慢病毒介导的shRNA靶向干扰I2PP2A胃癌稳定细胞株的建立

Establishment of a stable gastric cancer cell line with lentivirus-mediated RNA interference for I2PP2A

背景与目的:蛋白磷酸酶2A抑制剂-2(inhibitor 2 of protein phosphatase 2A,I2PP2A)在包括胃癌的多种肿瘤中过度表达,提示其可能在胃癌的发生中发挥重要作用。为进一步探讨I2PP2A的功能及其在胃癌发生中的作用,建立稳定抑制I2PP2A基因表达的人胃癌BGC823细胞株。方法:筛选出I2PP2A基因的RNA干扰(RNA interference,RNAi)有效靶序列,合成靶序列的Oligo DNA并构建p GLV2_sh RNA_I2PP2A慢病毒载体,酶切和测序鉴定正确后,经病毒包装,感染BGC823细胞,经嘌呤霉素筛选稳定表达细胞株,通过实时定量PCR(real-time PCR,RT-PCR)和蛋白[质]印迹法(Western blot)鉴定I2PP2A的表达。结果:重组慢病毒质粒经测序鉴定正确;RT-PCR和Western blot证实干扰I2PP2A后,BGC823细胞株中I2PP2A表达水平明显降低,抑制率约为90%。结论:成功构建了I2PP2A sh RNA慢病毒表达载体,建立了稳定抑制I2PP2A基因表达的人胃癌BGC823细胞株,为进一步研究I2PP2A在胃癌发生中的作用提供了可靠的细胞模型。

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.