Screening of differentially expressed genes and gene pathways in hepatitis C virus 1b type nonstructural protein 4B stably expressed L02 cell line

Abstract

To screen differentially expressed genes and gene pathways in L02 cell line stably expressing hepatitis C virus (HCV) Ib type nonstructural protein 4B (NS4B) mediated by lentiviral system, and to provide a basis for further research of molecular biological mechanism of NS4B gene in chronic hepatitis C and hepatocarcinogenesis. NS4B stably overexpressed L02 cell line and negative control stable L02 cell line, designated as L02-NS4B and L02-mkate2 respectively, were resurrected and amplified in vitro. The differentially expressed genes between L02-NS4B and L02-mkate2 were determined by gene expression microarray from Human Gene 1.0ST. The significant pathways of the differential genes were selected by the Fisher's exact test and χ2 test according to kyoto encyclopedia of genes and genomes (KEGG) database. The differential expression levels of 5 selected genes including protein kinase C delta binding protein (PRKCDBP), tumor protein p53 (TP53), v-akt murine thymoma viral oncogene homolog 1 (AKT1), baculoviral IAP repeat containing 3 (BIRC3) and B-cell lymphoma 2-like1 (BCL2L1) from cDNA microarray data were further verified by real-time quantitative polymerase chain reaction (real-time QPCR). Between L02-NS4B and L02-mkate2, the genes with flurescence intensity ratio >1.2 or <0.8 were considered as differentially expressed genes. A total of 2 682 differentially expressed genes in the known 28 869 human genes were detected in L02-NS4B, 1 446 genes were upregulated and 1 236 genes were downregulated. A total of 41 involved pathways of up-regulated differential genes were identified by KEGG database, mainly including apoptosis, extracellular matrix receptor interaction, cell cycle, pathways in cancer and Toll-like receptor signaling pathway; and 20 involved pathways of down-regulated differential genes were identified, mainly including pathways in cancer, Wnt signaling pathway and cell cycle pathway. Of the 5 upregulated genes selected from cDNA microarray data, 3 genes showed the same differential expression pattern by real-time QPCR as that shown in cDNA microarray data, namely AKT1, BIRC3 and BCL2L1. The confirmation rate of real-time QPCR was 60%. HCVNS4B can up-regulate or down-regulate the expression of many genes in L02 cells, thus affecting multiple signaling pathways relevant to cell apoptosis, cell cycle and carcinogenesis.