摘要
目的探究胆汁酸(bile acids,BAs)和MAPK信号通路对HepG2细胞中人类过氧化物酶体增殖物激活受体α(human peroxisome proliferation activated receptorα,hPPARα)转录表达的调控作用。方法PCR扩增hPPARα基因启动子片段P1(-764~+228 bp)、P2(-2090~-744 bp)、P2-1(-744~-1287 bp)、P2-2(-1548~-1265 bp)、P2-3(-2090~-1540 bp),将扩增得到的片段分别插入荧光素酶报告基因质粒(pGL4-luc2P-Hygro)的启动子上游。将包含hPPARα基因启动子片段的荧光素酶报告基因质粒转染HepG2细胞,分别用U0126(ERK1/2信号通路选择性小分子抑制剂)、SP600125(JNK1/2信号通路选择性小分子抑制剂)、石胆酸(lithodeoxycholic acid,LCA)、脱氧胆酸(deoxycholic acid,DCA)、鹅去氧胆酸(chenodeoxycholic acid,CDCA)、甘氨脱氧胆酸(glycodeoxycholic acid,GDCA)、熊去氧胆酸(ursodeoxycholic acid,UDCA)进行处理,每次处理均以DMSO处理组作为对照,通过双荧光素酶报告基因试验分别检测ERK1/2、JNK1/2信号通路以及胆汁酸对hPPARα启动子片段的调控作用。另外,在HepG2细胞中共转染荧光素酶报告基因质粒与人类法尼酯X受体(human farnesoid X receptor,hFXR)过表达质粒,用GW4064激活过表达的hFXR,通过双荧光素酶报告基因试验检测胆汁酸的生理受体hFXR对hPPARα启动子片段的调控作用。利用数据库预测片段中相关转录因子的结合位点,通过重组PCR定点突变以及双荧光素酶报告基因试验验证所预测位点的作用。结果与DMSO对照组相比,U0126组P1、P2片段的转录活性无显著变化;SP600125组P2片段的转录活性被显著抑制(P<0.05)。进一步研究表明,SP600125对P2-1、P2-2、P2-3片段的转录活性均有抑制作用(P<0.01)。在P2-1片段中预测得到两个c-Jun结合位点,其中位点1突变减弱了SP600125的抑制作用(P<0.0001),而位点2突变没有影响SP600125的抑制作用。在5种胆汁酸的处理中,与DMSO对照相比,LCA处理显著抑制P2片段的转录活性(P<0.05),而CDCA�
Objective To investigate the regulation role of bile acids(BAs)and MAPK signaling pathway in the transcription of human nuclear receptor of peroxisome proliferation activated receptorα(hPPARα).Methods The DNA region at the promoter of human PPARαgene was amplified by PCR,named as P1(from-764 bp to+228 bp),P2(from-2090 bp to-744 bp),P2-1(from-744 bp to-1287 bp),P2-2(from-1548 bp to-1265 bp),and P2-3(from-2090 bp to-1540 bp),and then the amplified fragments were inserted into the upstream of the luciferase gene in the pGL4-luc2P-Hygro plasmid to construct various luciferase reporter plasmids.HepG2 cells transfected with luciferase reporter plasmid were treated with U0126(a selective small molecule inhibitor of the ERK1/2 signaling pathway),SP600125(a selective small molecule inhibitor of the JNK1/2 signaling pathway),and bile acids including lithodeoxycholic acid(LCA),deoxyoxycholic acid(DCA),chenodeoxycholic acid(CDCA),glycodeoxycholic acid(GDCA),and deoxycholic acid(DCA).The roles of signaling pathways of JNK1/2 and ERK1/2,and bile acids in the regulation of PPARαtranscription were detected by a dual luciferase reporter gene assay.In addition,the luciferase reporter gene plasmid and human farnesoid X receptor(hFXR)overexpression plasmid were co-transfected into HepG2 cells,and the overexpressed hFXR was activated with GW4064.The regulatory effect of hFXR(a physiological receptor of bile acid)on the hPPARαpromoter fragment was detected by a dual luciferase reporter gene assay.The binding sites of transcription factors in the genome region was predicted by bioinformatics,and the role of binding sites was verified by PCR site-directed mutagenesis.Results Compared to DMSO control group,there was no significant change in the transcriptional activities of the P1 and P2 fragments in U0126 group,and the transcriptional activity of the P2 fragment was significantly inhibited in SP600125 group.Further studies revealed that SP600125 had the inhibitory effect on the transcriptional activities of P2-1,P2-2 and P2-3 fragm
作者
黄娜娜
庞碧滢
黄晓霞
李馨
熊文婷
孔波
刘吉升
HUANG Nana;PANG Biying;HUANG Xiaoxia;LI Xin;XIONG Wenting;KONG Bo;LIU Jisheng(Metabolic Disease Laboratory,School of Life Sciences,Guangzhou University,Guangzhou 510006,China;Teaching and Research Section of Biotechnology,School of Life Sciences,Guangzhou University)
出处
《山西医科大学学报》
CAS
2023年第10期1295-1306,共12页
Journal of Shanxi Medical University
基金
国家自然科学基金面上项目(81973376)。
