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Transforming growth factor-β-induced lncRNA-Smad7 inhibits apoptosis of mouse breast cancer JygMC(A) cells.

Arase M, Horiguchi K, Ehata S, Morikawa M, Tsutsumi S, Aburatani H, Miyazono K, Koinuma D - Cancer Sci. (2014)

Bottom Line: Transforming growth factor (TGF)-β exhibits both pro-apoptotic and anti-apoptotic effects on epithelial cells in a context-dependent manner.The anti-apoptotic effect of lncRNA-Smad7 appeared to occur independently of the transcriptional regulation by TGF-β of anti-apoptotic DEC1 and pro-apoptotic Bim proteins.Small interfering RNA for lncRNA-Smad7 did not alter the process of TGF-β-induced epithelial-mesenchymal transition, phosphorylation of Smad2 or expression of the Smad7 gene, suggesting that the contribution of this lncRNA to TGF-β functions may be restricted to apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

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Cloning of lncRNA-Smad7. (a) RNA-sequencing data from NMuMG cells at the mouse Smad7 gene locus. Black and grey tags are aligned to the sense and antisense genomic sequences, respectively. Vertical axis: mapped tag numbers. Smad7 gene is shown in green, and its exons are shown as boxes (thin boxes, untranslated regions; thick boxes, coding regions) connected by horizontal lines representing introns. (b) Relative positions of the Smad7 gene and EST clones with direction of transcription. Positions of the gene-specific primers used for RACE are shown at the back ends of the blue arrows. (c) Northern blotting of lncRNA-Smad7. NMuMG cells were treated with TGF-β or left untreated for 24 h. Left panel: Ethidium bromide staining of the gel to show the positions of 28s/18s rRNA and equal loading of the samples. Right panel: Northern blotting with an EST sequence (CX230377) as a probe. (d) Schematic representation of the identified lncRNA-Smad7 transcripts. V3 transcript was identified by quantitative RT-PCR (qRT-PCR) analysis of JygMC(A) and confirmed by sequencing of the PCR product. V3 had a longer exon 2 and a shorter exon 3 than V2 transcript.
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fig01: Cloning of lncRNA-Smad7. (a) RNA-sequencing data from NMuMG cells at the mouse Smad7 gene locus. Black and grey tags are aligned to the sense and antisense genomic sequences, respectively. Vertical axis: mapped tag numbers. Smad7 gene is shown in green, and its exons are shown as boxes (thin boxes, untranslated regions; thick boxes, coding regions) connected by horizontal lines representing introns. (b) Relative positions of the Smad7 gene and EST clones with direction of transcription. Positions of the gene-specific primers used for RACE are shown at the back ends of the blue arrows. (c) Northern blotting of lncRNA-Smad7. NMuMG cells were treated with TGF-β or left untreated for 24 h. Left panel: Ethidium bromide staining of the gel to show the positions of 28s/18s rRNA and equal loading of the samples. Right panel: Northern blotting with an EST sequence (CX230377) as a probe. (d) Schematic representation of the identified lncRNA-Smad7 transcripts. V3 transcript was identified by quantitative RT-PCR (qRT-PCR) analysis of JygMC(A) and confirmed by sequencing of the PCR product. V3 had a longer exon 2 and a shorter exon 3 than V2 transcript.

Mentions: We performed RNA sequencing to quantitatively screen for lncRNA downstream of TGF-β signaling in NMuMG cells. To this end, we calculated changes in the expression of the reported lncRNA after the cells were treated with TGF-β (Table S1).(14) We focused on a TGF-β-induced and highly expressed lncRNA transcribed from an upstream and antisense strand of the Smad7 gene, which encodes a known target and inhibitor of TGF-β signaling (Fig. 1a). Distribution of the sequenced tags fell within two spliced EST, CX230377 and CF356854, and did not overlap with Smad7 (Fig. 1b). Northern blotting using CX230377 as a probe resulted in the detection of an approximately 3-kb transcript that was strongly induced by TGF-β treatment (Fig. 1c). We then cloned the transcript by RACE using gene-specific primers designed from CX230377 sequences and identified two transcripts, which we designated lncRNA-Smad7 V1 (2685 bp) and V2 (2801 bp) (Fig. 1d). In addition, variant 3 of lncRNA-Smad7 (V3) was identified through RT-PCR analysis of JygMC(A) cells (data not shown). There were only short open reading frames (ORF) encoding <97 amino acids in lncRNA-Smad7. These ORF did not have a canonical Kozak sequence (data not shown).


Transforming growth factor-β-induced lncRNA-Smad7 inhibits apoptosis of mouse breast cancer JygMC(A) cells.

Arase M, Horiguchi K, Ehata S, Morikawa M, Tsutsumi S, Aburatani H, Miyazono K, Koinuma D - Cancer Sci. (2014)

Cloning of lncRNA-Smad7. (a) RNA-sequencing data from NMuMG cells at the mouse Smad7 gene locus. Black and grey tags are aligned to the sense and antisense genomic sequences, respectively. Vertical axis: mapped tag numbers. Smad7 gene is shown in green, and its exons are shown as boxes (thin boxes, untranslated regions; thick boxes, coding regions) connected by horizontal lines representing introns. (b) Relative positions of the Smad7 gene and EST clones with direction of transcription. Positions of the gene-specific primers used for RACE are shown at the back ends of the blue arrows. (c) Northern blotting of lncRNA-Smad7. NMuMG cells were treated with TGF-β or left untreated for 24 h. Left panel: Ethidium bromide staining of the gel to show the positions of 28s/18s rRNA and equal loading of the samples. Right panel: Northern blotting with an EST sequence (CX230377) as a probe. (d) Schematic representation of the identified lncRNA-Smad7 transcripts. V3 transcript was identified by quantitative RT-PCR (qRT-PCR) analysis of JygMC(A) and confirmed by sequencing of the PCR product. V3 had a longer exon 2 and a shorter exon 3 than V2 transcript.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig01: Cloning of lncRNA-Smad7. (a) RNA-sequencing data from NMuMG cells at the mouse Smad7 gene locus. Black and grey tags are aligned to the sense and antisense genomic sequences, respectively. Vertical axis: mapped tag numbers. Smad7 gene is shown in green, and its exons are shown as boxes (thin boxes, untranslated regions; thick boxes, coding regions) connected by horizontal lines representing introns. (b) Relative positions of the Smad7 gene and EST clones with direction of transcription. Positions of the gene-specific primers used for RACE are shown at the back ends of the blue arrows. (c) Northern blotting of lncRNA-Smad7. NMuMG cells were treated with TGF-β or left untreated for 24 h. Left panel: Ethidium bromide staining of the gel to show the positions of 28s/18s rRNA and equal loading of the samples. Right panel: Northern blotting with an EST sequence (CX230377) as a probe. (d) Schematic representation of the identified lncRNA-Smad7 transcripts. V3 transcript was identified by quantitative RT-PCR (qRT-PCR) analysis of JygMC(A) and confirmed by sequencing of the PCR product. V3 had a longer exon 2 and a shorter exon 3 than V2 transcript.
Mentions: We performed RNA sequencing to quantitatively screen for lncRNA downstream of TGF-β signaling in NMuMG cells. To this end, we calculated changes in the expression of the reported lncRNA after the cells were treated with TGF-β (Table S1).(14) We focused on a TGF-β-induced and highly expressed lncRNA transcribed from an upstream and antisense strand of the Smad7 gene, which encodes a known target and inhibitor of TGF-β signaling (Fig. 1a). Distribution of the sequenced tags fell within two spliced EST, CX230377 and CF356854, and did not overlap with Smad7 (Fig. 1b). Northern blotting using CX230377 as a probe resulted in the detection of an approximately 3-kb transcript that was strongly induced by TGF-β treatment (Fig. 1c). We then cloned the transcript by RACE using gene-specific primers designed from CX230377 sequences and identified two transcripts, which we designated lncRNA-Smad7 V1 (2685 bp) and V2 (2801 bp) (Fig. 1d). In addition, variant 3 of lncRNA-Smad7 (V3) was identified through RT-PCR analysis of JygMC(A) cells (data not shown). There were only short open reading frames (ORF) encoding <97 amino acids in lncRNA-Smad7. These ORF did not have a canonical Kozak sequence (data not shown).

Bottom Line: Transforming growth factor (TGF)-β exhibits both pro-apoptotic and anti-apoptotic effects on epithelial cells in a context-dependent manner.The anti-apoptotic effect of lncRNA-Smad7 appeared to occur independently of the transcriptional regulation by TGF-β of anti-apoptotic DEC1 and pro-apoptotic Bim proteins.Small interfering RNA for lncRNA-Smad7 did not alter the process of TGF-β-induced epithelial-mesenchymal transition, phosphorylation of Smad2 or expression of the Smad7 gene, suggesting that the contribution of this lncRNA to TGF-β functions may be restricted to apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus