DNA Methylation Is Involved in the Expression of miR-142-3p in Fibroblasts and Induced Pluripotent Stem Cells

Figure 3

Expression of miR-142-3p was regulated by DNA methylation. (a) Left panel shows schematic representation of luciferase constructs. Luciferase analysis using plasmids containing indicated length fragments of the 5′ upstream region of miR-142-3p-luciferase was done. Plasmid was transfected into 3T3 cells, and, after 6 hours, samples were treated with DMSO or 5-aza-dC (10 μM) and cultured for additional 3 days. Then cells were harvested, and luciferase activities were examined. Values are average of 3 times independent experiments with standard deviation. value, ∗∗ < 0.01 and n.s. > 0.05, was calculated by Student’s -test. (b–g) CpG methylation of 5′ upstream region of miR-142-3p was examined by bisulfite conversions. Genomic DNAs extracted from 3T3, MEF in the presence or absence of 5-aza-dC, iPS, or EB prepared from iPS were subjected to bisulfite sequence. 5-Aza-dC was present in the culture medium of 3T3 or MEF 72 hours before harvesting cells for genomic DNA extraction (e, f). (h) 3T3 cells were transfected with expression plasmid of Oct4, Sox2, Klf4, or Myc with −540 Luc. For control sample, empty expression plasmid and −540 Luc were transfected. Cells were harvested after 3 days of culture, and luciferase analysis was conducted. (i) 3T3 cells were transfected with indicated expression plasmid, and, after 3 days, cells were harvested, and total RNA was extracted. Expression level of endogenous miR-142-3p was examined by RT-qPCR. (h, i) Values are relative to control vector transfected samples and average of 4 independent samples with SD.