| Objective:A model of human umbilical vein endothelial cells(HUVECs)injury induced by oxidized low density lipoprotein(Ox-LDL)was established to observe the effect of Ox-LDL on the function of vascular endothelial cells,and explore the effect of 27nt-miRNA that derived from the 27 th base repeat sequence of the fourth intron of the endothelial nitric oxide synthase(e NOS)on the damage of HUVECs induced by Ox-LDL and its underlying molecular mechanism,which may have important implications theoretical 27nt-miRNA as a potential therapy for atherosclerotic disease.Methods:1.Culturing and 27nt-miRNA lentivirus vectors transfection of HUVECsThe 27nt-miRNA high expression,anti-27nt-miRNA and negative control lentivirus vectors were designed,synthesized and constructed base on the27nt-miRNA sequence: 5’-GAAGTCTAGACCTGCTGCAGGGGTGAG-3’,which were transfected into HUVECs to establish stable transfected cell lines.To determine the establishment of 27nt-miRNA cell lines,fluorescence microscopy was used to observe the expression of green fluorescence in cells,and the transfection efficiency of lentivirus was detected by flow cytometry,and the expression level of 27nt-miRNA was measured by real-time quantitative poly-merase chain reaction(q RT-PCR).2.The effect of 27nt-miRNA on the apoptosis,proliferation,migration and angiogenesis of HUVECs induced by Ox-LDLHUVECs were treated with 10 μg/m L,20 μg/m L,40 μg/m L,80 μg/m L and160 μg/m L Ox-LDL for 48 h respectively.The apoptotic rate and cell viability of HUVECs were examined by flow cytometry and MTT assay respectively.According to the above results,the conditions for Ox-LDL induced HUVECs injury were determined.HUVECs were divided into the normal control group,Ox-LDL group,27nt-miRNA+Ox-LDL group,anti-27nt-miRNA+Ox-LDL group and miRNA-NC+Ox-LDL group.Among which,the Ox-LDL group was treated with Ox-LDL at 40 μg/m L for 48 h,while the normal control group was untreated but cultured normally.HUVECs in 27nt-miRNA+Ox-LDL group,anti-27nt-miRNA+Ox-LDL group and miRNA-NC+Ox-LDL group were transfected with their corresponding lentiviral vectors under the same procedure,following were treated with Ox-LDL at 40 μg/m L for 48 h to induce HUVECs injury.The cell viability of HUVECs was measured by MTT assay.The migration capacity of HUVECs was detected by scratch assay and transwell assay.The apoptotic rate of HUVECs was examined by Hoechst 33258 and flow cytometry.The ability of tube formation of HUVECs was measured by artificial basement membrane.The expression of 27nt-miRNA,e NOS,VEGF,Caspase-3,Bax and Bcl-2 at m RNA level or protein level were assayed by q RT-PCR or western blot respectively.The NO production of HUVECs was determined by nitrate reductase assay.3.27nt-miRNA modulates HUVECs injury induced by Ox-LDL through the PI3K/Akt pathwayHUVECs were divided into the Ox-LDL group,anti-27nt-miRNA+Ox-LDL group,LY294002+Ox-LDL group and LY294002+anti-27nt-miRNA+Ox-LDL group.The Ox-LDL group and anti-27nt-miRNA+Ox-LDL group were treated with Ox-LDL at 40 μg/m L for 48 h to induce HUVECs injury.The LY294002+Ox-LDL group and LY294002+anti-27nt-miRNA+Ox-LDL group were treated with 0.45 μg/m L LY294002 for 0.5 h,and then treated with Ox-LDL at 40 μg/m L for 48 h to induce HUVECs injury.The cell viability of HUVECs was measured by MTT assay.The migration capacity of HUVECs was detected by scratch assay and transwell assay.The apoptotic rate of HUVECs was examined by Hoechst 33258 and flow cytometry.The ability of tube formation of HUVECs was measured by artificial basement membrane.The expression of 27nt-miRNA was assayed by q RT-PCR.The protein expression levels of p-Akt,e NOS,VEGF,Caspase-3,Bax and Bcl-2 were analyzed by western blot.The NO production of HUVECs was determined by nitrate reductase assay.Results:1.Fluorescence microscopy showed that the transfected cells had strong green fluorescence,and flow cytometry showed that the transfection efficiency of the three types of lentivirus was more than 90%.In addition,as shown in q RT-PCR assay,compared with the normal control group,the expression of27nt-miRNA was increased in the 27nt-miRNA group(P<0.05),and the expression of 27nt-miRNA was decreased in the anti-27nt-miRNA group(P<0.05).These results indicated that lentivirus successfully transfected HUVECs and cell lines were successfully constructed.2.In this study,the injury model was established on HUVECs exposuring to 40 μg/m L Ox-LDL for 48 h and used in the following experiment.Compared with the normal control group,the expression of 27nt-miRNA and the apoptotic rate was increased(all P<0.05),and the cell viability,migration ability and angiogenesis ability were decreased(all P<0.05)in the Ox-LDL group.The apoptotic rate was significantly increased(P<0.05),and the cell viability,migration ability and angiogenesis ability were significantly decreased(all P<0.05)by over-expression of 27nt-miRNA in HUVECs.The m RNA and protein expression levels of e NOS,VEGF and Bcl-2 were significantly down-regulated(all P<0.05),and the m RNA and protein expression levels of Bax and Caspase-3 were up-regulated(all P<0.05),and the NO production was reduced(P<0.05)by over-expression of 27nt-miRNA in HUVECs.In contrast,the apoptotic rate was significantly decreased(P<0.05),and the cell viability,migration ability and angiogenesis ability were significantly increased(all P<0.05)by inhibiting the expression of 27nt-miRNA in HUVECs.At the same time,the m RNA and protein expression levels of e NOS,VEGF and Bcl-2 were significantly up-regulated(all P<0.05),and the m RNA and protein expression levels of Bax and Caspase-3 were down-regulated(all P<0.05),and the NO production was increased(P<0.05)by inhibiting the expression of 27nt-miRNA in HUVECs.3.Compared with the normal control group,the protein expression level of p-Akt was obviously decreased in the Ox-LDL group(P<0.05).The protein expression level of p-Akt was significantly down-regulated in the 27nt-miRNA group(P<0.05).Meanwhile,the protein expression level of p-Akt was significantly up-regulated in the anti-27nt-miRNA group(P<0.05).The expression of 27nt-miRNA and cell apoptotic rate were increased(P<0.05),and the cell viability,migration ability and angiogenesis ability were significantly decreased(all P<0.05)of HUVECs that were treated with PI3K/Akt inhibitor LY294002.The protein expression of p-Akt,e NOS,VEGF and Bcl-2 were obviously down-regulated(all P<0.05),and the protein expression of Bax and Caspase-3 were significantly up-regulated(all P<0.05),and the production of NO was obviously reduced(P<0.05)in HUVECs which were treated with PI3K/Akt inhibitor LY294002.Conclusion:1.27nt-miRNA promotes Ox-LDL-induced apoptosis of HUVECs and inhibits cell proliferation,migration and angiogenesis.2.27nt-miRNA promotes Ox-LDL-induced apoptosis of HUVECs and inhibits cell proliferation,migration and angiogenesis through negative regulating the PI3K/Akt signaling pathway. |
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