MicroRNA-34c suppresses proliferation of vascular smooth muscle cell via modulating high mobility group box protein 1

Abstract

Background: Atherosclerosis is the most frequent pathological process that causes cardiovascular diseases.

Objective: The present study aimed to confirm miRNAs associated with atherosclerosis and explore the molecular mechanism of miR-34c and its target high mobility group box protein 1 (HMGB1) in the control of growth of smooth muscle cells in the development of atherosclerosis.

Methods: Real-time PCR was firstly performed to confirm miRNA correlation with atherosclerosis, and computational analysis and luciferase assay were performed to explore the target of miR-34c, Western blot, and real-time PCR were also utilized to reveal the effect of whether high glucose (HG) and miR-34c affect miR-34c, HMGB1 levels, NF-κB p65 and TNF-α levels, and the role of miR-34c on vascular smooth muscle cells (VSMCs) viability induced by HG. Students' unpaired t test was performed to compare data between two groups.

Results: MiR-34c level was associated with atherosclerosis with different expression between VSMCs treated with high glucose or normal VSMCs. Then, HMGB1 is a virtual target of miR-34c with predicted binding site resided in HMGB1 3'UTR and further verified by that miR-34c remarkably reduced luciferase activity of wild HMGB1 3'UTR under a concentration-dependent fashion, and miR-34c cannot influence luciferase activity of mutant HMGB1 3'UTR.

Conclusions: The results suggested miR-34c might be a novel therapeutic strategy in the management of atherosclerosis by suppressing the expression of HGMB1 and its downstream effectors.

Keywords: MicroRNA-34c; atherosclerosis; high glucose; high mobility group box protein1; proliferation; vascular smooth muscle cell.

Figure 1

VSMCs treated with different dose of glucose (5.5, 10, 20, 30 nmol/L), only high glucose (≥20 nmol/L) enhanced proliferation of VSMCs

Figure 2

Real‐time PCR was performed to detect eight miRNAs (miR‐34c, miR‐144‐3p, miR‐224‐SNP, miR‐675, miR‐320b, miR‐378, miR‐328, miR‐23b‐3p) levels between VSMCs treated with high glucose or normal VSMCs, only miR‐34c level was obviously different between these groups

Figure 3

A, target of miR‐34c with predicted binding site resided in HMGB1 3′UTR. B, miR‐34c remarkably suppressed luciferase activity of wild HMGB1 3′UTR. C, Luciferase activities of the cells transfected with mutant HMGB1 3′UTR

Figure 4

Real‐time PCR and Western blot were performed to detect effect of miR‐34c and HG on miR‐34c and HMGB1 levels. A, MiR‐34c remarkably increased miR‐34c expression. B, HG apparently increased HMGB1 level, but miR‐34c and HMGB1 siRNA remarkably decreased HMGB1 expression

Figure 5

BrdU cell proliferation assay kit was utilized to measure effect of miR‐34c and HG on cell viability, and miR‐34c suppressed proliferation of VSMC caused by high glucose

Figure 6

The influence of HG and miR‐34c on relative expressions of NF‐κB p65 was examined using Western blot analysis and real‐time PCR, and miR‐34c reduced expressions of NF‐κB p56 in VSMCs by high glucose

Figure 7

The impact of HG and miR‐34c on relative expressions of TNF‐α was measured by Western blot analysis and real‐time PCR, and miR‐34c reduced expressions of TNF‐α in VSMCs caused by high glucose