Construction Of A Gastric Cancer Cell Strain BGC823 With MMP-9 Gene Silencing Using RNA Inteference

A series of enzymes involved in the process of tumor cells and endothelial cells's migration across basement membrane and extracellular matrix. As a kind of gelatinase, matrix metalloproteinase-9(MMP-9) can degradate extracellular matrix and basement membrane, which will facilitate the invasion, metastasis and interstitial angiogenesis. Since the research of the invasion and metastasis becomes an important field, MMP-9 attracts more and more focus. With the development of RNAi,this technology also applied extensively. This article discussed the expression of MMP-9 in the gastric cancer cells, and established a new gastric cancer cell strain with MMP-9 gene silencing using RNAi.BGC823 express relative large amount of MMP-9, and most MMP-9 locates in the cytoplasm,some on the membrane as granulas. In gelatin zymography, MMP-9 in the cuture mediem could digest the gelatin, which indicates that tumor cells can secrete MMP-9 to extracellualr environment and degrade ECM to facilitate the invasion and metastasis of tumor cells.In this study, a stable RNAi system mediated by pGenesil plasmid vector was transfected into poorly differentiated gastric adenocarcinoma cell BGC823 by liposome. The best condition for transfection in BGC823 was groped after optimized the cells density, the dose and ratio of plasmid and liposome. In 24 wells plate,1×10⁵ cells planted before transfection, and a dose of 1.2μg pGenesil and 1μl Sofast^TM can lead to the best transfection efficiency,about 30%. A efficiency higher than 90% can be obtained by adding G418 at a concentration of 500μg/ml to the cells after transfection for clone screeningtin in order to essay the effect of MMP-9 gene silencing by RNAi. EGFP can still be observed after five months, which indicates that the RNA inferecence mediated by this vector has a long effect.As we know, the effect of gene silencing by siRNAs shows highly selectivity. In this study, three shRNAs aiming different MMP-9 target sequence was designed by professional company and ligated to pGenesil plasmid. The three shRNA all can downregulate the expression of MMP-9 after essaying the MMP-9 mRNA , protein and activity in the RNAi-cells and non-RNAi cells by RT-PCR, immunofluorescence and gelatin zymography. The different gene silencing effect shows the most effective shRNA sequnce, that is MMP-9₁, which was screened to construct the MMP-9 gene silencing BGC823 cell strain after stable transfection. The modal would be important for next invasion essay.This study tried to silence MMP-9 gene in gastric cancer cells by RNAi, and first construct a MMP-9 gene silencing BGC823 cell strain. It's important to gain a high transfection efficiency and effect in RNA interference. Cell clones with satisfactory transfection efficiency and a shRNA with better interference effect were obtained in this research. This cell strain would be beneficial for further research of gastric cancer invasion and metastasis in vivo and in vitro.