TGF-Beta upregulates URG-4/URGCP gene expression in Hepatoma cells

Abstract

URG-4/URGCP has been found to contribute importantly tomultistep carcinogenesis. Over-expression of URG4/URGCPaccelerated tumor development in nude mice and also induced insome cancer cell types such as hepatocellular carcinoma, osteo-sarcoma, gastric and leukemia. However, the mechanism ofURG-4 regulation by TGF-b1 and its significance in cancer pro-gression remains largely unknown. To our knowledge, this is thefirst study analyzing mRNA and protein level changes of URG-4/URGCP gene in the presence or absence TGF-b1 in HepatomaCell line. Hep3B (Human Hepatoma Cells) cells were cultured inDMEM supplemented with 10% heat-inactivated Fetal CalfSerum and 2 mML-Glutamine. For cytokine treatment, serum-starved cells were treated with 10, 100, 200 and 500 U/ml TGF-b1 cytokine at certain time points (1, 3, 6, 24, 48 and 72 h). Weobtained the effect of TGF-b1 on URG-4/URGCP expression atmRNA level with Real-Time PCR analysis and protein level withWestern blotting. Our results showed an increased mRNA levelof URG-4 gene during early (1–3 h) and late (48 h) time with200 U/ml TGF-b1 treatment. In addition, we investigated dose-response of TGF-b1 on URG-4/URGCP expression. Basically,100 and 200 U/ml of TGF-bupregulated URG-4 gene with 2-and 4-fold, respectively. Consistent with the mRNA results, thelevel of the URG-4 protein was significantly increased. Quantita-tive analysis shows a two-fold increase of the URG-4 proteinlevel 48 h after TGF-b1 induction.