Effects Of Silencing ERβ Of HUVEC

Research Objective:Estrogen receptor a(ERα)and Estrogen receptor β(ERβ)have differences in both gene and protein structure.Which provides a theoretical fundament for its different functions.To distinguish the specific functions of ERa and ERβ,this study mainly uses RNA interference(RNAi)technology to specifically silence the expression of ERβ.To explore the effect of shRNA/ERβ in HUVEC on cell cycle and cell viability.Research Methods:According to the principle of RNAi design,pGFP-U6-Neo-shRNA/ERβ expression vector was constructed,and the plasmid shRNA was transfected into HUVEC lines.ERβ mRNA and protein expression level were determined by qRT-PCR and Western blotting.Flow cytometry and CCK-8 were used to detect the cell cycle and survival of HUVEC after ERβ silencing.The experimental data were expressed with(x+s)and analyzed by SAS 9.1 3 statistic software.Research Results:The results of restriction enzyme digestion and DNA sequencing demonstrate that the pGFP-U6-Neo-shRNA/ERβ eukaryotic expression vector was successfully constructed.The pGFP-U6-Neo-shRNA/NC and pGFP-U6-Neo-shRNA/ERβ were transfected into HUVEC cells,identification of RT-PCR,western blotting,the pGFP-U6-Neo-shRNA/ERβ/HUVEC vetor were established successfully.The results of CCK8 have showed that the cell viability among pGFP-U6-Neo-shRNA/ERβ/HUVEC、pGFP-U6-Neo-shRNA/NC/HUVEC and untransfected HUVEC has no significant difference.The cell cycle research results have showed that ERβ shRNA decreased the cells in G0/G1 phase and increased the cells in S phase,compared with pGFP-U6-Neo-shRNA/NC/HUVEC and untransfected HUVEC.Research Conclusion:①Three vector-shRNA-ERpwere constructed.Plasmid by enzyme digestion and sequence analysis confirmed that the purpose of identification of sequence has been inserted into the site which is expected to meet the design requirements.②The vector was transient transfected into HUVEC,the results of transient transfection show that pGFP-U6-Neo-shRNA3 could decrease the ERβboth expression and protein level,the inhibition rate of pGFP-U6-Neo-shRNA3 is the best(P<0.05).③After HUVECs were transfected with pGFP-U6-Neo-shRNAl,pGFP-U6-Neo-shRNA2,pGFP-U6-Neo-shRNA/NC for 48 h,cell cycle was measured by flow cytometry.The results of cell cycle showed that the proportion of cells in G1 phase in ERβ gene expression silencing group decreased,while the proportion of cells in S phase increased,the difference was no significant(P>0.05).