丙型肝炎病毒NS4B蛋白调控MMP-2和Bcl-2表达的信号通路研究

发布时间：2018-09-14 18:05

【摘要】：丙型肝炎病毒(HCV)是一种带有包膜的黄病毒科家族RNA病毒,它包含一条长为9600个碱基的正义单链RNA基因组。目前全世界有超过1亿7000万的人遭受着HCV慢性感染,并且它也是导致慢性肝病甚至肝癌的一个主要原因。由于丙肝所引发的疾病负担和人员死亡正成倍增长,丙肝和艾滋病一样,都严重摧残着人类的身心健康,导致严重的公共卫生安全和社会问题。因此,进一步展开丙肝病毒感染引起肝癌的内在分子机制的研究显得尤为迫切和重要。本研究中,我们发现在HCV感染的病人的外周血单个核细胞中,信号转导与转录活化子-3(STAT3),基质金属蛋白酶-2(MMP-2)以及B细胞淋巴因子-2(Bcl-2)的mRNA表达水平相对于健康人群都有显著的上调。通过体外JFH-1亚型的HCV活病毒感染Huh7.5.1细胞实验,结果表明：HCV能刺激STAT3,MMP-2, Bcl-2以及细胞外信号调节激酶(ERK),C-Jun氨基末端激酶(JNK)的表达,并呈现出时间和剂量依赖效应.我们进一步对HCV基因组的10个蛋白进行功能性筛选,发现非结构蛋白4B(NS4B)能激活STAT3信号通路,上调MMP-2以及Bcl-2的表达,同时也能抑制细胞信号转导抑制因子-3(SOCS3)的表达。通过细胞核抽提,荧光共聚焦以及过表达实验,我们发现NS4B蛋白能促进STAT3的入核,显著上调STAT3的磷酸化水平。当我们分别用激酶(PKC, JNK, ERK)的干扰RNA转染细胞后,发现STAT3的磷酸化水平受到抑制,MMP-2以及Bcl-2的表达水平也相应下调。证明PKC, JNK以及ERK在NS4B激活STAT3信号通路中扮演重要角色。此外,由于已有研究表明NS4B的C末端结构域(CTD)与HCV的复制相关,能和细胞因子相互作用,我们进一步研究发现NS4B的CTD能显著激活STAT3, ERK以及JNK,上调MMP-2, BCL-2的表达,其中从第227到第250这24个氨基酸区域尤为重要,同时我们通过点突变实验证实第237,239,245这三个氨基酸残基的改变,能极大的抑制NS4B上调STAT3通路,表明第237,239,245这三个氨基酸残基对NS4B的功能的发挥起着重要作用。由于MMP-2和BCL-2与细胞转化,肿瘤形成有重要关系,本研究阐明了一种新的HCV感染的致病机制,为临床疾病诊断以及新药靶标设计提供了新的思路和实验依据。
[Abstract]:Hepatitis C virus (HCV) is a family of RNA virus with envelope. It contains a sense single-stranded RNA genome with a length of 9600 bases. At present, more than 100 million people worldwide suffer from chronic HCV infection, and it is also a major cause of chronic liver disease and even liver cancer. As the burden of disease and death caused by hepatitis C is increasing exponentially, both hepatitis C and AIDS seriously destroy the physical and mental health of human beings, leading to serious public health safety and social problems. Therefore, it is urgent and important to further study the intrinsic molecular mechanism of liver cancer caused by hepatitis C virus infection. In this study, we found that the expression of signal transduction and transcriptional activator 3 (STAT3), matrix metalloproteinase-2 (MMP-2) and B-cell lymphoid factor-2 (Bcl-2) in peripheral blood mononuclear cells (PBMC) of patients with HCV infection were significantly up-regulated than those of healthy controls. In vitro, HCV subtype of JFH-1 subtype of HCV infected Huh7.5.1 cells. The results showed that HCV could stimulate the expression of STAT3,MMP-2, Bcl-2 and extracellular signal-regulated kinase (ERK) C-Jun amino-terminal kinase (JNK) in a time-and dose-dependent manner. We further screened 10 proteins in the HCV genome and found that non-structural protein 4B (NS4B) could activate the STAT3 signaling pathway, up-regulate the expression of MMP-2 and Bcl-2, and inhibit the expression of cell signal transduction inhibitor 3 (SOCS3). Through nuclear extraction, fluorescence confocal and overexpression experiments, we found that NS4B protein can promote the entry of STAT3 and significantly up-regulate the phosphorylation level of STAT3. After transfection of RNA with kinase (PKC, JNK, ERK) interference, we found that the phosphorylation level of STAT3 was inhibited and the expression of MMP-2 and Bcl-2 were down-regulated. It is demonstrated that PKC, JNK and ERK play an important role in the activation of STAT3 signaling pathway by NS4B. In addition, it has been shown that the C-terminal domain (CTD) of NS4B is related to the replication of HCV and can interact with cytokines. We further found that CTD of NS4B can significantly activate STAT3, ERK and JNK, upregulate the expression of MMP-2, BCL-2. The 24-amino acid region from 227th to 250th is particularly important, and we confirmed by point mutation experiment that the change of the three amino acid residues of 237239245 can significantly inhibit the up-regulation of STAT3 pathway by NS4B. These three amino acid residues 237239245 play an important role in the function of NS4B. Because MMP-2 and BCL-2 have important relationship with cell transformation and tumor formation, this study clarifies a new mechanism of HCV infection, and provides new ideas and experimental basis for clinical disease diagnosis and new drug target design.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R373

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