miR-30e通过抑制SIRT1/PGC-1α信号通路诱导线粒体功能障碍和足细胞损伤

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. Inhibition of SIRT1 (silent mating type information regulation 2 homolog 1)/PGC-1α (peroxisome proliferator activated receptor-gamma coactivator-1α) axis activation plays an important role in the mitochondrial dysfunction. However, the molecular mechanism involved in SIRT1/PGC-1α axis regulation remains unclear. To determine which miRNA species may be involved in inhibiting the activation of SIRT1/PGC-1α axis, we first compared miRNA expression profiles in renal cortex from control and aldosterone-infused mice. Microarray analysis identified 7 upregulated and 8 downregulated miRNAs from 960 analyzed miRNAs in renal cortex, including upregulated miR-30e. Further real-time PCR analysis demonstrated a previously unidentified alteration, namely, the consistent overexpression of miR-30e. Moreover, upregulation of miR-30e precedes the mitochondrial dysfunction and podocyte injury. Overexpression of miR-30e reduced SIRT1 and PGC-1α expression and induced mitochondrial dysfunction and podocyte injury in cultured podocytes. Informatic analysis predicted 3'-UTR of SIRT1 and PGC-1α contains conserved critical nucleotides that may serve as a legitimate target of miR-30e. Therefore, we hypothesized that overexpression of miR-30e in CKD induced mitochondrial dysfunction and podocyte injury by inhibition of SIRT1/PGC-1α axis activation. To test this hypothesis, we firstly identify that miR-30e directly targets the 3'-UTR of SIRT1 and PGC-1α and inhibits the activation of SIRT1/PGC-1α axis. We next detect whether inhibition of SIRT1/PGC-1α axis activation mediates miR-30e-induced mitochondrial dysfunction and podocyte injury by overexpression and RNAi strategy. Finally, we generate mice with podocyte conditional knockout of miR-30e and examine whether miR-30e deletion prevents podocyte injury by blocking of mitochondrial dysfunction. Together, the present study highlights a new role for miR-30e in mitochondrial dysfunction and podocyte injury, reveals a new molecular mechanism underlying SIRT1/PGC-1α axis regulation, and could aid in the design of new therapies for the prevention of podocyte injury in chronic kidney disease.

足细胞损伤是导致慢性肾脏病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，而SIRT1/PGC-1α轴活性抑制是导致MtD的主要原因，但SIRT1/PGC-1α调控机制尚不清楚。课题组应用miRNA芯片结合定量PCR研究发现miR-30e表达增加先于MtD和足细胞损伤，过表达miR-30e可显著抑制SIRT1/PGC-1α表达，并导致MtD和足细胞损伤，生物信息学分析提示SIRT1/PGC-1α是其潜在靶基因。由此设想：CKD高表达的miR-30e通过抑制SIRT1/PGC-1α轴活性，诱导MtD和足细胞损伤。本研究将鉴定miR-30e靶基因SIRT1和PGC-1α，并应用过表达和沉默技术阐明其在miR-30e介导MtD和足细胞损伤中的作用；制备足细胞条件性miR-30e敲除小鼠，鉴定其在体内的生物学功能，为足细胞损伤机制的研究提供新视点。

足细胞损伤是导致慢性肾脏病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在足细胞损伤中发挥重要作用，但线粒体功能障碍的分子机制尚不清楚。近年来有研究发现，miRNA参与线粒体功能调控。本研究应用AB miRNA低密度芯片检测正常和醛固酮灌注小鼠肾皮质线粒体以及醛固酮刺激的体外培养的肾小球足细胞中miRNA表达谱的变化，发现miR-30e在醛固酮灌注小鼠肾组织以及刺激的足细胞中均呈低表达。定量PCR的检测结果进一步证实醛固酮可呈时间和剂量依赖性的抑制miR-30e表达。正常培养的肾小管上皮细胞中，敲低miR-30e可诱导肾小球足细胞线粒体功能障碍和足细胞凋亡。过表达miR-30e可阻断醛固酮诱导的肾小球足细胞线粒体功能障碍和细胞凋亡。生物信息学分析发现，BNIP3L（BCL2/adenovirus E1B interacting protein 3-like）是miR-30e的靶基因。醛固酮可显著上调BNIP3L表达，而应用siRNA敲低BNIP3L阻断线粒体诱导的线粒体功能障碍和足细胞凋亡。过表达BNIP3L直接诱导线粒体损伤和足细胞凋亡。上调miR-30e可抑制醛固酮诱导的BNIP3L表达。这些研究结果揭示了miR-30e通过作用于靶基因BNIP3L，保护线粒体功能，减轻足细胞损伤。我们随后又进一步观察了miR-30e在肾小管上皮细胞中的作用。我们发现miR-30e组成型表达于肾小管上皮细胞中，TGF-β1呈剂量依赖性降低miR-30e的表达，过表达miR-30e阻断TGF-β1诱导的肾小管上皮细胞表型转化，上调E-cadherin表达，而抑制a-SMA和vimentin的表达。荧光素酶报告实验显示Snail1是miR-30e的直接靶基因，并定位到Snail1基因的3UTR第720-726位。过表达miR-30e降低Snail1的表达。TGF-β1显著上调Snail1的表达，而过表达miR-30e降低TGF-β1诱导的Snail1表达。过表达Snail1加重了TGF-β1诱导的表型转化，而miR-30e mimic阻断Snail1对TGF-β1诱导表型转化的促进作用，而Snail1过表达则阻断了miR-30e mimic的作用，提示miR-30e作用于靶基因Snail1，阻断TGF-β1诱导的肾小管上皮细胞表型转化。