Effects Of Hypoxia On The Expressions Of Dec1and HIF-1α In Hepatocellular Carcinoma

Objective:To investigate the differential expression of differentiated embryo-chondrocyte expressed gene1(DEC1) in both normal liver cell line and hepatocellular carcinoma (HCC) cell lines and the role of DEC1in the development of HCC by culturing the cell lines under normoxia. To study the effect of hypoxia on the expressions of DECl and hypoxia-inducible factor-1α (HIF-1α) in HCC by culturing the HCC cell lines under hypoxia. To explore the regulation of HIF-1a on DEC1through inhibiting the expression of HIF-1a protein under hypoxia.Methods:1. Cell culture under normoxia:Human normal liver cell line QSG-7701, HCC cell lines BEL-7402and SMMC-7721were cultured in RPMI1640containing10%fetal bovine serum (FBS),100U/ml penicillin and100μg/ml streptomycin at37℃in5%CO2. Then collecting cells which were in logarithmic growth phase for RT-PCR and western blot experiments.2. Cell culture under hypoxia:Hypoxic condition was induced by CoCl2. HCC cell lines BEL-7402and SMMC-7721were cultured in RPMI1640containing200uM CoCl2for different time (0,2,4,6,24and48h). Then collecting cells for RT-PCR and western blot experiments.3. Cell culture with HIF-1a inhibitor YC-1:HCC cell line SMMC-7721was cultured in RPMI1640containing200μM CoCl2and50μM YC-1for4h, the other two groups were treated with (hypoxia) or without (normoxia)200μM CoCl2for4h. Then collecting cells for RT-PCR and western blot experiments. 4. RT-PCR was used to measure the mRNA expression of HIF-1a and DEC1.5. Western blot was used to determine the protein expression of HIF-1a and DEC1.6. The data were analyzed by SPSS17.0statistical software. Differences between the mean values of two subgroups were evaluated using Student’s t-test. Differences between the mean values of more than three subgroups were compared by one-way analysis of variance, further differences between any two subgroups were evaluated using LSD-t test. A level of P<0.05was considered statistically significant.Results:1. The experimental result under normoxia showed that high level of DEC1expression were detected in all cell lines examined, particularly the HCC cell lines (BEL-7402, SMMC-7721) as compared with normal liver cell line QSG-7701, P<0.05.2. The experimental result under hypoxia showed that hypoxia could not affect the transcription of HIF-1α (P>0.05). However, the protein level of HIF-1α was upregulated under hypoxia in a time-dependent manner, which could also be seen in DEC1mRNA and protein levels (P<0.01).3. High level of HIF-1α mRNA expression were detected in all three groups (normoxia group, hypoxia group, hypoxia+YC-1group), but there were no statistically significant, P>0.05. However, the mRNA level of DEC1and the protein level of HIF-1α and DEC1were lower in hypoxia+YC-1group than hypoxia group, P<0.01.Conclusion:1. Our research confirmed that the mRNA and protein expression of DEC1in HCC cell lines was significantly higher than that in normal liver cell line, which implied that DEC1played an important role in the carcinogenesis and development of HCC.2. Using CoCl2hypoxia model, we firstly showed that hypoxia could induce a higher expression of HIF-1α protein, DEC1mRNA and DEC1protein in HCC cell lines. Meanwhile, the expression of HIF-1α and DEC1were remained at high levels when the hypoxia time had lasted for24h and48h. These suggested that HIF-1α and DEC1played an important role in the adaption to hypoxic microenvironment of liver cancer cells.3. To further identify the relationship of HIF-1α and DEC1, we used YC-1to inhibit expression of HIF-1α protein. The result showed that hypoxia can induce a higher expression of DEC1by up-regulating the expression of HIF-1α protein.