Kozak序列对TIMP1基因在人乳腺癌细胞MCF-7中表达的影响  

Effect of Kozak Sequence on Expression of Gene Encoding Tissue Inhibitor 1 of Metalloproteinase in Human Breast Cancer MCF-7 Cells

作　　者：马艳娇[1,2] 李鹏[1,2] 周海峰[2] 阮楠[2] 宫鹏涛[2] 李建华[2] 欧阳红生[2] 张西臣[2] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]吉林大学畜牧兽医学院,长春130062

出　　处：《中国生物制品学杂志》2010年第7期687-691,共5页Chinese Journal of Biologicals

基　　金：国家重大基础研究计划资助项目(2006CB910505);黑龙江省研究生创新科研项目(YHSCX2009-131HLJ)

摘　　要：目的探讨Kozak序列对基质金属蛋白酶抑制剂1(TIMP1)基因在人乳腺癌细胞MCF-7中表达的影响。方法构建pcDNA3.1-TIMP1和pcDNA3.1-TIMP1-K(含有Kozak序列)重组真核表达质粒,用FugeneHD转染试剂将重组表达质粒转染MCF-7细胞,采用荧光定量PCR和Western blot检测pcDNA3.1-TIMP1和pcDNA3.1-TIMP1-K在MCF-7细胞中表达的差异。结果重组真核表达质粒经PCR、双酶切及测序鉴定,证明构建正确。质粒pcDNA3.1-TIMP1-K转染细胞比空白对照细胞TIMP1基因mRNA表达量高约0.95倍,蛋白表达量高0.43倍;而质粒pcDNA3.1-TIMP1转染细胞比空白对照细胞mRNA表达量高0.37倍,蛋白表达量高0.25倍。结论质粒pcDNA3.1-TIMP1-K与pcDNA3.1-TIMP1在MCF-7细胞中mRNA和蛋白表达水平均存在差异,Kozak序列提高了TIMP1基因的表达。Objective To investigate the effect of Kozak sequence on the expression of gene encoding tissue inhibitor 1 of metalloproteinase（TIMP1） in human breast cancer MCF-7 cells.Methods Recombinant plasmids pcDNA3.1-TIMP1 and pcDNA3.1-TIMP1-K containing Kozak sequence were constructed then transfected to MCF-7 cells using Fugene HD transfection agent,and their expressions were determined by fluorescent quantitative PCR and Western blot.Results PCR,restriction analysis and sequencing proved that recombinant plasmids pcDNA3.1-TIMP1 and pcDNA3.1-TIMP1-K were constructed correctly.The transcription level of TIMP1 mRNA and the expression level of TIMP1 protein in the MCF-7 cells transfected with pcDNA3.1-TIMP1-K increased by about 0.95 and 0.43 folds,while those in the MCF-7 cells transfected with pcDNA3.1-TIMP1 increased by 0.37 and 0.25 folds,respectively,as compared with those in blank control cells.Conclusion The expressions of recombinant plasmids pcDNA3.1-TIMP1 and pcDNA3.1-TIMP1-K in MCF-7 cells were significantly different at both mRNA and protein levels,indicating that Kozak sequence enhanced the expression of TIMP1 gene.

关 键 词：KOZAK序列 基质金属蛋白酶 基质金属蛋白酶抑制剂1 乳腺肿瘤 MCF-7细胞 

分 类 号：Q786[生物学—分子生物学] R737.9[医药卫生—肿瘤]