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: Then, to assess whether the GC-Boxes 2 and 3 bind Sp1 for the regulation of the CerS1 promoter, we performed EMSA using nuclear extracts isolated from UM-SCC-1 cells, and the [32P]-labelled oligonucleotide (22-mer) containing a conserved Sp1-recognition sequence as a probe with and without unlabelled oligonucleotides containing the Sp1 (cold probe), GC-Boxes 1, 2 or 3 sequences or their mutated variants as competitors. Incubation of the labelled Sp1 probe with the UM-SCC-1 nuclear extracts generated two main shifted bands (complexes 1 and 2), which were competed off when unlabelled wt-probe, but not its mutant, were used as competitors (Fig 4A, lanes 2–4, respectively). As expected, co-incubation of the labelled Sp1 probe with the wt- or mutant GC-Box-1 oligonucleotides had no effect on probe binding with C1 or C2 compared to probe-only control (Fig 4A, lanes 5–6 and 2, respectively). Conversely, while unlabelled oligonucleotides containing wt-GC-Boxes 2 and 3 almost completely prevented the Sp1-C1/C2 binding, their mutated isoforms had no effect compared to probe-only control (Fig 4A, lanes 7–10, compared to lane 2, respectively). We also confirmed that C1/C2 complexes indeed associate with the Sp1 transcription factor using a super-shift assay in the presence of an anti-Sp1 or anti-IgG antibodies in EMSA assays. As seen in Fig 4B, pre-incubation of nuclear extracts with the anti-Sp1 antibody, but not the anti-IgG antibody, caused a super-shift on the mobility of the probe-protein complex (C1), indicating that the C1 contains Sp1-probe complex (Fig 4B). Taken together, both functional (promoter mutations) and biochemical (EMSA) studies suggest that Sp1 induces CerS1 core promoter with the specific involvement of GC-rich sequences within the −177 and −139 regions (GC-Boxes 2 and 3) of the promoter.
Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, SC, USA.