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Volume 139, Issue 2 , Pages 632-643.e4, August 2010

C/EBPα Is Up-regulated in a Subset of Hepatocellular Carcinomas and Plays a Role in Cell Growth and Proliferation

Guo–Dong Lu
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Carol Ho–Wing Leung
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Benedict Yan
- Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Colyn Mui–Yeong Tan
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Sie Yieh Low
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Myat Oo Aung
- Department of Medicine, National University Hospital Health Systems, Singapore
Manuel Salto–Tellez
- Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Seng Gee Lim
- Department of Medicine, National University Hospital Health Systems, Singapore
Shing Chuan Hooi
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- Reprint requests Address requests for reprints to: Dr Shing Chuan Hooi, Department of Physiology, National University of Singapore, Singapore. Blk MD9, 2 Medical Drive, Singapore 117597. fax: (65) 6778-8161

Received 3 August 2009; accepted 18 March 2010. published online 29 March 2010.

Background & Aims

C/EBPα (cebpa) is a putative tumor suppressor. However, initial results indicated that cebpa was up-regulated in a subset of human hepatocellular carcinomas (HCCs). The regulation and function of C/EBPα was investigated in HCC cell lines to clarify its role in liver carcinogenesis.

Methods

The regulation of C/EBPα expression was studied by quantitative reverse transcription–polymerase chain reaction (qRT-PCR), Western blotting, immunohistochemistry, methylation-specific PCR, and chromatin immunoprecipitation assays. C/EBPα 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/EBPα, recapitulating the pattern of expression observed in HCCs. No mutations were detected in the CEBPA gene in HCCs and cell lines. C/EBPα 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/EBPα is up-regulated in a subset of HCCs and has growth-promoting activities in HCC cells. Novel oncogenic mechanisms involving C/EBPα may be amenable to epigenetic regulation to improve treatment outcomes.

Keywords: C/EBPα, Hepatocellular Carcinoma, Growth Promotion, Epigenetic Regulation

Abbreviations used in this paper: 5-aza dC, 5-aza-2′-deoxycytidine, ChIP, chromatin immunoprecipitation, HBV, hepatitis B, HCC, hepatocellular carcinoma, HCV, hepatitis C, HDAC, histone deacetylase, LY, LY294002, OA, okadaic acid, PI3K, phosphoinositol-3 kinase, qRT-PCR, quantitative reverse-transcription–polymerase chain reaction, shRNA, short hairpin RNA, siRNA, small interfering RNA, TSA, trichostatin A