【摘要】 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(dsControl);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p21mRNA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p21mRNA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、96h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、96h,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。更多还原

【Abstract】 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 experimental 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 conducted 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 observed 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 significantly(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 formation 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 demonstrated that,when detected at 48,72,96 hafter 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 proliferation 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. Conclusions MiR-370-5p could up-regulate the expression of p21 by RNA activation pathway and inhibit the proliferation of prostate cancer cells.更多还原