| Objective:In this experiment,we measured the expression of microRNA33 and the apoptosis of human umbilical vein endothelial cells(HUVECs)after the intervention of oxidized low density lipoprotein(ox-LDL).The expression of miR-33 and the apoptosis of endothelial cells,as well as the cholesterol outflow of endothelial cells and the protein expression of caspase-3,bax and bcl-2 were also measured after inhibiting the expression of miR-33 to study on the role and mechanism of miR-33 in the apoptosis of endothelial cells induced by ox-LDL.Methods:The first part of the experiment: HUVECs were subcultured and treated with different concentrations of ox-LDL(0,50,100,200?g / ml)and then divided into A(Control)group,B(ox-LDL-LD)group,C(ox-LDL-MD)group and D(ox-LDL-HD)group.The four groups were successively added with ox-LDL 0?g / ml,50 ?g / ml,100 ?g / ml,200?g/ml,and intervened endothelial cells for 24 hours.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression of miR-33 in each group and the apoptosis was detected by flow cytometry.The second part of the experiment: subcultured HUVECs were divided into A(control)group,B(ox-LDL)group,C(ox-LDL + NC)group,D(ox-LDL + mir-33 inhibitor)group,B,C,D group added 100 ?g/ ml ox-LDL and cultured for 24 hours;then miR-33 inhibitor negative control(NC)/ Lipofectamine? 2000 compounds was added to group C culture medium,mir-33 inhibitor / Lipofectamine? 2000 compounds was added into group D culture medium.After 4-6 hours of incubation,the complexes were removed and cultured for 48 hours.qRT-PCR was used to detect the expression of miR-33 in 4 groups,apoptosis was detected by flow cytometry,cholesterol content in the culture environment was detected by ELISA,and protein expression of caspase-3,bax,and bcl-2 was detected by western blot.Results:The first part of the experiment: different concentrations of ox-LDL(50,100,200 ?g / ml)interfered with HUVECs for 24 hours.The results showed that as the concentration of ox-LDL increased,the expression of miR-33 in HUVECs gradually increased compared with the control group,and it increased in a concentration-dependent manner,and the difference was statistically significant(P <0.05).The expression of miR-33 was the highest at 200 ?g / ml ox-LDL.The apoptosis rates of A(Control)group,B(ox-LDL-LD)group,C(ox-LDL-MD)group,and D(ox-LDL-HD)group were 8.49%,15.04%,23.71% and 34.92%.The rate of apoptosisafter intervention with different concentrations of ox-LDL(50,100,200?g / ml)increased compared with the control group,and showed a concentration-dependent increase.In this experiment,when 200 ?g / ml ox-LDL was used to interfere with HUVECs,the apoptosis rate was the highest.The second part of the experiment: Compared with the control group,the expression of miR-33 in group B with only 100 ?g / ml ox-LDL increased significantly(P <0.01).Compared with group B,the expression of miR-33 in group C with empty vector was not significantly different(P>0.05),while the expression of miR-33 in group D with miR-33 inhibitor was significantly lower(P < 0.01).Compared with the control group,the apoptosis rate of group B with only 100?g / ml ox-LDL increased by 19.18%;compared with group B,there was no significant difference in the apoptotic rate of group C with empty carrier,while the apoptotic rate of group D with mir-33 inhibitor decreased by8.64%.Compared with the control group,the cholesterol efflux rate in group B with only 100 ?g / ml ox-LDL decreased by 9.98%,and the difference was statistically significant(P <0.05).Compared with group B,there was no significant difference in cholesterol efflux rate in group C with empty vehicle(P> 0.05),while the cholesterol efflux rate in group D with miR-33 inhibitor increased by 15.29%,the difference was statisti cally significant(P<0.01).Compared with the control group,the proteinexpression of bax and caspase-3 in group B with only ox-LDL increased.Compared with group B,there was no significant difference in the protein expression of bax and caspase-3 in group C with empty vector,while the protein expression of bax and caspase-3 in group D with mir-33 inhibitor decreased.In contrast to bax and caspase-3,ox-LDL decreased the protein expression of bcl-2,while mir-33 inhibitor increased the protein expression of bcl-2.Conclusion:1.Ox-LDL promotes the expression of miR-33 and the apoptosis of endothelial cells;2.Inhibition of miR-33 can promote cholesterol efflux,decrease the protein expression of caspase-3 and bax,increase the protein expression of bcl-2,and decrease the apoptosis of endothelial cells,suggesting that inhibition of miR-33 is one of the ways to regulate the apoptosis of endothelial cells. |
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