ER/miR-140信号通路调控软骨基质代谢的分子机制研究

Estrogen deficiency is closely related to the development of menopausal arthritis. The underlying mechanism is still unclear. Our previous study showed that estrogen (17β-estradiol) suppressed MMP-13 expression in human articular chondrocytes. We also found that estrogen significantly increased the expression level of miR-140. MiR-140 over-expression inhibited ERα expression. Bioinformatics analysis showed a molecular basis of estrogen receptor (ER) and miR-140 complex. Therefore, we hypothesize the ER/miR-140 signal transduction pathway and the negative feedback loop are involved in menopausal arthritis. The aim of this project is to explore the binding mechanism between ER and miR-140 in vitro and in vivo. Furthermore, we will confirm whether there is a negative feedback between miR-140 and ER signaling pathway. Finally, we will develop novel selective estrogen receptor modulators and miR-140 gene therapy on menopausal arthritis animal models. Results from this project will contribute to elucidating the pathogenesis of menopausal arthritis and providing new targets and peptides for menopausal arthritis prevention and treatment.

雌激素水平低下与绝经后关节炎的发病率明显增加有密切关联，但其确切机制尚未阐明。本项目前期发现：雌激素激活人软骨细胞中雌激素受体(ER), 上调miR-140，抑制软骨基质降解因子MMP-13；过表达miR-140则抑制ERα；生物信息学分析提示miR-140启动子区域存在雌激素反应元件，且ERα3ˊUTR存在miR-140结合位点。提示ERα/miR-140信号通路及其负反馈环路在维持软骨基质代谢平衡中发挥重要的调控作用。本研究拟：1）采用体外软骨细胞及基因敲除、转基因动物模型，阐明ERα和miR-140相互结合的机制，验证结合位点；2）采用绝经后关节炎动物模型，研究新型多肽类选择性ER调节剂及miR-140转基因治疗效果及功能机制。本项目通过揭示雌激素/ERα/miR-140信号通路异常可致软骨基质代谢失衡，为阐明绝经后关节炎的发病机制提供新的研究视角，并为其防治提供新的靶点和多肽药物。

本研究采取细胞培养、实时定量PCR、蛋白印迹和荧光素酶报告基因等分子生物学手段和OA 动物模型，观察雌激素调控软骨基质代谢的相关指标，探讨E2/ER/miR-140信号在调控软骨基质分解代谢中的作用，并初步探讨miR-140过表达对软骨合成基质代谢的作用。研究发现：（1）雌激素可抑制软骨基质分解代谢；（2）雌激素通过雌激素受体结合元件上调miRNA-140的表达水平；(3)E2能降低miR-140靶基因（IGFBP-5, SMAD3, SMAD4和ADAMTS-5 ）的表达水平；（4）miR-140受到抑制后， 雌激素抑制MMP-13表达的能力被抑制， 过表达miR-140后， 能加强雌激素对miR-140靶基因MMP-13的抑制作用，MMP-13被进一步抑制；（5）雌激素/ER/miR-140抑制IL-1β所诱导的MMP-13表达；（6）通过关节腔注射过表达miR-140的脐带间充质干细胞可促进OA软骨病变缺损修复。上述结果表明miR-140在调控软骨基质合成和分解代谢中均发挥重要的调控作用。