mRNA前体剪切因子19蛋白对乙型肝炎病毒感染Huh7细胞病毒复制的影响  

Influence of pre-mRNA processing 19 on viral replication in hepatitis B virus-infected Huh7 cells

作　　者：马新平 郑欣 黄迎丽 李雨轩 丁辉 韩双印 孙锁锋 MA Xin-ping;ZHENG Xin;HUANG Ying-li;LI Yu-xuan;DING Hui;HAN Shuang-yin;SUN Suo-feng(Department of Infectious Diseases,the Second Affiliated Hospital of Zhengzhou University,Zhengzhou,Henan 450000,China;Department of Digestive Diseases,Zhengzhou University People's Hospital,Henan Provincial People's Hospital,Zhengzhou,Henan 450003,China)

机构地区：[1]郑州大学第二附属医院感染性疾病科,河南郑州450000 [2]郑州大学人民医院河南省人民医院消化内科,河南郑州450003

出　　处：《中华实用诊断与治疗杂志》2022年第9期900-904,共5页Journal of Chinese Practical Diagnosis and Therapy

基　　金：河南省自然科学基金面上项目(212300410390)。

摘　　要：目的 观察mRNA前体剪切因子19(pre-mRNA processing 19, PRP19)蛋白对乙型肝炎病毒(hepatitis B virus, HBV)感染Huh7细胞HBV核心启动子、EnhⅡ/基本核心启动子(basal core promoter, BCP)活性的影响,探讨其调控HBV复制的可能机制。方法 取对数生长期Huh7细胞分为PRP19过表达组(转染pcDNA3.1-Flag-PRP19过表达质粒和HBV B基因型表达质粒pUC19-HB-Bj),PRP19敲低组(转染PRP19-siRNA 36 h后转染pUC19-HB-Bj),空白对照组(转染NC-siRNA 36 h后转染pUC19-HB-Bj),正常对照组(转染pUC19-HB-Bj)。转染72 h, 4组采用Western blot法检测PRP19蛋白相对表达量,采用实时荧光定量PCR法检测HBV前基因组RNA(pregenomic RNA, pgRNA)和HBV-DNA相对表达量;采用Western blot法检测PRP19过表达组、正常对照组HBsAg、HBcAg蛋白相对表达量。取PRP19过表达组和正常对照组细胞,分别转染HBV核心启动子及EnhⅡ/BCP驱动的海肾荧光素酶报告质粒,转染48 h,测定2组HBV核心启动子、EnhⅡ/BCP荧光素酶活性;采用DNA pull down法检测PRP19与HBV核心启动子、EnhⅡ/BCP的关系。结果 PRP19过表达组PRP19蛋白相对表达量(0.92±0.11)高于正常对照组(0.08±0.06)(t=27.941,P<0.001),PRP19敲低组(0.13±0.05)低于空白对照组(0.51±0.10)(t=5.494,P=0.005);PRP19过表达组pgRNA相对表达量(0.40±0.22)低于正常对照组(1.07±0.05)(t=7.173,P=0.002),PRP19敲低组(1.61±0.14)高于空白对照组(1.05±0.08)(t=10.504,P=0.005)。PRP19过表达组HBsAg(0.35±0.09)、HBcAg(0.20±0.09)蛋白及HBV-DNA(2.60×10~7±0.28)相对表达量均低于正常对照组(0.83±0.06、0.78±0.12、5.50×10~7±0.15)(t=13.204,P<0.001;t=12.346,P<0.001;t=11.232,P<0.001)。PRP19过表达组HBV核心启动子(0.28±0.23)、EnhⅡ/BCP(0.16±0.12)荧光素酶活性均低于正常对照组(1.10±0.09、1.08±0.07)(t=9.864,P=0.005;t=21.342,P<0.001)。PRP19可与生物素标记的EnhⅡ/BCP探针相结合。结论 PRP19通过与HBV核心启动子上的EnhⅡ/BCP区相互作用,负向调控HBV核心启动子活�Objective To observe the influences of pre-mRNA processing 19(PRP19) protein on the activities of core promoter and enhancer Ⅱ(EnhⅡ)/basal core promoter(BCP) of hepatitis B virus(HBV)-infected Huh7 cells, and to explore the possible mechanism of regulating HBV replication. Methods Huh7 cells in logarithmic growth phase were divided into PRP19 overexpression group(transfected with pcDNA3.1-Flag-PRP19 overexpression plasmid and HBV B genotype expression plasmid pUC19-HB-Bj), PRP19 knockdown group(transfected with PRP19-siRNA for 36 h and then transfected with pUC19-HB-Bj), blank control group(transfected with NC-siRNA for 36 h transfected and then transfected with pUC19-HB-Bj) and normal control group(transfected with pUC19-HB-Bj). After 72 h of transfection, the relative expressions of PRP19 protein was detected by Western blot, and the relative expressions of HBV pregenomic RNA(pgRNA) and HBV-DNA were detected by real-time fluorescence quantitative PCR. The relative expressions of HBsAg and HBcAg proteins were detected by Western blot in PRP19 overexpression and normal control groups. PRP19 overexpression group and control group were transfected with HBV core promoter and EnhⅡ/BCP driven renilla luciferase reporter plasmid for 48h,and HBV core promoter and EnhⅡ/BCP luciferase activities were detected.The relationships of PRP19with HBV core promoter and EnhⅡ/BCP were detected by DNA pull-down assay.Results The relative expression of PRP19protein was higher in PRP19overexpression group(0.92±0.11)than that in normal control group(0.08±0.06)(t=27.941,P<0.001),and lower in PRP19knockdown group(0.13±0.05)than that in blank control group(0.51±0.10)(t=5.494,P=0.005).The relative expression of pgRNA was lower in PRP19overexpression group(0.40±0.22)than that in normal control group(1.07±0.05)(t=7.173,P=0.002),and higher in PRP19knockdown group(1.61±0.14)than that in blank control group(1.05±0.08)(t=10.504,P=0.005).The relative expressions of HBsAg and HBcAg proteins as well as HBV-DNA were lower in PRP19o

关 键 词：乙型肝炎病毒 mRNA前体剪切因子19 核心启动子 增强子EnhⅡ 

分 类 号：R512.62[医药卫生—内科学]