Molecular mechanism of leiomyoma growth and regression

描述（由申请人提供）：平滑肌瘤是一种良性子宫肿瘤，由子宫肌层细胞转化而成，病因不明。通过基因组和蛋白质组学分析，我们已经确定了一些差异表达和调控的基因在平滑肌瘤相比，子宫肌层，具有不同的生物学功能，包括细胞转化，增殖，促炎/促纤维化活动，如IL-13和TGF-2。ZEB 1是一种转录因子，也是E-钙粘蛋白的主要阻遏物，在细胞向间充质表型转化的过程中丢失，与子宫肌层相比，在平滑肌瘤中过表达。基于差异表达，激素调节和TGF-2和IL-13的调节，我们提出ZEB 1和ZEB 2也是子宫肌层表型转化为平滑肌瘤的关键调节因子。我们进一步提出ZEB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、调节和功能导致平滑肌瘤的发展和生长的假设，我们提出了以下具体目标。目标#1将检验以下假设：与子宫肌层相比，ZEB和E-钙粘蛋白在平滑肌瘤中差异表达和调节，并且它们的表达与TGF-2和IL-13的表达直接/负相关，并且在接受激素治疗以抑制平滑肌瘤生长的患者中改变。此外，HDAC 2、4和EZH 2在来自上述群组的平滑肌瘤和子宫肌层中的表达谱与它们作为靶向其表达的ZEB和miRNA的表达的表观遗传调节剂的作用相关，并且与高加索人相比，在来自非洲裔美国人的组织中差异表达。目标#2将检验以下假设：促炎/促纤维化分子（如IL-13和TGF-2）直接调节子宫肌层平滑肌细胞（MSMC）中ZEB的表达，导致E-钙粘蛋白丢失导致表型转化，并且在用GnRHa、RU-486、Depo-Provera和促纤维化/炎症介质抑制剂曲尼司特治疗后，其作用被阻止。通过用siRNA和miR-200 b/c处理MSMC和LSMC中的ZEB的进一步功能活性，从而改变其下游靶基因，包括E-钙粘蛋白。目标#3将检验以下假设：ZEB的表达通过表观遗传修饰和子宫肌层细胞中miRNA的作用来调节，并且该机制的改变导致转化为平滑肌瘤细胞。这将通过评估HDAC和EZH 2以及靶向其在子宫肌层和平滑肌瘤细胞中表达的miRNA的表达模式来测试;在用IL-13和TGF-2单独和组合治疗后，以及在用曲尼司特、miRNA功能获得和HDAC和CpG甲基化抑制剂治疗后。为了实现这些目标，我们将使用平滑肌瘤和匹配的子宫肌层，并利用生物化学，分子和细胞生物学方法的组合。我们预计，这项拟议的研究将导致进一步确定促纤维化/炎症衍生介质在子宫肌层细胞转化为平滑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，以防止平滑肌瘤的生长和特异性治疗。 公共卫生相关性：纤维瘤是良性子宫肿瘤，估计在70%的女性中发展，更具体地说是在非洲裔美国人的生育年龄。症状性子宫肌瘤引起慢性盆腔疼痛和异常子宫出血，这是接受子宫切除术的妇女的主要指征。该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.