BACKGROUND Literature reports that mature microRNA (miRNA) can be methylated at adenosine, guanosine and cytosine. However, the molecular mechanisms involved in cytosine methylation of miRNAs have not yet been fully elucidated. Here we investigated the biological role and underlying mechanism of cytosine methylation in miRNAs in glioblastoma multiforme (GBM). METHODS RNA immunoprecipitation with the anti-5methylcytosine (5mC) antibody followed by Array, ELISA, dot blot, incorporation of a radio-labelled methyl group in miRNA, and miRNA bisulfite sequencing were perfomred to detect the cytosine methylation in mature miRNA. Cross-Linking immunoprecipiation qPCR, transfection with methylation/unmethylated mimic miRNA, luciferase promoter reporter plasmid, Biotin-tagged 3'UTR/mRNA or miRNA experiments and in vivo assays were used to investigate the role of methylated miRNAs. Finally, the prognostic value of methylated miRNAs was analyzed in a cohorte of GBM pateints. RESULTS Our study reveals that a significant fraction of miRNAs contains 5mC. Cellular experiments show that DNMT3A/AGO4 methylated miRNAs at cytosine residues inhibit the formation of miRNA/mRNA duplex and leading to the loss of their repressive function towards gene expression. In vivo experiments show that cytosine-methylation of miRNA abolishes the tumor suppressor function of miRNA-181a-5p miRNA for example. Our study also reveals that cytosine-methylation of miRNA-181a-5p results is associated a poor prognosis in GBM patients. CONCLUSION Together, our results indicate that the DNMT3A/AGO4-mediated cytosine methylation of miRNA negatively.

中文翻译：

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成熟microRNA的胞嘧啶甲基化会抑制其功能，并与多形性胶质母细胞瘤的预后不良有关。

背景技术文献报道，成熟的微小RNA（miRNA）可以在腺苷，鸟苷和胞嘧啶上被甲基化。但是，尚未完全阐明与miRNA胞嘧啶甲基化有关的分子机制。在这里，我们调查了胶质母细胞瘤（GBM）中miRNA中胞嘧啶甲基化的生物学作用和潜在机制。方法采用抗5甲基胞嘧啶（5mC）抗体进行RNA免疫沉淀，然后进行Array，ELISA，斑点印迹，在miRNA中掺入放射性标记的甲基以及进行miRNA亚硫酸氢盐测序，以检测成熟miRNA中的胞嘧啶甲基化。交联免疫沉淀qPCR，用甲基化/未甲基化的模拟miRNA转染，荧光素酶启动子报告质粒，生物素标记的3' 使用UTR / mRNA或miRNA实验和体内测定法研究甲基化miRNA的作用。最后，在一组GBM患者中分析了甲基化miRNA的预后价值。结果我们的研究表明，很大一部分miRNA含有5mC。细胞实验表明，胞嘧啶残基处的DNMT3A / AGO4甲基化miRNA抑制了miRNA / mRNA双链体的形成，并导致其对基因表达的抑制功能丧失。体内实验表明，miRNA的胞嘧啶甲基化消除了例如miRNA-181a-5p miRNA的肿瘤抑制功能。我们的研究还显示，miRNA-181a-5p结果胞嘧啶甲基化与GBM患者预后不良有关。结论在一起，我们的结果表明DNMT3A / AGO4介导的miRNA胞嘧啶甲基化呈阴性。