miR-101-3p在雞毒支原體感染中的作用研究
發(fā)布時間：2018-12-13 03:24
【摘要】：雞毒支原體(Mycoplasma gallisepticum,簡稱MG)是雞慢性呼吸道疾病的病原體,主要通過定殖于宿主呼吸道建立慢性感染,引起呼吸道組織的損傷和一系列病理變化。該病廣泛分布于世界各地,給家禽養(yǎng)殖業(yè)造成了巨大的經濟損失,迄今仍未找到有效的防控措施。深入研究MG的致病機制,有助于該病的預防及治療方法的尋找。大量研究表明,mi RNA在病原致病中發(fā)揮著重要的作用,本研究旨在探討高通量測序篩選出的gga-mi R-101-3p在MG感染中的作用,為闡明MG的致病機理提供科學依據。實驗以雞毒支原體HS株(MG-HS)感染雞胚及DF-1細胞為對象,運用Real-time Quantitative PCR(q PCR)方法檢測感染與非感染組織、細胞中gga-mi R-101-3p及其靶基因的表達量;應用雙熒光素酶報告基因系統(tǒng)對gga-mi R-101-3p的靶基因進行驗證;進一步通過gga-mi R-101-3p的超表達和抑制實驗來確證gga-mi R-101-3p與靶基因的調控關系。利用MTT法檢測DF-1細胞的增殖及流式細胞儀檢測細胞周期的變化,了解gga-mi R-101-3p對DF-1細胞生物學的影響。主要成果如下:1.檢測了gga-mi R-101-3p在MG-HS感染與非感染雞胚(12-20日齡)肺組織及DF-1細胞中的表達量。gga-mi R-101-3p在17 d、18 d、19 d MG-HS感染雞胚的肺組織中的表達量極顯著高于正常對照組(p0.01),而13 d、14 d時極顯著低于正常對照組(p0.01),20 d時顯著低于正常對照組(p0.05)。MG-HS感染DF-1細胞中gga-mi R-101-3p在的表達量極顯著高于正常對照組(p0.01)。2.利用在線軟件mi RDB、Mir Target2和Target Scan等預測了gga-mi R-101-3p的靶基因,通過RNAhybrid等軟件對gga-mi R-101-3p與EZH2 3’UTR靶序列的堿基配對情況、二者結合形成RNA雙鏈的二級結構和自由能,以及靶序列在不同物種間保守性等主要生物信息學指標進行了歸納分析,并結合功能分析初步確定EZH2為gga-mi R-101-3p的靶基因。3.通過雙熒光素酶報告系統(tǒng)分析,gga-mi R-101-3p能顯著抑制EZH2 3’UTR雙熒光素酶報告基因的熒光素酶活性,揭示了gga-mi R-101-3p是通過與EZH2 3’UTR結合而發(fā)揮作用,初步確定EZH2可能是gga-mi R-101-3p的靶基因。4.gga-mi R-101-3p超表達和抑制實驗表明,在DF-1細胞中轉染gga-mi R-101-3p mimics能顯著抑制EZH2基因m RNA和蛋白的表達;而轉染gga-mi R-101-3p inhibitor后,EZH2基因m RNA和蛋白的表達則顯著上調。進一步證實了EZH2是gga-mi R-101-3p的靶基因,且gga-mi R-101-3p負向調控EZH2的表達。5.q PCR分析了EZH2在MG-HS感染與非感染雞胚(12-20日齡)肺組織、DF-1細胞中的表達量。結果顯示,18 d、19 d MG-HS感染雞胚的肺組織中EZH2的相對表達量極顯著低于與正常對照組(p0.01),20 d時極顯著高于正常對照組(p0.01);且在MG-HS感染DF-1細胞中,EZH2的表達量極顯著低于正常對照組(p0.01),說明EZH2與gga-mi R-101-3p的表達量呈明顯負相關。6.超表達gga-mi R-101-3p能顯著抑制DF-1細胞的增殖,且使細胞周期停滯在G1期,不能進入S期進行DNA的合成與復制。
[Abstract]:Mycoplasma gallinis (Mycoplasma gallisepticum,) is the pathogen of chronic respiratory diseases in chickens. Chronic infection is established by colonization of host respiratory tract, which results in the injury of respiratory tract tissue and a series of pathological changes. The disease is widely distributed all over the world, causing huge economic losses to poultry industry, so far no effective prevention and control measures have been found. Further study on the pathogenesis of MG is helpful to the prevention and treatment of MG. A large number of studies have shown that, mi RNA plays an important role in pathogenicity. The purpose of this study was to explore the role of gga-mi R-101-3p screened by high-throughput sequencing in MG infection, and to provide scientific basis for elucidating the pathogenesis of MG. The expression of gga-mi R-101-3p and its target genes in infected and non-infected tissues and cells were detected by Real-time Quantitative PCR (q PCR) method in chicken embryo and DF-1 cells infected with Mycoplasma Chicken HS strain (MG-HS). The target gene of gga-mi R-101-3p was verified by double luciferase reporter gene system, and the regulatory relationship between gga-mi R-101-3p and target gene was confirmed by gga-mi R-101-3p overexpression and inhibition experiments. MTT assay was used to detect the proliferation of DF-1 cells and flow cytometry was used to detect the changes of cell cycle. The effect of gga-mi R-101-3p on the cell biology of DF-1 cells was investigated. The main results are as follows: 1. The expression of gga-mi R-101-3p in lung tissues and DF-1 cells of MG-HS infected and non-infected chicken embryos (12-20 days old) was detected. Gga-mi R-101-3p was expressed at 17 days and 18 days after infection. The expression of MG-HS in the lung tissue of chicken embryo infected with MG-HS on day 19 was significantly higher than that of the normal control group (p0.01), and was significantly lower than that of the control group at the 14th day of 13 days (p0.01). The expression of gga-mi R-101-3p in DF-1 cells infected with MG-HS was significantly higher than that in normal controls (p0.01). The target genes of gga-mi R-101-3p were predicted by on-line software mi RDB,Mir Target2 and Target Scan, and the base pairs of gga-mi R-101-3p and EZH2 3'UTR target sequences were analyzed by RNAhybrid and other software. The secondary structure and free energy of RNA double strands and the conservation of target sequences among different species were summarized and analyzed. Combined with functional analysis, EZH2 was identified as the target gene of gga-mi R-101-3p. Through the analysis of double luciferase report system, gga-mi R-101-3p can significantly inhibit the luciferase activity of EZH2 3'UTR double luciferase reporter gene, which indicates that gga-mi R-101-3p acts by combining with EZH2 3'UTR. EZH2 may be the target gene of gga-mi R-101-3p. The overexpression and inhibition of 4.gga-mi R-101-3p in DF-1 cells showed that transfection of gga-mi R-101-3p mimics could significantly inhibit the expression of EZH2 gene m RNA and protein. After transfection of gga-mi R-101-3p inhibitor, the expression of m RNA and protein of EZH2 gene was significantly up-regulated. It was further confirmed that EZH2 was the target gene of gga-mi R-101-3p, and gga-mi R-101-3p negatively regulated the expression of EZH2. 5.q PCR was used to analyze the lung tissues of MG-HS infected and non-infected chicken embryos (12-20 days old). The amount of expression in DF-1 cells. The results showed that the relative expression of EZH2 in the lung tissue of chicken embryo infected with MG-HS on day 18 and 19 was significantly lower than that of the control group (p0.01), and at 20 days it was significantly higher than that of the normal control group (p0.01). The expression of EZH2 in DF-1 cells infected with MG-HS was significantly lower than that in normal controls (p0.01), indicating that the expression of EZH2 and gga-mi R-101-3p was negatively correlated. 6. Overexpression of gga-mi R-101-3p could significantly inhibit the proliferation of DF-1 cells and arrest the cell cycle in G1 phase, and could not enter S phase for DNA synthesis and replication.
【學位授予單位】：華中農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.31
本文編號：2375782
[Abstract]:Mycoplasma gallinis (Mycoplasma gallisepticum,) is the pathogen of chronic respiratory diseases in chickens. Chronic infection is established by colonization of host respiratory tract, which results in the injury of respiratory tract tissue and a series of pathological changes. The disease is widely distributed all over the world, causing huge economic losses to poultry industry, so far no effective prevention and control measures have been found. Further study on the pathogenesis of MG is helpful to the prevention and treatment of MG. A large number of studies have shown that, mi RNA plays an important role in pathogenicity. The purpose of this study was to explore the role of gga-mi R-101-3p screened by high-throughput sequencing in MG infection, and to provide scientific basis for elucidating the pathogenesis of MG. The expression of gga-mi R-101-3p and its target genes in infected and non-infected tissues and cells were detected by Real-time Quantitative PCR (q PCR) method in chicken embryo and DF-1 cells infected with Mycoplasma Chicken HS strain (MG-HS). The target gene of gga-mi R-101-3p was verified by double luciferase reporter gene system, and the regulatory relationship between gga-mi R-101-3p and target gene was confirmed by gga-mi R-101-3p overexpression and inhibition experiments. MTT assay was used to detect the proliferation of DF-1 cells and flow cytometry was used to detect the changes of cell cycle. The effect of gga-mi R-101-3p on the cell biology of DF-1 cells was investigated. The main results are as follows: 1. The expression of gga-mi R-101-3p in lung tissues and DF-1 cells of MG-HS infected and non-infected chicken embryos (12-20 days old) was detected. Gga-mi R-101-3p was expressed at 17 days and 18 days after infection. The expression of MG-HS in the lung tissue of chicken embryo infected with MG-HS on day 19 was significantly higher than that of the normal control group (p0.01), and was significantly lower than that of the control group at the 14th day of 13 days (p0.01). The expression of gga-mi R-101-3p in DF-1 cells infected with MG-HS was significantly higher than that in normal controls (p0.01). The target genes of gga-mi R-101-3p were predicted by on-line software mi RDB,Mir Target2 and Target Scan, and the base pairs of gga-mi R-101-3p and EZH2 3'UTR target sequences were analyzed by RNAhybrid and other software. The secondary structure and free energy of RNA double strands and the conservation of target sequences among different species were summarized and analyzed. Combined with functional analysis, EZH2 was identified as the target gene of gga-mi R-101-3p. Through the analysis of double luciferase report system, gga-mi R-101-3p can significantly inhibit the luciferase activity of EZH2 3'UTR double luciferase reporter gene, which indicates that gga-mi R-101-3p acts by combining with EZH2 3'UTR. EZH2 may be the target gene of gga-mi R-101-3p. The overexpression and inhibition of 4.gga-mi R-101-3p in DF-1 cells showed that transfection of gga-mi R-101-3p mimics could significantly inhibit the expression of EZH2 gene m RNA and protein. After transfection of gga-mi R-101-3p inhibitor, the expression of m RNA and protein of EZH2 gene was significantly up-regulated. It was further confirmed that EZH2 was the target gene of gga-mi R-101-3p, and gga-mi R-101-3p negatively regulated the expression of EZH2. 5.q PCR was used to analyze the lung tissues of MG-HS infected and non-infected chicken embryos (12-20 days old). The amount of expression in DF-1 cells. The results showed that the relative expression of EZH2 in the lung tissue of chicken embryo infected with MG-HS on day 18 and 19 was significantly lower than that of the control group (p0.01), and at 20 days it was significantly higher than that of the normal control group (p0.01). The expression of EZH2 in DF-1 cells infected with MG-HS was significantly lower than that in normal controls (p0.01), indicating that the expression of EZH2 and gga-mi R-101-3p was negatively correlated. 6. Overexpression of gga-mi R-101-3p could significantly inhibit the proliferation of DF-1 cells and arrest the cell cycle in G1 phase, and could not enter S phase for DNA synthesis and replication.
【學位授予單位】：華中農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.31
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