Impact of HBV infection on TGF-β signaling in primary human hepatocytes

TGF-β is a profibrotic cytokine playing a pivotal role in cell signaling in liver. Chronical HBV infection leads to fibrosis and often ends in HCC. As TGF-β is frequently upregulated in HBV-infected patients, it is important to elucidate its role in the course of HBV infection. Primary human hepatocytes from 3 donors were infected with HBV. Replication of HBV in infected cells was investigated by HBsAg values in the supernatant. Infection efficiency was about 100% as investigated by IF staining against HBV core protein. Cells were cultured for 7 days until TGF-β treatment for 1 or 24hrs, respectively. cRNA was hybridized to Affymetrix Human Genome 1.0 ST Array according to protocols of the manufacturer. DAVID Database was used to determine significant differential expression after prefiltering of genes. Difference of expression values of comparison groups was set at >1.3-fold or <–1.3-fold, and the significant difference of a Student's t test between the comparison group was set at P < 0.1. Upon HBV infection 60, 63 or 121 genes are up-, whereas 64, 40 or 160 genes are downregulated in the different donors. Genes commonly upregulated are e.g. p21, SPATA18, and PGA5, whereas no genes are commonly downregulated. CYP7A1, ZNF737, SNORD36A, and TGF-β2 are strongly downregulated, SNORD68, HIST1H2BE, and BHLHA9 represent most upregulated genes in individual patients. Smad7 and GADD45β are most upregulated upon TGF-β treatment in infected and non-infected cells. However, ADM and OCM2 are only regulated upon TGF-β in non-infected cells. In donor 1, TGF-β induced PRR13 downregulation in HBV-infected cells, but upregulation in non-infected cells. We will now perform a more detailed gene analysis to identify (i) changed genes associated to TGF-β signaling upon HBV infection in general and donor specifically as well as (ii) short and long term impact of enhanced TGF-β signaling on gene expression in HBV-infected hepatocytes versus non-infected hepatocytes.