title: Effects of hypoxia-inducible factor-1α silencing on the proliferation of CBRH-7919 hepatoma cells.
abstract: OBJECTIVE
  To study the effects of hypoxia-inducible factor-1α (HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells.
  
  
  METHODS
  The CBRH-7919 rat hepatoma cell line was used in this study and the hypoxic model was constructed using CoCl2. The HIF-1α-specific RNAi sequences were designed according to the gene coding sequence of rat HIF-1α obtained from GeneBank. The secondary structure of the HIF-1α gene sequence was analyzed using RNA draw software. The small interfering RNA (siRNA) transfection mixture was produced by mixing the siRNA and Lipofectamine2000(TM), and transfected into the hypoxic hepatoma cells. Real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assay were used to detect the expression levels of mRNA and protein. HIF-1α and vascular endothelial growth factor (VEGF) mRNA was determined using real time RT-PCR; the protein expression levels of AKT, p-AKT, p21 and cyclinD1 were determined using Western blotting. The proliferation of hepatoma cells was observed using the methyl thiazolyl tetrazolium (MTT) assay and the bromodeoxyuridine (BrdU) incorporation cell proliferation assay.
  
  
  RESULTS
  Under induced hypoxia, the viability of the hepatoma cells reached a minimum at 800 μmol/L CoCl2; the viability of the cells was relatively high at CoCl2 concentrations between 100 μmol/L and 200 μmol/L. Under hypoxia, the mRNA and protein expression levels of HIF-1α and VEGF were significantly higher than that of hepatoma cells that were cultured in normaxia. HIF-1α-specific RNAi sequences were successfully transfected into hepatoma cells. The transfection of specific siRNAs significantly inhibited the mRNA and protein expression levels of HIF-1α and VEGF, along with the protein expression levels of p-AKT and cyclinD1; the protein expression of p21 was significantly increased, and there was no significant difference in the expression of AKT. The MTT assay showed that the amount of hepatoma cells in S phase in the siRNA transfection group was obviously smaller than that in the control group; in the siRNA transfection group, the amount of hepatoma cells in G1 phase was more than that in the control group. The BrdU incorporation assay showed that the number of BrdU positive hepatoma cells in the siRNA transfection group was less than that in the control group. The data of the MTT assay and BrdU incorporation assay suggested that HIF-1α silencing using siRNAs significantly inhibited the proliferation of hepatoma cells.
  
  
  CONCLUSIONS
  Hypoxia increases the expression of HIF-1α, and HIF-1α silencing significantly inhibits the proliferation of hypoxic CBRH-7919 rat hepatoma cells.
authors.affiliation: Department of Interventional Radiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510210, Guangdong Province, China.
authors.forename: Lin-Feng
authors.initials: LF
authors.lastname: Xu
authors.forename: Jia-Yan
authors.initials: JY
authors.lastname: Ni
authors.forename: Hong-Liang
authors.initials: HL
authors.lastname: Sun
authors.forename: Yao-Ting
authors.initials: YT
authors.lastname: Chen
authors.forename: Yu-Dan
authors.initials: YD
authors.lastname: Wu
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D059447:Cell Cycle Checkpoints; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D003035:Cobalt / Q000494:pharmacology; D019938:Cyclin D1 / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D013997:Time Factors; D059016:Tumor Microenvironment; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: C530106:Ccnd1 protein, rat; C493958:Cdkn1a protein, rat; D050759:Cyclin-Dependent Kinase Inhibitor p21; C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat; D019938:Cyclin D1; D003035:Cobalt; D051057:Proto-Oncogene Proteins c-akt; C018021:cobaltous chloride
keywords: CBRH-7919 hepatoma cells; Hypoxia-inducible factor-1α; Protein kinase B; RNA interference; Vascular endothelial growth factor
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