Function of Long Non-Coding RNA MD1 in Leiomyoma Pathogenesis

子宫肌瘤(肌瘤)在70%以上的妇女中发生在生殖年龄段；然而，其病因 仍然不为人知。我们的团队已经确定了两个miRNAs，miR-200c和miR-29c，它们对 调控与细胞周期、细胞转化、炎症和免疫相关的基因 细胞外基质积聚对肌瘤的发病具有重要意义。我们最新的发现 使用下一代测序提供了非编码RNA的全面配置文件，包括长的非 编码RNA(LncRNAs)以及新型的小非编码RNA群(SncRNAs)。我们的预赛 本研究发现Linc-MD1和miR-135家族在子宫肌瘤中异常表达，因此 这项提议的推动力。使用qRT-PCR检测到Linc-MD1水平显著降低，而 MiR-135家族在子宫肌瘤中的高表达。LINC-MD1和OUR序列 初步数据表明，Linc-MD1可以作为miR-135家族(miR-135a/-135b)的miRNA海绵 平滑肌瘤的平滑肌细胞。因此，根据这一初步数据，我们假设平滑肌瘤 与子宫肌层相比，Linc-MD1通过miRNA引导的表达降低 MiR-135参与功能调控特定靶基因表达的机制 与Wnt/β-Catenin信号通路相关，已知该信号通路是肌瘤的中枢 发病机制。为了检验我们的核心假设，我们提出了两个具体目标。在目标1中，我们将确定 用RNA免疫沉淀和RNA下拉实验研究Linc-MD1与miR-135家族的相互作用。我们 将Linc-MD1在LSMC或MSMC球状细胞中过表达或不表达及其对miR-135的影响 已知的调节β-连环蛋白的家族及其下游靶基因Gsk3、Gsk3、Apc和Apc 退化。在这些实验中，β-连环蛋白、其磷酸化形式及其核定位将被 在体外对过度表达和基因敲除研究做出反应而确定。建立临床相关性 和Linc-MD1的治疗潜力在目标2中我们将确定Linc-MD1过表达对人肝癌细胞的影响 子宫肌瘤动物模型中，子宫肌瘤细胞对肌瘤进展的影响。此翻译建议解决了 对新的lncRNA-miRNA网络在肌瘤发病机制中的作用的认识存在显著差距 这是美国国立卫生研究院的优先研究领域，并可能成为治疗目的的目标 肌瘤。

Uterine leiomyoma (fibroids) develop during reproductive years in over 70% of women; however, their etiology remains unknown. Our group has identified two miRNAs, miR-200c and miR-29c, which are critical to regulation of genes functionally associated with cell cycle, cellular transformation, inflammation and extracellular matrix accumulation and thus of great significance to fibroid pathogenesis. Our recent findings using next generation sequencing provided a comprehensive profile of non-coding RNAs including long non- coding RNAs (lncRNAs) as well as novel groups of small non-coding RNAs (sncRNAs). Our preliminary findings here have identified aberrant expression of linc-MD1 and miR-135 family in fibroids and thus the impetus for this proposal. Using qRT-PCR we detected markedly reduced levels of linc-MD1 while significantly higher expression of miR-135 family in leiomyomas. The sequences of the linc-MD1 and our preliminary data suggest that linc-MD1 can act as miRNA sponge for miR-135 family (miR-135a/-135b) in leiomyoma smooth muscle cells. Therefore, based on this preliminary data we hypothesize that leiomyoma as compared to myometrium displays reduced expression of linc-MD1 which through a miRNA-guided mechanism involving miR-135 regulates the expression of specific target genes functionally associated with Wnt/β-catenin signaling pathway which is known to be central to leiomyoma pathogenesis. To test our core hypothesis we propose 2 specific aims. In Aim 1 we will determine the interaction between linc-MD1 and miR-135 family by RNA immunoprecipitation and RNA pull-down assay. We will over or under express linc-MD1 in LSMC or MSMC spheroid cells and determine its effect on miR-135 family and its downstream target genes namely GSK3β and APC which are known to regulate β-Catenin degradation. In these experiments β-Catenin, its phosphorylated form and its nuclear localization will be determined in response to overexpression and knockdown studies in vitro. To establish the clinical relevance and therapeutic potential of linc-MD1 in Aim 2 we will determine the effect of linc-MD1 overexpression in leiomyoma cells on fibroid progression in a leiomyoma animal model. This translational proposal addresses a significant gap in knowledge on the role of a novel lncRNA-miRNA network in the pathogenesis of fibroids which is a priority area of investigation for NIH, and could potentially be targeted for therapeutic purposes for fibroids.