EVI1, a target gene for amplification at 3q26, antagonizes transforming growth factor-β-mediated growth inhibition in hepatocellular carcinoma.
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.
Affiliation: Department of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto, Japan.Show MeSH
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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.
Affiliation: Department of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto, Japan.