Construction And Identification Of Recombinant EA.hy926 Cells Overexpressing A Triple Mutant Hypoxia-Inducible Factor-1?

ObjectiveTo construct recombinant lentiviral vector that can overexpress a triple mutant hypoxia-inducible factor-1?and to evaluate the effect of HIF-1?gene on the proliferation,cell cycle and apoptosis of EA.hy926 cells,so as to lay a foundation for further study on the regulatory mechanism of HIF-1?and coactivators.Methods1.A triple mutant HIF-1?gene fragment was obtained by PCR that used the plasmid pcDNA3.1~+-HIF1-Ala402-Ala564-Ala803 as template.The HIF-1?gene and the plasmid pHS-BVC-B027 were digested by NotI/SpeI restriction endonuclease.The digested HIF-l?and plasmid pHS-BVC-B027 were connected to form an entry vector by Gibson assembly method.The entry vector containing the HIF-1?gene was link with lentivirus-associated vectors by the Gateway technology to construct a triple mutant HIF-1?lentiviral vector(PLV_CMV-HIF1a-2A-EGFP-2A-Puro).The lentiviral vector was transformed into E.coli DH5?competent cells.After the processes of screening for positive clones,shaking culture,and plasmid extraction,the plasmid was identified by double enzyme digestion and sequencing to determine whether the vector construction was correct.2.The recombinant lentiviral vector and the packaging plasmid were cotransfected into HEK293T cells to get recombinant lentivirus.3.Thehumanumbilicalveinendothelialcells(EA.hy926)overexpressing HIF-1?were obtained after infection withthe lentiviral particles and screening with 1?g/ml puromycin.The efficiency of viral infection was observed by enhanced green fluorescent protein(EGFP).Real-time PCR and Western blot were used to verify the overexpression of HIF-1?in EA.hy926cells.4.CCK8 assay was used to assess proliferation of EA.hy926 cells.5.The change of cell cycle and apoptosis after HIF-1?overexpression was analyzed by flow cytometry.Results1.Identification by enzyme digestion and gene sequencing confirmed that the recombinant lentiviral vector was successfully constructed.2.Lentivirus with high efficiency infection was produced by transfection to HEK293T cells after48h.ObviousexpressionofEGFPwasobservedunderfluorescent microscope.3.The EGFP could be observed after the lentiviruses were transfected into EA.hy926 cells and screening with 1?g/ml puromycin.The results of Real-time PCR showed that the expression of HIF-1?mRNA in Lenti-HIF-1?group was higher than that in Lenti-negative group(P<0.001).Western blot results showed that the expression of HIF-1?protein in Lenti-HIF-1?group was significantly higher than that in Lenti-negative group(P<0.001).4.The results of cell proliferation detected by CCK8 assay showed that there was no significant difference in cell proliferation between Lenti-HIF-1?group and Lenti-negative group after 24,48 and 72 hours of cell culture(P>0.05).5.The results of cell cycle assay by flow cytometry showed that there was no significant difference between the Lenti-HIF-1?group and the Lenti-negative group in the G0/G1 phase(P>0.05).The Lenti-HIF-1?group had less cells in the S+G2/M phase than in the Lenti-negative group,and the difference was statistically significant(P<0.05).6.Flow cytometry results showed that the apoptosis rate of Lenti-HIF-1?group was higher than that of Lenti-negative group,and the difference was statistically significant(P<0.01).Conclusion1.The recombinant lentiviral vector was successfully constructed.2.The EA.hy926 cells overexpressing HIF-1?were obtained.3.HIF-1?plays a pro-apoptotic role in EA.hy926 cells.