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Wig-1 regulates cell cycle arrest and cell death through the p53 targets FAS and 14-3-3σ.

Bersani C, Xu LD, Vilborg A, Lui WO, Wiman KG - Oncogene (2014)

Bottom Line: We identified 2447 transcripts with >fourfold differential expression between Wig-1 and control small interfering (si)RNA-treated HCT116 cells.We found that Wig-1 regulates FAS and 14-3-3σ mRNA independently of p53.We show that Wig-1 binds to FAS mRNA 3'-UTR and decreases its stability through an ARE in the 3'-UTR.

View Article: PubMed Central - PubMed

Affiliation: Cancer Center Karolinska (CCK), Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden.

ABSTRACT
Wig-1, also known as ZMAT3, is a p53 target gene that encodes an RNA-binding zinc-finger protein involved in the regulation of mRNA stability through binding to AU-rich elements (AREs). We have used microarray analysis to identify novel Wig-1 target mRNAs. We identified 2447 transcripts with >fourfold differential expression between Wig-1 and control small interfering (si)RNA-treated HCT116 cells. Several p53 target genes were among the deregulated transcripts. We found that Wig-1 regulates FAS and 14-3-3σ mRNA independently of p53. We show that Wig-1 binds to FAS mRNA 3'-UTR and decreases its stability through an ARE in the 3'-UTR. Depletion of Wig-1 was associated with increased cell death and reduced cell cycle arrest upon DNA damage. Our results suggest a role of Wig-1 as a survival factor that directs the p53 stress response toward cell cycle arrest rather than apoptosis through the regulation of FAS and 14-3-3σ mRNA levels.

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Microarray target validation after Wig-1 knockdown. List of targets selected for validation with their microarray gene expression levels (a). Wig-1 knockdown (siW1, siW2) in HCT116 p53+/+ cells leads to increased FAS, WNT1, AKT3 and APP (b, c) and decreased 14-3-3σ and PPP2CB protein levels (c, d), confirming the microarray results. Arrows indicate the two major Wig-1 species. Protein-level quantifications are shown in (f). The same effect on 14-3-3σ and FAS protein levels after Wig-1 knockdown in HCT116 p53+/+ cells is also observed after stress induction by cisplatin (5 μM for 24 h) (e). Protein level quantifications are shown in (g). Wig-1 knockdown leads to decreased levels of 14-3-3σ mRNA and increased levels of FAS mRNA in HCT116 p53+/+ and p53−/− (h) cells in the presence and absence of cisplatin (5 μM 24 h), as assessed by qRT–PCR. The figure shows 14-3-3σ and FAS mRNA levels relative to mRNA levels in control siRNA-transfected untreated cells. Representative image from one of three independent experiments are shown in (b–e). Columns and error bars in (f–h) represent the mean±s.d.; n=3; ***P<0.001; **P<0.01; *P<0.05.
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fig1: Microarray target validation after Wig-1 knockdown. List of targets selected for validation with their microarray gene expression levels (a). Wig-1 knockdown (siW1, siW2) in HCT116 p53+/+ cells leads to increased FAS, WNT1, AKT3 and APP (b, c) and decreased 14-3-3σ and PPP2CB protein levels (c, d), confirming the microarray results. Arrows indicate the two major Wig-1 species. Protein-level quantifications are shown in (f). The same effect on 14-3-3σ and FAS protein levels after Wig-1 knockdown in HCT116 p53+/+ cells is also observed after stress induction by cisplatin (5 μM for 24 h) (e). Protein level quantifications are shown in (g). Wig-1 knockdown leads to decreased levels of 14-3-3σ mRNA and increased levels of FAS mRNA in HCT116 p53+/+ and p53−/− (h) cells in the presence and absence of cisplatin (5 μM 24 h), as assessed by qRT–PCR. The figure shows 14-3-3σ and FAS mRNA levels relative to mRNA levels in control siRNA-transfected untreated cells. Representative image from one of three independent experiments are shown in (b–e). Columns and error bars in (f–h) represent the mean±s.d.; n=3; ***P<0.001; **P<0.01; *P<0.05.

Mentions: To validate the microarray findings, eight targets were selected based on the extent of changes in expression in the microarray analysis, the presence of AREs in their 3′-UTR and known involvement in the most affected pathways (Figure 1a). We analyzed the protein levels of the selected eight targets in HCT116 cells transfected with control siRNA or two different siRNAs against Wig-1 (siW1, siW2), using the same conditions as for the microarray experiments. We were able to confirm changes of FAS, WNT1, AKT3, APP, 14-3-3σ and PPP2CB at the protein level (Figures 1b–d and f). The changes in protein levels for FZD8 and CDC42 were inconsistent with the microarray data (Supplementary Figure 1).


Wig-1 regulates cell cycle arrest and cell death through the p53 targets FAS and 14-3-3σ.

Bersani C, Xu LD, Vilborg A, Lui WO, Wiman KG - Oncogene (2014)

Microarray target validation after Wig-1 knockdown. List of targets selected for validation with their microarray gene expression levels (a). Wig-1 knockdown (siW1, siW2) in HCT116 p53+/+ cells leads to increased FAS, WNT1, AKT3 and APP (b, c) and decreased 14-3-3σ and PPP2CB protein levels (c, d), confirming the microarray results. Arrows indicate the two major Wig-1 species. Protein-level quantifications are shown in (f). The same effect on 14-3-3σ and FAS protein levels after Wig-1 knockdown in HCT116 p53+/+ cells is also observed after stress induction by cisplatin (5 μM for 24 h) (e). Protein level quantifications are shown in (g). Wig-1 knockdown leads to decreased levels of 14-3-3σ mRNA and increased levels of FAS mRNA in HCT116 p53+/+ and p53−/− (h) cells in the presence and absence of cisplatin (5 μM 24 h), as assessed by qRT–PCR. The figure shows 14-3-3σ and FAS mRNA levels relative to mRNA levels in control siRNA-transfected untreated cells. Representative image from one of three independent experiments are shown in (b–e). Columns and error bars in (f–h) represent the mean±s.d.; n=3; ***P<0.001; **P<0.01; *P<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: Microarray target validation after Wig-1 knockdown. List of targets selected for validation with their microarray gene expression levels (a). Wig-1 knockdown (siW1, siW2) in HCT116 p53+/+ cells leads to increased FAS, WNT1, AKT3 and APP (b, c) and decreased 14-3-3σ and PPP2CB protein levels (c, d), confirming the microarray results. Arrows indicate the two major Wig-1 species. Protein-level quantifications are shown in (f). The same effect on 14-3-3σ and FAS protein levels after Wig-1 knockdown in HCT116 p53+/+ cells is also observed after stress induction by cisplatin (5 μM for 24 h) (e). Protein level quantifications are shown in (g). Wig-1 knockdown leads to decreased levels of 14-3-3σ mRNA and increased levels of FAS mRNA in HCT116 p53+/+ and p53−/− (h) cells in the presence and absence of cisplatin (5 μM 24 h), as assessed by qRT–PCR. The figure shows 14-3-3σ and FAS mRNA levels relative to mRNA levels in control siRNA-transfected untreated cells. Representative image from one of three independent experiments are shown in (b–e). Columns and error bars in (f–h) represent the mean±s.d.; n=3; ***P<0.001; **P<0.01; *P<0.05.
Mentions: To validate the microarray findings, eight targets were selected based on the extent of changes in expression in the microarray analysis, the presence of AREs in their 3′-UTR and known involvement in the most affected pathways (Figure 1a). We analyzed the protein levels of the selected eight targets in HCT116 cells transfected with control siRNA or two different siRNAs against Wig-1 (siW1, siW2), using the same conditions as for the microarray experiments. We were able to confirm changes of FAS, WNT1, AKT3, APP, 14-3-3σ and PPP2CB at the protein level (Figures 1b–d and f). The changes in protein levels for FZD8 and CDC42 were inconsistent with the microarray data (Supplementary Figure 1).

Bottom Line: We identified 2447 transcripts with >fourfold differential expression between Wig-1 and control small interfering (si)RNA-treated HCT116 cells.We found that Wig-1 regulates FAS and 14-3-3σ mRNA independently of p53.We show that Wig-1 binds to FAS mRNA 3'-UTR and decreases its stability through an ARE in the 3'-UTR.

View Article: PubMed Central - PubMed

Affiliation: Cancer Center Karolinska (CCK), Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden.

ABSTRACT
Wig-1, also known as ZMAT3, is a p53 target gene that encodes an RNA-binding zinc-finger protein involved in the regulation of mRNA stability through binding to AU-rich elements (AREs). We have used microarray analysis to identify novel Wig-1 target mRNAs. We identified 2447 transcripts with >fourfold differential expression between Wig-1 and control small interfering (si)RNA-treated HCT116 cells. Several p53 target genes were among the deregulated transcripts. We found that Wig-1 regulates FAS and 14-3-3σ mRNA independently of p53. We show that Wig-1 binds to FAS mRNA 3'-UTR and decreases its stability through an ARE in the 3'-UTR. Depletion of Wig-1 was associated with increased cell death and reduced cell cycle arrest upon DNA damage. Our results suggest a role of Wig-1 as a survival factor that directs the p53 stress response toward cell cycle arrest rather than apoptosis through the regulation of FAS and 14-3-3σ mRNA levels.

Show MeSH
Related in: MedlinePlus