Effects of hypoxia-inducible factor-1α silencing on the proliferation of CBRH-7919 hepatoma cells

AIM:To study the effects of hypoxia-inducible factor1α(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 Lipofectamine2000TM,and transfected into the hypoxic hepatoma cells.Real time reverse transcription-polymerase chain reaction(RTPCR) 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 contr