YTHDF3 modulates the progression of breast cancer cells by regulating FGF2 through mA methylation.

INTRODUCTION

Breast cancer (BC) is a prevailing malignancy among women, and its inconspicuous development contributes significantly to mortality. The RNA N6-methyladenosine (mA) modification represents an emerging mechanism for gene expression regulation, with the active involvement of the YTH N6-methyladenosine RNA binding protein 3 (YTHDF3) in tumor progression across multiple cancer types. Nonetheless, its precise function in breast cancer necessitates further investigation.

METHODS

The expression of YTHDF3 in both cell lines and patient tissues was examined using Western blotting, reverse transcription quantitative PCR (RT-qPCR), and immunohistochemistry (IHC) techniques. Bioinformatics analysis of methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptome RNA sequencing (RNA-seq) data was employed to screen for the target genes of YTHDF3. The main focus of this study was to investigate the biological functions of YTHDF3. The specific binding of YTHDF3 to its target genes and its correlation with mA methylation were studied through RNA pull-down, RNA immunoprecipitation, and co-immunoprecipitation experiments. The protein regulatory mechanisms of downstream genes of YTHDF3 were assessed using protein stability analysis. Furthermore, the biological functions of YTHDF3 and its target genes in breast cancer cells were validated through CRISPR-Cas9 technology and rescue experiments.

RESULTS

By constructing a risk model using the TCGA database, YTHDF3 was identified as a high-risk factor among mA methylation factors. Subsequent investigations revealed its elevated expression in various subtypes of breast cancer, accompanied by poor prognosis. MeRIP-seq analysis further revealed fibroblast growth factor 2 (FGF2) as a downstream gene of YTHDF3. Knockdown of YTHDF3 in breast cancer cells led to significant inhibition of cell self-renewal, migration, and invasion abilities . Mechanistically, YTHDF3 specifically recognized the methylated transcript of FGF2 within its coding sequence (CDS) region, leading to the inhibition of FGF2 protein degradation. Moreover, depletion of FGF2 markedly suppressed the biological functions of breast cancer cells, while reducing FGF2 expression in YTHDF3-overexpressing breast cancer cell lines substantially alleviated the malignant progression.

CONCLUSIONS

In summary, our study elucidates the role of YTHDF3 as an oncogene in maintaining FGF2 expression in BC cells through an mA-dependent mechanism. Additionally, we provide a potential biomarker panel for prognostic prediction in BC.