Construction Of GPERshRNA Expression Vector And Its Effect On Vascular Endothelial Cell Proliferation

Objective:To observe the effect of GPER on HVECs and MVECs proliferation, short hairpin RNA (shRNA) eukaryotic expression plasmid of human and mouse G-protein-coupled receptor 30(GPER) was constructed, and identified its effects on GPER expression in HVECs and MVECs.Methods:Two different shRNA oligonucleotides targeting the coding sequence of human GPER mRNA and mouse GPER mRNA were designed and synthesized in vitro, while a missense non-specific sequence was synthesized as negative control,then, they were annealed and connected into the pSuper plasmids which they were linearized by BglⅡ-HindⅢ. GPER shRNA expression plasmids were transformed into E. Coli.Positive clones were picked.Plasmids were extracted and identified by the EcoR I, HindⅢrestriction enzyme digestion and sequence analysis.HVECs and MVECs were cultured.then the recombinant plasmids were transfected into HVECs and MVECs via Lipofectamine 2000. Expression of GPER protein was detected by Western blot. After the recombinant plasmid was transfected into HVECs and MVECs, proliferation of HVECs and MVECs were detected by MTT.Results:GPER shRNA expression plasmids were successfully constructed and identified by restriction enzyme and sequence analysis. Target nucleotide sequences were successfully inserted into the expected sites and sequences were accurate.Compared with control group, after the GPER shRNA expression plasmids were transfected into HVECs and MVECs, the expression of GPER protein were downregulated (P<0.05), especially, GPER shRNA2 plasmid had stronger downregulation effect. The expression of GPER protein had the strongest inhibitory effect by 84.2% at 48h.The proliferation of HVECs and MVECs could be inhibited after the recombinant plasmid had been transfected into HVECs and MVECs. Conclusion:The GPER shRNA expression plasmids are successfully constructed, they can target the coding sequence of GPER mRNA in human and mouse.and they can effectively inhibit the expression of GPER protein in HVECs and MVECs. After the recombinant plasmid was transfected into HVECs and MVECs,the proliferation of HVECs and MVECs can be inhibited.