Increased FTO Expression Demethylates XBP1 m6A, Thereby Regulating XBP1-C/EBPα and Promoting Hepatocellular Carcinoma Growth

Abstract

Background: N6-methyladenosine (m⁶A) modification predominantly occurs in cancer cells mRNA. The X-box binding protein 1 (XBP1) influences hepatocellular carcinoma (HCC) progression, but its m⁶A regulatory mechanism remains unclear. Furthermore, the dysregulation of CCAAT/enhancer binding proteins alpha (C/EBPα) in liver cancer is influenced by fat mass and obesity-associated protein (FTO) and acts downstream of XBP1. Therefore, this study aims to investigate how FTO catalyzes XBP1 m⁶A demethylation in HCC regulation. Methods: Initially, HepG2 cells were used to construct FTO overexpression and knockdown cells. The cells were divided into the FTO overexpression group (oe-FTO), overexpression control group (oe-NC), FTO knocked-down group (sh-FTO), and control of FTO knocked-down group (sh-NC) groups. RNA immunoprecipitation quantitative polymerase chain reaction (RIP-qPCR) was used to determine the interaction between FTO and XBP1. Furthermore, quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting (WB) analysis were utilized to assess the expression levels of XBP1 and C/EBPα. Additionally, subcutaneous transplanted tumor models were constructed and the tumor size, weight, and occurrence time were monitored. Moreover, Hematoxylin-Eosin (H&E) staining was employed to observe the pathological changes of tumors. m⁶A immunoprecipitation (MeRIP)-qPCR was used to evaluate the XBP1 m⁶A modification levels. qRT-PCR and WB analysis were used to determine the expression levels of XBP1 and C/EBPα. Results: We observed that FTO specifically binds to XBP1 mRNA in HCC cells, indicating a potential regulatory role at the RNA level. At the cellular level, compared to the sh-NC and oe-NC groups, the m⁶A methylation level of XBP1 was significantly increased in the sh-FTO group, while it was decreased in the oe-FTO group (p < 0.05). Furthermore, the mRNA and protein expression levels of FTO, XBP1, and C/EBPα were altered following FTO manipulation. Functional assays demonstrated that FTO overexpression promoted cell proliferation and invasion while inhibiting apoptosis. Conversely, FTO knockdown resulted in decreased cell proliferation and invasion and increased apoptosis. In a mouse xenograft tumor model, we observed rapidly growing tumors in the oe-FTO group, whereas sh-FTO tumors exhibited slower growth. Histological analysis revealed distinct patterns of tumor growth and damage. Collectively, these findings suggest that FTO plays a crucial role in HCC progression through its effects on XBP1 and C/EBPα, providing insights into the potential therapeutic intervention of FTO in hepatocellular carcinoma. Conclusion: FTO overexpression leads to m⁶A demethylation of XBP1, thereby modulating the expression of XBP1-C/EBPα and suppressing cell apoptosis. This, in turn, facilitates the progression of hepatocellular carcinoma by promoting cell growth.