Overexpression of microRNA-506-3p aggravates the injury of vascular endothelial cells in patients with hypertension by downregulating Beclin1 expression

Abstract

The aim of the present study was to measure the expression of microRNA (miRNA)-506-3p in the peripheral blood of patients with hypertension and to determine the biological functions and mechanisms of action of miR-506-3p. A total of 61 patients with primary hypertension were included in the present study. Peripheral blood was collected from all patients, as well as 31 healthy subjects who were included in a control group. The expression of miR-506-3p in peripheral blood was determined by reverse transcription-quantitative polymerase chain reaction. Human umbilical vein endothelial cells (HUVECs) were transfected with miR-506-3p mimics or miR-506-3p inhibitor. The proliferation and migration of HUVECs were determined using cell-counting kit 8 and Transwell assays, respectively. The cell cycle and apoptosis of HUVECs were detected by flow cytometry. The expression of Beclin1 (BECN1) protein, a potential target of miR-506-3p, was measured using western blotting. A dual-luciferase reporter assay was performed to determine the interaction between BECN1 and miR-506-3p. It was demonstrated that miR-506-3p expression in the peripheral blood of patients with patients was upregulated and dependent on the severity of hypertension. miR-506-3p overexpression inhibited the proliferation and migration of HUVECs. In addition, miR-506-3p inhibited the transition from the G1 phase to the S-phase in HUVECs. Overexpression of miR-506-3p promoted the apoptosis of HUVECs. Notably, miR-506-3p downregulated the expression of BECN1 by directly binding to its 3'-untranslated region. The present study demonstrated that miR-506-3p expression is elevated in the peripheral blood of patients with hypertension and is associated with the severity of hypertension. By downregulating BECN1 expression, miR-506-3p aggravates injury in vascular endothelial cells by inhibiting the proliferation and migration of HUVECs, as well as promoting their apoptosis.

Keywords: Beclin1; hypertension; microRNA-506-3p.

Figure 1.

Bioinformatics prediction of direct interactions between miR-506-3p and BECN1. Bioinformatics prediction is a powerful tool for the study of the functions of miRNAs. To understand the regulatory mechanism of BECN1 in hypertension, miRanda (http://www.microma.org/rnicroma/home.do), TargetScan (http://www.targetscan.org), PiTa (http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html), RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/) and PICTA (http://pictar.mdc-berlin.de/) were used to predict the miRNA molecules that may regulate BECN1. It was demonstrated that miR-506-3p was able to regulate BECN1. miRNA/miR, microRNA; BECN1, Beclin1.

Figure 2.

Relative expression of miR-506-3p in peripheral blood as determined by reverse transcription-quantitative polymerase chain reaction. (A) Relative expression of miR-506-3p in healthy subjects and patients with hypertension. *P<0.05 vs. control. (B) Relative expression of miR-506-3p in patients with hypertension at grades I, II and III. *P<0.05 vs. grade I and ^#P<0.05 vs. grade II. miR, microRNA.

Figure 3.

Proliferation of HUVECs at 24 and 48 h after transfection. A cell counting kit-8 assay was used to determine the proliferation of HUVECs transfected with miR-506-3p mimics or miR-506-3p inhibitor. The absorbance of each well was measured at 490 nm with a microplate reader and cell proliferation curves were plotted. *P<0.05 vs. NC group. miR, microRNA; HUVECs, human umbilical vein endothelial cells; NC, negative control.

Figure 4.

Effect of miR-506-3p on the migration ability of HUVECs. (A) Images of migrated HUVECs (magnification, ×200). (B) The number of migrated HUVECs. A Transwell assay was conducted to determine the migration ability of HUVECs transfected with miR-506-3p mimics or miR-506-3p inhibitor. *P<0.05 vs. NC group. HUVECs, human umbilical vein endothelial cells; NC, negative control; miR, microRNA.

Figure 5.

Effect of miR-506-3p on the cell cycle of HUVECs. Flow cytometry was used to detect the proportion of cells in each phase. HUVECs were transfected with miR-506-3p mimics or miR-506-3p inhibitor. *P<0.05 vs. NC group. HUVECs, human umbilical vein endothelial cells; NC, negative control; miR, microRNA.

Figure 6.

Effect of miR-506-3p on the apoptosis of HUVECs. (A) Flow cytometry plots of HUVECs transfected with miR-506-3p mimics or miR-506-3p inhibitor. (B) The apoptotic rate of HUVECs transfected with miR-506-3p mimics or miR-506-3p inhibitor. *P<0.05 vs. NC group. HUVECs, human umbilical vein endothelial cells; NC, negative control; miR, microRNA.

Figure 7.

Effect of miR-506-3p on the expression of BECN1. (A) Western blots of BECN1 protein. (B) Relative expression of BECN1 protein in HUVECs transfected with miR-506-3p mimics or miR-506-3p inhibitor. *P<0.05 vs. NC group. HUVECs, human umbilical vein endothelial cells; NC, negative control; miR, microRNA; BECN1, Beclin1.

Figure 8.

Fluorescence values of HUVECs transfected with WT or mutant 3′-UTR DNA sequences of BECN1 as well as miR-506-3p. A dual-luciferase reporter assay was used to evaluate the interaction between miR-506-3p and BECN1. *P<0.05 vs. NC group. HUVECs, human umbilical vein endothelial cells; NC, negative control; miR, microRNA; WT, wild-type; 3′-UTR, 3′-untranslated region; BECN1, Beclin1.