Fig. 6. YTHDF2 mediates the downregulation of FGF14-AS2.

a RIP assay was performed using indicated cell lysates and anti-YTHDF2 antibodies, and the coprecipitated RNAs were subjected to qRT-PCR to detect FGF14-AS2. The fold enrichment of FGF14-AS2 in the YTHDF2 pellet is shown relative to its matching IgG control. b FGF14-AS2 levels were detected by qRT-PCR in MDA-MB-231 cells transfected with YTHDF2 expression plasmids, YTHDF2 siRNA and their respective controls in the presence of actinomycin D (2 μg/mL). c Schematic description of the YTHDF2 domains and mutant construction. d MDA-MB-231 cells were transiently transfected with luciferase reporter constructs containing m⁶A modification sites of FGF14-AS2 together with wild-type or mutated YTHDF2 expression plasmids (YTHDF2-5A-mut), and Firefly and Renilla luciferase activities were evaluated using the Dual-Luciferase Reporter Assay system (Promega). Normalised data were calculated as the quotient of Renilla/Firefly luciferase activities. e FGF14-AS2 levels were detected by qRT-PCR in MDA-MB-231 cells transfected with YTHDF2 wild-type, YTHDF2-5A-mut expression plasmids, or pcDNA3.1 plasmids (control) in the presence of actinomycin D (2 μg/mL). f Expression levels of YTHDF2 in breast cancer tissues and normal tissues were analysed using TCGA and GSE109169 datasets. g YTHDF2 and FGF14-AS2 levels in 39 pairs of breast cancer tissues and corresponding normal tissues were examined using qRT-PCR, respectively. h Spearman correlation analysis of the association between YTHDF2 and FGF14-AS2 expression in 39 breast cancer tissues. i Kaplan–Meier analysis of the correlation of the expression levels of YTHDF2 and FGF14-AS2 with distant metastasis-free survival (DMFS) based on TCGA data. The data are shown as the mean ± s.d. of at least three independent experiments. *P < 0.05, **P < 0.005, ***P < 0.001 and ns, no significance. j Diagram of the proposed mechanism showing the role of FGF14-AS2 as a suppressor in BCa osteolytic metastasis. The upregulated YTHDF2 promotes the degradation of m⁶A-modified FGF14-AS2. The decrease of FGF14-AS2 promotes eIF4E/eIF4G complex formation and inhibits the ubiquitination-mediated degradation of EIF2AK2, which leads to the increase of eIF4E phosphorylation via the p38/Mnk1 pathway. The enhanced eIF4E/eIF4G complex formation and eIF4E phosphorylation results in an increase in RUNX2 mRNA translation, thereby increasing RANKL transcription and eventually promoting osteoclast differentiation and bone metastasis of BCa.