HBV DNA聚合酶在介导HBV相关肝细胞癌肿瘤细胞免疫逃逸中的作用  

Role of HBV DNA polymerase in mediating the immune escape of tumor cells in HBV-related hepatocellular carcinoma

作　　者：李鸿侠 孙一萌 张鸿涛 韩树旺 张德林 尚海涛 郭午 刘军舰 李忠廉 LI Hongxia;SUN Yimeng;ZHANG Hongtao;HAN Shuwang;ZHANG Delin;SHANG Haitao;GUO Wu;LIU Junjian;LI Zhonglian(Department of Hepatopancreatobiliary Surgery,Tianjin Hospital of Integrated Traditional Chinese and Western Medicine,Tianjin 300100,China;Graduate School of Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]天津市中西医结合医院肝胆胰外科,天津300100 [2]天津医科大学研究生院,天津300070

出　　处：《临床肝胆病杂志》2023年第12期2858-2866,共9页Journal of Clinical Hepatology

基　　金：国家自然科学基金面上项目(81273952);天津市卫生健康委员会中医中西医结合科研课题(2021042);天津市教委科研计划项目(2022KJ271)。

摘　　要：目的本研究旨在明确HBV DNA聚合酶是否与T淋巴细胞功能衰竭相关,进而介导HBV相关肝细胞癌(HCC)肿瘤细胞的免疫逃逸以及探寻具体的分子机制。方法稳定转染带有Flag标签的HBV DNA聚合酶表达质粒(FlagHBV-P)和细胞间黏附分子-1(ICAM1)的肝癌细胞系Huh7、HepG2与Jurkat细胞共培养,MTT、qRT-PCR、酶联免疫吸附测定(ELISA)实验分别检测Jurkat细胞增殖、活化(CD69表达)以及细胞因子IFN-γ分泌情况。RNA-seq筛选稳转细胞系与对照细胞中表达差异的免疫相关分子,mRNA半衰期及蛋白半衰期实验确定HBV DNA聚合酶对免疫相关分子具体在何种水平产生影响。网站预测此免疫相关分子启动子区域可能结合的转录因子,Western Blot实验验证转录因子对免疫相关分子的影响,挽救实验确定HBV DNA聚合酶是否通过此转录因子影响免疫相关分子的表达水平。两组间比较采用成组t检验。结果与对照组相比,实验组细胞(Huh7、HepG2)增殖、活化及细胞因子分泌明显降低(P值均<0.01)。与对照细胞相比,实验组细胞(Huh7、Hep G2)的ICAM1 mRNA和蛋白水平均降低(P值均<0.01)。网站预测ICAM1启动子并初步筛选锚定了NFKB1、RELA和STAT3。与对照组相比,实验组细胞(Huh7、Hep G2)p65蛋白的表达水平明显降低(P值均<0.01)。过表达p65后,ICAM1蛋白表达水平明显升高,降表达p65后,ICAM1蛋白表达水平明显降低(P值均<0.01),挽救实验中过表达p65后,对照组与实验组细胞的ICAM1表达水平无明显差异(P值均>0.05)。过表达ICAM1后,对照组与实验组细胞(Huh7、Hep G2)的增殖、活化及细胞因子分泌无明显差异(P值均>0.05)。结论HBV DNA聚合酶通过抑制NF-κB中p65的表达来下调ICAM1的水平以介导HCC免疫逃逸。Objective To determine whether HBV DNA polymerase is associated with T-cell failure and thus mediates the immune escape of HBV-related hepatocellular carcinoma(HCC)tumor cells,and to investigate the specific molecular mechanisms.Methods Liver cancer cell lines Huh7 and HepG2 stably transfected with HBV DNA polymerase expression plasmid with Flag(Flag-HBV-P)and intercellular adhesion molecule-1(ICAM1)were co-cultured with Jurkat cells,and MTT assay,qRT-PCR,and ELISA were used to measure Jurkat cell proliferation,activation(CD69 expression),and secretion of the cytokine IFN-γ.RNA-seq was used to screen for differentially expressed immune-associated molecules between stably transfected cell lines and control cells,and mRNA half-life and protein half-life assays were used to determine the specific levels of the immune-associated molecules that were affected by HBV DNA polymerase.Related websites were used to predict the transcription factors that may bind to the promoter region of this immune-associated molecule,Western blot was used to verify the effect of transcription factors on the immune-associated molecule,and rescue experiment was used to determine whether HBV DNA polymerase affects the expression level of the immune-associated molecule through this transcription factor.The independent-samples t test was used for comparison between two groups.Results The experimental group had significant reductions in Jurkat cell proliferation,activation,and cytokine secretion compared with the control group(all P<0.01).Compared with the control group,the experimental group(Huh7 and HepG2 cell lines)had significant reductions in the mRNA and protein expression levels of ICAM1(all P<0.01).Website prediction identified the ICAM1 promoter and preliminarily highlighted NFKB1,RELA,and STAT3.Compared with the control group,the experimental group(Huh7 and HepG2 cell lines)had a significant reduction in the protein expression level of p65(all P<0.01).After p65 overexpression,there was a significant increase in the protein expression

关 键 词：癌 肝细胞 肿瘤逃逸 指导DNA的DNA聚合酶 细胞黏附分子 

分 类 号：R735.7[医药卫生—肿瘤]