郑大勇,张彤彤,常兴,吴英良,左代英
ZHENG Dayong,ZHANG Tongtong,CHANG Xing,WU Yingliang,ZUO Daiying

• 1:沈阳药科大学生命科学与生物制药学院
• 2:华北理工大学药学院

摘要(Abstract)：

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(KeyWords)： 乳腺癌;MDM2;肿瘤转移
breast cancer;MDM2;tumor metastasis

Abstract:

Keywords:

基金项目(Foundation): 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)

作者(Author): 郑大勇,张彤彤,常兴,吴英良,左代英
ZHENG Dayong,ZHANG Tongtong,CHANG Xing,WU Yingliang,ZUO Daiying

DOI: 10.14066/j.cnki.cn21-1349/r.2021.0819

参考文献(References)：

• [1] WEI W Q,ZENG H M,ZHENG R S,et al.Cancer registration in China and its role in cancer prevention and control[J].Lancet Oncol,2020,21:e342-349.
• [2] ZHENG R S,SUN K X,ZHANG S W,et al.Report of cancer epidemiology in China,2015[J].Chinese Journal of Oncology(中华肿瘤杂志),2019,41(1):19-28.
• [3] SIEGEL R L,MILLER K D,FUCHS H E,et al.Cancer statistics,2021[J].CA Cancer J Clin,2021,71(1):7-33.
• [4] DESANTIS C E,MA J M,SAUER A G,et al.Breast cancer statistics,2017,racial disparity in mortality by state[J].CA Cancer J Clin,2017,67(6):439-448.
• [5] FIDLER I J,KRIPKE M L.The challenge of targeting metastasis[J].Cancer Metastasis Rev,2015,34:635-641.
• [6] MICHAEL D,OREN M.The p53 and MDM2 families in cancer[J].Curr Opin Genet Dev,2002,12(1):53-59.
• [7] CHEN Y,WANG B Z.Expressions of TBX2 and MDM2 and their clinical significance in breast invasive ductal carcinoma[J].Chinese Journal of Clinical and Experimental Pathology(临床与实验病理学杂志),2012,28(11):1211-1214
• [8] BALIOU E,NONNI A,KERAMOPOULOS D,et al.Deregulation of p53-MDM2 auto-regulatory pathway in breast carcinoma[J].J Buon,2016,21(5):1099-1103.
• [9] CHOUAYEKH K S,BACCOUCHE S,KHABIR A,et al.Prognostic significance of p16INK4a/p53 in Tunisian patients with breast carcinoma[J].Acta Histochem,2011,113:508-513.
• [10] TURBIN D A,CHEANG M C,BAJDIK C D,et al.MDM2 protein expression is a negative prognostic marker in breast carcinoma[J].Mod Pathol,2006,19:69-74.
• [11] SONG H L,GOU W F,MA Y H,et al.Construction and expression of EML4-ALK-G1202R point-mutated plasmid in vitro[J].Journal of Shenyang Pharmaceutical University(沈阳药科大学学报),2018,35(9):776-780.
• [12] LIU Y,GOU W F,XU X B,et al.Construction and verification of CD74-ROS1-L1951R point-mutated plasmid[J].Journal of Shenyang Pharmaceutical University(沈阳药科大学学报),2019,36(8):729-734.
• [13] SPANO D,HECK C,ANTONELLIS D P,et al.Molecular networks that regulate cancer metastasis[J].Semin Cancer Biol,2012,22:234-249.
• [14] GOLDHIRSCH A,WINER E P,COATES A S,et al.Personalizing the treatment of women with early breast cancer:highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2013[J].Ann Oncol,2013,24:2206-2223.
• [15] ABUBAKAR M,FIGUEROA J,ALI H R,et al.Combined quantitative measures of ER,PR,HER2,and KI67 provide more prognostic information than categorical combinations in luminal breast cancer[J].Mod Pathol,2019,32(9):1244-1256.
• [16] SCHMIDT O K,LOKSHIN M,PRIVES C.The roles of MDM2 and MDMX in cancer[J].Annu Rev Pathol,2016,11:617-644.
• [17] WADE M,LI Y C,WAHL G M.MDM2,MDMX and p53 in oncogenesis and cancer therapy[J].Nat Rev Cancer,2013,13(2):86-96.
• [18] OLINER J D,SAIKI A Y,CAENEPEEL S.The role of MDM2 amplification and overexpression in tumorigenesis[J].Cold Spring Harb Perspect Med,2016,6(6):a026336.
• [19] HAUCK P M,WOLF E R,OLIVOS D J,et al.Early-stage metastasis requires MDM2 and not p53 gain of function[J].Mol Cancer Res,2017,15:1598-1607.
• [20] WOLF E R,MABRY A R,DAMANIA B,et al.MDM2 mediated neddylation of pVHL blocks the induction of anti-angiogenic factors[J].Oncogene,2020,39(29):5228-5239.