Fig. 5

Arecoline and NNK induced DNMT3B activity and repressed ADHFE1, ALDH1A2 and miRNAs expression. a qRT–PCR analysis of miR-30a and miR-379 expression level after treatment with arecoline (50 μM) or NNK (10 μM) for indicated days. b RT-PCR analysis of ADHFE1, ALDH1A2 level and western blot analysis of DNMT3B level in DOK cells after treatment with arecoline (50 μM) or NNK (10 μM) for indicated times. GAPDH and α-tubulin were used as internal control. c ChIP assay of ADHFE1 and ALDH1A2 promoter region was performed with DOK cells using anti-DNMT3B antibody after treatment with vehicle control (DMSO, 10 nM), arecoline (50 μM) or NNK (10 μM) for 5 days. Mouse IgG (mIgG) antibody was used as negative control. d RT-PCR analysis of ADHFE1 and ALDH1A2 level in DOK cells after treatment with arecoline (50 μM) or NNK (10 μM) alone or combined with 5-aza-dC (5 μM) for 5 days. GAPDH was used as internal control. e qRT-PCR analysis of ADHFE1 and ALDH1A2 level in DOK cells after treatment with vehicle control (C), or arecoline (50 μM) plus control mimics (NC), miR-30a (20 nM), miR-379 (20 nM), or NNK (10 μM) plus control mimics (NC), miR-30a (20 nM), miR-379 (20 nM) for 5 days. GAPDH was used as an internal control. All data are presented as mean ± SD; **p < 0.01; ***p < 0.001