Figure 8. Entinostat demethylates miR-203 promoter in NSCLC cells correlated with the downregulation of both protein and mRNA levels of DNMT1 in vitro.

A549 and H460 cells were untreated or treated with entinostat (3 μmol/L for A549, 1 μmol/L for H460) for 24 or 48 h. (A) Some cells were collected and subjected to DNA extraction. Methylation-specific PCR (MSP) and unmethylation-specific PCR (USP) analyses of miR-203 promoter were performed as described in Methods. Blank control, double distilled H₂O as PCR template. Negative control, unconverted genomic DNA of A549 or H460 cells as PCR template. (B) Some cells were subjected to western blot analyses of DNMT1, DNMT3A, DNMT3B, or β-actin. The densitometry analyses of DNMT1 signals were shown underneath, and the arbitrary numbers indicate the intensities of each cell line relative to controls, defined as 1.0. (C and D) One third of the cells were subjected to total RNA extraction. The mRNA levels of DNMT1, DNMT3A, and DNMT3B were measured by qRT-PCR. All results were normalized with the internal control β-actin. Bars, S.D. Data show the representative of three independent experiments. NS, no significance.