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.Interference with HDAC1 and miR-574-5p reconstituted CerS1-2 expression and C(18) -ceramide generation in multiple human cancer cell lines, which subsequently inhibited proliferation and anchorage-independent growth.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: The optimal core promoter DNA of CerS1 spanning between the −200 and −80 region contains four conserved GC-box Sp1-recognition sequences centred at the −200, −177, −139 and −107 regions, which were referred to as GC-Boxes 1–4, respectively (Fig 3A). To delineate specific roles of these four Sp1-recognition sites in the regulation of the CerS1 promoter, we first generated serial deletion mutants of these GC-box sequences (Fig 3A), and then determined their effects on CerS1 promoter activity in UM-SCC-22A cells using luciferase reporter assays, as described above. Data indicate that deletion of GC-Box 1 had no significant effect on CerS1 core promoter activity (spanning the +1 to −200 region), whereas, deletion of the GC-Boxes 1–2 and 1–2–3 significantly suppressed the CerS1 promoter in an additive manner (about 2.5- and 5-fold, respectively, p < 0.05) compared to the wt (−200) and GC-Box-1-deleted controls (Fig 3B). Deletion of the GC-Box 4 had no additional effect on CerS1 promoter inhibition, compared to the deletions of GC-Boxes 1–2–3 (Fig 3B). Thus, these data suggest that Sp1-recognition sites referred to as GC-Boxes 2 and 3, but not 1 and 4, play important roles in the activation of the core CerS1 promoter, under basal expression conditions. It should also be noted that other transcription factors, such as E2A, CREB, HIF-1 or STAT, whose recognition sites are present on the CerS1 minimal promoter or other factors that associate with the promoter distant regulatory sites, might affect CerS1 transcription in various other cell types and stress conditions.
Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, SC, USA.