摘要
目的:筛选上调尾型同源盒基因2(caudal type homeobox gene 2,CDX2)基因表达时人的胃癌细胞SGC-7901中差异表达的miRNA并预测其靶基因,并观察其对胃癌细胞生物学行为的影响.方法:提取瞬时转染48 h后空载体组(转染P E G F P-N1组)和转染组(转染P E G F P-N1-C D X2组)细胞的总R N A,应用m i R N A芯片检测差异表达的miRNA,并通过Miranda、TargetScan、Mirtarget2软件预测其靶基因,通过Gene Ontology(GO)和KEGG pathway分析了解靶基因功能.CCK8法检测细胞增殖能力,划痕试验、Transwell小室、细胞黏附实验检测各组细胞体外迁移黏附能力.结果:PEGFP-N1-CDX2组较空载体组有59种差异表达的miRNA,其中25种miRNA发生2倍以上表达上调,34种miRNA发生2倍以上表达下调.通过Gene Ontology(GO)和KEGG pathway分析得到部分miRNA靶基因功能,这些靶基因参与了肿瘤的发生、发展、转移及预后.与对照组相比,PEGFP-N1-CDX2组的胃癌细胞生长、迁移和黏附能力均明显受到抑制(P<0.05).结论:上调CDX2基因表达可明显抑制人胃癌细胞SGC-7901的生长,抑制其迁移黏附能力,CDX2基因的抗肿瘤作用可能与miRNA有关.
AIM： To screen differentially expressed miR- NAs in human gastric carcinoma cells with up- regulated caudal type homeobox gene 2 （CDX2） expression and to analyze their effect on biologi- cal behavior of cells.METHODS： A recombinant eukaryotic expres- sion plasmid carrying the CDX2 gene was con- structed. SGC-7901 cells were divided into three groups： non-transfected cells, cells transfected with PEGFP-N1 or PEGFP-N1-CDX2. The ex- pression of GFP was observed by fluorescent microscopy. Expression of CDX2 mRNA and EGFP-CDX2 fusion protein was detected by qRT-PCR and Western blot, respectively. Cell proliferation was measured using CCK8 assay. In vitro cell migration and adhesion were mea- sured by cell scratch assay, Transwell assay and cell adhesion assay. Differentially expressed miRNAs were detected using a miRNA chip, and their target genes were forecasted using Mi- randa, TargetScan and Mirtarget2 software. RESULTS： The eukaryotic expression vector PEGFP-N1-CDX2 was successfully constructed. The expression levels of CDX2 mRNA and protein were higher in SGC-7901 transfected with PEGFP-N1-CDX2 than in control cells. In SGC-7901 cells transfected with PEGFP-N1- CDX2, cell proliferation, invasion and adhesion were significantly inhibited （all P 〈 0.05） com- pared with non-transfected cell or cells transfect- ed with PEGFP-N1. Of 59 identified differential- ly expressed miRNAs between cells transfected PEGFP-N1-CDX2 and those transfected with PEGFP-N1, 25 had 〉 2-fold up-regulation and 34 had 〉 2-fold down-regulation in cells transfected with PEGFP-N1-CDX2. Many target genes of these differentially expressed miRNAs were pre- dicted using miRNA target predication tools. CONCLUSION： Up-regulation of CDX2 expres- sion inhibits the growth, migration and adhesion of SGC-7901 cells. The antitumor effect of CDX2 may be associated with miRNA expression.
出处
《世界华人消化杂志》
CAS
北大核心
2013年第23期2241-2249,共9页
World Chinese Journal of Digestology
