Alternative splicing of KDM6A regulated by 25(OH)D promotes proliferation and stemness by increasing the transcription of TRAP1 in breast cancer

25-hydroxyvitamin D (25(OH)D), a vitamin D metabolite, has been shown to have anticancer effects. However, its anticancer effect through the regulation of alternative splicing has rarely been investigated. Our study found that 25(OH)D promotes exon 13 skipping of full length isoform of KDM6A (+exon13,NM_001291415.2,KDM6A-L) to generate the short isoform (-exon13,NM_001291416,KDM6A-S). Further experiments demonstrated that, compared to KDM6A-L, KDM6A-S lost its effect to promote proliferation and stemness of tumour cells, and lacks H3K27 demethylase activity.Anti-H3K27me3 CUT&Tag assay was conducted in MDA-MB-453 cells overexpressed KDM6A-L, KDM6A-S and Vector (n=3). Data showed that KDM6A-S overexpression group lost its demethylation of H3K27me3 in the transcriptional start site (TSS) of TRAP1. RNA-seq of three groups (n=3 per group) showed that mRNA expression of TRAP1 is significantly lower in KDM6A-S group than KDM6A-L group. Besides, GO enrichment of RNA-seq data showed a reduction in TGF-b/Smad pathway. ChIP-qPCR and WB assays confirmed that KDM6A-S isoform lost its demethylation of H3K27me3 in the promoter region of TRAP1, resulting in the inability to activate TRAP1 transcription and a reduced level of phosphorylation of the downstream pathway Smad protein. Our study validated that 25(OH)D inhibiting the proliferation and stemness of breast cancer cells through KDM6A exon13 skipping event, and explored the molecular mechanism.