丙型肝炎病毒感染促进53BP1蛋白通过泛素-蛋白酶体途径降解的研究

Hepatitis C virus infection promotes the ubiquitination and degradation of 53BP1

目的探讨丙型肝炎病毒(Hepatitis C virus,HCV)对53BP1表达的影响及机制。方法建立HCV体外感染Huh7细胞模型,采用Western blot检测HCV感染对53BP1总蛋白、胞浆和胞核蛋白表达的影响,qPCR检测53BP1的mRNA表达,采用免疫荧光(Immunofluorescence,IF)方法检测HCV感染对53BP1亚细胞定位的影响;采用免疫组织化学(immunohistochemistry,IHC)法定量检测HCV相关肝癌中53BP1蛋白的表达,并与癌旁组织及正常肝脏相比较;采用免疫共沉淀(Co-Immunoprecipitation,CO-IP)方法检测HCV对53BP1蛋白泛素化水平的影响。结果HCV感染导致Huh7细胞53BP1蛋白的相对表达水平为(0.41±0.03),明显低于对照组(1.00±0.08),差异有统计学意义(P<0.01);在HCV感染后,胞核53BP1蛋白相对表达水平为(0.35±0.03),明显低于对照组(1.00±0.09),差异有统计学意义(P<0.01);而胞浆53BP1相对表达水平为(1.20±0.18),对照组相对表达水平为(1.00±0.17),两者表达无显著改变(P>0.05);HCV感染可导致53BP1蛋白,尤其是胞核53BP1蛋白的表达下降。免疫荧光结果显示HCV感染导致53BP1从胞核到胞浆的亚细胞定位改变;免疫组化结果显示,与正常肝脏相比,53BP1在HCV相关HCC的癌组织(P<0.01)和癌旁组织(P<0.05)中显著降低。机制分析表明HCV感染促进53BP1的泛素-蛋白酶体途径降解。结论HCV感染可致Huh7细胞53BP1发生由细胞核到细胞质的定位改变,并促进其通过泛素-蛋白酶体途径降解。

Objective To investigate the effect of hepatitis C virus(HCV)infection on the expression of 53BP1 and its mechanism.Methods Total,cytoplasmic,and nuclear 53BP1 protein after HCV infection were detected by western blotting.The expression of 53BP1 in normal liver and HCV-related HCC tissues was quantified by immunohistochemistry.The subcellular localization of 53BP1 cells were detected by immunofluorescence staining and the ubiquitination level of 53BP1 was determined by Co-Immunoprecipitation.Results The relative expression level of 53BP1 protein in Huh7 cells caused by HCV infection was(0.41±0.03),significantly lower than that in the control group(1.00±0.08),the difference was statistically significant(P<0.01);After HCV infection,the relative expression level of nuclear 53BP1 protein was(0.35±0.03),significantly lower than that in the control group(1.00±0.09),the difference was statistically significant(P<0.01).The relative expression level of cytoplasmic 53BP1 was(1.20±0.18)and that of control group was(1.00±0.17);There was no significant change in the expression of both groups(P>0.05).HCV infection can decrease the expression of 53BP1 protein,especially in the nucleus.Immunofluorescence showed that HCV infection could lead to a striking re-localization of 53BP1 from nucleus to cytoplasm.Compared with the normal liver tissues,significantly lower levels of 53BP1 were found in the HCC tumor(P<0.01)and adjacent tissues(P<0.05).Mechanism analysis showed that HCV infection promoted the ubiquitination and degradation of 53BP1.Conclusion HCV infection shifts the localization of 53BP1 from nucleus to cytoplasm and promote its ubiquitination and degradation.