Aims
C/EBPalpha (cebpa) is a putative tumor suppressor. However, initial
Background & aims
C/EBPalpha (cebpa) is a putative tumor suppressor. However, initial
Conclusions
C/EBPalpha is up-regulated in a subset of HCCs and has growth-promoting activities in HCC cells. Novel oncogenic mechanisms involving C/EBPalpha may be amenable to epigenetic regulation to improve treatment outcomes.
Methods
The regulation of C/EBPalpha expression was studied by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, immunohistochemistry, methylation-specific PCR, and chromatin immunoprecipitation assays. C/EBPalpha expression was knocked-down by small interfering RNA or short hairpin RNA. Functional assays included colony formation, methylthiotetrazole, bromodeoxyuridine incorporation, and luciferase-reporter assays.
Results
Cebpa was up-regulated at least 2-fold in a subset (approximately 55%) of human HCCs compared with adjacent nontumor tissues. None of the up-regulated samples were positive for hepatitis C infection. The HCC cell lines Hep3B and Huh7 expressed high, PLC/PRF/5 intermediate, HepG2 and HCC-M low levels of C/EBPalpha, recapitulating the pattern of expression observed in HCCs. No mutations were detected in the CEBPA gene in HCCs and cell lines. C/EBPalpha was localized to the nucleus and functional in Hep3B and Huh7 cells; knocking-down its expression reduced target-gene expression, colony formation, and cell growth, associated with a decrease in cyclin A and CDK4 concentrations and E2F transcriptional activity. Epigenetic mechanisms including DNA methylation, and the binding of acetylated histone H3 to the CEBPA promoter-regulated cebpa expression in the HCC cells. Conclusions: C/EBPalpha is up-regulated in a subset of HCCs and has growth-promoting activities in HCC cells. Novel oncogenic mechanisms involving C/EBPalpha may be amenable to epigenetic regulation to improve treatment outcomes.