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LncRNA GAS5 regulated by FTO-mediated m6A demethylation promotes autophagic cell death in NSCLC by targeting UPF1/BRD4 axis

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Abstract

Long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) has been shown to be a regulator for many cancers, including non-small cell lung cancer (NSCLC). Therefore, its role and mechanism in the process of NSCLC deserve to be further revealed. The expression levels of GAS5, fat mass and obesity-associated protein (FTO) and bromodomain-containing protein 4 (BRD4) were detected by quantitative real-time PCR. Western blot analysis was used to examine the protein expression of FTO, BRD4, up-frameshift protein 1 (UPF1) and autophagy-related markers. Methylated RNA immunoprecipitation was used to assess the m6A level of GAS5 regulated by FTO. Cell proliferation and apoptosis were determined using MTT assay, EdU assay and flow cytometry. Autophagy ability was assessed by immunofluorescence staining and transmission electron microscope. Xenograft tumor model was constructed to explore the effects of FTO and GAS5 on NSCLC tumor growth in vivo. The interaction between UPF1 and GAS5 or BRD4 was confirmed by pull-down assay, RIP assay, dual-luciferase reporter assay, and chromatin immunoprecipitation. Fluorescent in situ hybridization was used to analyze the co-localization of GAS5 and UPF1. Actinomycin D treatment was employed to evaluate BRD4 mRNA stability. GAS5 was downregulated in NSCLC tissues and was associated with poor prognosis in NSCLC patients. FTO was highly expressed in NSCLC, and it inhibited GAS5 expression by reducing GAS5 m6A methylation level. GAS5 suppressed by FTO could promote the autophagic death of NSCLC cells in vitro and inhibit NSCLC tumor growth in vivo. In addition, GAS5 was able to interact with UPF1 to reduce the mRNA stability of BRD4. Knockdown of BRD4 reversed the inhibition of GAS5 or UPF1 silencing on the autophagic cell death of NSCLC. The findings of the study showed that lncRNA GAS5 mediated by FTO could contribute to the autophagic cell death of NSCLC by interacting with UPF1 to reduce BRD4 mRNA stability, suggesting that GAS5 might be a vital therapy target for NSCLC progression.

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All data generated or analyzed during this study are included in this published article.

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Funding

This study was supported by Hainan Province Science and Technology Special Fund (Grant No. ZDYF2023SHFZ133) and Hainan Provincial Natural Science Foundation of China (Grant No. 823MS142).

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  1. Yihui Fu and Lirong Liu are co-first authors.

Authors and Affiliations

  1. Department of Respiratory and Critical Care Medicine, Hainan General Hospital (Hainan Affiliated Hospital of Hainan Medical University), Haikou, 570311, People’s Republic of China

    Yihui Fu, Lirong Liu, Haihong Wu & Yamei Zheng

  2. Department of Pharmacy, Hainan General Hospital (Hainan Affiliated Hospital of Hainan Medical University), Haikou, People’s Republic of China

    Huijuan Zhan

  3. Department of Thoracic Surgery, Hainan General Hospital (Hainan Affiliated Hospital of Hainan Medical University), No. 19, Xiuhua Road, Xiuying District, Haikou, 570311, Hainan, People’s Republic of China

    Liang Li

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YHF and LRL conceived and designed research. YHF and HHW conducted experiments. HHW, YMZ and HJZ analyzed data. LL wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Liang Li.

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Each patient signed written informed consent, and the research was approved by the Ethics Committee of the Hainan Affiliated Hospital of Hainan Medical University.

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Fu, Y., Liu, L., Wu, H. et al. LncRNA GAS5 regulated by FTO-mediated m6A demethylation promotes autophagic cell death in NSCLC by targeting UPF1/BRD4 axis. Mol Cell Biochem 479, 553–566 (2024). https://doi.org/10.1007/s11010-023-04748-6

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