TRAF1基因干扰质粒的构建及其对胃癌细胞生物学功能的影响

摘要

目的:构建TRAF1基因干扰重组质粒及建立TRAF1基因干扰瞬时转染胃癌细胞模型,然后通过干扰目标胃癌细胞中的TRAF1基因来检测TRAF1对胃癌细胞的生物学功能的影响.方法:首先通过real-time PCR和Western印迹验证TRAF1在胃癌细胞系BGC823,SGC7901,MGC803中的表达,筛选出TRAF1表达量最高的一株细胞作为干扰转染的目标细胞；设计及构建3个TRAF1的shRNA表达载体pLVX-shRNA-TRAF1-shRNA1～3,将其分别转染至目标胃癌细胞系中,应用real-time PCR和Western印迹筛选出干扰效率最强的shRNA,最后通过MTF及流式细胞术检测TRAF1对胃癌细胞凋亡能力的影响,通过Transwell小室迁移实验来检测对其迁移功能的影响.结果:与GES-1比较,TRAF1基因在BGC823,SGC7901,MGC803中的表达均有上调(P＜0.05),其中以BGC823表达最高；3个TRAF1 shRNA对TRAF1基因均有抑制作用,以pLVX-shRNA-TRAF1-shRNA2干扰效果最强；干扰胃癌细胞BGC823 中的TRAF1可使细胞生长活力减弱,凋亡明显增加,但对其迁移功能无明显影响.结论:TRAF1基因在胃癌细胞系BGC823中上调最明显；pLVX-shRNA-TRAF 1-shRNA2可成功干扰BGC823中TRAF1的表达；TRAF1可抑制BGC823细胞的凋亡.%Objective: To construct the RNAi targeting tumor necrosis factor receptor associated factor (TRAF1) gene, and to explore the effect of interference targeting TRAF Ion the biological behavior of gastric cancer cells.Methods: We detected the expression of TRAF1 in BGC823, SGC7901, and MGC803 gastric cancer cell lines through the real-time PCR and Western blot; then we constructed three pLVX-shRNA-TRAFl-shRNAs expression vector targeting TRAF1. When TRAF1 was interfered successfully) we selected the strongest interference efficiency ShRNA by real-time PCR and Western blot. Based on interference targeting TRAF1 on gastric cancer, we tested the cell proliferation activity and apoptosis through MTT assay and flow cytometry, and the cell migration by transwell migration assay.Results: The expression of TRAF1 was increased in BGC823, SGC7901, and MGC803 gastric cancer cell lines compared with gastric epithelial cells (P<0.05), and the highest expression was in BGC823 gastric cell line. In the three TRAF1 shRNAs, the strongest interference efficiency shRNA was pLVX-shRNA-TRAFl-shRNA2. When the gene TRAF1 of BGC823 was interfered, the cell growing power was weakened and the apoptosis rate increased, and the cell migration had no difference.Conclusion: The expression of TRAF1 is up-regulated in gastric cancer cell lines BGC823, SGC7901, and MGC803, and the most obvious one is BGC823. The interference targeting TRAF1 can successfully inhibit the expression of TRAF1 in gastric cancer cell line BGC823. TRAF1 can inhibit the apoptosis of BGC823 cells.