miR200a调控β-catenin在肾间质纤维化中的作用研究

Renal interstitial fibrosis is the final common pathway of many progressive chronic kidney disease. Our late studies in which the expression of β-catenin was down-regulated by shRNA suggested that Wnt/β-catenin signaling pathway might involve the process of renal interstitial fibrosis. Bio-information analysis revealed that β-catenin is among the predicted target molecules of miR-200a with one potentially conserved binding site in its 3'UTR. Therefore, we hypothesized that regulation of miR-200a which affectsβ-catenin and its downstream molecules might be beneficial to relief of renal interstitial fibrosis. In this proporsal, dual luciferase reporter assay system will be used to verify whether miR-200a can regulateβ-catenin. The alters of miR-200a expression will be monitored in vitro(TGF-β1-induced human proximal tubular epithelium cell line HK-2 )and in vivo(UUO mice model)by TaqMan stem-loop qPCR. The expression of miR-200a was up-regulated or down-regulated to testify its effects on expression of Wnt/β-catenin signaling pathway. Further evidence that miR-200a regulates Wnt/β-catenin pathway would get by blockades of expression of Wnt/β-catenin in the cell line HK-2. This study would be helpful to elucidate the initiative and progressive mechanisms of renal interstitial fibrosis, and to find its new intervention targets.

肾脏纤维化是各种慢性肾脏病发展的共同通路。我们前期研究发现shRNA干扰β-catenin可抑制肾小管上皮细胞间质转分化，提示Wnt/β-catenin信号通路参与了肾间质纤维化过程。β-catenin 为miR-200a的预选靶基因，调控miR-200a对β-catenin及下游信号能否缓解肾间质纤维化，目前尚无文献报道。本课题拟首先用双荧光素酶报告系统验证miR-200a预测靶基因；继之用TGF-β1诱导的肾小管上皮细胞株HK-2及建立UUO鼠模型，TaqMan stem-loop qPCR体内外检测miR-200a表达的变化；外源性干预miR-200a的表达，观察对Wnt/β-catenin信号通路影响；阻断Wnt/β-catenin信号通路，反证miR200a调控β-catenin在肾间质纤维化的作用。本项目有助于阐明肾间质纤维化发生、发展机制，并可望为干预这一进程提供新的靶点。

肾间质纤维化是各种慢性肾脏疾病发展的共同通路。研究发现shRNA干扰β-catenin可抑制肾小管上皮细胞间质转分化，提示wnt/β-catenin信号通路参与了肾间质纤维化的过程。本课题通过构建双荧光素酶报告基因载体野生型和突变型验证miR-200a的靶基因，检测结果表明miRNA-200a能直接与β-catenin的3’UTR结合并抑制其表达，验证l了β-catenin为miRNA-200a的靶基因。随之用TGF-β1诱导的肾小管上皮细胞株及建立UUO模型，检测miR-200a的表达，外源性干预miR-200a的表达,观察对wnt/β-catenin信号通路的影响。阻断wnt/β-catenin信号通路，反证miR-200a在肾间质纤维化中的作用。细胞实验结果表明：miR-200a在TGF-β1诱导下HK-2细胞表达逐渐降低；同时β-catenin的表达逐渐升高；HK-2细胞发生上皮间质转分化。转染agomir能使细胞过表达miR-200a，减弱β-catenin表达，抑制EMT发生，转染antagomir抑制miR-200a的表达；增强β-catenin表达， 提示β-catenin在miR-200a调控HK-2细胞上皮间质转分化中起着重要作用。动物实验从建立UUO模型开始，然后检测miR-200a及wnt、β-catenin、ɑ-SMA、FN在UUO模型中的表达。结果表明miR-200a在UUO模型肾中呈降低趋势，同时wnt、β-catenin、ɑ-SMA、FN的表达逐渐升高，说明Wnt/β-catenin信号通路在肾间质纤维化中激活。我们进一步将agomir打入小鼠体内，观察miR-200a对Wnt/β-catenin信号通路和肾间质纤维化的影响，结果表明agomir能提高miR-200a在肾中的表达，并且减弱wnt、β-catenin、ɑ-SMA、FN的表达，提示在肾脏中表达miR-200a能抑制Wnt/β-catenin信号通路，同时缓解肾间质纤维化,改善肾脏功能。本项目探讨了肾间质纤维化的发生、发展的可能机制，并可望为干预这一进程提供新的靶点。