HBV rtA181T突变对Huh7细胞自噬的影响

时景仁; 郭向华; 王洁琳; 王珊珊; 乔录新; 陈德喜

doi:10.3969/j.issn.1001-5256.2016.11.020

HBV rtA181T突变对Huh7细胞自噬的影响

DOI: 10.3969/j.issn.1001-5256.2016.11.020

首都医科大学附属北京佑安医院北京市肝病研究所

基金项目:

国家自然科学基金(81361120401)；首都卫生发展科研专项项目(首发2014-1-1151)；肿瘤侵袭和转移机制研究北京市重点实验室开放课题(2015ZLQX05)；

详细信息

中图分类号: R512.62
计量
- 文章访问数: 2010
- HTML全文浏览量: 31
- PDF下载量: 428
- 被引次数: 0
出版历程
- 出版日期: 2016-11-20

Effect of hepatitis B virus rtA181T mutation on autophagy of Huh7 hepatoma cells

Research funding:

摘要

摘要: 目的探讨HBV逆转录酶区A181T（rtA181T）位点突变对Huh7细胞自噬的影响。方法 Huh7细胞分别转染1.3mer HBV野生型质粒（WT组）、1.3mer HBV rtA181T突变型质粒（181T组）及1∶1共转WT和rtA181T突变型质粒（WT+181T组）。Western Blot、免疫荧光检测细胞内HBV表面蛋白（HBs）表达水平,ELISA检测细胞外HBs Ag表达水平;Western Blot分析内源性自噬相关蛋白微管相关蛋白轻链3-Ⅱ（LC3-Ⅱ）及P62表达水平,Real-time PCR检测自噬相关基因Atg5 mRNA、Beclin1 mRNA的转录水平;同时转染绿色荧光蛋白LC3后,免疫荧光检测自噬颗粒。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果免疫荧光观察181T组、WT组及WT+181T组细胞内均有HBs表达,Western Blot结果显示181T组细胞内的HBs表达高于WT组和WT+181T组。ELISA检测3组细胞培养上清的HBs Ag水平差异有统计学意义（F=66.739,P<0.001）,其中18...
- 肝炎病毒,乙型 /
- 突变 /
- 自噬
Abstract: Objective To investigate the effect of reverse transcriptase A181T( rtA181T) mutation in hepatitis B virus( HBV) on autophagy of Huh7 hepatoma cells. Methods Huh7 hepatoma cells were transfected with wild- type( WT) 1. 3mer HBV plasmids( WT group),1. 3mer HBV rt A181 T mutant plasmids( 181 T group),or WT and rt A181 T mutant plasmids at a ratio of 1∶ 1( WT + 181 T group). Western blot and immunofluorescence assay were performed to measure the expression of intracellular HBV surface protein( HBs),and ELISA was used to measure the expression of extracellular HBs Ag. Western blot was used to measure the expression of endogenous autophagy- related proteins microtubule- associated light chain 3- II( LC3- Ⅱ) and P62,and Real time PCR was used to measure the mRNA expression of autophagy- related genes Atg5 and Beclin1. After transfection with green fluorescent protein- LC3,immunofluorescence assay was used to measure autophagy particles. An analysis of variance was used for comparison of continuous data between multiple groups and the LSD- t test was used for further comparison between any two groups. Results Immunofluorescence assay showed that there was expression of HBs in the 181 T group,WT group,and WT + 181 T group,and the 181 T group had significantly higher expression of intracellular HBs than the WT group and WT + 181 T group. The results of ELISA showed that there were significant differences in the level of HBs Ag in cell culture supernatant between the three groups( F = 66. 739,P < 0. 001),and the 181 T group had a significantly lower level of extracellular HBs Ag than the WT group and WT + 181 T group( 0. 111 ± 0. 056 vs 3. 157 ± 0. 490 /2. 240 ± 0. 797,both P < 0. 001). The WT group,181 T group,and WT + 181 T group all showed the aggregation of LC3- II autophagy particles; compared with the WT group and WT + 181 T group,the181 T group had significantly higher expression of LC3- II and significantly lower expression of P62( both P < 0. 05). The 181 T group had significantly higher mRNA expression of autophagy- related genes Atg5 and Beclin1 than the WT group and WT + 181 T group( P < 0. 05).
- hepatitis B virus /
- mutation /
- autophagy

HTML全文

参考文献(18)

[1]YEH CT,CHIEN RN,CHU CM,et al.Clearance of the original hepatitis B virus YMDD-motif mutants with emergence of distinct lamivudine-resistant mutants during prolonged lamivudine therapy[J].Hepatology,2000,31(6):1318-1326.

[2]POLLICINO T,CACCIOLA I,SAFFIOTI F,et al.Hepatitis B virus PreS/S gene variants:pathobiology and clinical implications[J].J Hepatol,2014,61(2):408-417.

[3]YEH CT,CHEN T,HSU CW,et al.Emergence of the rtA 181T/sW 172*mutant increased the risk of hepatoma occurrence in patients with lamivudine-resistant chronic hepatitis B[J].BMC Cancer,2011,11:398.

[4]LI J,LIU Y,WANG Z,et al.Subversion of cellular autophagy machinery by hepatitis B virus for viral envelopment[J].J Virol,2011,85(13):6319-6333.

[5]TAKAMURA A,KOMATSU M,HARA T,et al.Autophagydeficient mice develop multiple liver tumors[J].Genes Dev,2011,25(8):795-800.

[6]TIAN Y,KUO CF,SIR D,et al.Autophagy inhibits oxidative stress and tumor suppressors to exert its dual effect on hepatocarcinogenesis[J].Cell Death Differ,2015,22(6):1025-1034.

[7]GAO G,XU YH,ZHANG FJ,et al.Analysis onrtA 181T mutant in HBV related and hepatocellular carcinoma in patients with liver cirrhosis[J/CD].Chin J Liver Dis:Electronic Edition,2015,7(1):95-101.(in Chinese)高歌,徐永红,张凤娟,等.肝硬化患者HBV rtA 181T突变与肝癌发生相关分析[J/CD].中国肝脏病杂志:电子版,2015,7(1):95-101.

[8]LAI MW,YEH CT.The oncogenic potential of hepatitis B virus rtA 181T/surface truncation mutant[J].Antivir Ther,2008,13(7):875-879.

[9]AHN SH,PARK YK,PARK ES,et al.The impact of the hepatitis B virus polymerase rtA 181T mutation on replication and drug resistance is potentially affected by overlapping changes in surface gene[J].J Virol,2014,88(12):6805-6818.

[10]ZHENG J,JIANG S,LU F.HBV multidrug resistant sW 172*truncated variant possibly involve in the ER stress pathway during hepatocarcinogenesis[J].Jpn J Infect Dis,2016,69(4):306-313.

[11]HUANG LW,XU LM.Relationship between liver diseases and autophagy[J].J Clin Hepatol,2014,30(2):186-188.(in Chinese)黄兰蔚,徐列明.细胞自噬在肝脏疾病中的作用[J].临床肝胆病杂志,2014,30(2):186-188.

[12]CZARNY P,PAWLOWSKA E,BIAIKOWSKA-WARZECHA J,et al.Autophagy in DNA damage response[J].Int J Mol Sci,2015,16(2):2641-2662.

[13]CHEN N,WANG SS,CHEN YH,et al.Effects of exogenous hydrogen sulfide on autophagy of HepG 2 cells[J].J Capit Med Univ,2015,36(1):116-120.(in Chinese)陈宁,王珊珊,陈玉涵,等.外源性硫化氢对肝癌细胞系HepG 2自噬的影响[J].首都医科大学学报,2015,36(1):116-120.

[14]LIN X,LI S,ZHAO Y,et al.Interaction domains of p62:a bridge between p62 and selective autophagy[J].DNA Cell Biol,2013,32(5):220-227.

[15]SIR D,TIAN Y,CHEN WL,et al.The early autophagic pathway is activated by hepatitis B virus and required for viral DNA replication[J].Proc Natl Acad Sci U S A,2010,107(9):4383-4388.

[16]LIU B,FANG M,HU Y,et al.Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation[J].Autophagy,2014,10(3):416-430.

[17]TIAN Y,WANG L,OU JH.Autophagy,a double-edged sword in hepatocarcinogenesis[J].Mol Cell Oncol,2015,2(4):e1004968.

[18]WARNER N,LOCARNINI S.The antiviral drug selected hepatitis B virus rtA 181T/s W172*mutant has a dominant negative secretion defect and alters the typical profile of viral rebound[J].Hepatology,2008,48(1):88-98.

施引文献

资源附件(0)

访问统计