Abstract
Bladder cancer is one of the most prevalent malignancies of the urinary system, and its global incidence is rising, particularly among males. Hypoxia-inducible factor 1α (HIF-1α) plays a central role in enabling tumor cells to adapt to hypoxic conditions; however, its specific mechanisms of action in bladder cancer remain incompletely understood. This study aimed to investigate the effects of HIF-1α knockdown on bladder cancer cell proliferation, apoptosis, migration, invasion, and angiogenesis. The T24 and 5637 bladder cancer cell lines were employed. Specific si-HIF-1α plasmids were constructed and transfected into the cells. A series of functional assays were conducted, including MTT viability tests, flow cytometry for apoptosis analysis, Transwell migration and invasion assays, and in vitro angiogenesis experiments. Western blotting was performed to analyze the expression of tissue inhibitor of metalloproteinase 3 (TIMP3). Subsequent functional rescue experiments were carried out by overexpressing TIMP3. Additionally, clinical tissue samples were collected to assess the levels of HIF-1α and TIMP3. The results showed that under hypoxic conditions, knockdown of HIF-1α enhanced bladder cancer cell proliferation, migration, invasion, and angiogenesis while suppressing apoptosis. Furthermore, hypoxia was found to suppress TIMP3 expression in HIF-1α-knockdown cells. Notably, overexpression of TIMP3 reversed the alterations in bladder cancer cell behavior induced by HIF-1α knockdown. Analysis of clinical samples revealed that HIF-1α levels were significantly elevated in bladder cancer tissues. These findings uncover a complex regulatory role for HIF-1α in bladder cancer and identify TIMP3 as a critical mediator of its effects. Further studies are warranted to elucidate the transcriptional regulation of TIMP3 by HIF-1α and to validate its role in vivo, which may contribute to the advancement of targeted therapeutic strategies for bladder cancer.
Data availability
The datasets generated and analyzed during the current study are not publicly available due to patient privacy considerations but are available from the corresponding author upon reasonable request. No sequencing, proteomics, or other high-throughput datasets were generated in this study.
References
Siegel, R. L. et al. Cancer statistics. CA Cancer J. Clin. 75 (1), 10–45. https://doi.org/10.3322/caac.21871 (2025).
Lobo, N. et al. Epidemiology, Screening, and prevention of bladder cancer. Eur. Urol. Oncol. 5 (6), 628–639. https://doi.org/10.1016/j.euo.2022.10.003 (2022).
Guo, C. C. et al. Molecular profile of bladder cancer progression to clinically aggressive subtypes. Nat. Rev. Urol. 21 (7), 391–405. https://doi.org/10.1038/s41585-023-00847-7 (2024).
Tassinari, E. et al. State of the Art of adjuvant immunotherapy in urothelial cancer: new developments and upcoming changes. Hum. Vaccin Immunother. 21 (1), 2440165. https://doi.org/10.1080/21645515.2024.2440165 (2025).
Weiner, A. B. et al. Predictors of use and overall survival for patients undergoing metastasectomy for bladder cancer in a National cohort. Int. J. Urol. 27 (9), 736–741. https://doi.org/10.1111/iju.14288 (2020).
Kawahara, I. et al. Targeting metabolic reprogramming to overcome drug resistance in advanced bladder cancer: insights from gemcitabine- and cisplatin-resistant models. Mol. Oncol. 18 (9), 2196–2211. https://doi.org/10.1002/1878-0261.13684 (2024).
Patel, V. G., Oh, W. K. & Galsky, M. D. Treatment of muscle-invasive and advanced bladder cancer in 2020. CA Cancer J. Clin. 70 (5), 404–423. https://doi.org/10.3322/caac.21631 (2020).
Zhao, Y., Xing, C., Deng, Y., Ye, C. & Peng, H. HIF-1α signaling: essential roles in tumorigenesis and implications in targeted therapies. Genes Dis. 11 (1), 234–251. https://doi.org/10.1016/j.gendis.2023.02.039 (2023).
Infantino, V., Santarsiero, A., Convertini, P., Todisco, S. & Iacobazzi, V. Cancer cell metabolism in hypoxia: role of HIF-1 as key regulator and therapeutic target. Int. J. Mol. Sci. 22 (11), 5703. https://doi.org/10.3390/ijms22115703 (2021).
Dong, S. et al. ROS/PI3K/Akt and Wnt/β-catenin signalings activate HIF-1α-induced metabolic reprogramming to impart 5-fluorouracil resistance in colorectal cancer. J. Exp. Clin. Cancer Res. 41 (1), 15. https://doi.org/10.1186/s13046-021-02229-6 (2022).
Lee, J. W., Bae, S. H., Jeong, J. W., Kim, S. H. & Kim, K. W. Hypoxia-inducible factor (HIF-1)alpha: its protein stability and biological functions. Exp. Mol. Med. 36 (1), 1–12. https://doi.org/10.1038/emm.2004.1 (2004).
Sun, J. et al. The tumor microenvironment mediates the HIF-1α/PD-L1 pathway to promote immune escape in colorectal cancer. Int. J. Mol. Sci. 25 (7), 3735. https://doi.org/10.3390/ijms25073735 (2024).
Clara, C. A. et al. Angiogenesis and expression of PDGF-C, VEGF, CD105 and HIF-1α in human glioblastoma. Neuropathology 34 (4), 343–352. https://doi.org/10.1111/neup.12111 (2014).
Saxena, K., Jolly, M. K. & Balamurugan, K. Hypoxia, partial EMT and collective migration: emerging culprits in metastasis. Transl Oncol. 13 (11), 100845. https://doi.org/10.1016/j.tranon.2020.100845 (2020).
Rankin, E. B. & Giaccia, A. J. The role of hypoxia-inducible factors in tumorigenesis. Cell. Death Differ. 15 (4), 678–685. https://doi.org/10.1038/cdd.2008.21 (2008).
Tiwari, A. et al. Loss of HIF1A from pancreatic cancer cells increases expression of PPP1R1B and degradation of p53 to promote invasion and metastasis. Gastroenterology 159 (5), 1882–1897e5. https://doi.org/10.1053/j.gastro.2020.07.046 (2020).
Han, X. G. et al. TIMP3 regulates osteosarcoma cell migration, invasion, and chemotherapeutic resistances. Tumour Biol. 37 (7), 8857–8867. https://doi.org/10.1007/s13277-015-4757-4 (2016).
Anania, M. C. et al. TIMP3 regulates migration, invasion and in vivo tumorigenicity of thyroid tumor cells. Oncogene 30 (27), 3011–3023. https://doi.org/10.1038/onc.2011.18 (2011).
Zhan, S. et al. TGFBI promotes proliferation and epithelial-mesenchymal transition in renal cell carcinoma through PI3K/AKT/mTOR/HIF-1α pathway. Cancer Cell. Int. 24 (1), 265. https://doi.org/10.1186/s12935-024-03454-7 (2024).
Zhou, J. et al. Clinical and prognostic significance of HIF-1α overexpression in oral squamous cell carcinoma: a meta-analysis. World J. Surg. Oncol. 15 (1), 104. https://doi.org/10.1186/s12957-017-1163-y (2017).
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Xiaoxian Wang 1,†, Jian Guo2,†, Ruoran Zhang3,†, Yang Dong1, Lin Hao1, Bo Chen1, Hao Xu4, Yuyang Ma5, Kun Pang1,*. † These authors have contributed equally to this work and share the first authorship.* Correspondence.
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Wang, X., Guo, J., Zhang, R. et al. Hypoxia-inducible factor 1α exerts dual roles in bladder cancer progression through TIMP3-mediated regulation of angiogenesis and invasion. Sci Rep (2026). https://doi.org/10.1038/s41598-026-39635-9
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DOI: https://doi.org/10.1038/s41598-026-39635-9
