m6A Demethylase FTO Promotes Lung Cancer Progression in Vitro and in Vivo by Inhibiting CLIC5

Abstract

Background: Lung cancer (LC), a leading cause of cancer-related mortality worldwide, is a major health concern. The N6-methyladenosine (m⁶A) methylation, a pivotal RNA modification, plays a crucial role in the progression of LC. Therefore, this study aimed to identify m⁶A modification-related mechanisms underpinning LC progression. Methods: Expression levels of chloride intracellular ion channel 5 (CLIC5), fat mass and obesity-associated protein (FTO), and the proto-oncogene tyrosine-protein kinase SRC (SRC) in LC cells and xenograft tumors were assessed utilizing bioinformatics tools, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and Western blot analysis. Furthermore, their interactions were predicted and validated using bioinformatics tools and/or Methylated RNA immunoprecipitation (MeRIP) assay. After CLIC5/FTO overexpression in LC cells, their roles in LC cell proliferation, migration, invasion, and tumorigenesis were examined employing cell counting kit-8 (CCK-8), 5-Ethynyl-2′-deoxyuridine (EdU), Transwell, and murine xenograft assays. Results: CLIC5 was downregulated in LC cells (p < 0.001). CLIC5 overexpression inhibited cell viability, proliferation, migration, invasion, and tumorigenesis in LC (p < 0.01). The m⁶A level was decreased while the FTO level was increased in LC cells (p < 0.001). FTO could interact with the m⁶A modification site on CLIC5, and the m⁶A methylation of CLIC5 was potentiated following FTO knockdown in LC cells (p < 0.001). Furthermore, FTO overexpression reversed the inhibitory effect of CLIC5 overexpression on the proliferation, migration, invasion, and tumorigenesis in LC (p < 0.05). Additionally, elevated SRC expression in LC could interact with CLIC5 (p < 0.001). CLIC5 overexpression diminished SRC expression, and the effect of CLIC5 on the SRC expression was reversed by FTO overexpression. Conclusions: The downregulation of CLIC5, induced by FTO-mediated m⁶A demethylation, facilitates LC progression both in vitro and in vivo.