miR‑125a‑5p inhibits the expression of NLRP3 by targeting CCL4 in human vascular smooth muscle cells treated with ox‑LDL

Abstract

Recent findings have revealed that aberrant miR‑125a‑5p expression is involved in the development of atherosclerosis. The present study aimed to investigate the precise mechanism of microRNA (miR)‑125a‑5p in atherosclerosis. Human vascular smooth muscle cells (HVSMCs) were treated with 20 µg/ml oxidized low‑density lipoprotein (ox‑LDL) for 24 h and were employed as in vitro models of atherosclerosis. Reverse transcription quantitative (RT‑qPCR) assays were used to detect miR‑125a‑5p levels. Immunofluorescence analysis was conducted to assess α‑smooth muscle actin (α‑SMA) expression. Western blotting and RT‑qPCR assays were performed to measure the expression levels of NACHT, LRR and PYD domains‑containing protein 3 (NLRP3), apoptosis associated speck‑like protein (ASC), caspase‑1, active interleukin (IL)‑1β and C‑C motif chemokine 4‑like (CCL4). Furthermore, the association between miR‑125a‑5p and CCL4 was assessed using a double luciferase analysis. In addition, VSMCs were transfected with miR‑125a‑5p mimics (30 nM), miR‑125a‑5p inhibitor (100 nM) or small interfering RNA against CCL4 (si‑CCL4, 50 pM), respectively to further investigate the function of miR‑125a‑5p in ox‑LDL‑treated HVSMCs. The present study found that the expression levels of miR‑125a‑5p were significantly downregulated in HVSMCs, whereas the expression levels of α‑SMA, NLRP3, ASC, caspase‑1, IL‑1β and CCL4 were markedly upregulated following ox‑LDL treatment. Overexpression of miR‑125a‑5p in the absence of ox‑LDL treatment decreased NLRP3, IL‑1β and CCL4 expression, whereas inhibition of miR‑125a‑5p exhibited the opposite effects. The results of double luciferase analysis confirmed that CCL4 was a direct target of miR‑125a‑5p. Moreover, transfection of si‑CCL4 into HVSMCs significantly decreased the ox‑LDL‑induced expression of NLRP3, ASC, caspase‑1 and IL‑1β proteins. Taken collectively, the results of the present study suggested that miR‑125a‑5p could negatively regulate the NLRP3 inflammasome by targeting CCL4 in ox‑LDL‑treated HVSMCs. The data provide new insight to the inhibition of atherosclerosis progression.