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目的构建高尔基体转运蛋白P115基因shRNA表达载体,探讨P115基因沉默对胃癌细胞株BGC-823中巨噬细胞移动抑制因子(Macrophage migration inhibitory factor,MIF)表达的影响。方法设计4对针对P115基因的shRNA序列,构建重组表达质粒,转染高表达P115的胃癌细胞株BGC-823。RT-PCR、Western blot和免疫细胞化学检测P115及MIF的mRNA和蛋白的表达。结果 4个P115基因shRNA质粒经单酶切和测序证实构建正确;转染BGC-823细胞后,均能抑制P115基因的表达,其中以pGPU6/GFP/Neo-shP115-2的沉默效果最好,其对P115基因mRNA表达的抑制率为75.07%,对P115蛋白表达的抑制率为70.97%;转染pGPU6/GFP/Neo-shP115-2后,MIF基因的mRNA及蛋白表达水平均明显降低(P<0.05)。结论 P115基因沉默后,BGC-823细胞MIF的表达明显降低,提示P115可能参与调节胃癌细胞MIF的表达,P115基因可作为研究胃癌发生发展分子机理的新靶点。
Objective To construct shRNA expression vector of Golgi transporter P115 gene and investigate the effect of P115 gene silencing on the expression of Macrophage migration inhibitory factor (MIF) in gastric cancer cell line BGC-823. Methods Four pairs of shRNA sequences targeting P115 gene were designed and constructed into recombinant plasmids. The recombinant plasmid was transfected into BGC-823 gastric cancer cell line P115. The mRNA and protein expression of P115 and MIF were detected by RT-PCR, Western blot and immunocytochemistry. Results Four plasmids of P115 gene were confirmed by single digestion and sequencing. The expression of P115 gene was inhibited after transfection with BGC-823 cells. Among them, pGPU6 / GFP / Neo-shP115-2 had the best silencing effect, The inhibitory rate of P115 gene mRNA expression was 75.07%, and the inhibitory rate of P115 protein expression was 70.97%. After transfection with pGPU6 / GFP / Neo-shP115-2, the mRNA and protein expression levels of MIF gene were significantly decreased (P <0.05). Conclusion The silencing of P115 gene significantly decreased the expression of MIF in BGC-823 cells, suggesting that P115 may be involved in the regulation of MIF expression in gastric cancer cells. P115 gene may serve as a new target for studying the molecular mechanism of gastric cancer progression.