Journal: Brain Pathology
Article Title: Role of micro RNA s Located on Chromosome Arm 10q in Malignant Gliomas
doi: 10.1111/bpa.12294
Figure Lengend Snippet: miR‐146b‐5p expression in 5‐aza‐2′‐deoxycytidine (5‐AZA‐dCR) and trichostatin A (TSA) treated glioma cell lines (A) as well as methylation of CpG sites located in putative SP1 transcription factor‐binding sites associated with the miR‐146b‐5p locus (B). (A) Relative expression levels of miR‐146b‐5p were determined by real‐time RT‐PCR using let‐7a as reference gene. Expression of the miRNA in the 5‐AZA‐dCR/TSA samples was calculated relative to miRNA expression in the controls (Co). A + T1: cell lines were grown in medium supplemented with 500 nM 5‐AZA‐dCR for 48 h, washed and grown for another 24 h in medium with 500 nM 5‐AZA‐dcr and 1 µM TSA; A + T2: like A + T1, except for a higher 5‐AZA‐dCR concentration (1 µM). Asterisks indicate significant difference when compared with the respective non‐treated controls (*P < 0.05; **P < 0.01; paired t‐test). Bars represent the mean, and error bars represent the standard deviation of three independent experiments, except for U138MG A + T2 with two independent experiments. (B) Methylation analysis of the seven CpG sites located from nt 104,195,358 to nt 104,195,483 on chr10 (UCSC genome browser, GRCh37/hg19 assembly) was performed by pyrosequencing of two PCR products generated from sodium bisulfite‐modified DNA. The three CpG sites located within each of the three putative SP1‐binding sequences are labeled by hachures. The methylation status at each CpG site was gray scale‐coded as follows: white square, 0–25% methylated alleles; light gray square, 26–50% methylated alleles; gray square, 51–75% methylated alleles; black square, 76–100% methylated alleles. methCo, methylation positive control (CpGenome™ Universal Methylated DNA, Merck Millipore, Darmstadt, Germany); AII, diffuse astrocytoma WHO grade II; AAIII, anaplastic astrocytoma WHO grade III; sGBIV, secondary glioblastoma WHO grade IV; pGBIV, primary glioblastoma WHO grade IV; NB, non‐neoplastic brain tissue; Meth% (mean), mean of the percentages of methylated allele derived from pyrosequencing at the seven investigated CpG sites. IDH mut: gray square, IDH1 or IDH2 mutation detected; white square, no IDH1 or IDH2 mutation detected. TERT mut: gray square, TERT promoter mutation detected; white square, no TERT promoter mutation detected. Note a significant difference in the mean percentage of methylated alleles between primary glioblastoma (mean: 5% methylated alleles) and secondary glioblastoma (mean: 30% methylated alleles; P < 0.01; Student's t‐test, two‐sided).
Article Snippet: Three different commercially available genomic DNA samples (BioChain) from human brain tissue served as reference for DNA methylation analyses.
Techniques: Expressing, Methylation, Binding Assay, Quantitative RT-PCR, Concentration Assay, Standard Deviation, Generated, Modification, Labeling, Positive Control, Derivative Assay, Mutagenesis
