miR-493-Stathmin通路在缺氧导致的肾小管上皮细胞G2/M期阻滞及肾脏纤维化的作用及机制

Hypoxia plays an important role in the development of renal fibrosis of patients with chronic kidney disease (CKD). We have previously demonstrated HIF-1α-Twist is essential for hypoxia-induced kidney fibrosis via inducing EMT. However, EMT was not observed in renal tubular epithelial cells overexpressing HIF-1α/Twist in vivo, indicating that there are other pathways which mediate hypoxia-induced renal fibrosis. G2/M arrest in renal tubular cells is an important pathway in CKD development. We have for the first time shown that hypoxia could induce G2/M arrest in renal tubular cells and upregulate miR-493. It was found that overexpression of miR-493 could induce G2/M arrest in HK2 cells and upregulate profibotic factors. Bioinformatics analysis showed that HIF-1α and Twist could bind to the promoter of miR-493. Thus, we postulated that HIF-1α/Twist may directly regulate the expression of miR-493, thereby regulating the downstream target genes and leading to G2/M arrest in renal tubular cells and renal fibrosis. This study aims to testify this postulation by in vitro and in vivo assays. The findings of this study will provide a novel insight into the pathogeneis of hypoxia-induced kidney injury and lay a foundation for the future development of new treatment modalities.

缺血缺氧是慢性肾脏病（CKD）肾脏纤维化进展的重要原因。我们前期发现HIF-1α-Twist可通过上皮细胞转分化参与缺氧导致的肾脏纤维化，但体内高表达HIF-1α/Twist的肾小管并未观察到EMT表型，提示缺氧导致的肾脏纤维化存在其他重要途径。肾小管上皮细胞G2/M期阻滞是CKD发展的重要途径，我们研究首次发现缺氧能诱导肾小管上皮细胞（HK2）G2/M期阻滞和高表达miR-493，miR-493mimic能诱导HK2细胞G2/M期阻滞并高表达促纤维化因子。进一步生物信息学预测：miR-493启动子区域存在HIF-1α和Twist结合位点。由此我们推测，缺氧后，HIF-1α/Twist可能直接转录miR-493的表达，进而调控其下游靶基因，导致肾小管上皮细胞G2/M期阻滞和纤维化。本课题将通过体内外实验证实这一推论，这将为阐明缺氧性肾损伤机制提供全新的视角，为探讨新的防治手段提供理论依据

缺氧在肾纤维化发展中起着重要的作用。肾小管细胞G2 / M期阻滞是慢性肾脏疾病（CKD）发展的重要途径。缺氧是否通过调控肾小管上皮细胞的G2 / M期阻滞导致肾纤维化尚不清楚。我们首次发现缺氧可诱导肾小管细胞发生G2 / M期阻滞，从而导致肾纤维化，microRNA可参与调控。我们比较了缺氧条件下HK2细胞中microRNAs的表达，并在缺氧24和48小时后鉴定发现miR-493的上升显著。并且miR-493在慢性肾脏病患者肾组织中表达显著高于正常肾组织。 miR-493的高表达降低了HK2细胞中细胞周期调控因子STMN1的表达，增加了G2 / M期细胞的百分比。用sh-RNA靶向STMN1产生与miR-493的过表达相似的效果。 miR-493抑制剂在体外动物模型逆转这些作用。与这些结果一致，miR-493海绵腺相关病毒（Adeno-associated virus，AAV）在体内降低profibotic因子和stathmin1的表达。总之，我们的研究结果表明，miR-493-stathmin通路的激活促进缺氧诱导的肾小管上皮细胞G2 / M阻滞和肾纤维化。消除G2 / M阻滞和阻断miR-493-STMN1途径将为新的抗纤维化治疗的未来发展提供新的见解。