Abstract

MicroRNA-219-1 (miR-219-1) acts as a tumor suppressor in a variety of cancers but, the regulatory epigenetic mechanism involved in its gene expression level has not been studied. Using real-time polymerase chain reaction (real-time PCR) and bisulfite genomic sequencing technology, promoter methylation level of miR-219-1 and gene expression levels of miR-219-5p and miR-219-1-3p were determined respectively, in glioblastoma multiforme (GBM) (n = 31), their adjacent normal tissues (n = 31), and GBM U87 cell line. Following treatment of GBM U87 cells with 5-aza-2′-deoxycitidine (5-aza-dC), miR-219-1 promoter methylation, their target mRNA, and protein levels were determined by genomic bisulfite modification, real-time-PCR, and ELISA techniques, respectively. Our results showed that gene expression levels of miR-219-5p and miR-219-1-3p were significantly lower in GBM patients relative to their adjacent normal tissues (p < 0.01). MiR-219-1 promoter had a high level of methylation in GBM tissues (p < 0.01) and a negative correlation was observed between the miRNAs gene expression and methylation levels in GBM tissues (p < 0.01). Treatment of GBM U87 cells by 5-aza-dC decreased the level of miR-219-1 methylation, amount of target mRNA, and levels of cyclin A2 and mucin 4 (MUC4) proteins, and increased the expression levels of miR-219-5p and miR-219-1-3p (p < 0.01). Using external miR-219-5p and miR-219-1-3p, the expression of cyclin A2 and MUC4 were suppressed and proliferative activity of the U87MG cell line was reduced (p < 0.01). These findings suggested that DNA methylation has a crucial role in the regulation of miR-219-1 gene expression and that hypermethylated miR-219-1 may be involved in GBM pathogenesis.

中文翻译：

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MicroRNA-219-1的表观遗传修饰及其与多形性胶质母细胞瘤的关联

摘要

MicroRNA-219-1（miR-219-1）在多种癌症中起着抑癌作用，但尚未研究涉及其基因表达水平的调控表观遗传机制。使用实时聚合酶链反应（实时PCR）和亚硫酸氢盐基因组测序技术，分别确定了miR-219-1的启动子甲基化水平和miR-219-5p和miR-219-1-3p的基因表达水平，在多形性胶质母细胞瘤（GBM）中（n = 31），其相邻的正常组织（n= 31），以及GBM U87细胞系。用5-氮杂-2'-脱氧胞苷（5-氮杂-dC）处理GBM U87细胞后，通过基因组亚硫酸氢盐修饰，实时PCR确定miR-219-1启动子甲基化，其靶mRNA和蛋白质水平和ELISA技术。我们的结果表明，GBM患者的miR-219-5p和miR-219-1-3p的基因表达水平相对于其相邻的正常组织明显降低（p <0.01）。MiR-219-1启动子在GBM组织中具有较高的甲基化水平（p <0.01），并且在miRNA基因表达与GBM组织中的甲基化水平之间存在负相关性（p<0.01）。5-氮杂-dC处理GBM U87细胞可降低miR-219-1甲基化水平，靶mRNA量以及细胞周期蛋白A2和粘蛋白4（MUC4）蛋白水平，并增加miR-219-mRNA的表达水平。 5p和miR-219-1-3p（p <0.01）。使用外部miR-219-5p和miR-219-1-3p，可抑制细胞周期蛋白A2和MUC4的表达，并降低U87MG细胞系的增殖活性（p <0.01）。这些发现表明，DNA甲基化在miR-219-1基因表达的调节中起着至关重要的作用，而高甲基化的miR-219-1可能与GBM发病有关。