miR-127-5p通过直接靶向SCARB2抑制EV71感染

发布时间：2018-02-09 05:59

  本文关键词： 手足口病 肠道病毒71型 SCARB2受体 miR-127-5p 病毒感染　出处：《南京大学》2017年硕士论文　论文类型：学位论文

【摘要】：手足口病是由多种肠道病毒感染引起的一个高度传染性疾病,主要在春季到秋季期间影响着婴幼儿的健康,它的临床表现范围广泛,从轻度和自限性到无菌性脑膜炎,脊髓灰质炎样急性松弛性麻痹,脑干脑炎等死亡率高的罕见致命表现和严重后遗症。手足口病最常见的是由柯萨奇病毒A16感染引起的,其次由肠道病毒71(EV71)感染引起。重要的是,EV71造成的手足口病往往与致命的并发症有关。EV71属于小核糖核酸病毒科家族的单股正链RNA病毒,首先从患有神经系统疾病的感染患者中被分离出来。EV71感染开始于与宿主细胞受体的结合,人类清道夫受体B类成员2(SCARB2)被证明是组织中广泛表达的EV71的受体。作为EV71所有病毒株的受体,SCARB2能够介导病毒结合,内化和脱壳,并且在EV71感染的早期步骤中起到关键作用。MicroRNA(miRNA)是一类在动物,植物和病毒中发现的19至25nt的高度保守的非编码RNA寡核苷酸。这些小RNA主要通过结合靶mRNA上的互补序列来调节特异性基因的表达,以调控其翻译或稳定性。在人类中有数百个miRNA编码基因,其调节蛋白质编码基因,越来越多的证据表明miRNA可以调节广泛的生物过程,包括发育,分化,细胞增殖,细胞凋亡和免疫应答。正链RNA病毒在复制过程的各个步骤中需要招募特定的宿主因子,这些宿主因子有助于病毒基因组复制,病毒蛋白质合成和防御宿主免疫反应。然而,最近有研究证实病毒感染可以改变其宿主细胞中miRNA的表达,并且miRNA可以通过靶向细胞因子来间接调节病毒的复制或通过直接靶向病毒mRNA来影响病毒复制,miRNA被认为在病毒和宿主之间的复杂相互作用的网络中作为一个关键的效应分子。然而,miRNA在EV71复制和发病机制中的作用尚不是很清楚,有研究报道miR-127-5p可以抑制SCARB2的表达,于是我们首先通过生物信息学分析,发现miR-127-5p在SCARB2 mRNA的3'UTR区域存在两个靶结合位点,然后,我们利用荧光素酶报告基因实验证实了 miR-127-5p可以与SCARB2 3'UTR上的两个靶位点结合来影响SCARB2的表达。SCARB2作为EV71的重要受体,于是我们想进一步探究,miR-127-5p在EV71感染过程是否会发挥作用。首先我们发现EV71感染细胞后,胞内miR-127-5p的水平发生了上升,值得注意的是,此时SCARB2总的蛋白水平相应的发生了微小的下调。另外,我们将细胞转染miR-127-5p模拟物后再感染EV71,发现EV71在宿主细胞内的复制和翻译均受到明显的抑制,同时我们用miR-127-5p inhibitor来抑制内源性的miR-127-5p的表达,发现这可以明显的促进接下来病毒的感染。此外,我们也探究了 miR-127-5p是否会与病毒的基因组直接结合而影响了病毒复制,结果表明miR-127-5p不会与病毒基因组结合,并不影响进入后病毒的复制。总之,我们的实验结果表明miR-127-5p通过直接靶向SCARB2 mRNA,介导SCARB2的表达,从而抑制EV71黏附和感染。
[Abstract]:Foot and mouth disease is a highly contagious disease caused by a variety of intestinal virus infection, mainly during the spring to fall affects the health of infants and young children, its clinical manifestations range from mild and self limiting to aseptic meningitis, poliomyelitis like acute flaccid paralysis, a rare fatal manifestation of brainstem encephalitis such a high mortality rate and serious sequelae. HFMD is most commonly caused by coxsackievirus A16 infection, followed by enterovirus 71 (EV71) infection. It is important that EV71 caused HFMD and often fatal complications related to.EV71 belongs to the Picornaviridae family of positive strand RNA virus. First of all from the infected patients suffering from neurological disorders have been separated with.EV71 infection began in the host cell receptor, human scavenger receptor class B member 2 (SCARB2) was shown to be widely in table As the EV71 receptor. All strains as EV71 receptor, SCARB2 can mediate virus binding, internalization and shelling, and in the early steps of EV71 infection plays a key role in the.MicroRNA (miRNA) is a kind of animal, plant and found 19 to 25nt virus in the highly conserved non RNA oligonucleotide encoding. These small RNA mainly through the combination of complementary sequences on target mRNA expression to regulate specific gene regulation, to the translation or stability. There are hundreds of miRNA encoding gene in humans, the regulation of protein encoding genes, there is growing evidence that miRNA can regulate the biological process, including extensive development, differentiation, cell proliferation, apoptosis and immune response. Positive strand RNA virus replication process needs at every step in the recruitment of host specific factors, these host factors contribute to the replication of the virus genome, protein synthesis and anti virus The host immune response. However, recent studies have confirmed that the virus infection can change the expression of miRNA in the host cell, and miRNA by targeting cytokines to indirectly regulate the replication of the virus or by directly targeting mRNA virus to affect viral replication, miRNA is considered as a key effector molecule of complex interactions between the virus and the host in the network. However, the role of miRNA in EV71 replication and pathogenesis are still not very clear, some studies reported that miR-127-5p can inhibit the expression of SCARB2, so we first through bioinformatics analysis, it is found that there are two target binding sites in the 3'UTR region, miR-127-5p SCARB2 mRNA and then we used luciferase reporter gene assays confirmed that miR-127-5p can be combined with the two target sites of SCARB2 3'UTR to SCARB2 as an important effect on the expression of.SCARB2 by EV71 The body, so we want to further explore the miR-127-5p in the course of EV71 infection might play a role. First, we found that EV71 infected cells, intracellular miR-127-5p levels rise, it is worth noting that the total SCARB2 protein level corresponding to the occurrence of tiny fall. In addition, we will re infection of cells transfected with EV71 analogue of miR-127-5p, found that EV71 replication in host cells and translation was inhibited, the expression at the same time we use miR-127-5p inhibitor to inhibit endogenous miR-127-5p, found that it could promote the virus infection. In addition, we also explored whether miR-127-5p directly binds to the viral genome and affect the virus the results showed that miR-127-5p replication does not bind with the viral genome does not affect after entering the replication of the virus. In conclusion, our results suggest that miR-127- 5p mediates the expression of SCARB2 by targeting SCARB2 mRNA directly, thus inhibiting the adhesion and infection of EV71.

【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.5
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本文编号：1497242