title: URG4/URGCP enhances the angiogenic capacity of human hepatocellular carcinoma cells in vitro via activation of the NF-κB signaling pathway.
abstract: BACKGROUND
  Angiogenesis is essential for tumor growth. Hepatocellular carcinoma (HCC) is characterized by hypervascularity; high levels of angiogenesis are associated with poor prognosis and a highly invasive phenotype in HCC. Up-regulated gene-4 (URG4), also known as upregulator of cell proliferation (URGCP), is overexpressed in multiple tumor types and has been suggested to act as an oncogene. This study aimed to elucidate the effect of URG4/URGCP on the angiogenic capacity of HCC cells in vitro.
  
  
  METHODS
  Expression of URG4/URGCP in HCC cell lines and normal liver epithelial cell lines was examined by Western blotting and quantitative real-time PCR. URG4/URGCP was stably overexpressed or transiently knocked down using a shRNA in two HCC cell lines. The human umbilical vein endothelial cell (HUVEC) tubule formation and Transwell migration assays and chicken chorioallantoic membrane (CAM) assay were used to examine the angiogenic capacity of conditioned media from URG4/URGCP-overexpressing and knockdown cells. A luciferase reporter assay was used to examine the transcriptional activity of nuclear factor kappa - light - chain - enhancer of activated B cells (NF-κB). NF-κB was inhibited by overexpressing degradation-resistant mutant inhibitor of κB (IκB)-α. Expression of vascular endothelial growth factor C (VEGFC), tumor necrosis factor-α (TNFα), interleukin (IL)-6, IL-8 and v-myc avian myelocytomatosis viral oncogene homolog (MYC) were examined by quantitative real-time PCR; VEGFC protein expression was analyzed using an ELISA.
  
  
  RESULTS
  URG4/URGCP protein and mRNA expression were significantly upregulated in HCC cell lines. Overexpressing URG4/URGCP enhanced - while silencing URG4/URGCP decreased - the capacity of HCC cell conditioned media to induce HUVEC tubule formation and migration and neovascularization in the CAM assay. Furthermore, overexpressing URG4/URGCP increased - whereas knockdown of URG4/URGCP decreased - VEGFC expression, NF-κB transcriptional activity, the levels of phosphorylated (but not total) IκB kinase (IKK) and IκB-α, and expression of TNFα, IL-6, IL-8 and MYC in HCC cells. Additionally, inhibition of NF-κB activity in HCC cells abrogated URG4/URGCP-induced NF-κB activation and angiogenic capacity.
  
  
  CONCLUSIONS
  This study suggests that URG4/URGCP plays an important pro-angiogenic role in HCC via a mechanism linked to activation of the NF-κB pathway; URG4/URGCP may represent a potential target for anti-angiogenic therapy in HCC.
authors.affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510000, P.R. China. sizhongx@hotmail.com.|Department of Gastroenterology, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510000, P.R. China. sizhongx@hotmail.com.|Department of Internal Medicine, Baoan People's Hospital, Shenzhen, 518101, P.R. China. sizhongx@hotmail.com.
authors.forename: Sizhong
authors.initials: S
authors.lastname: Xing
authors.affiliation: Department of Medical Imaging, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510000, P.R. China. lihecihange@163.com.
authors.forename: Bing
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Department of Oncology, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510000, P.R. China. huaruixi@163.com.
authors.forename: Ruixi
authors.initials: R
authors.lastname: Hua
authors.affiliation: Center for Cancer and Inflammation Research, Institute of Integrated Bioinformedicine and Translational Science, Hong Kong Baptist University, Hong Kong, S.A.R., China. wcstai@hkbu.edu.hk.
authors.forename: William Chi-shing
authors.initials: WC
authors.lastname: Tai
authors.affiliation: Department of Gastroenterology, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510000, P.R. China. xanksu@126.com.
authors.forename: Zhirong
authors.initials: Z
authors.lastname: Zeng
authors.affiliation: Department of Hepatobiliary Surgery, the First Affiliated Hospital of Gannan Medical University, Ganzhou, 341000, P.R. China. binhuix@163.com.
authors.forename: Binhui
authors.initials: B
authors.lastname: Xie
authors.affiliation: Department of Internal Medicine, Baoan People's Hospital, Shenzhen, 518101, P.R. China. chengh773@tom.com.
authors.forename: Chenghui
authors.initials: C
authors.lastname: Huang
authors.affiliation: Department of Internal Medicine, Baoan People's Hospital, Shenzhen, 518101, P.R. China. jusuxue@tom.com.
authors.forename: Jisu
authors.initials: J
authors.lastname: Xue
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester, UK. shiqiuxiong@126.com.
authors.forename: Shiqiu
authors.initials: S
authors.lastname: Xiong
authors.affiliation: Department of Medical Imaging, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510000, P.R. China. jianyongyang0899@hotmail.com.
authors.forename: Jianyong
authors.initials: J
authors.lastname: Yang
authors.affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510000, P.R. China. side743@126.com.
authors.forename: Side
authors.initials: S
authors.lastname: Liu
authors.affiliation: Department of Medical Imaging, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510000, P.R. China. hepingli7408@yahoo.com.|Department of Oncology, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510000, P.R. China. hepingli7408@yahoo.com.
authors.forename: Heping
authors.initials: H
authors.lastname: Li
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis; D016209:Interleukin-8 / Q000096:biosynthesis; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology; D016328:NF-kappa B / Q000096:biosynthesis* / Q000235:genetics; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D015398:Signal Transduction; D015533:Transcriptional Activation / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D042461:Vascular Endothelial Growth Factor A
chemical_list: C508600:IL6 protein, human; D015850:Interleukin-6; D016209:Interleukin-8; D016328:NF-kappa B; D009363:Neoplasm Proteins; C439524:TNF protein, human; D014409:Tumor Necrosis Factor-alpha; C470990:URGCP protein, human; D042461:Vascular Endothelial Growth Factor A