Construction of SRY-box transcription factor 7(SOX7)lentiviral plasmid and its effects on proliferation and migration of HUVECs under hypoxia
Objective To construct lentiviral plasmid overexpressing SRY-box transcription factor 7(SOX7),and to establish human umbilical vein endothelial cells(HUVECs)which stably expressed SOX7.The effects of overexpression of SOX7 on migration and proliferation of HUVECs were investiga-ted under normal oxygen and hypoxic conditions.Methods Polymerase chain reaction(PCR)technology was used to amplify theSOX7 gene,and SOX7-pHAGE recombinant plasmid using pHAGE as vector was constructed through homologous recombination.Real-time fluorescence quantitative PCR(qPCR)and Western blot were used to detect the mRNA expression and protein level of the target gene.The SOX7-pHAGE recombinant plasmid and packaging plasmid were co-transfected into 293T cells to obtain SOX7-pHAGE lentiviral suspension,and then lentivirus was concentrated and infected with HUVECs to con-struct a SOX7 stably infected cell line.qPCR and Western blot were used to detect the mRNA expression and protein level of SOX7 gene.Under normoxic and hypoxic conditions,the effects of overexpression of SOX7 on the migration and proliferation of HUVECs were detected through cell scratch experiments and CCK-8 assay.Results After sequencing of the SOX7-pHAGE recombinant plasmid,the sequence align-ment was correct.qPCR and Western blot successfully detected that 293T cells transfected with SOX7-pHAGE recombinant plasmid overexpressed 111.4 times approximately at the mRNA level(P=0.002 8),and overexpression bands appeared at the protein level.qPCR and Western blot successfully detected HU-VECs infected with SOX7-pHAGE lentivirus significant increased approximately 68.2 times at the mRNA level(P=0.003 0)and significant increased approximately 1.7 times at the protein level(P=0.004 3).The SOX7-pHAGE recombinant plasmid and SOX7 stably infected cell lines were successfully construc-ted.The migration ability of SOX7 stably infected cell lines did not change under normoxic conditions(P＞0.999 9),but the migration ability under hypoxic conditions was enhanced by about 1.2 times(P=0.004 4).The proliferation ability of SOX7 stably infected cell lines was enhanced by about 1.3 times un-der normoxic conditions(P=0.008 7),and the proliferation ability under hypoxic conditions was also sig-nificantly enhanced by about 1.4 times(P=0.022 8).Conclusion SOX7 stably infected cell lines can pro-mote the proliferation of HUVECs.Under hypoxic conditions,SOX7 stably infected cell lines restored the inhibited migration and proliferation abilities of HUVECs.
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维普
