Abstract
To explore the role of forkhead box protein O1 (FOXO1) in the progression of glioblastoma multiforme (GBM) and related drug resistance, we deciphered the roles of FOXO1 and miR-506 in proliferation, apoptosis, migration, invasion, autophagy, and temozolomide (TMZ) sensitivity in the U251 cell line using in vitro and in vivo experiments. Cell viability was tested by a cell counting kit-8 (CCK8) kit; migration and invasion were checked by the scratching assay; apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and flow cytometry. The construction of plasmids and dual-luciferase reporter experiment were carried out to find the interaction site between FOXO1 and miR-506. Immunohistochemistry was done to check the protein level in tumors after the in vivo experiment. We found that the FOXO1-miR-506 axis suppresses GBM cell invasion and migration and promotes GBM chemosensitivity to TMZ, which was mediated by autophagy. FOXO1 upregulates miR-506 by binding to its promoter to enhance transcriptional activation. MiR-506 could downregulate E26 transformation-specific 1 (ETS1) expression by targeting its 3’-untranslated region (UTR). Interestingly, ETS1 promoted FOXO1 translocation from the nucleus to the cytosol and further suppressed the FOXO1-miR-506 axis in GBM cells. Consistently, both miR-506 inhibition and ETS1 overexpression could rescue FOXO1 overactivation-mediated TMZ chemosensitivity in mouse models. Our study demonstrated a negative feedback loop of FOXO1/miR-506/ETS1/FOXO1 in GBM in regulating invasiveness and chemosensitivity. Thus, the above axis might be a promising therapeutic target for GBM.
摘要
为了探索FOXO1在胶质母细胞瘤(GBM)进展和化疗耐药中的作用和机制, 本研究采用体外细胞学实验和动物实验分析FOXO1和miR-506对GBM细胞系U251增殖、 凋亡、 迁移、 侵袭、 自噬和替莫唑胺(TMZ)化疗敏感性的影响, 并通过双荧光素酶报告实验分析FOXO1与miR-506相互作用的靶点. 结果显示: FOXO1-miR-506轴可抑制GBM细胞侵袭和迁移能力, 并促进其对TMZ的化疗敏感性, 且自噬参与其中; FOXO1作为转录因子可与miR-506启动子区域相结合并上调其表达; 此外, miR-506可结合在E26转录因子-1(ETS-1)3’-UTR区并下调ETS1表达, 而ETS1可促进FOXO1从细胞核向细胞浆转移进而抑制FOXO1-miR-506轴. 裸鼠动物实验结果显示, 过表达FOXO1可促进GBM对TMZ的化疗敏感性, 但miR-506抑制剂和过表达ETS1均可逆转该现象. 综上所述, FOXO1/miR-506/ETS1/FOXO1环路参与GBM侵袭性和化疗敏感性的调节, 可作为GBM治疗的潜在靶点.
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Data availability statement
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (Nos. 81402076, 81872072, and 82073274) and the Science Technology Commission of Shanghai Municipality (No. 20S11900700). We thank Dr. Yingmei LI (HaploX Biotechnology Co., Ltd., Shenzhen, China) for manuscript editing.
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Juxiang CHEN and Yufeng SHI conceived and designed the project. Chao CHEN and Yu’e LIU conducted experiments and drafted the manuscript. Hongxiang WANG performed data calculation and validation. Xu ZHANG collected samples and did all the bioinformatic work. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Chao CHEN, Yu’e LIU, Hongxiang WANG, Xu ZHANG, Yufeng SHI, and Juxiang CHEN declared that they have no conflict of interest.
All institutional and national guidelines for the care and use of laboratory animals were followed. The animal experiments were carried out according to the guidelines of the Committee on Ethics of Medicine of Second Military Medical University (No. 20190122), Shanghai, China.
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FOXO1-miR-506 axis promotes chemosensitivity to temozolomide and suppresses invasiveness in glioblastoma through a feedback loop of FOXO1/miR-506/ETS1/FOXO1
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Chen, C., Liu, Y., Wang, H. et al. FOXO1-miR-506 axis promotes chemosensitivity to temozolomide and suppresses invasiveness in glioblastoma through a feedback loop of FOXO1/miR-506/ETS1/FOXO1. J. Zhejiang Univ. Sci. B 24, 698–710 (2023). https://doi.org/10.1631/jzus.B2200503
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DOI: https://doi.org/10.1631/jzus.B2200503
Key words
- Glioblastoma
- Forkhead box protein O1 (FOXO1)
- MiR-506
- E26 transformation specific-1 (ETS1)
- Chemosensitivity