Jiang Yan, Wang Wei, Liu Zong-Yang, Xie Yi, Qian Yuan, Cai Xue-Ni
Department of Nephrology, The Cancer Hospital of Guizhou, Guiyang, Guizhou 550003, P.R. China.
Department of Nephrology, The 455 Hospital of Chinese PLA, Nephrology Center of Nanjing Military Area Command of Chinese PLA, Shanghai 200052, P.R. China.
Exp Ther Med. 2018 Jan;15(1):1021-1028. doi: 10.3892/etm.2017.5465. Epub 2017 Nov 8.
Tumor necrosis factor (TNF)-α has been reported to be important in glomerulonephritis, which is closely associated with podocyte dysfunction and apoptosis. However, the precise mechanisms by which TNF-α expression are regulated remain unclear. The purpose of the present study was to investigate the role of microRNA (miR)-130a-3p/301a-3p in the post-transcriptional control of TNF-α expression and high glucose (HG)-induced podocyte dysfunction. Mice MPC5 podocytes were incubated with HG and transfected with miR-130a-3p/301a-3p mimics or inhibitors, reactive oxygen species (ROS) levels were measured by flow cytometry assay, and the mRNA and protein levels were assayed by using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The targeted genes were predicted by a bioinformatics algorithm and verified using a dual luciferase reporter assay. It was observed that miR-130a-3p/301a-3p was a novel regulator of TNF-α in mouse podocytes. miR-130a-3p/301a-3p mimics inhibited TNF-α 3'-untranslated region luciferase reporter activity, in addition to endogenous TNF-α protein expression. Furthermore, forced expression of miR-130a-3p or miR-301a-3p resulted in the downregulation of ROS and malondialdehyde (MDA) and the upregulation of superoxide dismutase (SOD) 1 in the presence of HG. Inhibition of TNF-α level prevented a remarkable reduction in SOD activity and a marked increase in ROS and MDA levels in HG-treated podocytes. Furthermore, TNF-α loss-of-function significantly reversed HG-induced podocyte apoptosis. These data demonstrated a novel up-stream role for miR-130a-3p/301a-3p in TNF-α-mediated podocyte dysfunction and apoptosis in the presence of HG.
据报道，肿瘤坏死因子（TNF）-α在肾小球肾炎中起重要作用，而肾小球肾炎与足细胞功能障碍和凋亡密切相关。然而，TNF-α表达调控的精确机制仍不清楚。本研究的目的是探讨微小RNA（miR）-130a-3p/301a-3p在TNF-α表达的转录后调控以及高糖（HG）诱导的足细胞功能障碍中的作用。将小鼠MPC5足细胞与HG孵育，并用miR-130a-3p/301a-3p模拟物或抑制剂转染，通过流式细胞术检测活性氧（ROS）水平，分别使用逆转录定量聚合酶链反应和蛋白质印迹法检测mRNA和蛋白质水平。通过生物信息学算法预测靶基因，并使用双荧光素酶报告基因检测进行验证。结果发现，miR-130a-3p/301a-3p是小鼠足细胞中TNF-α的新型调节因子。miR-130a-3p/301a-3p模拟物除了抑制内源性TNF-α蛋白表达外，还抑制TNF-α 3'-非翻译区荧光素酶报告基因活性。此外，在存在HG的情况下，miR-130a-3p或miR-301a-3p的强制表达导致ROS和丙二醛（MDA）下调，超氧化物歧化酶（SOD）1上调。抑制TNF-α水平可防止HG处理的足细胞中SOD活性显著降低以及ROS和MDA水平显著升高。此外，TNF-α功能丧失显著逆转了HG诱导的足细胞凋亡。这些数据证明了miR-130a-3p/301a-3p在存在HG的情况下，在TNF-α介导的足细胞功能障碍和凋亡中具有新的上游作用。
