野生型RASSF1A基因抑制肝癌细胞增殖的分子机制

Inhibitory Effect of Wild-type RASSF1A Gene Expression on Proliferation of Hepatocellular Carcinoma QGY-7703 Cells

背景与目的:RASSF1A(ras association domain family1A)基因抑制肿瘤增殖的分子机制目前尚未完全明确,本文将探讨野生型RASSF1A基因抑制肝癌细胞增殖的分子机制。方法:利用已构建成功的稳定表达野生型和突变型RASSF1A基因的肝癌细胞株QGY-7703,使用流式细胞仪对其进行细胞周期的测定,Western blot分析其cyclin D1、cyclin E及P21蛋白的表达水平,通过检测荧光素酶报告基因活性及RT-PCR检测野生型RASSF1A基因的表达来分析RASSF1A基因对cyclin D1启动子和mRNA表达水平的影响。结果:野生型RASSF1A基因的表达可使QGY-7703细胞的细胞周期发生G1/S期阻滞,使其cyclin D1蛋白的表达水平下调,但不影响cyclinE和P21蛋白的表达水平。外源性cyclin D1的表达可使野生型RASSF1A基因引起的QGY-7703细胞G1/S期阻滞消失。野生型RASSF1A基因表达不影响QGY-7703细胞cyclin D1启动子的活性和cyclin D1 mRNA的表达水平。结论:野生型RASSF1A基因通过转录后机制负性调控cyclin D1蛋白的表达,使肝癌细胞株QGY-7703细胞周期阻滞在G1/S期。

BACKGROUND ＆ OBJECTIVE： The potential molecular mechanisms of the antitumor role of Ras association domain family 1 A （RASSF1A） has not been well understood. This study was to explore the molecular mechanisms of proliferation-suppressing ability of wild type RASSF1A in hepatocellular carcinoma （HCC） QGY-7703 cells. METHODS. Vectors containing wild type or mutant RASSF1A were transfected into QGY- 7703 cells. Cell cycle was determined by flow cytometry （FCM）. Western blot was used to examine the protein levels of Cyclin D1, Cyclin E and P21. Luciferase activity assay was performed to study the effect of wild type RASSFIA expression on cyclin D1 promoter activity in QGY-7703 cells. Reverse transcription-polymerase chain reaction （RT-PCR） was performed to detect the mRNA level of cyclin D1. RESULTS： Wild type RASSF1A expression resulted in G1/S arrest in QGY-7703 cells, decreased the protein level of Cyclin D1, but had no effect on the protein levels of Cyclin E and P21, the promoter activity and mRNA level of cyclin D1. The exogenous expression of cyclin D1 rescued the G1 phase arrest induced by wild type RASSF1A. CONCLUSION： Wild type RASSF1A expression could induce G1 phase arrest in QGY-7703 cells, which is accompanied by a down-regulation of Cyclin D1 protein expression through a post-transcriptional mechanism.