Journal: Oncotarget

Article Title: miRNA array screening reveals cooperative MGMT-regulation between miR-181d-5p and miR-409-3p in glioblastoma

doi: 10.18632/oncotarget.8618

Figure Lengend Snippet: MGMT 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.

Article Snippet: The membranes were washed three times for 5 min each with 25 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with MGMT primary mouse monoclonal antibody (1:1000) (Cat. GTX27045, GeneTex International Corporation, Alton Pkwy, CA, USA) in 15 ml primary antibody dilution buffer with gentle agitation over-night at 4°C.

Techniques: Methylation, Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: miRNA array screening reveals cooperative MGMT-regulation between miR-181d-5p and miR-409-3p in glioblastoma

doi: 10.18632/oncotarget.8618

Figure Lengend Snippet: ( 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.

Article Snippet: The membranes were washed three times for 5 min each with 25 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with MGMT primary mouse monoclonal antibody (1:1000) (Cat. GTX27045, GeneTex International Corporation, Alton Pkwy, CA, USA) in 15 ml primary antibody dilution buffer with gentle agitation over-night at 4°C.

Techniques: Expressing

Journal: Oncotarget

Article Title: miRNA array screening reveals cooperative MGMT-regulation between miR-181d-5p and miR-409-3p in glioblastoma

doi: 10.18632/oncotarget.8618

Figure Lengend Snippet: ( 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).

Article Snippet: The membranes were washed three times for 5 min each with 25 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with MGMT primary mouse monoclonal antibody (1:1000) (Cat. GTX27045, GeneTex International Corporation, Alton Pkwy, CA, USA) in 15 ml primary antibody dilution buffer with gentle agitation over-night at 4°C.

Techniques: Methylation

Journal: Oncotarget

Article Title: miRNA array screening reveals cooperative MGMT-regulation between miR-181d-5p and miR-409-3p in glioblastoma

doi: 10.18632/oncotarget.8618

Figure Lengend Snippet: T98G glioblastoma cells were transfected with 200 nM of the pre-miRs indicated in the figure labels for each microRNA or scrambled sequence (−). ( A ) MGMT mRNA expression was detected with TaqMan probes, as indicated in the Method section, and the relative expression normalized to that of T98G cells transfected with scrambled sequence. ( B ) MGMT protein expression as detected by Western analysis is shown in the inset panel (using β-actin as reference). Band density was plotted and normalized to that of the T98G cells transfected with the scrambled sequence. Statistical significance in A and B at the levels of p < 0.05 (*) and p < 0.01 (**) is relative to cells transfected with scrambled sequence.

Article Snippet: The membranes were washed three times for 5 min each with 25 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with MGMT primary mouse monoclonal antibody (1:1000) (Cat. GTX27045, GeneTex International Corporation, Alton Pkwy, CA, USA) in 15 ml primary antibody dilution buffer with gentle agitation over-night at 4°C.

Techniques: Transfection, Sequencing, Expressing, Western Blot

Journal: Oncotarget

Article Title: miRNA array screening reveals cooperative MGMT-regulation between miR-181d-5p and miR-409-3p in glioblastoma

doi: 10.18632/oncotarget.8618

Figure Lengend Snippet: MGMT 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 ). On the opposite, MGMT expression is downregulated by different mechanisms, namely methylation of the CpG islands in the promoter , di-methylation of histone H3K9 and microRNAs. The graph represents the central role of miR-181d-5p 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 , and 409-3p cooperate with miR-181d-5p in MGMT mRNA degradation, whereas miR-648 , miR-767-3p , -124-3p and 127-3p could play an ancillary role.

Article Snippet: The membranes were washed three times for 5 min each with 25 ml of TBS/T (TBS, 0.1% Tween-20) and incubated with MGMT primary mouse monoclonal antibody (1:1000) (Cat. GTX27045, GeneTex International Corporation, Alton Pkwy, CA, USA) in 15 ml primary antibody dilution buffer with gentle agitation over-night at 4°C.

Techniques: Expressing, Binding Assay, Methylation