小分子双链 RNAs 联合应用抑制膀胱癌细胞增殖的实验研究

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、CDK6 mRNA 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p21 mRNA 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,CDK4/6 mRNA 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 Blad-der cancer cells were divided into:①negative control group (dsControl),dsP21-322 group,dsP21-555 group and dsP21-322 + dsP21-555 group.② dsControl 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 CDK4/6 proteins. Cell proliferation assay was performed to evaluate the proliferation capacity of transfected cells.Colo-ny 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,dsP21-322 group,dsP21-555 group and dsP53-285 group up-regulated the expressions of P21 mRNA (P <0.01)in T24 and EJ cells.Compared with trans-fection of dsP21-322,or dsP21-555 alone,the expression of p21 mRNA showed no significantly difference in combined trans-fection of dsP21-322 and dsP21-555 group (P >0.05).Compared with dsControl group,dsP53-285 activated p53 mRNA ex-pression.And the expression of p53 mRNA in dsP21-322 + dsP53-285 group and dsP53-285 group showed no difference.Com-pared with dsControl group,dsP21-322 and dsP53-285 dramatically reduced CDK4/6 mRNAs expression (P <0.05).Besides, compared with transfection of dsP21-322,dsP53-285 alone,the expression of p21,CDK4/6 mRNAs were suppressed in com-bined transfection of dsP21-322 and dsP53-285 group (P <0.05 ).Western blot assay verified the different expression of p21 and CDK4/6 genes 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 dsP53-285 together decreased significantly (P <0.05).Colo-ny formation assay showed that the numbers of colonies formed in the dsP21-322 +dsP53-285 group were fewer than that in the dsP21-322 group and the dsP53-285 group. Conclusions The proliferation of bladder cancer cells could significantly sup-pressed when transfected with dsP21-322 and dsP53-285 together.