miRNA-17-92基因簇对大肠癌HT29细胞周期调控机制研究

Regulatory Mechanism of miRNA-17-92 Cluster for Colon Cancer HT29 Cell Cycle

【目的】探讨miRNA(miR)-17-92基因簇对大肠癌HT29细胞周期的调控机制,为大肠癌临床诊断和治疗提供潜在靶点。【方法】体外培养人结肠癌HT29细胞,转染miR-17-5p和miR-20a后,采用实时荧光定量PCR验证转染效果,采用四甲基偶氮唑盐(MTT)法检测转染后细胞增殖情况,流式细胞术检测细胞周期分布情况,Western印记法检测E2F1蛋白表达水平。【结果】miR-17-5p和miR-20a分别以50、100 nmol/L浓度转染24 h后的HT29细胞用于后续实验;两者均可促进HT29细胞的增殖,具有时间依赖性;能够将细胞阻滞在S期,抑制靶基因E2F1蛋白的表达水平。【结论】miR-17-92基因簇通过抑制E2F1的翻译促进大肠癌细胞的增殖,提示miR-17-92基因簇在大肠癌细胞周期调控中可能发挥癌基因的作用。

Objective To study the regulatory mechanism of miRNA-17-92 cluster for cell cycle of colon cancer cell line HT29, thus to reveal the potential diagnostic and therapeutic target. Methods After miR-17-5p and miR-20a were transfected into in-vitro cultured HT29 cells, transfection efficiency was tested by real-time fluorescence quantitative polymerase chain reaction （PCR） , the proliferation of HT29 cells was measured by methyl thiazolyl tetrazolium （MTT） assay, the distribution of cell cycle was detected by flow cytometry, and E2F1 protein expression was examined by western blotting method. Results HT29 cells transfected with 50 nmol/L miR-17-5p and 100 nmol/L miR-20a for 24h were used for the subsequent research. Both clusters could increase the proliferation of HT29 cells time-dependently, block HT29 in the S phase, and decrease E2F1 protein expression. Conclusion miR-17-92 cluster can promote the proliferation of colorectal cancer cell line HT29 through down-regulation of E2F1 protein expression. It is indicated that miR-17-92 cluster plays a carcinogenic role in the modulation of cell cycle of colon cancer cell HT29, and can used as a potential target for the diagnosis and treatment of colorectal cancer.