Construction of eukaryotic expression vector of shRNA targeting CIP2 A gene and its inhibition effect on BGC-823 cell proliferation / 基础医学与临床

ABSTRACT

Objective To construct eukaryotic vectors expressing short hairpin RNAs ( shRNAs ) targeting at the CIP2 A gene and to explore its effects on gastric cell line BGC-823 .Methods Four oligonucleotides targeting the CIP2A gene were synthesized and cloned into the eukaryotic expression plasmid pGPU 6.The recombinant plas-mids, pGPU6/GFP/Neo-CIP2A-shRNA-1, 2, 3 and 4, were introduced into BGC-823 cells by lipofectamine-me-diated transfection and the infection rate was observed by fluorescence microscope .The gene silencing efficiency was measured by real-time PCR and Western blot .The effects on proliferation of BGC-823 cells were detected by CCK-8 .Results DNA sequencing and enzyme digestion analysis confirmed the identity of the four recombinant shRNA expression vectors .Immunofluorescsence demonstrated that transfection efficiency was above 70%.Trans-fection of shRNA-1, 2, 3 and 4, significantly knocked down the expression of CIP 2A mRNA and protein at 24, 48 and 72 h after transfection .Compared with the 2 , 3 and 4 , shRNA-1 had the more strong inhibitory effect on the expression of CIP2A mRNA and protein.The CCK-8 assay showed that the anti-proliferation effect on BGC-823 cells was significant ( P<0.05 ) .Conclusions The recombinant vector may effectively inhibit the expression of CIP2A in BGC-823 cells and depress the proliferation of BGC-823 cells.