title: Inhibition of connective tissue growth factor overexpression decreases growth of hepatocellular carcinoma cells in vitro and in vivo.
abstract: BACKGROUND
  We have previously found that connective tissue growth factor (CTGF) is highly expressed in a rat model of liver cancer. Here, we examined expression of CTGF in human hepatocellular carcinoma (HCC) cells and its effect on cell growth.
  
  
  METHODS
  Real-time PCR was used to observe expression of CTGF in human HCC cell lines HepG2, SMMC-7721, MHCC-97H and LO2. siRNA for the CTGF gene was designed, synthesized and cloned into a Plk0.1-GFP-SP6 vector to construct a lentivirus-mediated shRNA/CTGF. CTGF mRNA and protein expression in HepG2 cells treated by CTGF-specific shRNA was evaluated by real-time PCR and Western blotting. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was utilized to evaluate the growth effect, and a colony formation assay was used for observing clonogenic growth. In vivo, tumor cell proliferation was evaluated in a nude mouse model of xenotransplantation. Statistical significance was determined by t test for comparison between two groups, or analysis of variance (ANOVA) for multiple groups.
  
  
  RESULTS
  Immunohistochemical staining of CTGF was seen in 35 of 40 HCC samples (87.5%). CTGF was overexpressed 5-fold in 20 HCC tissues, compared with surrounding non-tumor liver tissue. CTGF mRNA level was 5 - 8-fold higher in HepG2, SMMC-7721 and MHCC-97H than in LO2 cells. This indicated that the inhibition rate of cell growth was 43% after knockdown of CTGF expression (P < 0.05). Soft agar colony formation assay showed that siRNA mediated knockdown of CTGF inhibited colony formation in soft agar of HepG2 cells (P < 0.05). The volume of tumors from CTGF-shRNA-expressing cells only accounted for 35% of the tumors from the scrambled control-infected HepG2 cells (P < 0.05).
  
  
  CONCLUSIONS
  CTGF was overexpressed in human HCC cells and downregulation of CTGF inhibited HCC growth in vitro and in vivo. Knockdown of CTGF may be a potential therapeutic strategy for treatment of HCC.
authors.affiliation: Department of Pathology, School of Medicine, Yangzhou University, Yangzhou, Jiangsu 225500, China.
authors.forename: Xiao-Qin
authors.initials: XQ
authors.lastname: Jia
authors.forename: Hai-Qing
authors.initials: HQ
authors.lastname: Cheng
authors.forename: Hong
authors.initials: H
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhu
authors.forename: Yu-Hua
authors.initials: YH
authors.lastname: Li
authors.forename: Zhen-Qing
authors.initials: ZQ
authors.lastname: Feng
authors.forename: Jian-Ping
authors.initials: JP
authors.lastname: Zhang
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000628:therapy*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics / Q000502:physiology; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000628:therapy*; D008297:Male; D051379:Mice; D008819:Mice, Nude; D034741:RNA, Small Interfering / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
chemical_list: D034741:RNA, Small Interfering; D055513:Connective Tissue Growth Factor