Abstract
Aim: The current in vitro study investigated the role of Period 2 (PER2) in aggressiveness and the acquisition of drug resistance in hepatocellular carcinoma (HCC). Methods: Parental PLC/PRF/5 cells, along with everolimus-resistant (EveR) and Sorafenib-resistant (SorR) cell lines, were used in this study. PER2 expression was silenced using siRNA knockdown (KD) and blocked using CRISPR/Cas9 Plasmid knockout (KO). PER2 expression levels were assessed by quantitative real-time reverse transcription polymerase chain reaction and immunofluorescence, together with markers of epithelial-mesenchymal transition, casein kinase 1ε (CK1ε), and tumor protein p53. Modulation of p53, p21, cellular myelocytomatosis oncogene, and mouse double minute 2 homolog was investigated by western blot. Mitochondrial activity was evaluated using the Seahorse System. The role of PER2 on the onset of aggressiveness was examined through assays of cell proliferation, migration, and colony formation. Results: PLC/PRF/5 everolimus-resistant (EveR), SorR, PER2 KD, and PER2 KO cells expressed significantly lower PER2 mRNA and protein levels compared to the parental PLC/PRF/5 cells. Remarkably, in PLC/PRF/5 EveR and SorR cells, PER2 protein was entirely localized in the cytoplasm, where it colocalized with CK1ε, in contrast to the parental cells. In PLC/PRF/5 EveR, PER2 KD and PER2 KO cells, but not in SorR cells, E-cadherin was significantly decreased while vimentin and ZEB1 protein levels were significantly increased across all modified cell models. Interestingly, p53 expression was reduced in PER2 KO cells and completely absent in PLC/PRF/5 EveR and SorR cells. Consistent with these findings, the inhibitory effect of everolimus (10-9 M) and sorafenib (5 × 10-6 M) on cell proliferation, migration, and colony formation observed in parental PLC/PRF/5 cells were reversed in PER2 KD and KO cells, which was accompanied by upregulation of oncogenes, downregulation of tumor suppressor genes, and alterations in mitochondrial activity. Conclusion: These results suggest that the acquisition of an aggressive phenotype is characterized by reduced PER2 expression and loss of its nuclear translocation, which, in turn, is associated with resistance to systemic therapy in hepatocellular carcinoma.
Keywords:
Clock genes; Period 2; drug resistance; epithelial-mesenchymal transition (EMT); everolimus; hepatocellular carcinoma; sorafenib.