TNF-alpha induces URG-4/URGCP gene expression in hepatoma cells through starvation dependent manner

Abstract

URG-4/URGCP is a gene that may be associated with the onset of tumorigenesis and cell cycle regulation. In the literature, there is no study about inflammatory cytokine-mediated URG-4/URGCP regulation. In this study, the effect of TNF-alpha cytokine was investigated on URG-4/URGCP expression in serum-starved and serum-cultured hepatoma cells. The effect of TNF-alpha on hepatoma cells was shown using MTT and Annexin-V/PI staining with flow cytometer analyses. As a result, TNF-alpha leads to the cytotoxicity of hepatoma cells in serum-starved condition whereas no decrease was detected from serum-cultured condition. TNF-alpha-mediated URG-4/URGCP expression was determined at mRNA and protein level with qRT-PCR analyses and Western blotting method. URG-4URGCP mRNA expression was upregulated in both serum-starved and serum-cultured hepatoma cells. The transfection studies were carried out with URG-4/URGCP promoter constructs for determining the transcriptional activity. TNF-alpha caused to the upregulation of the activities of URG/URGCP promoter constructs. The basal activities of the URG-4/URGCP promoter conditions are differential according to serum conditions. In addition, some pathway inhibitors were added into hepatoma cells for blocking specific pathways to find out TNF-alpha-mediated URG-4/URGCP upregulation at mRNA and protein level. TNF-alpha used JNK and PI3K pathways for regulating URG-4/URGCP gene at serum-starved Hep3B cells. In serum-cultured condition, wortmannin (PI3K inhibitor), MEK-1 (MAPK inhibitor), and SP600125 (JNK inhibitor) did not inhibit the activation response of TNF-alpha on URGCP.