miR expression microarray analysis on M2 macrophages treated with epigenetic modifiers

Epigenetic regulation of gene expression involves DNA methylation, histone methylation or acetylation, and microRNA (miRNA)-mediated mRNA degradation. We studied, if epigenetic modifiers namely 5-aza-2'-deoxycytidine and trichostatin A could remodulate the miR expression in M2 macrophage and enable M2-M1 reprogramming. Bone marrow cells were isolated from tibias and femurs of Balb/c mice and cultured in DMEM supplemented with 10 ng/mL of colony-stimulating factor-1 (CSF-1; Gibco, Carlsbad, CA, USA) and 10% FBS for 7 d. The culture medium was changed every other day. Next, the cells were incubated with 100 ng/mL of lipopolysaccharide and 20 ng/mL of recombinant mouse IFN-γ (R&D Systems, Minneapolis, MN, USA), for 24–48 h to achieve M1 polarization. For M2 polarization, BMDMs were incubated with 20 ng/mL of recombinant mouse IL-4 for 24–48 h. M2-polarized macrophages were treated with either 50 nM 5-aza-dC (Merck, Rahway, NJ, USA) for 72 h or 25 nM TSA (Merck) for 48 h. For treatment with the combination, M2-polarized macrophages were treated with 5-aza-dC for 24 h followed by treatment with 5-aza-dC and TSA for 48 h.RNAs for miRNA profiling were isolated using the miRNeasy® Mini Kit. Microarray analysis of miRNAs was performed using an Affymetrix GeneChip miRNA 4.0 array (Affymetrix, Santa Clara, CA, USA) by Macrogen (Seoul, Korea).