HOXA5基因真核表达质粒的构建及在乳腺癌细胞中的功能研究

Construction of HOXA5 eukaryotic expression plasmid and its biological significance in breast cancer cells

目的:构建HOXA5基因真核表达重组质粒,并转染乳腺癌细胞株观察其对细胞迁移能力的影响。方法:实时定量聚合酶链反应(RT-QPCR)检测MCF7、BT474及MDA-MB-231等3种乳腺癌细胞中HOXA5 mRNA的表达。采用全基因合成法合成HOXA5编码区序列,克隆入pcDNA3.1(+)载体,构建重组质粒pcDNA3.1-HOXA5。重组质粒转染乳腺癌细胞,采用Western blot及RT-QPCR检测HOXA5的表达效率。观察细胞形态并通过细胞划痕实验检测细胞的迁移能力。结果:3株乳腺癌细胞中,MDA-MB-231细胞中HOXA5 mRNA表达水平较低。其基因测序结果显示与预期片段大小一致,表明HOXA5重组质粒构建成功。将HOXA5重组质粒瞬时转染MDA-MB-231细胞后,检测到其mRNA和蛋白表达水平均明显上调,MDA-MB-231细胞的迁移能力降低,EMT相关转录因子Twist的表达显著下调。结论:成功构建HOXA5过表达重组质粒,在MDA-MB-231中过表达HOXA5抑制乳腺癌的迁移能力。

Objective： To construct eukaryotic expression plasmid of pcDNA3.1-HOXA5 and examine its effect on the migration of the breast cancer cell line. Methods： The HOXA5 mRNA levels were detected in MCF7,BT47g and MDA-MB-231 cells, respectively, by realtime quantitative PCR. The full-length coding sequence of HOXA5 was amplified using standard RT-QPCR. The amplified HOXA5 was cloned into pcDA3.1 vector. And the eukaryotic expression plasmid pcDNA3.1-HOXA5 was transfected into breast cancer cells. RT-QPCR and Western blot were performed to measure the expression level of HOXA5. The cell morphology changes were observed. The migration ability of the cells was also observed through scratch assay. Results： The HOXA5 mRNA expression level of MDA-MB-231 was low in MDA-MB-231 ceils. Western blot and RT-QPCR indicated that transfection of HOXA5 expression vector into MDA-MB-251 cells resulted in enhanced mRNA/protein levels of HOXA 5. The forced HOXA5 expression significantly decreased cell migration of MDA-MB- 231 and down-regulated the expression of Twist. Conclusion： The eukaryotic expression plasmid of pcDNA3.1-HOXA5 can be successfully constructed. Over-expression of HOXA5 inhibits the migration ability of MDA-MB-231 cells.