ABCA1基因启动子区VNTR-ZNF和-14nt位点变异对HepG2细胞中基因转录活性的影响

Effect of transcription activity regulated by VNTR-ZNF and --14C/T variants in the promoter region of ATP-binding cassette transporter 1 in HepG2 cells

目的通过体外报告基因实验观察腺苷三磷酸结合盒转运体A1（ATP—binding cassette transporter A1，ABCAI）基因启动子区VNTR—ZNF位点和-14nt位点变异对转录活性的影响。方法采用基因重组技术将含ABCAl启动子区域VNTR—ZNF位点ACCCC插入型或缺失型DNA片段和同时含VNTR—ZNF位点插入／缺失变异与-14ntC或T等位基因的DNA片段分别插 入携带荧光素酶报告基因的表达载体PGL2，构建重组质粒。以重组质粒转染HepGz细胞后培养48h，收集并裂解细胞，测定细胞液中荧光素酶活性，观察和分析启动子部位VNTR—ZNF和-14nt位点的序列变异对于转录活性的影响。结果成功构建了ABCA1基因启动子VNTR—ZNF单一位点表达载体（PGL2-ZNF-ACCCCDel、PGL2-ZNF-ACCCCIns）及VNTR—ZNF和一14nt双位点表达载体（PGL2-ZNFDel-14C、PGL2-ZNFDel-14T、PGL2-ZNFIns-14C和PGL2-ZNFIns-14T）。报告基因表达结果显示：经转染HepGz细胞培养48h，单一位点表达载体PGLz—ZNF-ACCCCDel荧光素酶活性明显高于PGL2-ZNF-AcccCIns，双位点表达载体中PGLz—ZNFDel-14C重组质粒荧光素酶活性明显高于PGL2-ZNFins-14C、PGL2-ZNFDe1-14T、PGL2-ZNFIns-14T.结论ABCA1基因启动子区域VNTR—ZNF位点ACCCC缺失型等位基因可明显增强ABCA1的转录活性，-14ntC等位基因与VNTR-ZNF缺失型等位基因具有协同作用，使ABCA1的转录活性进一步加强。

Objective To explore the effect of VNTR-ZNF and - 14C/T variants of the promoter region of the ABCA1 gene on the transcription activity of genes in vitro. Methods The recombinants were constructed by ligating DNA fragment containing VNTR-ZNF ACCCC inserted/deleted allele with or without -14C/T substitution fragments with a PGL2-basic vector containing luciferase reporter gene. The recombinants were then transfected into HepG2 cells using the cationic lipid method. After 48 h, transfected cells were collected and used to detect the luciferase activity. Results Luciferase activity of PGL2-ZNF- ACCCCDel was greater than that of PGL2-ZNF-ACCCCIns. Luciferase activity of PGL2-ZNFDel-14C was greater than that of PGL2-ZNFDel-14T, PGL2-ZNFIns-14C, PGL2-ZNFIns-14T. Conclusion Compared with the insertion type, the ACCCCdeleted type of VNTR-ZNF can significantly enhance the transcription activity of ABCA1. And co-transfection of - 14 C allele can further enhance this activity.