DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3
Abstract Background The RAR-related orphan receptor alpha (RORA), a circadian clock molecule, is highly associated with anti-oncogenes. In this paper, we defined the precise action and mechanistic basis of RORA in ESCC development under hypoxia. Methods Expression analysis was conducted by RT-qPCR,...
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BMC
2024-12-01
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| Series: | Journal of Translational Medicine |
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| Online Access: | https://doi.org/10.1186/s12967-024-05960-8 |
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| author | Wenjian Yao Linlin Shang Yinghao Wang Lei Xu Yu Bai Mingyu Feng Xiangbo Jia Sen Wu |
| author_facet | Wenjian Yao Linlin Shang Yinghao Wang Lei Xu Yu Bai Mingyu Feng Xiangbo Jia Sen Wu |
| author_sort | Wenjian Yao |
| collection | DOAJ |
| description | Abstract Background The RAR-related orphan receptor alpha (RORA), a circadian clock molecule, is highly associated with anti-oncogenes. In this paper, we defined the precise action and mechanistic basis of RORA in ESCC development under hypoxia. Methods Expression analysis was conducted by RT-qPCR, western blotting, immunofluorescence (IF), and immunohistochemistry (IHC) assays. The functions of RORA were assessed by detecting its regulatory effects on cell viability, motility, invasion, and tumor growth. DNA pull-down assay and proteomic analysis were employed to identify proteins bound to the RORA promoter. The promoter methylation level of RORA was detected by DNA pyrosequencing. RNA-seq analysis was performed to explore the downstream mechanisms of RORA, and the transcriptional regulation of RORA on SLC2A3 was verified by ChIP-qPCR and dual-luciferase reporter assay. Glycolysis was assessed by detecting the consumption of glucose and the production of lactic acid and ATP. Results In vitro, RORA was shown to suppress ESCC cell viability, motility, and invasion under hypoxic condition. In vivo, increased RORA expression in mouse xenografts impeded tumor growth. DNMT1 was identified to widely exist in the RORA promoter, increasing DNA methylation and reducing RORA expression in hypoxia-induced KYSE150 ESCC cells. Mechanistically, RORA was found to inactivate the transcription of glucose transporter protein SLC2A3 by interacting with its promoter F1 region. Furthermore, rescue experiments revealed that RORA-mediated suppressive effects on ESCC cell migration and invasion were largely based on its negative regulation of SLC2A3 and glycolysis. Conclusion DNMT1-driven methylation of RORA promotes ESCC progression largely through affecting SLC2A3 transcription and glycolysis. These findings turn RORA into potential target of anti-cancer therapeutic agents. |
| format | Article |
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| institution | Kabale University |
| issn | 1479-5876 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | BMC |
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| series | Journal of Translational Medicine |
| spelling | doaj-art-6bb9e9ca326a4d5faa6c5825a1002b212025-01-05T12:44:18ZengBMCJournal of Translational Medicine1479-58762024-12-0122111710.1186/s12967-024-05960-8DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3Wenjian Yao0Linlin Shang1Yinghao Wang2Lei Xu3Yu Bai4Mingyu Feng5Xiangbo Jia6Sen Wu7Department of Thoracic Surgery, School of Clinical Medicine, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, Henan UniversityDepartment of Thoracic Surgery, Zhengzhou University People’s Hospital, Henan Provincial People’s HospitalDepartment of Thoracic Surgery, Henan University People’s Hospital, Henan Provincial People’s HospitalDepartment of Thoracic Surgery, School of Clinical Medicine, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, Henan UniversityDepartment of Pathology, Xinxiang Medical UniversityDepartment of Education, Xuanwu Hospital, Capital Medical UniversityDepartment of Thoracic Surgery, School of Clinical Medicine, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, Henan UniversityDepartment of Thoracic Surgery, School of Clinical Medicine, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, Henan UniversityAbstract Background The RAR-related orphan receptor alpha (RORA), a circadian clock molecule, is highly associated with anti-oncogenes. In this paper, we defined the precise action and mechanistic basis of RORA in ESCC development under hypoxia. Methods Expression analysis was conducted by RT-qPCR, western blotting, immunofluorescence (IF), and immunohistochemistry (IHC) assays. The functions of RORA were assessed by detecting its regulatory effects on cell viability, motility, invasion, and tumor growth. DNA pull-down assay and proteomic analysis were employed to identify proteins bound to the RORA promoter. The promoter methylation level of RORA was detected by DNA pyrosequencing. RNA-seq analysis was performed to explore the downstream mechanisms of RORA, and the transcriptional regulation of RORA on SLC2A3 was verified by ChIP-qPCR and dual-luciferase reporter assay. Glycolysis was assessed by detecting the consumption of glucose and the production of lactic acid and ATP. Results In vitro, RORA was shown to suppress ESCC cell viability, motility, and invasion under hypoxic condition. In vivo, increased RORA expression in mouse xenografts impeded tumor growth. DNMT1 was identified to widely exist in the RORA promoter, increasing DNA methylation and reducing RORA expression in hypoxia-induced KYSE150 ESCC cells. Mechanistically, RORA was found to inactivate the transcription of glucose transporter protein SLC2A3 by interacting with its promoter F1 region. Furthermore, rescue experiments revealed that RORA-mediated suppressive effects on ESCC cell migration and invasion were largely based on its negative regulation of SLC2A3 and glycolysis. Conclusion DNMT1-driven methylation of RORA promotes ESCC progression largely through affecting SLC2A3 transcription and glycolysis. These findings turn RORA into potential target of anti-cancer therapeutic agents.https://doi.org/10.1186/s12967-024-05960-8RORAEsophageal squamous cell carcinomaDNA methylationGlycolysisDNMT1SLC2A3 |
| spellingShingle | Wenjian Yao Linlin Shang Yinghao Wang Lei Xu Yu Bai Mingyu Feng Xiangbo Jia Sen Wu DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3 Journal of Translational Medicine RORA Esophageal squamous cell carcinoma DNA methylation Glycolysis DNMT1 SLC2A3 |
| title | DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3 |
| title_full | DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3 |
| title_fullStr | DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3 |
| title_full_unstemmed | DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3 |
| title_short | DNMT1-driven methylation of RORA facilitates esophageal squamous cell carcinoma progression under hypoxia through SLC2A3 |
| title_sort | dnmt1 driven methylation of rora facilitates esophageal squamous cell carcinoma progression under hypoxia through slc2a3 |
| topic | RORA Esophageal squamous cell carcinoma DNA methylation Glycolysis DNMT1 SLC2A3 |
| url | https://doi.org/10.1186/s12967-024-05960-8 |
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