EZH2、UTX与天然产物EGCG在HIV-1转录激活作用中的研究
发布时间：2018-12-18 21:26
【摘要】：Ⅰ型人免疫缺陷病毒（HIV-1）编码的反式激活蛋白（Tat）可通过多种途径激活病毒的基因转录，组蛋白H3K27甲基化酶EZH2和去甲基化酶UTX在肿瘤形成、细胞分化和器官形成、病毒感染复制和潜伏感染等方面发挥重要作用。表没食子儿茶素没食子酸酯（EGCG）是绿茶中的主要茶多酚成分，具有抗氧化、抗炎、抗病毒的特性。本课题主要研究EZH2、UTX在Tat介导的HIV-1病毒长末端重复序列（LTR）转录激活的作用及其潜在机制。同时探究了EGCG抑制Tat介导的LTR转录激活的作用及途径。 应用质粒定点突变技术，构建了一系列包括酶失活在内的质粒突变体；应用多核活化半乳糖苷酶指示剂（MAGI）实验检测转染的Tat质粒对TZM-bl细胞中LTR的激活程度。应用实时定量PCR（RT-PCR）和western blot技术检测mRNA和蛋白表达水平。 基因测序显示质粒突变体构建成功；使用siRNA干扰EZH2的表达或EZH2的抑制剂DZNep增强了Tat蛋白介导的LTR的激活作用；细胞内过表达EZH2质粒及其突变体EZH2S21A、EZH2H694A则减弱了Tat蛋白这种作用。使用siRNA干扰UTX的表达减弱了Tat蛋白介导的LTR激活作用，，细胞内过表达UTX质粒增强了Tat蛋白的这种作用。Tat质粒转染不影响TZM-bl细胞中EZH2和UTX的mRNA水平，Tat质粒转染分别下调了TZM-bl细胞中EZH2的蛋白水平、H3K27me2和H3K27me3水平；Tat质粒上调了UTX蛋白水平；Tat突变体Tat-C30G也有较弱的类似功能。同时发现EGCG能够上调细胞核内Nrf2蛋白水平，激活AMPK信号通路，抑制AKT信号通路，抑制了Tat介导的HIV-1LTR的转录激活。对EZH2、UTX、天然产物EGCG与Tat介导的转录激活关系的研究可以使我们对HIV-1的致病和潜伏感染机理有更深入的认识，从而为HIV-1的治疗方法和药物筛选提供新的启示。
[Abstract]:The transactivator protein (Tat) encoded by human immunodeficiency virus type I (HIV-1) can activate the gene transcription of the virus in a variety of ways. Histone H3K27 methylase EZH2 and demethylase UTX are involved in tumor formation, cell differentiation and organ formation. Viral infection replication and latent infection play an important role. Epigallocatechin gallate (EGCG) is the main tea polyphenol in green tea, which has the characteristics of antioxidation, anti-inflammation and anti-virus. In this study, we investigated the role of EZH2,UTX in the transcriptional activation of long terminal repeat (LTR) of HIV-1 virus mediated by Tat and its potential mechanism. At the same time, we explored the role and pathway of EGCG in inhibiting Tat mediated activation of LTR transcription. A series of plasmid mutants including enzyme inactivation were constructed by site-directed mutagenesis, and the activity of LTR in TZM-bl cells was detected by polynuclear activated galactosidase indicator (MAGI) assay. Real-time quantitative PCR (RT-PCR) and western blot techniques were used to detect the expression of mRNA and protein. Gene sequencing showed that the plasmid mutants were successfully constructed, that the expression of EZH2 was interfered by siRNA or that DZNep, the inhibitor of EZH2, enhanced the activation of LTR mediated by Tat protein, and that the overexpression of EZH2 plasmid and its mutant EZH2S21A,EZH2H694A weakened the effect of Tat protein. Using siRNA to interfere the expression of UTX attenuated the activation of LTR mediated by Tat protein, and the overexpression of UTX plasmid enhanced the effect of Tat protein. Tat plasmid transfection did not affect the mRNA levels of EZH2 and UTX in TZM-bl cells. The levels of EZH2 protein, H3K27me2 and H3K27me3 in TZM-bl cells were down-regulated by Tat plasmid transfection. Tat plasmid upregulated the level of UTX protein and Tat mutant Tat-C30G had weak similar function. At the same time, it was found that EGCG could up-regulate the level of Nrf2 protein in the nucleus, activate the AMPK signaling pathway, inhibit the AKT signal pathway, and inhibit the transcriptional activation of HIV-1LTR mediated by Tat. The study of the relationship between EZH2,UTX, natural product EGCG and transcriptional activation mediated by Tat can give us a deeper understanding of the pathogenesis and latent infection mechanism of HIV-1 and provide new inspiration for the treatment of HIV-1 and drug screening.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.91
本文编号：2386523
[Abstract]:The transactivator protein (Tat) encoded by human immunodeficiency virus type I (HIV-1) can activate the gene transcription of the virus in a variety of ways. Histone H3K27 methylase EZH2 and demethylase UTX are involved in tumor formation, cell differentiation and organ formation. Viral infection replication and latent infection play an important role. Epigallocatechin gallate (EGCG) is the main tea polyphenol in green tea, which has the characteristics of antioxidation, anti-inflammation and anti-virus. In this study, we investigated the role of EZH2,UTX in the transcriptional activation of long terminal repeat (LTR) of HIV-1 virus mediated by Tat and its potential mechanism. At the same time, we explored the role and pathway of EGCG in inhibiting Tat mediated activation of LTR transcription. A series of plasmid mutants including enzyme inactivation were constructed by site-directed mutagenesis, and the activity of LTR in TZM-bl cells was detected by polynuclear activated galactosidase indicator (MAGI) assay. Real-time quantitative PCR (RT-PCR) and western blot techniques were used to detect the expression of mRNA and protein. Gene sequencing showed that the plasmid mutants were successfully constructed, that the expression of EZH2 was interfered by siRNA or that DZNep, the inhibitor of EZH2, enhanced the activation of LTR mediated by Tat protein, and that the overexpression of EZH2 plasmid and its mutant EZH2S21A,EZH2H694A weakened the effect of Tat protein. Using siRNA to interfere the expression of UTX attenuated the activation of LTR mediated by Tat protein, and the overexpression of UTX plasmid enhanced the effect of Tat protein. Tat plasmid transfection did not affect the mRNA levels of EZH2 and UTX in TZM-bl cells. The levels of EZH2 protein, H3K27me2 and H3K27me3 in TZM-bl cells were down-regulated by Tat plasmid transfection. Tat plasmid upregulated the level of UTX protein and Tat mutant Tat-C30G had weak similar function. At the same time, it was found that EGCG could up-regulate the level of Nrf2 protein in the nucleus, activate the AMPK signaling pathway, inhibit the AKT signal pathway, and inhibit the transcriptional activation of HIV-1LTR mediated by Tat. The study of the relationship between EZH2,UTX, natural product EGCG and transcriptional activation mediated by Tat can give us a deeper understanding of the pathogenesis and latent infection mechanism of HIV-1 and provide new inspiration for the treatment of HIV-1 and drug screening.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.91
【参考文献】
相关期刊论文 前1条
1 滕竞飞;潘卫;;HIV-1 Tat蛋白的生物学特性及其致病效应[J];中国生物制品学杂志;2009年04期
本文编号：2386523
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