Abstract: Objective To construct eukaryotic vectors expressing short hairpin RNAs (shRNAs) targeting the CIP2A gene and 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 pGPU6. The recombinant plasmids, pGPU6/GFP/Neo-CIP2A-shRNA-1, 2, 3 and 4, were introduced into BGC-823 cells by lipofectamine-mediated transfection and the infection rate were 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%. Transfection of shRNA-1, 2, 3 and 4, significantly knocked down the expression of CIP2A mRNA and protein at 24 h, 48 h 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 were significant (P < 0.05). Conclusion The recombinant vector could effectively inhibit the expression of CIP2A in BGC-823 cells and depress the proliferation of BGC-823 cells.

Key words: CIP2A, Short hairpin RNA, Gastric cancer, PP2A