Fig 2: EZH2-mediated regulation of histone methylation may be the main reason for expression inhibition of multiple miRNAs on chromosome 5 in prostate cancer cells.A Western blotting analysis of EZH2, H3K27me3, H3K4me3, DNMT1, DNMT3a, and DNMT3b in the DU145 and LNcap cells treated with inhibitor GSK343 or gamma-Oryzanol or not. B Western blotting analysis of EZH2, H3K27me3, H3K4me3, DNMT1, DNMT3a, and DNMT3b in the DU145 and LNcap cells with EZH2 overexpression or knockdown. C Western blotting analysis of H3K27me3 in the DU145 and LNcap cells treated with EED226 or UNC1999 for H3K27me3 knockdown or treated with H3K4me3 inhibitor CPI-455 HCl. D QPCR analysis for the expression of miR-340-5p, miR-145-5p, and miR-143-3p in DU145 and LNcap cells treated with inhibitor GSK343 or gamma-Oryzanol or not. E QPCR analysis for the expression of miR-340-5p, miR-145-5p, and miR-143-3p in DU145 and LNcap cells with EZH2 overexpression or knockdown. F QPCR analysis for the expression of miR-340-5p, miR-145-5p, and miR-143-3p in DU145 and LNcap cells treated with EED226 or UNC1999 for H3K27me3 knockdown or treated with H3K4me3 inhibitor CPI-455 HCl. The results are presented as the mean ± SD. *P < 0.05, **P < 0.01. EZH2 Enhancer of Zeste Homolog 2, H3K27me3 histone 3 lysine 27 tri-methylation, H3K4me3 histone 3 lysine 4 tri-methylation, DNMT DNA methyltransferases.

Fig 3: Diagram of the DNMT3B/miR-152-3p/NCAM1 pathway. DNMT3B regulates the A549 cell proliferation through miR-152-3p/NCAM1 pathway tested by MTT assay, colony formation, apoptosis and western blotting.

Fig 4: Knockdown of DNMT3B increases miR-152-3p expression and delays cell proliferation. (A) Cell survival rate of A549 cell lines transfected with sh-DNMT3B and or miR-152-3p inhibitor, as indicated by the MTT assay. (B) Colony formation assay was used to detect the proliferative ability of the indicated groups. (C) Reverse transcription-quantitative PCR demonstrated the different expression levels of miR-152-3p and NCAM1 in the indicated groups. (D) Expression levels of NCAM1, cleaved-caspase-3 and cleaved-PARP proteins in A549 cells detected via western blotting. One-way ANOVA was used for statistical analysis. *P<0.05, **P<0.01, ***P<0.001. DNMT, DNA methyltransferase; miR, microRNA; NC, negative control; NCAM1, neural cell adhesion molecule 1; sh, short hairpin.

Fig 5: DNMT3B binds to and methylates the miR-152-3p core region. (A) Bisulfite treated DNA samples from A549, A549/DDP and A549/ADM cells were amplified with methylated and non-methylated primers at the same time, and the amplification level was detected by PCR. (B) Bisulfite sequencing was used to detect the methylation level of miR-152-3p in A549, A549/DDP and A549/ADM cells. Five respective clones from each group were sequenced. (C) ChIP assay detected the binding of DNMT3B, DNMT3A and DNMT1 proteins to the core region of miR-152-3p. (D) Bisulfite sequencing results of the methylation levels in the core region of miR-152-3p in A549 cells were detected after treatment with DNMT3B/DNMT1 methylase inhibitors in three independent experiments. (E) Relative mRNA expression levels of miR-152-3p and NCAM1 in A549 cells treated with DNMT3B methylase inhibitor. Two-tailed Student's t-test was used for statistical analysis. *P<0.05, ***P<0.001. DNMT, DNA methyltransferase; miR, microRNA; IP, immunoprecipitation, ChIP, chromatin IP; NCAM1, neural cell adhesion molecule 1; U, unmethylated; M, methylated.