论文部分内容阅读
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。
In this study, microRNA-1 (miR-1) was transfected into human colon cancer cells HT-29 and HCT 116 by cationic liposome-mediated method to overexpression. MTS assay, clonogenic assay and flow cytometry were used to detect cell proliferation and cell cycle changes after transfection. The results showed that miR-1 could arrest cell cycle of HT-29 and HCT 116 cells in G1 phase, Proliferation; Prediction of target genes and luciferase analysis and prediction of cyclin D1 (CCND1) and CDK6 (cyclin dependent kinases 6) are targets of miR-1. The results of western blot showed that miR-1 down-regulated the expressions of CDK2, CDK4, p-Rb (retinoic acid receptor) and E2F1 (E2F transcription factor 1) in HT-29 and HCT 116 cells , P-Cdc2 (cell division cycle 2) and other protein levels. To study the effect of miR-1 on the tumorigenicity of human colon cancer cells in human colon cancer nude mice model, we found that miR-1 can significantly inhibit the proliferation of human colon cancer cells in vivo. This study shows that miR-1 downregulates the phosphorylation of Rb and Cdc2, which are the key proteins in cell cycle progression, by down-regulating the expression of target genes cyclin D1, CDK6 and affecting the expression of CDK2 and CDK4. E2F1 protein levels, so that cell cycle retention in the G1 phase, thereby inhibiting cell proliferation.