E2F1 promotes LIHC malignant phenotype via NEK2-mediated Wnt/β-catenin and Notch activation and EMT.

[BACKGROUND] Hepatocellular carcinoma (LIHC) is the most common primary liver cancer, with high recurrence and metastasis rates, and its molecular mechanisms remain incompletely understood.

[METHODS] The cancer genome atlas (TCGA) database was used to obtain LIHC-related information. TNMplot, gene expression profiling interactive analysis (GEPIA), and kaplan-meier plotter were used for differential gene expression analysis, visualization, and assessing the relationship between genes and patient survival. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) were used to detect gene and protein expression levels. Cell transfection was used for gene knockdown and overexpression. Transwell and wound healing assays were used to evaluate cell migration and invasion. The jaspar database was used to predict transcription factor-gene interactions. Chromatin immunoprecipitation (ChIP) and dual-luciferase assays were used to validate transcription factor-gene binding.

[RESULTS] Never in mitosis gene a related kinase 2 (NEK2) was highly expressed in LIHC, and its high expression potentially had a negative impact on patient survival. NEK2 knockdown reduced the migration and invasion abilities of LIHC cells, decreased the expression of epithelial-mesenchymal transition (EMT) markers neural cadherin (N-cadherin) and vimentin, and increased epithelial cadherin (E-cadherin) expression, whereas NEK2 overexpression showed the opposite effects. E2F transcription factor 1 (E2F1) was also highly expressed in LIHC and positively regulated NEK2, exhibiting similar regulatory effects on LIHC cells. Moreover, NEK2 was positively correlated with Wnt/β-catenin marker catenin beta 1 (CTNNB1), as well as Notch signaling markers notch receptor 1 (NOTCH1), jagged canonical notch ligand 1 (JAG1), HES family BHLH transcription factor 1 (HES1), and HES-related family BHLH transcription factor with YRPW motif 1 (HEY1).

[CONCLUSION] The E2F1/NEK2 axis promotes the activation of Wnt/β-catenin and Notch signaling pathways and induces EMT to drive malignant behavior of LIHC cells.