Gene expression profiles in an hepatitis B virus transfected hepatoblastoma cell line and differentially regulated gene expression by interferon-alpha

Abstract

Aim: To study interactions between hepatitis B virus (HBV) and interferon-alpha in liver- derived cells.

Methods: mRNAs were separately isolated from an HBV-transfected cell line (HepG(2)2.2.15) and its parental cell line (HepG(2)) pre- and post-interferon-alpha (IFN-alpha) treatment at 6, 24 and 48 h, followed by hybridization with a cDNA microarray filter dotted with 14 000 human genes. After hybridization and scanning of the arrays, the data were analyzed using ArrayGauge software. The microarray data were further verified by Northern blot analysis.

Results: Compared to HepG(2) cells, 14 genes with known functions were down-regulated 3 to 12- magnitudes, while 7 genes were up-regulated 3-13 magnitudes in HepG(2)2.2.15 cells prior to IFN-alpha treatment. After interferon-alpha treatment, the expression of four genes (vascular endothelial growth factor, tyrosine phosphate 1E, serine protein with IGF-binding motif and one gene of clathrin light chain) in HepG(2)2.2.15 were up-regulated, while one gene encoding a GTP-binding protein, two genes of interferon-induced kinases and two proto-oncogenes were further down- regulated. Interestingly, under IFN-alpha treatment, a number of differentially regulated genes were new ESTs or genes with unknown functions.

Conclusion: The up-regulated genes in HepG(2)2.2.15 cell line suggested that under IFN-alpha treatment, these repressed cellular genes in HBV infected hepatocytes could be partially restored, while the down- regulated genes were most likely the cellular genes which could not be restored under interferon treatment. These down-regulated genes identified by microarray analysis could serve as new targets for anti-HBV drug development or for novel therapies.

Figure 1

RNA slot analyses of IFN-inducible P27, KIAA0919 (Sec15B), beta3gal-T5 and FLJ12673 genes in HepG₂ 2.2.15 be-fore and after IFN treatment Four genes (interferon related genes P27, KIAA0919, β-3-gal-T5 gene and FLJ12673 human cDNA clone) were selected and tested by RNA slot analysis to confirm the altered expression level indicated by the results of the microarrays.10 μg/sample total cellular RNA was blotted to the nylon membrane. The membrane was then hybridized with [α-³²P]dCTP labeled cDNA probes. Autoradiographs were exposed to X-ray film overnight at -70 °C. The β-actin gene was used as the control to normalize the total RNA quantity.