miRNA-449a调控Mfn2表达促进肾间质纤维化的研究

Renal interstitial fibrosis (RIF) is characterized by accumulation of excessive Collagen IV and other extracellular matrix components, Mitofusin -2 (Mfn2) overexpression can decrease the expression of Collagen IV, inhibiting RIF. However, its mechanism is unknown. Expression of miRNA449a was markedly up-regulated, and expression of krüppel-like factor 4 (Klf4) was down-regulated in the mouse model of unilateral ureteral obstruction (UUO) in our preliminary test. Studies have shown that Klf4 regulate Mfn-2 expression positively, and Klf4mRNA was one of predicted target genes of miRNA449a.Thus, we presume that miRNA-449a modulate the expression of Mfn2 through Klf4, promoting Collagen IV expression, resulting in the initial development of RIF. In order to verify the hypothesis, we explore it from molecular, cellular, tissue and animal level by RT-PCR, Western blot, lentivirus and other means. This study will clarify whether miRNA-449a involved in the regulation of RIF and its mechanisms, providing a theoretical basis and experimental evidence for RIF prevention.

肾间质纤维化(RIF)的特征是IV型胶原蛋白（Collagen IV）等细胞外基质成分的过度堆积，线粒体融合蛋白-2(Mfn-2)高表达可下调Collagen IV的表达，抑制RIF，但其机制不明。预实验示正常小鼠肾组织低表达miRNA-449a；UUO小鼠肾组织高表达miRNA-449a，低表达Kruppel-like因子4（Klf4）。研究表明，Klf4正调控Mfn-2表达,而Klf4mRNA是miRNA-449a预测靶基因之一。为此，我们假设，miRNA-449a通过Klf4调节Mfn2的表达，促进Collagen IV的表达，促进RIF的发生、发展。为了验证这一假说，我们采用RT-PCR、Western blot、慢病毒转染等手段从分子、细胞、组织及整体动物水平等多方面进行探讨。本研究将阐明miRNA-449a是否参与RIF的调节及其机制，为RIF的防治提供理论基础及实验依据。

肾间质纤维化（renal interstitial fibrosis, RIF)是各种不同病因CKD进行性发展至ESRD的共同病理状态。大量临床实践和实验数据表明，RIF轻重程度是决定肾脏疾病预后的重要因素[3]。线粒体融合蛋白-2(Mfn-2)高表达可下调Collagen IV的表达，抑制RIF，但其机制不明。 预实验示正常小鼠肾组织低表达miRNA-449a；UUO小鼠肾组织高表达miRNA-449a，低表达Krupp el-like因子4（Klf4）。本项目发现：（1）Klf4参与了肾间质纤维化进展；且Klf4的敲除可通过调控Mfn2表达来促进肾间质纤维化的发生发展；（2）双荧光素酶验证了Klf4 mRNA是miRNA-449a的靶基因；miRNA-449a通过负性调控Klf4而下调Mfn2的表达，从而促进肾间质纤维化；（3）虽然，本课题组在UUO模型小鼠中证实miRNA-449a可通过抑制Klf4和Mfn2来促进肾纤维化进展，但miRNA-449a在慢性肾脏病患者肾脏组织中较正常肾脏组织无显著差异。因此，本课题组调整实验方案，重新探索调控Klf4的上游因子；（4）circPlekha7通过靶向miRNA-493-3p/Klf4抑制肾纤维化。综上所述，本项目表明，circPlekha7通过靶向miRNA-493-3p抑制Klf4/Mfn2的表达，抑制肾小管上皮细胞间充质转化和纤维化。