稳定转染靶向autotaxin的shRNA对胃癌细胞株AGS增殖、迁移及侵袭力的影响

Effects of Stably Expressing shRNA-targeted Autotaxin on Proliferation,Migratory and Invasive Capability of Gastric Carcinoma AGS Cells

目的研究shRNA抑制人胃癌细胞株AGS中自分泌运动因子Autotaxin表达对胃癌细胞增殖、迁移及侵袭能力的影响。方法通过基因序列设计合成特异性ATX-shRNA及随机阴性对照mock-shRNA,构建相应的表达质粒pSUPER-ATX及pSUPER-mock,在阳离子脂质体的介导下将此表达质粒及空载质粒pSUPER-control转染至人胃癌细胞株AGS。于转染后24、48、72h收集细胞,用RT-PCR和Western blot法检测转染后各组细胞及野型AGS细胞的内源性ATX mRNA和蛋白的表达;体外实验(MTT法、transwell法及matrigel法)测定肿瘤细胞增殖、体外迁移及侵袭能力。结果 shRNA表达载体pSUPER-ATX经限制性酶切及序列分析证明基因插入正确。转染pSUPER-ATX后的胃癌细胞ATX mRNA和蛋白较其他组表达明显降低(P<0.01),其增殖、体外迁移及侵袭力也明显降低。结论 shRNA能有效持续抑制人胃癌细胞株AGS的ATX基因与蛋白的表达,并降低癌细胞的迁移及侵袭力。

Objective To explore the effect of shRNA targeting autotaxin on the migratory and invasive capability of human gastric cancer cell AGS. Methods The pSUPER - ATX and pSUPER - mock （ non - specific） , which were constructed in corresponding to the ATX - shRNA and negative control mock - shRNA synthesized on basis of gene sequence, were transfected with blank plasmid pSUPER - control into human gastric cancer cell AGS by Lipofectamine. At 24h, 48h and 72h post -transfection, the cells were harvested and ana- lyzed. The endogenous ATX mRNA and protein of different group AGS cells were detected by RT - PCR and western blot assays. The cell proliferation was measured by MTT assay. In vitro transwell test and matrigel test were used to detect the cell migratory and invasive capa- bility. Results PCR and Western blot analyses confirmed that the recombinant plasmids pSUPER - ATX, pSUPER - mock have been successfully constructed. The mRNA and protein level of ATX in pSUPER - ATX group were both significantly down - regulated （P 〈 0. 01 ） , and the cell proliferation,migration and invasive capability were significantly deceased as well. Conclusion The specific shRNA targeting ATX clown - regulated the endogenous ATX of human gastric cancer AGS cells, and could inhibite the AGS cell proliferation, mi- gratory and invasive capability.