微小核糖核酸miR-370-5p对前列腺癌细胞增殖影响的研究

目的 探讨微小核糖核酸miR-370-5p对前列腺癌细胞系PC3和DU145细胞增殖的影响.方法 将前列腺癌细胞系PC3和DU145分为2组:阴性对照组-转染随机序列(dsCon-trol);实验组-转染miR-370-5p或miR-370-5p+siP21.实时荧光定量聚合酶链反应(qPCR)检测各组细胞中p21 mRNA的表达及前列腺癌细胞系中miR-370-5p的基础表达情况;蛋白质印迹法检测p21蛋白的表达;集落形成实验检测各组单个细胞克隆增殖情况;细胞增殖实验检测转染后各组细胞的增殖能力.结果 与正常前列腺上皮细胞(RWPE-1)比较,前列腺癌细胞系PC3和DU145中miR-370-5p表达下降;与阴性对照组相比,实验组PC3和DU145细胞中p21 mRNA的相对表达量分别提高了(2.457±0.392)倍和(1.844±0.295)倍.实验组PC3和DU145细胞中p21蛋白的相对表达分别为(0.52±0.09)和(0.63±0.14),阴性对照组为(0.18±0.06)和(0.24±0.05),两组比较,差异有统计学意义(P<0.05).阴性对照组和实验组中PC3和DU145细胞的集落形成数目分别为(0.281±0.024)、(0.084±0.016)、(0.293±0.017)和(0.310±0.041)、(0.088±0.019)、(0.300±0.032),实验组在转染miR-370-5p后,细胞集落形成数目均较阴性对照组少;而在miR-370-5p+siP21共转染后,与转染miR-370-5p组相比较,PC3和DU145细胞集落形成数目均显著恢复,差异均有统计学意义(P<0.05).细胞增殖实验结果显示,实验组转染miR-370-5p后,PC3和DU145细胞在48、72、96 h的存活情况(用吸光度OD值表示)分别为(0.395±0.040)、(0.691±0.042)、(0.874±0.045)和(0.437±0.044)、(0.700±0.051)、(0.875±0.052),与阴性对照组相比,实验组细胞增殖能力明显下降(P<0.05);miR-370-5p+siP21共转染后48、72、96 h,PC3和DU145细胞的OD值分别为(0.675±0.041)、(1.072±0.124)、(1.323±0.136)和(0.633±0.106)、(1.072±0.167)、(1.337±0.102),与转染miR-370-5p比较,差异均有统计学意义(P<0.05).结论 miR-370-5p能够通过上调p21蛋白的表达抑制前列腺癌细胞的增殖.

Effect of miR-370-5p on proliferation of human prostate cancer cell lines

Objective To study the effects of a synthetic miR-370-5p mimics onprostate cancer cell lines of PC3 and DU145 in vitro. Methods PC3 and DU145 cells were cultured in vitro and treated with two different processing:negative control group (infection with dsControl)and the ex-perimental group (infection with miR-370-5p or miR-370-5p+siP21)from November 2016 to April 2017.Real-time fluorescent quantitative PCR (qPCR)was performed to detect the expression of p21 mRNA and the expression of miR-370-5p in prostate cancer cell lines.Western Blot method was con-ducted to evaluate the expression of p21 protein.Colony formation assay was used to test the ability of single cancer cell clone proliferation.Cell proliferation assay (CCK-8)was implemented to be ob-served the inhibitive effect of cell proliferative potential. Results Result of qPCR showed that, compared with normal prostate epithelial cell (RWPE-1),miR-370-5p decreased in PC3 and DU145;the relative expression of p21 messenger in PC3 and DU145 cells in the experimental group was sig-nificantly (2.457±0.392)times and (1.844±0.295)times higher than that in the negative control group respectively.Western Blot analysis testified that the expressions of p21 in PC3 and DU145 cells were respectively (0.52±0.09)and (0.63±0.14),the difference was statistically significant between two groups (P <0.05).Cell colony formation assay certified that the colony for-mation rates were (0.084±0.016)and (0.088 ±0.019)in the negative control group,less than in that of miR-370-5p group (0.281±0.024)and (0.310 ±0.041 )(P <0.05 );however,the colony formation rates were (0.293 ±0.017)and (0.300 ± 0.032)in the groups of miR-370-5p+siP21,restored in that of miR-370-5p group (P <0.05).Cell proliferation assay demon-strated that,when detected at 48,72,96 h after transfected with miR-370-5p,the cell survival rates were respectively (0.395 ±0.040),(0.691±0.042),(0.874±0.045)and (0.437±0.044),(0.700±0.051),(0.875±0.052),indicating that cell prolif-eration ability decreased obviously when transfected with miR-370-5p (P <0.05),compared with dsControl group;meanwhile, when transfected with siP21 and miR-370-5p,the cell survival rates were respectively (0.675 ± 0.041 ),(1.072 ± 0.124), (1.323±0.136)and (0.633±0.106),(1.072 ±0.167),(1.337 ±0.102),indicating that cell proliferation ability was restored when co-transfected with siP21 and miR-370-5p (P <0.05),compared with only transfected with miR-370-5p group. Conclu-sions MiR-370-5p could up-regulate the expression of p21 by RNA activation pathway and inhibit the proliferation of prostate cancer cells.