肌细胞增强因子2A基因真核表达质粒的构建及对乳腺癌细胞MCF-7增殖能力的影响

目的：构建肌细胞增强因子2A（MEF2A）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增殖能力的影响。方法：实时定量聚合酶链反应（RT-QPCR）检测MCF-7、BT474及MDA-MB-231等3种乳腺癌细胞中MEF2A mRNA的表达。采用全基因合成法合成MEF2A编码区序列,克隆入pcDNA3.1（＋）-HA载体,构建重组质粒pcDNA3.1-MEF2A-HA。重组质粒转染乳腺癌细胞,采用Western blot及RT-QPCR检测MEF2A的表达效率。细胞计数实验检测细胞的增殖能力。结果：3株乳腺癌细胞中,MDA-MB-231细胞中MEF2A 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MEF2A重组质粒构建成功。将MEF2A重组质粒瞬时转染MDA-MB-231细胞后,MEF2A表达质粒组与对照组比较,其MEF2A表达升高,细胞增殖能力增强。结论：成功构建MEF2A过表达重组质粒,在MDA-MB-231中过表达MEF2A促进细胞的增殖能力。

Construction of myocyte enhancer factor 2A eukaryotic expression plasmid and its effects on cell proliferation in breast cancer cell line MCF7

Objective： To construct eukaryotic expression plasmid of pcDNA3.1-MEF2A and examine its effect on the proliferation of breast cancer cell line. Methods： The MEF2A mRNA levels were detected in MCF-7, BT474 and MDA-MB-231 cells, respectively by real-time quantitative PCR（RT-QPCR）. The full-length coding sequence of MEF2A was amplified by standard RT-QPCR. The amplified MEF2A was cloned into pcDNA3.1 vector. The eukaryotic expression plasmid pcDNA3.1-MEF2A-HA was transfected into breast cancer cells. RT-QPCR and western blot were performed to measure the expression level of MEF2A. Cell counting was performed to evaluate the effects of MEF2A expression on cell proliferation. Results： The MEF2A mRNA expression level was low in MDA-MB-231 cells. Western blot and RT-QPCR indicated that transfection of MEF2A expression vector into MDA-MB-231 cells resulted in increased mRNA/protein levels of MEF2A. The forced MEF2A expression significantly enhanced cell proliferation of MDA-MB-231. Conclusion： The eukaryotic expression plasmid of pcDNA3.1-MEF2A-HA is constructed successfully. Over-expression of MEF2A enhances proliferation ability of MDA-MB-231 cells.