| AIM Coronary atherosclerosis is the basis of coronary heart disease,and hyperlipidemia is an important environmental factor that promotes coronary atherosclerosis by causing coronary endothelial dysfunction.Damage of coronary endothelium caused by Low-density lipoprotein(ox-LDL)occurs throughout the pathogenesis of coronary heart disease,eventually leading to myocardial infarction.Therefore,it is of great significance to prevent coronary heart disease and reduce the mortality rate of coronary heart disease via reducing and preventing ox-LDL-induced coronary endothelial cell damage.At present,a variety of Micro-RNA have been found to be involved in the occurrence and development of coronary atherosclerosis.For instance,it has been found that the expression of miR-130 a is abnormal in patients with coronary heart disease,and the role of cardiomyocyte injury in acute myocardial infarction has been clearly defined.It may also play an important role in coronary endothelial dysfunction.The purpose of this study was to investigate the effect of miR-130 a on the regulation of PPAR-? in the inflammation of HCAECs.It may provide a new research direction for the diagnosis and treatment of coronary atherosclerotic heart disease.METHODS Primary human coronary endothelial cells(HCAECs)were cultured and subcultured in vitro.(1)Using different concentrations of ox-LDL stimulated normal HCAECs for 24 hours to induce cell damage,and cell morphology confirmed the damage model was established.RT-q PCR and Western blot was used to detect difference concentrations of miR-130 a and PPAR-? expression at RNA and protein levels among different groups.CCK-8 method was employed to detect the relationship between ox-LDL stimulation concentration and the activity of HCAECs.(2)Cell lines with low-expression and high-expression of miR-130 a were constructed by liposomes transfection,and the above two cell lines were stimulated with 80 ?g/m L ox-LDL for 24 hours.RT-q PCR and Western blot were adopted to detect the difference in expression of PPAR-? in the miR-130a high/low-expression groups,and the enzyme-linked immunosorbent assay(ELISA)was used to detect the downstream inflammatory factors IL-1? and IL-6 and TNF-? expression.(3)After introducing PPAR-? agonist pioglitazone for 24 hours,cells were stimulated with ox-LDL at a concentration of 80 ?g / m L for 24 hours.Western blot was used to detect the expression of PPAR-?,and CCK-8 was used to detect the change of cell viability.ELISA was used to detect the inflammatory factors IL-1? and IL-6 and TNF-? expression.This part of the experiment is to prove the effect of PPAR-? on inflammation of HCAECs induced by ox-LDL injury.Use image J software for band grayscale analysis,SPSS22.0 software for statistical analysis,measurement data are expressed as mean ± standard deviation,P <0.05 indicates that the difference is statistically significant.RESULTS The main results were as follows:(1)compared with the normal coronary artery endothelial cell group,the cell survival rate were(91.58 ±1.37)%,(84.58 ±1.14)%,(62.66 ±1.94)% and(50.28±1.38)% respectively,after stimulated by ox-LDL at concentrations of 40,80,120 and 160 ?g/m L,and there was significant difference among the four groups(P < 0.01).It is suggested that oxLDL stimulation can reduce the activity of coronary artery endothelial cells in a concentration-dependent manner.(2)Western blot and PCR results showed that with the increase of ox-LDL stimulation concentration,the expression level of miR-130 a in HCAECs gradually increased,while the expression level of PPAR-? gradually decreased,the results were statistically significant(P <0.01).(3)Compared with the control group,the expression of miR-130 a was increased while the mRNA and protein expression of PPAR-? were significantly reduced in the miR-130 a mimic group.Mi R-130 a inhibitors group showed an opposite result that the expression of miR-130 a was reduced.The mRNA and protein expression level of PPAR-? in the miR-130 a inhibitor group was significantly increased,with significant differences(P <0.05).(4)The results of Western blot and ELISA showed that the expression of inflammatory cytokines IL-1?,IL-6 and TNF-? in miR-130 a mimic group were increased than that in control group,while the expression of inflammatory cytokines in HCAECs cells in miR-130 a inhibitor group was significantly decreased than that in control group.The difference was statistically significant(P <0.01).(5)Compared with the ox-LDL group,the PPAR-? agonist drug group increased the expression of PPAR-? protein and the cell survival rate of HCAECs,while the expression of inflammatory cytokines IL-1?,IL-6 and TNF-? were decreased.The difference was statistically significant(P <0.05).CONCLUSION Ox-LDL can induce the decrease of cell activity and miR-130-3p overexpression in HCAECs.Low expression of miR-130-3p promotes the expression of PPAR-?,and it can also protect the HCAECs cells from injury induced by ox-LDL through down-regulating the expression of downstream inflammatory factors.In this study,it is the first time to explore the role of miR-130a/PPAR-? in antiapoptosis and anti-inflammation in damage of primary coronary artery endothelial cells caused by ox-LDL,which may provide a new theoretical basis for molecular targeted therapy of coronary heart disease. |
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