N6-methyladenosine modification of CENPF mRNA facilitates gastric cancer metastasis via regulating FAK nuclear export

Background: N6-methyladenosine (m⁶ A) modification is the most common modification that occurs in eukaryotes. Although substantial effort has been made in the prevention and treatment of gastric Cancer (GC) in recent years, the prognosis of GC patients remains unsatisfactory. The regulatory mechanism between m⁶ A modification and GC development needs to be elucidated. In this study, we examined m⁶ A modification and the downstream mechanism in GC.

Methods: Dot blotting assays, The Cancer Genome Atlas analysis, and quantitative Real-Time PCR (qRT-PCR) were used to measure the m⁶ A levels in GC tissues. Methylated RNA-immunoprecipitation Sequencing and RNA Sequencing were performed to identify the targets of m⁶ A modification. Western blotting, Transwell, wound healing, and angiogenesis assays were conducted to examine the role of centromere protein F (CENPF) in GC in vitro. Xenograft, immunohistochemistry, and in vivo metastasis experiments were conducted to examine the role of CENPF in GC in vivo. Methylated RNA-immunoprecipitation-qPCR, RNA immunoprecipitation-qPCR and RNA pulldown assays were used to verify the m⁶ A modification sites of CENPF. Gain/loss-of-function and rescue experiments were conducted to determine the relationship between CENPF and the mitogen-activated protein kinase (MAPK) signaling pathway in GC cells. Coimmunoprecipitation, mass spectrometry, qRT-PCR, and immunofluorescence assays were performed to explore the proteins that interact with CENPF and elucidate the regulatory mechanisms between them.

Results: CENPF was upregulated in GC and facilitated the metastasis of GC both in vitro and in vivo. Mechanistically, increased m⁶ A modification of CENPF was mediated by methyltransferase 3, and this modified molecule could be recognized by heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1), thereby promoting its mRNA stability. In addition, the metastatic phenotype of CENPF was dependent on the MAPK signaling pathway. Furthermore, CENPF could bind to FAK and promote its localization in the cytoplasm. Moreover, we discovered that high expression of CENPF was related to lymphatic invasion and overall survival in GC patients.

Conclusions: Our findings revealed that increased m⁶ A modification of CENPF facilitates the metastasis and angiogenesis of GC through the CENPF/FAK/MAPK and epithelial-mesenchymal transition axis. CENPF expression was correlated with the clinical features of GC patients; therefore, CENPF may serve as a prognostic marker of GC.

CENPF; FAK; MAPK; N6-methyladenosine; epithelial-mesenchymal transition; gastric cancer; metastasis.