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Concerted functions of HDAC1 and microRNA-574-5p repress alternatively spliced ceramide synthase 1 expression in human cancer cells.

Meyers-Needham M, Ponnusamy S, Gencer S, Jiang W, Thomas RJ, Senkal CE, Ogretmen B - EMBO Mol Med (2011)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, SC, USA.

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Reconstitution of CerS1-2 expression by inhibition of HDAC1 and miR-574-5pA.CerS1 protein in response to MS-275 (lanes 2 and 4) in the absence/presence of siRNA against miR-574-5p (lanes 3 and 4), compared to controls (lanes 1 and 3) was detected by Western blotting, and normalized to actin in UM-SCC-22A or UM-SCC-1 cells (left and right panels, respectively).B. Levels of C17–C18-ceramide in response to MS-275 in the absence/presence of siRNA against miR-574-5p were detected in UM-SCC cells using RT-PCR and LC/MS/MS, respectively.C-D. Effects of MS-275 and knockdown of miR-574-5p using siRNA on the growth of UM-SCC-22A cells were measured by trypan blue exclusion (C), or in UM-SCC-1 cells by Cyquant (D) in the absence/presence of Scr or CerS1 siRNAs.E. Regulation of anchorage-independent growth of UM-SCC-22A cells by combination of MS-275 and miR-574-5p siRNA treatment was assessed by measurement of growth on soft agar. These data represent at least two-independent trials performed as duplicates. The error bars represent the standard deviations.F. Suggested novel mechanism for the regulation of CerS1 expression and C18-ceramide generation by the coordinated functions of HDAC1 and miR-574-5p in human cancer cells is shown.
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fig09: Reconstitution of CerS1-2 expression by inhibition of HDAC1 and miR-574-5pA.CerS1 protein in response to MS-275 (lanes 2 and 4) in the absence/presence of siRNA against miR-574-5p (lanes 3 and 4), compared to controls (lanes 1 and 3) was detected by Western blotting, and normalized to actin in UM-SCC-22A or UM-SCC-1 cells (left and right panels, respectively).B. Levels of C17–C18-ceramide in response to MS-275 in the absence/presence of siRNA against miR-574-5p were detected in UM-SCC cells using RT-PCR and LC/MS/MS, respectively.C-D. Effects of MS-275 and knockdown of miR-574-5p using siRNA on the growth of UM-SCC-22A cells were measured by trypan blue exclusion (C), or in UM-SCC-1 cells by Cyquant (D) in the absence/presence of Scr or CerS1 siRNAs.E. Regulation of anchorage-independent growth of UM-SCC-22A cells by combination of MS-275 and miR-574-5p siRNA treatment was assessed by measurement of growth on soft agar. These data represent at least two-independent trials performed as duplicates. The error bars represent the standard deviations.F. Suggested novel mechanism for the regulation of CerS1 expression and C18-ceramide generation by the coordinated functions of HDAC1 and miR-574-5p in human cancer cells is shown.

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.


Concerted functions of HDAC1 and microRNA-574-5p repress alternatively spliced ceramide synthase 1 expression in human cancer cells.

Meyers-Needham M, Ponnusamy S, Gencer S, Jiang W, Thomas RJ, Senkal CE, Ogretmen B - EMBO Mol Med (2011)

Reconstitution of CerS1-2 expression by inhibition of HDAC1 and miR-574-5pA.CerS1 protein in response to MS-275 (lanes 2 and 4) in the absence/presence of siRNA against miR-574-5p (lanes 3 and 4), compared to controls (lanes 1 and 3) was detected by Western blotting, and normalized to actin in UM-SCC-22A or UM-SCC-1 cells (left and right panels, respectively).B. Levels of C17–C18-ceramide in response to MS-275 in the absence/presence of siRNA against miR-574-5p were detected in UM-SCC cells using RT-PCR and LC/MS/MS, respectively.C-D. Effects of MS-275 and knockdown of miR-574-5p using siRNA on the growth of UM-SCC-22A cells were measured by trypan blue exclusion (C), or in UM-SCC-1 cells by Cyquant (D) in the absence/presence of Scr or CerS1 siRNAs.E. Regulation of anchorage-independent growth of UM-SCC-22A cells by combination of MS-275 and miR-574-5p siRNA treatment was assessed by measurement of growth on soft agar. These data represent at least two-independent trials performed as duplicates. The error bars represent the standard deviations.F. Suggested novel mechanism for the regulation of CerS1 expression and C18-ceramide generation by the coordinated functions of HDAC1 and miR-574-5p in human cancer cells is shown.
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Related In: Results  -  Collection

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fig09: Reconstitution of CerS1-2 expression by inhibition of HDAC1 and miR-574-5pA.CerS1 protein in response to MS-275 (lanes 2 and 4) in the absence/presence of siRNA against miR-574-5p (lanes 3 and 4), compared to controls (lanes 1 and 3) was detected by Western blotting, and normalized to actin in UM-SCC-22A or UM-SCC-1 cells (left and right panels, respectively).B. Levels of C17–C18-ceramide in response to MS-275 in the absence/presence of siRNA against miR-574-5p were detected in UM-SCC cells using RT-PCR and LC/MS/MS, respectively.C-D. Effects of MS-275 and knockdown of miR-574-5p using siRNA on the growth of UM-SCC-22A cells were measured by trypan blue exclusion (C), or in UM-SCC-1 cells by Cyquant (D) in the absence/presence of Scr or CerS1 siRNAs.E. Regulation of anchorage-independent growth of UM-SCC-22A cells by combination of MS-275 and miR-574-5p siRNA treatment was assessed by measurement of growth on soft agar. These data represent at least two-independent trials performed as duplicates. The error bars represent the standard deviations.F. Suggested novel mechanism for the regulation of CerS1 expression and C18-ceramide generation by the coordinated functions of HDAC1 and miR-574-5p in human cancer cells is shown.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, SC, USA.

Show MeSH
Related in: MedlinePlus