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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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Related in: MedlinePlus

CerS1-2 mRNA containing a miR-574-5p target site within its 3′UTR region is predominantly expressed in HNSCC versus keratinocytesA.CerS1/GDF1 bicistronic mRNA transcript with six potential polyA signal sequences within the intronic region between exons 6 and 7 of the CerS1 mRNA transcript is shown. The miR-574-5p target site is located upstream of the polyA sequence 5.B. Expression of CerS1-1 spanning exons 2–7, or CerS1-exon 7/GDF1-exon 1 juncture were detected using RT-PCR in keratinocytes, UM-SCC-1 and/or UM-SCC-22A cells (left and right panels, lanes 1–3 or 1 and 2, respectively).C. Expression of CerS1-2 containing 3′UTR sequences down-stream of polyA signal sequences without (3′UTR #1, upper panel), or without a potential miR-574-5p binding site (3′UTR #5, middle panel) were measured using RT-PCR in keratinocytes versus UM-SCC-1 and UM-SCC-22A cells.D. Relative expression of CerS1-1, CerS1-2 containing 3′UTR #1 and CerS1-2 containing 3′UTR #5 mRNAs were measured using RT-PCR in UM-SCC-1 and UM-SCC-22A cells compared to immortalized keratinocytes.E-F. Levels of CerS1 (E) and miR-574-5p (F) mRNAs were measured with Q-PCR and RT-PCR in primary HNSCC tumour tissues compared to the paired and adjacent pathologically non-cancerous head and neck tissues, respectively. These data represent at least two-independent trials performed as duplicates. The error bars represent the standard deviations.
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fig06: CerS1-2 mRNA containing a miR-574-5p target site within its 3′UTR region is predominantly expressed in HNSCC versus keratinocytesA.CerS1/GDF1 bicistronic mRNA transcript with six potential polyA signal sequences within the intronic region between exons 6 and 7 of the CerS1 mRNA transcript is shown. The miR-574-5p target site is located upstream of the polyA sequence 5.B. Expression of CerS1-1 spanning exons 2–7, or CerS1-exon 7/GDF1-exon 1 juncture were detected using RT-PCR in keratinocytes, UM-SCC-1 and/or UM-SCC-22A cells (left and right panels, lanes 1–3 or 1 and 2, respectively).C. Expression of CerS1-2 containing 3′UTR sequences down-stream of polyA signal sequences without (3′UTR #1, upper panel), or without a potential miR-574-5p binding site (3′UTR #5, middle panel) were measured using RT-PCR in keratinocytes versus UM-SCC-1 and UM-SCC-22A cells.D. Relative expression of CerS1-1, CerS1-2 containing 3′UTR #1 and CerS1-2 containing 3′UTR #5 mRNAs were measured using RT-PCR in UM-SCC-1 and UM-SCC-22A cells compared to immortalized keratinocytes.E-F. Levels of CerS1 (E) and miR-574-5p (F) mRNAs were measured with Q-PCR and RT-PCR in primary HNSCC tumour tissues compared to the paired and adjacent pathologically non-cancerous head and neck tissues, respectively. These data represent at least two-independent trials performed as duplicates. The error bars represent the standard deviations.

Mentions: We then determined the mechanisms involved in the decreased stability of CerS1 mRNA in HNSCC cells compared to keratinocytes. It is known that CerS1 has two possible alternatively spliced variants. CerS1-1 is a bi-cistronic mRNA containing both CerS1 and GDF1 (Wang et al, 2007), growth and differentiation factor 1, coding sequences (Fig 6A). CerS1/GDF1 bi-cistronic mRNA is controlled by a common promoter element at the upstream of the 5′-end of the CerS1 gene (exons1–7), and by a 3′UTR poly-adenylation (polyA) sequence (AATAAA), present at the 3′-end of the GDF1 gene. According to mirBase, there are no known miR-target sequences at the 3′UTR of the CerS1/GDF1 bicistronic pre-mRNA (CerS1-1). The alternatively spliced variant of CerS1-2 contains exons 1–6, but not exon 7 and the intronic sequence between exons 6 and 7 contains six putative poly(A)-signals (A1–A6), indicating that this intronic sequence might be utilized as a 3′UTR of the CerS1-2 mRNA (Fig 6A). In fact, analysis of the CerS1-2 3′UTR sequence revealed that there is a hsa-miRNA-574-5p-target sequence upstream of the putative A5–A6 poly-A sequences (Fig 6A and Fig S4A of Supporting Information). This finding suggested that the expression of CerS1-2 mRNA might be regulated by miR-574-5p.


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)

CerS1-2 mRNA containing a miR-574-5p target site within its 3′UTR region is predominantly expressed in HNSCC versus keratinocytesA.CerS1/GDF1 bicistronic mRNA transcript with six potential polyA signal sequences within the intronic region between exons 6 and 7 of the CerS1 mRNA transcript is shown. The miR-574-5p target site is located upstream of the polyA sequence 5.B. Expression of CerS1-1 spanning exons 2–7, or CerS1-exon 7/GDF1-exon 1 juncture were detected using RT-PCR in keratinocytes, UM-SCC-1 and/or UM-SCC-22A cells (left and right panels, lanes 1–3 or 1 and 2, respectively).C. Expression of CerS1-2 containing 3′UTR sequences down-stream of polyA signal sequences without (3′UTR #1, upper panel), or without a potential miR-574-5p binding site (3′UTR #5, middle panel) were measured using RT-PCR in keratinocytes versus UM-SCC-1 and UM-SCC-22A cells.D. Relative expression of CerS1-1, CerS1-2 containing 3′UTR #1 and CerS1-2 containing 3′UTR #5 mRNAs were measured using RT-PCR in UM-SCC-1 and UM-SCC-22A cells compared to immortalized keratinocytes.E-F. Levels of CerS1 (E) and miR-574-5p (F) mRNAs were measured with Q-PCR and RT-PCR in primary HNSCC tumour tissues compared to the paired and adjacent pathologically non-cancerous head and neck tissues, respectively. These data represent at least two-independent trials performed as duplicates. The error bars represent the standard deviations.
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Related In: Results  -  Collection

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fig06: CerS1-2 mRNA containing a miR-574-5p target site within its 3′UTR region is predominantly expressed in HNSCC versus keratinocytesA.CerS1/GDF1 bicistronic mRNA transcript with six potential polyA signal sequences within the intronic region between exons 6 and 7 of the CerS1 mRNA transcript is shown. The miR-574-5p target site is located upstream of the polyA sequence 5.B. Expression of CerS1-1 spanning exons 2–7, or CerS1-exon 7/GDF1-exon 1 juncture were detected using RT-PCR in keratinocytes, UM-SCC-1 and/or UM-SCC-22A cells (left and right panels, lanes 1–3 or 1 and 2, respectively).C. Expression of CerS1-2 containing 3′UTR sequences down-stream of polyA signal sequences without (3′UTR #1, upper panel), or without a potential miR-574-5p binding site (3′UTR #5, middle panel) were measured using RT-PCR in keratinocytes versus UM-SCC-1 and UM-SCC-22A cells.D. Relative expression of CerS1-1, CerS1-2 containing 3′UTR #1 and CerS1-2 containing 3′UTR #5 mRNAs were measured using RT-PCR in UM-SCC-1 and UM-SCC-22A cells compared to immortalized keratinocytes.E-F. Levels of CerS1 (E) and miR-574-5p (F) mRNAs were measured with Q-PCR and RT-PCR in primary HNSCC tumour tissues compared to the paired and adjacent pathologically non-cancerous head and neck tissues, respectively. These data represent at least two-independent trials performed as duplicates. The error bars represent the standard deviations.
Mentions: We then determined the mechanisms involved in the decreased stability of CerS1 mRNA in HNSCC cells compared to keratinocytes. It is known that CerS1 has two possible alternatively spliced variants. CerS1-1 is a bi-cistronic mRNA containing both CerS1 and GDF1 (Wang et al, 2007), growth and differentiation factor 1, coding sequences (Fig 6A). CerS1/GDF1 bi-cistronic mRNA is controlled by a common promoter element at the upstream of the 5′-end of the CerS1 gene (exons1–7), and by a 3′UTR poly-adenylation (polyA) sequence (AATAAA), present at the 3′-end of the GDF1 gene. According to mirBase, there are no known miR-target sequences at the 3′UTR of the CerS1/GDF1 bicistronic pre-mRNA (CerS1-1). The alternatively spliced variant of CerS1-2 contains exons 1–6, but not exon 7 and the intronic sequence between exons 6 and 7 contains six putative poly(A)-signals (A1–A6), indicating that this intronic sequence might be utilized as a 3′UTR of the CerS1-2 mRNA (Fig 6A). In fact, analysis of the CerS1-2 3′UTR sequence revealed that there is a hsa-miRNA-574-5p-target sequence upstream of the putative A5–A6 poly-A sequences (Fig 6A and Fig S4A of Supporting Information). This finding suggested that the expression of CerS1-2 mRNA might be regulated by miR-574-5p.

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