LncRNA Snhg16 Inhibits ABCA1 Expression Through MiR-506-3p/EGR1 Pathway To Promote Atherosclerosis

[Background and Objective]:Cardiovascular disease（CVD）is the most important cause of morbidity and mortality in the world,and atherosclerosis（As）is one of the main pathological bases of cardiovascular disease,so the occurrence of As will undoubtedly seriously affect human life and health.As is a chronic pathological process characterized by lipid accumulation and inflammatory reaction in arterial wall,and the formation of foam cells caused by lipid accumulation in macrophages is an important reason for the occurrence and development of As.Therefore,reducing foam cell formation and promoting cholesterol efflux from macrophages is an important means to inhibit As.ATP-binding cassette transporter A1（ABCA1）is a kind of cell membrane protein,which can mediate the efflux of intracellular free cholesterol and phospholipid to lipid-poor apolipoprotein A-I（apo A-I）.High-density lipoprotein（HDL）is formed,and then transported to liver through blood circulation for metabolism and excretion,thus reducing intracellular lipid accumulation and foam cell formation.Therefore,exploring the mechanism that affects the expression of ABCA1 is an important target to reduce As.The lncRNA SNHG16belongs to the family of small nucleolarRNA host genes.recent literature shows that lncRNA SNHG16 is highly expressed in the plasma of patients with atherosclerosis,and participates in regulating the proliferation of aortic smooth muscle cells,promoting the proliferation and inflammatory reaction of macrophages in patients with atherosclerosis.All of these indicate that lncRNA SNHG16 is an important molecule in the occurrence and development of As.Other studies have shown that lncRNA SNHG16is related to the regulation of lipid metabolism genes,but the mechanism on cholesterol efflux and lipid accumulation is not clear.Therefore,we explored the mechanism of lncRNA SNHG16 regulating ABCA1expression and cholesterol efflux.[Methods]:Firstly,lncRNA SNHG16 was overexpressed in apoE^-/-mice by using lentiviral vectors.The aortic sinus plaques of the mice were evaluated by Oil red O,HE and Masson staining.The changes of plasma lipid of the mice were collected to detect by oxidase method.The RCT efficiency of the mice was detected by[³H]labeled cholesterol.The q PCR and Western Blot were used to detect the expression of ABCA1 in mouse aorta and peritoneal macrophages.Sencondly,the THP-1 macrophage-derived foam cells were cultured in vitro and transfected with lentiviral vector of lncRNA SNHG16.The expression of ABCA1 was detected by q PCR and Western Blot,cholesterol efflux was detected by liquid scintillation count,lipid accumulation was detected by Oil red O,and intracellular lipid content was detected by high performance liquid chromatography.Next,the bioinformatics website and the luciferase reporter gene were used to verify the binding of transcription factor（EGR1）and ABCA1 promoter region.The mRNA and protein expression of ABCA1 were detected by q PCR and Western Blot in macrophages co-treated with lncRNA SNHG16 overexpression vector and EGR1knockdown vector.Then,bioinformatics websites and luciferase reporter genes were used to predict and analyze the binding of miR-506-3p and EGR1.The miR-506-3p mimics were used to treat the cells and further verify the targeted regulation effect of miR-506-3p on EGR1 by q PCR and Western Blot.Finally,bioinformatics,luciferase reporter gene andRNA Pull down assay were used to analyze the binding of lncRNA SNHG16with miR-506-3p.The miR-506-3p mimics were transfected in macrophages overexpressed by lncRNA SNHG16 to the q PCR and Western Blot were used detect the mRNA and protein expression of EGR1and ABCA1 in the cells treated with lncRNA SNHG16 vectors and miR-506-3p mimics.[Results]:The expression of lncRNA SNHG16 in aorta and peritoneal macrophages of the apoE^-/-mouse were detected by q PCR and the results showed that lncRNA SNHG16 was significantly increased,indicating successful overexpression of lncRNA SNHG16 in vivo.The Oil red O staining of mouse aorta showed that lipid deposition was significantly increased.The tissue staining of mouse aortic sinus showed that lncRNA SNHG16 could increase the plaque area and lipid deposition of the mouse aortic sinus,but didn’t affect the collagen content.The plasma lipid analysis of the mice showed that lncRNA SNHG16 overexpression of mice significantly decreased HDL-C,but significantly increased TC and non-HDL;The[³H]content in plasma,liver and feces of mice was detected and the results showed that lncRNA SNHG16 could reduce the RCT efficiency.The expression of ABCA1 in aortic and peritoneal macrophages was detected by q PCR and Western Blot and the result showed that lncRNA SNHG16 could reduce the ABCA1 expression.The expression of ABCA1,cholesterol efflux and lipid accumulation were detected in cells with lncRNA SNHG16 overexpression vectors and the results showed that ABCA1 expression and cholesterol efflux were decreased in lncRNA SNHG16 overexpression cells,while intracellular TC,FC,CE and lipid accumulation were increased.Bioinformatics analysis and luciferase reporter gene showed that ABCA1 promoter region existed the binding sites of EGR1.ABCA1 expression was significantly increased in the lncRNA SNHG16 overexpression vector and EGR1 siRNA treated cells compared with the lncRNA SNHG16 overexpression group alone,indicating that lncRNA SNHG16 reduces ABCA1 expression through EGR1.The binding of miR-506-3p to EGR1 3’UTR was analyzed by bioinformatics and luciferase reporter gene and the results showed that there were binding sites between miR-506-3p and EGR1 3’UTR.The cells were treated with miR-506-3p mimics and the q PCR and Western Blot results showed that miR-506-3p inhibited the expression of EGR1.Bioinformatics,luciferase reporter gene andRNA pull-down showed that the there are binding sites between lncRNA SNHG16 and miR-506-3p.Lentiviral vector of overexpressed lncRNA SNHG16 and miR-506-3p were used to treat cells.The results showed that,the effects of increasing EGR1 and decreasing ABCA1 expression were reversed in the group that overexpressed lncRNA SNHG16 and miR-506-3p mimics were co-treated compared with the lncRNA SNHG16 alone treatment group.These results indicated that lncRNA SNHG16 could up-regulate the expression of EGR1and down-regulate the expression of ABCA1 by sponging miR-506-3p.[Conclusion]:lncRNA SNHG16 sponges miR-506-3p to upregulate EGR1 level,inhibit ABCA1 expression,reduce cholesterol efflux,leading to lipid accumulation in macrophage and As progression.