Cytosine methylation of mature microRNAs inhibits their functions and is associated with poor prognosis in glioblastoma multiforme

Abstract

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 cytosinemethylation 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.

Advances in RNA cytosine-5 methylation: detection, regulatory mechanisms, biological functions and links to cancer

“…miRNAs are also common m 5 C substrates, and the methylation of mature miR-181a-5p results in the abolishment of its tumor suppressor effect and is negatively correlated with poor prognosis in GBM. Mechanistically, mediated by the complex comprising DNMT3a and AGO4 (a miRNA-induced silencing complex), miR-181a-5p methylation impedes the formation of the miRNA-181a-5p/mRNA BIM duplex, which was originally defined to induce apoptosis by interacting with antiapoptotic Bcl-2 or Bcl-xl [14]. As a result, reduced apoptosis, as well as enhanced proliferation and invasion, was observed in cancer cells.…”

“…As a result, reduced apoptosis, as well as enhanced proliferation and invasion, was observed in cancer cells. In addition, as estimated by Kaplan-Meier analysis, the methylation level of miR-181a-5p is associated with a poor survival rate, suggesting the profound potential for attenuating m 5 C methylation and restoring normal miRNA function for cancer treatment [14].…”

“…With the advances in RNA m 5 C detection techniques, including bisulfite sequencing, m 5 C RNA immunoprecipitation sequencing (m 5 C-RIP-seq), 5-AZAcytidinemediated RNA immunoprecipitation sequencing (Aza-IP-seq) and methylation-individual nucleotide resolution crosslinking immunoprecipitation sequencing (miCLIPseq), over 10,000 potential sites of m 5 C modification were detected within the whole human transcriptome [7], and the existence of m 5 C was found in diverse RNA species from various organisms, not only in ribosomal RNA (rRNA), messenger RNA (mRNA) and transfer RNA (tRNA) but also in viral RNA, vault RNA, small nuclear RNA, small nucleolar RNA, pseudogenes, natural antisense transcripts, enhancer RNA, long noncoding RNA (lncRNA) and microRNA (miRNA) [8][9][10][11][12][13][14][15]. The distribution of m 5 C differs among different organisms, as it has been common to find m 5 C methylation substrates in the tRNA and mRNA of eukaryotes and archaea, while no m 5 C methylation substrates have been detected in bacteria [16].…”

“…The alteration of m 5 C modification as well as its regulators has been proven to influence RNA stability, gene expression, and protein synthesis and thus has a great impact on various cellular and physical processes [15,[17][18][19][20][21][22]. Additionally, to date, RNA m 5 C modification and its regulators have been confirmed to play crucial roles in the pathogenesis of bladder cancer [23], hepatocellular carcinoma (HCC) [24], glioblastoma multiforme (GBM) [14] and leukemia [18], indicating the promising prospect of m 5 C modification in cancer treatment.…”

Construction of circRNA-based ceRNA Network to Reveal the Role of circRNAs in the Progression and Prognosis of Hepatocellular Carcinoma

“…The results showed that the transcription level of microRNAs was related to the tolerance and effectiveness of immunotherapy. Research of Cheray et al showed that many members of miRNA contain 5mc, and some fragments of cytosine residues could methylate miRNA, and inhibit the formation of miRNA/mRNA duplexes, resulting in the loss of the duplex's function of suppressing gene expression, and further led to the occurrence of glioblastoma multiforme (GBM) [22]. Explaining the interaction mechanism of miRNA and exosomes is also a hot topic.…”