摘 要: 为了筛选人线粒体转录终止因子3 (human mitochondrial transcription termination factor 3, hMTERF3)基因各段启动子的活性, 探讨肝癌细胞HepG2和BEL-7402中hMTERF3基因启动子活性受DNA甲基化的影响, 首先利用PCR技术扩增hMTERF3基因启动子区, 克隆至荧光素酶基因报告载体中, 构建pGL6-hMTERF3重组质粒; 随后运用荧光素酶检测法检测肝癌细胞中各段hMTERF3启动子对荧光素酶报告基因的启动活性, 并采用DNA甲基化酶SssⅠ处理肝癌细胞, 观察各报告基因的启动子活性变化。结果显示, 成功构建了7个携带hMTERF3基因启动子的重组荧光素酶报告基因载体, 重组子经NheⅠ和BglⅡ双酶切及PCR鉴定正确。双荧光素酶报告基因检测法显示, 与pGL6相比, 含有P523的3个启动子区段P523、P866、P932有强启动子活性, 组间比较差异无统计学意义(P>0.05)。此外, 与对照组比较, SssⅠ甲基化酶处理的P523、P866和P932启动子活性显著降低(P<0.05)。以上研究提示, hMTERF3启动子活性受DNA甲基化的影响, 其中P523可能是hMTERF3启动子核心, 为进一步研究肝癌细胞中hMTERF3基因表达的表观遗传学机制奠定了实验基础。

Abstract: To construct a reporter vector regulated by the hMTERF3 promoter to determine the effect of DNA methylation on the promoter activity of hMTERF3 gene, PCR amplification was performed to obtain seven potential hMTERF3 promoter fragments, which were then cloned into the vector to obtain recombinant constructs. Promoter activities of different hMTERF3 fragments in cancer cells were detected by luciferase assay to identify the potential promoter area. The activity of the hMTERF3 promoter with or without treatment of methylase M. SssⅠwas also evaluated by luciferase assay. It showed that three fragments (P523, P866 and P932), all of which contained the P523 sequence, had stronger promoter activity than the pGL6 control. Their promoter activities decreased significantly after methylase treatment (P<0.05). A luciferase reporter gene system containing the hMTERF3 promoter was successfully constructed. P523 is the potential core area of the hMTERF3 promoter. These results provide a basis for studying the epigenetic mechanism of hMTERF3 gene expression in hepatoma cells.