Research Paper Volume 13, Issue 14 pp 18310—18330

BMI1 activates P-glycoprotein via transcription repression of miR-3682-3p and enhances chemoresistance of bladder cancer cell

Interacted with p53, BMI1 epigenetically repressed transcription of miR-3682-3p. (A) Co-IP assay showing the interaction of BMI1 with p53 in T24 cells. (B) ChIP assay showing the nucleotide regions of miR-3682-3p promoter that are physically associated with p53. Upper panel: schematic illustration of predicted p53-bound sites and PCR-amplified fragments of the miR-3682-3p promoter; lower panel: ChIP assays were performed using p53 antibody to validate p53-bound miR-3682-3p promoter regions. IgG was used as a negative control. (C) ChIP-qPCR analysis showing enrichment of p53 at miR-3682-3p promoter in the indicated cells. (D) miR-3682-3p promoter luciferase reporter plasmids, Renilla pRL-TK plasmids, vector, or BMI1 were transfected into T24 cells. After 48 h, cells were subjected to a luciferase reporter assay. (E, F) ChIP-qPCR analysis of BMI1 (E) and H2AK119ub1 (F) at promoter of miR-3682-3p in T24 cells. (G) Cell proliferation changes of T24/DDP&GEM cells transfected with miR-3682-3p mimics, ABCB1 inhibitor XR-9576, or negative control were assessed by cell counting kit-8 assays after treatment with 2 μg/ml DDP. (H) Apoptosis of the indicated cells detected by the Annexin V/flow cytometric apoptosis assay after treatment with 2 μg/ml DDP. (I) The retention rate of Rhodamine 123 in the indicated cells detected by flow cytometry. *P P P

Figure 5. Interacted with p53, BMI1 epigenetically repressed transcription of miR-3682-3p. (A) Co-IP assay showing the interaction of BMI1 with p53 in T24 cells. (B) ChIP assay showing the nucleotide regions of miR-3682-3p promoter that are physically associated with p53. Upper panel: schematic illustration of predicted p53-bound sites and PCR-amplified fragments of the miR-3682-3p promoter; lower panel: ChIP assays were performed using p53 antibody to validate p53-bound miR-3682-3p promoter regions. IgG was used as a negative control. (C) ChIP-qPCR analysis showing enrichment of p53 at miR-3682-3p promoter in the indicated cells. (D) miR-3682-3p promoter luciferase reporter plasmids, Renilla pRL-TK plasmids, vector, or BMI1 were transfected into T24 cells. After 48 h, cells were subjected to a luciferase reporter assay. (E, F) ChIP-qPCR analysis of BMI1 (E) and H2AK119ub1 (F) at promoter of miR-3682-3p in T24 cells. (G) Cell proliferation changes of T24/DDP&GEM cells transfected with miR-3682-3p mimics, ABCB1 inhibitor XR-9576, or negative control were assessed by cell counting kit-8 assays after treatment with 2 μg/ml DDP. (H) Apoptosis of the indicated cells detected by the Annexin V/flow cytometric apoptosis assay after treatment with 2 μg/ml DDP. (I) The retention rate of Rhodamine 123 in the indicated cells detected by flow cytometry. *P < 0.05. **P < 0.01. ***P < 0.001. Co-IP: co-immunoprecipitation; ChIP: chromatin immunoprecipitation; DDP: cisplatin.