转录因子GRHL3调控GPR108基因表达及其作用机制

Transcription factor GRHL3 regulates the expression of GPR108 gene and its mechanism

目的研究转录因子果蝇头状因子(grainyhead-like,GRHL)3调控G蛋白偶联受体108(G protein-coupled receptor 108,GPR108)表达的分子机制。方法利用生物信息学分析在各种癌症组织的肿瘤细胞中,GRHL3和GPR108的表达水平以及相互关系;运用反转录定量聚合酶链反应(reverse transcription quantitative polymerase chain reaction,RT-qPCR)检测GRHL3和GPR108基因在人皮肤鳞癌细胞系(A-431)、人肝癌细胞系(Hep G2)以及人神经母细胞瘤细胞系(SH-SY5Y)的表达;采用分子克隆技术构建含有GPR108基因启动子和突变型GPR108基因启动子的荧光素酶报告基因表达载体;通过双荧光素酶报告基因检测系统,观察GRHL3对GPR108基因启动子的调控作用;通过染色质免疫共沉淀(chromatin immunoprecipitation assay,ChIP)分析GRHL3和GPR108启动子结合位点之间的关系。结果生物信息学分析在各种组织的肿瘤细胞中,GRHL3的表达较低,而GPR108的表达较高。利用分子克隆技术成功构建了GPR108基因启动子和突变型GPR108基因启动子荧光素酶报告基因表达载体;GRHL3对荧光素酶报告基因GPR108的启动子具有明显的抑制作用。将GPR108启动子上的结合位点删除突变后,GRHL3对GPR108基因启动子的负调控作用减弱;ChIP结果显示GRHL3能与GPR108启动子结合位点结合。结论转录因子GRHL3通过结合GPR108启动子上的保守序列5′-AACCGGTG-3′抑制GPR108表达。

Objective To study the molecular mechanism of transcription factor grainyhead-like(GRHL3)involved in the regulation of GPR108 expression.Methods Bioinformatics was used to analyze the expression level and relationship of GRHL3 and G protein-coupled receptor 108(GPR108)in various cancer cells.Reverse transcription quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of GRHL3 and GPR108 genes in human epidermoid carcinoma cell line(A-431),human hepatocellular liver carcinoma cell line(Hep G2)and human neuroblastoma cell line(SH-SY5Y).The luciferase reporter gene expression vector containing GPR108 promoter and mutant GPR108 promoter was constructed by molecular cloning technology.The regulatory effect of GRHL3 on GPR108 promoter was observed by double luciferase reporter gene detection system;The relationship between GRHL3 and GPR108 promoter binding sites was analyzed by chromatin immunoprecipitation assay(ChIP).Results Bioinformatics analysis showed that the expression of GRHL3 was lower and GPR108 was higher in various cancer cells.The luciferase reporter gene expression vectors of GPR108 promoter and mutant GPR108 promoter were successfully constructed by molecular cloning technology.Luciferase results showed that GRHL3 significantly inhibited the promoter of GPR108.After deleting the binding site on the GPR108 promoter,the negative regulatory effect of GRHL3 on the GPR108 promoter was weakened;ChIP assay showed that GRHL3 could interact with GPR108 promoter binding site.Conclusion The transcription factor GRHL3 can be negatively regulated by binding to the conserved sequence 5′-AACCGGTG-3′on the GPR108 promoter.