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目的构建人CARMA3真核表达载体,探讨其在乳腺癌MCF-7细胞中的表达及其与肿瘤发生发展的关系。方法以T7-CARMA3为模版,利用特异PCR引物扩增CARMA3基因全长编码序列,将其定向克隆至TMp3×FLAG-cmv-10载体,融合蛋白表达载体命名为Flag-CARMA3。将Flag-CARMA3转染到MCF-7乳腺癌细胞系中,Western blot和Real-Time PCR法检测基因和蛋白表达。MTT检测过表达Flag-CARMA3的细胞生长能力。结果成功构建Flag-CARMA3真核表达载体,Flag-CARMA3融合基因和蛋白在MCF-7乳腺癌细胞系中表达。MTT实验证实Flag-CARMA3过表达增强MCF-7乳腺癌细胞生长能力。结论 CARMA3促进乳腺肿瘤细胞生长。
Objective To construct human CARMA3 eukaryotic expression vector and investigate its expression in breast cancer MCF-7 cells and its relationship with tumor development. Methods Using T7-CARMA3 as a template, the full-length coding sequence of CARMA3 gene was amplified by specific PCR primers and cloned into TMp3×FLAG-cmv-10 vector. The fusion protein expression vector was named Flag-CARMA3. Flag-CARMA3 was transfected into MCF-7 breast cancer cell lines and the expression of genes and proteins was detected by Western blot and Real-Time PCR. MTT detects the ability of cells expressing Flag-CARMA3 to grow. Results The eukaryotic expression vector of Flag-CARMA3 was successfully constructed and the Flag-CARMA3 fusion gene and protein were expressed in MCF-7 breast cancer cell line. MTT experiments confirmed that overexpression of Flag-CARMA3 enhanced the growth of MCF-7 breast cancer cells. Conclusion CARMA3 promotes the growth of breast tumor cells.