miR-15a调控的自噬在顺铂所致急性肾损伤中的保护作用及其机制研究

Cisplatin is a widely used and highly effective cancer chemotherapeutic agent.One of the limiting side effects of cisplatin use is nephrotoxicity. The most serious and one of the more common presentations is acute kidney injury (AKI). Pathologically, AKI is characterized by tubular cell injury and death. Recent studies have demonstrated that autophagy is rapidly induced during AKI to protect tubular cells from injury and death and PI3K/Akt/mTOR pathway has an important role in autophagy. miR-15a play an important role in the inflammatory responses and proliferation, migration of malignant tumor. Recently, miR-15a was also reported to play a role in autophagy of malignant tumor. In kidney, the miR-15a overexpression inhibited EMT. However, little is known about its role in AKI..Our previous studies showed that miR-15a was well expressed in renal tubular epithelial cells and its expression was significantly upregulated when treated with cisplatin. The miR-15a overexpression in tubular epithelial cells could down-regulate p62 and up-regulate LC3II protein expression. These data suggested that miR-15a might promote cisplatin-induced acute kidney injury by inducing autophagy via PI3K/Akt/mTOR signalling..We are going to verify this hypothesis by the following studies. Firstly, we are going to detect the changes of autophagy by miR-15a mimics and inhibitor transfection followed by cisplatin treatment in renal epithelial cells. Secondly, we will examine whether miR-15a promote cisplatin-induced acute kidney injury by inducing autophagy via PI3K/Akt/mTOR signaling by using PI3K inhibitor. Thirdly, we are going to confirm these changes in vivo by transfection of miR-15a agomir and antagomir.

急性肾损伤（AKI）增加住院患者死亡率和致残率，肾小管上皮细胞损伤是其主要病理表现；自噬在AKI中具有保护作用，PI3K/Akt/mTOR信号是调控自噬的重要通路。miR-15a可调控细胞的凋亡、炎症损伤；有报道其可促进肿瘤细胞自噬。但其在AKI功能不清。我们发现顺铂刺激人近端肾小管上皮细胞（HK2）后miR-15a表达显著上升，细胞自噬上调；HK2细胞过表达miR-15a可促进细胞自噬，且I型PI3K水平上调。因此假设miR-15a经PI3K/Akt/mTOR通路促进细胞自噬从而保护顺铂所致急性肾损伤。我们将分别通过在细胞和动物水平上调、下调miR-15a的表达，观察其对顺铂所致AKI及自噬变化的影响；通过抑制PI3K/Akt/mTOR通路探讨miR-15a影响细胞自噬的机制。本研究将为临床防治AKI提供治疗新思路。

研究背景 急性肾损伤（AKI）增加患者死亡率和致残率，肾小管上皮细胞损伤是其主要病理表现；自噬在AKI中具有保护作用，PI3K/Akt/mTOR信号是调控自噬的重要通路。miR-15a可调控细胞的凋亡、炎症损伤；有报道其可促进肿瘤细胞自噬。但其在AKI功能不清。主要研究内容 本课题主要目的是研究miR-15a在顺铂诱导急性肾损伤及自噬变化中的作用及可能的机制研究。1.体外研究:采用顺铂刺激大鼠肾小管上皮细胞株，转染miR-15a mimics上调miR-15a表达，观察细胞损伤及自噬水平的变化以及PI3K/Akt/mTOR信号通路的变化。2.动物体内实验部分：小鼠腹腔注射顺铂建立急性肾损伤模型，体内转染miR-15a模拟物上调miR-15a的表达，观察小鼠肾功能变化及肾脏病理改变；并检测小鼠肾脏组织细胞自噬水平变化及PI3K/Akt/mTOR信号通路的变化。主要结果 本课题体外实验发现顺铂刺激大鼠近端肾小管上皮细胞损伤后伴随有细胞自噬上调；过表达miR-15a可减轻小管上皮细胞的损伤，且进一步增强细胞自噬。大鼠腹腔注射顺铂致急性肾损伤后，也可观察到肾组织自噬上调；而过表达miR-15a可改善顺铂导致的急性肾损伤，并且进一步增强了肾组织的自噬水平的上调。体内体外实验均证实miR-15a可改善顺铂导致的急性肾损伤，该作用很可能通过上调细胞自噬水平而实现。然而无论体内、体外实验均发现miR-15a过表达与否，Akt/mTOR通路的磷酸化水平均未发生显著变化，提示miR-15a并非通过Akt/mTOR通路上调细胞自噬水平。 结论 miR-15a可改善顺铂导致的急性肾损伤，该作用很可能通过上调细胞自噬水平而实现。