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Mol Cancer. 2020 Feb 25;19(1):36. doi: 10.1186/s12943-020-01155-z.

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

Cheray M1,2,3, Etcheverry A4,5,6,7, Jacques C8, Pacaud R1,2,9,10, Bougras-Cartron G1,9,11,12, Aubry M4,5,6, Denoual F7, Peterlongo P13, Nadaradjane A1,2,11,12, Briand J1,2,9,11,12, Akcha F12,14, Heymann D1,2,9, Vallette FM1,2,9,10, Mosser J4,5,6,7,11, Ory B8,11,12, Cartron PF15,16,17,18,19,20,21.

Author information

1
CRCINA, INSERM, Université de Nantes, Nantes, France.
2
Faculté de Médecine, Université de Nantes, Nantes, France.
3
Present address: Department of Oncology-Pathology, Cancer Centrum Karolinska (CCK), R8:03, Karolinska Institutet, SE-171 76, Stockholm, Sweden.
4
CNRS, UMR 6290, Institut de Génétique et Développement de Rennes (IGdR), F-35043, Rennes, France.
5
Université Rennes1, UEB, UMS 3480 Biosit, Faculté de Médecine, F-35043, Rennes, France.
6
Plate-forme Génomique Environnementale et Humaine Biosit, Université Rennes1, F-35043, Rennes, France.
7
CHU Rennes, Service de Génétique Moléculaire et Génomique, F-35033, Rennes, France.
8
INSERM, UMR 1238, équipe labellisée ligue 2012, 1 Rue Gaston Veil, 44035, Nantes, France.
9
LaBCT, Institut de Cancérologie de l'Ouest, Saint Herblain, France.
10
LabEx IGO "Immunotherapy, Graft, Oncology", Nantes, France.
11
Cancéropole Grand-Ouest, réseau Epigénétique (RepiCGO), Nantes, France.
12
EpiSAVMEN, Epigenetic consortium Pays de la Loire, Nantes, France.
13
IRISA Inria Rennes Bretagne Atlantique, équipe GenScale, Campus de Beaulieu, 35042, Rennes, France.
14
Ifremer, Laboratoire d'Ecotoxicologie, Rue de l'Ile d'Yeu, BP21105, cedex 03 44311, . Nantes, France.
15
CRCINA, INSERM, Université de Nantes, Nantes, France. pierre-francois.cartron@inserm.fr.
16
Faculté de Médecine, Université de Nantes, Nantes, France. pierre-francois.cartron@inserm.fr.
17
LaBCT, Institut de Cancérologie de l'Ouest, Saint Herblain, France. pierre-francois.cartron@inserm.fr.
18
LabEx IGO "Immunotherapy, Graft, Oncology", Nantes, France. pierre-francois.cartron@inserm.fr.
19
Cancéropole Grand-Ouest, réseau Epigénétique (RepiCGO), Nantes, France. pierre-francois.cartron@inserm.fr.
20
EpiSAVMEN, Epigenetic consortium Pays de la Loire, Nantes, France. pierre-francois.cartron@inserm.fr.
21
Institut de Cancérologie de l'Ouest, CRCINA INSERM U1232, Equipe 9 -Apoptose et Progression tumorale, LaBCT, Boulevard du Pr J Monod, 44805, Saint-Herblain, France. pierre-francois.cartron@inserm.fr.

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.

KEYWORDS:

AGO4; Cytosine-methylation; DNMT3A; Epigenetics; Epitranscriptomics; Glioblastoma; miRNA

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