GALNT14对人乳腺癌MCF-7细胞迁移的影响

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

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。

The recombinant plasmid-pcDNA 3. 1 enzyme digestion and DNA sequencing, pcDNA 3. -Flag-T14 was constructed and identified by restriction 1-Flag-T14 was transfeeted 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.