Cytosine methylation of mature microRNAs inhibits their functions and is associated with poor prognosis in glioblastoma multiforme.
Animals
Apoptosis
Argonaute Proteins
/ genetics
Biomarkers, Tumor
/ genetics
Cell Proliferation
Cytosine
/ chemistry
DNA (Cytosine-5-)-Methyltransferases
/ genetics
DNA Methylation
DNA Methyltransferase 3A
Eukaryotic Initiation Factors
/ genetics
Gene Expression Regulation, Neoplastic
Glioblastoma
/ genetics
Humans
Mice
Mice, Nude
MicroRNAs
/ genetics
Prognosis
Promoter Regions, Genetic
Survival Rate
Tumor Cells, Cultured
Xenograft Model Antitumor Assays
AGO4
Cytosine-methylation
DNMT3A
Epigenetics
Epitranscriptomics
Glioblastoma
miRNA
Journal
Molecular cancer
ISSN: 1476-4598
Titre abrégé: Mol Cancer
Pays: England
ID NLM: 101147698
Informations de publication
Date de publication:
25 02 2020
25 02 2020
Historique:
received:
04
10
2019
accepted:
13
02
2020
entrez:
27
2
2020
pubmed:
27
2
2020
medline:
2
2
2021
Statut:
epublish
Résumé
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). 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. 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. Together, our results indicate that the DNMT3A/AGO4-mediated cytosine methylation of miRNA negatively.
Sections du résumé
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.
Identifiants
pubmed: 32098627
doi: 10.1186/s12943-020-01155-z
pii: 10.1186/s12943-020-01155-z
pmc: PMC7041276
doi:
Substances chimiques
AGO4 protein, human
0
Argonaute Proteins
0
Biomarkers, Tumor
0
DNMT3A protein, human
0
Dnmt3a protein, mouse
0
Eukaryotic Initiation Factors
0
MIrn181 microRNA, human
0
MicroRNAs
0
Cytosine
8J337D1HZY
DNA (Cytosine-5-)-Methyltransferases
EC 2.1.1.37
DNA Methyltransferase 3A
EC 2.1.1.37
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
36Références
Cancer Cell. 2014 Apr 14;25(4):442-54
pubmed: 24656771
PLoS One. 2013 Nov 18;8(11):e79044
pubmed: 24260150
Nucleic Acids Res. 2019 Jan 8;47(D1):D175-D180
pubmed: 30371818
Nucleic Acids Res. 2015 Jul 13;43(12):6112-24
pubmed: 25990724
Biology (Basel). 2016 Jan 06;5(1):
pubmed: 26751487
Methods. 2007 Oct;43(2):162-5
pubmed: 17889804
Carcinogenesis. 2013 Feb;34(2):446-53
pubmed: 23125218
J Biol Chem. 2002 Apr 5;277(14):11735-45
pubmed: 11821381
Front Cell Neurosci. 2013 Dec 25;7:279
pubmed: 24399935
Prog Mol Subcell Biol. 2010;50:1-20
pubmed: 19841878
RNA. 2008 Aug;14(8):1663-70
pubmed: 18567810
Nat Rev Genet. 2007 Feb;8(2):93-103
pubmed: 17230196
Anim Genet. 2014 Aug;45 Suppl 1:15-24
pubmed: 24990588
J Clin Invest. 2013 Jan;123(1):150-63
pubmed: 23241956
Cell Death Dis. 2012 Nov 15;3:e421
pubmed: 23152057
Essays Biochem. 2013;54:17-28
pubmed: 23829524
Epigenetics. 2011 Nov;6(11):1354-61
pubmed: 22048249
Dev Biol. 2007 Feb 1;302(1):1-12
pubmed: 16989803
Curr Biol. 2005 Aug 23;15(16):1501-7
pubmed: 16111943
Nat Methods. 2006 Dec;3(12):995-1000
pubmed: 17072308
Nat Commun. 2019 Aug 29;10(1):3888
pubmed: 31467274
Nucleic Acids Res. 2011 Sep 1;39(16):6845-53
pubmed: 21652644
Nucleic Acids Res. 2015 Jan;43(Database issue):D141-5
pubmed: 25392422
Genome Med. 2013 Dec 30;5(12):111
pubmed: 24373327
J Biol Chem. 2010 Jun 4;285(23):17442-52
pubmed: 20353945
J Cell Biol. 2012 Apr 16;197(2):201-8
pubmed: 22492723
Genome Biol. 2011 Dec 30;12(12):R126
pubmed: 22208850
PLoS One. 2015 Feb 27;10(2):e0118438
pubmed: 25723394
Chembiochem. 2011 Jan 24;12(2):206-22
pubmed: 21243710
Cell Res. 2014 Feb;24(2):177-89
pubmed: 24407421
Mol Cell Biol. 2002 Feb;22(3):704-23
pubmed: 11784849
Front Genet. 2013 Dec 03;4:258
pubmed: 24348513
Cancer Sci. 2012 May;103(5):837-45
pubmed: 22320679
EMBO Rep. 2011 Jul 01;12(7):647-56
pubmed: 21660058
Cell. 2012 Oct 12;151(2):278-88
pubmed: 23063121
Biosci Rep. 2016 Aug 05;36(4):
pubmed: 27364355
BMC Biochem. 2005 Mar 30;6:6
pubmed: 15799776
Nat Rev Cancer. 2013 Jul;13(7):455-65
pubmed: 23783119
Mol Cell. 2019 Jun 20;74(6):1278-1290.e9
pubmed: 31031083
Neurochem Int. 2014 Nov;77:68-77
pubmed: 24937770
Proc Natl Acad Sci U S A. 2011 Nov 1;108(44):18061-6
pubmed: 22011581
Gene. 2016 Apr 1;579(2):133-8
pubmed: 26743126
Hepatology. 2017 Feb;65(2):529-543
pubmed: 27774652
Science. 2006 Jan 20;311(5759):395-8
pubmed: 16424344
Exp Cell Res. 2013 Feb 1;319(3):173-84
pubmed: 23211718
Genes (Basel). 2017 Sep 05;8(9):
pubmed: 28872587
Cell. 2004 Jan 23;116(2):281-97
pubmed: 14744438
Nature. 2015 Mar 26;519(7544):482-5
pubmed: 25799998