miR-99a与miR-146c在鸡毒支原体感染中的作用研究

发布时间：2018-03-15 02:32

本文选题：鸡毒支原体　切入点：gga-miR-99a　出处：《华中农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：鸡毒支原体(Mycoplasma gallisepticum, MG)是禽类支原体感染中最重要的病源微生物之一,引起禽类的慢性呼吸道疾病(chronic respiratory disease, CRD),该病广泛分布于世界上所有养禽的国家,对家禽业造成严重的经济损失。microRNAs是一类长度18-25nt的非编码微小分子RNAs,主要通过识别特殊序列进行转录后基因表达的调控。当外界病原微生物进入宿主后,宿主体内miRNAs呈现不同丰度的表达,且在病原微生物感染不同时期以及由于病原微生物种类的不同,宿主体内miRNAs表达量发生变化。因此,通过研究宿主细胞感染病原前后miRNAs的表达变化及其靶标功能,不仅有助于揭示了该病的致病机制,还为实现基因治疗提供理论依据。本实验组前期通过MG感染鸡胚,深度测序发现,处理组中gga-miR-99a的表达量显著下调,gga-miR-146c的表达量显著上调,表明gga-miR-99a、gga-miR-146c可能在MG引起的疾病中发挥调控作用。本研究主要运用Q-PCR研究不同时期MG感染鸡胚肺组织中miR-99a和miR-146c的表达量；通过生物信息学方法预测靶基因,并进行验证；利用超表达和RNAi技术研究靶基因的功能,初步阐明gga-miR-99a、gga-miR-146c在MG感染中的调控机制。本研究的主要成果如下：(1)采用Q-PCR检测不同时期MG感染鸡胚肺组织中miR-99a的表达量,13d、14d、16d、17d肺组织中gga-miR-99a的相对表达量在感染MG后极显著上调(p0.01),12d、15d、19d、20d肺组织中gga-miR-99a的相对表达量在感染MG后极显著下调(p0.01)。(2)通过TargetScan和miRDB预测gga-miR-99a的靶基因,初步选定SMARCA5作为gga-miR-99a的靶基因。进一步运用双荧光素酶报告基因验证,结果显示,与NC组、突变载体组、空载体组相比,gga-miR-99a能够极显著抑制SMARCA5 3'UTR的双荧光素酶报告基因的表达(p0.01)。同时,DF-1细胞中超表达gga-miR-99a后,与NC组相比,SMARCA5相对表达量极显著下调(p0.01)；抑制gga-miR-99a后,与Inhibitor NC相比,SMARCA5相对表达量极显著上调(p0.01)。此外,12d感染组肺组织中SMARCA5的相对表达量极显著上调(p0.01)；16d感染组肺组织中SMARCA5的相对表达量极显著下调(p0.01),该结果与同时期肺组织中gga-miR-99a的表达趋势相反。(3)DF-1细胞中转染gga-miR-99a 24h、48h、72h,结果发现,转染72h后,DF-1细胞增殖率显著下降(P0.05)；同时,转染gga-miR-99a后检测DF-1细胞周期,与阴性对照相比,G1期水平明显升高(P0.05), G2期基本保持不变(P0.05)。(4)采用Q-PCR检测不同时期MG感染鸡胚肺组织中miR-146c的表达量,13d、14d极显著下调(p0.01),而18d、19d、20d极显著上调(p0.01)；16d、17d显著上调(p0.05)。(5)通过TargetScan和miRDB预测gga-miR-146c的靶基因,初步选定MMP16、 TRAF7作为gga-miR-146c的靶基因。进一步运用双荧光素酶报告基因验证,结果显示,与NC组、突变载体组、空载体组相比,gga-miR-146c能够极显著抑制MMP16、 TRAF7 3'UTR的双荧光素酶报告基因的表达(p0.01)。同时,DF-1细胞中超表达gga-miR-146c后,与NC组相比,MMP16、TRAF7的相对表达量极显著下调(p0.01)；抑制gga-miR-146c后,与Inhibitor NC相比,MMP16、TRAF7相对表达量极显著上调(p0.01)。(6)DF-1细胞中超表达gga-miR-146c后,与NC组相比,TLR6、TLR2、Myd88、 TNF-a相对表达量极显著上调(p0.01), Apoal无显著性差异；抑制gga-miR-146c后,与Inhibitor NC相比,Apoa I相对表达量极显著上调(p0.01), TLR6、TLR2、 Myd88、TNF-α无显著性差异。
[Abstract]:Mycoplasma gallisepticum (Mycoplasma gallisepticum MG) is one of the most important infection of pathogenic microorganisms of poultry mycoplasma, caused by avian chronic respiratory disease (chronic respiratory, disease, CRD), the disease is widely distributed in the world all countries of poultry, poultry industry caused serious economic losses in.MicroRNAs is non encoding small molecule RNAs the length of 18-25nt, mainly through the identification of special sequence of regulation of gene expression after transcription. When the external pathogenic microorganisms into the host, the host miRNAs showed different expression in abundance, and infection of pathogenic microorganisms in different periods and the different kinds of pathogenic microorganisms, the host miRNAs expression change. Therefore, the pathogen and expression the change of miRNAs and its target function of host cell infection, not only helps to reveal the pathogenic mechanism of the disease, but also for real We provide a theoretical basis for gene therapy. The experimental group by the early MG infected chicken embryo, deep sequencing found that the expression of gga-miR-99a in the treatment group were significantly reduced, the expression of gga-miR-146c was significantly up-regulated, indicating that gga-miR-99a, play to control the possible role of gga-miR-146c in the disease caused by MG. This research mainly uses the Q-PCR expression of different periods of MG infection miR-99a and miR-146c in lung tissues of chicken embryo in quantity; through the method of bioinformatics prediction of target genes, and verified; using overexpression and RNAi technology to study the target gene function, elucidate the regulatory mechanism of gga-miR-146c gga-miR-99a in MG infection. The main results of this study are as follows: (1) the expression of Q-PCR detection in different stages of MG infection of miR-99a in lung tissue of chick embryos in 13D, 14d, 16d, relative expression of gga-miR-99a 17D in lung tissue infected with MG significantly up-regulated (P0.01), 12D, 15d The relative expression of gga-miR-99a, 19d, 20d in lung tissue was significantly reduced in MG after infection (P0.01). (2) the target gene TargetScan and miRDB prediction gga-miR-99a, selected SMARCA5 as the target gene of gga-miR-99a. Further results show that using the dual luciferase reporter verified, with the NC group, the mutant vector group. Compared with the empty vector group, gga-miR-99a can significantly inhibit the expression of luciferase reporter gene SMARCA5 of 3'UTR (P0.01). At the same time, the expression of gga-miR-99a in DF-1 cells, compared with the NC group, the relative expression of SMARCA5 was significantly reduced (P0.01); the inhibition of gga-miR-99a, compared with Inhibitor NC, the relative expression of SMARCA5 was significantly up-regulated (P0.01). In addition, the relative expression of SMARCA5 12D in the lung tissues of the infected group was significantly increased (P0.01); the relative expression of SMARCA5 16d in the lung tissues of the infected group were significantly reduced (P0.01), and the results The trend of the expression of gga-miR-99a in lung tissue at the same time the opposite. (3) DF-1 cell transfer 24h 48h, 72h gga-miR-99a staining, results showed that, after 72h transfection significantly decreased DF-1 cell proliferation rate (P0.05); at the same time, detection of cell cycle of DF-1 cells after transfection of gga-miR-99a, compared with the negative control, G1 level increased significantly (P0.05), G2 (P0.05) remained unchanged. (4) the expression of Q-PCR, MG in the different stages of miR-146c infection detection of lung tissue in chicken 13D, 14d was significantly reduced (P0.01), 18D, 19d, 20d was significantly increased (P0.01); 16d, 17D were significantly increased (5 (P0.05). The target gene TargetScan and miRDB) of gga-miR-146c, selected MMP16, TRAF7 as a target gene of gga-miR-146c. Further results show that using the dual luciferase reporter verified, with the NC group, compared with the mutant vector group, vector group, gga-miR-146c significantly inhibited MMP16, TRAF7 3'UTR The expression of the dual luciferase reporter gene (P0.01). At the same time, the expression of gga-miR-146c in DF-1 cells, compared with group NC, MMP16, TRAF7 expression was significantly reduced (P0.01); the inhibition of gga-miR-146c, compared with Inhibitor, NC MMP16, the relative expression was significantly up-regulated the expression of TRAF7 (P0.01) (6). The expression of gga-miR-146c in DF-1 cells, compared with group NC, TLR6, TLR2, Myd88, relative expression was significantly up-regulated the expression of TNF-a (P0.01), no significant difference in Apoal; the inhibition of gga-miR-146c, compared with Inhibitor NC, Apoa I relative expression was very significantly increased (P0.01), TLR6, TLR2, Myd88, no significant differences of TNF- alpha.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855

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