The Mechanism Of MicroRNA-182 Silenced HDAC9 Influence On Lipid Accumulation And Inflammatory Factor Secretion In THP-1 Macrophages

[Background and Objective]: It is well known that both lipid accumulation and infiltration of inflammatory cells play critical roles in the initiation and development of Atherosclerosis(As).It was reported that macrophage-derived Lipoprotein lipase(LPL)can promote macrophage accumulation of lipid and inflammatory cytokine secretion,to promote the formation of foam cell formation and play a pro-atherosclerosis effect.Recent studies have shown the roles of miRNAs in lipid metabolism and atherosclerosis.As a polyphenic miRNA,miR-182 is known to be involved in lipid metabolism,inflammation and angiogenesis.Interestingly,miR-182 has been connected to the differentiation process of T cells and circulating miR-182 may be an important biomarker of coronary artery disease(CAD).However,it remains unclear whether miR-182 affects thedevelopment of atherosclerosis.Reversible histone acetylation has been characteristically linked to histone and chromatin-dependent processes.It was reported that a genetic variant in the loci corresponding to histone deacetylase 9(HDAC9)is associated with large vessel stroke.The up-regulated expression of HDAC9 is found in human atherosclerotic plaques in different arteries.However,the role of HDAC9 in atherosclerosis has not yet been fully elucidated.The aim of this study was to explore the influences and underlying mechanisms of miR-182 on the expression of LPL and LPL-induced lipid accumulation and inflammatory factor secretion in THP-1 macrophages.Our results showed that miR-182 might up-regulation of LPL expression via silencing of HDAC9 in THP-1 macrophage-derived foam cells,which led to a marked increase in lipid accumulation and pro-inflammatory cytokine secretion.Taken together,these data suggest that miR-182 might act as a novel pharmacologic target for atherosclerosis therapy.[Methods] : Using bioinformatics analyses and dual-luciferase reporter assay,we analyzed miR-182 potential gene targets,predict binding sites for miR-182 and HDAC9,combined with a grade and free energy score,etc.Moreover,we tested the effects of miR-182 on the expression of HDAC9 in human THP-1 macrophages through transfection of miR-182 mimic or miR-182 inhibitor at different concentrations(0,10,20,40,80 n M)and different time(0,6,12,24 h).The m RNA and protein levels of HDAC9 were determined by RT-PCR and western blot analysis,respectively.The effect of miR-182 on HDAC9 activity in macrophages was detected by histone deacetylase activity colorimetric assay kit.Next,we transfected human THP-1macrophages with HDAC9,sh HDAC9,miR-182 mimic,mimic-neg,miR-182 inhibitor,inhibitor-neg or treatment with Trichostatin A(an inhibitor of HDAC9),respectively.Human THP-1 macrophages LPL core histone H4K16 deacetylation level and LPL expression levels were determined by RT-PCR and western blot analysis.We next investigated whether miR-182 affected lipid contents in ox LDL-treated human THP-1macrophages using HPLC.We determined the effects of miR-182 on the NF-?B pathway,the relative protein levels were examined by western blot analysis.The levels of IL-6,IL-1?,MCP-1 and TNF-? in response to treatment of cells with miR-182 mimic as determined by ELISA.[Results]:Bioinformatics prediction results showed that miR-182 is highly conserved in different species evolution,miR-182 in combination with HDAC9 seed sequence and having a low free combination energy,these results strongly suggest that miR-182 has the potential targeting with HDAC9.Using Target Scan,we found that LPL 3' UTR had no binding site for miR-182,suggesting that miR-182 had no direct effect on LPL.Co-transfection of miR-182 mimic and p-HDAC9 WT 3'UTR miRNA luciferase reporter vector into HEK 293 T cells potently inhibitedluciferase activity,but the luciferase activity of the mutant was significantly rescued.Human THP-1 macrophages transfection with miR-182 mimic decreased HDAC9 m RNA and protein levels in both concentration-and time-dependent manners.However,transfection with miR-182 inhibitor had an opposite effect.Overexpression of HDAC9 significantly decreased the levels of LPL core histone H4K16 acetylation and down-regulated the expression of LPL m RNA and protein expression.In contrast,HDAC9 knockdown or treatment with Trichostatin A(an inhibitor of HDAC9)increased the levels of LPL core histone H4K16 acetylation,LPL m RNA and protein.Treatment with miR-182 mimic significantly increased LPL core histone H4K16 acetylation levels and up-regulated LPL expression in THP-1 macrophages.Overexpression of HDAC9 abolished the effect of miR-182 mimic on LPL expression in THP-1 macrophages.Consistently,miR-182 mimic markedly enhanced LPL activity,which was reversed in the presence of HDAC9 overexpression.Human THP-1 macrophages transfection with miR-182 mimic evidently up-regulated the levels of TC,FC and CE in human THP-1 macrophages.Also,overexpression of HDAC9 decreased TC,FC and CE levels in human THP-1 macrophages.However,the levels of TC,FC and CE in human THP-1 macrophages treated with miR-182 mimic combined with overexpression of HDAC9 were not significantly different from those in control cells.The levels of P65 protein phosphorylationlevels were significantly increased in response to treatment with miR-182 mimic.We also found that the levels of IL-6,IL-1?,MCP-1 and TNF-?were significantly increased in response to treatment of cells with miR-182 mimic as determined by ELISA.[Conclusion] : Micro RNA-182-promoted atherogenesis might be associated with up-regulation of LPL expression via silencing of HDAC9 in THP-1 macrophage-derived foam cells,which led to a marked increase in lipid accumulation and pro-inflammatory cytokine secretion.