MDM2基因真核表达质粒构建及对人乳腺癌MCF-7细胞运动与侵袭能力的影响  

Construction of eukaryotic expression plasmid of MDM2 gene and its effect on cell motility and invasion in human breast cancer MCF-7 cells

作　　者：郑大勇[1,2] 张彤彤 常兴 吴英良 左代英[1] ZHENG Dayong;ZHANG Tongtong;CHANG Xing;WU Yingliang;ZUO Daiying(Scholl of Life Science and Biopharmaceutical,Shenyang Pharmaceutical Universitiy,Shenyang 110016,China;School of Pharmacy,North China University of Science and Technology,Tangshan 063210,China)

机构地区：[1]沈阳药科大学生命科学与生物制药学院,辽宁沈阳110016 [2]华北理工大学药学院,河北唐山063210

出　　处：《沈阳药科大学学报》2023年第1期57-62,共6页Journal of Shenyang Pharmaceutical University

基　　金：国家自然科学基金资助项目(81872394);河北省自然科学基金资助项目(H2020209284);河北省高等学校科学技术研究项目(QN2021120)。

摘　　要：目的构建MDM2的pcDNA3.1真核表达质粒(pcDNA3.1-MDM2),并将其转染至人乳腺癌MCF-7中,考察MDM2蛋白过表达对乳腺癌转移的影响。方法采用PCR方法体外克隆MDM2基因全序列,并将其用同源重组技术连接到真核pcDNA3.1质粒载体上,构建真核表达质粒,并进行测序鉴定,利用脂质体法转染至MCF-7细胞中。通过G418抗生素筛选出单克隆高表达细胞系,使用免疫印迹法考察MDM2蛋白表达。同时,通过划痕试验及transwell法来检测细胞的运动与侵袭能力。结果测序结果表明pcDNA3.1-MDM2基因序列准确无误,免疫印迹法表明稳定转染细胞系构建成功,划痕试验及transwell试验表明MDM2高表达细胞系的运动与侵袭能力增加。结论成功构建了pcDNA3.1-MDM2真核表达质粒,转染MCF-7细胞后,可稳定表达,并促进了MCF-7细胞的运动及侵袭,为研究MDM2蛋白表达对人乳腺癌转移的分子机制及作为诊疗的生物标志物奠定了基础。Objective To construct the pcDNA3.1 eukaryotic expression plasmid carrying MDM2 gene,transfect to breast cancer MCF-7 cells,and to investigate the effect of overexpression of MDM2 on breast cancer cell metastasis.Methods The full sequence of MDM2 gene was amplified in vitro by PCR,and cloned into the eukaryotic pcDNA3.1 plasmid vector by homologous recombination technology.The recombinant plasmid was identified by sequencing,then transfected into MCF-7 cells using Lipo6000 reagent.Monoclonal high-expressing cell lines were screened by G418,and the expression of MDM2 protein was determined by western blotting.Meanwhile,the wound healing and the transwell assays were performed to analysis cell motility,migration and invasion capabilities.Results The sequencing results showed that the pcDNA3.1-MDM2 gene sequences were correct.The stable transfected cell line was identified by western blotting.The wound healing and transwell assays showed that the motility,migration and invasion abilities of MDM2 overexpressing cell line were increased.Conclusion The pcDNA3.1-MDM2 eukaryotic expression plasmid is successfully constructed,which can be stably overexpressed after transfection into MCF-7 cells,and promote the motility,migration and invasion of MCF-7 cells.This study lays the foundation for further molecular mechanism of MDM2 expression on breast cancer metastasis and as a biomarker for diagnosis and treatment.

关 键 词：乳腺癌 MDM2 肿瘤转移 

分 类 号：R96[医药卫生—药理学]