【摘要】 目的运用全转录组芯片测序技术结合生物信息学筛选缝隙连接蛋白CX43的下游差异性表达基因并进行功能分析。方法采用TransIntro^TM EL将重组pCX43质粒转染至人肝癌SMMC-7721细胞,构建CX43过表达组,对照组仅转染空载质粒。采用RT-PCR、Western blot检测转染后细胞CX43 mRNA和蛋白的表达情况,两组CX43 mRNA和蛋白表达量比较采用t检验。运用全转录组芯片Clariomtm D Assays筛选CX43的下游差异表达基因;利用DAVID和STRING数据库对差异性表达基因进行基因功能、信号通路和蛋白相互作用分析,确定中心节点蛋白。采用Kaplan-Meier曲线和Cox回归分析靶点蛋白在生存预后中的价值。结果过表达组CX43 mRNA和蛋白表达量分别为363±64、1.36±0.02,明显高于对照组的1、0.81±0.01（t=5.67,19.12;P<0.05）。筛选CX43下游差异表达基因,其中上调基因394个,下调基因534个。STRING数据库分析出9个中心节点蛋白。Kaplan-Meier曲线和Cox回归分析显示,ENO1、RALA、SRC高表达患者总体生存率较差（HR=2.29,1.72,1.75;95%CI:1.61～3.27,1.20～2.46,1.22～2.53;P<0.05）。结论 ENO1、RALA、SRC为CX43的下游作用靶点,3个靶点蛋白为生存预后的独立影响因素,靶点蛋白高表达患者生存预后差。更多还原

【Abstract】 Objective To screen the downstream differentially-expressed genes of gap junction protein CX43 by using the whole transcriptome sequencing combined with bioinformatics and to analyze its function. Methods Human liver cancer SMMC-7721 cells were transfected with recombinant pCX43 plasmid with TransIntro^TM EL to construct the CX43 over-expression group, and the cells were transfected with empty plasmid in control group. The expression of CX43 mRNA and protein level were quantitatively measured by RT-PCR and Western blot, and were compared between two groups by t test. The downstream differentially-expressed genes of CX43 were screened by whole transcriptome Clariomtm D Assays. The gene function, signaling pathway and transactional function of protein in differentially-expressed genes were analyzed with DAVID and STRING databases to determine the central node proteins. The predictive value of target protein in survival and prognosis was analyzed with Kaplan-Meier curve and Cox’s regression analysis. Results The expression levels of CX43 mRNA and protein in overexpression group were 363±64 and 1.36±0.02, significantly higher than 1 and 0.81±0.01 in control group（t=5.67,19.12; P<0.05）. The downstream differentially-expressed genes of CX43 were screened, of which 394 genes were up-regulated and 534 were down-regulated. 9 central node proteins were analyzed by STRING Database. Kaplan-Meier curve and Cox’s regression analysis demonstrated that the overall survival of patients with high expression of ENO1, RALA and SRC was poor（HR=2.29, 1.72, 1.75; 95%CI: 1.61-3.27, 1.20-2.46, 1.22-2.53; P<0.05）.Conclusions ENO1, RALA and SRC are the downstream effect targets of CX43. Three target proteins are the independent factors affecting the survival and prognosis. Patients with high expression of target proteins have poor clinical prognosis.更多还原