Establishment of a stable gastric cancer cell line with lentivirus-mediated RNA interference for I2PP2A / 中国癌症杂志

ABSTRACT

Background and purpose:Overexpression of inhibitor of protein phosphatase 2 A-2 (I2PP2A) in many tumors including gastric cancer suggests that I2PP2A may contribute to the development of gastric cancer. To further study the biological function of I2PP2A and its role in gastric cancer, we established a BGC823 cell line for stable expression of shRNA targeting human I2PP2A gene. Methods: A double-stranded shRNA targeting the I2PP2A was designed, synthesized and was inserted into a lentivirus vector (pGLV2), and the insertion was identiifed by restriction endonuclease analysis and DNA sequencing. BGC823 cells were then transfected with the packaged recombinant lentivirus, and resistant cell clones were selected with puromycin. The expression of I2PP2A was examined using real-time PCR (RT-PCR) and Western blot. Results:Sequencing result proved that recombinant lentivirus vector pGLV2-shRNA-I2PP2A was constructed correctly. RT-PCR and Western blot results conifrmed that the expression of I2PP2A was signiifcantly down-regulated in this infected BGC823 cell line. The efifciency of siRNA interference of I2PP2A could be up to about 90%. Conclusion:A lentiviral vector carrying a shRNA targeting the I2PP2A gene is successfully constructed, and a BGC823 cell line stably expressing I2PP2A shRNA is established with this lentiviral system.