)检测各组细胞p21 mRNA及细胞周期依赖性激酶CDK4、CDK6mRNA的表达;蛋白印迹法(Western blot)检测P21蛋白和CDK4、CDK6蛋白的表达;细胞增殖实验检测转染后细胞增殖能力;集落形成实验检测单个细胞克隆增殖能力。结果qPCR结果显示,与dsControl组相比,dsP21-322组、dsP2l-555组和dsP53-285组均分别上调T24和EJ细胞中p21mRNA的表达(P<0.01);而与单独转染dsP21-322、dsP2l-555相比,同时转染dsP21-322+dsP21-555,差异无统计学意义(P>0.05)。与dsControl组相比,dsP53-285能够上调p53基因的表达(P<0.05);与单独转染dsP53-285相比,p53 mRNA表达在同时转染dsP21-322和dsP53-285组中差异无统计学意义(P>0.05)。与单独转染dsP21-322、dsP53-285相比,dsP21-322+dsP53-285能够显著增加p21 mRNA的表达(P<0.05)。与dsControl相比,转染dsP21-322、dsP53-285分别使T24和EJ细胞中CDK4、CDK6 mRNA的表达下调(P<0.05);与单独转染dsP21-322、dsP53-285相比,CDK4/6mRNA的表达在dsP21-322+dsP53-285组中明显被抑制(P<0.05)。Western blot检测结果验证了组间p21和CDK4/6基因表达的差异。细胞增殖实验结果显示,同时转染dsP21-322和dsP53-285比单独转染抑制膀胱癌细胞增殖能力更明显。细胞集落形成实验中,dsP21-322+dsP53-285组中形成的集落数量显著少于单独转染组。结论同时转染dsP21-322和dsP53-285能够显著抑制膀胱癌细胞的增殖。Objective To investigate the different effects between combined application of two different synthetic small double-stranded RNAs（dsRNAs）dsP21-322＋dsP21-555,dsP21-322＋dsP53-285 and one dsRNA on proliferation of bladder cancer cell lines T24 and EJ. Methods Bladder cancer cells were divided into：①negative control group（dsControl）,dsP21-322 group,dsP21-555 group and dsP21-322＋dsP21-555 group.②dsControl group,dsP21-322 group,dsP53-285 group and dsP21-322＋dsP53-285 group.Real-time fluorescent quantitative polymerase chain reaction（qPCR）was conducted to detect the expressions of p21 mRNA and cyclin-dependent kinases 4/6（CDK4/6）mRNA.Western blot was operated to verify the expression of p21 and CDK4/6proteins.Cell proliferation assay was performed to evaluate the proliferation capacity of transfected cells.Colony formation assay was carried out to analyze the proliferative ability of single cancer cell. Results qPCR results showed that,compared with the negative dsControl group,dsP21-322group,dsP21-555 group and dsP53-285 group up-regulated the expressions of P21mRNA（P〈0.01）in T24 and EJ cells.Compared with transfection of dsP21-322,or dsP21-555 alone,the expression of p21 mRNA showed no significantly difference in combined transfection of dsP21-322 and dsP21-555group（P〉0.05）.Compared with dsControl group,dsP53-285 activated p53mRNA expression.And the expression of p53 mRNA in dsP21-322 ＋ dsP53-285 group and dsP53-285 group showed no difference.Compared with dsControl group,dsP21-322 and dsP53-285 dramatically reduced CDK4/6mRNAs expression（P〈0.05）.Besides,compared with transfection of dsP21-322,dsP53-285 alone,the expression of p21,CDK4/6mRNAs were suppressed in combined transfection of dsP21-322 and dsP53-285group（P〈0.05）.Western blot assay verified the different expression of p21 and CDK4/6genes among groups.Cell proliferation assay showed that,compared with dsP21-322 group and dsP53-285 group,the proliferative capacities of cells transfected with dsP21-322 and

关 键 词：膀胱肿瘤 dsRNAs P21 RNA激活 

分 类 号：R737.14[医药卫生—肿瘤]