【作者】 张立民；

【导师】 曾小峰； 张文； 李梦涛；

【作者基本信息】 中国协和医科大学 ， 内科学风湿免疫， 2010， 博士

【摘要】 背景MicroRNAs(miRNAs)属非编码RNA大家族,高度保守,广泛存在于从病毒、线虫、植物到动物体内,参与生物体的生长、发育、衰老、凋亡的调控。成熟miRNA通过核酸序列互补识别特定的目标mRNA,使之降解或抑制其翻译,从而抑制蛋白质的合成,达到调控基因表达的目的。生物信息学预测约1／3的mRNA受miRNA调控。在免疫细胞中表达的miRNA超过100个,在固有免疫及适应性免疫中发挥重要作用。近年来已经有研究显示在一些疾病中某些特定的miRNA与疾病的发生发展和预后密切相关,miRNA表达谱可以作为疾病的诊断和预后的指标。系统性红斑狼疮(SLE)是最为经典的自身免疫病,其特征为多系统受累,体内多种自身抗体产生、免疫复合物沉积在多种组织器官引起的免疫病理损伤,但具体的发病机制仍不清楚。已经有许多研究证实T、B细胞的功能异常在SLE的发病机制中发挥着重要作用。鉴于miRNA在调节T、B等免疫细胞中起重要作用,因此我们推测1miRNA可能在SLE的发病中同样发挥着重要作用。外周血PBMC是研究miRNA在SLE免疫细胞作用很好的靶点,本研究从高通量筛选miRNA在SLE患者PBMC中表达差异入手,对miRNA在SLE发病机制中的作用进行了初步研究。目的本研究通过筛选SLE患者PBMC中miRNA表达谱差异,寻找与SLE发病相关的miRNA,分析miRNA相对表达量与SLE临床谱的相关性,初步探讨了miR-125b在SLE中的可能的作用机制。方法1 MicroRNA表达谱芯片技术检测847个成熟miRNAs在初治SLE和正常对照组间表达水平差异,应用分层聚类分析筛选并获得一批SLE特异性表达的1niRNA分子,采用Real-timePCR方法对芯片的结果进行验证。2进一步扩大样本量,采用Real-timePCR方法对miR-125b在SLE患者、正常对照组及疾病对照组的表达水平进行检测,其中包括58例SLE患者、26例正常对照、8例类风湿关节炎(RA)患者、8例干燥综合征(pSS)患者,分析其与SLE临床谱的相关性；采用流式细胞仪分选免疫细胞,Real-timePCR方法检测miR-125b在不同免疫细胞中的相对表达量。3应用生物信息学方法预测miR-125b的靶基因。构建靶基因表达载体,通过双荧光报告转染实验,体外运用荧光报告系统检测过表达miR-125b后对双荧光素酶报告基因活性的影响。结果1对847个成熟miRNAs的表达谱检测结果表明存在SLE特异性的miRNA表达谱。筛选并获得了一批SLE特异性表达的miRNA分子,其中有37个miRNA在SLE和正常人PBMC中表达差异显著,26个表达下调,11个表达上调,揭示了SLE患者PBMC中存在着特异的miRNA表达谱。进一步用Real-time PCR对上述结果进行了验证,得出相同结论。2扩大样本量进一步证实了miR-125b在SLE患者外周血PBMC中表达量明显减低,与正常对照比较有明显差异,且降低幅度与SLE脏器受累相关。比较miR-125b在T、B细胞及非T、B细胞表达量中的差异发现miR-125b在T细胞表达差异最为明显。3运用TargetScan等软件对miR-125b的靶基因进行了预测,根据软件评分及基因功能选择了Ets1、TNFAIP3和STAT3作为候选靶基因。构建了Ets1、TNFAIP3和STAT3的双荧光酶报告基因载体,体外实验证实miR-125b可与Ets1、TNFAIP3及STAT3相互作用,减弱报告基因的表达,推测Ets1、TNFAIP3和STAT3可能是1niR-125b的靶基因。结论该研究结果表明miRNA在SLE发病中发挥重要作用,miRNA表达信号谱可作为SLE诊断和病情评价重要生物标志物。1niR-125b通过作用于Etsl等靶基因影响T细胞功能参与SLE的致病机制,miR-125b可能成为SLE治疗新的药物干预靶点。更多还原

【Abstract】 MicroRNAs(miRNAs) are small, singal-stranded noncoding RNAs, many of which have been highly conserved throughout evolution. Currently, miRNA is known to regulate cellular processes such as differentiation, cell cycle, apoptosis and immune functions. The known function of miRNAs is the post-transcriptional regulation of certain subsets of messenger RNAs(mRNAs) by binding to their 3’untranslated region(UTR) thus targeting them for degradation or translational repression. To date, the miRNA sequence database, miRbase, includes over 9000 predicted miRNA in numerous species of plants, animals, and viruses. For humans alone, miRBase lists over 1000 predicted miRNAs, and other bioinformatics predictions indicate that as much as one-third of all mRNAs may be regulated by miRNA. More than 100 different miRNAs are expressed by cells of the immune system. They have the potential to broadly influence the molecular pathways that control the development, and function of innate and adaptive immune response by regulating target mRNA expression. The latest research demonstrated that some miRNAs are closely correlated with occurrence, development and prognosis of some diseases, which can be adopted as diagnostic and prognostic index for disease. SLE is a prototypic autoimmune disease with a diverse array of clinical manifestations, which is characterized by the production of antibodies to components of the cell nucleus. The pathogenesis of SLE is poorly understood, and current therapies are based on nonspecific immunosuppression. To date, little is known about the SLE-specific miRNAs expression profile and the roles of miRNAs in SLE pathogenesis. To study SLE-related miRNAs, an important srarting point is to examine expression profile of miRNAs in PBMC. In this study, we developed a novel miRNA-specific microarray, screened a set of SLE-related miRNAs, and further predicted their targets by bioinformatics and valited it by dual-luciferase reporter transfection assay.Objective This study was undertaken to explore microRNA expression profile in peripheral blood cells of patients with SLE, and to study the potential biological functions of the associated miRNA in the pathogenesis of SLE.Methods In this study, a total of 58 patients with available clinical data and 26 normal controls were enrolled to study the role of miRNA in SLE.4 patients and 4 normal controls were used for miRNA microarray analysis to detecting the levels of 847 miRNAs in peripheral blood cells. The rest were used for qRT-PCR confirmation and fulfill miRNA functional study. Reporter gene assay was conducted to determine the biological function of miR-125b.Results We performed a miRNA profiling analysis to 4 SLE patients using the 847 human mature miRNAs and obtained a set of SLE-specific miRNAs, such as miR-125b, miR-326, miR-26a ect. Eleven miRNAs were up-regulated, twenty-six miRNAs were down-regulated. The difference of expression in these miRNAs suggests that a unique SLE-specific miRNA profiling exists. Further analysis showed that underexpression of miR-125b mainly in T cell negatively correlated with organ involvement. To further demonstrate the role of miR-125b, we predicted its putative target using TargetScan software and chosen several potential targets. Then we validated Etsl, TNFAIP3 and STAT3 were targets of miR-125b by dual-luciferase reporter transfection assay.Conclusion Our findings revealed that miRNA expression profile may serve as a new biomarker of SLE. Underexpression of miR-125b mainly in T cells contributed to SLE progression, by targeting the gene Ets1, TNFAIP3 and STAT3 which would not only elucidate the novel pathogenic machanism in SLE, but also lay a foundation for new drug development based on miR-125b.更多还原