ABSTRACT
microRNA-199a (miR-199a) is a highly conserved miRNA, always deregulated in numerous human tumors. The results of microarray analysis indicated that miR-199a was frequently downregulated in hepatocellular carcinoma (HCC). The expression levels of miR-199a in 11 pairs of matched HCC neoplastic and adjacent non-neoplastic tissues, 5 HCC cell lines and liver cell line L02 were examined by quantitative real-time PCR analysis. We found miR-199a was significantly down-regulated in HCC tissues when compared with pair-matched adjacent non-tumor tissues. We also found the expression level of miR-199a was also substantially decreased in several human HCC cell lines including SMMC-7721, BEL-7402, BEL-7701, MHCC97H, and HepG2. To investigate the role of miR-199a in tumorigenesis, we developed a lentiviral vector for the expression of pre-miR-199a (Lenti-miR-199a). Lenti-miR-199a inhibited HCC cell proliferation in vitro and in vivo. Compared to parental cells or cells transfected with a control vector, the overexpression of microRNA-199a in the HCC cell lines HepG2 stably was showed to reduce cell proliferation in vitro and in vivo. Luciferase reporter assay revealed the regulation of miR-199a on 3'-UTR of HIF-1α. Further investigation confirmed that miR-199a significantly reduced the endogenous protein level of HIF-1α in hypoxia. MiR-199a inhibits cell proliferation in vitro and in vivo partly through down-regulation of HIF-1α in human HCC. Thus, these studies provide an important new insight into the pathogenesis of human HCC and it may open a new perspective for the development of effective gene therapy for human HCC.
Subject(s)
Carcinoma, Hepatocellular/pathology , Lentivirus/genetics , Liver Neoplasms/pathology , MicroRNAs/genetics , MicroRNAs/metabolism , 3' Untranslated Regions , Animals , Cell Hypoxia , Cell Line, Tumor , Cell Proliferation , Genetic Vectors/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Mice, Nude , Transplantation, HeterologousABSTRACT
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.
