miR-101-3p在鸡毒支原体感染中的作用研究
发布时间：2018-12-13 03:24
【摘要】：鸡毒支原体(Mycoplasma gallisepticum,简称MG)是鸡慢性呼吸道疾病的病原体,主要通过定殖于宿主呼吸道建立慢性感染,引起呼吸道组织的损伤和一系列病理变化。该病广泛分布于世界各地,给家禽养殖业造成了巨大的经济损失,迄今仍未找到有效的防控措施。深入研究MG的致病机制,有助于该病的预防及治疗方法的寻找。大量研究表明,mi RNA在病原致病中发挥着重要的作用,本研究旨在探讨高通量测序筛选出的gga-mi R-101-3p在MG感染中的作用,为阐明MG的致病机理提供科学依据。实验以鸡毒支原体HS株(MG-HS)感染鸡胚及DF-1细胞为对象,运用Real-time Quantitative PCR(q PCR)方法检测感染与非感染组织、细胞中gga-mi R-101-3p及其靶基因的表达量;应用双荧光素酶报告基因系统对gga-mi R-101-3p的靶基因进行验证;进一步通过gga-mi R-101-3p的超表达和抑制实验来确证gga-mi R-101-3p与靶基因的调控关系。利用MTT法检测DF-1细胞的增殖及流式细胞仪检测细胞周期的变化,了解gga-mi R-101-3p对DF-1细胞生物学的影响。主要成果如下:1.检测了gga-mi R-101-3p在MG-HS感染与非感染鸡胚(12-20日龄)肺组织及DF-1细胞中的表达量。gga-mi R-101-3p在17 d、18 d、19 d MG-HS感染鸡胚的肺组织中的表达量极显著高于正常对照组(p0.01),而13 d、14 d时极显著低于正常对照组(p0.01),20 d时显著低于正常对照组(p0.05)。MG-HS感染DF-1细胞中gga-mi R-101-3p在的表达量极显著高于正常对照组(p0.01)。2.利用在线软件mi RDB、Mir Target2和Target Scan等预测了gga-mi R-101-3p的靶基因,通过RNAhybrid等软件对gga-mi R-101-3p与EZH2 3’UTR靶序列的碱基配对情况、二者结合形成RNA双链的二级结构和自由能,以及靶序列在不同物种间保守性等主要生物信息学指标进行了归纳分析,并结合功能分析初步确定EZH2为gga-mi R-101-3p的靶基因。3.通过双荧光素酶报告系统分析,gga-mi R-101-3p能显著抑制EZH2 3’UTR双荧光素酶报告基因的荧光素酶活性,揭示了gga-mi R-101-3p是通过与EZH2 3’UTR结合而发挥作用,初步确定EZH2可能是gga-mi R-101-3p的靶基因。4.gga-mi R-101-3p超表达和抑制实验表明,在DF-1细胞中转染gga-mi R-101-3p mimics能显著抑制EZH2基因m RNA和蛋白的表达;而转染gga-mi R-101-3p inhibitor后,EZH2基因m RNA和蛋白的表达则显著上调。进一步证实了EZH2是gga-mi R-101-3p的靶基因,且gga-mi R-101-3p负向调控EZH2的表达。5.q PCR分析了EZH2在MG-HS感染与非感染鸡胚(12-20日龄)肺组织、DF-1细胞中的表达量。结果显示,18 d、19 d MG-HS感染鸡胚的肺组织中EZH2的相对表达量极显著低于与正常对照组(p0.01),20 d时极显著高于正常对照组(p0.01);且在MG-HS感染DF-1细胞中,EZH2的表达量极显著低于正常对照组(p0.01),说明EZH2与gga-mi R-101-3p的表达量呈明显负相关。6.超表达gga-mi R-101-3p能显著抑制DF-1细胞的增殖,且使细胞周期停滞在G1期,不能进入S期进行DNA的合成与复制。
[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.
【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】：S858.31
本文编号：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.
【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】：S858.31
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