丙型肝炎病毒感染人肝细胞系对多种唾液酸转移酶和半乳糖基转移酶表达的影响

摘要

目的 探讨丙型肝炎病毒(hepatitis C virus,HCV)感染人肝癌细胞系Huh7.5.1细胞后与肿瘤相关的唾液酸转移酶家族和半乳糖基转移酶家族的35个糖基转移酶的表达变化.方法 利用HCV感染的Huh7.5.1肝细胞模型,采用Western blot检测HCV感染后HCV非结构蛋白NS3的表达,定量即时聚合酶链锁反应(quantitative real-time polymerase chain reaction,qRT-PCR)检测HCV感染后HCV-RNA的表达及HCV感染对Huh7.5.1细胞中35个糖基转移酶mRNA表达水平的影响.凝集素染色实验测定HCV感染对Huh7.5.1细胞中糖苷表达水平的影响.结果 在HCV感染后的Huh7.5.1 细胞中,发现4 个唾液酸转移酶(ST3Gal Ⅲ、ST6GalNAC Ⅲ、ST8Sia Ⅲ和ST8Sia Ⅳ)和5个半乳糖基转移酶(B3GALT 1、B3GALT 2、B3GALT 3、B3GALT 4和B4GALNT 2)的mRNA水平明显升高11~45倍,其中变化最大的是B3GALT 1,上升45倍,其次是ST8Sia Ⅲ和ST8Sia Ⅳ,分别上升37倍和39倍.进一步采用凝集素染色实验验证,发现结合末端唾液酸糖苷Neu5Acα6Gal的接骨木凝集素(elderberry lectin,EBL)在HCV感染后升高1.97倍,结合末端半乳糖苷Galβ1,3GalNAc的尾穗苋凝集素(amaranthus caudatus agglutinin,ACA)和结合Galβ1,4GalNAc的鸡冠刺桐凝集素(erythrina cristagalli agglutinin,ECA)分别升高3倍和4.3倍.结论 HCV感染导致人肝细胞中特定唾液酸转移酶和半乳糖基转移酶及其相应糖苷表达水平呈现明显上升.这些结果对研究HCV感染的治疗靶点和诊断标志物具有一定参考价值.%Objective To investigate the altered glycotransferases and glycan profile in HCV-infected cells, the expression levels of 35 kinds of sialyltransferases and galactosyltransferases were evaluated in hepatitis C virus (HCV) infected Huh7.5.1 cell cultured model.Methods Western blot was used to measure the expressions of HCV-nonstructural protein NS3 and and quantitative real-time PCR (qRT-PCR) was performed to determine HCV-RNA levels and mRNA levels of 35 kinds of sialyltransferases and galactosyltransferases.Lectin microarray was used to compare the glycan profiles of sialyltransferases-and galactosyltransferases-associated glycan-profile in Huh7.5.1 cells and HCV infected Huh7.5.1 cells.Results Among the 35 kinds of sialyltransferases and galactosyltransferases, the mRNA levels of four sialyltransferases (including ST3Gal Ⅲ, ST6GalNAC Ⅲ, ST8Sia Ⅲ and ST8SiaⅣ) and five galactosyltransferases (including B3GALT1, B3GALT 2, B3GALT3, B3GALT4 and B4GALNT2) were found to be 11-45 fold higher in HCV-infected cells compared with naive Huh7.5.1 cells.The up-regulated level of B3GALT 1 was the most evident (45-fold change), followed by ST8Sia Ⅳ and ST8Sia Ⅲ, with 39-and 37-fold respectively.Consistently, lectin microarray showed that glycans recognized by EBL (binding terminal sialic acid, Neu5Acα6Gal, 1.97-fold change), ECA letin (binding terminal galactose, Galβ1,4GalNAc,4.3-fold change), and ACA(binding terminal galactose, Galβ1-3GalNAc, 3-fold change) were elevated in glycoprotein products of sialyltransferase and galactosyltransferase respectively.Conclusion HCV infection causes the increased expression levels of mutiple sialyltransferases and galactosyltransferases in Huh7.5.1 cell line and hence the upregulation of glycan profiles of the glycoproteins.These results provide potential therapeutic targets and diagnostic markers for HCV infection and insights on the molecular mechanism of HCV infection.