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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 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.

SUBMITTER: Cheray M

PROVIDER: S-EPMC7041276 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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

Cheray Mathilde M Etcheverry Amandine A Jacques Camille C Pacaud Romain R Bougras-Cartron Gwenola G Aubry Marc M Denoual Florent F Peterlongo Pierre P Nadaradjane Arulraj A Briand Joséphine J Akcha Farida F Heymann Dominique D Vallette François M FM Mosser Jean J Ory Benjamin B Cartron Pierre-François PF

Molecular cancer 20200225 1

<h4>Background</h4>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).<h4>Methods</h4>RNA immunoprecipitation with the anti-5methylcytosine (5mC) antibody followed by Array, ELISA, dot blot, incorporat ...[more]

PMID: 32098627

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Project description:PurposeHOXD10 is a tumor modulator that can either be a tumor-suppressor or a tumor-promoting gene. However, the role of HOXD10 in glioblastoma multiforme (GBM) remains unclear.MethodsImmunohistochemistry (IHC) was applied to detect protein expression of HOXD10 in GBM and normal brain tissue patients. Clinicopathological characteristics with GBM were recorded, and a Kaplan-Meier curve was plotted. Additionally, the mRNA expression of HOXD10 and its effect on prognosis were analyzed using the online tool GEPIA and the Cancer Genome Atlas (TCGA), Chinese Glioma Genome Atlas (CGGA), and the Gene Expression Omnibus (GEO) databases. Based on the mRNA expression of HOXD10, GBM patients from TCGA database were divided into low- and high-HOXD10 expression groups to analyze the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and construct a lncRNA-miRNA-mRNA network and a protein-protein interaction (PPI) network.ResultsThe mRNA expression of HOXD10 was up-regulated in GBM according to GEPIA, while the protein expression of HOXD10 in GBM was down-regulated according to IHC analysis of samples from patients collected from our hospital. Correlation analysis showed that HOXD10 expression was significantly related to IDH1 status. Univariate analysis revealed that low HOXD10 expression, complete surgical resection, postoperative radiotherapy, postoperative temozolomide chemotherapy and IDH1 mutation were all beneficial prognostic factors. Further multivariate analysis revealed that only complete surgical resection and postoperative radiotherapy were independent prognostic factors. GO and KEGG enrichment analyses indicated that HOXD10 expression is mainly involved in cytokine-cytokine receptor interactions. In the ceRNA network, 89 nodes, containing 45 mRNAs, 39 miRNAs and five lncRNAs associated with prognosis were involved. The PPI network revealed a tight interaction between HOXD10 and HOXD8, HOXD9, HOXD11, HOXD13 and HOXB3.ConclusionBased on our experimental data, although HOXD10 expression is low in GBM compared with normal brain tissue, GBM patients with high HOXD10 expression have a worse prognosis. HOXD10 may play different or even opposite roles in different stages of GBM occurrence and development. For patients with GBM, HOXD10 may be a valid predictor of prognosis.

Project description:BACKGROUND:Lycorine has been revealed to inhibit the development of many kinds of malignant tumors, including glioblastoma multiforme (GBM). Although compelling evidences demonstrated Lycorine's inhibition on cancers through some peripheral mechanism, in-depth mechanism studies of Lycotine's anti-GBM effects still call for further exploration. Epidermal Growth Factor Receptor (EGFR) gene amplification and mutations are the most common oncogenic events in GBM. Targeting EGFR by small molecular inhibitors is a rational strategy for GBM treatment. METHODS:The molecular docking modeling and in vitro EGFR kinase activity system were employed to identify the potential inhibitory effects of Lycorine on EGFR. And the Biacore assay was used to confirm the direct binding status between Lycorine and the intracellular EGFR (696-1022) domain. In vitro assays were conducted to test the suppression of Lycorine on the biological behavior of GBM cells. By RNA interference, EGFR expression was reduced then cells underwent proliferation assay to investigate whether Lycorine's inhibition on GBM cells was EGFR-dependent or not. RT-PCR and western blotting analysis were carried out to investigate the underlined molecular mechanism that Lycorine exerted on EGFR itself and EGFR signaling pathway. Three different xenograft models (an U251-luc intracranially orthotopic transplantation model, an EGFR stably knockdown U251 subcutaneous xenograft model and a patient-derived xenograft model) were performed to verify Lycorine's therapeutic potential on GBM in vivo. RESULTS:We identified a novel small natural molecule Lycorine binding to the intracellular EGFR (696-1022) domain as an inhibitor of EGFR. Lycorine decreased GBM cell proliferation, migration and colony formation by inducing cell apoptosis in an EGFR-mediated manner. Furthermore, Lycorine inhibited the xenograft tumor growths in three animal models in vivo. Besides, Lycorine impaired the phosphorylation of EGFR, AKT, which were mechanistically associated with expression alteration of a series of cell survival and death regulators and metastasis-related MMP9 protein. CONCLUSIONS:Our findings identify Lycorine directly interacts with EGFR and inhibits EGFR activation. The most significant result is that Lycorine displays satisfactory therapeutic effect in our patient-derived GBM tumor xenograft, thus supporting the conclusion that Lycorine may be considered as a promising candidate in clinical therapy for GBM.

Dataset Information

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

Publications

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

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