Concerted functions of HDAC1 and microRNA-574-5p repress alternatively spliced ceramide synthase 1 expression in human cancer cells.
Bottom Line: Moreover, an alternatively spliced variant CerS1 mRNA (CerS1-2) was detected mainly in cancer cells or primary tumour tissues compared to controls, which was targeted by miR-574-5p for degradation.A specific 3'UTR-targeting site, localized within the retained intron between exons 6 and 7, was identified, and its mutation, or miR-574-5p knockdown prevented the degradation of CerS1-2 mRNA.Accordingly, knockdown of CerS1 partially protected cancer cells from MS-275/miR-574-5p siRNA-mediated growth inhibition.
Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, SC, USA.Show MeSH
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Mentions: These data suggested that CerS1 transcription is regulated by both epigenetic repression and post-transcriptional mRNA degradation via HDAC1 and miR-574-5p, respectively, and to optimally increase CerS1 expression, both of these negative regulators might require simultaneous inhibition. Indeed, knockdown of miR-574-5p and HDAC1, but not HDAC3, using siRNAs increased CerS1 mRNA approximately 4-fold compared to controls (Fig 8A) in UM-SCC-1 cells. Accordingly, treatment of multiple human cancer cells (UM-SCC-1 or UM-SCC-22A-HNSCC, K562-chronic myeloid leukaemia or Daoy-medulloblastoma) cells with HDAC1 inhibitor MS-275 in the presence of the siRNA against miR-574-5p, which effectively decreased miR-574-5p by approximately 70% in these cell lines (Fig S5A–D of Supporting Information), increased CerS1 mRNA approximately 2.0-, 1.7-, 22- and 2.5-fold, respectively (Fig 8B–E). Indeed, combination treatment with MS-275 and knockdown of miRNA-574-5p using siRNA increased CerS1-2 mRNA around 2- and 4-fold in UM-SCC-22A and Daoy cells compared to controls (Fig 8F). These data were also consistent with increased CerS1-2 protein expression around 1.9- or 3.0-fold compared to controls in UM-SCC-22A or UM-SCC-1 cells, respectively (Fig 9A). Importantly, treatment of UM-SCC-22A cells with MS-275 in combination with knockdown of miRNA-574-5p using siRNA increased (2.5-fold) C17–C18-ceramide (Spassieva et al, 2007) generation (Fig 9B). There were no significant changes in C17–C16-ceramide, which is generated by CerS5 and CerS6 (Fig S5E of Supporting Information). Treatment with MS-275 or siRNA against miRNA-574-5p alone had no significant effect on ceramide generation (Fig 9B). Accordingly, ectopic expression of CerS1-2-FLAG was as effective as CerS1-1-FLAG (their equal expression was determined by Western blotting using anti-FLAG antibody, see Fig S6A of Supporting Information) in the generation of C18- and C18:1-ceramides, but not other ceramide species (Fig S6B and C of Supporting Information), confirming that CerS1-2 plays a role in the selective generation of C18-ceramide as CerS1-1. Overall, these data suggest that interference with HDAC1 and miR-574-5p in combination reconstitutes CerS1-2, which increases C18-ceramide generation.
Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, SC, USA.