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. 2021 Jul 16;13(14):18310-18330.
doi: 10.18632/aging.203277. Epub 2021 Jul 16.

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

Ming-Kun Chen  1 , Jun-Hao Zhou  1 , Peng Wang  1 , Yun-Lin Ye  2 , Yang Liu  1 , Jia-Wei Zhou  1 , Zi-Jian Chen  1 , Jian-Kun Yang  1 , De-Ying Liao  1 , Zhi-Jian Liang  1 , Xiao Xie  1 , Qi-Zhao Zhou  1 , Kang-Yi Xue  1 , Wen-Bin Guo  1 , Ming Xia  1 , Ji-Ming Bao  1 , Cheng Yang  1 , Hai-Feng Duan  1 , Hong-Yi Wang  1 , Zhi-Peng Huang  1 , Zi-Ke Qin  2 , Cun-Dong Liu  1
Affiliations

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

Ming-Kun Chen et al. Aging (Albany NY). .

Abstract

Chemoresistance is the most significant reason for the failure of cancer treatment following radical cystectomy. The response rate to the first-line chemotherapy of cisplatin and gemcitabine does not exceed 50%. In our previous research, elevated BMI1 (B-cell specific Moloney murine leukemia virus integration region 1) expression in bladder cancer conferred poor survival and was associated with chemoresistance. Herein, via analysis of The Cancer Genome Atlas database and validation of clinical samples, BMI1 was elevated in patients with bladder cancer resistant to cisplatin and gemcitabine, which conferred tumor relapse and progression. Consistently, BMI1 was markedly increased in the established cisplatin- and gemcitabine-resistant T24 cells (T24/DDP&GEM). Functionally, BMI1 overexpression dramatically promoted drug efflux, enhanced viability and decreased apoptosis of bladder cancer cells upon treatment with cisplatin or gemcitabine, whereas BMI1 downregulation reversed this effect. Mechanically, upon interaction with p53, BMI1 was recruited on the promoter of miR-3682-3p gene concomitant with an increase in the mono-ubiquitination of histone H2A lysine 119, leading to transcription repression of miR-3682-3p gene followed by derepression of ABCB1 (ATP binding cassette subfamily B member 1) gene. Moreover, suppression of P-glycoprotein by miR-3682-3p mimics or its inhibitor XR-9576, could significantly reverse chemoresistance of T24/DDP&GEM cells. These results provided a novel insight into a portion of the mechanism underlying BMI1-mediated chemoresistance in bladder cancer.

Keywords: BMI1; P-glycoprotein; bladder cancer; chemoresistance; miR-3682-3p.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Elevated BMI1 in GC-chemoresistant bladder cancer conferred poor prognosis. (A) BMI1 mRNA expression in bladder cancer tissues of patients partial response to chemotherapy versus patients complete response to chemotherapy from TCGA-BLCA database. (B) Relapse-free survival of patients in TCGA-BLCA dataset with low versus high levels of BMI1 mRNA. (C) IHC analysis of BMI1 protein expression in bladder cancer tissues of patients resistant to GC chemotherapy and that sensitive to GC chemotherapy, magnification, ×200 & ×400. (D) Statistical quantification of the IHC score of BMI1 staining in bladder cancer specimens from patients resistant versus sensitive to GC chemotherapy. (E) Relapse-free survival of patients with bladder cancer with low versus high BMI1 expression. (F) Progression-free survival of patients with bladder cancer with low versus high BMI1 expression. *P < 0.05. GC: Cisplatin and Gemcitabine; TCGA: The Cancer Genome Atlas; BLCA: Bladder Urothelial Carcinoma; IHC: Immunohistochemistry; TPM: Transcripts Per Million.
Figure 2
Figure 2
Inhibition of BMI1 overexpression reversed the chemoresistance in GC-resistant T24 cells. (A) Cell proliferation changes of T24 and T24/DDP&GEM cells assessed by cell counting kit-8 assays after treatment with 2 μg/ml DDP or 25 μg/ml GEM. (B) Apoptosis of T24 and T24/DDP&GEM cells detected by the Annexin V/flow cytometric apoptosis assay after treatment with 2 μg/ml DDP or 25 μg/ml GEM. (C) Western Blot analysis of BMI1 protein in T24 and T24/DDP&GEM cells. (D) Western Blot detection of BMI1 protein in T24/DDP&GEM cells upon BMI1 knockout using CRISPR/Cas9 system. (E) Cell proliferation changes of T24/DDP&GEM cells upon knockout of BMI1 were assessed by cell counting kit-8 assays after treatment with 2 μg/ml DDP. (F) Apoptosis of T24/DDP&GEM-sgBMI1 and T24/DDP&GEM cells detected by the Annexin V/flow cytometric apoptosis assay after treatment with 2 μg/ml DDP. (G) Under observation in inverted fluorescence microscope, the fluorescent intensity of dye in indicated cells after transfection with rhodamine 123. (H) The retention rate of rhodamine 123 dye in down-regulating BMI1 or vector control T24/DDP&GEM cells detected by flow cytometry. *P < 0.05. **P < 0.01. ***P < 0.001. GC: cisplatin and gemcitabine; DDP: cisplatin; GEM: gemcitabine.
Figure 3
Figure 3
BMI1 enhanced the chemoresistance of bladder cancer cells. (A, B) Western blot analysis of BMI1 in the indicated BMI1-transduced, BMI1-silenced, or vector control cells. GAPDH was used as the loading control. (C, D) Cell proliferation changes of BMI1-overexpressing, BMI-silenced or vector control cells assessed by cell counting kit-8 assays after treatment with 2 μg/ml DDP. (E, F) Apoptosis of T24 and BIU-87 cells upon BMI1 up-regulation or knock-down was determined by the Annexin V/flow cytometric apoptosis assay after treatment with 2 μg/ml DDP for 72h. (G, H) The retention rate of rhodamine 123 in up-regulating BMI1, down-regulating BMI1 or vector control cells detected by flow cytometry. *P < 0.05. GAPDH: glyceraldehyde3-phosphate dehydrogenase; DDP: cisplatin.
Figure 4
Figure 4
BMI1 activated P- glycoprotein via suppression of miR-3682-3p. (A) qRT-PCR detection of 12 human ABC-transporters, which are associated with drug transport, in T24/DDP&GEM cells upon BMI1 knockdown. (B) Western blot detection of BMI1 and P-GP in T24/DDP&GEM and T24/DDP&GEM-sgBMI1 cells. (C, D) Western blot analysis of BMI1 and P-GP in overexpressing, downregulating BMI1 or vector control cells (T24 and BIU-87). (E) A Venn diagram showing the overlap of candidate miRNAs that were predicted by miRWalk2.0 to potentially bind to the ABCB1 3'-UTR. (F) Heatmap of 6 candidate miRNAs expression in miRNA microarray assay analysis between T24/DDP&GEM and T24/DDP&GEM-sgBMI1 cells. (G) Quantification analysis of miR-3682-3p expression by q-RT-PCR in T24/DDP&GEM and T24/DDP&GEM-sgBMI1 cells. (H) Detection of miR-3682-3p expression by q-RT-PCR in overexpressing, downregulating or vector control cells (T24 and BIU-87). (I) Results of luciferase reporter assay in HEK293T cells with co-transfection of ABCB1 3'-UTR vector (H9688) or mutant control vector (H9689). *P < 0.05. **P < 0.01. ***P < 0.001. DDP: cisplatin; GEM: gemcitabine; P-GP: P-glycoprotein.
Figure 5
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.
Figure 6
Figure 6
Clinical relevance among BMI1/miR-3682-3p/P-GP axis in human bladder cancer. (A) qRT-PCR or Western Blot detection of BMI1, P-GP and miR-3682-3p expression in 8 freshly collected human bladder cancer samples. (B) Correlation analyses among BMI1/miR-3682-3p/P-GP in these 8 bladder cancer tissues. P-GP: P-glycoprotein.
Figure 7
Figure 7
Aberrant BMI1 amplification contributed to BMI1 overexpression and chemoresistance in bladder cancer. (A, B) Analysis of BMI1 copy number variant (CNV) in bladder cancer patients resistant to chemotherapy (A) and that sensitive to chemotherapy (B) in TCGA-BLCA data sets. (C, D) BMI1 gene CNV and corresponding mRNA expression in bladder cancer patients resistant chemotherapy (C) vs. that sensitive to chemotherapy (D) in TCGA-BLCA data sets (P = 0.0254). (E) BMI1 gene CNV and corresponding mRNA expression in a TCGA bladder cancer data set (P < 0.001). (F) Kaplan-Meier analysis of overall survival for patients with amplified or non-amplified BMI1 expression (P = 0.0383). *P < 0.05. **P < 0.01. ***P < 0.001. CNV: copy number variation; TCGA: The Cancer Genome Atlas; BLCA: Bladder Urothelial Carcinoma.
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