miR-214：肾小球足细胞线粒体功能调控的新机制

Podocyte injury is one of the critical events leading to proteinuria and glomerulosclerosis. Our previous study indicated that mitochondrial dysfunction is an early event and iniates podocyte injury. However, the molecular mechanism involved in mitochondrial dysfunction remains unclear. To determine which miRNA species may be involved in inducing mitochondrial dysfunction, we first compared miRNA expression profiles in renal cortex cytosol and mitochondria from aldosterone-infused mice. Microarray analysis and further real-time PCR analysis indicated that miR-214 was enriched and unregulated in mitochondria. Moreover, upregulation of miR-214 precedes the mitochondrial dysfunction and podocyte injury. Overexpression of miR-214 by lentivirus-miR-214 induced mitochondrial dysfunction and podocyte injury in cultured podocytes. Informatic analysis predicted 3'-UTR of ATPase contains conserved critical nucleotides that may serve as a legitimate target of miR-214. In a series of preliminary experiments, we found that the promoter of miR-214 gene contains three binding sites for transcription factor Oct-1. Overexpression of Oct-1 could increase mitochondrial miR-214 expression in podocytes. Therefore, we hypothesized that activation of Oct-1 in CKD unregulated mitochondrial miR-214 expression, which induced mitochondrial dysfunction and podocyte injury by inhibition of mitochondrial ATPase8 expression. To test this hypothesis, we firstly identify that miR-214 directly targets the 3'-UTR of ATPase8 and induces mitochondrial dysfunction and podocyte injury by overexpression and RNAi strategy. Secondly, it will be determined whether Oct-1 binds to miR-214 promoter by using independent Chromosome Immunoprecipitation (ChIP) PCR, Electrophoretic Mobility Shift Assay (EMSA) and luciferase reporter system assay. Finally, the effect of miR-214 will be observed on Oct-1-reduced MtD and podocyte injury by specific anti-miR-214 inhibitor. Together, the present study highlights a new role for Oct-1/miR-214 signaling pathway in mitochondrial dysfunction and podocyte injury, which may guide us in therapeutic strategies for CKD.

足细胞损伤是导致蛋白尿和肾小球硬化的重要病理基础。我们前期研究发现线粒体功能障碍(MtD)是足细胞损伤的起始事件，但MtD的发生机制尚不清楚。课题组研究发现miR-214富集于线粒体中，且在醛固酮灌注小鼠肾组织中表达显著增加，过表达miR-214诱导MtD和足细胞凋亡，生物信息学分析提示线粒体ATPase8是其潜在靶基因；miR-214启动子中含有Oct-1结合位点，过表达Oct-1促进线粒体miR-214表达。由此设想CKD时肾组织中Oct-1活化，诱导miR-214表达，miR-214从胞浆转运并富集到线粒体中，抑制线粒体ATPase8表达，诱导MtD和足细胞损伤。本研究将鉴定miR-214靶基因ATPase8，并应用过表达和沉默技术阐明miR-214在诱导MtD和足细胞损伤中的作用及其机制；明确Oct-1对miR-214表达调控及其是否通过诱导miR-214而促进MtD和足细胞损伤。

慢性肾脏病（CKD）是临床常见多发病，发病率高达10%左右。其中相当一部分病人会进展到终末期肾病。到目前为止，CKD的发病机制还不十分清楚，临床上也缺少满意的治疗措施。近年来，足细胞损伤被认为是CKD发生和发展的重要病理因素，因此，足细胞损伤机制及干预成为近年来的研究热点。我们发现，在足细胞损伤过程中，miR-214的表达显著上调，并且其表达的增加主要发生在线粒体。进一步的体外研究发现，miR-214可以从足细胞胞浆转入线粒体，并作用在线粒体基因ATPase8、ND1、ND4L及ND6，提示miRNA-214 可能通过调控多个线粒体内的靶基因而发挥作用，导致线粒体功能障碍，进而影响足细胞的功能及表型改变，甚至导致细胞的凋亡。在体内，我们制备了足细胞特异性miR-214基因敲除小鼠，发现在足细胞敲除miR-214后，小鼠出现了明显的蛋白尿，同时足细胞的特异表达蛋白WT1及podocin的水平也显著降低，提示发生了显著的足细胞损伤。此外，我们也发现，不仅仅在成年小鼠，在新生小鼠，电镜检测也显示了显著的足细胞形态学异常，高度提示了足细胞发育障碍。除上述研究外，我们也制备了肾小管细胞miR-214基因敲除小鼠，正常情况下，我们并未发现显著的肾小管的结构及功能障碍，但是在梗阻性肾病及缺血再灌注诱导的CKD模型，在小管敲除miR-214可以显著减轻肾脏纤维化及线粒体的形态学异常，高度提示在CKD情况下，足细胞及肾小管的miR-214均参与了CKD的发生和发展。本研究不但揭示了miR-214在CKD中的重要病理作用，也为临床治疗CKD提供了新的靶标。