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

Abstract

Transforming growth factor (TGF)-β exhibits both pro-apoptotic and anti-apoptotic effects on epithelial cells in a context-dependent manner. The anti-apoptotic function of TGF-β is mediated by several downstream regulatory mechanisms, and has been implicated in the tumor-progressive phenotype of breast cancer cells. We conducted RNA sequencing of mouse mammary gland epithelial (NMuMG) cells and identified a long non-coding RNA, termed lncRNA-Smad7, which has anti-apoptotic functions, as a target of TGF-β. lncRNA-Smad7 was located adjacent to the mouse Smad7 gene, and its expression was induced by TGF-β in all of the mouse mammary gland epithelial cell lines and breast cancer cell lines that we evaluated. Suppression of lncRNA-Smad7 expression cancelled the anti-apoptotic function of TGF-β. In contrast, forced expression of lncRNA-Smad7 rescued apoptosis induced by a TGF-β type I receptor kinase inhibitor in the mouse breast cancer cell line JygMC(A). 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. Our findings suggest a complex mechanism for regulating the anti-apoptotic and tumor-progressive aspects of TGF-β signaling.

Keywords: Apoptosis; Smad7; breast cancer; long non-coding RNA; transforming growth factor-β.

Fig. 1

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.

Fig. 2

Upregulation of expression of lncRNA-Smad7 by TGF-β. (a) Effect of TGF-β on expression of lncRNA-Smad7 in mouse JygMC(A) and 4T1 breast cancer cells. Cells were treated with 1 ng/mL TGF-β for the indicated periods, and expression of lncRNA-Smad7 relative to that of TATA box binding protein (Tbp) was determined by quantitative RT-PCR (qRT-PCR). (b) Effect of TGF-β and the TGF-β type I receptor kinase inhibitor SB431542 (SB) on lncRNA-Smad7 expression. Cells were treated with 1 ng/mL TGF-β or 10 μM SB431542 for 48 h or left untreated. Relative expression of lncRNA-Smad7 was determined as in (a). Error bars: standard deviations.

Fig. 3

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.

Fig. 4

Forced expression of lncRNA-Smad7 partially inhibits apoptosis of JygMC(A) cells. (a) Cells were infected with either adenoviral lncRNA-Smad7 expression vector (Ad-lncRNA-Smad7) or Ad-LacZ as a control. Twelve hours after infection, cells were serum starved and treated with 1 ng/mL TGF-β or 10 μM SB431542 (SB) for 48 h. Expression of lncRNA-Smad7 was determined by quantitative RT-PCR (qRT-PCR). (b) Effect of Ad-lncRNA-Smad7 on cleaved PARP level was determined by immunoblotting. Cells were treated as in (a) and lysed for SDS-PAGE. The bottom graph shows quantification of cleaved PARP normalized to α-tubulin. **P < 0.001. (c) TUNEL staining of JygMC(A) cells. Cells were treated as in (a). TUNEL, red; DAPI, blue. (d) The percentage of TUNEL-positive cells among DAPI-positive cells was determined. Error bars: standard deviations.

Fig. 5

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.

Fig. 6

Knockdown of lncRNA-Smad7 represses tumor-forming ability of JygMc(A) cells. (a) Effect of siRNA for lncRNA-Smad7 (silncRNA-1) on the size of xenografted tumors (n = 6). Photos were taken 8 d after tumor inoculation. Representative images are shown in this figure. (b) Sequential tumor volume measurement after inoculation. Error bars: standard deviations.