miR-145-5p在类风湿关节炎中的表达及功能研究
发布时间：2018-09-08 06:10
【摘要】：目的类风湿关节炎(RA)属于风湿性疾病的一种,也是全世界范围内最为严重和普遍的自身免疫性疾病。RA通常起病隐匿,发病痛苦,病程漫长;且病患个体差异性大,缺乏彻底治愈的治疗药品和手段;病情的恶化常引发关节畸形,严重者甚至致残。目前,关于RA的发病机制所公认的说法是免疫系统的紊乱,该过程中涉及到多个细胞因子在滑膜中的非正常作用,与许多复杂免疫反应有较大关联。对RA的表观遗传研究陆续发现有部分miRNA,不仅受到某些炎性细胞因子的调节,也可通过调控某些细胞因子或靶向某些免疫相关的基因的表达来影响RA的发生和发展。目前,RA领域已有数个miRNA的作用被一步步揭示出来,而miR-145在RA中的作用却未曾有人关注。癌症中miR-145的研究成果的较突出,可调节多种恶性表型相关的基因,抑制其转录或蛋白功能的正常行使,发挥抑制肿瘤的作用。因此,本课题把miR-145-5p作为研究对象,以期发现miR-145-5p在RA中的调控作用。方法本实验首先采用荧光定量PCR技术检测了20例RA患者PBMC细胞中miR-145-5p的相对表达量,并以20例普通人的PBMC中的含量作为对比,得到miR-145-5p的差异表达的结果。随后在RA患者的RA-FLS中转染miR-145-5p的mimic或inhibitor实现对miR-145-5p的过表达和敲减之后,进行RA-FLS细胞的增殖、周期、凋亡实验,得出miR-145-5p对细胞表型的影响。并通过荧光定量PCR实验检测过表达miR-145-5p后RA相关炎性因子基因的表达。接着运用生物信息学软件和通路分析来寻找与细胞增殖、周期和凋亡有关或与骨代谢通路相关的基因作为候选研究的靶基因,接着采用Q-PCR实验在转录水平上进行初步检测,最后经由western blot对待验靶在翻译水平上进行验证,最终明确miR-145-5p在RA中的功能及作用机制。结果1.对20例RA患者和20例正常人PBMC细胞中miR-145-5p的表达检测发现,RA病人PBMC中miR-145-5p的表达量比正常人PBMC中显著升高。2.RA-FLS中,过表达miR-145-5p后,细胞的增殖能力的降低比较明显;敲减miR-145-5p后,细胞的增殖能力的上调比较明显。该结果提示,miR-145-5p可能通过调节RA-FLS细胞的增值能力发挥对RA的作用。3.RA-FLS中,miR-145-5p含量升高后,可显著发现G2期的细胞数目减少;敲减miR-145-5p后,可显著发现G2期的细胞数目增多。分析上述可得出,miR-145-5p可能通过影响RA-FLS细胞的周期进程来影响细胞的增殖能力。4.RA-FLS中,过表达miR-145-5p的样本,凋亡的细胞数目比对照组的数目明显要高;敲减miR-145-5p的样本,凋亡的细胞数目比对照组显著减少。分析上述可得出,miR-145-5p可以影响RA-FLS的细胞凋亡过程。5.RA-FLS中,miR-145-5p水平的升高,可造成MMP9表达的下调。6.生物信息学预测出ADAM17为miR-145-5p的靶基因,其mRNA和蛋白含量在过表miR-145-5p后下调,敲减miR-145-5p后上调。结论miR-145-5p在RA患者PBMC中表达升高。在RA-FLS中,miR-145-5p可能通过靶基因ADAM17的表达来调节TNF-α、细胞增殖相关通路以及MMP9的表达,从而影响细胞的增殖、凋亡和细胞周期进程,对滑膜的炎症程度造成影响。
[Abstract]:Objective Rheumatoid arthritis (RA) is one of the most serious and widespread autoimmune diseases in the world. RA usually has a hidden onset, painful onset, and a long course of disease. The individual variability of RA patients is large, and the lack of thorough treatment drugs and means. The deterioration of RA often leads to joint deformities, and even serious cases. Disability. At present, it is generally accepted that the pathogenesis of RA is the disorder of the immune system, which involves the abnormal action of many cytokines in the synovium, and is closely related to many complex immune responses. At present, the role of several microRNAs in RA has been revealed step by step. However, the role of microRNAs in RA has not been paid attention to. In cancer, microRNAs-145 can regulate many malignant phenotypes. In order to discover the regulatory role of microRNAs-145-5p in RA, we first detected the relative expression of microRNAs-145-5p in PBMC cells of 20 patients with RA by fluorescence quantitative PCR. The results of differentially expressed microRNAs-145-5p were obtained by comparing the contents of PBMC in a normal person.Mimic or inhibitor transfected with microRNAs-145-5p in RA-FLS of RA patients overexpressed and knocked down microRNAs-145-5p.The proliferation, cycle and apoptosis experiments of RA-FLS cells were carried out to determine the effect of microRNAs-145-5p on cell phenotype. The expression of RA-related inflammatory factor gene was detected by photoquantitative polymerase chain reaction (PCR) assay after the expression of microRNAs-145-5p. Bioinformatics software and pathway analysis were used to identify genes related to cell proliferation, cycle and apoptosis or bone metabolism pathway as candidate genes. Q-PCR assay was used to detect these genes at transcriptional level. Results 1. The expression of microRNA-145-5p in PBMC cells of 20 RA patients and 20 normal subjects was detected. The expression of microRNA-145-5p in PBMC cells of RA patients was significantly higher than that in normal PBMC. Mi-145-5p significantly decreased the proliferation of RA cells, and the up-regulation of cell proliferation was more obvious after the knockdown of Mi-145-5p. The results suggested that Mi-145-5p might play a role in RA by regulating the proliferation of RA-FLS cells. 3. In RA-FLS, when the content of Mi-145-5p increased, the number of G2 cells decreased significantly. Mi-145-5p could significantly increase the number of G2 cells after reducing the expression of Mi-145-5p. These results suggest that Mi-145-5p may affect the proliferation of RA-FLS cells by influencing the cell cycle progression. 4. In RA-FLS, the number of apoptotic cells was significantly higher than that in control group when the expression of Mi-145-5p was over-expressed; Mi-145-5p knocked down, apoptotic cells were observed. The number of cells was significantly lower than that of the control group. The results showed that the expression of MMP9 in RA-FLS was down-regulated by increasing the level of microRNAs-145-5p. Bioinformatics predicted that ADAM17 was the target gene of microRNAs-145-5p, and its mRNA and protein contents were down-regulated after overexpression of microRNAs-145-5p, knocking down microRNAs-145-5p. Conclusion The expression of microRNAs-145-5p in PBMC of RA patients is elevated. In RA-FLS, microRNAs-145-5p may regulate the expression of TNF-alpha, cell proliferation-related pathways and MMP9 through the expression of target gene ADAM17, thereby affecting cell proliferation, apoptosis and cell cycle progression, and affecting the degree of synovial inflammation.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.22
[Abstract]:Objective Rheumatoid arthritis (RA) is one of the most serious and widespread autoimmune diseases in the world. RA usually has a hidden onset, painful onset, and a long course of disease. The individual variability of RA patients is large, and the lack of thorough treatment drugs and means. The deterioration of RA often leads to joint deformities, and even serious cases. Disability. At present, it is generally accepted that the pathogenesis of RA is the disorder of the immune system, which involves the abnormal action of many cytokines in the synovium, and is closely related to many complex immune responses. At present, the role of several microRNAs in RA has been revealed step by step. However, the role of microRNAs in RA has not been paid attention to. In cancer, microRNAs-145 can regulate many malignant phenotypes. In order to discover the regulatory role of microRNAs-145-5p in RA, we first detected the relative expression of microRNAs-145-5p in PBMC cells of 20 patients with RA by fluorescence quantitative PCR. The results of differentially expressed microRNAs-145-5p were obtained by comparing the contents of PBMC in a normal person.Mimic or inhibitor transfected with microRNAs-145-5p in RA-FLS of RA patients overexpressed and knocked down microRNAs-145-5p.The proliferation, cycle and apoptosis experiments of RA-FLS cells were carried out to determine the effect of microRNAs-145-5p on cell phenotype. The expression of RA-related inflammatory factor gene was detected by photoquantitative polymerase chain reaction (PCR) assay after the expression of microRNAs-145-5p. Bioinformatics software and pathway analysis were used to identify genes related to cell proliferation, cycle and apoptosis or bone metabolism pathway as candidate genes. Q-PCR assay was used to detect these genes at transcriptional level. Results 1. The expression of microRNA-145-5p in PBMC cells of 20 RA patients and 20 normal subjects was detected. The expression of microRNA-145-5p in PBMC cells of RA patients was significantly higher than that in normal PBMC. Mi-145-5p significantly decreased the proliferation of RA cells, and the up-regulation of cell proliferation was more obvious after the knockdown of Mi-145-5p. The results suggested that Mi-145-5p might play a role in RA by regulating the proliferation of RA-FLS cells. 3. In RA-FLS, when the content of Mi-145-5p increased, the number of G2 cells decreased significantly. Mi-145-5p could significantly increase the number of G2 cells after reducing the expression of Mi-145-5p. These results suggest that Mi-145-5p may affect the proliferation of RA-FLS cells by influencing the cell cycle progression. 4. In RA-FLS, the number of apoptotic cells was significantly higher than that in control group when the expression of Mi-145-5p was over-expressed; Mi-145-5p knocked down, apoptotic cells were observed. The number of cells was significantly lower than that of the control group. The results showed that the expression of MMP9 in RA-FLS was down-regulated by increasing the level of microRNAs-145-5p. Bioinformatics predicted that ADAM17 was the target gene of microRNAs-145-5p, and its mRNA and protein contents were down-regulated after overexpression of microRNAs-145-5p, knocking down microRNAs-145-5p. Conclusion The expression of microRNAs-145-5p in PBMC of RA patients is elevated. In RA-FLS, microRNAs-145-5p may regulate the expression of TNF-alpha, cell proliferation-related pathways and MMP9 through the expression of target gene ADAM17, thereby affecting cell proliferation, apoptosis and cell cycle progression, and affecting the degree of synovial inflammation.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.22
【参考文献】
相关期刊论文 前10条
1 邵勤;高洪燕;周小莉;曹春辉;;IL-22在类风湿关节炎中的表达及临床意义[J];中国免疫学杂志;2017年01期
2 刘丽丽;毛艳艳;高柳滨;;类风湿关节炎全球药物研发状况分析[J];科技导报;2016年24期
3 武永红;孙静;刘蓓;;类风湿关节炎患者血清IgG-IgM型类风湿因子免疫复合物检测的临床意义[J];标记免疫分析与临床;2016年07期
4 黄林芳;肖欣悦;张p，
本文编号：2229610
本文链接：https://www.wllwen.com/yixuelunwen/nfm/2229610.html
最近更新
教材专著
