摘要
目的:探讨吉西他滨对HepG2.2.15细胞中乙肝病毒前基因组RNA(pregenomic RNA,pgRNA)转录以及乙肝病毒复制的影响及其可能的作用机制。方法:使用不同浓度吉西他滨处理HepG2.2.15细胞,分别使用CCK-8法、流式细胞术、实时定量聚合酶链式反应(real-time polymerase chain reaction,RT-PCR)、酶联免疫吸附测定(enzyme-linked immuno sorbent assay,ELISA)法检测细胞活性、细胞凋亡、细胞周期、细胞内乙肝病毒pgRNA、细胞培养上清液中乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)、乙型肝炎e抗原(hepatitis B e antigen,HBeAg)、乙肝病毒DNA(hepatitis B virus DNA,HBV DNA);实时定量PCR法检测吉西他滨作用下HepG2.2.15细胞中与pgRNA转录有关的调控基因肝细胞核因子4α(hepatocyte nuclear factor4,HNF4α)、P38蛋白激酶(P38 mitogen-activated protein kinase,P38MAPK)、环磷酸腺苷效应元件结合蛋白1(CAMP responsive element binding protein 1,CREB1)、核转录因子p65(nuclear factor-kappa B p65,NF-κb p65)、组蛋白去乙酰化酶1(histone deacetylase 1,HDAC1)、过氧化物酶体增殖物激活受体γ辅助激活因子-1α(peroxisome proliferator-activated receptor gamma coactivator 1-alpha,PGC-1α)、叉头转录因子O1(forkhead box protein O1,FOXO1)、相关调控基因沉默信息调节因子2相关酶1(sirtuin 1,SIRT1)、P53 mRNA水平以及pgRNA的表达量,分析这些基因随吉西他滨浓度变化与pgRNA随浓度变化的相关性,找出与pgRNA变化相关性最高的基因,并使用Western blot在蛋白质水平验证吉西他滨对此基因蛋白质表达量的影响。结果:吉西他滨对HepG2.2.15细胞的生长显示出浓度以及剂量依赖性的抑制作用;吉西他滨使细胞凋亡率增高;吉西他滨使细胞更多的细胞停滞在G0G1期,而处于S期细胞比例降低;吉西他滨明显增加HepG2.2.15细胞内乙肝病毒pgRNA,72 h之内呈现明显的时间与剂量依赖性;吉西他滨作用细胞24 h会使细胞培养上清液中的HBeAg增加;吉西他滨促进HNF4αmRNA、HNF4α蛋白质表达量增加,且在所检测的与乙肝病毒转录调节有关的基因中,HNF4αmRNA变化趋势与HBV pgRNA变化趋势相关性最高。结论:吉西他滨可在细胞水平促进乙肝病毒pgRNA转录以及乙肝病毒的复制,其机制为通过促进乙肝病毒转录因子HNF4α的表达实现的。
Objective:To investigate the effect of gemcitabine on hepatitis B virus(HBV)pregenomic RNA(pgRNA)transcription and HBV replication in HepG2.2.15 cells and its possible mechanisms.Methods:HepG2.2.15 cells were treated with different concentrations of gemcitabine,and cell viability,apoptosis,cell cycle,intracellular HBV pgRNA,and HBsAg,HBeAg,HBV DNA in the cell culture supernatant were measured by CCK8 assay,flow cytometry,qRT-PCR,and ELISA respectively.The m RNA expression levels of pgRNA transcription-related regulatory genes,hepatocyte nuclear factor 4α(HNF4α),P38 mitogen-activated protein kinase(P38 MAPK),CAMP responsive element binding protein 1(CREB1),nuclear factor-kappa B p65(NF-κb p65),histone deacetylase 1(HDAC1),peroxisome proliferator-activated receptorγcoactivator 1α(PGC-1α),forkhead box protein O1(FOXO1),sirtuin1(SIRT1),and P53,and pgRNA in HepG2.2.15 cells treated with gemcitabine were determined by real-time quantitative PCR.The correlations between these genes and pgRNA with changes in gemcitabine concentration were analyzed to identify the gene with the highest correlation with pgRNA,and the effect of gemcitabine on the protein expression of the gene was verified at the protein level using Western blot.Results:Gemcitabine showed a concentration-and dose-dependent inhibitory effect on HepG2.2.15 cell growth;gemcitabine increased apoptosis;gemcitabine caused more cells to arrest in G0 G1 phase,and the proportion of cells in S phase decreased.Gemcitabine significantly increased HBV pgRNA in hepG2.2.15 cells in a significant time-and dose-dependent manner within 72 h;treatment of cells with gemcitabine for 24 hours increased HBeAg in the cell culture supernatant;gemcitabine promoted the mRNA and protein expression of HNF4α,and among all the genes involved in the regulation of HBV transcription we detected,the changing trend of HNF4αmRNA expression was the most correlated with that of HBV pgRNA.Conclusion:Gemcitabine can increase HBV pgRNA transcription and HBV replication at the cellular level by promoting the expression of the HBV transcription factor HNF4α.
作者
叶雨笙
常凯
朱紫衣
刘晨霞
江忠勇
熊杰
Ye Yusheng;Chang Kai;Zhu Ziyi;Liu Chenxia;Jiang Zhongyong;Xiong Jie(Department of Clinical Laboratory,Chengdu Military General Hospital)
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2019年第12期1564-1570,共7页
Journal of Chongqing Medical University
基金
四川省科技厅资助项目(编号:2013JY0173)
