慢病毒介导5HRE－CEAp调控RASSF1A表达对胃癌细胞SGC7901抑制的研究

摘要

目的:探讨5拷贝低氧反应元件(5HRE)联合癌胚抗原启动子(CEAp)调控抑癌基因RAS相关域家族1A(RASSF1A)的慢病毒载体(LV－5HRE－CEAp－RASSF1A)对胃癌细胞SGC7901的体外抑制作用.方法:采用qRT－PCR方法检测空白对照组细胞SGC7901、阴性对照组细胞SGC7901/NC及慢病毒载体感染的实验组细胞SGC7901/5HRE－CEAp－RASSF1A中RASSF1A mRNA表达水平;通过CCK－8实验检测各组细胞的生长曲线;通过流式细胞技术检测各细胞的凋亡及细胞周期.结果:qRT－PCR结果显示,与其他组比较,实验组细胞的RASSF1A mRNA水平在低氧条件下明显升高(P＜0.01);CCK－8实验结果显示从第3天开始,低氧条件下实验组细胞的OD值开始低于其它各组细胞(P＜0.05);流式细胞检测结果显示与其它各组细胞比较,低氧条件下实验组细胞凋亡比例明显增加(P＜0.01),并且其细胞周期G1期的细胞百分比明显增加(P＜0.05).结论:慢病毒载体LV－5HRE－CEAp－RASSF1A具有在低氧诱导下显著抑制胃癌细胞株SGC7901增殖的作用.%Objective:To explore the inhibition effect of RAS association domain family 1 isoform A(RASSF1A)regulated by 5 copies hypoxia－responsive element(5HRE)and carcinoembryonic antigen promoter(CEAp)on gas-tric cancer SGC7901 cells in vitro.Methods:Cells were divided into 2 groups:Normoxia group(without CoCl2)and hypoxia group(with CoCl2),RASSF1A mRNA expression in SGC7901,negative control cells SGC7901/NC and infec-tion cells SGC7901/5HRE－CEAp－RASSF1A were detected by real time－PCR(qRT－PCR).The viability of cells was assessed by cell counting Kit－8(CCK－8)assay,the apoptosis and cell cycle were investigated by flow cytome-try.Results:SGC7901/5HRE－CEAp－RASSF1A cells showed very high levels of RASSF1A mRNA under hypoxic condition,but only slightly elevated levels of RASSF1A mRNA under normoxic conditions(P＜0.01).CCK－8 assay showed that RASSF1A expression under hypoxic condition significantly reduced SGC7901/5HRE－CEAp－RASSF1A cell viability when compared with either SGC7901 or SGC7901/NC cell viability under both normoxic and hypoxic conditions(P＜0.05).The cell apoptosis assay showed that the rate of apoptosis was increased in SGC7901/5HRE－CEAp－RASSF1A cells under hypoxic condition(P＜0.01).Cell cycle analysis revealed that SGC7901/5HRE －CEAp－RASSF1A cells accumulated at the G1phase of the cell cycle under hypoxic conditions(P＜0.05).Conclu-sion:The restoration of RASSF1A expression using a hypoxia－inducible and CEA promoter－driven lentivirus vector suppressed aggressive phenotypes of gastric cancer cells SGC7901 in vitro.