Pathway analysis for the gene expression profile in the HBx induced HCC mouse model and validation on human and zebrafish animal model

Ting-Fen Tsai², Shwu-Rong Grace Shieh³, Pei-Ting Lin¹, Yu-I Lin¹, Jeng-Wei Lu¹, Hsaio-Chen Tu¹, Sreeja Sarasamma¹, Shih-Feng Tsai¹, and Chiou-Hwa Yuh^{1, 4, 5}

蔡亭芬²,謝叔蓉³, 林佩婷¹,林佑怡¹,盧正偉¹,凃曉蓁¹, Sreeja¹,蔡世峰¹,喻秋華^{1, 4, 5}

¹Division of Molecular and Genomic Medicine, National Health Research Institutes, ²Faculty of Life Science and Institute of Genome Sciences, Yang Ming University, ³Institute of Statistical Science, Academia Sinica, ⁴College of Life Science and Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, ⁵ Department of Biological Science & Technology, National Chiao Tung University

Hepatoarcinogenesis is a slow and multistep process. In this study, we systematically delineate the events in five stages of carcinogenesis using HBx transgenic mouse model. We identify the biomarkers, describe in details the interrelationships responsible for hepatocellular carcinogenesis, and identify the pathways in different stages of tumor formation. At each specific stage, we analyze the genes whose expression level have two fold changes in HBx carried HCC verse wild type mouse liver samples by GeneSpring 7.3 software. Two statistics to capture genes that have increasing or decreasing trend are derived, and these genes may be associated with disease progress. The relevant pathways have been analyzed using Pathway Studio 5.0 software. Different positive feedback loops drive the activation of genes in 1.5 months, 8 months and 12 months stage of HCC formation. Intrastage cross-inhibiting interactions are identified between the differentially up and down-regulated genes. Furthermore, we identify the interactions between earlier stages to later stages and they may play an important role in the transition of stages. Notably, we identified the important links between 12M and cancer. A few genes up-regulated in 12M, such as edn1, bmp7, pcsk6 may promote the carcinogenesis by up-regulating precancer-oncogenes. This study shows that many genes are activated during the carcinogenesis, in a continuous and tumor specific manner, and these genes are involved in ATM, Caspases, Apoptosis, Insulin, Integrin signaling, MAPK-ERK, and STAT pathways. Pathways that are activated throughout the carcinogenesis include: TGF-beta, Wnt pathway, antiapoptotic, AP-1 and PTEN signaling pathways. Furthermore, genome wide study using antibody against methylated DNA for chromatin-immunoprecipitation coupled with 10kb promoter array is ongoing for the epigenomic profiling. This systematic approach provides the fundamental basis for the HCC carcinogenesis.

These identified biomarkers may shed light on molecular targets for the treatment of human HCC. Human HCC samples will be analyzed for the biomarkers identified from mouse HCC model. Furthermore, over expression of selected candidate genes will be tested in Zebrafish animal model for their effect on carcinogenesis. Using this comparative oncogenomic study, we will find out the carcinogenesis pathways, the usefulness of finding a new diagnostic biomarkers as well as the mechanism of the oncogenesis.