转录抑制因子ZHX2对AFP启动子的抑制作用

Human ZHX2＇s transcriptional repression function on AFP promoter

目的：探讨转录抑制因子ZHX2对人甲胎蛋白（AFP）启动子的抑制作用。方法PCR扩增人ZHX2基因，构建ZHX2与绿色荧光蛋白和HA-tag融合表达载体pEGFP-ZHX2、pcDNA-ZHX2-HA，共转染后通过荧光显微镜及Western blot检测融合基因的表达。扩增人AFP核心启动子269bp插入pGl3-Basic，构建报告质粒phAF269。将pcDNA-ZHX2-HA和phAF269共转染HepG2细胞。48h后裂解细胞，双荧光检测分析ZHX2对AFP启动子的抑制作用。结果荧光显微镜及westem blot检测证实pEGFP-ZHX2和pcDNA-ZHX2-HA可在体外有效表达ZHX2融合蛋白，双荧光实验表明报告质粒phAF269具有AFP启动子活性，且pcDNA-ZHX2-HA与phAF269共转染HepG2后可显著抑制AFP启动子的转录作用，此抑制作用具有剂量依赖性。结论：成功构建两种新型转录抑制因子ZHX2融合表达载体，并证实其对人AFP启动子的抑制作用，为进一步探讨肝癌发生中AFP表达调控机制及提高AFP介导的靶向肿瘤基因治疗提供基础资料。

Objective To investigate the transcriptional repression function of ZHX2 on AFP promoter in hepatecellular carcinoma cells HepG2. Methods Human ZHX2 was amplified with RT-PCR and cloned. Two expression vector pEGFP-ZHX2 and pcDNA-ZHX2-HA which can express EGFP-ZHX2 and ZHX2-HA fusion proteins were constructed.The expression of fusion protein in COS-7 cells was identified by fluorescence microscopy and Western blot.AFP core promoter with size of 269 bp was amplified and cloned into pGL3-Basic to construct the reporter plamfid phAF269. Transient transfection was used to test the phAF269-cloned AFP promoter activity in HepG2 cells. Different dosage of pcDNA-ZHX2-HA was cotransfected with phAF259 into HepG2 cells and dual-luciferase reporter assay system was applied to detect the activity of AFP promoter.Results The recombinant plasraids, pEGFP-ZHX2, pcDNA-ZHX2-HA and phAF269 were constructed and verified by enzyme analysis,PCR amplification and DNA sequencing.Using fluorescence microscopy and Westem blot,the expression of pEGFP- ZHX2 and ZHX2-HA could be detected in COS-7 cells 48 h after transfection,phAF269 promoted luciferase expression in HepG2 cells.After cotransfecfion of peDNA-ZHX2-HA and pliAF269, dual-lucifernse reporter assay system confirmed that ZHX2 had transcriptional repression function on AFP promoter in HepG2 cells. The inhibition was dosage dependant. Conclusion Human ZHX2 genc was successful expressed in COS-7 cells and could repress AFP promoter effectively in HepG2 cells.