Downregulation of miRNA-328 promotes the angiogenesis of HUVECs by regulating the PIM1 and AKT/mTOR signaling pathway under high glucose and low serum condition

Abstract

Vascular complications are the primary reason for disability and mortality associated with diabetes mellitus (DM), and numerous microRNA s (miRNA s/miRs) are involved in the process, such as miR- 122, miR- 24 and miR- 423. It has been reported that miR- 328 regulates DM and cardiovascular disease; however, the role and mechanism of action underlying miR- 328 in HUVEC s is not completely understood. The present study aimed to investigate the role and mechanism of action underlying the effects of miR- 328 on the functions of HUVEC s. To simulate hyperglycemia combined with ischemia-induced tissue starvation, HUVEC s were cultured in endothelial cell medium with 25 mmol/l D- glucose and 2% FBS for 24 h [high glucose (HG) + 2% FBS group]. HUVEC miR- 328 expression levels were detected by reverse transcription-quantitative PCR . Cell migration, cytotoxicity and tube-like structure formation were analyzed using wound healing, Cell Counting Kit-8 and tube formation assays, respectively. Following transfection with miR- 328 inhibitor, miR- 328 expression was downregulated in HUVEC s. Protein expression levels were determined by western blotting. Compared with the control group, the migration and tube-like structure formation of HUVEC s were decreased, and cell cytotoxicity was increased in the HG + 2% FBS group. The protein expression levels of vascular endothelial growth factor were also decreased, and the expression levels of miRNA- 328 in the HG + 2% FBS group were increased compared with the control group. However, miRNA- 328 downregulation reversed the aforementioned effects. Further experiments indicated that the AKT signaling pathway was inhibited in the HG + 2% FBS group; however, miR- 328 downregulation activated the AKT/mTOR signaling pathway, which was blocked by the AKT signaling pathway inhibitor, perifosine. Gene prediction and western blotting demonstrated that miR- 328 displayed a regulatory role via Pim-1 proto-oncogene, serine/threonine kinase (PIM1). In conclusion, miR- 328 expression was upregulated and angiogenesis was inhibited when HUVEC s were subjected to high glucose and low serum conditions. miR- 328 downregulation enhanced angiogenesis by increasing PIM1 expression and activating the AKT/mTOR signaling pathway in HUVEC s under high glucose and low serum conditions.