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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 plasmid of human SIAH2 gene and test its effect on the proliferation of breast cancer MDA-MB-231 cells. Methods The full-length sequence of SIAH2 gene was amplified by PCR from MDA-MB-231 cells and cloned into expression vector pcDNA-3.1-hisB to construct recombinant eukaryotic expression vector pcDNA-3.1-hisB-SIAH2. -231 cells. The expression of SIAH2 protein was detected by Western blot. The localization of SIAH2 protein in the cells was detected by immunofluorescence. The cell cycle distribution was detected by flow cytometry. The proliferation activity was detected by MTT assay. Results The recombinant plasmid pcDNA-3.1-hisB-SIAH2 was confirmed by EcoRⅠ / XhoⅠ sequencing and sequencing. The expression of SIAH2 protein in transfected cells was significantly increased (P <0.05) , And expressed in the nucleus, the proportion of cells in the S phase was significantly increased; 48,72 and 96 h after transfection of the recombinant plasmid, the cell proliferation activity was significantly enhanced. Conclusion The SIAH2 gene eukaryotic expression plasmid was successfully constructed and SIAH2 protein was expressed in MDAMB-231 cells. SIAH2 protein can significantly promote the proliferation of breast cancer MDA-MB-231 cells, suggesting that SIAH2 play a role in the occurrence and progression of breast cancer An important role, is expected to become a new target for the development of breast cancer drug development.