Construction of lentiviral vector-mediated siRNA knockdown of ER-α36 and its action on gastric cancer cell growth / 中国病理生理杂志

ABSTRACT

[ ABSTRACT] AIM:To construct a lentiviral vector for stable delivery of the ER-α36 gene and to detect its effect on SGC7901 cell growth.METHODS: The efficient RNAi targeting sequences identified for the ER-α36 gene were screened.The Oligo DNA was synthesized with target sequences and annealed to form double-stranded DNA.Then it was digested by Xho I and EcoR I and connected with GV307 vector to produce LV-ER-α36-RNAi lentiviral vector.PCR was used to screen the positive clones and sequence.The LV-ER-α36-RNAi, pHelper 1.0 and pHelper 2.0 plasmids were co-transfected into 293T cells for producing lentiviral vector and infecting SGC7901 cell line.Fluorescence microscopy, real-time PCR and Western blotting were used to detect the transfection efficiency and gene silencing effect.17β-estrodial at concentration of 1 ×10 -10 mol/L was used to stimulate the recombinant cell line, and the action on the growth of gastric cancer cells and the expression of Src, ERK1/2 and cyclin D1 were determined.RESULTS: DNA sequencing analysis confirmed the identity of recombinant shRNA expression vectors.Immunofluorescence assay demonstrated that transfection efficiency was above 80%.Transfection of LV-ER-α36-RNAi significantly knocked down the expression of ER-α36 at mR-NA and protein levels with tetracycline ( TeT) simulating as revealed by real-time PCR and Western blotting.Compared with control group, the growth of the recombinant cell line declined and the expression of Src, ERK1/2 and cyclin D1 and the activation of Src decreased (P<0.05).CONCLUSION: Lentiviral vectors that silence ER-α36 expression are con-structed successfully and can be used to study the role of ER-α36 in gastric cancer.The ER-α36 is related with many kinds of cancer cell growth, including gastric cancer cells.