PBC患者PBMCs中miR-20a-5p的表达及其功能研究

发布时间：2018-10-23 19:43

【摘要】：原发性胆汁性肝硬化（primary biliary cirrhosis，PBC）是一种慢性肝脏疾病，以肝内中小胆管损伤、胆汁淤积以及部分患者出现肝硬化为特征，约90~95%的患者血清可出现高度特异的抗线粒体抗体（antimitochondrial autoantibodies，AMAs）[1]。PBC好发于中年女性，近年来其发病率逐年升高，但其发病机制仍不明确。在治疗方面，熊去氧胆酸（ursodeoxycholic acid，UDCA）是目前唯一公认的治疗药物，PBC患者对免疫抑制剂的治疗反应性差。微小RNA（microRNA，miRNA）作为一类序列短、不具有编码功能的小分子RNA，广泛参与基因的转录后调控[2]。已经有不少研究者证实了miRNA在类风湿性关节炎（rheumatoid arthritis，RA）、系统性红斑狼疮（systemic lupus erythematosus，SLE）、多发性硬化症（multiple sclerosis，MS）以及溃疡性结肠炎（ulcerative colitis，UC）等一系列自身免疫性疾病的发生、发展以及治疗过程中发挥着重要作用，近年来miRNA在PBC中的作用也逐渐得到关注。本实验小组之前已经就PBC患者的外周血单个核细胞（peripheral blood mononuclear cells，PBMCs）进行了差异性表达的miRNA芯片筛查，miR-17家族的miR-20a-5p、miR-106b和miR-93表达均显著上调，且位于miRNA-gene-network位于中心位置。白介素-8（interleukin-8，IL-8）是一种重要的炎症介质，Zimmermann H W等发现慢性肝脏疾病（chronic liver disease，CLD）患者血清IL-8水平升高，其中胆汁淤积性肝脏疾病和酒精性肝硬化患者IL-8水平升高最为显著，且IL-8水平与CLD的疾病进展以及非侵袭性肝硬化指标呈正相关[3]。本课题针对PBC患者miR-20a-5p和IL-8的表达水平进行检测，并对二者之间可能存在的调控关系进行探讨。第一部分：PBC患者miR-20a-5p和IL-8表达水平的检测我们收集长征医院PBC、RA、SLE、病毒性肝炎肝硬化（Viral hepatitis cirrhosis，VHC）住院患者和正常对照各20例，利用RT-PCR检测miR-20a-5p、IL-8在各类疾病PBMCs中的表达差异，血浆IL-8的表达水平采用ELISA方法检测。结果显示PBC患者的PBMCs中miR-20a-5p和IL-8的相对表达水平均较正常对照和其他疾病对照组显著升高，差异均有统计学意义（P0.05）。miR-20a-5p的相对表达量为正常对照组的6.33倍，IL-8的相对表达量为正常对照的7.96倍，PBC患者血浆IL-8的平均水平为89.5±36.6ng/L。第二部分：THP-1细胞中转染miR-20a-5p对IL-8表达水平的影响用100ng/ml的LPS对THP-1细胞进行预处理，6小时后转染miR-20a-5p的mimic和inhibitor，分别利用RT-PCR和ELISA检测IL-8表达水平的变化。结果显示转染了miR-20a-5p的mimic后，THP-1细胞IL-8的相对表达量降低，为空白对照的0.25倍，而转染了miR-20a-5p的inhibitor后THP-1细胞IL-8的相对表达量升高，为空白对照的4.02倍，差异均有统计学意义（P 0.05）。第三部分：miR-20a-5p对IL-8调控机制的探究为了进一步探究miR-20a-5p对IL-8表达水平调控的机制，我们在miRDB、TargetScan以及MicroRNA.org数据库对miR-20a-5p的靶基因进行预测，并利用双荧光素酶报告实验进行了miR-20a-5p与预测靶基因干扰素调节因子9（IFN regulatoryfactor9，IRF9）之间靶向作用关系的验证，最后针对IRF9设计了小干扰RNA（smallinterfering RNA，siRNA），利用RT-PCR和ELISA检测转染siRNA后IL-8表达的改变情况。MicroRNA.org数据库显示miR-20a-5p和IRF9之间有9个连续互补配对的碱基，存在紧密的靶向作用关系，，而将miR-20a-5p的mimic与构建好的报告基因载体共转后，报告基因相对荧光值下调了30.3%（P 0.05），也说明miR-20a-5p对IRF9有靶向调节作用。在LPS预处理的THP-1单核细胞株内转染IRF9的siRNA以后，细胞和培养上清内IL-8的表达均下调，其改变趋势与转染了miR-20a-5p的mimic之后改变一致。转染miR-20a-5p以及siRNA后IRF9的蛋白表达水平均用Westernblot实验进行检测，结果与RT-PCR检测结果一致。结论PBC患者外周血miR-20a-5p和IL-8的表达均升高；在THP-1细胞株内miR-20a-5p对IL-8的表达有抑制作用，而这种抑制作用可能是通过靶作用于IRF9的表达而实现的。miR-20a-5p有可能作为一种保护性因素抑制IL-8的持续升高，而IRF9通过何种途径影响IL-8的表达有待进一步研究。
[Abstract]:Primary biliary cirrhosis (PBC) is a kind of chronic liver disease, characterized by small and medium-sized bile duct injury, bile siltation and partial cirrhosis. About 90-95% of patients have high specific anti-mitochondrial antibody (AMAs)[1]. PBC is a middle-aged woman, whose incidence has increased year by year, but its pathogenesis is still unclear. In the treatment aspect, ursodic acid (UDCA) is the only recognized therapeutic drug, and PBC patients have poor therapeutic response to immunosuppressive agents. MicroRNA (miRNA) is a kind of short-molecule RNA with short sequence and no coding function, and is widely involved in the post-transcriptional regulation of gene. 2] Many researchers have demonstrated that miRNA is a series of autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), and chronic colitis (UC). In recent years, the role of miRNA in PBC has also been gradually obtained. The results showed that the expression of miR-20a-5p, miR-106b and miR-93 in the peripheral blood mononuclear cells (PBMNC) of PBC patients was significantly increased, and the expression of miR-20a-5p, miR-106b and miR-93 was significantly increased, and the miRNA-gene-network was located at the center. Interleukin-8 (IL-8) is an important inflammatory mediator, and the levels of IL-8 in serum of patients with chronic liver disease (CLD) are elevated in patients with chronic liver disease (CLD). The highest and most significant, and the level of IL-8 was positive with the disease progression of CLD and the non-invasive liver cirrhosis index.[3]. The objective of this study was to detect the expression levels of miR-20a-5p and IL-8 in patients with PBC. Regulatory relationships. Part 1: PBC patient miR-20a-5p The levels of IL-8 expression in patients with PBC, RA, SLE, viral hepatitis cirrhosis (VHC) and 20 normal controls were collected by RT-PCR, and miR-20a-5p and IL-8 were detected by RT-PCR. Differences in expression in PBRER and Table of plasma IL-8 The results showed that the relative expression levels of miR-20a-5p and IL-8 in PBMNC of PBC patients were significantly higher than those of normal control group and other control group (P <0.05). The relative expression of miR-20a-5p was 6.33 times that of normal control group. The expression level was 7. 96 times the normal control, and the level of plasma IL-8 in PBC patients was higher than that of normal control. miR-1 cells were transfected with miR-The effect of 20a-5p on the expression level of IL-8 was pretreated with 100ng/ ml LPS and then transfected into mimeic and intreitor of miR-20a-5p after 6 hours, respectively using RT-PC. The expression level of IL-8 was detected by R and ELISA. The results showed that the relative expression of IL-8 of IL-8 decreased in the transfected cells of miR-20a-5p, the relative expression of IL-8 was 0. 25 times of that of blank control, but the relative expression of IL-8 was increased after transfection of inactivator of miR-20a-5p. 02-fold, difference was statistically significant (P 0.05). In order to further explore the mechanism of miR-20a-5p on the regulation of IL-8 expression levels, we used miRDB, TargetScan and

MicroRNA. org database, and the relationship between miR-20a-5p and the target gene interferon regulatory factor 9 (IF9) was verified by double luciferase reporter assay. Finally, small interfering RNA was designed for IRF9. The changes of IL-8 expression after transfection of siRNA by RT-PCR and ELISA showed that there were 9 consecutive complementary pairs of bases between miR-20a-5p and IRF9 by RT-PCR and ELISA. The relative fluorescence value of reporter gene decreased by 30.3% (P 0.05). It is also indicated that miR-20a-5p has targeted regulation effect on IRF9. After transfected with IRF9 siRNA, the expression of IL-8 in cells and cultured supernatant is down regulated. After transfection with mimeic transfected with miR-20a-5p, the expression level of IRF9 after transfection of miR-20a-5p and siRNA was expressed by Western blot. The results showed that the expression of miR-20a-5p and IL-8 in peripheral blood of PBC was increased, and the expression of miR-20a-5p in human IL-1 cell line was inhibited by RT-PCR. This inhibition may be achieved by the expression of the target acting on IRF9. miR-20a-5p is likely to inhibit the continuous rise of IL-8 as a protective facto
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.22

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