摘要
目的:探索顺铂诱导乙型肝炎病毒(hepatitis B virus,HBV)再激活的机制。方法:将稳定表达HBV的肝癌细胞系HepAD38分为2组,即磷酸盐缓冲液(phosphate buffered saline,PBS)对照组及顺铂(Cisplatin)处理组,分别检测cAMP应答元件结合蛋白质-1(cAMP responsive element binding protein 1,CREB1)、乙肝病毒蛋白表达(HBsAg、HBcAg)及病毒复制水平(HBV DNA、HBV mRNA);同样在成功构建稳定敲低CREB1(shCREB1)的HepAD38细胞中,同正常表达HBV的肝癌细胞系HepAD38进行对照,检测经顺铂处理后的HBV复制水平(HBV DNA、HBV m RNA)及蛋白表达水平(HBsAg、HBcAg)。结果:稳定表达HBV的肝癌细胞系HepAD38中,Western blot检测显示,与PBS对照组相比,Cisplatin处理组中CREB1相对表达量为1.355±0.049(P=0.010);HBsAg蛋白相对表达量为1.679±0.060(P=0.003);HBcAg蛋白相对表达量为1.488±0.047(P=0.005)。qRTPCR结果显示经顺铂处理后,HBV DNA绝对表达量为249.600±54.400(P=0.006);HBV m RNA相对表达量为3.084±0.256(P=0.000);以上结果表明在HepAD38细胞中乙肝病毒蛋白表达水平以及病毒复制水平较PBS对照组明显上调,均具有统计学差异;与此同时,选择转入shCREB1质粒的HepAD38细胞,即shCREB1细胞,以及转入对照质粒且能正常表达CREB1的HepAD38细胞,即shControl细胞,2种细胞组内均设置PBS对照组和Cisplatin处理组,分别检测乙肝病毒蛋白表达以及病毒复制水平。qRT-PCR结果显示,HBV DNA在shControl细胞的Cisplatin处理组相对表达量为518.300±3.458;而在shCREB1细胞的Cisplatin处理组相对表达量为265.300±3.125 (P=0.000);HBV m RNA在shControl细胞的Cisplatin处理组相对表达量为2.713±0.318;而在shCREB1细胞的Cisplatin处理组相对表达量为1.263±0.056(P=0.000)。Western blot分析显示shControl细胞的Cisplatin处理组中的HBsAg、HBcAg相对表达量分别为1.254±0.001、2.238±0.041;而在shCREB1细胞的Cisplatin处理组中的HBsAg、HBcAg相对表达量分别为1.121±0.036(P=0.021)、1.681±0.015(P=0.000)。结论:顺铂可以通过CREB1促进乙肝病毒复制水平以及蛋白表达水平上调。
Objective:To investigate the mechanism of cisplatin-induced hepatitis B virus (HBV)reactivation.Methods:HepAD38 hepatoma cells with stable expression of HBV were divided into phosphate buffered saline(PBS)control group and cisplatin treatment group.The expression of cAMP responsive element binding protein 1(CREB1)and HBV proteins(HBsAg and HBcAg)and the level of HBV replication(HBV DNA and HBV mRNA)were measured.HepAD38 cells with stable knock-down of CREB1 (shCREB 1)were compared with those with stable expression of HBV,and the level of HBV replication (HBV DNA and HBV rnRNA)and protein expression level after cisplatin treatment were measured.Results :As for HepAD38hepatoma cells with stable expression of HBV,Westem blot showed that compared with the PBS control group,the cisplatin treatment group had a relative expression level of CREB1of 1.355±0.049(P=0.010), a relative expression level of HBsAg protein of 1.679±0.060 (P=0.003),and a relative expression level of HBcAg protein of 1.488±0.047(P=0.005).The results of qRT-PCR showed that compared with the PBS contrbl group,the cisplatin treatmen.t group had an absolute expression level of HBV DNA of 249.600±54.400(P= 0.006)and a relative expression level of HBV mRNA of 3.084±0.256(P=0.000),suggesting that compared with the PBS control group,the cisplatin treatment group had significantly higher expression levels of HBV proteins and level of HBV replication.In addition,. HepAD38cells transfected with shCREB1plasmid(shCREB1cells)and HepAD38cells transfeeted with control plasmid with normal CREB1expression(shControl cells)were divided into PBS control group and cisplatin treatment group,and the expression of HBV proteins and the level of HBV replication were measured.The results of qRT-PCR showed that compared with the cisplatin treatment group of shCREB1cells,the cisplatin treatment group of shControl cells had significantly higher relative expression levels of HBV DNA(518.300±3.458vs.265.300±3.125,P=0.000)and HBV mRNA(2.713±0.318vs.1.263±0.056,P=0.000).According to the results of Western blot,the cisplatin treatment group of shControl cells had a relative expression level of HBsAg of 1.254± 0.001 and a relative expression level of HBcAg of 2.238±0.041,while the cispiatin treatment group of shCREB1 cells had a relative expression level of HBsAg of 1.121±0.036 and a relative expression level of HBcAg of 1.681±0.015;there were significant differences in the relative expression levels of HBsAg and HBcAg between the two groups(P=0.021 and 0.000).Conclusion:Cisplatin can increase the level of HBV replication and the expression levels Of HBV proteins via CREB1.
作者
潘娥
陈雪梅
高庆祝
魏杰
胡杰
梁利
汪凯
黄爱龙
唐霓
Pan E;Chen Xuemei;Gao Qingzhu;Wei Jie;Hu Jie;Liang Li;Wang Kai;Huang Ailong;Tang Ni(The Key Laboratory of Molecuar Biology on Infection Diseases ,Ministry of Education,Chongqing Medical University)
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2018年第11期1409-1415,共7页
Journal of Chongqing Medical University
基金
国家自然科学基金资助项目(编号:81471946
81572683)
重庆市科委基础与前沿重点资助项目(编号:cstc2015jcyj BX0011)
重庆市高校创新团队建设计划资助项目(编号:CXTDX201601015)
重庆市科技领军人才支持计划资助项目(CSTCCXLJRC201719)
