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. 2014 Jun 30;9(6):e100581. doi: 10.1371/journal.pone.0100581

Figure 3. The possible involvement of epigenetic modifications in the downregulation of miR-31 in lung cancer cell lines was investigated.

Figure 3

Cells treated with vehicle control (CTL), 5-azacytidine (AZA), trichostatin A (TRC), or a combination of AZA and TSA (AZ/TR) were examined for the restoration of miR-31 expression using quantitative RT-PCR. The relative level of miR-31 normalized to those of U6 snRNA was calculated. Two independent experiments were performed. Technical replicates of PCR analysis were performed in triplicate. Means and standard deviations (error bars) are shown (A). The two sites (S1 and S2) from the putative promoter of miR-31 were PCR-amplified with primers specific for either methylated (M) or unmethylated (UM) DNA using sodium bisulfate-modified genomic DNA as a template, according to the method described in a previous study [21]. Representative results are presented (B). The region of the miR-31 promoter containing CpG sites was PCR-amplified using sodium bisulfate-modified genomic DNA as a template, according to the method described in a previous study [21]. Eight subclones of PCR products were analyzed for their methylation status using DNA sequencing. Open (○) and filled (•) circles indicated unmethylated and methylated cytosine at the indicated CpG sites, respectively (C). AEC, airway epithelial cells (non-cancerous cells); ADC, adenocarcinoma; SQC, squamous cell carcinoma; LCC, large cell carcinoma; SCC, small cell carcinoma.