结直肠癌中AEG-1对血管生成相关因子HIF-1α和miR-34a的影响  

AEG-1 affects expression of HIF-1α and miR-34a in colorectal cancer cells

作　　者：黄素军 吴斌文[2] 李东风[2] 刘宝龙[1,2] 邓罡[2] 张凯军[2] 

机构地区：[1]南方医科大学,广东省广州市510515 [2]广东省人民医院广东省医学科学院,广东省广州市510080

出　　处：《世界华人消化杂志》2014年第18期2532-2538,共7页World Chinese Journal of Digestology

基　　金：广东省自然科学基金资助项目;No.10151008008000005;广东省科技计划基金资助项目;Nos.2011B031800001;2011B031800004~~

摘　　要：目的:构建携带星形胶质细胞升高基因-1(astrocyte elevated gene-1,AEG-1)的慢病毒载体并进行慢病毒包装,感染结直肠癌SW1116细胞建立稳定株,检测AEG-1改变后对血管生成相关因子缺氧诱导因子-1α(hypoxiainducible factor-1α,HIF-1α)和miR-34a表达的影响.方法:采用Western blot方法筛选合适的结直肠癌细胞株;构建携带AEG-1的慢病毒载体,经PCR筛选阳性克隆及测序鉴定后,用psPAX、pMD2.G慢病毒包装系统共转染包装细胞293FT,包装产生慢病毒,用以感染筛选出来的结肠癌细胞,细胞提取mRNA和蛋白进行qRT-PCR和Western blot分析验证细胞模型建立是否成功.分别采用qRT-PCR和Western blot检测miR-34a和HIF-1α在AEG-1转染前后的表达变化情况.结果:蛋白质印迹结果显示SW1116在7个结直肠癌中的表达量最低(P<0.05);成功构建AEG-1的慢病毒载体,对结肠癌SW1116细胞株AEG-1表达的上调作用显著(0.53±0.44vs 2.02±0.22,P<0.05;0.71±0.14 vs 2.02±0.22,P<0.05),上调AEG-1的表达会抑制miR-34a(P<0.01)和促进HIF-1α(P<0.01)的表达.结论:成功构建AEG-1慢病毒载体,AEG-1在结直肠癌中可以抑制血管生成相关因子miR-34a和促进HIF-1α的表达,我们推测AEG-1可能通过抑制miR-34a促进HIF-1α的表达,从而发挥促进肿瘤血管新生的作用.AIM： To construct a stable expression system for astrocyte elevated gene-1（AEG-1） using lentiviral vector in colorectal cancer（CRC） cells and assess its relationship with miR-34a and hypoxiainducible factor-1α（HIF-1α）. METHODS： AEG-1 expression in CRC cells was examined by Western blot analysis. A len-tiviral vector carrying AEG-1 was constructed, identified by PCR and DNA sequencing, and then transfected into 293 FT cells using lentiviral packaging systems. SW1116 cells were infected with the virus and analyzed by qRT-PCR and Western blot. Expression of miR-34a and HIF-1α was detected using qRT-PCR and Western blot.RESULTS： Western blot analysis showed that AEG-1 expression was the lowest in SW1116 among 7 CRC cell lines（P〈0.05）. AEG-1 lentiviral vector was constructed successfully as revealed by DNA sequencing. The stable expression of AEG-1 was validated by qRT-PCR and Western blot（0.53 ± 0.44 vs 2.02 ± 0.22, P〈0.05; 0.71 ± 0.14 vs 2.02 ± 0.22, P〈0.05）. Up-regulation of AEG-1 inhibited miR-34a expression（P〈0.01） but increased HIF-1α expression（P〈0.01）. CONCLUSION： An AEG-1 lentiviral vector has been successfully constructed. AEG-1 can downregulate the expression of miR-34a and upregulate the expression of HIF-1α.

关 键 词：星形胶质细胞升高基因-1 MIR-34A 缺氧诱导因子-1Α 慢病毒 结直肠癌 

分 类 号：R735.34[医药卫生—肿瘤]