EPCR基因mRNA在人胃癌细胞系中的表达及其shRNA干扰载体内源性筛靶研究  

Expression of human EPCR mRNA in gastric cancer cell lines and the endogenous target of their short hairpin RNA plasmids

作　　者：刘庆茹 王庆苓[2] 嵇玮嘉 吴永平[2] 

机构地区：[1]徐州医学院附属医院病理科,江苏徐州221002 [2]徐州医学院病理教研室,江苏徐州221002

出　　处：《中国现代医学杂志》2014年第26期1-6,共6页China Journal of Modern Medicine

基　　金：国家自然科学青年基金(No:81101493)

摘　　要：目的检测人胃癌细胞株中内皮细胞蛋白C受体(endothelial protein creceptor,EPCR)基因的mRNA表达水平,构建EPCR基因特异的短发夹RNA(shRNA)干扰载体,并通过内源性筛靶实验检测EPCR mRNA表达,筛选出最佳的干扰靶序列。方法通过逆转录-聚合酶链反应(RT-PCR)及蛋白免疫印迹(Western blot)检测胃癌细胞系(SGC7901、MGC803、HGC27、AGS)中EPCR mRNA和蛋白的表达,筛选出EPCR高表达的细胞株;设计5对EPCR基因特异性短发夹RNA(shRNA)干扰片段pGPH1/GFP/Neo-EPCR,用Lipofectamine 2000将pGPH1/GFP/Neo-EPCR转染入EPCR高表达的人胃癌细胞,以pGPH1/GFP/Neo空载体作对照;转染后通过观察绿色荧光蛋白来判断转染的效率,选出最优质粒/脂质体比;转染后收集样本,采用RT-PCR及Western blot进行检测,筛选出有效的干扰片段。结果 EPCR在MGC803细胞中表达量最高;最佳质粒:脂质体比值为1μg︰1μL;pGPH1/GFP/Neo-EPCR-homo-555为最有效的干扰质粒。结论成功筛选出针对EPCR基因特异的shRNA干扰载体,转染细胞后可下调EPCR的表达,为进一步研究EPCR的功能和肿瘤的基因治疗奠定了基础。[ Objectives] To detect the expression of EPCR mRNA in gastric cancer cell lines and con- struct the vector of a short hairpin RNA （shRNA）, to select the best RNAi target sequence through detecting the expression of EPCR mRNA in the endogenous target experiment. [Methods] The expression of EPCR mRNA and protein were detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western Blot in several gastric cancer cell lines including SGC7901, MGCS03, HGC27 and AGS to select the highest expression cell line. Five pairs of EPCR shRNA were designed and then transfected by liposome into the highest expression gastric cancer cell line with the empty one as control. The transfection efficiency of plas- mids was analyzed by observing the fluorescence amount. And select the best proportion of plasmid to lipo- some. After transfection, RT-PCR and Western Blot were used to select the effective interference fragment. [Results] MGCS03 is the highest expression level of EPCR mRNA among the above cell lines. The best proportion of plasmid to liposome is 1 μg/1 μL. The best efficiency sequence is pGPH1/GFP/Neo-EPCR-ho- mo-555. [Conclusion] Plasmid vector expressing small hairpin RNA against EPCR can be successfully pro-duced and it can down-regulate EPCR expression after transfection into cells, which will lay a foundation for further studies of EPCR function and its application in tumor gene therapy.

关 键 词：内皮细胞蛋白C受体(EPCR)基因 胃癌细胞 短发夹RNA(shRNA) 转染条件 

分 类 号：R735.2[医药卫生—肿瘤] Q782[医药卫生—临床医学]