microRNA-101通过靶定ATP5B调控HSV-1的复制

发布时间：2018-05-03 13:21

本文选题：微小RNA + miR-101　；参考：《天津医科大学》2011年硕士论文

【摘要】：目的:微小RNA(microRNA,miRNA)是一类非编码调控性单链小分子RNA,能够与特异性的靶mRNA通过碱基配对结合,对靶基因进行转录后水平调控。近年来的研究表明miRNA不仅参与正常细胞功能的调控,也在病毒与宿主之间的作用以及病毒复制等方面发挥着重要的作用。单纯疱疹病毒Ⅰ型(Herpes simpleX virus type 1,HSV-1)是一种DNA病毒,它可以引起口唇疱疹等疾病,并可以潜伏在三叉神经节形成潜伏感染。目前已经发现疱疹病毒本身可以编码病毒的miRNA,而且其表达的改变对病毒自身的复制有一定的作用,但宿主miRNA与病毒复制的关系并不是很明确,此研究的目的即探寻是否有宿主的miRNA参与了病毒复制的调控过程,这一调控过程是通过哪些基因实现的。方法:首先在HeLa细胞中,过表达和封闭miR-101后,利用病毒滴定空斑形成实验和real-time PCR分别检测病毒复制能力的变化。而后利用生物信息学方法筛选出miR-101的候选靶基因,并利用荧光报告载体实验验证miR-101对靶基因的直接调控作用。利用real-time PCR和western blot实验技术检测miR-101对靶基因mRNA和蛋白水平的表达变化。然后过表达靶基因的全长,进行挽救实验,进一步验证miR-101对靶基因的调控作用。利用RNA干扰技术敲除该靶基因后,通过病毒滴定空斑形成实验和real-time PCR实验检测靶基因对病毒复制的影响,进一步研究了该靶基因的功能。最后用real-time PCR检测HSV-1感染HeLa细胞后内源的miR-101和ATP5B的表达水平。结果:在HeLa细胞中,过表达miR-101后,病毒的复制明显受到抑制,病毒空斑的形成明显降低,病毒的DNA聚合酶基因的水平也明显降低。封闭miR-101后,病毒的复制增强,病毒的DNA聚合酶基因的水平也增加。靶基因筛选结果表明,ATP5B是miR-101的候选靶基因,其3'非翻译区包含miR-101的潜在结合位点。荧光报告载体实验证实,miR-101能够通过作用于ATP5B基因的3'非翻译区对其表达进行负性调节。在HeLa细胞中过表达miR-101,ATP5B的mRNA和蛋白水平都有明显降低;封闭miR-101后,ATP5B的mRNA和蛋白水平升高。此外,过表达不含3'UTR的ATP5B基因可以减弱miR-101对病毒增殖的抑制作用,病毒复制能力得到恢复。同时,在HeLa细胞中,敲降ATP5B后,病毒空斑的形成和病毒的DNA含量明显降低,与过表达miR-101相一致。在HSV-1感染HeLa细胞后,随着感染时间延长,miR-101的水平逐渐增加,其靶基因ATP5B的水平逐渐降低,二者呈现负相关性。结论:miRNA作为细胞基因转录后水平调节的内源性分子,参与调控病毒与宿主细胞的相互作用。本研究首次发现宿主细胞编码的miR-101具有抗病毒作用。miR-101通过降低靶基因ATP5B的表达而抑制HSV-1在HeLa细胞中增殖。在HSV-1感染HeLa细胞后,miR-101的水平逐渐增加,其靶基因ATP5B的水平逐渐降低,说明HSV-1可以通过抑制宿主的miRNA抗病毒作用来维持自身的增殖。首次确定miR-101及其靶基因对HSV-1复制的调控作用,有利于我们对病毒感染的发生和发展进行深入的了解,同时也为病毒感染的诊断和治疗提供新依据。
[Abstract]:Objective: RNA (microRNA, miRNA) is a kind of non coding regulatory single strand small molecule RNA, which can be combined with specific target mRNA through base pairs to regulate the target gene after transcriptional level. In recent years, the research shows that miRNA not only participates in the regulation of normal cell function, but also the role of the virus and the host and the replication of the virus. The herpes simplex virus type I (Herpes simpleX virus type 1, HSV-1) is a DNA virus that can cause diseases such as lip herpes and lurk in the trigeminal ganglion. The herpes virus itself has been found to be able to codec miRNA, and its expression changes from the virus to the virus. The reproduction of the body has a certain effect, but the relationship between the host miRNA and the virus replication is not very clear. The purpose of this study is to explore whether the host miRNA participates in the regulation process of the virus replication. This regulation process is realized through which genes. First, after overexpressing and blocking the miR-101 in the HeLa cells, the virus drops are used. The plaque formation experiment and real-time PCR were used to detect the changes in the replication ability of the virus. Then the candidate target genes of miR-101 were screened by bioinformatics, and the direct regulation of miR-101 on the target gene was verified by the fluorescence report carrier experiment. The real-time PCR and Western blot experimental techniques were used to detect the mRN of the target gene to the target gene. The expression changes of A and protein levels. Then overexpress the full length of the target gene, carry out the rescue experiment, further verify the regulatory role of miR-101 on the target gene. After the target gene was knocked out by RNA interference technique, the effect of the target base on the replication of the virus was examined by the virus titrated plaque formation experiment and the real-time PCR test. The function of the target gene. Finally, real-time PCR was used to detect the expression level of miR-101 and ATP5B in HSV-1 infected HeLa cells. Results: in HeLa cells, the replication of the virus was obviously inhibited after the miR-101 was overexpressed, the formation of the virus's plaque was obviously reduced and the level of the virus DNA polymerase gene decreased obviously. After closed miR-101, the virus was closed to the virus. The replication enhancement, the level of the virus DNA polymerase gene also increased. Target gene screening results showed that ATP5B was a candidate target gene for miR-101, and its 3'non translation region contained miR-101 potential binding site. The fluorescence report carrier experiment confirmed that miR-101 could negatively regulate its expression through the 3' non translation region acting on the ATP5B gene. The overexpression of miR-101 in La cells, the level of mRNA and protein in ATP5B decreased significantly, and the mRNA and protein level of ATP5B increased after blocking miR-101. In addition, the overexpression of the ATP5B gene without 3'UTR could weaken the inhibitory effect of miR-101 on the proliferation of the virus, and the replication ability of the virus was restored. The formation and DNA content of the virus decreased significantly, consistent with the overexpressed miR-101. After HSV-1 infection of HeLa cells, the level of miR-101 gradually increased with the prolongation of the infection time, the level of the target gene ATP5B gradually decreased, and the two had a negative correlation. Conclusion: miRNA is an endogenous molecule regulating the level of the cell gene after transcription, and participates in the modulation. The interaction between virus control and host cells. This study for the first time found that the host cell encoded miR-101 has the antiviral effect of.MiR-101 by reducing the expression of the target gene ATP5B and inhibiting the proliferation of HSV-1 in HeLa cells. After HSV-1 infection of HeLa cells, the level of miR-101 gradually increases, and the level of the target gene ATP5B gradually decreases, indicating HSV-1. For the first time, the regulation of miR-101 and its target gene on HSV-1 replication can be maintained by inhibiting the miRNA antiviral effect of the host. It is beneficial for us to understand the occurrence and development of the virus infection and provide new evidence for the diagnosis and treatment of virus infection.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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