靶向CIP2A的shRNA对人胃癌BGC-823细胞增殖、凋亡及侵袭的影响

Effects of shRNA Targeting CIP2A Gene on the Proliferation,Apoptosis and Invasion of Human Gastric Cancer Cells(BGC-823)

目的探讨shRNA介导的蛋白磷酸酶2A的癌性抑制因子(cancerous inhibitor of protein phosphatase 2A,CIP2A)基因表达抑制对人胃癌细胞株BGC-823细胞增殖、凋亡及侵袭的影响。方法用已构建的shRNA-CIP2A重组质粒转染BGC-823细胞,荧光显微镜下观察转染效率;采用实时荧光定量PCR和Western blot技术检测重组质粒对CIP2AmRNA和蛋白表达的影响;分别用WST-1法、流式细胞术和Transwell小室法测定重组质粒转染对细胞增殖、凋亡及侵袭能力的影响。结果 shRNA-CIP2A转染效率达80%。转染shRNA-CIP2A后,可明显抑制BGC-823细胞内CIP2AmRNA和蛋白的表达(均P<0.05),与阴性对照组相比,转染shRNA-CIP2A的BGC-823细胞增殖明显受到抑制[(1.880±0.050)vs.(0.794±0.042),P<0.05],侵袭出的细胞数量明显少于对照组[(378.0±5.6)vs.(206.0±15.6),P<0.05],BGC-823细胞的增殖和侵袭能力明显下降,凋亡细胞比例增加[(5.33±0.51)%vs.(7.40±0.09)%,P<0.05]。结论 shRNA下调CIP2AmRNA和蛋白的表达,可有效抑制BGC-823细胞的增殖、侵袭,同时诱导细胞发生凋亡,提示CIP2A基因可能成为一个潜在的胃癌治疗靶标。

Objective To investigate the effects of shRNA mediated cancerous inhibitor of protein phosphatase 2A（CIP2A） gene silencing on the proliferation,apoptosis and invasion of human gastric cancer BGC-823 cells.Methods The specific recombinant vector shRNA-CIP2A was transfected into BGC-823 cells with lipofectamine 2000.Transfection efficiency was examined by fluorescence microscopy and the expression levels of CIP2A mRNA and protein were determined by real-time PCR and Western blot,respectively.Cell proliferation,apoptosis and invasion were determined by WST-1 assay,flow cytometry（FCM） and Transwell assay,respectively.Results The efficiency of shRNA transfection into human BGC-823 cells was 80%.The shRNA against CIP2A gene significantly reduced the mRNA and protein expression of CIP2A in BGC-823 cells（P〈0.01）.The BGC-823 cells transfected with shRNA-CIP2A showed a significant decrease of cell proliferation[（1.880± 0.050） vs.（0.794 ± 0.042）,P〈0.05]and invasion [（378.0±5.6） vs.（206.0± 15.6）,P〈0.05]when compared with the control cells.The apoptotic rate was much higher in CIP2A shRNA group than that in negative control group[（5.33 ± 0.51）% vs.（7.40 ± 0.09）%,P〈0.05].Conclusion CIP2A knockdown effectively inhibited the proliferation and invasion of gastric cancer cells and induced their apoptosis,indicating that CIP2A may become a potential therapeutic target for gastric cancer.