Molecular mechanism of leiomyoma growth and regression

描述(申请人提供)：子宫肌瘤是一种良性子宫肿瘤，由子宫肌层细胞转化而来，病因不明。通过基因组和蛋白质组分析，我们已经鉴定了一些在肌瘤中与肌层相比差异表达和调控的基因，这些基因具有不同的生物学功能，包括细胞转化、增殖和促炎/促纤维化活性，如IL-13和转化生长因子-2。ZEB1是一种转录因子和E-钙粘蛋白的主要抑制因子，在细胞向间充质表型转变过程中丢失，与子宫肌层相比，在肌瘤中过表达。根据差异表达、激素调节以及转化生长因子-2和IL-13的调节，我们认为ZEB1和ZEB2也是子宫肌瘤表型转化的关键调节因子。我们进一步认为，Zebs的表达和稳定性分别受到表观遗传和microRNA的调控。为了验证Zebs的表达、调控和功能导致肌瘤发生和生长的假设，我们提出了以下具体目标。目的1验证Zebs和E-cadherin在子宫肌瘤中的差异表达和调控，它们的表达与转化生长因子-2和IL-13的表达直接/负相关，并在接受激素治疗以抑制子宫肌瘤生长的患者中发生改变。此外，上述队列中的HDAC 2、4和EZH2在肌瘤和子宫肌层中的表达谱与它们作为针对其表达的ZeB和miRNAs表达的表观遗传调节因子的作用相关，并且与高加索人相比在非裔美国人的组织中差异表达。目的#2将验证一种假说，即促炎症/促纤维化分子，如IL-13和转化生长因子-2，直接调节子宫肌层平滑肌细胞(MSMC)中Zebs的表达，导致E-钙粘素丢失而导致表型转化，而GnRHa、RU-486、Depo-Provera和促纤维化/炎症介质抑制剂曲尼司特可阻止它们的作用。在MSMC和LSMC中，通过siRNAs和miR-200b/c的处理改变了ZeBS的进一步功能活性，从而改变了它们下游的靶基因，包括E-钙粘蛋白。目的#3将验证这一假说，即Zebs的表达是通过表观遗传修饰和miRNAs在子宫肌层细胞中的作用来调节的，这种机制的改变会导致转化为肌瘤细胞。这将通过评估HDACs和EZH2以及针对其在子宫肌层和肌瘤细胞中表达的miRNAs的表达模式进行测试；在单独和联合使用IL-13和转化生长因子-2治疗后，以及在曲尼司特治疗之后，miRNAs分别获得HDACs和CpG甲基化的功能和抑制剂。为了实现这些目标，我们将使用肌瘤和匹配的子宫肌层，并利用生化、分子和细胞生物学方法的组合。我们期望这项拟议的研究将导致进一步确认促纤维化/炎性衍生介质在子宫肌层细胞转化为肌瘤细胞中的直接作用，并有助于全面开发一种新的治疗方法来防止子宫肌瘤的生长和特异性治疗。 公共卫生相关性：子宫肌瘤是一种良性子宫肿瘤，估计在70%的女性中发生，更具体地说是在非裔美国人的生育期。有症状的子宫肌瘤导致慢性盆腔疼痛和异常子宫出血，这是接受子宫切除术的主要适应症。这项提案将调查肌瘤是如何发展和生长的，以及预防它们的方法，包括它们的症状。

DESCRIPTION (provided by applicant): Leiomyomas are benign uterine tumors which develop from transformation of myometrial cells without a known etiology. Through genomic and proteomic analysis we have identified a number of differentially expressed and regulated genes in leiomyomas as compared to myometrium, with diverse biological functions, including cellular transformation, proliferation, and pro-inflammatory/pro-fibrotic activities such as IL-13 and TGF-2. ZEB1, a transcription factor and major repressor of E-cadherin, which is lost during cellular transition into mesenchymal phenotype, is overexpressed in leiomyomas as compared to myometrium. Based on differential expression, hormonal regulation and regulation by TGF-2 and IL-13, we propose that ZEB1 and ZEB2 also serve as key regulators of myometrial phenotypic transformation into leiomyomas. We further propose that expression and stability of ZEBs are subject to epigenetic and microRNA regulation, respectively. To test the hypothesis that the expression, regulation and function of ZEBs result in development and growth of leiomyoma, we propose the following specific aims. Aim#1 will test the hypothesis that ZEBs and E-cadherin are differentially expressed and regulated in leiomyomas as compared to myometrium, and their expression directly/inversely correlates with the expression of TGF-2 and IL-13 and altered in patients who receive hormonal therapies to suppress leiomyoma growth. Furthermore, the expression profiles of HDAC's 2, 4, and EZH2 in leiomyoma and myometrium from the above cohorts correlate with their role as epigenetic regulators of the expression of ZEBs and miRNAs that target their expression, and are differentially expressed in tissues from African Americans as compared to Caucasians. Aim#2 will test the hypothesis that pro-inflammatory/pro-fibrotic molecules, such as IL-13 and TGF-2 directly regulate the expression of ZEBs in myometrial smooth muscle cells (MSMC) resulting in phenotypic transformation as a consequence of loss of E-cadherin, and their actions are prevented following treatments with GnRHa, RU-486, Depo-Provera and Tranilast, an inhibitor of pro-fibrotic/inflammatory mediators. Further functional activity of ZEBs is altered through treatments with siRNAs and miR-200b/c in MSMC and LSMC thus their downstream target genes, including E-cadherin. Aim#3 will test the hypothesis that ZEBs expression is regulated via epigenetic modifications and by the action of miRNAs in myometrial cells, and alteration in this mechanism results in transformation into leiomyoma cells. This will be tested by assessing the patterns of expression of HDACs and EZH2, as well as miRNAs that target their expression in myometrial and leiomyoma cells; following treatments with IL-13 and TGF-2, alone and in combination, and following treatment with Tranilast, miRNAs gain-of functions and inhibitors of HDACs and CpG methylation, respectively. To achieve these Aims, we will use leiomyoma and matched myometrium and utilize a combination of biochemical, molecular and cell biological approaches. We anticipate that this proposed research will lead to further identification of a direct role of pro-fibrotic/inflammatory derived mediators in myometrial cellular transformation into leiomyoma cells and help to work toward the overall development of a novel therapeutic approach to prevent the growth and specifically treat leiomyomas. PUBLIC HEALTH RELEVANCE: Fibroids are benign uterine tumors estimated to develop in 70% of women, more specifically among African Americans during their reproductive years. Symptomatic fibroids cause chronic pelvic pain and abnormal uterine bleeding which are the main indications for women undergoing hysterectomy. This proposal will investigate how fibroids are developed and grow, and ways to prevent them, including their symptoms.