The Mechanism Of Homocysteine Induces The MiR-33 Inhibiting Cholesterol Reverse Transport By The PPAR?-LXR? Pathway

Objective:To investigate whether homocysteine(HCY)induces miR-33 to inhibit cholesterol reverse transport(RCT)and promote lipid aggregation by oxidase proliferator-activated receptor ?(PPAR?)-liver X nuclear receptor ?(LXR?)pathway and promotes lipid accumulation,leading to atherosclerosis(AS).Methods:Method one:(1)RAW264.7 macrophages were induced by oxidized low density lipoprotein(ox-LDL)to establish foam cell model.Oil red “O” staining was used to determine whether the model was established successfully.(2)Homocysteine(HCY)in different concentrations gradients(control blank group?0.5 mmol/L,1.0 mmol/L?2.0 mmol/L and 5.0 mmol/L)were given after induction.(3)The intracellular lipid droplets was observed by oil red “O” staining.(4)The expression of ABCA1,ABCG1 and PPAR?,LXR? at protein and mRNA levels were determined by western blot and real-time PCR.(5)The cellular cholesterol content was analyzed by HPLC.(6)Effluent rate of cholesterol was detected by the method of liquid scintillation counting.Method two:(1)RAW264.7 macrophages were induced by oxidized low densitylipoprotein(ox-LDL)to establish foam cell model.Oil red “O” staining was used to determine whether the model was established successfully.(2)miR-33 mimics and miR-33 inhibitor were transfected into the cells by lipofectaminTM 2000,and was exposed to HCY for 24 hours.(3)The intracellular lipid droplets was observed by oil red “O” staining.(4)The expression of ABCA1 and ABCG1 and PPAR?,LXR? at protein and mRNA levels were determined by western blot and real-time PCR.(5)Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-33 mRNA.(6)The cellular cholesterol content was analyzed by HPLC.(7)Effluent rate of cholesterol was detected by the method of liquid scintillation counting.Results:Results one(1)Compared with the blank control group,the expression of ABCA1,ABCG1 and PPAR?,LXR? at mRNA and protein levels was decreased in HCY intervention group(P<0.05),the intracellular cholesterol content increased(P<0.05),and the intracellular cholesterol efflux rate decreased(P<0.05).(2)With the increase of HCY concentration,the expression of ABCA1,ABCG1 and PPAR?,LXR? at mRNA and protein levels was lower(P<0.05),the intracellular cholesterol content was higher(P<0.05),and the intracellular cholesterol outflow rate was lower(P<0.05).Results two(1)As the concentration of HCY increased,the comparison between the groups showed that the higher the concentration,the higher the expression of miR-33 mRNA,the difference was statistically significant(P<0.05).(2)Compared with control group,the lipid content was increased,and the expression of ABCA1,ABCG1 and PPAR?,LXR? at mRNA and protein levels was decreased(P<0.05),the intracellular cholesterol content was increased gradually(P<0.05),the cellular cholesterol efflux rate was gradually decreased(P<0.05)in miR-33 mimics group.(3)Compared with control group,the lipid content was decreased,and the expression of ABCA1,ABCG1 and PPAR?,LXR? at mRNA and protein levels was increased(P<0.05),the intracellular cholesterol content was decreased gradually(P<0.05),the cellular cholesterol efflux rate was gradually increased(P<0.05)in miR-33 inhibitor group.(4)No difference of the above indexes between blank group,miR-33 mimics-NC group and miR-33 inhibitor-NC group was observed(P>0.05).Conclusion:1.HCY reduces the efficiency of reverse cholesterol transport(RCT)by inhibiting the protein and mRNA expression of ABCA1 and ABCG1 and eventually lead to the atherosclerosis(AS)in RAW264.7 macrophages derived foam cells.2.HCY reduces the efficiency of reverse cholesterol transport(RCT)by inhibiting PPAR?-LXR? pathway,which can reduce the protein and mRNA expression of ABCA1 and ABCG1 and eventually lead to atherosclerosis(AS)in RAW264.7 macrophages derived foam cells.3.Homocysteine(HCY)can induce an increase in miR-33 content,suggesting a correlation between the two.4.HCY inhibit the protein and mRNA expression of ABCA1 and ABCG1 through miR-33 signaling,and reduces the efficiency of RCT in RAW264.7 macrophages derived foam cells.5.HCY inhibit the protein and mRNA expression of ABCA1 and ABCG1 through inhibiting PPAR?-LXR? pathway by miR-33,and reduces the efficiency of RCT inRAW264.7 macrophages derived foam cells.