Study On The Effect And Mechanism Of Microrna-33 On Cholesterol Metabolism In Endothelial Cells

Objective:In this study,to investigate the regulation of miR-33 on cholesterol metabolism in endothelial cells after ox-LDL intervention,and to explore that if CS is involved in the regulation of endothelial cell cholesterol metabolism,we tested the expression changes of microRNA-33 ?adenosine triphosphate combined box transporter A1(ABCA1)? citrate synthase(CS)in umbilical vein endothelial cells(HUVECs)after oxidized low-density lipoprotein(ox-LDL)intervention,and the changes of cholesterol efflux rate?ABCA1 and CS in HUVECs after inhibition of miR-33 expression.It provides new ideas and more theoretical basis for miR-33 regulation of lipid metabolism and mechanism of action in atherosclerosis(AS).Methods:Part I of the experiment: after subculture of HUVECs,the experimental group was added into ox-LDL with final concentration of 50 ug/ml(ox-LDL +LD),100 ug/ml(ox-LDL +MD)and 200 ug/ml(ox-LDL +HD)for 24 hours,and was divided into 4 groups: group A(control),group B(ox-LDL +LD),group C(ox-LDL+MD)and group D(ox-LDL +HD).The expression levels of miR-33 were detected by real-time fluorescence quantitative PCR(qRT-PCR).Protein contents of ABCA1 and CS in 4groups were determined by Western blot.Part ? of the experiment: subculture HUVECs into four groups:group A(control),group B(ox-LDL),group C(ox-LDL+ miR-33 inhibitor negtive control),group D(ox-LDL + miR-33 inhibitor),To join in group C cultures of miR-33 inhibitor negative control(NC)/Lipofectamine ? 2000 compounds,join in group D cultures of miR-33 inhibitor/Lipofectamine ? 2000 compounds,incubation after 4to 6 hours,remove compounds,group B?C?D replacement for the 100ug/ml ox-LDL completely medium for 24 h,collecting cells.The expressions of miR-33,CS and ABCA1 in each group were detected by qRT-PCR,the protein amounts of ABCA1 and CS in each group were detected by Western blot,and the cholesterol outflow rate and CS expression in groups B ? C and D were detected by enzyme-linked immunosorbent assay(ELISA).Results: 1.Part I: the expression of miR-33 in HUVECs was increased after the intervention of ox-LDL at different concentrations.The relative expression of miR-33 detected by qRT-PCR in group B,C and D was1.36,2.04,3.94 times of the control group,respectively.The expression level of miR-33 was the highest when the ox-LDL concentration was 200ug/ml.While the expression of ABCA1 and CS decreased,the relative expression of ABCA1 detected by qRT-PCR in group B,C and D was0.91,0.76,0.41 times of the control group,respectively.The relative expression of CS detected by qRT-PCR in group B,C and D was 0.72,0.49,and 0.14 times of the control group,respectively.When 200 ?g/ml ox-LDL was used to intervene in HUVECs,the expression levels of ABCA1 and CS were the lowest,and the differences were statistically significant.The protein content of ABCA1 and CS in each group was detected by Western-blot.Compared with group A,the bands of ABCA1 and CS in group B,C and D gradually became lighter.The integrated optical density(IOD)ratio is reduced gradually.2.Part ?: the relative expression of miR-33 detected by qRT-PCR in group B,C and D was 1.99,2.08,0.20 times of the control group,respectively.the relative expression of ABCA1 detected by qRT-PCR in group B,C and D was 0.60,0.70,2.65 times of the control group,respectively,and the relative expression of CS detected by qRT-PCR in group B,C and D was 0.80,0.70,2.28 times of the control group,respectively.Compared with group C,the expression of miR-33 was significantly down-regulated in group D,and the expression of ABCA1 and CS was up-regulated,and the differences were statistically significant.Western-blot detected the protein content of ABCA1 and CS in each group,the results showed that compared with group A,the color of the bands of ABCA1 and CS in group B and C became lighter,the integral optical density(IOD)ratio decreased,while the color of the bands in group D became deeper,the integrated optical density(IOD)ratio increases.The expression of CS in ELISA assay showed that theexpressions of CS in group A,B,C and D were 64.01,36.08,37.69 and100.24pg/ml,respectively.Compared with group C,the expression of CS in group D increased significantly.The difference was statistically significant.The mean cholesterol efflux rates of group B,group C and group D were 15.94%,17.65% and 31.23%,respectively.Compared with group C,the cholesterol efflux rate of group D increased by 13.58%,the difference was statistically significant.Conclusion:1.miR-33 is up-regulated in endothelial cells after ox-LDL intervention and inhibits cholesterol efflux in endothelial cells.2.CS may be involved in the regulation of endothelial cell cholesterol metabolism.