Hepatitis B virus X protein upregulates survivin expression in hepatoma tissues

Abstract

The hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). The relationship was examined between HBV antigens and IAP (inhibitor of apoptosis) family in development of HCC. The expression levels of HBV antigens (HBsAg, HBcAg, and HBxAg) and members of the IAP family (survivin, XIAP, cIAP-1, and cIAP-2) were detected immunohistochemically in tissues from 34 cases of HCC and 30 cases of liver cirrhosis. The positive rate of survivin was higher than these three molecules in all three tissue types (P < 0.05). The positive rates of HBxAg and survivin were high in HCC (76.5% and 88.2%), paratumor (85.3% and 91.2%), and liver cirrhosis (100% and 93.3%) tissues, with no significant differences between the survivin- and HBxAg-positive rates (each P > 0.05). To examine the effect of HBx on survivin expression, plasmid pCMV-X (encoding the HBx gene) was transfected transiently with or without plasmid pcDNA3-sur (encoding the survivin gene) into H7402 hepatoma cells and L-O2 human normal liver cells. Cells over-expressing HBx alone showed increased apoptosis along with a dose-dependent increase in survivin levels. However, co-expression of survivin inhibited the HBx-induced apoptosis. To examine the effect of HBx on survivin in hepatoma cells without apoptosis, plasmid pCMV-X was transfected stably into human hepatoma H7402 cells and L-O2 cells. These H7402-X and L-O2-X cells showed high-level expression of both HBx and survivin, but did not show apoptosis. The addition of pSilencer 3.0-X, an RNAi vector targeting the HBx gene, reduced the expression levels of survivin protein in H7402-X cells. Collectively, these data demonstrate that HBx upregulates survivin expression in hepatoma tissues, suggesting that HBx and survivin may both be involved in carcinogenesis of HCC.

Gene expression profiles of human liver cells mediated by hepatitis B virus X protein

“…Previously, we demonstrated that HBx upregulated both the expression and the activity of hTERT in hepatoma [8] . We also found that HBx was able to upregulate the expression of survivin, which suggests that survivin may be involved in the carcinogenesis of HCC that is mediated by HBx [9] . cDNA microarray is a powerful tool for identifying disease-related gene expression profiles in biological samples.…”

“…Cell culture The human liver L-O2 cell line (purchased from Nanjing KeyGen Biotech Co Ltd, Nanjing, China), which originated histologically from normal human liver tissue that had been immortalized by stable transfection of the hTERT gene, was used previously [9,[11][12][13] . L-O2-P (cell line from L-O2 cells stably transfected with empty pcDNA3 plasmid) and L-O2-X (cell line from L-O2 cells stably transfected with the HBx gene) were established as described previously [13] .…”

“…The following primers were used: sense, 5′-GATCCCGGTCTTACA-TAAGAGGACTTTCAAGAGAAGTCCTCTTATGT-AAGACCTTTTTTGGAAA-3′; antisense, 5′-AGC TTTT-CCAAAAAAGGTCTTACATAA GAGGACTTCTCTT-GAAAGTCCTCCTTATGTAAGACCGGG-3′ [9] . The purified vector was transfected using Lipofectamine into L-O2-X cells for 36 h, as described previously [9] . The expression level of the HBx protein in L-O2-X cells was examined by Western blot analysis.…”

“…Following electro-transfer onto polyvinylidene difluoride membranes, the membranes were blocked with 5% non-fat dry milk in 0.1% Triton X 100-TBS (TTBS) and incubated overnight at 4°C with specific primary antibodies. The following primary antibodies were used as described previously [9,15] : PCNA (NeoMarkers, Fremont, CA, USA, 1:1000 dilution); Bcl-2 (NeoMarkers, Fremont, CA, USA, 1:500 dilution); HBx (obtained from the Fox Chase Institute for Cancer Research, Philadelphia, Pa, USA, 1:50 000 dilution); COX-2 (NeoMarkers, Fremont, CA, USA, 1:200 dilution); and β-actin (NeoMarkers, Fremont, CA, USA, 1:1000 dilution). Staining was performed with an HRP-linked secondary antibody (GE Healthcare Bio-Science, USA).…”

Hepatitis B virus X protein promotes hepatoma cell proliferation via upregulation of MEKK2

“…RNA interference pSilencer-HBx (pSi-HBx) was used to produce small interfering RNAs (siRNAs) to target HBx mRNA, and pSilencercontrol (pSi-con) was used as a negative control [27,28] . siRNA duplexes targeting human MEKK2 were synthesized and purified by RiboBio (Guangzhou, China) [29] .…”