The Role of miR-17~92 in Autoimmune Diseases

描述(申请人提供)：microRNAs(MiRNAs)最近成为一类主要的反式因子，通过其3‘UTRs调节蛋白质编码基因的表达，从而控制包括细胞分化、增殖和凋亡在内的各种生物学过程。MiRNAs与其靶基因的mRNAs配对，通常的结果是通过翻译抑制、mRNAs切割或促进mRNAs的降解而下调蛋白质的表达。已在哺乳动物中发现了数百个miRNAs，其中许多在进化上是保守的，其中一小部分分子在免疫系统中表现出高度特异的、受调控的表达模式。我们和其他研究小组的遗传学研究表明，miRNAs在淋巴细胞发育、免疫反应和淋巴肿大中发挥关键作用。然而，对miRNAs在自身免疫性疾病中的作用知之甚少。在人类患者中发现了miRNA编码基因的大量遗传和表观遗传学改变。其中包括miR-17~92基因的扩增，该基因编码6个不同的miRNAs，以及携带该基因扩增的细胞中miR-17~92miRNAs的表达上调。我们成功构建了miR-17~92转基因细胞，该基因可被Cre重组酶有条件地激活表达。引人注目的是，当使用hCD2-iCre在B和T淋巴细胞中启动转基因时，转基因小鼠患上了淋巴增殖性和自身免疫性疾病，并过早死亡。我们的初步研究表明，在抗IgM大自身(5b-ms)超抗原转基因模型中，转基因miR-17~92的表达打破了B细胞的耐受性。我们推测，转基因miR-17~92的表达主要通过破坏B细胞耐受而导致自身免疫性疾病。我们现在建议进行研究，以阐明miR-17~92介导的B细胞耐受破坏和自身免疫性疾病发生的细胞和分子机制，并阐明miRNA表达的变化如何促进自身免疫以及miRNA簇如何发挥其功能。我们将确定转基因miR-17~92在B细胞中的表达在自身免疫性疾病中的作用(目标1)，评估转基因miR-17~92表达对B细胞耐受检查点的影响(目标2)，剖析miR-17~92簇中单个miRNAs在打破B细胞耐受性中的功能贡献(目标3)，并在miR-17~92转基因小鼠中识别导致自身免疫性疾病的靶基因和分子通路(目标4)。我们将结合遗传、蛋白质组、基因组、分子和生物信息学方法来实现这些目标。 与公共卫生相关：这项拟议的研究将阐明miRNA表达的变化有助于自身免疫的方式。一旦miRNA失控与人类自身免疫性疾病之间的因果关系被确定，这些miRNA本身就可以成为有价值的诊断标记物和可能的治疗靶点。此外，miRNA靶基因以及miRNAs与其关键靶mRNAs之间的物理相互作用可能成为治疗干预的理想靶点。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) have recently emerged as a major class of trans-factors that regulate expression of protein coding genes through their 3'UTRs, thereby controlling a diverse range of biological processes including cell differentiation, proliferation, and apoptosis. miRNAs pair with mRNAs of their target genes and the usual consequence is the downregulation of protein expression by translational repression, mRNA cleavage, or promotion of mRNA decay. Hundreds of miRNAs, many of them evolutionarily conserved, have been identified in mammals, and a fraction of these molecules exhibit highly specific, regulated expression patterns in the immune system. Genetic studies from us and other groups have demonstrated that miRNAs play critical roles in lymphocyte development, immune responses, and lymphomagenesis. However, little is known about the roles of miRNAs in autoimmune diseases. A large amount of genetic and epigenetic alterations in miRNA coding genes have been found in human patients. Among them is the amplification of the miR-17~92 gene, which encodes six distinct miRNAs, and the elevated expression of miR-17~92 miRNAs in cells carrying this gene amplification. We have generated a miR-17~92 transgene whose expression can be turned on conditionally by Cre recombinase in mice. Strikingly, the transgenic mice developed a lymphoproliferative and autoimmune disease, and died prematurely, when this transgene was turned on in both B and T lymphocytes using hCD2-iCre. Our preliminary studies showed that transgenic miR-17 ~ 92 expressions broke B cell tolerance in an anti-IgM macroself (5b-ms) superantigen transgenic model. We hypothesize that transgenic miR-17~92 expression causes autoimmune diseases mainly by breaking B cell tolerance. We now propose studies to elucidate the cellular and molecular mechanisms underlying miR-17~92 mediated breaking of B cell tolerance and development of autoimmune diseases, and to illustrate how alterations in miRNA expression contribute to autoimmunity and how miRNA clusters carry out their functions. We will determine the contribution of transgenic miR-17~92 expression in B cells to the autoimmune disease (Aim 1), assess the impact of transgenic miR-17~92 expression on B cell tolerance checkpoints (Aim 2), dissect the functional contribution of individual miRNAs in the miR-17~92 cluster to the breaking of B cell tolerance (Aim 3), and identify target genes and molecular pathways whose deregulation leads to the autoimmune disease in miR-17~92 transgenic mice (Aim 4). We will combine genetic, proteomic, genomic, molecular, and bioinformatic approaches to achieve these goals. PUBLIC HEALTH RELEVANCE: The proposed study will shed light on the ways by which alterations in miRNA expression contribute to autoimmunity. Once the causative relationship between miRNA deregulation and human autoimmune diseases has been established, those miRNAs themselves can become valuable diagnostic markers and possible targets of therapeutics. Furthermore, miRNA target genes and the physical interactions between miRNAs and their key target mRNAs could make ideal targets for therapeutic interventions.