细胞体外恶性转化过程中差异甲基化基因的筛选

A genome-wide screen for promoter-specific sites of differential DNA methylation during human cell malignant transformation in vitro

目的 筛选细胞体外恶性转化过程中差异甲基化基因,为毒作用效应以及肿瘤化学预防、生物监测寻找潜在的表观遗传生物标志物.方法 利用苯并[a]芘[B（a）P]诱导水生化人支气管上皮细胞（HBER）转化模型和猿猴空泡病毒小T抗原（SV40 ST）诱导HBER转化模型,采用甲基化DNA免疫沉淀-全基因组扩增法富集基因组甲基化DNA,应用甲基化芯片技术和信息学分析方法,寻找不同作用因素诱导细胞转化的共同差异甲基化基因,通过RT-PCR法检测这些基因在B（a）P诱导HBER细胞转化过程中mRNA表达水平的变化.结果 芯片检测发现,HBER、染毒后末转化细胞株（HBERNT）和转化细胞株（HBERT）中甲基化基因的数目分别为733、661、738个,83个基因在染毒前呈非甲基化状态,而在转化前和转化后呈甲基化状态,在这83个基因中,有25个基因在SV40ST诱导HBER转化细胞株（HBERST）中同样发生高甲基化.其中,序列相似性家族178A（family with sequence similarity 178,member A,FAM178A）、视黄酸受体应答子（他扎罗汀诱导）[retinoic acid receptor responder（tazarotene induced）,RARRES1]、泛素特异性肽酶28（ubiquitin specific peptidase 28,USP28）、Scm-like with four mbt domains 2（SFMBT2）、序列相似性家族59A（family with sequence similarity 59,member A,FAM59A）和核受体亚家族4组A3（nuclear receptor subfamily 4,group A,member 3,NR4A3）等6个基因在B（a）P诱导HBER细胞转化过程中mRNA表达水平下降.结论 细胞体外转化过程中筛查出来的特定基因高甲基化,可能成为化学毒物暴露监测及肿瘤预测的生物标志物.

Objective To explore potential epigenetic biomarkers for toxic effects, tumor-related chemical prevention and biological monitor by a genome-wide screening for differential DNA methylation during human cell malignant transformation in vitro. Methods The two in vitro cell transformation models included B（ a）P-induced human bronchial epithelial cell introduced by H-Ras （HBER） cell transformation and simian vacuolating virus 40 small T antigen induced （SV40 ST-induced ） HBER cell transformation. Methylated genes were collected by methylated DNA immunoprecipitation and whole genome amplification （MeDIP-WGA） at three time points during cell transformation which represented different transformation stage. Then, CpG island microarray was used to screen differentially methylated genes. The mRN A levels of hypermethylated genes were also observed by RT-PCR. Results The CpG island microarray showed that the number of hypermethylated genes in HBER,HBERNT,HBERT cells were 733,661 and 738 respectively. 83 genes were hypermethylated in pre-transformed cell and transformed cell. Moreover, 25 of 83 genes were also hypermethylated in SV40 ST-transformed cell （HBERST）. We further confirmed that the mRNA expression of six of these 25 genes, namely family with sequence similarity 178, member A （ FAM178A ）, retinoic acid receptor responder （ tazarotene induced ） （ RARRES1 ）, ubiquitin specific peptidase 28 （USP28） ,Scm-like with four mbt domains 2 （SFMBT2）, family with sequence similarity 59,member A（FAM59A） and nuclear receptor subfamily 4 ,group A,member 3 （NR4A3） were supressed during B（a） P-induced transformation. Conclusion The abnormal hypermethylation of specific genes was a common event in the two kinds of human cell transformation models, which shed light on the study for chemical exposure monitor and tumor-related epigenetic biomarkers.