microRNA expression signatures in colorectal cancer cell lines with demethylating treatment. Homo sapiens

To screen for epigenetically silenced miRNAs, wecarried out miRNA microarray analysis in three colorectal cancer (CRC) cell lines (HCT116, DLD-1 and RKO) treated with or without 5-aza-2'-deoxycytidine (aza). HCT116 and RKO cells were also treated with aza plus 4-phenylbutyric acid (PBA). In addition, we analyzed HCT116 cells in which the DNA methyltransferase genes DNMT1 and DNMT3B were genetically disrupted (double knockout; DKO cells), thereby abrogating DNA methylation. Expression of a majority of miRNAs was downregulated in all three CRC cell lines tested, as compared to normal colonic mucosa. DAC treatment upregulated expression of a large number of miRNAs in all three CRC cell lines, and combination treatment with DAC plus PBA induced even greater numbers of miRNAs in CRC cells. The most profound effect on the miRNA expression profile was induced by genetic disruption of DNMT1 and DNMT3B in HCT116 cells. Overall design: CRC cells were treated with 5-aza-2’-deoxycytidine (aza) or aza plus 4-phenylbutyrate (PBA). Nomal colon RNA was purchased from BioChain. Expression of 470 miRNAs was analyzed using Human miRNA Microarray V1 (G4470A; Agilent technologies, Santa Clara, CA, USA).

为筛选表观遗传沉默的微小RNA（miRNAs），我们对经5-氮杂-2'-脱氧胞苷（5-aza-2'-deoxycytidine，简称aza）处理或未处理的三株结直肠癌（CRC）细胞系（HCT116、DLD-1及RKO）开展了miRNA芯片分析。HCT116与RKO细胞还同时接受了aza联合4-苯基丁酸（4-phenylbutyric acid，简称PBA）的处理。此外，我们对DNA甲基转移酶基因DNMT1和DNMT3B发生基因敲除的HCT116细胞（双敲除细胞；DKO细胞）进行了分析，该细胞系的DNA甲基化功能已被完全消除。相较于正常结肠黏膜，本研究检测的三株结直肠癌细胞系中绝大多数miRNA的表达均出现下调。5-氮杂-2'-脱氧胞苷处理可上调三株结直肠癌细胞中大量miRNA的表达，而aza联合PBA处理则可诱导结直肠癌细胞中更多miRNA的表达上调。对HCT116细胞中DNMT1和DNMT3B进行基因敲除，对miRNA表达谱产生的影响最为显著。总体实验设计如下：结直肠癌细胞经5-氮杂-2'-脱氧胞苷（aza）或aza联合4-苯基丁酸（PBA）处理。正常结肠RNA购自BioChain。本研究采用人类miRNA芯片V1（G4470A；安捷伦科技公司，美国加利福尼亚州圣克拉拉市）对470种miRNA的表达水平进行了检测。