Figure 3. The knockdown of DNMT3B increased miR-34a expression and decreased the methylation of the miR-34a promoter. EJ and UMUC3 cells were infected with lentiviral vectors expressing shRNAs targeting DNMT3B (shRNA)to construct DNMT3B-knockdown cells, termed as the shRNA group. EJ and UMUC3 cells infected with empty lentivirus were used as the negative control (NC) group. (A) Expression of DNMT3B in bladder cancer cells measured by qRT-PCR. (B, C) Expression of DNMT3B in the NC and shRNA groups as detected by qRT-PCR (B) and western blot analysis (C). (D) The levels of miR-34a in the NC and shRNA groups as detected by qRT-PCR. (E) The methylation ratio of the miR-34a promoter as determined by bisulfite genomic sequencing. DNMT3B knockdown decreased methylation in the promoter of miR-34a. (F–G) The effects of DNMT3B knockdown on the transcription activity of the miR-34a promoter. (A) luciferase reporter plasmid containing the miR-34a promoter (pGL3-miR-34a) was transfected into the shRNA groups of EJ and UMUC3 cells. The empty vector pGL3 was used as NC. The relative luciferase activity was calculated using the ratio of firefly and Renilla luciferase activities. Data were presented as means±SD. *p<0.05 between NC and shRNA. DNMT3B, DNA methyltransferase 3B; qRT-PCR, quantitative reverse transcription polymerase chain reaction; NC, negative control; SD, standard deviation.