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牙鲆microRNA pol-miR-194a和pol-miR-3p-2的调控机制及在迟缓爱德华氏菌感染过程中的作用研究
管晓璐
Subtype博士
Thesis Advisor孙黎
2019-05-17
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学博士
KeywordMicrorna,牙鲆,迟缓爱德华氏菌,i型干扰素,自噬
Abstract

MicroRNA (miRNA) 作为转录后调控的重要手段,广泛参与生物体多种生理过程的调控。在病原与宿主的博弈过程中,miRNA同样扮演着重要角色。实验室前期研究发现了大量免疫调控相关的牙鲆miRNAs。在此基础上,本研究鉴定了两条参与迟缓爱德华氏菌(Edwardsiella tarda, E. tarda)感染过程的牙鲆miRNAspol-miR-194apol-miR-3p-2,并探索了二者的调控机制及在E. tarda感染过程中的免疫调控作用。

我们首先检测了pol-miR-194aE. tarda感染的响应。通过实时荧光定量PCRqRT-PCR)的方法,发现在E. tarda感染24 h后,牙鲆的肝脏、脾脏和鳃中pol-miR-194a的表达量均显著上调。利用荧光素酶报告实验,我们发现pol-miR-194a能够与干扰素调节因子7Interferon regulatory factor 7 IRF7)的3’UTR相互作用,表明IRF7pol-miR-194a的一个靶基因。同时,通过对IRF7靶序列及pol-miR-194a种子序列的突变实验,证明了pol-miR-194aIRF7-3’UTR的相互作用依赖于完整的种子序列。进一步研究发现,在牙鲆鳃细胞系FG-9307细胞中过表达pol-miR-194a后,内源性IRF7的蛋白表达水平显著降低。为了验证pol-miR-194aIRF7下游信号通路的调控,将pol-miR-194a mimicpol-miR-194a模拟物)和I型干扰素的启动子报告质粒共转染到FG-9307细胞中,结果发现pol-miR-194a mimic能够显著抑制I型干扰素启动子的活性。通过体外过表达实验和胞内复制实验,我们发现pol-miR-194a能够增强E. tarda在宿主细胞内的复制。以上实验表明,pol-miR-194a表达受E. tarda调控,而pol-miR-194a下调牙鲆IRF7I型干扰素的表达,进而降低牙鲆的抗感染免疫应答,最终促进E. tarda的胞内复制。

自噬在多种生理过程中发挥重要作用,如生长发育、肿瘤发生、炎症反应以及病原感染与宿主的免疫调控等。在本研究中,我们发现E. tarda感染能够诱导牙鲆脾脏免疫细胞和牙鲆FG-9307细胞系的自噬现象。p53在自噬过程中发挥着重要作用,同时被预测为牙鲆pol-miR-3p-2的靶基因。利用qRT-PCR实验,我们发现在E. tarda感染24 h后,牙鲆的肝脏、脾脏和鳃中pol-miR-3p-2的表达量均显著上调。通过荧光素酶报告实验和western blot分析,我们确认p53pol-miR-3p-2的靶基因。在FG-9307细胞中过表达pol-miR-3p-2能够激活细胞自噬。进一步研究发现,在FG-9307细胞中敲降p53后,自噬关键蛋白Beclin的表达水平上调;在HEK293T细胞中异源表达实验进一步确认p53敲降能够上调Beclin的表达。通过体外过表达实验结合胞内复制实验,我们发现pol-miR-3p-2能够抑制E. tarda的胞内复制。以上结果表明,E. tarda感染能够促进pol-miR-3p-2的表达,进而下调p53蛋白表达,并上调Beclin的表达,从而促进牙鲆免疫细胞发生自噬,抑制E. tarda的胞内复制。

综上所述,我们阐释了两条牙鲆miRNAs, pol-miR-194apol-miR-3p-2,的调控机制,并揭示了二者在E. tarda感染过程中的作用。这些研究结果促进了我们对鱼类miRNAs作用机制以及E. tarda感染机制的理解。

Other Abstract

As an important mechanism of post-transcriptional modulation, microRNA (miRNA) participates in the regulation of multi-biological processes. During the battle between pathogen invasion and host defense, miRNAs also play a crucial role. A number of Japanese flounder miRNAs have been discovered in previous studies in our laboratory.  In this thesis, two flounder miRNAs identified before, i.e. pol-miR-194a and pol-miR-3p-2, were selected for the study of their regulation mechanisms and involvement in the infection of Edwardsiella tarda.

Firstly, we examined the response of pol-miR-194a expression during E. tarda infection. By quantitative real time PCR (qRT-PCR) method, we discovered that the expression level of pol-miR-194a in liver, spleen and gill of flounder infected by E. tarda was significantly up-regulated at 24 hours post infection. Luciferase report assay revealed interaction between pol-miR-194a and the 3’UTR of interferon regulatory factor 7 (IRF7), indicating that IRF7 is a target gene of pol-miR-194a. Mutation analysis showed that the interaction between pol-miR-194a and the 3’UTR of IRF7 relied on the specific seed sequence of pol-miR-194a.  Western blot analysis showed that over-expression of pol-miR-194a in flounder FG-9307 cells significantly decreased the protein level of endogenous IRF7. When pol-miR-194a mimic and the promoter reporter plasmid of type I interferon (IFN) were co-transfected into FG-9307 cells, the promoter activity of type I IFN was markedly inhibited. By in-vitro over-expression and intracellular replication assay, we discovered that pol-miR-194a could enhance the intracellular replication of E. tarda. The above results indicated that the expression of pol-miR-194a was regulated by E. tarda; the up-regulated pol-miR-194a could then inhibit the expression of IRF7 and type I interferon, leading to an attenuated immune response, which finally enhanced the intracellular replication of E. tarda.

Autophagy plays an important role in many biological processes including growth and development, tumorigenesis, inflammatory response, pathogen infection and host immune response. In this study, we found that the infection of E. tarda induced autophagy in the spleen cells of Japanese flounder and in FG-9307 cells, a cell line of Japanese flounder. p53, which plays an important role in autophagy process, was predicted to be a target gene of a flounder miRNA named pol-miR-3p-2. By qRT-PCR method, we found that in flounder infected with E. tarda, the expression levels of pol-miR-3p-2 in the liver, spleen and gill of the fish were significantly up-regulated at 24 hours post infection. Through luciferase reporter assay and western blot analysis, p53 was confirmed to be a target of pol-miR-3p-2. When over-expressed in FG-9307 cells, pol-miR-3-2 could induce the autophagy process. Further experiment discovered that, when p53 was knocked-down in FG-9307 cells, the autophagy related gene of beclin was significantly up-regulated. Similar results were obtained in the experiment with HEK293T cells. By  in vitro over-expression and intracellular replication assays, we found that pol-miR-3p-2 could inhibit the intracellular replication of E. tarda. These results indicated that, infection of E. tarda promoted the expression of pol-miR-3p-2, which decreased p53 protein level and enhanced the expression of beclin, which finally promoted autophagy in Japanese flounder and inhibited the intracellular replication of E. tarda.

In summary, we have elucidated the regulation mechanisms of  two flounder microRNAs, pol-miR-194a and pol-miR-3p-2, and revealed their effects on E. tarda infection. The results of this study add new insights into the function of fish miRNAs and the infection mechanism of E. tarda.

MOST Discipline Catalogue理学::海洋科学 ; 理学::生物学
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/156819
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
管晓璐. 牙鲆microRNA pol-miR-194a和pol-miR-3p-2的调控机制及在迟缓爱德华氏菌感染过程中的作用研究[D]. 中国科学院海洋研究所. 中国科学院大学,2019.
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