MicroRNA130a通过调节干扰素信号通路及丙酮酸代谢调控HCV及HBV复制的机制研究

发布时间：2017-12-30 17:53

本文关键词：MicroRNA130a通过调节干扰素信号通路及丙酮酸代谢调控HCV及HBV复制的机制研究　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：研究背景:乙肝病毒(HBV)和丙肝病毒(HCV)感染是导致严重肝脏疾病的主要原因之一。目前,全球约有HBV慢性感染者2.57亿人,HCV慢性感染者7100万人,对公共健康造成严重的负担。目前,乙肝的治疗药物主要有干扰素和核苷类似物两种,干扰素治疗应答率低,而核苷类药物治疗易复发。丙肝治疗的传统方法为长效干扰素联合利巴韦林,但毒副作用大,且对HCV基因Ⅰ型病毒应答率低。近年,靶向HCV蛋白酶和聚合酶的直接抗病毒药物(Direct-actingAntivirals,DAAs)的飞速发展极大的提高了抗病毒应答率,但是一些不足之处也逐渐显现,如在HBV/HCV共感染的患者中,DAAs药物易造成HBV的复发,加之DAAs药物价格昂贵,因此,寻找新的抗病毒策略,尤其是能同时抵抗HCV和HBV两种病毒的广谱抗病毒策略,仍然是非常有必要的。保持自身的代谢和激发免疫应答是宿主细胞在受到外界刺激,如病毒感染时,维持内稳态的两个方面。microRNAs在调节宿主代谢和免疫应答中均起着极为重要的作用。有研究显示,在HCV及HBV感染的病人肝组织及体外细胞培养模型中,miR-130a的表达发生明显的改变;同时有研究显示,miR-130a表达的改变对HCV及HBV的复制具有调节作用。我们在研究中发现miR-130a能调节Ⅰ型干扰素及其下游干扰素刺激基因的表达,同时miR-130a靶作用于丙酮酸激酶PKLR,调节PKLR的表达及其催化的丙酮酸生成反应。但是针对miR-130a调节HCV和HBV的复制及宿主免疫是否与丙酮酸代谢存在关联,目前尚不清楚。研究目的:1、探索miR-130a对HCV和HBV复制的影响;2、鉴定miR-130a作用的靶基因;3、阐述miR-130a调控HCV和HBV复制的分子机制。研究内容:第一部分miR-130a通过调节Ⅰ型干扰素信号通路调控HCV复制的研究;第二部分miR-130a与维生素D协同抗HCV作用研究;第三部分miR-130a通过调节靶基因PKLR的表达调控HCV和HBV复制的研究;第四部分PKLR及丙酮酸调控HCV和HBV复制的研究。研究方法:1、将miR-130a模拟物(mimic)转染HCV复制子细胞(Con1b细胞)及JFH1感染的Huh7.5.1细胞(JFH1细胞),使miR-130a高表达,检测miR-130a高表达对内源性IFNα/β生成、干扰素刺激基因(ISGs)表达及HCVRNA复制的影响;在干扰素受体缺陷的细胞U5A中及其母细胞2fTGH中转染miR-130a模拟物,检测两种细胞中,ISGs的表达,明确miR-130a对ISGs表达的影响是否依赖于干扰素与其受体的结合。2.在Con1b及JFH1感染的Huh7.5.1细胞中,加入骨化三醇(Calcitriol,VD的活性形式)处理、转染miR-130a模拟物或转染miR-130a模拟物的同时加入骨化三醇处理,检测骨化三醇、miR-130a对HCV复制、IFNα/β表达的影响,并检测二者是否具有协同作用。3、采用四种预测软件(TargetScan、miRanda、RNAhybrid 及 PITA)对 miR-130a 可能发挥作用的靶基因进行预测,通过双荧光素酶报告基因实验对可能作用的三个靶基因(IL18BP、PKLR及LDLR)进行验证。在HCV复制子细胞OR6、JFH1感染的Huh7.5.1、JFH1感染的人原代肝细胞(PHHs)中或者HBV细胞系HepAD38、HBV 感染的 pNTCP-PHHs、HBV 感染的 pNTCP-Huh7.5.1 细胞中,转染 miR-130a模拟物(mimic)或者 miR-130a表达抑制物(inhibitor)或者 CRISPR/Cas/9gRNA,考察miR-130a过表达、抑制或基因沉默对HCV RNA复制、HBV DNA复制及对PKLR表达的影响。4、靶基因功能研究,通过构建PKLR过表达质粒,将质粒转染细胞进行过表达或者通过小分子干扰RNA(siRNA)或CRISPR/Cas9技术使其沉默,检测PKLR过表达及沉默对HCV及HBV复制的影响。同时考察PKLR催化反应产物丙酮酸对HCV、HBV复制的影响,以及补充丙酮酸是否对miR-130a高表达及PKLR沉默引起的病毒复制的抑制具有营救效果。研究结果:1、JFH1感染的Huh7.5.1及HCV复制子细胞Con1b中,miR-130a过表达后,HCV RNA复制显著降低,Ⅰ型干扰素(IFNα/β)及一些ISGs基因(Mx1,CH25H)的表达显著增加,但miR-130a的表达不受IFNα刺激的影响。此外,在干扰素受体IFNAR2c缺失的U5A细胞中,miR-130a高表达对ISGs基因无上调作用,提示miR-130a对ISGs的调节是通过干扰素信号通路实现的。2、骨化三醇不影响miR-130a的表达;miR-130a过表达及骨化三醇均显著抑制HCV RNA复制,两者具有一定的叠加作用。miR-130a过表达及骨化三醇均显著促进IFNα/β的表达,但在miR-130a过表达的同时加入骨化三醇,IFNα/β的表达低于单独处理组。miR-130a过表达及骨化三醇均显著抑制HCV入胞受体LDLR的表达及HCV的入胞吸附过程,且同时处理具有一定的叠加作用。这些结果提示,miR-130a过表达与骨化三醇的抗病毒叠加效应与Ⅰ型干扰素信号通路无关,很可能是通过调节病毒的吸附入胞实现的。3、通过四种不同的软件对miR-130a可能作用的靶基因进行预测,我们获得2152个潜在的靶基因,其中有534个基因同时由2种以上的软件获得;通过文献检索的方法,我们从中筛选了 116个与病毒感染及肝脏疾病相关的基因进行研究,通过荧光定量PCR的方法,我们发现,在miR-130a高表达的细胞中,PKLR(编码丙酮酸激酶,广泛存在于肝脏及红细胞中),IL18BP(编码白介素18结合蛋白),LDLR(编码低密度脂蛋白受体)三个基因的表达显著降低。对这三个基因,我们进行了双荧光素酶报告基因实验,结果证实PKLR为miR-130a的靶基因,而IL18BP和LDLR不是。miR-130a高表达显著抑制PKLR的表达和HCV、HBV的复制;miR-130a表达抑制或基因沉默显著促进PKLR的表达及HCV和HBV的复制。4、PKLR过表达显著促进HCV及HBV的复制;PKLR基因沉默显著抑制HCV及HBV的复制。无丙酮酸培养基中培养的细胞,当向培养基中加入丙酮酸溶液后,HCV及HBV的复制显著增加,且在miR-130a过表达及PKLR沉默的实验组中加入丙酮酸,丙酮酸对miR-130a高表达及PKLR沉默导致的HCV、HBV复制的抑制具有营救作用。但是PKLR及丙酮酸处理均不影响miR-130a的表达,提示PKLR及丙酮酸位于miR-130a调节通路的下游。研究结论:1、miR-130a过表达显著抑制HCV及HBV的复制;而miR-130a表达抑制或基因沉默显著促进HCV及HBV的复制。miR-130a高表达与骨化三醇对HCV复制的抑制具有叠加效应。2、miR-130a过表达上调IFNα/β的表达,通过与细胞表面的干扰素受体结合,激活下游ISRE及ISGs的表达。但miR-130a与骨化三醇的抗病毒叠加作用与Ⅰ型干扰素信号通路无关。3、PKLR为miR-130a直接作用的靶基因,miR-130a通过调节PKLR的表达,影响其催化的丙酮酸生成过程,进而影响HCV和HBV的复制。4、miR-130a对Ⅰ型干扰素通路的调控与PKLR的表达及丙酮酸生成无显著的关联。
[Abstract]:Background: hepatitis B virus (HBV) and hepatitis C virus (HCV) infection is a major cause of serious liver disease. At present, there are about 257 million people worldwide with chronic HBV infection, chronic HCV infected 71 million people, causing serious burden on public health. At present, there are two kinds of interferon and nucleoside analogues treatment hepatitis B, interferon treatment rate is low, and the antiviral treatment of recurrence. The traditional method for the treatment of hepatitis C interferon combined with Leigh Bhave Lin, but the side effects, and the HCV genotype virus response rate is low. In recent years, targeting direct antiviral HCV protease and polymerase (Direct-actingAntivirals, DAAs) the rapid development greatly improve the antiviral response rate, but some shortcomings appear gradually, such as co infection in HBV/HCV patients, relapse DAAs drugs to cause HBV, DAAs and drug price Therefore, expensive, looking for new antiviral strategies, especially broad-spectrum antiviral strategy can also resist the HCV and HBV two kinds of viruses, is still very necessary. Maintain their metabolism and stimulate the immune response in host cells is stimulated by the outside world, such as viral infection, maintaining the homeostasis of the two aspects in.MicroRNAs regulation of host metabolism and immune response plays a very important role. Studies have shown that in HCV and HBV infection in patients with liver tissue and in vitro cell culture model, the expression of miR-130a changed obviously; while the study showed that the replication of miR-130a expression on the HCV and HBV play a role in the regulation. We found that miR-130a can regulate the expression of type I interferon and its downstream interferon stimulated genes, and miR-130a target effect on pyruvate kinase PKLR, pyruvate formation regulate the expression of PKLR and its catalytic activity Whether the replication and host immune response. But according to the miR-130a regulation of HCV and the presence of HBV is associated with pyruvate metabolism is unclear. Objective: To explore the influence of miR-130a on HCV 1, and HBV replication; 2, target gene identification miR-130a; 3, HCV and miR-130a on regulation of HBV replication on molecular mechanism. Content: the first part is miR-130a through the regulation of type I interferon signaling pathway to regulate HCV replication; Study on synergistic effect of anti HCV second part miR-130a and vitamin D; the third part of miR-130a through the research on the regulation of HCV expression and HBV replication regulation of target gene PKLR; fourth PKLR and pyruvate regulation of HCV and HBV replication study. Methods: 1, the analogue of miR-130a (mimic) transfected with HCV replicon cells (Con1b cells) and JFH1 infected Huh7.5.1 cells (JFH1 cells), the high expression of miR-130a, detection of miR-130a high expression of endogenous IFN alpha Beta / generation, interferon stimulated gene (ISGs) expression and effect of HCVRNA replication and its parent cell 2fTGH; transfection of miR-130a mimics in interferon receptor deficient cells U5A, detection of two kinds of cells, the expression of ISGs, miR-130a clear influence on the expression of ISGs is dependent on interferon receptor binding in Con1b and.2. JFH1 infected Huh7.5.1 cells, with ossification in three alcohol (the active form of VD Calcitriol), transfection of miR-130a mimics or transfected with miR-130a mimics with ossification in three alcohol treatment, detection of ossification in three alcohol, miR-130a of HCV replication, IFN alpha / beta expression, and detect whether the two has a synergistic effect.3, with four kinds of prediction software (TargetScan, miRanda, RNAhybrid and PITA) to predict the target gene miR-130a may play a role, through the dual luciferase assay of three target base effect Because of (IL18BP, PKLR and LDLR) for verification. In the HCV replicon cell OR6, JFH1 infected Huh7.5.1, JFH1 infection of primary human liver cells (PHHs) or HBV cell lines HepAD38, HBV infected pNTCP-PHHs, HBV infection in pNTCP-Huh7.5.1 cells transfected with miR-130a mimics (mimic) or miR-130a expression inhibitor (inhibitor) or CRISPR/Cas/9gRNA, over expression study miR-130a, inhibition or gene silencing on HCV RNA replication, HBV DNA replication and its effect on the expression of PKLR.4 gene function research target, through the construction of PKLR expression plasmid, the plasmid transfected cells by overexpression or by small interfering RNA (siRNA) or CRISPR/Cas9 technology make it silence, detection of PKLR overexpression and silencing of HCV and influence the replication of HBV. The combined effect of PKLR catalytic reaction products of pyruvate on HCV effect of HBV replication, and supplement the high expression of miR-130a and PK on Inhibition of virus replication LR silencing induced with the rescue effect. Results: 1, JFH1 infected Huh7.5.1 and HCV replication in Con1b cells, overexpression of miR-130a, HCV RNA replication significantly decreased (IFN, type I interferon alpha / beta) and ISGs gene (Mx1, CH25H) significantly increased the level of influence, but miR-130a expression was not stimulated with IFN. In addition, the interferon receptor deficiency in IFNAR2c U5A cells, miR-130a high expression up regulation of ISGs gene, suggesting that miR-130a regulation of ISGs by interferon signal pathway.2, ossification in three alcohol does not affect the expression of miR-130a; overexpression of miR-130a and ossification in three alcohol HCV significantly inhibited the replication of RNA, two havesuperimposed..miR-130a overexpression and ossification in three alcohol significantly promote the expression of IFN alpha / beta, but overexpression of adding ossification in three alcohol in miR-130a, IFN alpha / beta expression is lower than the Office The expression of HCV and overexpression of.MiR-130a group and ossification in three alcohol significantly inhibited HCV cell entry into the cell receptor LDLR in the adsorption process, and also dealing with superposition effect. These results suggest that the overexpression of miR-130a and ossification in three alcohol signal pathway of antiviral effect and superposition of type I interferon independent, is likely to be realized by modulating the virus into the cell adsorption of.3 target genes may effect on miR-130a through four different forecasting software, we obtained 2152 potential target genes, including 534 genes at the same time by more than 2 kinds of software; through the method of literature retrieval, we selected from 116 related with the virus infection and liver disease gene study by fluorescence quantitative PCR method, we found that the high expression of miR-130a cells, PKLR (encoding pyruvate kinase, widely exists in the liver and red blood cells), IL 18BP (encoding interleukin 18 binding protein (LDLR), encoding low density lipoprotein receptor) expression of three genes was significantly reduced. The three genes, we performed a dual luciferase reporter assay, the results confirmed that PKLR was the target gene of miR-130a, IL18BP and LDLR are not the high expression of.MiR-130a significantly inhibited the expression of HCV and PKLR, HBV replication; inhibition of miR-130a gene expression and gene silencing significantly promote the replication of.4 PKLR and the expression of HCV and HBV, overexpression of PKLR significantly promoted HCV and HBV replication; PKLR gene silencing significantly inhibited HCV and HBV replication. No pyruvate medium cells when added to the culture solution. Based in HCV, and the replication of HBV increased significantly, and the experimental group added pyruvate expression and PKLR silencing in miR-130a, pyruvate leads to high expression of miR-130a and PKLR silencing HCV, inhibition of HBV replication with rescue. But PKLR and pyruvate treatments did not affect the expression of miR-130a, suggesting that PKLR and pyruvate in miR-130a pathway downstream. Conclusions: 1. The overexpression of miR-130a significantly inhibited the replication of HBV and HCV; and the inhibition of miR-130a gene expression and gene silencing significantly promote the high expression of HCV and HBV and copy.MiR-130a to inhibit the replication of HCV ossification in three alcohol with the superposition effect of.2, miR-130a up-regulated IFN alpha / beta, through the combination of interferon receptor and cell surface expression, and activate the downstream ISRE and ISGs. But the antiviral effect and superposition of type I interferon signaling pathway miR-130a with ossification in three alcohol is independent of the.3, PKLR as the target gene of miR-130a directly, by miR-130a regulating the expression of PKLR, pyruvate formation process affect its catalytic effect, and then copy the.4 HCV and HBV, and the expression of pyruvate and regulation of PKLR miR-130a on type I interferon pathway. There is no significant association.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R373

【相似文献】