Function of Long Non-Coding RNA MD1 in Leiomyoma Pathogenesis

长链非编码RNA MD1在平滑肌瘤发病机制中的作用

• 批准号: 10156935
• 负责人: OMID A. KHORRAM
• 金额: $ 7.71万
• 依托单位: LUNDQUIST INSTITUTE FOR BIOMEDICAL INNOVATION AT HARBOR-UCLA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-15 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10156935
• 关键词: Address; African American; Age; Animal Model; Area; Benign; Biological Assay; Cell Cycle; Cell Proliferation; Cells; Characteristics; Code; Data; Development; Disease; Epigenetic Process; Etiology; Extracellular Matrix; Family; Female Genital Neoplasms; Fibroid Tumor; Fibrosis; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Hemorrhage; High Prevalence; Immunoprecipitation; In Vitro; Inflammation; Investigation; Kidney; Knowledge; Laboratories; Leiomyoma; Lentivirus; Link; MicroRNAs; Modeling; Mus; Nuclear; Pain; Pathogenesis; Pathway interactions; Pelvic Pain; Porifera; Preclinical Testing; Proteins; Quantitative Reverse Transcriptase PCR; RNA; Repression; Role; Signal Pathway; Signal Transduction; Smooth Muscle Myocytes; Symptoms; Testing; Therapeutic; Time; Tissues; Transplantation; United States National Institutes of Health; Untranslated RNA; Uterine Fibroids; Uterine Neoplasms; Uterine hemorrhage; Woman; Work; angiogenesis; base; beta catenin; capsule; clinically relevant; differential expression; experimental study; in vivo imaging; in vivo monitoring; knock-down; metaplastic cell transformation; myometrium; next generation; next generation sequencing; novel; overexpression; pressure; protein expression; reproductive; response; targeted treatment; transcriptome sequencing; tumor growth; tumorigenesis

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.

子宫平滑肌瘤（肌瘤）发展在生育年龄超过70%的妇女;然而，其病因， 仍然未知。我们的研究小组已经鉴定出两种miRNAs，miR-200 c和miR-29 c，它们对 调节与细胞周期、细胞转化、炎症和 细胞外基质积累，因此对纤维瘤发病具有重要意义。我们最近的发现 使用下一代测序提供了非编码RNA的全面概况，包括长的非编码RNA， 编码RNA（lncRNA）以及新的一类小的非编码RNA（sncRNA）。我们的初步 这些发现已经确定了linc-MD 1和miR-135家族在子宫肌瘤中的异常表达， 推动这一提议。使用qRT-PCR，我们检测到linc-MD 1水平显著降低， miR-135家族在平滑肌瘤中的表达显著更高。linc-MD 1的序列和我们的 初步数据表明，linc-MD 1可以作为miR-135家族（miR-135 a/-135b）的miRNA海绵， 平滑肌瘤平滑肌细胞因此，基于这些初步数据，我们假设平滑肌瘤 与子宫肌层相比，显示linc-MD 1表达减少， 涉及miR-135的机制在功能上调节特定靶基因的表达 与Wnt/β-catenin信号通路相关，该信号通路已知是平滑肌瘤的中心 发病机制为了验证我们的核心假设，我们提出了两个具体目标。在目标1中，我们将确定 通过RNA免疫沉淀和RNA pull-down测定linc-MD 1和miR-135家族之间相互作用。我们 将在LSMC或MSMC球状体细胞中过表达或低表达linc-MD 1，并确定其对miR-135的影响 家族及其下游靶基因，即已知调节β-Catenin的GSK 3 β和APC 降解在这些实验中，β-连环蛋白、其磷酸化形式及其核定位将被 在体外响应于过表达和敲低研究中确定。确定临床相关性 目的2中linc-MD 1的治疗潜力，我们将确定linc-MD 1过表达在 平滑肌瘤细胞对平滑肌瘤动物模型中纤维瘤进展的影响。这一翻译建议涉及 关于新型lncRNA-miRNA网络在纤维瘤发病机制中的作用， 这是NIH的优先研究领域，可能用于治疗目的， 纤维瘤