IGF2BP1 knockdown associates with alternative splicing changes of transcription factor genes in neuroblastoma cells

Abstract

IGF2BP1 is an oncofetal RNA-binding protein with oncogenic functions in neuroblastoma, but whether IGF2BP1 knockdown is associated with alternative splicing (AS) changes of transcription factor (TF) genes remains unclear. We re-analyzed RNA-seq data from IGF2BP1-knockdown BE(2)-C cells (three knockdown and three control samples). Differentially expressed genes (DEGs) and AS events (ASEs) were identified, followed by GO/KEGG enrichment. Differentially spliced genes were intersected with a human TF list. Isoform-specific qRT-PCR was performed in control and IGF2BP1-knockdown BE(2)-C cells to validate PSI changes. RIP-qPCR was used to test binding of IGF2BP1 to candidate TF mRNAs. ENCODE eCLIP-seq data from HepG2 and K562 cells were analyzed as indirect supportive evidence. We identified 807 DEGs and 3, 053 significant ASEs. Among genes with significant splicing changes, 64 were annotated as TFs; 20 splicing events corresponding to 18 genes passed stringent filtering. Independent qRT-PCR validated the PSI changes of LMO4 (A5SS), TCF7L2 (cassette exon), and EHMT2 (exon skipping), which correlated with RNA-seq predictions. RIP-qPCR confirmed that IGF2BP1 binds to LMO4, TCF7L2, and EHMT2 mRNAs in cells, but not to NCOR2. eCLIP-seq data from non-neuroblastoma cell lines provided indirect supporting evidence. IGF2BP1 knockdown is associated with widespread AS changes of TF genes in neuroblastoma cells, and IGF2BP1 binds to LMO4, TCF7L2, and EHMT2 mRNAs. These findings provide a correlative resource and hypotheses for future mechanistic studies; direct causality remains to be established.