人GATA1真核表达载体的构建及对乳腺癌细胞生长的影响

Construction of human GATA1 eukaryotic expression vector and its effects on the growth of breast cancer cells

目的构建人GATA1基因的真核表达载体,研究其对乳腺癌细胞生长的影响。方法以人乳腺文库为模板,采用PCR技术扩增出GATA1编码序列,将其插入到pc DNA3.0-FLAG载体中;将重组质粒与空载体分别转染293T细胞,Western印迹检测GATA1表达情况,并通过生长曲线研究GATA1对乳腺癌细胞生长的影响,细胞划痕实验检测GATA1对细胞迁移的影响。结果双酶切实验以及测序结果表明,pc DNA3.0-FLAG-GATA1的真核表达载体构建成功;Western印迹发现,GATA1在293T细胞中成功表达;生长曲线实验得出,GATA1可促进乳腺癌细胞的生长,细胞划痕实验表明GATA1促进细胞迁移。结论成功构建了pc DNA3.0-FLAG-GATA1的真核表达载体,并发现GATA1可促进乳腺癌细胞的生长和迁移。

Objective To construct the eukaryotic expression vector of human GATA1 and study its effects on the growth of breast cancer cells. Methods The coding sequences of GATA1 were amplified from the breast library by PCR and cloned into the pc DNA3. 0-FLAG vector. The recombinant plasmid and empty vector were transfected into 293 T cells,GATA1 expression was detected by Western blotting,and the growth curve was performed to study the effects of GATA1 on the growth of ZR75-1 cells and MCF-7 cells. Wound healing assays were performed to study the effects of GATA1 on the migration of ZR75-1 cells and MCF-7 cells. Results The pc DNA3. 0-FLAG-GATA1 eukaryotic expression vector was constructed by double digestion identification. Western blotting showed that the recombinant plasmid could express GATA1 protein in 293 T cells. High expression of GATA1 could markedly enhance the proliferation and migration of ZR75-1 cells and MCF-7 cells. Conclusion The pc DNA3. 0-FLAG-GATA1 eukaryotic expression vector is successfully constructed,and GATA1 may promote the proliferation and migration of breast cancer cells.