A genome-wide miRNA screen revealed miR-603 as a MGMT-regulating miRNA in glioblastomas

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.

Prognosis of glioblastoma with faint MGMT methylation-specific PCR product

“…Several miRNAsmiR181d, miR767-3p, miR-648 and miR-603 have been identified as significant post-transcriptional regulators of MGMT in GBMs [34,35]. Inverse correlation of miR-603 and miR-181d levels with MGMT expression has also been demonstrated in clinical GBM specimens [35]. From our study, it was also noted that although the methylation level of the MSP± group was much lower than that of the fully methylated (MSP?)…”

“…The above changes could cause incomplete downregulation of MGMT protein expression. In addition to methylation of CpG islands in the promoter region, which is the most significant mechanism of MGMT regulation that suppresses MGMT transcription [26,27], other mechanisms, such as MGMT genetic changes [28,29], histone modifications [30,31], p53 derangement [32,33] and post-transcriptional regulation [34,35] could also lead to MGMT silencing. Absence of MGMT protein expression (IHC-) in 15.8 % of our unmethylated (MSP-) GBM cases (Table 3) would be the result of other MGMT silencing mechanisms.…”

“…This finding also signaled the limitation of MGMT promoter methylation assay, as one should not use an unmethylated result to preclude the use of TMZ in GBM patients because other MGMT silencing mechanisms could occur and patients might still benefit from this treatment. Recent studies concerning post-transcriptional regulation of MGMT have demonstrated that it could be regulated by miRNA via direct and indirect regulations [35][36][37]. Several miRNAsmiR181d, miR767-3p, miR-648 and miR-603 have been identified as significant post-transcriptional regulators of MGMT in GBMs [34,35].…”

“…Recent studies concerning post-transcriptional regulation of MGMT have demonstrated that it could be regulated by miRNA via direct and indirect regulations [35][36][37]. Several miRNAsmiR181d, miR767-3p, miR-648 and miR-603 have been identified as significant post-transcriptional regulators of MGMT in GBMs [34,35]. Inverse correlation of miR-603 and miR-181d levels with MGMT expression has also been demonstrated in clinical GBM specimens [35].…”

Targeting DNA Repair Mechanisms to Treat Glioblastoma

“…Evidence suggests that mechanisms of post-transcriptional regulation alter MGMT protein expression since protein analysis of MGMT does not correlate with mRNA [76]. Recent data suggest that miRNA regulation of MGMT may explain these discrepancies and miRNAs are currently being investigated as therapeutic targets [70,82,83]. Because MGMT expression appears be predictive of progression free and overall survival [74], adequate assessment of tumors may need to include MGMT mRNA and/or protein expression.…”