METTL3 and METTL14 drive pancreatic adenocarcinoma progression via m6A-dependent alternative splicing in PANC-1 cells.

Unlabelled: METTL3 and METTL14, key components of the m6A methyltransferase complex, have been extensively studied in the context of various cancers. However, their roles in regulating alternative splicing in pancreatic cancer remain largely unexplored. In this study, we analyzed high-throughput RNA-seq data (GSE146806), alongside CLIP-seq datasets (GSE132306 and SRP163326), to investigate the molecular mechanisms underlying METTL3_14-mediated regulation. The results of differentially expressed genes (DEG) showed that that METTL3_14 knockdown significantly altered the expression of genes associated with canonical tumor-related pathways. Alternative splicing analysis identified METTL3_14-regulated alternatively spliced genes (RASGs) enriched in pathways such as protein processing in the endoplasmic reticulum, the spliceosome, and HIF-1 signaling, which are closely related to the progression of pancreatic cancer. Reanalysis of CLIP-seq data showed that METTL3_14 preferentially binds to the 3′UTR and coding sequences (CDS) regions. A total of 17 genes overlapped between DEGs and m6A-modified genes, while 59 genes overlapped with RASGs. Validation by qPCR confirmed significant regulatory effects on seven genes, especially EIF4A2, whose splicing was directly modulated by METTL3_14. These findings indicate that METTL3_14 may regulate gene expression and alternative splicing via m6A modifications in pancreatic cancer, particularly in pathways involved in transcription and DNA damage repair. This study provides novel mechanistic insights and potential targets for clinical diagnosis and treatment of pancreatic cancer.

Supplementary Information: The online version contains supplementary material available at 10.1007/s12672-025-03393-3.