人SIAH2基因真核表达质粒的构建及其对乳腺癌MDA-MB-231细胞增殖的影响

Construction of eukaryotic expression vector for human SIAH2 gene and its effect on proliferation of breast cancer MDA-MB-231 cells

目的构建人SIAH2基因真核表达质粒,并检测其对乳腺癌MDA-MB-231细胞增殖的影响。方法采用PCR技术从MDA-MB-231细胞中扩增SIAH2基因全长序列,克隆至表达载体pcDNA-3.1-hisB中,构建重组真核表达质粒pcDNA-3.1-hisB-SIAH2,转染MDA-MB-231细胞,Western blot法检测SIAH2蛋白在细胞中的表达;免疫荧光法检测SIAH2蛋白在细胞中的定位;流式细胞术检测细胞细胞周期的分布;MTT法检测细胞增殖活力的变化。结果重组真核表达质粒pcDNA-3.1-hisB-SIAH2经双酶切(EcoRⅠ/XhoⅠ)和测序证实构建正确;重组质粒转染细胞后,细胞中SIAH2蛋白的表达水平明显升高(P<0.05),且在细胞核内表达,处于S期的细胞比例明显增加;重组质粒转染细胞后48、72和96 h,细胞的增殖活力明显增强。结论成功构建了SIAH2基因的真核表达质粒,并在MDAMB-231细胞中表达了SIAH2蛋白;SIAH2蛋白能明显促进乳腺癌MDA-MB-231细胞增殖,提示其在乳腺癌的发生和进展中发挥了重要作用,有望成为治疗乳腺癌药物开发的新靶点。

Objective To construct the eukaryotic expression vector for human SIAH2 gene and investigate its effect on the proliferation of breast cancer MDA-MB-231 cells. Methods Full-length SIAH2 gene was amplified from MDA-MB- 231 ceils by PCR and cloned into expression vector pcDNA3. 1-hisB. The constructed recombinant plasmid pcDNA-3. 1- hisB-SIAH2 was transfected to MDA-MB-231 cells. The expression of SIAH2 protein in cells was determined by Western blot, while the location by IFA, the distribution of cell cycle by flow cytometry, and the proliferation activity of cells by MTr assay. Results Restriction analysis （EcoR Ⅰ /Xho Ⅰ ） and sequencing proved that recombinant plasmid pcDNA3. 1- hisB-SIAH2 was constructed correctly. The expression level of SIAH2 protein in the ceils transfected with the recombinant plasmid increased significantly （P 〈 0. 05）. The expressed protein was located in nuclei, of which the per- centage at S phase increased significantly. The proliferation activity of MDA-MB-231 cells 48, 72 and 96 h after transfection increased significantly. Conclusion The eukaryotic expression vector for SIAH2 gene was constructed successfully, and SIAH2 protein was expressed in MDA-MB-231 cells. SIAH2 protein promoted the proliferation of MDA- MB-231 cells significantly, indicating its important role in the pathogenesis and progress of breast cancer.