The miR-200 family and targets, ZEB1 and ZEB2, modulate uterine quiescence and contractility during pregnancy and labor

Throughout most of pregnancy, uterine quiescence is maintained by increased progesterone receptor (PR) transcriptional activity, while spontaneous labor is initiated/facilitated by a concerted series of biochemical events that activate inflammatory pathways and negatively impact PR function. In this study, we uncovered a new regulatory pathway whereby miRNAs serve as hormonally-modulated and conserved mediators of contraction-associated genes in the pregnant uterus from mouse to human. Using miRNA and gene expression microarray analyses of uterine tissues, we identified a conserved family of miRNAs, the miR-200 family, that is highly induced at term in both mice and humans, as well as two coordinately downregulated targets, zinc finger E-box binding homeobox proteins, ZEB1 and ZEB2, which act as transcriptional repressors. We also observed upregulation of the miR-200 family and downregulation of ZEB1 and ZEB2 in two different mouse models of preterm labor. We further demonstrated that ZEB1 is directly upregulated by the action of P4/PR at the ZEB1 promoter. Excitingly, we observed that ZEB1 and ZEB2 inhibit expression of the contraction- associated genes, oxytocin receptor and connexin-43 and block oxytocin-induced contractility in human myometrial cells. Together, these findings implicate the miR-200 family and their targets ZEB1 and ZEB2 as novel progesterone/PR- mediated regulators of uterine quiescence and contractility during pregnancy and labor, and shed new light on the molecular mechanisms involved in preterm birth. RNA was purified from mouse myometrium (miRNeasy kit, Qiagen). miRNA microarray was performed (LC Sciences) on 18 biological replicates of murine myometrium at 15.5 dpc and an equal number of replicates at 18.5 dpc. Gene expression microarray assays were performed (UT Southwestern Medical Center) on the same 36 samples as detailed further in SI Materials and Methods.妊娠的绝大多数阶段，子宫静息状态（uterine quiescence）均依靠孕酮受体（progesterone receptor, PR）的转录活性增强得以维持；而自发性分娩的启动与促进，则依赖一系列协同级联的生化事件——这些事件会激活炎症通路并损害孕酮受体的功能。本研究揭示了一条全新的调控通路：微小RNA（microRNA, miRNA）可作为激素调控且保守的介导因子，参与妊娠子宫中收缩相关基因的表达调控，且该调控通路在小鼠至人类中均保守存在。通过对子宫组织开展微小RNA与基因表达微阵列分析，我们鉴定出一个保守的微小RNA家族——miR-200家族，该家族在足月妊娠的小鼠与人类子宫组织中均显著上调；同时还发现了两个协同下调的靶标：锌指E盒结合同源框蛋白ZEB1与ZEB2，二者均可作为转录抑制因子。在两种不同的早产小鼠模型中，我们同样观察到miR-200家族的上调以及ZEB1、ZEB2的下调。我们进一步证实，孕酮（P4）/孕酮受体可直接通过作用于ZEB1的启动子区域上调其表达。令人振奋的是，我们发现ZEB1与ZEB2可抑制收缩相关基因——催产素受体（oxytocin receptor）与连接蛋白43（connexin-43）的表达，并可阻断人子宫肌层细胞中催产素诱导的收缩反应。综上，本研究结果表明miR-200家族及其靶标ZEB1、ZEB2可作为孕酮/孕酮受体介导的妊娠与分娩过程中子宫静息与收缩状态调控的新型调控因子，同时为早产发生的分子机制提供了全新的见解。我们使用Qiagen公司的miRNeasy试剂盒从小鼠子宫肌层中提取RNA。针对妊娠15.5天（dpc）与妊娠18.5天（dpc）的小鼠子宫肌层，各取18份生物学重复样本，由LC Sciences公司完成微小RNA微阵列检测。同样的36份样本交由德克萨斯大学西南医学中心完成基因表达微阵列检测，详细实验流程参见补充材料与方法（SI Materials and Methods）。