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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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Knockdown of lncRNA-Smad7 induces apoptosis of JygMC(A) cells. (a) Effect of siRNA for lncRNA-Smad7 in JygMC(A) cells. Cells were transfected with the indicated siRNA and stimulated with 1 ng/mL TGF-β for 48 h after transfection. silncRNA-1 was used for knockdown of all variants of lncRNA-Smad7, while silncRNA-2 was designed for knockdown of V1 only. Relative expression of lncRNA was determined as in Figure 2a. NTF, no transfection control; siNC, negative control siRNA. (b) Live-cell counting of JygMC(A) cells treated with siRNA for lncRNA-Smad7. Cells were transfected with siRNA as indicated and treated with 1 ng/mL TGF-β or 10 μM SB431542 with serum starvation 48 h after transfection. Live cells were counted using TC20 (BioRad, Hercules, California, USA.) after 48 h. (c) TUNEL staining of JygMC(A) cells treated with siRNA for lncRNA-Smad7. Cells were treated as in (b). TUNEL, red; DAPI, blue. (d) The percentage of TUNEL-positive cells among DAPI-positive cells was determined. (e) Immunoblot analysis of cleaved PARP. Cells were treated as in (b) and lysed for SDS-PAGE. An arrow head shows nonspecific band. The graph in the bottom shows quantification of cleaved PARP normalized to α-tubulin. Error bars: standard deviations. *P < 0.05, **P < 0.001.
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fig03: Knockdown of lncRNA-Smad7 induces apoptosis of JygMC(A) cells. (a) Effect of siRNA for lncRNA-Smad7 in JygMC(A) cells. Cells were transfected with the indicated siRNA and stimulated with 1 ng/mL TGF-β for 48 h after transfection. silncRNA-1 was used for knockdown of all variants of lncRNA-Smad7, while silncRNA-2 was designed for knockdown of V1 only. Relative expression of lncRNA was determined as in Figure 2a. NTF, no transfection control; siNC, negative control siRNA. (b) Live-cell counting of JygMC(A) cells treated with siRNA for lncRNA-Smad7. Cells were transfected with siRNA as indicated and treated with 1 ng/mL TGF-β or 10 μM SB431542 with serum starvation 48 h after transfection. Live cells were counted using TC20 (BioRad, Hercules, California, USA.) after 48 h. (c) TUNEL staining of JygMC(A) cells treated with siRNA for lncRNA-Smad7. Cells were treated as in (b). TUNEL, red; DAPI, blue. (d) The percentage of TUNEL-positive cells among DAPI-positive cells was determined. (e) Immunoblot analysis of cleaved PARP. Cells were treated as in (b) and lysed for SDS-PAGE. An arrow head shows nonspecific band. The graph in the bottom shows quantification of cleaved PARP normalized to α-tubulin. Error bars: standard deviations. *P < 0.05, **P < 0.001.

Mentions: We next knocked down the expression of lncRNA-Smad7 by two different siRNA; that is, silncRNA-1 and silncRNA-2 (Fig. 3a). silncRNA-1 was designed to knock down all three variants, while silncRNA-2 was designed to knock down only V1. As shown in Figure 3a, expression of lncRNA-Smad7 was repressed efficiently by silncRNA-1 and moderately by silncRNA-2 in JygMC(A) cells. We found that live cell counts were decreased by silncRNA-1 in vitro (Fig. 3b). JygMC(A) cells underwent apoptosis upon serum starvation, and the apoptosis was induced by SB431542 and inhibited by TGF-β.(4) The role of lncRNA-Smad7 in TGF-β-induced anti-apoptosis was then evaluated. Inhibition of apoptosis by TGF-β was partially cancelled by suppression of lncRNA-Smad7 expression, as determined by TUNEL staining (Fig. 3c,d). Consistent with the silencing efficiencies, the effects of silncRNA-2 were less marked than those of silncRNA-1 (Fig. 3c,d). Cleaved PARP levels in TGF-β-stimulated cells were also increased following treatment with silncRNA-1 and silncRNA-2 (Fig. 3e).


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)

Knockdown of lncRNA-Smad7 induces apoptosis of JygMC(A) cells. (a) Effect of siRNA for lncRNA-Smad7 in JygMC(A) cells. Cells were transfected with the indicated siRNA and stimulated with 1 ng/mL TGF-β for 48 h after transfection. silncRNA-1 was used for knockdown of all variants of lncRNA-Smad7, while silncRNA-2 was designed for knockdown of V1 only. Relative expression of lncRNA was determined as in Figure 2a. NTF, no transfection control; siNC, negative control siRNA. (b) Live-cell counting of JygMC(A) cells treated with siRNA for lncRNA-Smad7. Cells were transfected with siRNA as indicated and treated with 1 ng/mL TGF-β or 10 μM SB431542 with serum starvation 48 h after transfection. Live cells were counted using TC20 (BioRad, Hercules, California, USA.) after 48 h. (c) TUNEL staining of JygMC(A) cells treated with siRNA for lncRNA-Smad7. Cells were treated as in (b). TUNEL, red; DAPI, blue. (d) The percentage of TUNEL-positive cells among DAPI-positive cells was determined. (e) Immunoblot analysis of cleaved PARP. Cells were treated as in (b) and lysed for SDS-PAGE. An arrow head shows nonspecific band. The graph in the bottom shows quantification of cleaved PARP normalized to α-tubulin. Error bars: standard deviations. *P < 0.05, **P < 0.001.
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fig03: Knockdown of lncRNA-Smad7 induces apoptosis of JygMC(A) cells. (a) Effect of siRNA for lncRNA-Smad7 in JygMC(A) cells. Cells were transfected with the indicated siRNA and stimulated with 1 ng/mL TGF-β for 48 h after transfection. silncRNA-1 was used for knockdown of all variants of lncRNA-Smad7, while silncRNA-2 was designed for knockdown of V1 only. Relative expression of lncRNA was determined as in Figure 2a. NTF, no transfection control; siNC, negative control siRNA. (b) Live-cell counting of JygMC(A) cells treated with siRNA for lncRNA-Smad7. Cells were transfected with siRNA as indicated and treated with 1 ng/mL TGF-β or 10 μM SB431542 with serum starvation 48 h after transfection. Live cells were counted using TC20 (BioRad, Hercules, California, USA.) after 48 h. (c) TUNEL staining of JygMC(A) cells treated with siRNA for lncRNA-Smad7. Cells were treated as in (b). TUNEL, red; DAPI, blue. (d) The percentage of TUNEL-positive cells among DAPI-positive cells was determined. (e) Immunoblot analysis of cleaved PARP. Cells were treated as in (b) and lysed for SDS-PAGE. An arrow head shows nonspecific band. The graph in the bottom shows quantification of cleaved PARP normalized to α-tubulin. Error bars: standard deviations. *P < 0.05, **P < 0.001.
Mentions: We next knocked down the expression of lncRNA-Smad7 by two different siRNA; that is, silncRNA-1 and silncRNA-2 (Fig. 3a). silncRNA-1 was designed to knock down all three variants, while silncRNA-2 was designed to knock down only V1. As shown in Figure 3a, expression of lncRNA-Smad7 was repressed efficiently by silncRNA-1 and moderately by silncRNA-2 in JygMC(A) cells. We found that live cell counts were decreased by silncRNA-1 in vitro (Fig. 3b). JygMC(A) cells underwent apoptosis upon serum starvation, and the apoptosis was induced by SB431542 and inhibited by TGF-β.(4) The role of lncRNA-Smad7 in TGF-β-induced anti-apoptosis was then evaluated. Inhibition of apoptosis by TGF-β was partially cancelled by suppression of lncRNA-Smad7 expression, as determined by TUNEL staining (Fig. 3c,d). Consistent with the silencing efficiencies, the effects of silncRNA-2 were less marked than those of silncRNA-1 (Fig. 3c,d). Cleaved PARP levels in TGF-β-stimulated cells were also increased following treatment with silncRNA-1 and silncRNA-2 (Fig. 3e).

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