SP1 is a transcriptional regulator of URG-4/URGCP gene in hepatocytes

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'-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.

URG4 mediates cell proliferation and cell cycle in osteosarcoma via GSK3β/β-catenin/cyclin D1 signaling pathway

“…URG4, a novel oncogene located on the short arm of chromosome 7p13, promotes cell proliferation via the MAPK, PI3K, Akt, and NF-kappa B pathways. Studies have shown that high levels of URG4 protein have been found in many solid tumors and are involved in tumorigenesis and tumor development, including hepatocellular carcinoma [6], gastric cancer [7], ovarian cancer [8], glioblastoma [9], glioma [10], breast carcinoma [11], nonsmall-cell lung cancer [12], and nasopharyngeal cancer [13]. Huang et al suggested that URG4 expression in osteosarcoma tissue is closely associated with recurrence, metastasis, and poor prognosis of osteosarcoma [14].…”

Upregulation of PSMD4 gene by hypoxia in prostate cancer cells

“…EMSA was carried out in accordance with Pierce manufacturer's instructions (Thermo Scientific) with slight modifications (Tokay and Kockar, 2016;Aydemir et al, 2018). Oligonucleotides used as a probe were 5'GGC ACC TAC AAC TCC CAG AAC GCA ACG TGG GGG ACG GAG GCG GAA GCA GCT GGC CAA GCC GAG GT'3 and 3'CCG TGG ATG TTG AGG GTC TTG CGT TGC ACC CCC TGC CTC CGC CTT CGT CGA CCG GTT CGG CTC CA'5.…”

Ssap-NtrB prokaryotik geninin ökaryotik ekspresyon vektörü pcDNA3.1 / V5 / His B içine klonlanması

“…Cloning of the Ssap-NtrB was performed in a three-step procedure (Figure 1). Firstly, the PCR product was cloned into the pGEM-T Easy vector (Promega) with T: A cloning system [13,14]. Subsequently, transformation was carried out.…”

“…For this purpose 3 µL of vector (1000 ng/µL), 23 µL of plasmid (780 ng/ µL), 1 µL of T4 DNA ligase Enzyme (5 U/µL) and buffer (1X) in a reaction volume of 30 µL. Ten microliters of ligation mixture were transferred into 200 µL E. coli XL1Blue (the competency at 1 x 10 6 colony-forming unit / μg DNA/100 μL cells) competent cell lines [13][14][15]. Overnight cultures were prepared from each of the resulting colonies.…”