In human glioblastomas transcript elongation by alternative polyadenylation and miRNA targeting is a potent mechanism of MGMT silencing

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Abstract
Favorable outcome after chemotherapy of glioblastomas cannot unequivocally be linked to promoter hypermethylation of the O 6-methylguanine-DNA methyltransferase (MGMT) gene encoding a DNA repair enzyme associated with resistance to alkylating agents. This indicates that molecular mechanisms determining MGMT expression have not yet been fully elucidated. We here show that glioblastomas are capable to downregulate MGMT expression independently of promoter methylation by elongation of the 3′-UTR of the mRNA, rendering the alternatively polyadenylated transcript susceptible to miRNA-mediated suppression. While the elongated transcript is poorly expressed in normal brain, its abundance in human glioblastoma specimens is inversely correlated with MGMT mRNA expression. Using a bioinformatically guided experimental approach, we identified miR-181d, miR-767-3p, and miR-648 as significant post-transcriptional regulators of MGMT in glioblastomas; the first two miRNAs induce MGMT mRNA degradation, the latter affects MGMT protein translation. A regression model including the two miRNAs influencing MGMT mRNA expression and the MGMT methylation status reliably predicts The Cancer Genome Atlas MGMT expression data. Responsivity of MGMT expressing T98G glioma cells to temozolomide was significantly enhanced after transfection of miR-181d, miR-767-3p, and miR-648. Taken together, our results uncovered alternative polyadenylation of the MGMT 3′-UTR and miRNA targeting as new mechanisms of MGMT silencing.

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