RNA干扰P115基因对胃癌细胞巨噬细胞移动抑制因子表达的抑制作用

Inhibitory Effect of P115 Gene Silencing by shRNA on Expression of Macroph-ge Migration Inhibitory Factor in Gastric Carcinoma Cells

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 the shRNA expression vector for Golgi-vesicular transport protein P115 and investigate the regulatory effect of P115 gene silencing on expression of macrophage migration inhibitory factor（MIF） in gastric carcinoma cell BGC-823 strain.Methods Four shRNA sequencing specific to P115 gene were designed,based on which recombinant expression vectors were constructed and transfected to BGC-823 cell strain for high expression of P115.The expressions of P115 and MIF mRNAs were determined by RT-PCR,while those of proteins by Western blot.Results All the four shRNA plasmids for P115 gene were constructed correctly as proved by restriction analysis and sequencing,and inhibited the expression of P115 gene in transfected BGC-823 cells.The silencing effect of pGPU6/GFP/Neo-shP115-2 was satisfactory,of which the inhibiting rate to expressions of P115 mRNA and protein were 75.07% and 70.97% respectively.Both the expression levels of MIF mRNA and protein in cells transfected with pGPU6/GFP/Neo-shP115-2 decreased significantly（P 0.05）.Conclusion The expression level of MIF in BGC-823 cells after P115 gene silencing decreased significantly,indicating that P115 might involve in regulating the expression of MIF in gastric carcinoma cells and P115 gene might be used as a novel target for study on the molecular mechanism of onset and progress of gastric carcinoma.