MicroRNA-1调控人结肠癌细胞周期及其机制的研究

The Mechanism of MicroRNA-1 Regulating the Cell Cycle in Human Colon Cancer

该研究通过阳离子脂质体介导的方法将microRNA-1(miR-1)转染入人结肠癌细胞HT-29和HCT 116,使其过表达。应用MTS法、克隆形成实验、流式细胞术检测转染后细胞增殖及细胞周期的变化情况,结果发现,miR-1能使HT-29和HCT 116细胞周期滞留在G1期,显著抑制细胞的增殖;通过靶基因预测及荧光素酶分析法预测并确定CCND1(cyclin D1)和CDK6(cyclin dependent kinases 6)是miR-1作用的靶基因。采用Western blot法检测细胞内相关蛋白质表达水平,结果发现,miR-1能下调HT-29和HCT 116中细胞周期相关蛋白CDK2、CDK4、p-Rb(retinoblastoma gene)、E2F1(E2F transcription factor 1)、p-Cdc2(cell division cycle 2)等蛋白质水平。通过人结肠癌裸鼠动物模型检测miR-1对结肠癌细胞体内成瘤能力的改变情况,研究发现,miR-1能在体显著抑制人结肠癌细胞增殖能力。该研究表明,miR-1通过下调靶基因cyclin D1、CDK6的表达,并影响细胞周期相关蛋白CDK2、CDK4的水平,下调了决定细胞周期进程的关键蛋白Rb及Cdc2的磷酸化水平,同时也降低了E2F1蛋白质水平,使细胞周期滞留在G1期,从而抑制了细胞增殖。

microRNA-1 was transfected into human colon cancer cell line HT-29 and HCT 116. The proliferation of HT-29 and HCT 116 cells was examined by MTS cell proliferation assay and colony formation assay. The cell cycle was analyzed by Flow Cytometry. The results showed that miR-1 could inhibit HT-29 and HCT 116 cells proliferation, colony formation, and induce G1-phase cell cycle arrest, and suppress tumor growth in a xenograft mouse model. Furthermore, we identified CCND1（cyclin D1） and CDK6（cyclin dependent kinases 6） as the direct targets of miR-1 by dual-luciferase activity assay and Western blot. miR-1 also down-regulated the expression of CDK2, CDK4, phosphorylated-Rb（retinoblastoma gene）, E2F1（E2F transcription factor 1）, phosphorylated-Cdc2（cell division cycle 2） indirectly. Our findings suggested that miR-1 may function as a novel tumor suppressor in human colon cancer.