分化抑制因子1在环氧合酶2介导的胃癌血管生成中的作用及机制研究

目的验证分化抑制因子1（Id-1）在环氧合酶2（COX-2）介导的胃癌血管生成中的作用及机制。方法建立高表达COX-2和表达COX-2特异性siRNA的胃癌SGC7901细胞,通过Western印迹和逆转录PCR方法检测两种亚细胞系中Id1的表达,ELISA方法分析两种细胞上清中血管内皮生长因子（VEGF）的表达差异。通过四唑盐比色（MTT）实验分析两种细胞亚系的上清对体外脐静脉内皮细胞（HU-LT）增殖的影响,并在SGC7901／COX-2中抑制Id1后观察培养上清中VEGF的变化以及对脐静脉内皮细胞（HU-LT）增殖的影响。裸鼠成瘤实验观察转染细胞的成瘤性及肿瘤新生微血管密度（MVD）。结果成功建立高表达COX-2以及表达特异性COX-2小干扰RNA（siRNA）的稳定克隆,并鉴定了不同克隆的转染效率。高表达COX-2的胃癌SGC7901细胞中Id1的蛋白及mRNA表达水平增高,而转染COX-2-siRNA的SGC7901细胞中Id1的表达则均低于对照组。COX-2高表达的SGC7901／COX-2细胞上清中VEGF的蛋白含量高于对照组（2060±42 vs 1248±28,P=0．000）,而SGC7901／COX-2-siRNA细胞上清中VEGF的蛋白含量低于对照组（1024±20,2033±27vsP=0．000）。在SGC7901／COX-2细胞条件培养基中,HU-LT细胞生长较对照组快;在SGC7901／COX-2-SiRNA细胞条件培养基中,HU-LT细胞生长较对照组缓。在SGC7901／COX-2中抑制Id1后,培养上清中VEGF的含量增加以及对HU-LT促进增殖的作用均被逆转。裸鼠成瘤试验显示抑制Id1后SGC／901／COX-2细胞成瘤性降低[（353±12）mg vs（1020±91）mg,P=0．038],MVD降低（8．8±1．6vs20±1．7,P=0．001）。结论COX-2可以上调SGC7901细胞中Id1的表达,并通过此途径影响内皮细胞的体外增殖能力。因此在胃癌发生发展中Id1参与了COX-2所介导的促血管生成过程,有可能成为胃癌抗血管治疗的新靶标。

The potential role of Id1 in COX-2 mediated angiogenesis in gastric cancer

OBJECTIVE: To investigate the potential role of inhibitor of DNA binding/differentiation 1 (Id1) in cyclooxygenase-2 (COX-2) mediated angiogenesis in gastric cancer. METHODS: Two human gastric cancer sub-cell lines (SGC7901/COX-2 and SGC7901/COX-2RNAi) highly expressing COX-2 and COX-2 RNAi respectively were established. Western blotting and RT-PCR were performed to detect the protein and mRNA expression levels of Id1 in these transfectants. ELISA was performed to detect the levels of VEGF protein in the supernatants of these cells. Humane umbilical vein endothelial cells of the line HU-LT were cultured in condition medium, supernatant of SGC7901/COX2 cells transfected with COX-2 sense strand, COX-2 SiRNA and Id1. MTT assay was used to examine the proliferation ability of these cells. Suspensions of SGC7901/COX-2 and SGC7901/COX-2RNAi cells were injected subcutaneously to nude mice respectively. Eight weeks later the mice were killed and the tumors were taken out to undergo immunohistochemistry and detection of mcrovessel density (MVD). RESULTS: Western blotting and RT-PCR showed that the expression levels of COX-2 protein and mRNA, as well as those of Id1 in the SGC7901/COX-2 cells were high, and the expression level of Id1 was down-regulated in the SGC7901/COX-2RNAi cells transfected with Id1 RNAi. The VEGF level of the SGC7901/COX-2 cells was 2060 +/- 42, significantly higher than that of the control SGC7901/PC cells (1248 +/- 28, P = 0.000) and VEGF level of the supernatant of then SGC7901/COX-2 RNAi cells was 1024 +/- 20, significantly lower than that of the SGC7901/COX-2/PC cells (2033 +/- 27, P = 0.000). The proliferation rate of the HU-LT cells cultured in SGC7901/COX-2 condition medium was higher than that of the HU-LT cells cultured in SGC7901/COX-2RNAi condition medium. The increase of VEGF in the SGC7901/COX-2 and the effect of contribution to the proliferation of HU-LT were both abrogated when Id1 was knocked out in the SGC7901/COX-2. The tumor weight of the COX-2 RNAi group was (353 +/- 12) mg, significantly lower than that of the SGC7901/COX-2 group [(1020 +/- 91) mg, P = 0.038]. The MVD of the tumor of the COX-2 RNAi group was 8.8 +/- 1.6, significantly lower than that of the SGC7901/COX-2 group (20 +/- 1.7, P = 0.001). CONCLUSION: COX-2 can stimulate VEGF and enhance the proliferation of endothelial cells by upregulating Id1, and blocking this pathway may be helpful to the tumor therapeutics, so COX-2 and Id1 can be exploited as therapeutic targets of gastric cancer.