实验性关节炎相关长链非编码RNAs的筛

发布时间：2018-07-06 13:56

本文选题：类风湿关节炎 + 实验性关节炎　；参考：《安徽医科大学》2017年博士论文

【摘要】：类风湿关节炎(rheumatoid arthritis,RA)是一种全身性、自身免疫性疾病,以慢性炎症、滑膜细胞异常增生、关节肿胀与压痛为其临床特征,可导致关节功能受限、工作能力丧失,给患者和社会带来沉重的负担。RA好发于35~50岁人群,女性发病率约为男性2~3倍。目前,RA具体发病机制尚不清楚,认为可能与免疫因素、环境因素、遗传等有关。因此,积极探索RA的发病机制,寻找潜在诊断标志物具有重要的理论与现实意义。以前关于RA机制的研究主要集中在编码基因。近年来,越来越多的研究表明长期以来被视为“噪音”和“暗物质”的非编码RNAs(non-coding RNAs,nc RNAs),具有复杂的生物学功能。nc RNAs中的长链非编码RNAs(long non-coding RNAs,Lnc RNAs)是由基因组中非编码序列转录生成的、长度大于200nt、不具有翻译成蛋白质能力的转录本,在表观遗传水平、转录水平、翻译水平、蛋白修饰过程中均可发挥重要的调控作用,与多种疾病密切相关。但目前对RA发生、发展过程中Lnc RNAs的调控作用仍知之甚少。目前,西医临床上对于RA的治疗,主要以减轻疼痛、控制症状为主,长期应用副反应明显,患者多不能耐受或依从性差,远程疗效不理想。因此,从传统中药中开发治疗RA的药物,越来越受到重视。黄芪为豆科植物蒙古黄芪Astragalus membranaceus(Fisch.)Bge.var.mongolicus(Bge.)Hsiao或膜荚黄芪Astragalus membranaceus(Fisch.)Bge.的干燥根,具有行滞通痹、补气升阳、利水消肿等功效,为中医临床实践中常用药材。黄芪总皂苷(Astragalosides,AST)是黄芪的有效部位群,主要含有黄芪皂苷I~VIII等。现代药理学研究表明,AST具有抗炎、抗氧化、免疫调节、抗衰老等多种药理作用。本课题拟在课题组前期研究的基础上,开展如下研究:(1)实验性关节炎相关长链非编码RNAs的筛选及黄芪总皂苷的干预作用;(2)基于Lnc RNA-mi RNA-m RNA共表达网络筛选实验性关节炎发病过程中关键Lnc RNAs;(3)Lnc RNA S56464.1靶向mi R-152/Wnt通路诱导成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)增殖及黄芪总皂苷干预作用等研究,以期系统、深入地揭示Lnc RNAs在实验性关节炎发生、发展中所扮演的角色及黄芪总皂苷的干预作用,从Lnc RNAs的角度揭示实验性关节炎的发病机理,并阐明黄芪总皂苷治疗实验性关节炎的可能机制。第一部分实验性关节炎相关长链非编码RNA的筛选及黄芪总皂苷的干预作用目的:筛选实验性关节炎发病过程中差异表达的长链非编码RNA(long non-coding RNAs,Lnc RNAs),探讨实验性关节炎可能的发病机制及黄芪总皂苷可能的作用靶点。方法:采用大鼠Lnc RNA基因芯片检测佐剂性关节炎(adjuvant arthritis,AA)大鼠滑膜组织中Lnc RNAs/m RNAs表达情况,以Fold change值1.5,P-value0.05筛选差异表达基因,进行基因本体论(Gene Ontology,GO)和信号通路(Pathway)分析,构建差异表达基因Lnc RNA-m RNA共表达网络,筛选关键差异Lnc RNAs并采用实时荧光定量PCR技术对其进行验证。结果:正常组和模型组共筛选出260个差异表达的LncRNAs,其中模型组中上调Lnc RNAs表达谱数据170个,下调Lnc RNAs表达谱数据90个;获得675个差异表达的m RNAs,模型组中上调m RNAs表达谱数据422个,下调m RNAs表达谱数据253个;共获得4163个GO条目,其中上调差异基因富集于2494个GO条目,下调差异基因富集于1669个GO条目;获得42条与差异基因相关的信号通路,其中上调差异基因富集于25条信号通路,下调差异基因富集于17条信号通路;6个关键Lnc RNAs(XR_008357,U75927,MRAK046251,XR_006457,DQ266363和MRAK003448)实时荧光定量PCR检测结果与基因芯片的结果相一致。正常组、模型组和AST组共筛选出75个差异表达的LncRNAs,与正常组和AST组相比,模型组上调Lnc RNAs表达谱数据41个,下调Lnc RNAs表达谱数据34个;筛选出247个差异表达的m RNAs,模型组中上调m RNAs表达谱数据171个,下调m RNAs表达谱数据76个;共获得135个GO条目,其中上调差异基因富集于117个GO条目,下调差异基因富集于18个GO条目;共获得17条与差异基因相关的信号通路,其中上调差异基因富集于14条信号通路,下调差异基因富集于3条信号通路;4个关键Lnc RNAs(MRAK012530、MRAK132628、MRAK003448、XR_00645)实时荧光定量PCR检测结果与基因芯片的结果相一致。结论:LncRNAs在实验性关节炎发病过程中存在差异表达,AST防治实验性关节炎的机制可能与调控差异表达的Lnc RNAs有关。第二部分基于Lnc RNA-mi RNA-m RNA共表达网络筛选实验性发病过程中关键Lnc RNAs目的:筛选实验性关节炎发病过程中关键Lnc RNAs,探讨其可能的发病机制。方法:mi RNAs基因表达谱数据来源于美国国立生物信息中心高通量基因表达数据库和已发表文献,Lnc RNAs/m RNAs基因表达谱数据来源于课题组前期已发表文章;以差异倍数2和P-value0.05为标准,筛选差异表达的Lnc RNAs、mi RNAs和m RNAs;借助miranda和targetscan预测差异mi RNAs靶向的m RNAs,借助RNAhybrid预测差异mi RNAs靶向的Lnc RNAs;将mi RNAs靶向的Lnc RNAs和m RNAs,分别与差异Lnc RNAs和m RNAs合并,取交集,得到共表达Lnc RNAs和m RNAs;以共表达Lnc RNAs、m RNAs和差异mi RNAs为基础,构建Lnc RNA-mi RNA-m RNA共表达网络,并进行功能信息学分析,筛选实验性关节炎发病过程中关键Lnc RNAs。结果:Lnc RNA-mi RNA-m RNA共表达网络由7个Lnc RNAs节点,24个mi RNAs节点,90个m RNAs节点,301个边缘组成;功能信息学分析结果表明,差异基因主要富集于147个GO条目,23条信号通路;LncRNA S56464.1,LncRNA XR_006437.1和LncRNA J01878被鉴定为实验性关节炎发病过程中的关键基因。结论:LncRNAs在实验性关节炎发病过程中具有重要的调节作用,LncRNA S56464.1,Lnc RNA XR_006437.1和Lnc RNA J01878可作为其潜在的诊断生物标志物和治疗靶点。第三部分Lnc RNA S56464.1靶向mi R-152/Wnt通路诱导FLS增殖及黄芪总皂苷干预作用目的:明确Lnc RNA S56464.1通过靶向mi R-152/Wnt通路诱导滑膜细胞的增殖作用,探讨黄芪总皂苷治疗实验性关节炎的可能机制。方法:组织块移植法分离、培养AA大鼠滑膜细胞,给予LncRNA S56464.1 siRNA和/或黄芪总皂苷干预,MTT法检测成纤维样滑膜细胞(fibroblast-like synoviocyte,FLS)的增殖反应;RT-q PCR技术检测Lnc RNA S56464.1干扰和/或AST作用下Lnc RNA S56464.1、mi R-152、β-连环蛋白(β-catenin)、原癌基因C-myc、细胞周期蛋白(Cyclin)D1、糖原合成激酶(GSK)-3β和分泌型卷曲相关蛋白(SFRP)4 m RNA表达变化;免疫荧光和Western Blot技术检测β-catenin、C-myc、Cyclin D1、GSK-3β、p-GSK-3β(Ser9)和SFRP4蛋白表达变化。结果:与模型组相比,Lnc RNA S6464.1干扰组和/或黄芪总皂苷组,不仅可明显抑制AA大鼠成纤维样滑膜细胞的增殖反应,还可显著降低Lnc RNA56464.1、β-catenin、C-myc、Cyclin D1 m RNA,明显抑制β-catenin、C-myc、Cyclin D1和p-GSK-3β(Ser9)/GSK-3β蛋白表达水平,升高mi R-152和SFRP4 m RNA和/或蛋白表达水平。结论:黄芪总皂苷对实验性关节炎具有一定的治疗作用,其机制可能与下调Lnc RNA S56464.1表达,调控mi R-152/Wnt信号通路,抑制成纤维样滑膜细胞增殖有关。
[Abstract]:Rheumatoid arthritis (RA) is a systemic, autoimmune disease, with chronic inflammation, dysplasia of synovial cells, joint swelling and pressure pain as its clinical features, which can lead to limited joint function, loss of working ability and a heavy burden on patients and society with.RA in 35~50 years of age, and the incidence of women is about approximately Male 2~3 times. At present, the specific pathogenesis of RA is not clear. It is believed that it may be related to immune factors, environmental factors and heredity. Therefore, it is of great theoretical and practical significance to actively explore the pathogenesis of RA and to find potential diagnostic markers. The previous research on the mechanism of RA should focus on the coding gene. In recent years, more and more studies have been made. The non coded RNAs (non-coding RNAs, NC RNAs), which has long been regarded as "noise" and "dark matter", has a complex biological function of the long chain non coded RNAs in.Nc RNAs (long non-coding RNAs, Lnc) is generated from a non coded sequence in the genome, with a length greater than that of a.Nc RNAs and does not have the ability to translate into protein. It can play an important regulatory role in epigenetic level, transcriptional level, translation level and protein modification, which is closely related to a variety of diseases. However, there is still little knowledge about the regulatory role of Lnc RNAs in the development of RA. At present, the treatment of RA in the clinic of Western medicine is mainly to reduce pain and control the symptoms. The side effect is obvious, the patient is not tolerable or poor compliance, and the remote effect is not ideal. Therefore, more and more attention has been paid to the development of RA drugs from traditional Chinese medicine. Astragalus membranaceus is the leguminous plant of Mongolia Astragalus Astragalus membranaceus (Fisch.) Bge.var.mongolicus (Bge.) Hsiao or membranous membranous Astragalus Astragalus membranaceus (Fisch.) Bge.. The dry root, which has the efficacy of stagnation, Qi Yang, and water elimination, is a common medicine in the clinical practice of traditional Chinese medicine. Astragalosides (AST) is an effective part of Astragalus, which mainly contains Astragalus saponins I~VIII. Modern pharmacological research shows that AST has many pharmacological effects, such as anti-inflammatory, antioxidant, immune regulation, anti aging, and so on. On the basis of the preliminary research of the project group, the following studies are carried out as follows: (1) screening of experimental arthritis related long chain noncoding RNAs and the intervention of Astragalus saponins; (2) screening the key Lnc RNAs in the pathogenesis of experimental arthritis based on the Lnc RNA-mi RNA-m RNA co expression network; (3) Lnc RNA S56464.1 targeting mi R-152/Wnt pathway To induce the proliferation of fibroblast-like synoviocytes (FLS) and the intervention of Astragalus saponins in order to systematically reveal the role of Lnc RNAs in the development of experimental arthritis and the role of the total saponins of Astragalus membranaceus, and to reveal the pathogenesis of experimental arthritis from the point of view of Lnc RNAs, and the mechanism of the pathogenesis of experimental arthritis is revealed. To elucidate the possible mechanism of Astragalus total saponins in the treatment of experimental arthritis. The first part of the experimental arthritis related long chain noncoding RNA and the intervention of Astragalus saponins: to screen the differential expression of long chain non coded RNA (long non-coding RNAs, Lnc RNAs) in the pathogenesis of experimental arthritis and to explore the possibility of experimental arthritis. The pathogenesis and the possible target of Astragalus saponins. Methods: the expression of Lnc RNAs/m RNAs in the synovial tissue of adjuvant arthritis (adjuvant arthritis, AA) rats was detected by Lnc RNA gene chip, and the Fold change value was 1.5, and the differential expression basis was screened by P-value0.05, and the gene ontology (Gene) and signal transduction were carried out. Lnc RNA-m RNA co expression network was constructed by Pathway analysis, and the key difference Lnc RNAs was screened and the real-time fluorescent quantitative PCR technique was used to verify it. Results: 260 differentially expressed LncRNAs were screened in the normal group and the model group, of which the Lnc RNAs expression data of the model group were 170, and the Lnc RNAs expression was downregulated. The data were 90, and 675 differentially expressed m RNAs were obtained. In the model group, 422 m RNAs expression data were up-regulated and 253 of M RNAs expression profiles were downregulated; 4163 GO entries were obtained. Among them, the difference genes were enriched in 2494 GO entries and 1669 GO items were enriched with the difference genes, and 42 signal pathways related to differential genes were obtained. The up-regulated genes were enriched in 25 signal pathways, and the differentially regulated genes were enriched in 17 signal pathways. The real-time quantitative PCR detection results of 6 key Lnc RNAs (XR_008357, U75927, MRAK046251, XR_006457, DQ266363 and MRAK003448) were consistent with the results of the gene chip. The normal group, the model group and the AST group screened 75 differential expressions of Lnc RNAs, compared with the normal group and the AST group, the model group up-regulated the Lnc RNAs expression data 41, downregulated the Lnc RNAs expression data, selected 247 differentially expressed m RNAs, the model group raised the m RNAs expression data 171, down the m RNAs expression data 76, and obtained 135 entries, among which 117 items were enriched by 117 entries. The differentially regulated genes were enriched in 18 GO items, and 17 signal pathways related to differential genes were obtained, in which the difference genes were enriched in 14 signal pathways and the differential genes were enriched in 3 signal pathways; the 4 key Lnc RNAs (MRAK012530, MRAK132628, MRAK003448, XR_00645) real-time fluorescent quantitative PCR detection results and gene chips The results are consistent. Conclusion: there is a differential expression of LncRNAs in the pathogenesis of experimental arthritis. The mechanism of AST for the prevention and control of experimental arthritis may be related to the differential expression of Lnc RNAs. The second part is based on the Lnc RNA-mi RNA-m RNA co expression network to screen the key Lnc RNAs in the process of experimental pathogenesis: screening experimental arthritis. The key Lnc RNAs in the pathogenesis was to discuss the possible pathogenesis. Methods: the MI RNAs gene expression profiles were derived from the high throughput gene expression database and published literature in the national biological information center of the United States. The data of Lnc RNAs/m RNAs gene expression profiles were published in the earlier period of the project group, and the standard of differential multiplier 2 and P-value0.05 as the standard Select the differentially expressed Lnc RNAs, MI RNAs and m RNAs, with the help of Miranda and targetscan to predict the m RNAs of differential mi RNAs. On the basis of S, m RNAs and differential mi RNAs, the Lnc RNA-mi RNA-m RNA co expression network is constructed and functional informatics analysis is used to screen the key Lnc RNAs. results in the pathogenesis of experimental arthritis. This network consists of 7 nodes, 24 nodes, 90 nodes, and 301 edges; functional information credits. The results showed that the difference gene was mainly enriched in 147 GO items and 23 signal pathways; LncRNA S56464.1, LncRNA XR_006437.1 and LncRNA J01878 were identified as the key genes in the pathogenesis of experimental arthritis. Conclusion: LncRNAs plays an important role in the pathogenesis of experimental arthritis, LncRNA S56464.1, Lnc RNA. 1 and Lnc RNA J01878 can be used as a potential diagnostic biomarker and therapeutic target. Third part of Lnc RNA S56464.1 target mi R-152/Wnt pathway to induce FLS proliferation and the intervention of Astragalus saponins: Lnc RNA S56464.1 to induce the proliferation of synovial cells by targeting the target pathway, and to explore the treatment experiment of Astragalus saponins The possible mechanism of sexual arthritis. Methods: tissue block transplantation was used to isolate the synovial cells of AA rats and to give the intervention of LncRNA S56464.1 siRNA and / or Astragalus saponins. The proliferation reaction of fibroblast-like synoviocyte (FLS) was detected by MTT, and RT-q PCR technique was used to detect Lnc RNA. 6464.1, MI R-152, beta catenin (beta -catenin), proto oncogene C-myc, cyclin (Cyclin) D1, glycogen synthase kinase (GSK) -3 beta and secretory curl related protein (SFRP) 4 m RNA expression changes. Compared with the model group, the Lnc RNA S6464.1 interference group and / or the total saponins of Astragalus membranaceus can not only significantly inhibit the proliferation of fibroblast like synovial cells in AA rats, but also significantly reduce the Lnc RNA56464.1, beta -catenin, C-myc, Cyclin D1 m RNA, and obviously inhibit the beta protein expression level and increase the level of beta protein. 52 and SFRP4 m RNA and / or protein expression level. Conclusion: the total saponins of Astragalus membranaceus have a certain therapeutic effect on experimental arthritis. The mechanism may be related to the regulation of the expression of Lnc RNA S56464.1, the regulation of MI R-152/Wnt signaling pathway and the inhibition of the proliferation of fibroid synovial cells.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R593.22

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