MicroRNA-146a Alleviates Oxidative Low Density Lipoprotein Induced Macrophage Migration

ObjectiveIn this study,RAW264.7 macrophages induced by low density lipoprotein cholesterol(LDL-C)were used AS atherosclerosis(AS)model,and RAW264.7macrophages were set AS blank control group.The RAW264.7 macrophages induced by ox-LDL were added with miR-146 a mimic and miR-146 a inhibitor,respectively.The expression level of miR-146 a gene,the expression level of interleukin-6(IL-6)gene and protein,and the migration ability of macrophages in each group were observed to explore the regulatory effect of miR-146 a on ox-LDL-induced RAW264.7 macrophages,in order to elaborate the regulatory effect of miR-146 a on AS.MethodsRAW264.7 macrophages were cultured in RPMI-1640 medium,10 m L fetal bovine serum,1m L penicillin streptomycin and 1m L glutamine were added into every100 m L medium and cultured in an incubator containing 5%CO2 at 37℃.RAW264.7macrophages were subcultured to 6-10 passage and divided into groups: group A,blank control group;Group B ox-LDL group;miR-146 a mimic+ox-LDL group in group C;miR-146 a inhibitor+ox-LDL group;The macrophages in group A were not treated with any treatment,and the macrophages in group B were treated with ox-LDL(50mg/L)for 24 h.The macrophages in group C were treated with ox-LDL(50mg/L)after transfection with miR-146 a mimic(final concentration0.5μm/L)for 24 h.The macrophages in group D were treated with ox-LDL(50mg/L)after transfection with miR-146 a inhibitor(final concentration 0.5μm/L)for 24 h.Western blot(WB)was used to detect the protein expression levels of interleukin-6(IL-6)in each group,Quantitative real-time PCR was used to detect the gene expression levels of miR-146 a and IL-6 in each group,and Transwell method was used to detect the migration ability of macrophages in each group.Results1.Expression levels and correlation analysis of miR-146 a and IL-6in group A and group BCompared with group A,the relative expression level of miR-146 a gene in group B was significantly decreased,and the relative expression level of IL-6 gene was significantly increased,with statistically significant differences(P<0.01).Compared with group A,the relative expression of IL-6 protein in group B was significantly increased,with A statistically significant difference(P<0.01).Compared with group A,the migration ability of macrophages in group B was significantly increased,with statistically significant difference(P<0.01).Correlation analysis of the relative expression levels of miR-146 a and IL-6 in group B showed that there was a linear negative correlation between miR-146 a and IL-6,r=–0.9466,P=0.0004..2.The relative expression of IL-6 protein in macrophages of each groupCompared with group A,the relative expression of IL-6 protein in group B was significantly increased,with statistically significant difference(P<0.01).Compared with group B,the relative expression of IL-6 protein in group C was significantly increased,with statistically significant difference(P<0.05).Compared with group B,the relative expression of IL-6 protein in group D was significantly decreased,with a statistically significant difference(P<0.01).3.The relative expression results of miR-146 a and IL-6 genes in each group of macrophagesCompared with group A,the relative expression level of miR-146 a gene in group B was significantly decreased,and the relative expression level of IL-6 gene was significantly increased,with statistically significant differences(P<0.01).Compared with group B,the relative expression level of miR-146 a gene in group C was significantly decreased,while the relative expression level of IL-6 gene was significantly increased,with statistically significant differences(P<0.01).Compared with group B,the relative expression level of miR-146 a gene in group D was significantly increased,and the relative expression level of IL-6 gene was significantly decreased,with statistically significant differences(P<0.01).4.Transwell migration results of macrophages in each groupCompared with group A,the migration ability of macrophages in group B was significantly increased,with statistically significant difference(P<0.01).Compared with group B,the migration ability of macrophages in group C was significantly increased,with statistically significant difference(P<0.01).Compared with group B,the migration ability of macrophages in group D was significantly decreased,with a statistically significant difference(P<0.01).Conclusion1.In the macrophage model exposed to ox LDL,not only the inflammatory cytokine IL-6 is involved,but also the gene epigenetics is involved in its occurrence and development.2.The expression of miR-146 a was down-regulated in macrophages exposed to ox LDL,and its ability to interfere with genes was decreased,which promoted the increased expression of the downstream inflammatory cytokine IL-6 gene and protein,suggesting that miR-146 a may be a protective factor of AS.3.Exogenous increase of miR-146 a can reduce the expression of downstream inflammatory factors and inhibit the migration of macrophages,while exogenous reduction of miR-146 a has exactly the opposite effect on the migration of downstream inflammatory factors IL-6 and macrophages.Through the cell function gain and loss experiment,it was confirmed that the changes of upstream miR-146 a were involved in the expression of downstream genes and played an important role in the intervention of the occurrence and development of AS.4.miR-146 a may become a targeted identification detection marker for early detection of AS,and early intervention of miR-146 a may become a new target for targeted therapy.This study provides data and experimental support for the early detection and prevention of AS.