EB病毒及其立早蛋白Rta对干扰素通路的转录调控机制

发布时间：2018-07-05 08:13

本文选题：EB病毒 + 立即早期蛋白Rta　；参考：《南京医科大学》2014年博士论文

【摘要】：EB病毒感染在儿童期多表现为传染性单核细胞增多症(Infectious mononucleosis, IM),部分可演变为严重的嗜血细胞综合症及淋巴瘤。干扰素调节因子3(interferon regulatory factor3, IRF-3)是调节I型干扰素(IFN-a/p)基因表达的关键转录因子,在固有免疫反应和获得性免疫反应中起重要作用。但是EB病毒调控机体干扰素表达以逃避宿主免疫的具体机制尚不十分清楚。本研究通过Real time PCR法检测临床EB病毒感染的IM病人与健康对照组相关因子差异表达,发现IM病人中立即早期蛋白复制与转录激活子(replication and transcription activator, Rta)、miRNA146a、miRNA23a、miRNA24表达显著高于健康对照组,而IRF-3及IFNβ表达显著低于健康对照组。为探索Rta与miRNA是否调控IRF-3及IFNβ表达及相关机制,我们将Rta过表达质粒与含IRF-3启动子区不同片段的萤光素酶报告质粒共转染于HeLa细胞,发现Rta对含-149至-93bp片段的IRF-3启动子重组质粒萤光素酶活性影响较显著。既往研究报道,转录因子E2F1可与IRF-3启动子-149/-93bp区域内E2F1位点结合,抑制IRF-3转录表达。本研究通过E2F1结合位点点突变、过表达E2F1、RNA干扰E2F1表达及染色质免疫沉淀实验证实,在HeLa细胞中Rta可通过上调E2F1表达、增加其与IRF-3启动子区的结合下调IRF-3的表达。通过miRNA过表达及干扰,发现miRNA146a、miRNA23a、miRNA24可下调IRF-3mRNA表达,而miRNA146a可下调IRF-3蛋白表达。我们分别将miRNA146a、 miRNA23a、miRNA24与IRF-3启动子重组萤光素酶报告质粒共转染,发现启动子活性无显著降低,提示上述miRNA并非通过启动子区发挥负性调控作用。进一步实验发现miRNA146a可下调IFNβ信号通路上游分子TRAF6、IRAK1、 NF-κB等表达。通过TRAF6过表达及干扰实验证实,TRAF6在miRNA146a调控IFNβ表达中起重要介导作用。丙戊酸(Valproic acid, VPA)作为组蛋白去乙酰化酶抑制剂,不同细胞中均可上调IRF-3mRNA及蛋白表达,深入研究发现丙戊酸可通过增加组蛋白H3、H4的乙酰化促进IRF-3转录,增加IRF-3基因表达,而免疫荧光实验进一步证实丙戊酸可明显增加293T细胞核磷酸化IRF-3的表达。本研究发现：Rta可通过上调E2F1表达、增加其与IRF-3启动子区的结合从而抑制IRF-3的表达；miRNA146a通过TRAF6抑制IFNβ转录表达；丙戊酸可通过增加组蛋白乙酰化促进IRF-3的转录表达及磷酸化。本研究拓展了EB病毒逃避宿主免疫机制的探索,完善了EB病毒诱导Rta及miRNA调控IRF-3的分子机制,且为今后临床治疗EB病毒相关疾病提供实验依据。
[Abstract]:Epstein-Barr virus (EBV) infection in childhood is characterized by infectious mononucleosis (IM), which can develop into severe haemophilic syndrome and lymphoma. Interferon regulatory factor 3 (interferon regulatory factor3 (IRF-3) is a key transcription factor that regulates the expression of IFN-ap gene and plays an important role in innate and acquired immune responses. However, the specific mechanism of EB virus regulating interferon expression to escape host immunity is unclear. In this study, the differential expression of related factors in IM patients infected with Epstein-Barr virus (EBV) and healthy controls was detected by Real time PCR. It was found that the expression of miRNA146ahmiRNA23aRNA24 in IM patients was significantly higher than that in healthy controls, and the expression of immediate early protein replication and activator of transcription (replication and transcription activator, Rta) in IM patients was significantly higher than that in healthy controls. The expression of IRF-3 and IFN- 尾 was significantly lower than that of healthy control group. In order to investigate whether Rta and miRNA can regulate the expression of IRF-3 and IFN- 尾, we co-transfected RTA overexpression plasmid and luciferase reporter plasmid containing different fragments of IRF-3 promoter into HeLa cells. It was found that RTA had a significant effect on the luciferase activity of the recombinant plasmid of IRF-3 promoter containing -149 to -93 BP fragments. It has been reported that transcription factor E2F1 can bind to the E2F1 site of IRF-3 promoter -149 / 93bp and inhibit the expression of IRF-3 transcription. In this study, the overexpression of E2F1RNA interfering with E2F1 expression and chromatin immunoprecipitation assay demonstrated that Rta could up-regulate the expression of E2F1 and down-regulate the expression of IRF-3 by up-regulating the expression of E2F1 in HeLa cells. By over-expression and interference of miRNA, it was found that miRNA146a miRNA23a miRNA24 could down-regulate the expression of IRF-3 mRNA, while miRNA146a could down-regulate the expression of IRF-3 protein. We cotransfected the recombinant luciferase reporter plasmids of miRNA146a, miRNA23ahmiRNA24 and IRF-3 promoter, and found that the promoter activity was not significantly decreased, suggesting that the miRNA does not play a negative regulatory role through the promoter region. It was further found that miRNA146a could down-regulate the expression of TRAF6- IRAK1 and NF- 魏 B upstream molecules in IFN- 尾 signaling pathway. The overexpression of TRAF6 and interference experiments confirmed that TRAF6 plays an important role in the regulation of IFN- 尾 expression by miRNA146a. Valproic acid (VPA), as a histone deacetylase inhibitor, can up-regulate the expression of IRF-3 mRNA and protein in different cells. It has been found that valproic acid can promote the transcription of IRF-3 and increase the expression of IRF-3 by increasing the acetylation of histone H3H4. Immunofluorescence assay further confirmed that valproic acid could significantly increase the expression of phosphorylated IRF-3 in 293T nucleus. In this study, we found that: Rta could inhibit the expression of IRF-3 by up-regulating the expression of E2F1, increasing its binding to IRF-3 promoter region and inhibiting the transcription of IFN- 尾 by TRAF6, and valproic acid could promote the transcription and phosphorylation of IRF-3 by increasing histone acetylation. This study expanded the mechanism of EBV evading host immunity, improved the molecular mechanism of EBV induced Rta and miRNA regulating IRF-3, and provided experimental evidence for clinical treatment of EBV related diseases.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R725.1

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