Fig 2: DNMT3B overexpression promotes proliferation and inhibits cell cycle arrest and apoptosis in cholangiocarcinoma cells. Analysis of DNMT3B mRNA (A) and protein (B) levels in QBC939 cells transfected with LV-DNMT3B or LV-control. (C–E) Effect of DNMT3B overexpression on QBC939 cell colony formation (C and D) and proliferation (E). (F) Cell cycle distribution was subjected by flow cytometry. (G) Quantified histograms display the effect of DNMT3B overexpression on cell cycle distribution. (H) Relative CDKN2B gene promoter methylation level in QBC939 cells transfected with LV-DNMT3B or LV-control. (I) Flow cytometry plots illustrating apoptosis in Annexin V/PI-stained QBC939 cells. (J) Quantified histograms display the effect of DNMT3B overexpression on the apoptosis of QBC939 cells. (K–L) Effect of DNMT3B knockdown on CDKN2B mRNA (K) and protein (L) levels. The values presented are means ± SD. *P<0.05 compared to the negative control group, as determined by analysis of one-way variance (ANOVA), followed by the repeated measures.

Fig 3: RARα divergently regulates TGFβ signaling and proliferation in a cluster-specific manner: transcriptional and functional validation.a siRARα RT-qPCR on CL1 and CL2 RA FLS. Boxplots of change in mRNA levels under RARα depletion (CL1: p = 0.041, CL2: p = 0.015) for TGFβ1 (CL2: p = 0.004) and canonical CDKN2B (CL1: p = 0.0009, CL2: p = 0.0002)/non-canonical signaling axis genes that undergo transcriptional regulation. Arrows (↑) illustrate activating and blunt ended lines (T) inhibiting/repressive effects. Cyan infill depicts the primary CL1 regulation logic, dark blue representing the primary CL2 regulation logic and black arrows representing both CL1 and CL2 logic. Boxplot of change in CDKN2B protein levels under RARα depletion (CL1: p = 0.048). p-values calculated by paired two-tailed Student’s t-test for siRARA vs CTL *p < 0.05. CL1 n = 3 and CL2 n = 3 biologically independent cell lines. b Heatmap of gene expression (z-score(TPM)) for genes with significant differential expression (p < 0.05 by two-tailed Student’s t-test) between clusters, related to cellular proliferation. Growth rate boxplot for CL1 and CL2 FLS. CL1 n = 5 and CL2 n = 4 biologically independent cell lines. c MTT proliferation assay boxplot for siRARα and siCtrl at 1 and 5 days after plating. CL1 n = 6 and CL2 n = 3 biologically independent cell lines. For all box plots, red center line for median, whiskers represent maximum and minimum values, box width from quartile 1 to quartile 3. Source data are provided as a Source Data file.

Fig 4: miR-29b reduces CDKN2B promoter methylation and increases CDKN2B expression by directly targeting DNMT3B. Effect of miR-29b overexpression on CDKN2B mRNA (A) and protein (B) levels in QBC939 cells. (C) MSP analysis of CDKN2B gene promoter methylation changes in QBC939 cells transfected with LV-miR-29b or LV-miR-NC. (D) Schematic representation of the putative miR-29b binding site in the DNMT3B 3’UTR. (E, F) Effect of miR-29b overexpression on DNMT3B mRNA (E) and protein (F) levels. (G) Dual-luciferase reporter assay results showing reduced WT-DNMT3B promoter-driven luciferase activity in QBC939 cells transfected with miR-29b mimics. (H) Relative expression of DNMT3B protein in QBC939 cells. (I) Relative expression of DNMT3B mRNA in clinical cholangiocarcinoma specimens. (J) Pearson correlation analysis between miR-29b and DNMT3B expression in cholangiocarcinoma specimens. The values presented are means ± SD. *P<0.05 compared to the negative control group, as determined by analysis of one-way variance (ANOVA), followed by the repeated measures.