SP1 is a transcriptional regulator of URG-4/URGCP gene in hepatocytes Esra Tokay 1 • Feray Kockar 1 Received: 30 June 2016 / Accepted: 22 September 2016 Ó Springer Science+Business Media New York 2016 Abstract URG-4/URGCP gene was implicated as an oncogene that contributes hepatocarcinogenesis regulated by Hepatitis-B-virus-encoded X antigen. However, the mechanism of transcriptional regulation of this gene remains largely unknown. For this reason, we focused on the functional analyses of URG4/URGCP promoter site. First, 545 bp of URG-4/URGCP, -482/?63, and three different 5 0 -truncated constructs, -109/?63, -261/?63, -344/?63 were cloned by PCR-based approach into pMetLuc luciferase reporter vector. Transient transfection assay showed that, -109/?63 construct has the highest activity. The promoter of URG-4/URGCP gene contained a CpG island region spanning 400 bp from translation start site. Many SP1/GC boxes, named GC-1 to GC-10 are present in 545 bp of URG-4/URGCP promoter. Because of presence of multiple SP1/GC boxes, promoter constructs were transiently co-transfected with SP1 expression vector to determine the effect of SP1 on URG-4/URGCP promoter activity. Co-transfection analyses induced the basal activity of -268/?63, -344/?63 and -482/?63 constructs. EMSA analysis of GC-4, GC-5, GC-6 and GC-7 binding sites located in -128/-148 bases, showed two DNA– protein binding complexes. Competition assay and super- shifted complexes indicated these complexes are resulted from SP1 binding. Also, site-directed mutagenesis of potential SP1 binding sites diminished both DNA–protein complexes and SP1-mediated upregulation of URG-4 promoter activity. These findings are valuable for understanding transcriptional regulation of URG4/URGCP that has a pivotal role in cancer progression. Keywords URG4/URGCP Á SP1 Á Transcriptional regulation Á Promoter Introduction URG4/URGCP was first found in hepatocellular carcinoma and was strongly expressed in Hepatitis B-infected liver when compared with uninfected liver. Overexpression of URG4/URGCP in HepG2 cells promoted hepatocellular growth and survival in tissue culture and in soft agar and accelerated tumor development in nude mice [1]. Overex- pression of this gene also upregulated cyclin D1 in gastric carcinoma (GES-1 cells), whereas repression of it in SGC7901 and MKN28 cells downregulated cyclin D1. Therefore, it was implicated that URG4/URGCP played an important role in the development of human gastric cancer by regulating the expression of cyclin D1 and might be used as a potential therapeutic target for gastric cancer [2]. The expression of this gene was examined in osteosarcoma tissues by Huang et al. [3]. They demonstrated that URG4 was highly expressed in osteosarcoma specimens and also increased in the specimens with recurrence and metastasis [3]. RNAi-mediated URG-4/URGCP gene silencing sup- pressed the entry of cells into S phase and cell growth in culture and downregulated cyclin D1 in HepG2 cells. Conversely, overexpression of URG4 gene upregulated cyclin D1 expression [4]. The expression of URG4 was upregulated in several types of cancers, namely, in leuke- mia cells [5], glioma tissue [6] and cervical cancer tissues [7]. Knockdown of URG4/URGCP in bladder cancer decreased, while overexpression increased, the resistance & Feray Kockar fkockar@balikesir.edu.tr 1 Department of Molecular Biology and Genetics, Faculty of Science and Literature, Balikesir University, Balıkesir, Turkey 123 Mol Cell Biochem DOI 10.1007/s11010-016-2826-7