Fig 2: Scatter plots showing the relation between the levels of expression of MGMT mRNA and miRNAs(A) miR-127-3p, (B) -409-3p, (C) -124-3p, (D) -181d-5p, (E) -648, (F) -767-3p and (G) -603 in WHO 2007 grade IV glioblastoma with non-methylated MGMT promoter (n = 26).

Fig 3: Schematic summary of major microRNAs reported to down-modulate MGMT expressionMGMT expression is increased by different nuclear transcription factors (Sp1, NF-kB, AP-1, CEBP and HIF-1a), together with the acetylation of histones H3 and H4 and the stabilization by binding of N-myc Downstream Regulated Gene 1 (NDRG1) protein (for review see [31]). On the opposite, MGMT expression is downregulated by different mechanisms, namely methylation of the CpG islands in the promoter [32], di-methylation of histone H3K9 [33] and microRNAs. The graph represents the central role of miR-181d-5p [15] in down regulating MGMT expression, by acting both on MGMT mRNA degradation and interference with protein translation. Other and this report suggest that miR-603 [20], and 409-3p cooperate with miR-181d-5p in MGMT mRNA degradation, whereas miR-648 [17], miR-767-3p [17], -124-3p and 127-3p could play an ancillary role.

Fig 4: Initial screening of miRNAs related to MGMT expression in glioblastoma ex vivoMGMT mRNA was quantified in FineFix–fixed and paraffin-embedded 57 samples of glioblastoma (WHO grade IV). MGMT gene promoter was non-methylated in all samples. MGMT mRNA relative expression normalized over GAPDH mRNA was analyzed by RT-qPCR with TaqMan probes. (A) Relative expression was ordered in a rank in order to select samples at the extremes of low (L) and high (H) MGMT mRNA expression. (B) Expression pattern of five miRNAs differentially expressed between MGMT-low and MGMT-high glioblastoma samples (WHO grade IV), displaying extreme levels of MGMT mRNA. MGMT gene promoter was non-methylated in all samples. (C) MiR-181 family expression in the same low- and high-MGMT glioblastoma samples.

Fig 5: Validation of miRNA expression by RT-qPCR(A) Quantitative analyses of miR-127-3p, (B) miR-409-3p, (C) miR-129-5p, (D) miR-124-3p, (E) miR-381-3p, (F) miR-181d-5p, (G) miR-181c-3p, (H) miR-181c-5p and (I) miR-181a-5p. The two groups of extreme samples refer the specimens expressing to low (n = 5) and high (n = 5) MGMT mRNA. Analyses were performed with TaqMan probes and each miRNA was normalized to the most stable miRNA of the array (miR-760). Mean (bold line), median (light line), 5th and 95th confidence interval (box limits) and outliers as single dots are reported.