He Xuemei, Du Chao, Zou Yan, Long Yang, Huang Can, Chen Feng, He Yanzheng, Zhou Xiangyu
Experimental Medicine Center, the Affiliated Hospital of Southwest Medical University, Luzhou, China.
Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease of Sichuan Province, Southwest Medical University, Luzhou, China.
Cell Physiol Biochem. 2018;49(5):2073-2087. doi: 10.1159/000493717. Epub 2018 Sep 21.
BACKGROUND/AIMS: Vascular complications are the main reasons for disability and mortality associated with type 2 diabetes mellitus (T2DM) and numerous microRNAs (miRNAs) are involved in this process. Our previous study demonstrated that miR-4463 was increased in the plasma of T2DM patients combined with arteriosclerosis of low extremity artery (ASO). However, the role of miR-4463 remains unclear.
miR-4463 expression in the vascular tissues of patients with ASO and T2DM and in human umbilical vein endothelial cells (HUVECs) was detected by qPCR. Cell survival and apoptosis was analyzed via Cell Counting Kit-8 and flow cytometry assays, respectively. Protein expression was determined by Western blot and protein subcellular localization was detected with immunofluorescence. A dual-luciferase assay was used to elucidate the target gene of miR-4463.
miR-4463 was elevated in the vascular tissues of patients with T2DM and ASO. In HUVECs, both 25 mmol/L glucose (high glucose, HG) and hypoxia induced miR-4463 expression. Downregulation of miR-4463 promoted HUVEC survival and reduced cell apoptosis under HG and/or hypoxic conditions by facilitating the expression of protein phosphatase-1 nuclear targeting subunit (PNUTS), X-linked inhibitor of apoptosis protein (XIAP), p-AKT, p-Bad, increased the Bcl-2/Bax ratio, as well as downregulated cleaved caspase 3 expression. Mechanistically, we identified PNUTS as a direct target gene of miR-4463. Both the inhibition of AKT phosphorylation and silencing of PNUTS diminished the effect of miR-4463 on HUVEC apoptosis. Moreover, downregulation of miR-4463 enhanced PNUTS to enable PTEN nuclear localization, which resulted in AKT phosphorylation.
Our results suggest that downregulation of miR-4463 attenuates cell apoptosis by directly enhancing PNUTS expression to promote PTEN nuclear localization, subsequently activating AKT signaling pathway in HUVECs under HG and/ or hypoxic conditions.
背景/目的：血管并发症是2型糖尿病（T2DM）导致残疾和死亡的主要原因，众多微小RNA（miRNA）参与了这一过程。我们之前的研究表明，在合并下肢动脉硬化（ASO）的T2DM患者血浆中，miR-4463水平升高。然而，miR-4463的作用仍不清楚。
采用qPCR检测ASO和T2DM患者血管组织以及人脐静脉内皮细胞（HUVECs）中miR-4463的表达。分别通过细胞计数试剂盒-8和流式细胞术分析细胞存活和凋亡情况。采用蛋白质印迹法测定蛋白质表达，并用免疫荧光法检测蛋白质亚细胞定位。使用双荧光素酶报告基因检测法阐明miR-4463的靶基因。
T2DM和ASO患者的血管组织中miR-4463水平升高。在HUVECs中，25 mmol/L葡萄糖（高糖，HG）和缺氧均诱导miR-4463表达。下调miR-4463可通过促进蛋白磷酸酶-1核靶向亚基（PNUTS）、X连锁凋亡抑制蛋白（XIAP）、p-AKT、p-Bad的表达，增加Bcl-2/Bax比值，并下调裂解的半胱天冬酶3表达，从而促进HG和/或缺氧条件下HUVECs的存活并减少细胞凋亡。机制上，我们确定PNUTS是miR-4463的直接靶基因。抑制AKT磷酸化和沉默PNUTS均减弱了miR-4463对HUVEC凋亡的影响。此外，下调miR-4463可增强PNUTS，使PTEN核定位，从而导致AKT磷酸化。
我们的结果表明，在HG和/或缺氧条件下，下调miR-4463可通过直接增强PNUTS表达以促进PTEN核定位，随后激活HUVECs中的AKT信号通路，从而减轻细胞凋亡。
