GALNT14对人乳腺癌MCF-7细胞迁移的影响

构建真核表达载体pcDNA 3.1-Flag-T14,重组质粒经酶切分析及测序鉴定后,利用脂质体将重组质粒转染人乳腺癌细胞系MCF-7细胞,经G418筛选并建立稳定转染GALNT14细胞株.应用半定量RT-PCR、Western blot检测稳定细胞株GALNT14 mRNA及蛋白表达水平,细胞划痕修复及穿膜试验检测GALNT14基因对MCF-7迁移能力的影响,同时RT-PCR检测GALNT14对MMP-2,MMP-9,TGF-β1及VEGF等肿瘤浸润转移相关因子表达的影响.结果显示成功构建了真核重组表达载体pcDNA 3.1-Flag-T14,经RT-PCR和Western blot检测显示成功获得了稳定表达GALNT14的MCF-7细胞株；GALNT14能够提高MCF-7细胞株的迁移能力,且能增加侵袭转移相关因子MMP-2,MMP-9,TGF-β1及VEGF的表达.结论:GALNT14可明显促进MCF-7细胞的迁移,可能在肿瘤侵袭转移中起重要作用.

Effect of GALNT14 on the Migration of Human Breast Cancer Cells MCF-7

The recombinant plasmid—pcDNA 3.1-Flag-T14 was constructed and identified by restriction enzyme digestion and DNA sequencing.pcDNA 3.1-Flag-T14 was transfected into MCF-7 cells with lipofectamine 2000,and positive clones were selected by G418.The expression of GALNT14 in MCF-7 was tested by RT-PCR and Western blot.Wound healing assay and transwell migration assay were performed to detect migration activity.The effect of GALNT14 on the expression of MMP-2,MMP-9,TGF-β1and VEGF was measured by semi-quantitative RT-PCR.The result showed that pcDNA 3.1-Flag-T14 was successfully constructed and GALNT14 was expressed stably in MCF-7 cells.GALNT14 can promote the migration of MCF-7 cells.RT-PCR showed that GALNT14 also increased the expressions of MMP-2,MMP-9,TGF-β1and VEGF mRNA.In conclusion,GALNT14 can significantly improve the migration of MCF-7 cells and it might play an important role in the invasion and metastasis of tumor.