子宫平滑肌细胞miR-200启动子甲基化状态改变在早产发病机制中的作用

Preterm is the main cause of perinatal motality and affect the prognosis，which brings heavy burden to society and family. Currently there are multiple epidemiological and experimental evidences that DNA methylation regulation plays an important role in the phothology of preterm, but the studies on the role of DNA methalytion in regulation mechanism of uterine smooth muscle (UMC) are quite few. Our project is to investigate if there is correlation between the reversible DNA methylation status of miR-200 promotors and mature miR-200 or pre-miR-200 expression in preterm labor uterine smooth muscle cell by MassARRAY. We construct miR-200 promotor reporter palsmids, methylate the costructs, cell transfection or treat 5-aza-2'-deoxycytidine in UMC in witro to detect the expression change of miR-200 and its pathway. We also analyze its effect on the biological behavior and contraction response capability of UMC. We aim to confirm that high expression of miR-200 in pathological mechanisms of preterm is caused by its promotor's hypomethylation. This is a key issue for understanding the epigenetic mechanism in preterm phothogenesis. We also provide the new theory for studies of preterm, which lays the foundation for further study and exploration on the therapy of preterm.

早产造成围产儿死亡且影响远期预后，对社会和家庭造成沉重的负担。目前研究认为DNA甲基化调节在早产发病中有重要作用，但涉及DNA甲基化与早产子宫收缩的分子机制研究很少。本项目拟通过亚硫酸氢盐修饰后测序或MassARRAY等方法，检测自发性早产患者子宫平滑肌细胞miR-200启动子甲基化状态与成熟miR-200及其前体的表达之间的相关性。利用体外过甲基化miR-200启动子后转染和5-aza-2'-氮唑胞苷去甲基化，处理培养的子宫平滑肌细胞，分析对miR-200表达及下游通路蛋白表达的影响；对子宫平滑肌细胞迁移、凋亡等生物学行为和收缩应答能力的改变。目的在于证实：miR-200启动子低甲基化造成miR-200的高表达诱发早产子宫平滑肌收缩。这对阐明表观遗传在早产发病机制中的作用有重要意义，为早产发病机制研究提供新的思路。为未来寻找更有效的早产干预手段奠定基础。

早产造成围产儿死亡且影响远期预后，对社会和家庭造成沉重的负担。目前研究认为 DNA 甲基化调节在早产发病中有重要作用，但涉及 DNA 甲基化与早产子宫收缩的分子机制研究很少。.本课题前期假设早产与低甲基化有关，miR-200启动子低甲基化是引起子宫平滑肌收缩的关键。.主要研究内容在于验证早产与低甲基化有关。作用的靶点研究在miR200启动子的低甲基化引起其表达升高，进一步调节收缩相关蛋白引起子宫平滑肌收缩。.本课题取六组自发早产临产子宫平滑肌为阳性组，其他医源性早产未临产子宫平滑肌为对照组。应用Dot blot比较发现阳性组较对照组全基因组甲基化水平显著降低。Real time PCR比较发现miR-200家族miR-141、miR429、miR-200C-3p成熟剪切本及原始剪切本在两组表达均较高，但无统计学差异。应用BSP方法检测miR-200家族甲基化，差别也没有显著性。发现miR-141\miR429、miR-200在早产分娩鼠子宫平滑肌有高表达。我们将焦点转移到基因后调节。MeCP2是甲基化调节蛋白，发挥转录抑制作用。首先Western blot检测发现阳性组较对照组MeCP2水平显著下降，体外siRNA转染抑制MeCP2引起子宫平滑肌收缩。结论：早产与低甲基化有关，甲基化调节蛋白MeCP2是低甲基化作用的关键因子。.本项目应用Microarray进行两组全基因组miRNA表达谱检测及实时定量PCR验证发现miR-212、miR223-3p存在差异表达。动物实验证实正常孕鼠从未孕到孕16.5天，miR-223-3p水平增加，孕16.5天之后miR-223-3p水平逐渐下降，存在时序性变化。LPS早产鼠模型对比同孕周未临产组鼠子宫平滑肌细胞中miR-223-3p水平显著降低。体外细胞实验证实转染miR-223-3p模拟体后子宫平滑肌细胞收缩功能显著减弱。证实：miR-223-3p在早产子宫平滑肌收缩机制中发挥作用。.为早产发病机制研究提供新的思路。为未来寻找更有效的早产干预手段奠定基础。