miR-370-5p对前列腺癌细胞周期及增殖的影响

Effects of miR-370-5p on Cell Cycle and Proliferation of Prostate Cancer Cells

目的研究miR-370-5p导入对前列腺癌细胞株DU-145和LNCaP细胞周期和增殖的影响。方法合成miR-370-5p(实验组)和ds Control(阴性对照组),分别转染至两个细胞株。利用Real-time PCR和Western blot法分别检测细胞转染后p21、CDK4、Cyclin D1 mRNA和蛋白的表达变化。流式细胞术分析细胞周期变化,利用MTT法和集落形成实验分析细胞活力和增殖能力。结果 Real-time PCR结果提示,转染miR-370-5p后DU-145和LNCa P细胞中p21 mRNA水平分别上调3.43倍(P<0.01)和3.06倍(P<0.01),CDK4 mRNA水平分别下调0.51倍(P<0.01)和0.43倍(P<0.01),Cyclin D1 mRNA水平分别下调0.31倍(P<0.01)和0.35倍(P<0.01)。Western blot法检测结果符合这一趋势。流式细胞术检测结果显示,转染miR-370-5p后,位于S期和G_2/M期的细胞比例下降,位于G_0/G_1期的细胞比例则上升,说明细胞周期被阻滞在G_0/G_1期。MTT分析结果显示,与ds Control组相比,转染mi R-370-5p后,DU-145和LNCaP细胞活力明显降低。集落形成实验显示,miR-370-5p组的集落数数量明显较少,细胞增殖能力降低。结论 mi R-370-5p能显著激活前列腺癌细胞中p21蛋白的表达,抑制前列腺癌细胞周期的进展和增殖。

Objective To study the effect of miR - 370 - 5p transfection on the cell cycle and proliferation of prostate cancer cell lines DU -145 and LNCaP. Methods miR -370 -5p （experimental group） and dsControl （negative control group） were transfeeted into two cell lines. Real - time PCR and Western blot were used to detect the expression of p21 mRNA and p21 protein after transfection. Flow cytometry, MTT and colony forming assay were conducted to analyze cell cycle, cell viability and proliferation ability. Results Real - time PCR results showed that p21 mRNA levels of DU - 145 and LNCaP cells were up - regulated by 3.43 - fold （P 〈 0.01 ） and 3.06 -fold （P〈0.01）, CDK4 mRNA levels were down-regulated by0.51 -fold （P〈0.01） and0.43 -fold （P〈0.01）, Cyelin D1 mRNA levels were down-regulated by 0.31 -fold （P〈0.01） and 0.35 -fold （P〈0.01）, respectively, after miR-370-5p transfeetion. Western blot results were consistent with this trend. The results of flow cytometry showed that the percentage of cells in S phase and G2/M phase decreased after transfection of miR -370 -5p, the proportion of cells in Go/Gl phase increased, indicating that the cell cycle was arrested in G0/G1 phase. MTT analysis showed that the viability of DU - 145 and LNCaP cells was significantly de- creased after miR- 370 -5p transfeetion compared with dsControl. The colony formation assay showed that the number of colonies in the miR -370 -5p group was significantly less and the cell proliferation ability decreased. Conclusion miR -370 -5p can significantly ac- tivate the expression of p21 protein in prostate cancer cells, and inhibit the cell cycle progression and proliferation ability of prostate cancer cells.