Identification of essential genes for the development of hepatitis B virus-associated hepatocellular carcinoma

Abstract

Background and Aim: Hepatocellular carcinoma (HCC) is a highly lethal and prevalent cancer, posing a grave threat to human health globally. Hepatitis B virus (HBV) infection is considered as a major risk factor for this cancer, especially in the Asia-Pacific region. Unfortunately, the molecular mechanisms of hepatocarcinogenesis remain obscure, which hinders the development of effective therapies for the disease. In the present study, we attempted to elucidate the molecular details of HBV-induced hepatocarcinogenesis by investigating differentially regulated genes at multiple developmental stages of HCC in a HBV transgenic mouse model. Materials and Methods: The transgenic mice which overproduced HBV large envelope polypeptide in hepatocytes and developed liver tumors spontaneously were used in this study. To unravel transcriptomics dynamics underlying hepatocarcinogenesis, RNA prepared from livers of both transgenic and wild type mice of different ages (at months 2, 12, 18 and 19) were subjected to RNA sequencing. Selected target genes were first validated by quantitative PCR (qPCR) using a larger set of mouse liver tissues (n=96) collected from 8 time points. Clinical implications of the selected genes were then explored in a set of human liver samples comprising 18 normal, 29 cirrhosis and 96 pairs of HCC. RNA and protein expression levels were determined by qPCR, immunohistochemical staining and Western blotting, respectively. Results: Upon analysis of 20,209 gene transcripts, 2574 and 1035 transcripts were found to be up-regulated (≥ 2 folds) and down-regulated (≤ 2 folds) in tumors, respectively, when compared with the wild type controls. Among these, 133 most prominent genes that exhibited concordant differential expression throughout the stages of tumor progression were chosen for validation in mouse liver tissues. Correlation analysis showed a high correlation between RNA sequencing and qPCR data (r=0.7495; P<0.0001), indicating a high validity of the data. Forty-six biologically informative genes were further validated in human liver samples. By Gene Ontology analysis, the target genes were revealed to play roles in a variety of biological processes including stress and inflammation responses, metabolic and apoptotic processes. Immunohistochemical staining and Western blotting demonstrated significant differential expression of these genes between HCC and non-tumorous livers. Statistical analyses revealed their significant correlation with clinicopathological parameters including venous infiltration, tumor size and overall survival, implicating their roles in hepatocarcinogenesis. Conclusion: This study has demonstrated a systematic strategy for identifying crucial genes for HBV-associated HCC, which may have profound implications in combating this deadly cancer.