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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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Regulation of CerS1 expression via decreased mRNA stability in HNSCC versus keratinocytesA. UM-SCC-1 cells in 6-well plates (4 × 105 cells/well) were treated with 10 µM BML-210, 10 µM dPAHA or 5–10 µM MS-275 for 24 h. CerS1 promoter activity was measured by luciferase assay.B. Effects of MS-275 (10 µM for 24 h) on the association of Sp1 with the endogenous CerS1 promoter DNA were determined using Q-ChIP. Relative quantities of Sp1 were normalized to 1% input DNA and IgG controls.C. Effects of knockdown of HDAC1, HDAC2, HDAC3 and HDAC8 using siRNAs on CerS1 promoter activity were measured in UM-SCC-22A cells.D-E. The half-life of CerS1 mRNA in immortalized keratinocytes versus UM-SCC-1 or UM-SCC-22A cells, or in primary NHEK versus UM-SCC-22A cells were measured using Q-PCR in the absence/presence of DMSO or Act D (25 µg/ml) for 0.5–6 h. GAPDH mRNA was used as a control. These data represent at least two-independent trials performed in duplicates. The error bars represent the standard deviations.
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fig05: Regulation of CerS1 expression via decreased mRNA stability in HNSCC versus keratinocytesA. UM-SCC-1 cells in 6-well plates (4 × 105 cells/well) were treated with 10 µM BML-210, 10 µM dPAHA or 5–10 µM MS-275 for 24 h. CerS1 promoter activity was measured by luciferase assay.B. Effects of MS-275 (10 µM for 24 h) on the association of Sp1 with the endogenous CerS1 promoter DNA were determined using Q-ChIP. Relative quantities of Sp1 were normalized to 1% input DNA and IgG controls.C. Effects of knockdown of HDAC1, HDAC2, HDAC3 and HDAC8 using siRNAs on CerS1 promoter activity were measured in UM-SCC-22A cells.D-E. The half-life of CerS1 mRNA in immortalized keratinocytes versus UM-SCC-1 or UM-SCC-22A cells, or in primary NHEK versus UM-SCC-22A cells were measured using Q-PCR in the absence/presence of DMSO or Act D (25 µg/ml) for 0.5–6 h. GAPDH mRNA was used as a control. These data represent at least two-independent trials performed in duplicates. The error bars represent the standard deviations.

Mentions: Moreover, we determined which HDAC was involved in the repression of the CerS1 promoter in HNSCC cells. First, UM-SCC-1 cells were treated with the known HDAC1-specific inhibitor MS-275, a pan class I-HDAC inhibitor BML-210 or a class II-HDAC inhibitor dPAHA (Yang & Seto, 2008), and then their effects on CerS1 promoter activity were examined. Whereas, the class II-HDAC inhibitor dPAHA had no significant effect, inhibition of class-I HDACs using BML-210 or HDAC1 using MS-275 enhanced the CerS1 promoter by about 3- or 4-fold, respectively (Fig 5A). Accordingly, MS-275 treatment increased Sp1 recruitment to the CerS1 promoter DNA compared to controls (Fig 5B), as determined by Q-ChIP. Thus, these data suggest that a class I HDAC, possibly HDAC1 might be involved in the repression of the CerS1 promoter in HNSCC cells. Indeed, knockdown of HDAC1, but not other class-I HDACs (HDAC2, HDAC3 or HDAC8) using siRNAs (decreasing their target gene mRNA expression by about 50–90%, as determined by Q-PCR, Fig S3A of Supporting Information) increased the activity of the CerS1 core promoter (Fig 5C). Thus, these data suggest that the CerS1 promoter is repressed mainly by an HDAC1-dependent mechanism, and that inhibition of HDAC1 using MS-275 or HDAC1-siRNAs activates the CerS1 promoter.


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)

Regulation of CerS1 expression via decreased mRNA stability in HNSCC versus keratinocytesA. UM-SCC-1 cells in 6-well plates (4 × 105 cells/well) were treated with 10 µM BML-210, 10 µM dPAHA or 5–10 µM MS-275 for 24 h. CerS1 promoter activity was measured by luciferase assay.B. Effects of MS-275 (10 µM for 24 h) on the association of Sp1 with the endogenous CerS1 promoter DNA were determined using Q-ChIP. Relative quantities of Sp1 were normalized to 1% input DNA and IgG controls.C. Effects of knockdown of HDAC1, HDAC2, HDAC3 and HDAC8 using siRNAs on CerS1 promoter activity were measured in UM-SCC-22A cells.D-E. The half-life of CerS1 mRNA in immortalized keratinocytes versus UM-SCC-1 or UM-SCC-22A cells, or in primary NHEK versus UM-SCC-22A cells were measured using Q-PCR in the absence/presence of DMSO or Act D (25 µg/ml) for 0.5–6 h. GAPDH mRNA was used as a control. These data represent at least two-independent trials performed in duplicates. The error bars represent the standard deviations.
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fig05: Regulation of CerS1 expression via decreased mRNA stability in HNSCC versus keratinocytesA. UM-SCC-1 cells in 6-well plates (4 × 105 cells/well) were treated with 10 µM BML-210, 10 µM dPAHA or 5–10 µM MS-275 for 24 h. CerS1 promoter activity was measured by luciferase assay.B. Effects of MS-275 (10 µM for 24 h) on the association of Sp1 with the endogenous CerS1 promoter DNA were determined using Q-ChIP. Relative quantities of Sp1 were normalized to 1% input DNA and IgG controls.C. Effects of knockdown of HDAC1, HDAC2, HDAC3 and HDAC8 using siRNAs on CerS1 promoter activity were measured in UM-SCC-22A cells.D-E. The half-life of CerS1 mRNA in immortalized keratinocytes versus UM-SCC-1 or UM-SCC-22A cells, or in primary NHEK versus UM-SCC-22A cells were measured using Q-PCR in the absence/presence of DMSO or Act D (25 µg/ml) for 0.5–6 h. GAPDH mRNA was used as a control. These data represent at least two-independent trials performed in duplicates. The error bars represent the standard deviations.
Mentions: Moreover, we determined which HDAC was involved in the repression of the CerS1 promoter in HNSCC cells. First, UM-SCC-1 cells were treated with the known HDAC1-specific inhibitor MS-275, a pan class I-HDAC inhibitor BML-210 or a class II-HDAC inhibitor dPAHA (Yang & Seto, 2008), and then their effects on CerS1 promoter activity were examined. Whereas, the class II-HDAC inhibitor dPAHA had no significant effect, inhibition of class-I HDACs using BML-210 or HDAC1 using MS-275 enhanced the CerS1 promoter by about 3- or 4-fold, respectively (Fig 5A). Accordingly, MS-275 treatment increased Sp1 recruitment to the CerS1 promoter DNA compared to controls (Fig 5B), as determined by Q-ChIP. Thus, these data suggest that a class I HDAC, possibly HDAC1 might be involved in the repression of the CerS1 promoter in HNSCC cells. Indeed, knockdown of HDAC1, but not other class-I HDACs (HDAC2, HDAC3 or HDAC8) using siRNAs (decreasing their target gene mRNA expression by about 50–90%, as determined by Q-PCR, Fig S3A of Supporting Information) increased the activity of the CerS1 core promoter (Fig 5C). Thus, these data suggest that the CerS1 promoter is repressed mainly by an HDAC1-dependent mechanism, and that inhibition of HDAC1 using MS-275 or HDAC1-siRNAs activates the CerS1 promoter.

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