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EVI1, a target gene for amplification at 3q26, antagonizes transforming growth factor-β-mediated growth inhibition in hepatocellular carcinoma.

Yasui K, Konishi C, Gen Y, Endo M, Dohi O, Tomie A, Kitaichi T, Yamada N, Iwai N, Nishikawa T, Yamaguchi K, Moriguchi M, Sumida Y, Mitsuyoshi H, Tanaka S, Arii S, Itoh Y - Cancer Sci. (2015)

Bottom Line: Knockdown of EVI1 resulted in increased induction of the cyclin-dependent kinase inhibitor p15(INK) (4B) by transforming growth factor (TGF)-β and decreased expression of c-Myc, cyclin D1, and phosphorylated Rb in TGF-β-treated cells.Consequently, knockdown of EVI1 led to reduced DNA synthesis and cell viability.Collectively, our results suggest that EVI1 is a probable target gene that acts as a driving force for the amplification at 3q26 in HCC and that the oncoprotein EVI1 antagonizes TGF-β-mediated growth inhibition of HCC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto, Japan.

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Effect of EVI1 knockdown on transforming growth factor-β (TGF-β)-mediated growth inhibition in JHH-1 hepatocellular carcinoma cells. (a) Immunoblot analysis of EVI1 and β-actin, an internal control, in JHH-1 and JHH-7 cells transfected with EVI1 (#1) or EVII (#2) siRNA as indicated or with control siRNAs. (b) Relative mRNA expression levels of the indicated genes in JHH-1 cells that were transfected with EVI1 (#1) or control (Cont) siRNAs and were then treated with TGF-β1 (10 ng/mL) or vehicle for 24 h. (c) Immunoblot analysis of the indicated proteins in JHH-1 and JHH-7 cells that were transfected with EVI1 siRNA (#1 or #2 as indicated) or with control siRNA and were then treated with TGF-β1 for 24 h. (d) BrdU incorporation as determined by ELISA. JHH-1 cells transfected with EVI1 siRNA (#1) or control siRNA were treated with TGF-β1 for 24 h and were then labeled with BrdU for 6 h. $P < 0.01. (e) Immunoblot analysis of EVI1 in SNU398 cells transfected with the EVI1 expression vector or an empty vector. JHH-7 cells were used as a positive control. (f, g) SNU398 cells transfected with the EVI1 expression vector were treated with TGF-β1 for 12 h then labeled with BrdU for 12 h. (f) Immunofluorescence. The cells were triple-labeled with anti-EVI1 (red), anti-BrdU (green), and DAPI (blue; nuclei). In this image, EVI1-positive cells were positive for BrdU, whereas EVI1-negative cells were negative for BrdU (arrows). (g) Percentage of BrdU-positive cells in EVI1-positive or -negative cells. More than 300 cells were counted for each group. $$P < 0.05. (h) JHH-1 cells were transfected with EVI1 siRNA (#1 or #2) or control siRNA then treated with TGF-β1. Relative cell viability (%) is shown at the indicated times after TGF-β1 treatment. $$P < 0.05.
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fig04: Effect of EVI1 knockdown on transforming growth factor-β (TGF-β)-mediated growth inhibition in JHH-1 hepatocellular carcinoma cells. (a) Immunoblot analysis of EVI1 and β-actin, an internal control, in JHH-1 and JHH-7 cells transfected with EVI1 (#1) or EVII (#2) siRNA as indicated or with control siRNAs. (b) Relative mRNA expression levels of the indicated genes in JHH-1 cells that were transfected with EVI1 (#1) or control (Cont) siRNAs and were then treated with TGF-β1 (10 ng/mL) or vehicle for 24 h. (c) Immunoblot analysis of the indicated proteins in JHH-1 and JHH-7 cells that were transfected with EVI1 siRNA (#1 or #2 as indicated) or with control siRNA and were then treated with TGF-β1 for 24 h. (d) BrdU incorporation as determined by ELISA. JHH-1 cells transfected with EVI1 siRNA (#1) or control siRNA were treated with TGF-β1 for 24 h and were then labeled with BrdU for 6 h. $P < 0.01. (e) Immunoblot analysis of EVI1 in SNU398 cells transfected with the EVI1 expression vector or an empty vector. JHH-7 cells were used as a positive control. (f, g) SNU398 cells transfected with the EVI1 expression vector were treated with TGF-β1 for 12 h then labeled with BrdU for 12 h. (f) Immunofluorescence. The cells were triple-labeled with anti-EVI1 (red), anti-BrdU (green), and DAPI (blue; nuclei). In this image, EVI1-positive cells were positive for BrdU, whereas EVI1-negative cells were negative for BrdU (arrows). (g) Percentage of BrdU-positive cells in EVI1-positive or -negative cells. More than 300 cells were counted for each group. $$P < 0.05. (h) JHH-1 cells were transfected with EVI1 siRNA (#1 or #2) or control siRNA then treated with TGF-β1. Relative cell viability (%) is shown at the indicated times after TGF-β1 treatment. $$P < 0.05.

Mentions: The oncoprotein EVI1 is known to suppress TGF-β signaling by inhibiting Smad3 and to activate the PI3K/AKT and RAS/ERK signaling pathways. To determine whether EVI1 affects any of these signaling pathways in HCC cells, EVI1 protein expression in JHH-1 cells was knocked down using two different siRNAs (#1 and #2). EVI1 siRNA (#1) was also used to knockdown the EVII protein in a second cell line, JHH-7, that had the second highest level of EVII expression (Fig.2). Immunoblot analysis indicated successful knockdown of EVI1 in all cases (Fig.4a). Knockdown of EVI1 using EVI1 siRNA (#1) had little effect on the phosphorylated levels of AKT or ERK (data not shown), suggesting that EVI1 does not regulate the PI3K/AKT or RAS/ERK signaling pathways. To assay if EVI1 affects TGF-β signaling in HCC cells, JHH-1 cells that were transfected with EVI1 siRNA (#1) or control siRNA were treated with TGF-β for 24 h, following which the mRNA expression of PAI-1, a classic TGF-β-responsive gene, was analyzed using RT-PCR. The expression of PAI-1 mRNA was induced by TGF-β and was higher in JHH-1 cells treated with EVI1 siRNA compared to those treated with control siRNA (Fig.4b), suggesting that EVI1 attenuates TGF-β-mediated gene induction in JHH-1 cells.


EVI1, a target gene for amplification at 3q26, antagonizes transforming growth factor-β-mediated growth inhibition in hepatocellular carcinoma.

Yasui K, Konishi C, Gen Y, Endo M, Dohi O, Tomie A, Kitaichi T, Yamada N, Iwai N, Nishikawa T, Yamaguchi K, Moriguchi M, Sumida Y, Mitsuyoshi H, Tanaka S, Arii S, Itoh Y - Cancer Sci. (2015)

Effect of EVI1 knockdown on transforming growth factor-β (TGF-β)-mediated growth inhibition in JHH-1 hepatocellular carcinoma cells. (a) Immunoblot analysis of EVI1 and β-actin, an internal control, in JHH-1 and JHH-7 cells transfected with EVI1 (#1) or EVII (#2) siRNA as indicated or with control siRNAs. (b) Relative mRNA expression levels of the indicated genes in JHH-1 cells that were transfected with EVI1 (#1) or control (Cont) siRNAs and were then treated with TGF-β1 (10 ng/mL) or vehicle for 24 h. (c) Immunoblot analysis of the indicated proteins in JHH-1 and JHH-7 cells that were transfected with EVI1 siRNA (#1 or #2 as indicated) or with control siRNA and were then treated with TGF-β1 for 24 h. (d) BrdU incorporation as determined by ELISA. JHH-1 cells transfected with EVI1 siRNA (#1) or control siRNA were treated with TGF-β1 for 24 h and were then labeled with BrdU for 6 h. $P < 0.01. (e) Immunoblot analysis of EVI1 in SNU398 cells transfected with the EVI1 expression vector or an empty vector. JHH-7 cells were used as a positive control. (f, g) SNU398 cells transfected with the EVI1 expression vector were treated with TGF-β1 for 12 h then labeled with BrdU for 12 h. (f) Immunofluorescence. The cells were triple-labeled with anti-EVI1 (red), anti-BrdU (green), and DAPI (blue; nuclei). In this image, EVI1-positive cells were positive for BrdU, whereas EVI1-negative cells were negative for BrdU (arrows). (g) Percentage of BrdU-positive cells in EVI1-positive or -negative cells. More than 300 cells were counted for each group. $$P < 0.05. (h) JHH-1 cells were transfected with EVI1 siRNA (#1 or #2) or control siRNA then treated with TGF-β1. Relative cell viability (%) is shown at the indicated times after TGF-β1 treatment. $$P < 0.05.
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fig04: Effect of EVI1 knockdown on transforming growth factor-β (TGF-β)-mediated growth inhibition in JHH-1 hepatocellular carcinoma cells. (a) Immunoblot analysis of EVI1 and β-actin, an internal control, in JHH-1 and JHH-7 cells transfected with EVI1 (#1) or EVII (#2) siRNA as indicated or with control siRNAs. (b) Relative mRNA expression levels of the indicated genes in JHH-1 cells that were transfected with EVI1 (#1) or control (Cont) siRNAs and were then treated with TGF-β1 (10 ng/mL) or vehicle for 24 h. (c) Immunoblot analysis of the indicated proteins in JHH-1 and JHH-7 cells that were transfected with EVI1 siRNA (#1 or #2 as indicated) or with control siRNA and were then treated with TGF-β1 for 24 h. (d) BrdU incorporation as determined by ELISA. JHH-1 cells transfected with EVI1 siRNA (#1) or control siRNA were treated with TGF-β1 for 24 h and were then labeled with BrdU for 6 h. $P < 0.01. (e) Immunoblot analysis of EVI1 in SNU398 cells transfected with the EVI1 expression vector or an empty vector. JHH-7 cells were used as a positive control. (f, g) SNU398 cells transfected with the EVI1 expression vector were treated with TGF-β1 for 12 h then labeled with BrdU for 12 h. (f) Immunofluorescence. The cells were triple-labeled with anti-EVI1 (red), anti-BrdU (green), and DAPI (blue; nuclei). In this image, EVI1-positive cells were positive for BrdU, whereas EVI1-negative cells were negative for BrdU (arrows). (g) Percentage of BrdU-positive cells in EVI1-positive or -negative cells. More than 300 cells were counted for each group. $$P < 0.05. (h) JHH-1 cells were transfected with EVI1 siRNA (#1 or #2) or control siRNA then treated with TGF-β1. Relative cell viability (%) is shown at the indicated times after TGF-β1 treatment. $$P < 0.05.
Mentions: The oncoprotein EVI1 is known to suppress TGF-β signaling by inhibiting Smad3 and to activate the PI3K/AKT and RAS/ERK signaling pathways. To determine whether EVI1 affects any of these signaling pathways in HCC cells, EVI1 protein expression in JHH-1 cells was knocked down using two different siRNAs (#1 and #2). EVI1 siRNA (#1) was also used to knockdown the EVII protein in a second cell line, JHH-7, that had the second highest level of EVII expression (Fig.2). Immunoblot analysis indicated successful knockdown of EVI1 in all cases (Fig.4a). Knockdown of EVI1 using EVI1 siRNA (#1) had little effect on the phosphorylated levels of AKT or ERK (data not shown), suggesting that EVI1 does not regulate the PI3K/AKT or RAS/ERK signaling pathways. To assay if EVI1 affects TGF-β signaling in HCC cells, JHH-1 cells that were transfected with EVI1 siRNA (#1) or control siRNA were treated with TGF-β for 24 h, following which the mRNA expression of PAI-1, a classic TGF-β-responsive gene, was analyzed using RT-PCR. The expression of PAI-1 mRNA was induced by TGF-β and was higher in JHH-1 cells treated with EVI1 siRNA compared to those treated with control siRNA (Fig.4b), suggesting that EVI1 attenuates TGF-β-mediated gene induction in JHH-1 cells.

Bottom Line: Knockdown of EVI1 resulted in increased induction of the cyclin-dependent kinase inhibitor p15(INK) (4B) by transforming growth factor (TGF)-β and decreased expression of c-Myc, cyclin D1, and phosphorylated Rb in TGF-β-treated cells.Consequently, knockdown of EVI1 led to reduced DNA synthesis and cell viability.Collectively, our results suggest that EVI1 is a probable target gene that acts as a driving force for the amplification at 3q26 in HCC and that the oncoprotein EVI1 antagonizes TGF-β-mediated growth inhibition of HCC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto, Japan.

Show MeSH
Related in: MedlinePlus