过表达外源ST3Gal I增加MCF-7细胞与胞外基质粘附和侵袭能力

为探讨过表达外源α2,3-唾液酸转移酶（ST3Gal Ⅰ）对乳腺癌MCF-7细胞粘 附和侵袭能力的影响，构建pEGFP-N1-ST3Gal I真核表达载体.采用GenEscortTM Ⅱ包裹后转染MCF-7细胞. MCF-7细胞为3组：未转染组 (M)、转染空质粒组 (P) 和转染ST3Gal I组 (ST3); 荧光显微镜观察融合蛋白EGFP ST3Gal I的表达.采用 半定量RT-PCR、Western印迹法分析转染后MCF-7细胞ST3Gal Ⅰ基因mRNA水平和 蛋白表达水平;流式细胞术分析ST3Gal Ⅰ下游产物细胞表面α2,3-唾液酸含量；采用细胞粘附实验及transwell小室检测转染前后细胞与基质胶Matrigel粘附、迁移和侵袭运动能力的变化.结果表明, 荧光显微镜下P组细胞内绿色荧光呈弥散分 布，而ST3组绿色荧光主要集中在细胞质中，RT-PCR与Western印迹也证实了外源 ST3Gal Ⅰ基因mRNA和蛋白表达均明显增加（P<0.05），其下游产物细胞表面 α2,3-唾液酸含量明显增加（P<0.05）；与M、P组相比，ST3组表现为粘附、迁移和侵袭能力明显增强（P<0.05）.利用转染技术可明显提高外源ST3Gal Ⅰ在MCF -7细胞表达，明显增加MCF-7细胞与胞外基质（ECM）粘附、迁移和侵袭能力，可形成肿瘤入侵表型，将有望成为治疗乳腺癌转移的新靶点.

Exogenetic Overexpression of ST3Gal I Increases the Ability of Adhesion and Invasion to ECM in Breast Cancer MCF-7 Cells

To study the effect of exogenetic overexpression of α 2,3 sialyltransferase (ST3Gal Ⅰ ) on the abilities of adhesion and invasion in MCF-7 cells, we constructed pEGFP-N1-ST3Gal Ⅰ eukaryotic expression vector. The recombinant vector was transfected into MCF-7 cells by GenEscortTMⅡ. MCF-7 cells were divided into 3 groups: mock cells (M),parental cells (P) and ST3Gal Ⅰ-overexpression（ST3）.The expression of fusion protein ST3Gal I EGFP in cells was observed using fluorescence microscope. The expression levels of ST3Gal Ⅰ mRNA and protein were examined by RT-PCR and Western blotting. The amount of α 2,3-sialic acids on the cell surface, which is the downstream product of ST3Gal I, was detected by flow cytometry. The ability of adhesion and invasion of MCF-7 cells to Matrigel was analyzed by using adhesion assay and transwell assay. The results suggested that fluorescence was observed in whole cell in P, whereas that is in the cytoplasm in ST3. The exogenetic expression levels of ST3Gal Ⅰ mRNA, protein and the amount of α 2,3-sialic acids on cell surface in ST3 group were significantly increased compared with that in the M group and P group ( P<0.05). The abilities of adhesion and invasion of the cells in ST3 group were markedly higher than those in the other 2 groups (P<0.05). The technology of transfection can effectively enhance the exogenetic expression of ST3Gal Ⅰ and increase cell adhesion, migration and invasion to Matrigel in MCF-7 cells and can promote tumor metastasis. This may be meaningful for seeking novel target for therapeutic approaches in breast cancer.