| Objective To investigate the role and mechanism of histone H3K9 acetylation in atherosclerosis induced by homocysteine(Hcy),confirm that histone deacetylase 1(HDAC1)is an important enzyme for the regulation of H3K9 acetylation,determine the effect of HDAC1 on lipid accumulation in THP-1 monocyte-derived foam cells,analyse the change of mi R-34 a expression in AS induced by Hcy,Clarifying the regulation mechanism of mi R-34 a to HDAC1,reveal the mechanism by which mi R-34 a regulates H3K9 acetylation by HDAC1 to promote Hcy-induced As.Methods Animal Model Replication: Six-week old Apo E-/-mice were randomly divided into three groups(n=10,each group),named model control group(Apo E-/-mice were given normal mouse diet,A-control group),Hyperhomocysteinemia group(Apo E-/-mice were given normal mouse diet plus 1.7% methionine,Meth group)and intervention group(Apo E-/-mice were given normal mouse diet plus 1.7% methionine + folic acid + Vitamin B12,Meth + F + V group),and normal control group(C57BL/6J mice were given normal mouse diet,N-control group).Kept in SPF environment for 16 weeks,then mice were sacrificed after anesthesia and their aortas were harved and cut into slices.Performed the aortic root sections with HE,Oil Red O and immunofluorescence staining to investigate lipid deposition in each group.The levels of mice serum Hcy were determined by automatic biochemical analyzer.Duplicate model of THP-1 monocyte-derived foam cells in vitro and inspect the model by Oil Red O staining.Divided the foam cells into control group(0μmol/L Hcy,control),Hcy treatment group(100μmol/L Hcy,Hcy)and vitamin B12 and folic acid intervention group(100μmol/L Hcy + 30μmol/L Folic acid + 30μmol/L Vitamine B 12,Hcy + F + V).The contents of total cholesterol(TC),free cholesterol(FC)and triglyceride(TG)in foam cells of each group were measured by ELISA after 48 hours intervention respectively.Oil red O staining and immunofluorescence were used to detect lipid deposition and expression of lipid-related proteins in foam cells.The expression of HDAC1 and H3K9 acetylation level in aorta and foam cells were detected by real-time quantitative PCR and Western blot.HDAC1 overexpression and interference were constructed and transfected into foam cells.Checked the transfection efficiency and verified HDAC1 m RNA and protein expression,and then detected the changing of TG/TC/FC.Determined lipid deposition and lipid-related protein expression in HDAC1 overexpression/interference foam cells by Oil red O staining and immunofluorescence,meanwhile detected H3K9 acetylation levels by western blot.Real-time quantitative PCR was used to detect the expression of mi R-34 a in aorta and foam cells in each group.The lentiviral vectors of mi R-34 a overexpression(LV-mi R-34a)and interference(LV-mi R-34a-sh RNA)were constructed and transfected into THP-1 monocytes.The transfection efficiency was observed under a fluorescence microscope and the cell line stably overexpressed / interfered with mi R-34 a was screened with puromycin.Verified mi R-34 a expression and HDAC1 m RNA expression by real-time quantitative PCR,then the levels of HDAC1 protein and H3K9 acetylation were determined by Western blot.Luciferase reporter lentiviral vector carrying wild type(wt)and mutant type(mut)HDAC1 3’UTR were constructed and transfected into foam cells overexpressing mi R-34 a to analyze whether mi R-34 a acts directly on the HDAC1 3’UTR region.Results1.HE and oil red O staining showed that the model group(meth group)mouse aortic root appeared obvious atherosclerotic plaque.Detect serum Hcy levels in each group of mice by automatic biochemical analyzer and find that,compared with the control group,serum Hcy level in high-methionine group was significantly increased(P<0.05).Immunofluorescence detected plaque lipid-related protein expression and results showed that lipid-related protein ADFP expression in plaque was significantly increased.2.After intervention of Hcy with different concentrations for 48 h,the content of TC,FC and TG were detected.Real time quantitative PCR and Western blot were used to detect the expression of HDAC1 m RNA and protein,the level of H3K9 acetylation.Subsequently,oil red O staining and immunofluorescence staining were performed to detect the lipid deposition and the expression of the lipid-related protein ADFP.The results showed that,compared with control group,the expression of HDAC1 m RNA and protein was significantly increased in the mice aorta of high-methionine group and Hcy-treated foam cells,while the level of H3K9 acetylation was significantly decreased(P<0.05,P<0.05).3.HDAC1 overexpression and interference adenovirus vector was constructed and transfected into foam cells.Transfection efficiency was observed under a fluorescence microscope.The expression of HDAC1 m RNA and protein and the level of acetylation of H3K9 were further verified by real-time PCR and Western blot.The contents of TC,FC and TG were detected by ELISA.Oil red O staining and immunofluorescence assay further validated the lipid deposition and expression of the lipid related protein ADFP.The results showed that,compared with control group,over-expression of HDAC1 significantly increased the levels of TC,FC and TG(P<0.05,P<0.05,P<0.05),decrease the acetylation of H3K9(P <0.05).Moreover,lipid deposition and ADFP also increased significantly,which could be reversed after HDAC1 was interfered(P<0.05,P<0.05,P<0.05,P<0.05).4.The results of real-time PCR showed that the expression of mi R-34 a was significantly decreased in the mice aorta of high methionine group and Hcy-treated foam cells(P<0.05,P<0.05).Then mi R-34 a overexpression/interference lentiviral vector was constructed and transfected into THP-1 monocytes.After that we determined the relationship between mi R-34 a and HDAC.The results showed that overexpression of mi R-34 a significantly inhibited the expression of HDAC1,accompanied with up-regulated the acetylation level of H3K9 and decreased the lipid deposition in foam cells(P<0.05,P<0.05).In contrast, inhibition of mi R-34 a significantly increased HDAC1 expression,followed with down-regulated H3K9 acetylation level and increased lipid accumulation in foam cells(P <0.05,P <0.05).A luciferase reporter lentiviral vector carrying the 3’UTR of wild-type or mutant HDAC1 m RNA was constructed and transfected into foam cells stably expressing LV-mi R-34 a.The results of luciferase report assay showed that the luciferase activity of foam cells transfected with wild type HDAC1 m RNA 3’UTR was significantly lower than that of mutant(P <0.05).Conclusions1.Hcy decreased the level of histone H3K9 acetylation by up-regulating the expression of HDAC1,leading to the increase of lipid content in foam cells.2.mi R-34 a can decrease the expression of HDAC1 by targeting HDAC1 3’UTR.Hcy can induce the down-regulation of mi R-34 a,leading to increase of HDAC1 and decrease of H3K9 acetylation level and finally to accelerate the accumulation of lipid in foam cells induced by Hcy. |
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