P53结合位点对人NOD8基因的调控  

Effect of P53 binding site on regulation of human NOD8 gene

作　　者：曾琪[1] 张芸[1] 田莉[1] 唐清亮[1] 胡巢凤[1] 柏志全[2] 

机构地区：[1]暨南大学医学院病理生理学系,国家中医药管理局重点实验室,广东广州510632 [2]暨南大学医学院生理学系,广东广州510632

出　　处：《中国病理生理杂志》2011年第12期2362-2367,共6页Chinese Journal of Pathophysiology

基　　金：广东省自然科学基金资助项目(No.06025159);广东省教育厅自然科学基金资助项目[No.126(2005)];暨南大学“211工程”三期预研项目;暨南大学重点实验室基金资助项目

摘　　要：目的:探讨P53结合位点在NOD8基因调控中的作用。方法:利用生物信息学方法,我们发现人与黑猩猩的NOD8基因核心启动子区域相似位置含有P53结合位点;以人基因组DNA为模板,PCR扩增含有人NOD8基因序列,构建含有/缺失人P53结合位点的NOD8基因启动子驱动的、表达绿色荧光蛋白-NOD8融合蛋白的质粒pNOD8(760 bp)-EGFP-NOD8和mpNOD8(750 bp)-EGFP-NOD8;将构建的重组质粒经阳离子聚合物JetPeiTM介导瞬时转染HEK293细胞中,并加入不同浓度的P53抑制剂pifithrin alpha(PFT-α)处理HEK293细胞,用RT-PCR和Westren blotting方法检测NOD8 mRNA和蛋白的表达;此外,用pNOD8(760 bp)-EGFP质粒转染HEK293细胞,利用染色质免疫共沉淀法(ChIP)观察P53是否与NOD8启动子结合。结果:经酶切鉴定和序列测定证实重组质粒构建成功。ChIP实验证实P53能与NOD8启动子结合。pNOD8(760 bp)-EGFP-NOD8转染组中NOD8 mRNA的表达显著高于pEGFP-C2转染组(P<0.05),并且NOD8 mRNA在缺失人P53结合位点的mpNOD8(750 bp)-EGFP-NOD8转染组中的表达明显降低(P<0.01);同时发现加入PFT-α的pNOD8(760bp)-EGFP-NOD8转染组NOD8 mRNA的表达显著下降,并呈剂量依赖关系,其中90μmol/L PFT-α对NOD8mRNA表达的抑制作用最为显著(P<0.01)。与mRNA检测结果一致的是pNOD8(760 bp)-EGFP-NOD8转染组NOD8蛋白的表达量显著高于对照组pEGFP-C2;而mpNOD8(750 bp)-EGFP-NOD8转染组NOD8蛋白的表达量明显低于pNOD8(760 bp)-EGFP-NOD8转染组和加入PFT-α的pNOD8(760 bp)-EGFP-NOD8转染组(P<0.01)。结论:P53结合位点在NOD8基因调控中起着重要的作用,并且P53结合位点与NOD8基因之间可能存在正反馈调节。AIM： To investigated the characterization and biological function of P53 binding element in the regulation of NOD8 gene.METHODS： Using the method of bioinformatics,we found a completely preserved P53 binding site in NOD8 core promoter both in humans and chimpanzees.NOD8 gene was amplified by PCR from human cDNA and correctly connected into the vector pNOD8（760 bp）-EGFP-C2/ mpNOD8（750 bp）-EGFP-C2.The constructed plasmids pNOD8（760 bp）-EGFP-NOD8 and mpNOD8（750 bp）-EGFP-NOD8 were transiently transfected into the cell line HEK293 by JetPeiTM and treated with pifithrin alpha（PFT-α,an inhibitor of P53） at different concentrations for 24 h.The mRNA and protein expression levels of NOD8 were detected by RT-PCR and Western blotting.In addition,chromatin immunoprecipitation（ChIP） assay was performed to determine the binding of P53 to the NOD8 promoter after recombinant plasmid pNOD8（760 bp）-EGFP was transfected into HEK293 cells.RESULTS： The plasmids pNOD8（760 bp）-EGFP-NOD8 and mpNOD8（750 bp）-EGFP-NOD8 were successfully constructed and conformed by restriction digestion and sequence analysis.The results of ChIP confirmed that P53 bound to the promoter of NOD8.The mRNA expression of NOD8 in the cells transfected with pNOD8（760 bp）-EGFP-NOD8 was stronger than that in the cells transfected with control vector pEGFP-C2（P0.05）.Furthermore,the mRNA expression of NOD8 was reduced in HEK293 cells transfectnt with the mutant plasmid mp NOD8（750 bp）-EGFP-NOD8 compared with the cells transfected with pNOD8（760 bp）-EGFP-NOD8.Meanwhile,PFT-α inhibited the mRNA expression of NOD8 in the cells transfected with pNOD8（760 bp）-EGFP-NOD8 in a concentration-dependent manner,and PFT-α at concentration of 90 μmol/L was the most effective in inhibiting NOD8 mRNA expression（P0.01）.As expected,the protein expression of NOD8 in pNOD8（760 bp）-EGFP-NOD8 group significantly increased compared with that in pNOD8-C2 group,the protein expression of NOD8 in mpNOD8（750 bp）-EGFP-N

关 键 词：启动子 基因 NOD8 P53结合位点 核苷酸结合寡聚结构域样受体 

分 类 号：R363[医药卫生—病理学]