人工合成小分子双链RNA通过激活P21蛋白对膀胱癌细胞生长的影响

Effect of synthetic small double-stranded RNA on the development of bladder cancer by activating P21 expression

目的通过转染人工合成的小分子双链RNA（dsRNA）dsP21-555至膀胱癌细胞系T24和EJ，观察其对膀胱癌细胞生长的作用。方法根据对膀胱癌细胞的不同处理分为3组：阴性对照组[转染随机序列（dsContr01）]，阳性对照组（转染相应的微小RNA即miR-370）和实验组（转染dsP21．555）。实时荧光定量聚合酶链反应（qPCR）检测3组细胞p21mRNA及细胞周期依赖性激酶（CDK）4、CDK-6mRNA的表达；Western印迹法检测P21蛋白和CDK4、CDK6蛋白的表达；流式细胞术测定转染后细胞周期分布；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，与转染dsControl相比，转染dsP21—555分别上调T24和EJ细胞中p21mRNA表达至2．46倍（P〈0．01）和2．60倍（P〈0．01）；转染dsP21—555与转染miR-370相比，p21mRNA表达差异无统计学意义（均P〉0．05）。与转染dsControl相比，转染dsP21-555分别使T24和EJ细胞中CDK4mRNA的表达下调43％（P〈0．01）和54％（均P〈0．01），CDK6mRNA的表达下调39％（P〈0．01）和36％（P〈0．01）；转染dsP21—555与转染miR-370相比，CDK4、CDK6mRNA的表达差异无统计学意义（P〉0．05）。Western蛋白印迹检测结果验证了组间p21和CDK4、6基因表达的差异。流式细胞术检测显示，与转染＆Control相比，转染miR-370或dsP21—555后，G0／G1期细胞比例明显升高，而S期和G2／M期细胞比例减少，细胞周期被阻滞在G0／G1期。细胞增殖实验结果显示，与转染dsControl相比，转染miR-370或dsP21-555后细胞增殖能力明显下降（均P〈0．05），且转染miR-370与转染dsP21-555间细胞增殖能力差异无统计学意义（P〉0．05）。细胞集落形成实验显示，转染miR-370和dsP21—555形成的集落数量均较转染dsControl少。结论dsP21—555能通过RNA激活作用激活P21蛋白的表达并显著抑制膀胱癌细胞的生长。

Objective To investigate the effects of a synthetic small double-stranded RNA （dsRNA） dsP21-555 on the development of bladder cancer cell lines T24 and EJ. Methods According to the different treatments, bladder cancer cells were divided into three groups： negative control group （transfeeted with dsControl）, positive control group （transfected with candidate mieroRNA, i.e. miR-370） and experimental group （transfeeted with dsP21-555 ）. Real-time fluorescent quantitative polymerase chain reaction （qPCR） was conducted to detect the expressions of p21 mRNA and eyclin-dependent kinases 4/6 （CDK4/6） mRNA; Western blot was operated to verify the expression of P21 and CDK4/6 proteins. Cell cycle distribution was measured by flow cytometry after transfection. Cell proliferation assay was performed to evaluate the proliferative capacity of transfeeted cells. Colony formation assay was carried out to analyze the proliferative ability of single cancer cells. Results qPCR showed that, compared with the negative control group, dsP21-555 up-regulated the expressions of p21 mRNA by 2. 46 times （ P 〈 0. 01 ） in T24 cells and2. 60 times （P 〈0. 01 ） in EJ cells; compared with the positive control group, the expression of p21 mRNA was no significantly different in the experimental group （ P 〉 0. 05 ）. Compared with the dsControl group, dsP21-555 suppressed the expressions of CDK4 mRNA by 43% （P 〈0. 01 ） in T24 and 54% （P 〈0. 01 ） in EJ cells, the expression of CDK6 mRNA by 39% （P 〈0. 01 ） in T24 and 36% （P 〈0. 01） in EJ cells; the differences in the expression of CDK4 and CDK6 mRNAs between the miR-370 and dsP21-555 groups were not statistically significant （P 〉 0.05 ）. Western blot verified the differences of p21 and CDK4/6 genes expression among groups. Flow cytometry revealed that the G0/G1 phase cells significantly increased while S and G2/M phase cells decreased in the miR-370 and the dsP21-555 groups, compared with the dsControl group. Cell proliferation assay showed that, compared with the dsControl group, the proliferative capacities of cells transfected with miR-370 or dsP21-555 decreased significantly （both P 〈 0. 05 ）, but the difference in proliferative capacities between the miR-370 and the dsP21-555 groups was no statistically significant （P 〉 0. 05）. Colony formation assay showed that the numbers of colonies formed in the miR-370 and the dsP21-555 groups were both smaller than that in the dsControl group. Conclusion dsP21-555 may activate the expression of P21 protein by RNA activation, thereby significantly inhibit the growth of bladder cancer cells.