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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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MiR-574-5p targets CerS1-2 3′UTR, decreasing mRNA stability in HNSCCA-C. Cells plated in 6-well plates (2 × 105 cells/well), and transfected with luciferase expression vector containing CerS1-2 3′UTR1-1000 bp (A), CerS1-2 3′UTR-574-5p (B) and CerS1-2-3′UTR-mutant 574-5p (C) sequences in the absence/presence of siRNA against miR-574-5p, with/without Act D (25 µg/ml, 2 h) or DMSO. Effects of 3′UTR sequences on luciferase mRNA were determined by Q-PCR. Actin was used as a loading control. These data represent at least two-independent trials performed as duplicates. The error bars represent the standard deviations. Two vertical lines on the map of the construct (C) represent the deletion mutations.
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fig07: MiR-574-5p targets CerS1-2 3′UTR, decreasing mRNA stability in HNSCCA-C. Cells plated in 6-well plates (2 × 105 cells/well), and transfected with luciferase expression vector containing CerS1-2 3′UTR1-1000 bp (A), CerS1-2 3′UTR-574-5p (B) and CerS1-2-3′UTR-mutant 574-5p (C) sequences in the absence/presence of siRNA against miR-574-5p, with/without Act D (25 µg/ml, 2 h) or DMSO. Effects of 3′UTR sequences on luciferase mRNA were determined by Q-PCR. Actin was used as a loading control. These data represent at least two-independent trials performed as duplicates. The error bars represent the standard deviations. Two vertical lines on the map of the construct (C) represent the deletion mutations.

Mentions: To define the involvement of the putative miRNA-574-5p target sequence for mRNA degradation, the 3′UTR sequences of CerS1-2 with or without the miRNA-574-5p target sequence were sub-cloned down-stream of the full-length luciferase cDNA, whose expression was constitutively activated by the CMV promoter (Fig 7A–C). We used UM-SCC-14A cells to examine the effects of CerS1-2 3′UTR elements on luciferase stability, with/without the miRNA-574-5p target sequence in the absence/presence of the miRNA-574-5p-siRNA (which reduced its expression approximately 75% compared to Scr siRNA controls, as measured by Q-PCR, Fig S5A–D of Supporting Information). The data showed that the stability of luciferase mRNA containing the 3′UTR of CerS1-2 without the miRNA-574-5p target sequence did not change significantly in the presence of Act D at 2 h, and was not affected by miRNA-574-5p siRNA (Fig 7A). However, the stability of the luciferase mRNA was decreased around 60% (at 2 h) in the presence of miRNA-574-5p target sequence in the 3′UTR of the CerS1-2, compared to controls in UM-SCC-14A (Fig 7B). Remarkably, knockdown of miRNA-574-5p expression using siRNA prevented the degradation of luciferase mRNA in the presence of Act D at 2 h compared to scr-siRNA-transfected controls (Fig 7B). Previous studies indicate that for miR to bind its target site, it must recognize a ‘seed sequence’ which is localized within first 2–8 bases of the target sequence in the 3′UTR of its target mRNA (Bartel, 2009; Carthew & Sontheimer, 2009; Huntzinger & Izaurralde, 2011). Thus, we generated a mutation with deletion of second and third bases of the miR-574-5p-target/seed sequence of the CerS1-2 3′UTR (Fig S4A of Supporting Information), and determined its effects on luciferase mRNA stability. The data showed that when the mutant miR-574-5p-target sequence was introduced, the luciferase mRNA did not decrease in the presence of Act D at 2 h (Fig 7C). Overall, these data suggest that an alternatively spliced variant CerS1-2, a major CerS1 isoform expressed in HNSCC cells or primary tumour tissues, is targeted for degradation by a specific miRNA-574-5p, whose target sequence is localized within the 3′UTR (in the intronic sequence between exons 6 and 7 of CerS1-2 pre-mRNA) containing the TACACACA/(CACA)6-seed/recognition sequence (Fig S4A of Supporting Information). These data suggest also that knockdown of miRNA-574-5p expression using a siRNA reduces the degradation of CerS1-2 mRNA in these cancer cells.


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)

MiR-574-5p targets CerS1-2 3′UTR, decreasing mRNA stability in HNSCCA-C. Cells plated in 6-well plates (2 × 105 cells/well), and transfected with luciferase expression vector containing CerS1-2 3′UTR1-1000 bp (A), CerS1-2 3′UTR-574-5p (B) and CerS1-2-3′UTR-mutant 574-5p (C) sequences in the absence/presence of siRNA against miR-574-5p, with/without Act D (25 µg/ml, 2 h) or DMSO. Effects of 3′UTR sequences on luciferase mRNA were determined by Q-PCR. Actin was used as a loading control. These data represent at least two-independent trials performed as duplicates. The error bars represent the standard deviations. Two vertical lines on the map of the construct (C) represent the deletion mutations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376837&req=5

fig07: MiR-574-5p targets CerS1-2 3′UTR, decreasing mRNA stability in HNSCCA-C. Cells plated in 6-well plates (2 × 105 cells/well), and transfected with luciferase expression vector containing CerS1-2 3′UTR1-1000 bp (A), CerS1-2 3′UTR-574-5p (B) and CerS1-2-3′UTR-mutant 574-5p (C) sequences in the absence/presence of siRNA against miR-574-5p, with/without Act D (25 µg/ml, 2 h) or DMSO. Effects of 3′UTR sequences on luciferase mRNA were determined by Q-PCR. Actin was used as a loading control. These data represent at least two-independent trials performed as duplicates. The error bars represent the standard deviations. Two vertical lines on the map of the construct (C) represent the deletion mutations.
Mentions: To define the involvement of the putative miRNA-574-5p target sequence for mRNA degradation, the 3′UTR sequences of CerS1-2 with or without the miRNA-574-5p target sequence were sub-cloned down-stream of the full-length luciferase cDNA, whose expression was constitutively activated by the CMV promoter (Fig 7A–C). We used UM-SCC-14A cells to examine the effects of CerS1-2 3′UTR elements on luciferase stability, with/without the miRNA-574-5p target sequence in the absence/presence of the miRNA-574-5p-siRNA (which reduced its expression approximately 75% compared to Scr siRNA controls, as measured by Q-PCR, Fig S5A–D of Supporting Information). The data showed that the stability of luciferase mRNA containing the 3′UTR of CerS1-2 without the miRNA-574-5p target sequence did not change significantly in the presence of Act D at 2 h, and was not affected by miRNA-574-5p siRNA (Fig 7A). However, the stability of the luciferase mRNA was decreased around 60% (at 2 h) in the presence of miRNA-574-5p target sequence in the 3′UTR of the CerS1-2, compared to controls in UM-SCC-14A (Fig 7B). Remarkably, knockdown of miRNA-574-5p expression using siRNA prevented the degradation of luciferase mRNA in the presence of Act D at 2 h compared to scr-siRNA-transfected controls (Fig 7B). Previous studies indicate that for miR to bind its target site, it must recognize a ‘seed sequence’ which is localized within first 2–8 bases of the target sequence in the 3′UTR of its target mRNA (Bartel, 2009; Carthew & Sontheimer, 2009; Huntzinger & Izaurralde, 2011). Thus, we generated a mutation with deletion of second and third bases of the miR-574-5p-target/seed sequence of the CerS1-2 3′UTR (Fig S4A of Supporting Information), and determined its effects on luciferase mRNA stability. The data showed that when the mutant miR-574-5p-target sequence was introduced, the luciferase mRNA did not decrease in the presence of Act D at 2 h (Fig 7C). Overall, these data suggest that an alternatively spliced variant CerS1-2, a major CerS1 isoform expressed in HNSCC cells or primary tumour tissues, is targeted for degradation by a specific miRNA-574-5p, whose target sequence is localized within the 3′UTR (in the intronic sequence between exons 6 and 7 of CerS1-2 pre-mRNA) containing the TACACACA/(CACA)6-seed/recognition sequence (Fig S4A of Supporting Information). These data suggest also that knockdown of miRNA-574-5p expression using a siRNA reduces the degradation of CerS1-2 mRNA in these 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.

View Article: PubMed Central - PubMed

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

Show MeSH
Related in: MedlinePlus