Abstract
MGMT expression is a critical determinant for therapeutic resistance to DNA alkylating agents. We previously demonstrated that MGMT expression is post-transcriptionally regulated by miR-181d and other miRNAs. Here, we performed a genome-wide screen to identify MGMT regulating miRNAs. Candidate miRNAs were further tested for inverse correlation with MGMT expression in clinical specimens. We identified 15 candidate miRNAs and characterized the top candidate, miR-603. Transfection of miR-603 suppressed MGMT mRNA/protein expression in vitro and in vivo; this effect was reversed by transfection with antimiR-603. miR-603 affinity-precipitated with MGMT mRNA and suppressed luciferase activity in an MGMT-3'UTR-luciferase assay, suggesting direct interaction between miR-603 and MGMT 3'UTR. miR-603 transfection enhanced the temozolomide (TMZ) sensitivity of MGMT-expressing glioblastoma cell lines. Importantly, miR-603 mediated MGMT suppression and TMZ resistance were reversed by expression of an MGMT cDNA. In a collection of 74 clinical glioblastoma specimens, both miR-603 and miR-181d levels inversely correlated with MGMT expression. Moreover, a combined index of the two miRNAs better reflected MGMT expression than each individually. These results suggest that MGMT is co-regulated by independent miRNAs. Characterization of these miRNAs should contribute toward strategies for enhancing the efficacy of DNA alkylating agents.
Highlights
O6-methylguanine methyl transferase (MGMT) encodes an evolutionarily conserved protein, the primary function of which is to repair guanine nucleotides that are alkylated at the O6 position [1]
To identify miRNAs that suppressed MGMT expression, we individually transfected 885 known miRNAs (Human miScript miRNA mimic 96 set, Qiagen) into T98G, a glioblastoma cell line that showed high expression of MGMT. 48 hours after transfection, the cells were assessed for viability by direct visual inspection (Supplementary Figure 1)
MGMT protein expression was measured by Western blotting (Figure 1A). miRNAs that suppressed MGMT expression more than 50% without significant cytotoxicity (>50% cell death) were identified
Summary
O6-methylguanine methyl transferase (MGMT) encodes an evolutionarily conserved protein, the primary function of which is to repair guanine nucleotides that are alkylated at the O6 position [1]. O6-methyl guanine constitutes the major cytotoxic lesion induced by DNA alkylating chemotherapeutic agents, including temozolomide [2]. High MGMT mRNA and protein expression have been associated with therapeutic resistance to DNA alkylating agents in a number of cancers [5]. A major mechanism of MGMT regulation involves methylation of CpG islands in the promoter region [6]. Methylation of these regions suppresses MGMT transcription [7, 8]. This mechanism of regulation is important in glioblastoma, the most common www.impactjournals.com/oncotarget form of primary brain cancer [9]. MGMT promoter methylation has been associated with favorable response to temozolomide in three randomized controlled trials, including EORTC-NCIC [10], NOA-8 [11], and the Nordic Trial [12]
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