Hypoxia disrupts the expression levels of circadian rhythm genes in hepatocellular carcinoma

Disturbance in the expression of circadian rhythm genes is a common feature in certain types of cancer, however the mechanisms mediating this disturbance remain to be elucidated. The present study aimed to investigate the effect of hypoxia on the expression of circadian rhythm genes in liver cancer cells and to identify the mechanisms underlying this effect in hepatocellular carcinoma (HCC). The HCC cell line, PLC/PRF/5. was treated With either a vehicle control or CoCl2 at 50, 100 or 200 mu M for 24 h. Following treatment, the protein expression levels of hypoxia-inducible factor (HIF)-1 alpha and HIF-2 alpha were detected by western blotting and the mRNA expression levels of circadian rhythm genes, including circadian locomotor output cycles kaput (Clock), brain and muscle Arnt-like 1 (Bmall), period (Per)1, Per2, Per3, cryptochrome (Cry)1, Cry2 and casein kinase l epsilon (CKI epsilon), were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Expression plasmids containing HIF-1 alpha or HIF-2 alpha were transfected into the PLC/PRF/5 cells using liposomes and RT-qPCR was used to determine the effects of the transfections on the expression levels of circadian rhythm genes. Following treatment with CoCl2, the protein expression levels of HIF-1 alpha and HIF-2 alpha were upregulated in a CoCl2 concentration-dependent manner. The mRNA expression levels of Clock, Bmall and Cry2 were increased, and the mRNA expression levels of Per1, Per2, Per3, Cry1 and CKI epsilon were decreased following CoCl2 treatment (P<0.05). In the PLC/PRF/5 cells transfected with the plasmid containing HIF-1 alpha, the mRNA expression levels of Clock, Bmall and Cry2 were increased, and the mRNA expression levels of Per1, Per2, Per3, Cry1 and CKI epsilon were decreased. In the PLC/PRF/5 cells transfected with the plasmid containing HIF-2 alpha, the mRNA expression levels of Clock, Bmall, Per1, Cry1, Cry2 and CKI epsilon were upregulated, and the mRNA expression levels of Per2 and Per3 were downregulated (P<0.05). A hypoxic microenvironment may contribute to the disturbance in the expression of circadian genes in HCC. HIF-1 alpha and HIF-2 alpha are involved in this process and have redundant, but not identical effects.