Mechanism of Long Non-coding RNAs Action in leiomyoma

子宫肌瘤(肌瘤)是一种良性肿瘤，困扰着相当数量的育龄妇女和 没有已知的原因。我们的实验室一直致力于了解miRNAs如何影响促炎因子 和在肌瘤中的促纤维化途径，并鉴定了两个miRNAsmiR-200C和miR-29c，它们主要是 靶细胞周期、炎症和细胞外基质(ECM)成分基因分别在 子宫肌瘤的发展。我们使用下一代测序的最新发现证明了 肌瘤中存在大量非编码RNA，包括长非编码RNA(LncRNAs)。IncRNAs 可以作为miRNAs的海绵，因此可能是肌瘤基因失调的驱动因素。基于 表达水平和功能与肿瘤发生和组织纤维化的关系我们选择了一组 LncRNAs以进一步表征。利用QRT-PCR检测到XIST和MIAT在小鼠体内显著过表达 肌瘤。这些lncRNAs的序列和初步数据表明它们可以作为miRNA海绵 对于miR-29c和miR-200c。因此，根据我们公布的初步数据，我们假设 与子宫肌层相比，平滑肌瘤表现出许多LncRNAs的表达改变，这些LncRNA是 转录调控，并独立或通过miRNA引导的机制调节 在功能上与细胞周期进程、炎症、 纤维化和表观遗传修饰都是肌瘤发病的中心环节。测试我们的 核心假设我们提出了三个具体目标。在目标1中，我们将建立在初步的RNA测序研究的基础上 并提供了lncRNAs、miRNAs的全面概况，以及在大型集合中的并发mRNAs表达 来自不同种族/民族的增生性和分泌性子宫肌瘤和肌层配对 月经周期的阶段。通过生物信息学分析，我们将确定lncRNAs亚型，它们的 子宫肌瘤中经常重排的基因座上的染色体位置和识别潜在作用的lncRNAs 作为miRNA海绵，或竞争的内源RNA(CeRNAs)。我们还将确定是否存在MED12 突变对这些ncRNA的图谱有任何影响。目标2将涉及以下方面的规定和功能 子宫肌瘤的MIAT和XIST。利用RNA免疫沉淀研究，我们将确定这些lncRNAs是否会 已知与miR-29c和miR-200c相互作用在纤维化形成中起重要作用。在细胞层面上，我们将评估 卵巢类固醇激素对其表达的作用，并通过基因敲除和敲入策略确定其 对细胞外基质和细胞周期基因表达的影响。建立临床相关性和治疗潜力 对于目标3中的lncRNAs，我们将确定改变XIST和MIAT的表达的效果 在肌瘤动物模型中肌瘤过度表达对肌瘤进展和/或建立的影响。这 翻译建议解决了肌瘤发病机制方面的一个重大知识缺口，即 NIH的优先研究领域，并可能在治疗肌瘤方面具有潜在的治疗应用。

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.