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目的:研究过氧化物酶体增殖物激活受体α(peroxisome proliferator-activated receptor alpha,PPARα)是否参与调控乙型肝炎病毒(hepatitis B virus,HBV)微染色体重塑与病毒复制。方法:HepG2细胞单独转染、共转染线性HBV单体与PPARα表达或PPARα沉默载体;Southern blot检测转染细胞质内HBV核心颗粒DNA;实时定量PCR定量检测细胞质HBV核心颗粒DNA及细胞核内HBV共价闭合环状DNA(covalently closed circular DNA,cccDNA)水平;酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测转染细胞上清中乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)和e抗原(hepatitis B e antigen,HBeAg);染色质免疫共沉淀技术(chromatin immunoprecipitation,ChIP)检测与cccDNA结合的PPARα、组蛋白和染色质修饰酶。结果:与单独转染线性HBV单体相比,共转染PPARα增加了PPARα与HBV cccDNA的结合,增加了HBV开环DNA(open circular DNA,OC DNA)及HBsAg和HBeAg水平,同时也增加了cccDNA微染色质中组蛋白H3和H4的乙酰化水平及乙酰转移酶p300的水平;共转染PPARα沉默载体则有相反的结果。结论:PPARα能直接与cccDNA结合并有助于乙酰转移酶p300募集到cccDNA上,从而调控HBV微染色质的表观遗传学和HBV复制。这一发现对HBV与宿主转录因子间相互作用的相关分子机制提供了新的机制。
Objective: To investigate whether peroxisome proliferator-activated receptor alpha (PPARα) is involved in the regulation of microchromosome remodeling and viral replication of hepatitis B virus (HBV). Methods: HepG2 cells were transfected alone, co-transfected with linear HBV and PPARα expression vector or PPARα silencing vector. Southern blot was used to detect HBV DNA in cytoplasm. Quantitative real-time PCR was used to detect cytoplasmic HBV DNA and HBV DNA The covalently closed circular DNA (cccDNA) levels were detected by enzyme-linked immunosorbent assay (ELISA). The levels of hepatitis B surface antigen (HBsAg) e antigen (HBeAg); chromatin immunoprecipitation (ChIP) detection of cccDNA-binding PPARα, histone and chromatin modifying enzyme. RESULTS: Co-transfection of PPARα increased the binding of PPARα to HBV cccDNA and increased the open circular DNA (OC DNA) and HBsAg and HBeAg levels as compared with transfection of linear HBV monomer alone The acetylation levels of histone H3 and H4 and the level of acetyltransferase p300 in cccDNA microchromatin; co-transfection with PPARα silencing vector had the opposite result. CONCLUSION: PPARα binds directly to cccDNA and facilitates recruitment of acetyltransferase p300 to cccDNA, thereby regulating the epigenetics and HBV replication of HBV microchromatin. This finding provides a new mechanism for the molecular mechanisms underlying the interaction between HBV and host transcription factors.