MircoRNA-224 Inhibits Vascular Smooth Muscle Cell Lipid Accumulation Via PCSK9/LOX-1 Pathway

Objective: To investigate the effects of MircoRNA-224(miR-224)on lipid accumulation in human vascular smooth muscle cells(VSMCs)and the underlying mechanisms.Methods:(1)miRNA expression in mouse atherosclerosis(As)plaques was examined using gene chip data in the GEO database;the expression of miR-224 in oxLDL-treated VSMCs was detected by qRT-PCR.(2)To observe the effect of miR-224 on lipid accumulation in VSMCs,the cells transfected with miR-224 mimics were incubated with DiI-oxLDL(20 ?g/ml)for 4 h or oxLDL(50?g/ml)for 24 hours,and then Dil-oxLDL uptake was assessed by fluorescence microscope,lipid droplet was stained by Oil Red O and intracellular total cholesterol was detected by Total Cholesterol assay kit.(3)VSMCs were transfected with miR-224,in the presence or absence of oxLDL,the expression of scavenger receptor LOX-1 in VSMCs were determined by qRT-PCR and Western blot,and adopting bioinformatics methods were used to analyze the binding of miR-224 to LOX-1 3'UTR.(4)miR-224 candidate target genes were screened using bioinformatics methods and then functional enrichment analysis performed.RNA interference technology was used to knock down the expression of miR-224 candidate target gene PCSK9 in VSMCs,and the expression of LOX-1 was examined by Western blot.The binding sites of miR-224 and PCSK9 3'UTR were analyzed using bioinformatics methods and confirmed by dual luciferase reporter gene systems.(5)VSMCs were treated with different concentrations of miR-224 mimic and treated for different time periods,or VSMCs were transfected with miR-224 mimics and then treated with oxLDL,the expression of PCSK9 in VSMCs was detected by qRT-PCR and Western blot.(6)To assess whether PCSK9 overexpression alters the regulation of miR-224 on lipid accumulation in VSMCs,the cells were cotransfected with miR-224 mimics and the LV-PCSK9,following by treating with Dil-oxLDL or oxLDL,the expression of LOX-1 levels was determined by qPCR and western blot respectively;fluorescence microscopy,oil red O staining,and TC assay kit were used to analyze lipid accumulation in VSMCs.Results:(1)miR-224 was increased in mouse atherosclerotic plaques compared with normal aortic tissue.Furthermore,the expression of miR-224 was significantly increased in VSMCs treated with oxLDL by qRT-PCR.(2)miR-224 reduced DiI-oxLDL uptake,lipid droplets and intracellular TC in VSMCs.(3)qPCR and Western blot results showed that miR-224 down-regulated LOX-1 mRNA and protein levels in VSMCs.oxLDL-induced up-regulation of LOX-1 expression in cells was also markedly inhibited by miR-224.However,bioinformatics analysis indicated that miR-224 did not directly target to LOX-1 3'UTR.(4)Bioinformatics analysis showed that PCSK9 was one of the miR-224 candidate target genes and we confirmed that miR-224 bound to the site at 322-327 on the 3'UTR of PCSK9.PCSK9 siRNA down-regulated LOX-1 protein expression level in VSMC.(5)miR-224 decreased PCSK9 mRNA and protein expression in dose-and time-dependent manner,and inhibited oxLDL-induced PCSK9 expression in VSMCs.(6)LV-PCSK9 reversed the inhibitory effects of miR-224 on LOX-1 expression;LV-PCSK9 ameliorated miR-224 downregulated lipid accumulation in VSMCs.Conclusion: miR-224 silences its expression by targeting binding to the binding site at 322-327 on the 3' UTR of PCSK9,resulting in decreased expression of LOX-1 and inhibition of lipid accumulation in VSMCs.