Susanna Khalil et al., miRNA array screening reveals cooperative MGMT-regulation between miR-181d-5p and miR-409-3p in glioblastoma. Oncotarget

Susanna Khalil1,*, Enrica Fabbri3,*, Alessandra Santangelo1,*, Valentino Bezzerri1, Cinzia Cantù1, Gianfranco Di Gennaro1, Alessia Finotti3, Claudio Ghimenton2, Albino Eccher2, Maria Dechecchi1, Aldo Scarpa1,2,5, Brian Hirshman6, Clark Chen6, Manuela Ferracin4, Massimo Negrini4, Roberto Gambari3,#, Giulio Cabrini1,2,#

1Department of Pathology and Diagnostics, Laboratory of Molecular Pathology, University Hospital, Verona, Italy
2Section of Pathology and Histology, University Hospital, Verona, Italy
3Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy
4Department of Morphology, Surgery and Experimental Medicine and Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy
5Applied Research on Cancer Network (ARC-NET), University and Hospital Trust, Verona, Italy
6Center for Theoretical and Applied Neuro-oncology, Moores Cancer Center, Department of Neurosurgery, University of California San Diego, San Diego, CA, USA
*These authors share co-first authorship
#These authors share co-senior authorship


The levels of expression of O6-methylguanine-DNA methyltransferase (MGMT) are relevant in predicting the response to the alkylating chemotherapy in patients affected by glioblastoma. MGMT promoter methylation and the published MGMT regulating microRNAs (miRNAs) do not completely explain the expression pattern of MGMT in clinical glioblastoma specimens. Here we used a genome-wide microarray-based approach to identify MGMT regulating miRNAs. Our screen unveiled three novel MGMT regulating miRNAs, miR-127-3p, miR-409-3p, and miR-124-3p, in addition to the previously identified miR-181d-5p. Transfection of these three novel miRNAs into the T98G glioblastoma cell line suppressed MGMT mRNA and protein expression. However, their MGMT- suppressive effects are 30–50% relative that seen with miR-181d-5p transfection. In silico analyses of The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) revealed that miR-181d-5p is the only miRNA that consistently exhibited inverse correlation with MGMT mRNA expression. However, statistical models incorporating both miR-181d-5p and miR-409-3p expression better predict MGMT expression relative to models involving either miRNA alone. Our results confirmed miR-181d-5p as the key MGMT-regulating miRNA. Other MGMT regulating miRNAs, including the miR-409-3p identified in this report, modify the effect of miR-181d-5p on MGMT expression. MGMT expression is, thus, regulated by cooperative interaction between key MGMT-regulating miRNAs.

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