5HRE联合CEAp靶向调控RASSF1A基因对SGC7901胃癌细胞株抑制作用的研究  

Inhibition Effect on Expression of RASSF1A Gene by 5HRE Combined with CEAp Element Targeted Regulation for Human Gastric Cancer SGC7901 Cells

作　　者：周培华[1] 孙学军[1] 郑见宝[1] 王炜[1] 王孝珑 陈南征[1] 魏光兵[1] 范渊[1] 李孝彬 王训凯 禄韶英[1] 

机构地区：[1]西安交通大学第一附属医院普外科,陕西西安710061

出　　处：《中国普外基础与临床杂志》2015年第10期1201-1208,共8页Chinese Journal of Bases and Clinics In General Surgery

基　　金：国家自然科学基金项目(项目编号:81172362);陕西省科技统筹创新工程计划项目(项目编号:2013KTCQ03-08)~~

摘　　要：目的探讨人5拷贝低氧反应元件(5HRE)联合癌胚抗原启动子(CEAp)调控元件的靶向性及调控RAS相关区域家族1A(RASSF1A)抑癌基因的慢病毒载体对SGC7901胃癌细胞的抑制作用。方法 1分别采用实时定量RT-PCR(qRT-PCR)、免疫组织化学SP染色及Western blot法检测SGC7901、MKN28和MCF-10A细胞株中癌胚抗原(CEA)mRNA及其蛋白,以及RASSF1A蛋白的表达水平,以确定实验细胞和阴性对照细胞。2构建p GL4.20-5HRE-CEAp-Luc载体以转染SGC7901、MKN28和MCF-10A细胞株,将各细胞株分为2组,分别加入(缺氧组)或不加入CoCl2(常氧组),比较各细胞株中缺氧组和常氧组细胞的启动活性。3将重组慢病毒载体(p LV-5HRE-CEAp-RASSF1A)及相应空病毒载体包装成病毒颗粒LV-5HC-RASSF1A(感染组)和LV-NC(阴性对照组),感染SGC7901细胞。以未感染任何病毒的SGC7901细胞作为空白对照组,将该3组细胞再分为2个亚组,分别加入(缺氧组)或不加入CoCl2(常氧组),比较缺氧组和常氧组细胞中RASSF1A蛋白的表达水平和细胞增殖抑制率。结果 1 q RT-PCR结果显示:SGC7901细胞株中CEA m RNA的表达水平高于MKN28及MCF-10A细胞株(P<0.05);免疫组织化学染色结果显示:CEA的表达水平在SGC7901、MKN28及MCF-10A细胞株中依次递减,两两比较差异均有统计学意义(P<0.05);Western blot结果显示:在SGC7901及MKN28细胞株中未检测到RASSF1A蛋白的电泳条带,而在MCF-10A细胞株中可检测到。据此确定SGC7901细胞株为实验细胞,MKN28细胞株为阴性对照细胞。2转染p GL4.20-5HRE-CEAp-Luc载体后,在SGC7901及MKN28细胞株中,同种细胞株内与常氧组比较,缺氧组细胞的活性倍数均升高(P<0.01);而在MCF-10A细胞株中,常氧组和缺氧组细胞的活性倍数比较差异无统计学意义(P>0.05)。在不加入CoCl2条件和加入CoCl2条件下,同等条件下与SGC7901细胞株比较,MKN28及MCF-10A细胞株的活性倍数均较低(P<0.05);与MKN28细胞株比较,MCF-10A细胞株的活性倍数�Objective To explore the effect of five copies hypoxia-responsive element （5HRE） and carcinoembr- yonic antigen promoter （CEAp） element, and to explore the inhibition effect of lentiviral vectors targeted RAS association domain family I isoform A （RASSFIA） gene on SGC7901 human gastric cancer cells. Methods （1）Expressions of carcinoem- bryonic antigen （CEA） mRNA and its protein, and RASSF1A protein in SGC7901, MKN28, and MCF-10A cells were detect by real time-PCR （qRT-PCR）, immunocytochemistry, and Western blot, to confirm the experimental and negative control cells. （2）The recombinant vectors of pGL4.20-5HRE-CEAp-Luc were constructed through molecular cloning technique to transfected SGC7901, MKN28, and MCF-10A cells. Each kind of cell was divided into 2 groups： one of them didn＇t add CoC12 （normoxia group）, and another group added COC12 （hypoxia group）. Comparison of the fold of activation was performed. （3）SGC7901 cells were infected by lentiviral vectors of pLV-5HRE-CEAp-RASSF1A （infection group） and negative virus （negative control group）, SGC7901 cells without any treatment as blank control group. Then SGC7901 cells of 3 groups were divided into 2 groups： one of them didn＇t add COC12 （normoxia group）, and another group added COC12 （hypoxia group）. The expression of RASSFIA protein was tested by Western blot, and the growth inhibition rate was confirmed by cell counting Kit-8 （CCK-8） assay. Comparisons of expression of RASSFIA protein and growth inhibition rate of each group were performed. Results （1） Results of qRT-PCR, immunocytochemistry and Western blot showed that, SGC7901 cells showed higher expression of CEA mRNA and positive expression of RASSF1A protein than corresponding index of MKN28 cells and MCF-10A cells （P〈0.05）, which were assigned as experimental cells; but MKN28 cells showed lower expression of CEA mRNA and negative expression of RASSF 1A protein, which were assigned as negative control cells. （2）In SGC7901

关 键 词：5拷贝低氧反应元件 癌胚抗原启动子 RAS相关区域家族1A基因 胃癌 基因治疗 

分 类 号：R735.2[医药卫生—肿瘤]