【作者】 李海涛；

【导师】 杨蓉娅；

【作者基本信息】 第三军医大学 ， 皮肤病与性病学， 2011， 博士

【摘要】 一、研究背景和目的系统性硬皮病(SSc)是一种病因不明、发病机制尚不明确、涉及多因素的复杂性疾病,临床上主要以皮肤和内脏器官纤维化、血管异常病变和免疫系统异常为主要特点的自身免疫性结缔组织病。其中纤维化是系统性硬皮病发病过程中最突出的特点,也是严重影响病人生活质量、甚至危及病人生命的主要威胁。在以往的研究中,系统性硬皮病的发病机理主要涉及纤维化、血管病变、免疫系统异常、双胞胎研究、遗传、种族、嵌合体等多个方面,也涉及了基因芯片、蛋白芯片等技术在基因组范围内进行研究,但均未能成功的解释其发病机理。MircoRNAs (miRNAs)是存在于许多生物体内长约18~25 nt的一类短序列、非编码、具调控功能的单链小分子RNA,几乎参与所有生命过程中一系列的重要进程,包括早期发育,细胞增殖、分化与凋亡,物质代谢等。目前已经明确,miRNAs是通过与其靶mRNA分子的3′端非编码区(3′-UTR)互补结合,在翻译水平上特异性抑制基因表达,在许多疾病的发病中扮演着重要角色。mircoRNA芯片是近年来出现的发展比较成熟的高通量基因芯片技术,能在特定的时间点筛选出患者与正常人之间的差异microRNA表达情况,已在不少复杂性疾病的研究中得到了广泛的应用,特别是恶性肿瘤的研究中应用最为广泛。目前多个研究已经证实microRNA与肿瘤的发病、转移、复发以及银屑病、系统红斑狼疮、恶性黑素瘤等疾病的发病密切相关,并找到了疾病特异性microRNA,如miR-203是银屑病特异性microRNA,miR-140与骨肉瘤密切相关等。SSc作为一种复杂性自身免疫性疾病,与肿瘤、银屑病、系统红斑狼疮有类似的地方,因此,我们推测在其发病机制中很可能也有其发病相关的microRNA。本研究用microRNA芯片技术和生物信息学的方法,在国内外首次对系统性硬皮病患者皮损和正常人皮肤中的差异microRNA进行分析筛选,并对筛选结果进行初步的分析和预测,以期获得与系统性硬皮病发病关系密切相关或特异性的microRNA,为进一步研究microRNA在系统性硬皮病发病中的分子机制、发现新的诊断标记与治疗靶点提供重要参考。二、对象与方法将研究对象分为SSc组和对照组。SSc组的纳入标准为:符合美国风湿病学会关于系统性硬皮病的诊断标准、发病较早(<2年)且未经过长期系统激素治疗(激素应用<3个月)、有全身皮肤的硬化及肺纤维化的证据但临床症状不明显的患者,正常对照组为包皮环切患者的皮肤或良性皮肤痣切除术患者边缘的正常皮肤;分别切取足量皮肤,提取总RNA,分离microRNA并进行荧光标记;然后进行杂交、清洗,之后进行microRNA芯片扫描;扫描结果用LuxScan3.0图像分析软件和SAM2.1软件进行差异microRNA的分析筛选;Real-time PCR方法随机验证筛选出的差异表达microRNA,以确保芯片结果的可靠性。生物信息学分析过程:首先,建立重点靶基因筛选库;其次,在microRNA数据库中(TargetScan数据库)进行其靶基因(mRNA)的搜索获得其功能注释;第三,将搜索到的靶基因与重点靶基因筛选库的基因进行比对,挑选出功能上与细胞外基质(ECM)、纤维化、血管异常、免疫异常等相关以及存在于重点靶基因筛选库中的基因,作为重点靶基因;第四,登录NCBI网站,将重点靶基因与SSc输入NCBI进行检索,检索式:“SSc and重点靶基因”;然后阅读检索出的文献,分析其是否与SSc发病相关;如有多个(≧5个)由同一个miRNA调节的靶基因与SSc发病相关,那么就可以初步预测出该miRNA与SSc的发病相关;生物信息学方法查询出差异表达microRNA所调节的靶mRNA,PubMed上检索出目前已发表文献中与系统性硬皮病发病相关的靶mRNA,即可初步预测出该microRNA与系统性硬皮病的发病相关。三、结果1.我们首次从系统性硬皮病患者皮损中筛选出24个差异表达microRNA,其中9个microRNA表达上调, 15个microRNA表达下调;并随机抽取三个microRNA——hsa-let-7g, hsa-miR-206和hsa-miR-125b,用Real-time PCR的方法进行可靠性验证,验证结果与芯片分析结果一致,表明芯片结果可靠;2.通过生物信息学(www.gogene.org,目前该网站在维护中,不能打开,可以通过http://www.targetscan.org进行microRNA靶mRNA的查询)和文献分析(http://www.ncbi.nlm.nih.gov/pubmed/)发现有6个差异表达的microRNA其调节的靶基因与SSc的发病相关,其中hsa-miR-206调控的584条靶基因中有7条与SSc发病密切相关;hsa-let-7g调控的819条靶基因中有24条与SSc发病密切相关;hsa-miR-133a调控的502条靶基因中有12条与SSc发病密切相关;hsa-miR-125b调控的604条靶基因中有8条与SSc发病密切相关;hsa-miR-140-5p调控的251条靶基因中有7条与SSc发病密切相关;hsa-miR-23b调控的480条靶基因中有14条与SSc发病密切相关。四、结论1.microRNA不仅参与了系统性硬皮病的发病过程,而且可能在系统性硬皮病的发病中扮演着重要的角色;2.hsa-miR-206等6个miRNA与SSc发病密切相关,并很可能在SSc的发病中起着较为重要的作用;3.microRNA的表达异常可能是SSc发生、发展以及演化的重要分子事件,特别是在该病的早期阶段,可能起着更为重要的作用;4.有意义的差异表达microRNA可能是系统性硬皮病潜在的诊断标记、治疗靶点或重要的致病因素,为进一步研究系统性硬皮病的分子机制提供重要参考。更多还原

【Abstract】 Background and ObjectivesSystemic Scleroderma is an autoimmunity connective tissue disease, which is a complex disease involved in multiple factor, its etiology and pathogenesis is unclear. In clinically, it is characterized by excessive and widespread fibrosis in the skin and internal organs, vasculopathy and abnormal activation of the immune system. And fibrosis is the most remarkable clinic character in the process of scleroderma, it is also the major threaten of influencing seriously on the life quality of patients, even the patients’life. In previous studies, the pathogenesis of SSc was focused mainly on fibrosis, vessels change, the immune system abnormalty, twins, heredity, race, chimeras, and others, including genome-wide studies using gene-chip and protein-chip technology, but all can’t elucidate the SSc’s pathogenesis.MiRNAs are short 18- to 25-nt noncoding single strand RNAs that serve a regulatory function and participate in many vital processes, including early development, cell proliferation, cell differentiation, apoptosis, substance metabolism and others. These miRNAs have been demonstrated to suppress the expression of specific genes at the translation level by complementary binding to the 3′noncoding region of target mRNA, which play important role in the pathogenesis of many disease. MicroRNA arrays are a newly developed high-throughput screening technology that can be used to detect different expression levels of miRNAs in patients and the healthy subjects at the specified time and space. which hsa been applied to the study of some complex diseases, especially in the study of malignant tumor. It was demonstrated from a lot of study that microRNA plays the important role on the pathogenesis, metastasis, relapse, of malignant tumor, and the pathogenesis of psoriasis, SLE, malignant melanoma and so on, and some disease-specificity microRNA were found, for example: miR-203 is the psoriasis-specificity microRNA, miR-140 expression is related to osteosarcoma, and so on. As a sort of complex autoimmunity disease, SSc hsa been similar to tumor, psoriasis, SLE. So we presumed that microRNA may also play important role in the pathogensis and otherelse of SSc. With microRNA chip technology and bioinformatics methods, For the first time in the world, we screened different expression microRNA between SSc patient’s skin lesion and health people, then analyzed and predicted the screening results so as to get the microRNA, which is intimate or related or specific to SSc. The study result may provide the important reference for studying molecular mechanism of microRNA in the SSc, finding new diagnosis biomarker and new therapy target.Objects and MethodsThe objects were divided to two group, SSc group (SSc G.) and Control group (Ctrol. G.). the selected criterion of SSc G. was the patients who fulfilled the American College of Rheumatology classification criteria for SSc, had suffered from the SSc within 2 years, had not received recent hormonal systemic therapy or no more than 3 month, had systemic skin sclerosis and some signs of lung fibrosis but without obvious clinical manifestations. The Ctrol. G. was skin tissues of circumcision and the normal skin bordering a melanin pigmented naevus ectomy. Incise enough skin tissue from the two group patients, extract their total RNA, Isolate microRNA and fluorescent Labeled, Then hybridizing, cleaning and scanning. The scanning results and the different expression microRNA were analyzed with LuxScan 3.0 image analysis software and SAM 2.1 software. The different expression microRNA were demonstrated randomly with Real-time PCR to ensure the reliability of chip results.The process of bioinformatics analysis: firstly, establishing screening base of the emphsais target gene; Secondly, searching the target genes in the microRNA database (Target Scan), and get their function annotation; Thirdly, comparing the target genes with the genes in the screening base of the emphsais target gene, choose out the genes whose function are related to the ECM, fibrosis, vassular abnormality, immunologic abnormality, or which are in the screening base of the emphsais target gene, it is regarded as emphsais target genes; Fourthly, logging the website of NCBI, searching the emphsais target gene and the SSc, the searching formula is“SSc and emphsais target gene”, then reading the literature and analyzing which is relate to SSc or not; If there have many target genes regulated by the same microRNA are related to SSc or participant in the pathogenesis of SSc, then we would initially predict this microRNA correction with SSc pathogenesis.Results 1.We firstly selected out 24 different expression microRNA were from SSc patient’s skin lesion, in which the 9 microRNAs were upregulation and the 15 microRNAs downregulation. the three differential expression of microRNAs (microRNA—hsa-let-7g, hsa-miR-206 and hsa-miR-125b) were selected outrandomly, and demonstrated with real-time PCR to ensure the reliability of chip results. The verification results were consistent with the chip results. 2.The 6 differential expression of microRNAs were demonstrated that they may be related to SSc pathogenesis with bioinformatics methods and document analysis(http://www.ncbi.nlm.nih.gov/pubmed/). (www.gogene.org, now this website is maintenance, can’t open it, you can inquire out the target mRNA regulated by microRNA to http://www.targetscan.org ), in which, The 7 genes in the 584 target genes regulated by hsa-miR-206 were correlation with SSc, The 24 genes in the 819 target genes regulated by hsa-let-7g were correlation with SSc, The 12 genes in the 502 target genes regulated by hsa-miR-133a were correlation with SSc, The 8 genes in the 604 target genes regulated by hsa-miR-125b were correlation with SSc, The 7 genes in the 251 target genes regulated by hsa-miR-140-5p were correlation with SSc, The 14 genes in the 480 target genes regulated by hsa-miR-23b were correlation with SSc.Conclusions1.MicroRNA not only participant in the process of SSc pathogenesis, but play the important role in the SSc etiopathogenisis;2.The hsa-miR-206 six miRNAs are closely correlation with SSc’pathogenesis and they may have more important function in the SSc pathogenesis, development and turnover;3.The expression abnormalty of microRNA may be the important molecular event during the process of SSc genesis, development and disease evolution, especially in the early stage of SSc, which may take more important function;4.The significant different expression microRNAs may be potentially diagnosis marker, therapy target or important causative agent, which will provide the important reference for studying SSc molecular mechanism.更多还原