摘要
目的构建人类长链非编码RNA母系表达基因3(MEG3)的真核表达载体,筛选MEG3稳定高表达的肝癌细胞株,分析MEG3过表达对p53转录活性的影响。方法根据GenBank中MEG3的基因序列设计合成特异引物,从肝癌细胞中扩增MEG3的cDNA序列,将其构建入pcDNA3.0真核表达载体中,得到pcDNA3.0-MEG3表达载体,转染肝癌细胞SK-Hep-1,用G418筛选稳定株,采用RT-PCR技术检测转染细胞中MEG3基因表达,采用荧光素酶报告基因技术分析MEG3过表达细胞中对p53介导的基因转录活性的影响。结果成功构建了MEG3真核表达质粒pcDNA3.0-MEG3,筛选获得了稳定高表达MEG3基因的SK-Hep-1肝癌细胞株,双荧光报告实验结果显示MEG3过表达能增强p53介导的转录激活作用。结论本研究获得了稳定高表达MEG3基因的SK-Hep-1肝癌细胞株,为深入分析MEG3的功能及其作用机制奠定了基础。
ObjectiveTo construct a maternally expressed gene 3（MEG3） eukaryotic expression vector,to screen stable hepatoma cell lines with high expression of MEG3,and to analyze the effect of MEG3 overexpression on p53 transcriptional activity.MethodsA complete exon sequence based on the sequence of MEG3 in the GenBank was designed and inserted into the eukaryotic expression vector pcDNA3.0 to construct recombinant plasmid pcDNA3.0-MEG3.It was identified by sequencing and tranfected into hepatoma SK-Hep-1 cells.The SK-Hep-1 cells with stable expression of MEG3 were selected by G418 and confirmed by RT-PCR.Dual-Luciferase Reporter Assay System was used to analyze p53-mediated transcriptional activity in MEG3 overexpression cells.ResultsThe eukaryotic expression vector pcDNA3.0-MEG3 was constructed.SK-Hep-1 cells stably expressing MEG3 were selected by G418 with 1000 mg/L at 2 week.Results of Dual-Luciferase Reporter Assay suggested that overexpression of MEG3 stimulated p53-mediated transactivation.ConclusionThe SK-Hep-1 hepatocellular carcinoma cell lines are obtained with high expression of MEG3,which can facilitate further evaluation of the function and mechanism of MEG3.
出处
《军事医学》
CAS
CSCD
北大核心
2011年第6期424-427,共4页
Military Medical Sciences
基金
国家自然科学基金资助项目(30870529
30873008)
"重大新药创制"科技重大专项资助项目(2009ZX090301-002)
国家"973"计划项目(2010CB912801)
