目的:构建甲状腺癌基因-1(TC-1)真核表达质粒,观察TC-1癌蛋白高表达对人乳腺癌细胞迁移的影响。方法:用蛋白质印迹方法检测TC-1癌蛋白在培养的人MCF-7和BT549乳腺癌细胞中表达;从MCF-7细胞中提取总RNA,通过实时PCR(RT-PCR)扩增人TC-1 cDNA全长开放读码框架(ORF),并插入真核表达载体Flag-pcDNA3.0中构建Flag-TC-1-pcDNA3.0重组质粒;经限制性内切酶和DNA测序鉴定后,该重组质粒用于转染BT549细胞,进行蛋白质印迹分析鉴定Flag-TC-1融合蛋白表达;进行细胞划痕实验,观察TC-1对BT549细胞迁移的影响。结果:TC-1蛋白在人MCF-7乳腺癌细胞中显著表达,而BT549细胞中表达极低;人MCF-7乳腺癌细胞TC-1全长ORF的RT-PCR产物为337 bp;重组质粒Flag-TC-1-pcDNA3.0经BamH Ⅰ和Xho Ⅰ双酶切出现特征性的327 bp片段;DNA测序证实TC-1全长ORF序列正确无误,以正确的阅读框架插入Flag-pcDNA3.0载体中。蛋白质印迹结果显示该重组质粒转染的BT549细胞表达的Flag-TC-1融合蛋白可被抗Flag抗体特异性识别,分子质量约为13.6 kDa,符合预期;划痕实验结果显示,高表达TC-1的BT549细胞迁移性增加。结论:内源性TC-1蛋白在人BT549乳腺癌细胞中表达极低,Flag-TC-1融合蛋白高表达促进BT549细胞迁移。 |
Objective:To investigate impact of thyroid cancer-1(TC-1) overexpression on the migration of human breast cancer cells after construction of the TC-1 eukaryotic expression plasmid. Methods:Western blot was used to detect the TC-1 expression in the cultured human MCF-7 and BT549 breast cancer cells. Subsequently, the total RNA was extracted from the MCF-7 cells, and the full length open reading frame(ORF) of TC-1 cDNA was amplified by RealTime-PCR(RT-PCR) and then inserted into the eukaryotic expression vector Flag-pcDNA3.0 to generate recombinant plasmid Flag-TC-1-pcDNA3.0. After identification by restriction enzyme digestion and DNA sequencing, the recombinant plasmid was transfected into the BT549 cells. The expression of Flag-TC-1 fusion protein in the transfected cells was monitored by using Western blot. Scratch-wound assays were performed to observe impact of TC-1 overexpression on migration of the BT549 cells. Results:Human MCF-7 breast cancer cells expressed TC-1 protein at a high level while the protein level was extremely low in BT549 cells. The RT-PCR product of TC-1 from the MCF-7 cells was 337 bp, which was consistant with the full length ORF of the gene. The recombinant plasmid Flag-TC-1-pcDNA3.0 was identified with double digestion using BamH Ⅰ and Xho Ⅰ, in which a characteristic fragment of 327 bp appeared, and the full length ORF of TC-1 cDNA sequence was confirmed by direct automated sequencing. After the BT549 cells were transfected with the recombinant plasmid, Flag-TC-1 fusion protein expressed in the cells was specifically recognized by anti-Flag antibody. As expected, the molecular weight of the fusion protein is about 13.6 kDa. Then, the scratch-wound assays were performed in the transfected cells and showed that overexpression of TC-1 increased migration of the BT549 cells. Conclusions:The endogenous TC-1 protein level is extremely low in human BT549 breast cancer cells and overexpression of Flag-TC-1 fusion protein stimulates migration of the cells. |
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