Wang Hui, Wang Zhengxia, Tang Qingbin
Department of Endocrinology, Affiliated Hospital of Taishan Medical University, Taian, Shandong 271000, P.R. China.
Clinical Skills Center, Affiliated Hospital of Taishan Medical University, Taian, Shandong 271000, P.R. China.
Exp Ther Med. 2018 Oct;16(4):3639-3645. doi: 10.3892/etm.2018.6655. Epub 2018 Aug 24.
The aim of the present study was to investigate the function and mechanism of action of microRNA (miRNA or miR)-199a-3p in vascular endothelial cell injury induced by type 2 diabetes mellitus (T2DM). A total of 36 patients with T2DM (26 males and 10 females; mean age, 52.5±7.0 years) and 20 healthy subjects (10 males and 10 females; mean age, 55.6±4.5 years) were included in the present study. Peripheral blood samples were obtained from all participants and total RNA was extracted Reverse transcription-quantitative polymerase chain reaction was performed to determine the expression of miR-199a-3p. Following the transfection of human umbilical vein endothelial cells (HUVECs) with a negative control (NC) miRNA or miR-199a-3p mimics, cell proliferation was assessed using a Cell Counting kit-8 assay. Cell migration was investigated using Transwell assays and flow cytometry was performed to detect the apoptosis of HUVECs. HUVECs were infected with Ad-GFP-LC3B and laser-scanning confocal microscopy was performed to observe autophagosomes in HUVECs. Western blotting was used to measure the expression of proteins associated with autophagy and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor (NF)-κB signaling pathway. MiR-199a-3p was downregulated in peripheral blood from patients with T2DM compared with healthy subjects. Transfection with miR-199a-3p mimics promoted the proliferation and migration of HUVECs. However, miR-199a-3p overexpression inhibited the apoptosis of HUVECs. MiR-199a-3p facilitated HUVEC autophagy by affecting autophagy-associated signaling pathways. Furthermore, miR-199a-3p regulated the biological functions of HUVECs via the PI3K/AKT/NF-κB signaling pathway. The results of the present study suggest that miR-199a-3p expression was reduced in patients with T2DM compared with healthy subjects and may be associated with vascular endothelial cell injury. In addition, miR-199a-3p promoted the proliferation, migration and autophagy of HUVECs, potentially by regulating the PI3K/AKT/NF-κB signaling pathway. Therefore, miR-199a-3p may function as protector of vascular endothelia.
本研究旨在探讨微小RNA(miRNA或miR)-199a-3p在2型糖尿病(T2DM)诱导的血管内皮细胞损伤中的作用及其作用机制。本研究共纳入36例T2DM患者(男26例,女10例;平均年龄52.5±7.0岁)和20例健康受试者(男10例,女10例;平均年龄55.6±4.5岁)。采集所有参与者的外周血样本并提取总RNA。进行逆转录-定量聚合酶链反应以测定miR-199a-3p的表达。用阴性对照(NC)miRNA或miR-199a-3p模拟物转染人脐静脉内皮细胞(HUVECs)后,使用细胞计数试剂盒-8法评估细胞增殖。使用Transwell试验研究细胞迁移,并进行流式细胞术检测HUVECs的凋亡。用Ad-GFP-LC3B感染HUVECs,并进行激光扫描共聚焦显微镜观察HUVECs中的自噬体。采用蛋白质印迹法检测自噬相关蛋白及磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)/核因子(NF)-κB信号通路相关蛋白的表达。与健康受试者相比,T2DM患者外周血中miR-199a-3p表达下调。转染miR-199a-3p模拟物可促进HUVECs的增殖和迁移。然而,miR-199a-3p过表达可抑制HUVECs的凋亡。miR-199a-3p通过影响自噬相关信号通路促进HUVECs自噬。此外,miR-199a-3p通过PI3K/AKT/NF-κB信号通路调节HUVECs的生物学功能。本研究结果表明,与健康受试者相比,T2DM患者miR-199a-3p表达降低,可能与血管内皮细胞损伤有关。此外,miR-199a-3p可能通过调节PI3K/AKT/NF-κB信号通路促进HUVECs的增殖、迁移和自噬。因此,miR-199a-3p可能作为血管内皮的保护因子发挥作用。