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目的 miR-1236为新发现的具有肿瘤抑制作用的RNA。本研究旨在探讨外源性miR-1236转染对前列腺癌细胞p21基因的激活作用及对其生长的影响。方法根据对前列腺癌细胞的不同处理分为阴性对照组(转染dsControl)和实验组(转染miR-1236)2组。qRT-PCR检测p21、细胞周期依赖性激酶CDK4和CDK6mRNA的表达变化。蛋白质印迹检测p21、CDK4和CDK6蛋白的表达变化。流式细胞术检测细胞周期分布。MTS法检测细胞活力,集落形成实验检测细胞增殖能力。结果与dsControl组相比,转染miR-1236后2种细胞中p21mRNA表达分别上调2.16(t=12.67,P<0.01)和2.26倍(t=10.93,P<0.01);CDK4mRNA的表达分别下调0.56(t=6.617,P<0.01)和0.43倍(t=5.698,P<0.01);CDK6mRNA的表达分别下调0.78(t=3.101,P<0.05)和0.71倍(t=3.841,P<0.01)。蛋白质印迹检测结果与qRT-PCR结果相符。流式细胞术结果显示,转染miR-1236后,位于S期和G2/M期的细胞比例明显下降,位于G0/G1期的细胞比例明显增大。MTS法显示,与dsControl组相比,转染miR-1236 2种细胞活力明显下降。dsControl组DU145和PC-3细胞形成的集落数分别为241±37.64和254.33±38.18;miR-1236组DU145和PC-3细胞形成的集落数明显减少,分别为99.67±34.2(t=3.939,P<0.05)和81±35.55(t=4.354,P<0.05),提示细胞增殖能力降低。结论外源性miR-1236能通过激活前列腺癌细胞中p21蛋白的表达并显著抑制其生长,可能成为前列腺癌基因靶向治疗的新靶点。
Purpose miR-1236 is a newly discovered tumor suppressor RNA. The purpose of this study was to investigate the effect of exogenous miR-1236 transfection on the activation of p21 gene and its effect on prostate cancer cells. Methods According to different treatment of prostate cancer cells, the cells were divided into 2 groups: negative control group (transfected with dsControl) and experimental group (transfected with miR-1236). qRT-PCR was used to detect the expression of p21, cyclin-dependent kinase CDK4 and CDK6 mRNA. Western blot analysis of p21, CDK4 and CDK6 protein expression changes. Flow Cytometry to detect cell cycle distribution. MTS assay cell viability, colony formation assay to detect cell proliferation. Results Compared with the dsControl group, the expression of p21mRNA in the two cell lines was up-regulated by 2.16 (t = 12.67, P <0.01) and 2.26-fold (t = 6.617, P <0.01) and 0.43 times (t = 5.698, P <0.01). The expression of CDK6 mRNA was down-regulated by 0.78 (t = 3.101, P <0.05) and 0.71 times (t = 3.841, P <0.01). Western blot results were consistent with qRT-PCR results. The results of flow cytometry showed that the percentage of cells in S phase and G2 / M phase decreased significantly after transfected with miR-1236, and the proportion of cells in G0 / G1 phase increased significantly. MTS assay showed that compared with the dsControl group, the viability of 2 kinds of cells transfected with miR-1236 decreased significantly. The numbers of colonies formed by DU145 and PC-3 cells in dsControl group were 241 ± 37.64 and 254.33 ± 38.18, respectively. The numbers of colonies formed by DU145 and PC-3 cells in miR-1236 group were significantly decreased, which were 99.67 ± 34.2 (t = 3.939, P <0.05) and 81 ± 35.55 (t = 4.354, P <0.05), suggesting that the cell proliferation ability decreased. Conclusion Exogenous miR-1236 may be a new target of targeted therapy of prostate cancer by activating the expression of p21 protein in prostate cancer cells and significantly inhibiting its growth.