Figure 9

Knockdown of DDX3 inhibits p53 transcription through activation of the DNMTs and DNA hypermethylation. (a) Depletion of DDX3 suppressed the mRNA expression of p53 and target genes, p21, TP53I3, GADD45A and MDM2 in HCT116 cells. Quantitative real-time PCR analysis of p53 and p53 target genes were normalized to GAPDH and shown as average value ± S.D. calculated from three independent experiments. ***P < 0.001; **P < 0.01. (b) Analysis of the p53 mRNA stability in control and DDX3-knockdown HCT116 cells by incubation with 10 μg/ml actinomycin D at indicated time periods. Data were normalized to GAPDH and shown as average value ± S.D. calculated from at least two independent experiments. (c) Western blot analysis reveals DDX3 knockdown promoted the expression of DNMT1, DNMT3A and DNMT3B in HCT116 cells. Original images of western blots were presented in Supplementary Fig. S7b. (d) Quantitative real-time PCR analysis reveals DDX3 depletion increased the mRNA expression of DNMT1, DNMT3A and DNMT3B in HCT116 cells. Data were normalized to GAPDH and shown as average value ± S.D. calculated from three independent experiments. ***P < 0.001; *P < 0.05. (e) Knockdown of DDX3 induced p53 promoter (R2) hypermethylation detected by bisulfide sequencing PCR analysis. Schematic representation of p53 promoter harboring two CpG islands R2 (from -1009 to −586 bp) and R3 (from −304 to + 198 bp) relative to transcription start site (TSS) (+1). Open circles denote unmethylated CG sites, filled circles are methylated CG sites. Ten independent clones were sequenced in each case. (f) DDX3 deficiency induced hypermethylation in p53 promoter (R2) detected using EpiMark methylated DNA enrichment kit. Data are shown as average value ± S.D. calculated from at least two independent experiments. **P < 0.01; *P < 0.05.