Regulation Of Macrophage Lipid Accumulation By Long Non-coding RNA DANCR Via MiR-33a

Objective: lnc RNA DANCR is closely related to atherosclerosis,but its mechanism is not clear.This study will explore the relationship between lnc RNA DANCR and mi R-33 a,ABCA1 and ABCG1,and provide new ideas and directions for further study of the pathogenesis and clinical diagnosis and treatment of atherosclerosis based on macrophage lipid accumulation.Methods: Experiment one: 1.THP-1 cells were induced to differentiate into macrophages.2.The macrophages were transfected with lentivirus to overexpress DANCR,sh DANCR or DANCR RNAi sc or DANCR over sc.The cells were divided into 5 groups: blank control group,DANCR RNAi,DANCR RNAi sc,DANCR Over and DANCR Over sc.3.Real-time PCR was used to detect the expression level of mi R-33 a and its target genes ABCA1 and ABCG1 m RNA.4.Western blot was used to detect the expression of ABCA1 and ABCG1.5.The effect of lnc RNA DANCR on the activity of mi R-33 a reporter gene was detected by double luciferase reporter gene system.Experiment two: 1.THP-1 cells were induced to differentiate into macrophages.2.Macrophages were transfected with lentivirus to over express DANCR,sh DANCR,mi R-33 a mimics and mi R-33 a inhibitor.The cells were divided into 7 groups: blank control group,DANCR RNAi,inh-mi R-33 a,DANCR RNAi + inh-mi R-33 a,DANCR Over,min-mi R-33 a,DANCR Over + min-mi R-33 a.3.Real-time PCR was used to detect the expression of ABCA1 and ABCG1 m RNA.4.Western blot was used to detect the expression of ABCA1 and ABCG1.5.The rate of cholesterol efflux was measured by liquid scintillation counter.6.Determination of total cholesterol,free cholesterol and cholesterol esters in macrophages by HPLC.Results: Experiment one: 1.The m RNA expression level of mi R-33 a increased,and the activity of reporter gene decreased,the expression level of target gene ABCA1 and ABCG1 decreased,the difference was statistically significant(P < 0.05).2.When lnc RNA DANCR was over expressed,the m RNA expression level of mi R-33 a decreased,and the activity of reporter gene increased,the expression of ABCA1 and ABCG1 increased,the difference was statistically significant(P < 0.05).Experiment two: 1.In lnc RNA DANCR group,the expression of ABCA1 and ABCG1 m RNA was significantly higher than that in mi R-33 a inhibitor group(P < 0.05).When lnc RNA DANCR overexpression and mi R-33 a mimics were transfected at the same time,the expression of ABCA1 and ABCG1 m RNA in lnc RNA DANCR group was significantly lower than that in lnc RNA DANCR group(P < 0.05).2.Compared with the cells transfected with mi R-33 a inhibitor,the decreased expression of lnc RNA DANCR increased cholesterol outflow(P < 0.05).The difference was statistically significant(P < 0.05).3.Compared with the cells transfected with mi R-33 a inhibitor,the levels of TC,FC and CE in macrophages decreased,while CE/TC increased(P < 0.05).When lnc RNA DANCR overexpression and mi R-33 a mimics were transfected at the same time,TC,FC and CE contents in macrophages increased,while CE/TC decreased compared with the cells only overexpressing lnc RNA DANCR(P < 0.05).Conclusions: Experiment one: lnc RNA DANCR up regulates the m RNA and protein expression of ABCA1 and ABCG1 by inhibiting the expression of mi R-33 a.Experiment two: mi R-33 a mediates the regulation of lnc RNA DANCR on lipid accumulation in macrophages.