微小RNA-206通过干扰CDK4和GAK的表达对前列腺癌细胞生长的影响

doi:10.3760/cma.j.issn.1673-422X.2017.07.002

摘要/Abstract

摘要： 目的探讨微小RNA-206(miR-206)对前列腺癌细胞中细胞周期蛋白依赖性激酶4（CDK4）和细胞周期素G相关蛋白激酶（GAK）表达的干扰作用及对前列腺癌细胞生长的影响。方法前列腺癌细胞株DU145和PC3为转染对象，转染miRNC为对照组，转染miR206为实验组。荧光实时定量PCR（qRTPCR）检测CDK4和GAK mRNA的表达水平。Western blotting检测CDK4和GAK蛋白的表达水平。流式细胞术检测细胞周期分布。EdU增殖实验和集落形成实验检测细胞增殖能力。结果在DU-145和PC-3细胞株中，miR-NC组CDK4 mRNA表达量分别为1.00±0.09、1.00±0.10，GAK mRNA表达量为1.00±0.05、1.00±0.06；与之相比，两细胞株miR-206组中CDK4 mRNA表达明显下降，分别为0.36±0.18（t=6.572，P=0.001）、0.43±0.17（t=5.794，P=0.001），GAK mRNA表达量亦明显下降，分别为0.23±0.04（t=22.420，P<0.001）、0.32±0.08(t=14.500，P<0.001)。Western blotting实验结果与qRTPCR结果一致。流式细胞术检测结果显示，分别与两细胞株miR-NC组比较，转染miR-206后前列腺癌细胞在S期（23.60%±5.68%∶32.53%±4.52%，t=2.462，P=0.049；22.09%±4.35%∶30.96%±4.86%，t=2.720，P=0.035）和G2M期（16.28%±7.12%∶26.63%±4.33%，t=2.484，P=0.048；14.60%±1.62%∶24.68%±7.13%，t=2.758，P=0.033）的细胞比例下降，在G0G1期（60.13%±5.82%∶40.84%±5.37%，t=4.872，P=0.003；63.31%±3.27%∶44.36%±3.82%，t=7.533，P<0.001）的细胞比例升高。EdU增殖实验结果显示，转染miR206的细胞增殖能力明显减弱（22.56±3.81∶38.90±8.51，t=3.503，P=0.013；25.12±6.42∶48.45±8.92，t=4.244，P=0.005）。集落形成实验结果显示，两细胞株miR-NC组形成的集落数分别为218.66±44.59、177.35±24.49，miR-206组形成的集落数分别为125.38±32.80(t=3.370，P=0.015)、82.65±14.05(t=6.708，P=0.001)，提示miR-206组细胞增殖能力降低。结论 miR-206可通过干扰CDK4和GAK的表达显著抑制前列腺癌细胞的生长，提示miR-206可能成为前列腺癌的分子靶向治疗工具。

关键词: 微RNAs, 前列腺肿瘤, 细胞增殖, 细胞周期蛋白依赖性激酶4, 细胞周期素G相关蛋白激酶

Abstract: ObjectiveTo investigate the effect of microRN206 (miR206) on the expression of Cyclindependent kinase 4 (CDK4) and Cyclin Gassociated protein kinase (GAK), and the growth of prostate cancer cells. MethodsProstate cancer cell lines DU145 and PC3 were transfected with miRNC (the control group) or miR206 (the experimental group). The expressions of CDK4 and GAK mRNA were detected by realtime quantitative PCR (qRTPCR). The expressions of CDK4 and GAK protein were detected by Western blotting. Cell cycle distribution was detected by flow cytometry. EdU proliferation assay and colony forming assay were used to analyze the cell proliferation ability. ResultsIn DU145 and PC3 cells, the expressions of CDK4 mRNA in miRNC group were 1.00±0.09, 1.00±0.10, the expressions of GAK mRNA were 1.00±0.05, 1.00±0.06. The expressions of CDK4 mRNA in miR206 group were significantly decreased in DU145 (0.36±0.18; t=6.572, P=0.001) and PC3 cell lines (0.43±0.17; t=5.794, P=0.001). The expressions of GAK mRNA were also significantly decreased in DU145 (0.23±0.04; t=22.420, P<0.001) and PC3 cell lines (0.32±0.08; t=14.500, P<0.001). Western blotting results were consistent with qRTPCR results. The results of flow cytometry showed that compared with the miRNC group of DU145 and PC3 cell lines, the percentage of cells in S phase (23.60%±5.68% vs. 32.53%±4.52%, t=2.462, P=0.049; 22.09%±4.35% vs. 30.96%±4.86%, t=2.720, P=0.035) and G2M phase (16.28%±7.12% vs. 26.63%±4.33%, t=2.484, P=0.048; 14.60%±1.62% vs. 24.68%±7.13%, t=2.758, P=0.033) decreased after transfection of miR206, and the percentage of cells in G0G1 phase (60.13%±5.82% vs. 40.84%±5.37%, t=4.872, P=0.003; 63.31%±3.27% vs. 44.36%±3.82%, t=7.533, P<0.001) increased. The results of EdU proliferation assay showed that the proliferation abilities were significantly attenuated after transfection of miR206 (22.56±3.81 vs. 38.90±8.51, t=3.503, P=0.013; 25.12±6.42 vs. 48.45±8.92, t=4.244, P=0.005). The results of colony formation experiments showed that the numbers of colonies formed by DU145 and PC3 in miRNC group were 218.66±44.59 and 177.35±24.49, respectively. The numbers of colonies formed in miR206 group were 125.38±32.80 (t=3.370, P=0.015) and 82.65±14.05 (t=6.708, P=0.001), suggesting that cell proliferation ability in miR206 group was reduced. ConclusionmiR206 significantly inhibits the growth of prostate cancer cells by interfering with the expressions of CDK4 and GAK, suggesting that miR206 may be a molecular targeted therapy tool for prostate cancer.

Key words: MicroRNAs, Prostatic neoplasms, Cell proliferation, Cyclin dependent kinase 4, Cyclin G associated protein kinase

黄耿，姜卫东，毛青，桂定文. 微小RNA-206通过干扰CDK4和GAK的表达对前列腺癌细胞生长的影响[J]. 国际肿瘤学杂志, 2017, 44(7): 485-489.

Huang Geng, Jiang Weidong, Mao Qing, Gui Dingwen. Effect of microRNA206 on the growth of prostate cancer cells by interfering with the expression of CDK4 and GAK[J]. Journal of International Oncology, 2017, 44(7): 485-489.

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