PEA3的新功能：通过抑制miR-21促进急性肾损伤后修复

This study will explore the role of PEA3 gene in the repair after acute kidney injury (AKI), and investigate its relationship with miR-21, MMP2 and MMP9. Our prior study have demonstrated that PEA3 played an important role in the nephrogenesis. Since recent research also have found that the process of the repair after AKI was similar to metanephrogenesis, we observed the expression of PEA3 in Gentamicin-induced nephrotoxicity rats, found that PEA3 might contribute to the repair after AKI. MicroRNAs (miRNAs) are endogenously produced, short RNAs of 21-25 nucleotides that are important regulators of gene expression at the posttranscriptional level. By regulating gene expression, miRNAs play critical roles in a variety of cellular and physiological activities. The role of miRNAs in the regulation of renal development, physiology, and pathology has emerged as an important and potentially fruitful area of research. Many miRNAs have been screened and identified in diabetic nephropathy, AKI and other renal pathophysiologies. PEA3 can inhibit the activity of miR-21, and induce the expression of MMP2 and MMP9 in many tumor research. Besides, miR-21 may downregulate the expression of MMP2 and MMP9 in tumor. Therefore, we hypothesize that PEA3 may block miR-21 to induce the expression of MMP2 and MMP9, and promote the repair after AKI. To verify our main hypothesis, proposed studies are designed and divided into three sections. First, the molecular mechanism of PEA3 on regulating miR-21, MMP2, MMP9 and renal epthelial cell injury will be tested via overexpression and RNAi suppression of PEA3. Second, it will be determined whether PEA3 inhibiting miR-21 by transfected with miR-21 virus vector. Finally, the effect of PEA3 inducing MMP2, MMP9 by inhibiting miR-21 was investigated in Gentamicin-induced nephrotoxicity rats, This study would reveal a novel underlying molecular mechanism on PEA3 protecting renal interstitium, and could aid in the design of new therapies for the prevention of AKI.

本课题主要探讨PEA3基因在急性肾损伤修复中的作用及其与miR-21及MMP-2、MMP-9的关系。业已证实PEA3基因在肾脏发育中起重要作用，鉴于某些肾脏损伤后修复过程类似胚胎期后肾发育过程重演的理论基础，我们制造了庆大霉素诱导的急性肾损伤与修复的经典模型，发现PEA3基因亦可能参与急性肾损伤与再生修复。microRNA参与多种疾病的发生是近年来的热点研究。肿瘤研究中发现PEA3可抑制miR-21活性，亦可上调MMP-2、MMP-9的表达，而miR-21是MMP-2、MMP-9的抑制子。本课题拟进一步通过过表达或干扰PEA3的表达，在细胞和在体水平深入探讨PEA3基因在急性肾损伤修复过程中的作用及可能的机制。并通过转入miR-21的病毒载体，明确miR-21是否为PEA3调控MMP-2、MMP-9的中心环节。该研究旨在为临床急性肾损伤的诊治提供新的靶标。

急性肾损伤发病率在全球正以惊人的速度上升，在ICU病人中的发生率超过30%，>20%的ICU的透析幸存者在3-5年内进展至慢性肾脏疾病CKD和终末期肾病ESRD。如何有效地控制、逆转急性肾损伤已经越来越受到广泛重视。本研究主要探讨PEA3基因在急性肾损伤中的作用及机制。肾脏缺血缺氧、药物毒物、重症感染、创伤等多种因素均可导致急性肾损伤。首先，我们制造经典的庆大霉素诱导肾毒性模型，qRT-PCR和免疫荧光检测发现庆大霉素注射后5天PEA3显著高表达于肾小管，随后其表达逐渐下降，至注射后29天PEA3几乎恢复至正常水平。表明PEA3可能在庆大霉素诱导的急性肾损伤及修复中起重要作用。进一步我们应用庆大霉素诱导小鼠肾小管上皮细胞（MPTCs），发现庆大霉素诱导可使PEA3 mRNA 表达增加，细胞出现显著凋亡，急性肾损伤标记分子KIM-1和NGAL 表达上调。接着我们构建PEA3真核表达载体转染MPTCs，发现过表达PEA3可显著减轻庆大霉素诱导的细胞凋亡和KIM-1、NGAL的表达上调。 接着我们在庆大霉素诱导及PEA3真核表达载体预处理、再予庆大霉素诱导的MPTCs 中检测了线粒体功能相关指标。发现庆大霉素可显著降低线粒体膜电位、减少mtDNA 拷贝数，增加ROS生成。过表达PEA3可显著减轻庆大霉素诱导的线粒体功能障碍（MtD）及肾小管上皮细胞损伤。而转染PEA3 siRNA降低内源性PEA3的表达，则可加重庆大霉素诱导的MtD及肾小管上皮细胞损伤。我们还制造了低糖缺氧诱导的MPTCs损伤模型，发现下调MPTCs内PEA3表达可促进细胞低糖缺氧诱导的线粒体功能障碍和急性肾损伤。最后，我们制作小鼠缺血再灌注（IR）肾损伤模型，尾静脉注射PEA3真核表达质粒，PEA3过表达组的PEA3核酸及蛋白水平明显增高。而与IR模型组相比，过表达PEA3可显著抑制IR诱导的血肌酐及尿素氮增加、急性肾小管坏死及KIM-1、NGAL的表达上调。综上所述，PEA3可能通过抑制线粒体功能障碍在急性肾损伤中起保护作用。