Fig. 4

Downregulated DNMT3a-mediated promoter hypomethylation, and miR-219a transcription and RhoGDIβ inhibition in UROtsa cells following a 2-month BBN exposure. A UROtsa^C2mo and UROtsa^BBN2mo cells were transiently transfected with miR-219a promoter-driven luciferase reporter to determine promoter transcriptional activity. Results are presented as miR-219a promoter activity relative to vehicle control (relative miR-219a promoter activity). Bars represent mean ± SD of three independent experiments. The asterisk indicates significant difference vs. vehicle control (p < 0.05). B Methylation status of miR-219a promoter in UROtsa^C2mo and UROtsa^BBN2mo cells was determined using methylation-specific PCR [MS-PCR]. A primer set (173bp) was used to evaluate methylated (M) and unmethylated (U) copies of the miR-219a DMR gene. Methylated DNA was used as positive control (P); unmethylated control was used as the negative control (N). C DNA methyl-transferase DNMT3a, DNMT3b, and DNMT1 expression were evaluated using Western blot in UROtsa cells exposed to BBN (400 μM) for indicated periods. β-Actin was used as a protein loading control. D, E Ectopic DNMT3a or DNMT3b with Myc-tag constructs were stably transfected into UROtsa^BBN2mo cells. The efficiency of over-expressed DNMT3a or DNMT3b on downstream RhoGDIβ protein expression was assessed using Western blot. F miR-219a levels in UROtsa^BBN2mo/Vector and UROtsa^BBN2mo/Myc-DNMT3a cells were determined by using real-time PCR. Results are presented as the mean ± SD of triplicate experiments. The asterisk indicates significant decrease vs. control (p < 0.05). G, H UROtsa^BBN2mo/Vector and UROtsa^BBN2mo/Myc-DNMT3a cells were subjected to soft agar in the presence of EGF (20 ng/ml). The number of colonies was scored, and outcomes presented as colonies/10⁴ seeded cells. The asterisk indicates significant decrease vs. vector cells (p < 0.05)