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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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lncRNA-Smad7 does not regulate phospho-Smad2 levels or TGF-β-induced EMT. (a) JygMC(A) cells were transfected with siRNA as indicated and stimulated with TGF-β for 48 h. Phospho-Smad2 and total Smad2/3 levels were determined by immunoblotting (top panel). The bottom graph shows quantification of phospho-Smad2 normalized to total Smad2/3. n.s.: not significant. (b) Effect of siRNA for lncRNA-Smad7 on Smad7 expression evaluated by quantitative RT-PCR (qRT-PCR). Transfected cells were stimulated with TGF-β for 48 h. NTF, no transfection; error bars, standard deviations. (c) NMuMG cells were reverse-transfected with siRNA as indicated. Twelve hours later, cells were stimulated with TGF-β for 24 h and fixed. F-actin formation was evaluated by phalloidin staining. (d) NMuMG cells were treated as in (c) and expression of EMT marker proteins was determined by immunoblotting.
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fig05: lncRNA-Smad7 does not regulate phospho-Smad2 levels or TGF-β-induced EMT. (a) JygMC(A) cells were transfected with siRNA as indicated and stimulated with TGF-β for 48 h. Phospho-Smad2 and total Smad2/3 levels were determined by immunoblotting (top panel). The bottom graph shows quantification of phospho-Smad2 normalized to total Smad2/3. n.s.: not significant. (b) Effect of siRNA for lncRNA-Smad7 on Smad7 expression evaluated by quantitative RT-PCR (qRT-PCR). Transfected cells were stimulated with TGF-β for 48 h. NTF, no transfection; error bars, standard deviations. (c) NMuMG cells were reverse-transfected with siRNA as indicated. Twelve hours later, cells were stimulated with TGF-β for 24 h and fixed. F-actin formation was evaluated by phalloidin staining. (d) NMuMG cells were treated as in (c) and expression of EMT marker proteins was determined by immunoblotting.

Mentions: lncRNA have several modes of function, including neutralizing miRNA and inhibiting transcription of target genomic regions.(8) Therefore, we next evaluated the effect of siRNA for lncRNA-Smad7 on the TGF-β-Smad signaling pathway. Knockdown of lncRNA-Smad7 did not change phospho-Smad2 expression in JygMC(A) cells (Fig. 5a). Moreover, expression of Smad7 by TGF-β stimulation was minimally inhibited by the siRNA for lncRNA-Smad7 (Fig. 5b). We then evaluated the effect of silncRNA on TGF-β-induced EMT in NMuMG cells. Actin stress fiber formation by TGF-β stimulation was not inhibited by the silncRNA, and silncRNA-2 showed a mild F-actin-inducing effect in the absence of TGF-β (Fig. 5c). Both downregulation of E-cadherin and induction of fibronectin and N-cadherin by TGF-β were not affected by the silncRNA (Fig. 5d). These results suggest that lncRNA-Smad7 does not have a marked effect on TGF-β signaling in general but does regulate anti-apoptosis-specific effects. Upregulation of Bhlhe40 (encoding the anti-apoptotic DEC1 protein) and inhibition of Bcl2l11 (encoding the pro-apoptotic BH3-only protein Bim) through downregulation of Foxc1 have been demonstrated as mechanisms of TGF-β-induced anti-apoptosis in breast cancer cells.(4,5) We examined the effect of lncRNA-Smad7 on the mRNA levels of these molecules, but the changes in their expression profiles could not explain the anti-apoptotic function of lncRNA-Smad7 (Fig. S2). We also searched for other pro-apoptotic and anti-apoptotic factors whose mRNA and protein expression levels were regulated by lncRNA-Smad7, but we were not able to identify any definite targets (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)

lncRNA-Smad7 does not regulate phospho-Smad2 levels or TGF-β-induced EMT. (a) JygMC(A) cells were transfected with siRNA as indicated and stimulated with TGF-β for 48 h. Phospho-Smad2 and total Smad2/3 levels were determined by immunoblotting (top panel). The bottom graph shows quantification of phospho-Smad2 normalized to total Smad2/3. n.s.: not significant. (b) Effect of siRNA for lncRNA-Smad7 on Smad7 expression evaluated by quantitative RT-PCR (qRT-PCR). Transfected cells were stimulated with TGF-β for 48 h. NTF, no transfection; error bars, standard deviations. (c) NMuMG cells were reverse-transfected with siRNA as indicated. Twelve hours later, cells were stimulated with TGF-β for 24 h and fixed. F-actin formation was evaluated by phalloidin staining. (d) NMuMG cells were treated as in (c) and expression of EMT marker proteins was determined by immunoblotting.
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fig05: lncRNA-Smad7 does not regulate phospho-Smad2 levels or TGF-β-induced EMT. (a) JygMC(A) cells were transfected with siRNA as indicated and stimulated with TGF-β for 48 h. Phospho-Smad2 and total Smad2/3 levels were determined by immunoblotting (top panel). The bottom graph shows quantification of phospho-Smad2 normalized to total Smad2/3. n.s.: not significant. (b) Effect of siRNA for lncRNA-Smad7 on Smad7 expression evaluated by quantitative RT-PCR (qRT-PCR). Transfected cells were stimulated with TGF-β for 48 h. NTF, no transfection; error bars, standard deviations. (c) NMuMG cells were reverse-transfected with siRNA as indicated. Twelve hours later, cells were stimulated with TGF-β for 24 h and fixed. F-actin formation was evaluated by phalloidin staining. (d) NMuMG cells were treated as in (c) and expression of EMT marker proteins was determined by immunoblotting.
Mentions: lncRNA have several modes of function, including neutralizing miRNA and inhibiting transcription of target genomic regions.(8) Therefore, we next evaluated the effect of siRNA for lncRNA-Smad7 on the TGF-β-Smad signaling pathway. Knockdown of lncRNA-Smad7 did not change phospho-Smad2 expression in JygMC(A) cells (Fig. 5a). Moreover, expression of Smad7 by TGF-β stimulation was minimally inhibited by the siRNA for lncRNA-Smad7 (Fig. 5b). We then evaluated the effect of silncRNA on TGF-β-induced EMT in NMuMG cells. Actin stress fiber formation by TGF-β stimulation was not inhibited by the silncRNA, and silncRNA-2 showed a mild F-actin-inducing effect in the absence of TGF-β (Fig. 5c). Both downregulation of E-cadherin and induction of fibronectin and N-cadherin by TGF-β were not affected by the silncRNA (Fig. 5d). These results suggest that lncRNA-Smad7 does not have a marked effect on TGF-β signaling in general but does regulate anti-apoptosis-specific effects. Upregulation of Bhlhe40 (encoding the anti-apoptotic DEC1 protein) and inhibition of Bcl2l11 (encoding the pro-apoptotic BH3-only protein Bim) through downregulation of Foxc1 have been demonstrated as mechanisms of TGF-β-induced anti-apoptosis in breast cancer cells.(4,5) We examined the effect of lncRNA-Smad7 on the mRNA levels of these molecules, but the changes in their expression profiles could not explain the anti-apoptotic function of lncRNA-Smad7 (Fig. S2). We also searched for other pro-apoptotic and anti-apoptotic factors whose mRNA and protein expression levels were regulated by lncRNA-Smad7, but we were not able to identify any definite targets (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