Mechanism of Long Non-coding RNAs Action in leiomyoma

子宫平滑肌瘤（肌瘤）是一种良性肿瘤，困扰着相当数量的育龄妇女， 没有已知的原因。我们的实验室专注于了解miRNAs如何影响促炎性细胞因子， 和促纤维化途径，并确定了两个miRNAs-miR-200 c和miR-29 c， 靶向细胞周期、炎症和细胞外基质（ECM）组分基因分别作为 平滑肌瘤的发展。我们最近的发现使用下一代测序已经证明了一个整体 包括长链非编码RNA（lncRNA）在内的非编码RNA宿主在纤维瘤中失调。lncRNAs 可以作为miRNAs的海绵，因此可能是平滑肌瘤中基因失调的驱动因素。基于 我们选择了一组与肿瘤发生和组织纤维化相关的表达水平和功能， lncRNA以进一步表征。使用QRT-PCR，我们检测到XIST和MIAT的显著过表达， 平滑肌瘤这些lncRNA的序列和初步数据表明它们可以作为miRNA海绵 对于miR-29 c和miR-200 c。因此，根据我们公布的初步数据，我们假设， 与子宫肌层相比，平滑肌瘤显示出许多lncRNA表达的改变， 转录调节和独立或通过miRNA引导的机制调节 与细胞周期进程，炎症， 纤维化和表观遗传修饰，所有这些都是平滑肌瘤发病机制的核心。来测试我们 核心假设我们提出三个具体目标。在目标1中，我们将建立在我们初步的RNA测序研究基础上 并提供了lncRNA、miRNAs的全面概况，以及在大组细胞中同时表达的mRNA。 来自不同人种/种族群体的成对平滑肌瘤和子宫肌层，来自增殖型和分泌型 月经周期的阶段。通过生物信息学分析，我们将识别lncRNA亚型， 在平滑肌瘤中，染色体位置经常发生重排，并鉴定出可能起作用的lncRNA。 如miRNA海绵，或竞争性内源RNA（ceRNA）。我们还将确定是否存在MED 12 突变对这些ncRNA的谱有任何影响。目标2将涉及 子宫肌瘤中的MIAT和XIST。使用RNA免疫沉淀研究，我们将确定这些lncRNA是否 与已知在纤维形成中重要的miR-29 c和miR-200 c相互作用。在细胞水平上，我们将评估 卵巢类固醇在其表达中的作用，并通过敲低和敲入策略鉴定其 对ECM和细胞周期基因表达的影响。确定临床相关性和治疗潜力 我们将确定改变XIST和MIAT表达的影响，XIST和MIAT表达在Aim 3中高度表达。 在肌瘤中过表达对肌瘤进展和/或在平滑肌瘤动物模型中建立的影响。这 翻译建议解决了一个显着的差距，在知识的发病机制，肌瘤是一个 NIH的优先研究领域，并且可能具有治疗纤维瘤的潜在治疗应用。

Uterine leiomyoma (fibroids) are benign tumors afflicting a significant number of reproductive age women and without a known cause. Our laboratory has focused on understanding how miRNAs impact pro-inflammatory and pro-fibrotic pathways in leiomyoma, and identified two miRNAsmiR-200c and miR-29c which primarily target cell cycle, inflammation and extracellular matrix (ECM) component genes respectively as key in the development of leiomyoma. Our recent findings using next generation sequencing has demonstrated a whole host of non-coding RNAs including long non-coding RNAs (lncRNAs) are dysregulated in fibroids. LncRNAs can act as sponge for miRNAs and therefore may be a driver of gene dysregulation in leiomyomas. Based on the level of expression and functional association with tumorigenesis and tissue fibrosis we selected a group of lncRNAs to further characterize. Using QRT-PCR we detected marked overexpression of XIST and MIAT in leiomyomas. The sequences of these lncRNAs and preliminary data suggest they can act as miRNA sponges for miR-29c and miR-200c. Therefore, based on our published and preliminary data we hypothesize that leiomyoma as compared to myometrium display an altered expression of a number of lncRNAs that are transcriptionally regulated and independently or through miRNA-guided mechanisms regulate the expression of specific target genes functionally associated with cell cycle progression, inflammation, fibrosis and epigenetic modification all of which are central to leiomyoma pathogenesis. To test our core hypothesis we propose 3 specific aims. In Aim 1 we will build on our preliminary RNA sequencing study and provide a comprehensive profile of lncRNAs, miRNAs, with concurrent mRNAs expression in large sets of paired leiomyoma and myometrium from different race/ethnic groups derived from proliferative and secretory phase of the menstrual cycle. Through bioinformatic analysis we will identify lncRNAs subtypes, their chromosomal locations at loci often rearranged in leiomyoma and identify lncRNAs which could potentially act as miRNA sponges, or competing endogenous RNAs (ceRNAs). We will also determine if presence of MED12 mutations has any impact on the profiles of these ncRNAs. Aim 2 will address the regulation and function of MIAT and XIST in leiomyoma. Using RNA immunoprecipitation studies we will determine if these lncRNAs will interact with miR-29c and miR-200c known to be important in fibrogenesis. At the cellular level we will assess the role of ovarian steroids on their expression and through knockdown and knock-in strategies identify their effects on expression of ECM and cell cycle genes. To establish the clinical relevance and therapeutic potential of lncRNAs in Aim 3 we will determine the effect of altering the expression of XIST and MIAT which are highly overexpressed in fibroids on fibroid progression and/or establishment in a leiomyoma animal model. This translational proposal addresses a significant gap in knowledge in the pathogenesis of fibroids which is a priority area of investigation for NIH, and could have potential therapeutic applications for treatment of fibroids.