Introduction

Hepadnavirus is a small hepatotropic DNA-containing virus that replicates through an RNA intermediate.¹ Human hepatitis B virus (HBV), a member of the hepadnavirus family, is responsible for both acute and chronic liver infection. Chronic HBV infection, which causes liver cirrhosis and hepatocellular carcinoma (HCC), is a major global public health problem. Although an effective vaccine has been available for 20 years, and attempts at universal vaccination are now underway in developed countries,² there are an estimated 350 million hepatitis B carriers. Current antiviral therapies for HBV carriers include -interferon or lamivudine treatment; however, long-term disease resolution is disappointing because of low rates of seroconversion and the development of drug-resistant viral mutants.³ There is an urgent need for more effective antiviral therapies that can completely reduce or eliminate viral infection with fewer side effects. Although there is a wealth of information about the virus and the possible role that host factors may play in the infectious life cycle,^{1, 4, 5, 6} the direct relationship between chronically infected patients and host–pathogen interactions is poorly understood. DNA microarray technology has been used to measure the mRNA levels of thousands of cellular genes under a variety of experimental conditions. This approach is being used increasingly to monitor cellular gene expression in response to viral infection, expression of viral genes, or treatment with antiviral compounds such as interferon.^{7, 8, 9}

In this study, we performed genomic gene expression profiling in HepG2 cells and HepG2.2.15 cells using the Human Whole Genome Bioarray. HepG2.2.15 cells that are transfected with the HBV genome stably secrete hepatitis B virions and express all HBV proteins. The change in gene expression in HepG2.2.15 cells both before and after lamivudine treatment was also analyzed. Microarray data further suggested that a set of genes is involved in the cellular responses to HBV infection. Finally, we compared the anti-HBV activity of antisense oligodeoxynucleotides (ASODNs) against selected genes at both the DNA and the protein levels to validate our observations from the gene profiling experiments. Potential HBV response genes and novel targets for anti-HBV drugs were identified.

Results

Effect of lamivudine on HBV replication

HepG2.2.15 cells were used as a blank control. As shown in Figure 1, 25 M lamivudine significantly attenuated HBV replication but had little effect on hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) production in HepG2.2.15 cells 8 days post-infection.

Figure 1.

The effect of lamivudine on HBV replication. HepG2.2.15 cells were exposed to 25 M lamivudine. Medium was changed every 4 days with fresh test compounds for 8 days. The medium was collected for HBsAg, HBeAg and HBV DNA assays. HBV DNA in the cell supernatant was analyzed by real-time PCR, whereas HBsAg and HBeAg levels were analyzed by ELISA. The experiments were performed in triplicate. The levels of HBsAg, HBeAg and HBV DNA in the medium were normalized by cell number and are shown as the means.d. (n=3).

Full figure and legend (66K)

Cytotoxicity of lamivudine

During lamivudine treatment, cell morphology was visualized each day using a microscope. No visible differences were observed between the morphology of the test and control cells. Cell counting results revealed no significant differences between them (P>0.05, n=3). Thus, no visible cytotoxicity was observed in response to 25 M lamivudine treatments in HepG2.2.15 and HepG2 cells.

Differentially expressed genes in the HepG2.2.15 and HepG2 cell lines

To study genes that were differentially expressed between the HepG2.2.15 cell line and its parental cell line, HepG2, we used Amersham CodeLink Human Whole Genome Bioarray to systematically compare the gene expression profiles of both cell lines. We found that 2978 genes were up- or downregulated by at least twofold. Of these 2978 genes, 1358 genes had defined functional activities (Table 1), whereas 1620 genes were new expressed sequence tags (ESTs) with unknown functions. Of the 1358 known genes, 759 were upregulated, whereas 599 genes were downregulated. Approximately half of these genes could be classified into functional categories, including signal transduction, transport, cell adhesion, cell cycle and cellular metabolism.

Table 1 - Functional categories of over twofold regulated genes in HepG2.2.15 cells compared with HepG2 cells, and in cell treated with lamivudine.

Full table

Effects of lamivudine on HepG2.2.15 cell gene expression

Eight days after lamivudine treatment, significant changes in mRNA expression were observed in the HepG2.2.15 cells, with 2465 genes up- or downregulated by at least twofold. Of the 2465 differentially regulated genes, many more were overexpressed (71%) than underexpressed (29%) in the HepG2.2.15 cells after lamivudine treatment. Over half (52%) of the differentially expressed genes had functions that have not been characterized. The remaining 48% had numerous biologic functions as assessed by Gene Ontology (GO), including cellular metabolism, transport, signal transduction, cell cycle, cell adhesion, growth, apoptosis and immune responses (Table 1).

Screening for potential HBV-infection-associated genes and possible targets for anti-HBV therapy

The differential gene expression induced by HBV and lamivudine treatment was compared with the screening of important HBV-infection-associated genes. We found that 22 genes were upregulated in the HepG2.2.15 cells and downregulated by lamivudine (Table 2). These may be important HBV-infection-associated genes and potential targets for anti-HBV drugs.

Table 2 - Differentially expressed genes in HepG2.2.15 cells compared with HepG2 cells, which was reverse regulated by lamivudine.

Full table

Validation of the mRNA expression of selected genes

To ensure the altered expression level, nine genes (KHDRBS3, EPC1, CDC34, ACVR2B, EREG, IGF2, TGFB1, RORA and ABHD2) were selected and tested using semiquantitative reverse transcription (RT)-PCR (Figure 2). These genes were upregulated in HepG2.2.15 cells and downregulated by lamivudine, mirroring the microarray data. In contrast, the levels of above nine genes did not change obviously in HepG2 cells treated with lamivudine.

Figure 2.

RT-PCR analysis of the mRNA expression of selected genes based on microarray results. mRNA was extracted from HepG2.2.15, HepG2 and HepG2, HepG2.2.15 cells that were treated with lamivudine for 8 days. GAPDH was used as a control. 2215 represents gene expression in HepG2.2.15 cells, G2 represents gene expression in HepG2 cells, G2+Lam represents gene expression in HepG2 cells that were treated with lamivudine and 2215+Lam represents gene expression in HepG2.2.15 cells that were treated with lamivudine.

Full figure and legend (104K)

Downregulation of target genes inhibits HBV replication

To determine whether novel cellular genes are essential for HBV propagation, we used ASODNs to downregulate ABHD2, ACVR2B, CDC34, EREG, KHDRBS3 and RORA levels in HepG2.2.15 cells. The effect of ASODNs on mRNA levels was investigated using semiquantitative RT-PCR. Treatment of HepG2.2.15 cells with 0.8 M AB3, AC2, C2, E3, K2 and R3 significantly downregulated mRNA levels (Figure 3). The effect on HBV production was also measured in HepG2.2.15 cells. Cells transfected with 8 g/ml lipofectin or treated with 25 mol/l lamivudine were used as negative and positive controls, respectively. Treatment of HepG2.2.15 cells with 0.8 mol/l AB3 and E3 against ABHD2 and EREG, severely reduced HBV-DNA, HBsAg and HBeAg in the cell culture media, respectively. However, treatment with 0.8 mol/l of the other ASODNs had no significant effect on HBV propagation (the average inhibitory rate was <50%, Figure 4). The average inhibition rates for HBV-DNA, HBsAg and HBeAg by 0.8 mol/l AB3 were 65.50, 67.47 and 53.04%, respectively. After treatment with 0.8 mol/l E3, HBV DNA, HBsAg and HBeAg were inhibited by 61.13, 59.19 and 35.36%, respectively. The lipofectin control had a minimal effect on HBV levels in the HepG2.2.15 cells. The positive control, 25 mol/l lamivudine, inhibited HBV DNA, HBsAg and HBeAg levels by 71.77, 22.79 and 20.85%, respectively. Thus, we selected ABHD2 and EREG as HBV-infection-associated genes.

Figure 3.

RT-PCR analysis of the mRNA expression of selected genes after ASODN treatment of HepG2.2.15 cells. ABHD2 (a), ACVR2B (b), KHDRBS3 (c), CDC34 (d), EREG (e) and RORA (f) mRNA in HepG2.2.15 cells treated with ASODNs (0.8 mol/l) and lipofectin (8 mg/l) for 72 h was analyzed by RT-PCR. GAPDH was used as a control to normalize for total RNA. C represents the negative control, Lip represents the lipofectin control, M represents the DNA marker. The number under each band is expressed as a percentage of the cell control, as normalized by corresponding GAPDH level.

Full figure and legend (78K)

Figure 4.

The effects of ASODN on HBV propagation in HepG2.2.15 cells. HepG2.2.15 cells were transfected with ASODNs (0.8 M), 3TC (lamivudine, 25 mol/l) or Lip (lipofectin, 8 g/ml) for 3 days. HBV DNA in the cell supernatant was analyzed by real-time PCR, whereas HBsAg and HBeAg were analyzed by ELISA. Lip represents the lipofectin control. The data represent the means.d. (n=3). Asterisks indicate a statistically significant difference as compared to the lipofectin control (^*P<0.05).

Full figure and legend (63K)

Discussion

Persistent HBV infection results from the interaction between HBV and host cells, and ultimately leads to the development of liver cirrhosis and HCC.¹⁰ Because the interaction between HBV and host cells is involved in many cellular signaling pathways, it has not been completely defined.^{1, 11, 12, 13, 14} Inhibiting such cellular processes would be expected to have an antiviral effect. Evidence for this extends across many diverse virus types, including poxviruses,¹⁵ herpesviruses,^{16, 17, 18} retroviruses,¹⁹ hepadnaviruses^{20, 21} and flaviviruses.²² Moreover, any licensed drug that targets a human disease process affects the functioning of a cell or organ system in some way. Thus, in effect we have a readymade pharmacy of antiviral agents with defined safety data profiles.

Microarray technology has provided an opportunity to analyze complex viral–host interactions at the global level. Gene-expression profiling using DNA microarrays has identified genes that are upregulated as part of an infected cell response and are known to be drug targets. In this study, differential expression of host-cell proteins induced by HBV was analyzed. Using the Human Whole Genome Bioarray, we obtained 1679 upregulated genes and 1299 downregulated genes in HepG2.2.15 cells. Simultaneously, Otsuka,²³ used cDNA arrays of 2034 genes to screen genes that were differentially expressed between HepG2.2.15 cell and HepG2 cells. He observed seven upregulated genes (0.30%) (including IGFII, AFP, ASGPR and FABP), and three downregulated genes (including GTR2-2 and MDM2) in HepG2.2.15 cells (0.13%). The results of IGFII and MDM2 downregulation are consistent with our results.

Lamivudine has been a leading drug in the treatment of chronic HBV infection for the past decade. It is an orally administered nucleoside analog that is highly effective at inhibiting HBV DNA synthesis. Lamivudine selectively targets the HBV viral reverse transcriptase and directly blocks replication of the HBV genome.²⁴ In this study, lamivudine was used as a tool to inhibit the differential expression of genes induced by non-HBV infection factors. To screen for host-cell genes that are altered by HBV infection, changes in gene expression were studied in lamivudine-treated HepG2.2.15 cells. We obtained 22 functional genes that were upregulated in HBV-infected HepG2.2.15 cells and downregulated after lamivudine treatment. Many of the selected genes are well characterized and have widely varying functions as cell-cycle components, ligands, receptors, kinases, and signal transduction molecules, metabolism components, transcription regulators, and transport and development proteins. For example, HBx interacted with the ATF3 transcription factor to improve its expression.²⁵ HBx stimulation of IGF-II gene expression and autocrine IGF-II production involves phosphorylation and activation of the Sp1 transcription factor using the protein kinase C and p44/p42 mitogen-activated kinase pathways, and increased IGF-II gene expression may be involved in HBV-associated chronic liver disease pathogenesis.^{26, 27} HBx also enhanced transforming growth factor-1 (TGF1) expression.²⁸ HBxAg promotes TGF1 activity by upregulating TGF1 and downregulating 2-macroglobulin.²⁹ A relationship between the remaining genes and HBV infection has not yet been reported.

We also validated the differential expression of 9 of the 22 genes (ABHD2, EREG, ACVR2B, CDC34, KHDRBS3, RORA, EPC1, TGFB1 and IGF2) using semiquantitative RT-PCR. All of these genes were upregulated by HBV and downregulated after lamivudine treatment of HepG2.2.15 cells.

Persistent HBV infection is shown to impact host-cell pathways and enzymes. Excluding genes that were previously reported to share a close relationship with HBV, we discovered that ABHD2, EREG, ACVR2B, CDC34, KHDRBS3 and RORA also shared a strong relationship with HBV virus infection and could be critical for supporting the propagation of HBV-infected hepatic cells. These proteins may serve as potential targets for anti-HBV drugs.

Viruses generally use host-cell enzymes for replication, including those with hydratase catalytic activity. Carboxypeptidase N, a plasma metalloprotease, can bind, and activated core promoter of HBV.³⁰ Moreover, the cellular enzyme, S-adenosyl-L-homocysteine hydrolase, plays an important regulatory role in S-adenosyl-L-methionine-dependent methylation reactions.³¹ S-adenosyl-L-homocysteine hydrolase inhibition has an antiviral effect because most plant and animal viruses require a methylated cap structure at the 5' terminus of their mRNA in order for viral replication to occur.³² Thus, by inhibiting S-adenosyl-L-homocysteine hydrolase, the virus-encoded methyltransferases involved in the formation of this methylated cap structure are inhibited and viral replication is slowed. ABHD2, a / hydrolase protein family member, has strong hydratase catalytic activity.³³ We speculate, therefore, that ABHD2 may participate in HBV replication. On the other hand, EREG is a member of epidermal growth factor family and is able to enhance STAT1 activity, increasing p21WAF1/CIP1 expression, and stagnating cells in the G1 stage.³⁴ Numerous investigations have indicated that both DHBV^{35, 36} and HBV replicate more efficiently in quiescent cells^{37, 38} than proliferating cells. Thus, we would speculate that by inhibiting EREG expression, cells would proliferate from the metabolic stage.

A reduction in gene expression may decrease HBV replication. To support our speculation, antiviral activity of selected genes was validated using ASODN technology. ASODNs are based on the complementarity of the constructs to the appropriate target mRNA. Several mechanisms of action exist that lead to antisense effects, such as the inhibition of transcription, modulation of RNA processing, inhibition of translation, and selective cleavage of the target mRNA using the cellular endonuclease, RNaseH.³⁹ We initially screened potential anti-HBV targets by ASODNs against the above six genes to inhibit gene expression and observe the antiviral effects against HBV. Downregulation of ABHD2 and EREG by ASODNs significantly inhibited HBV propagation, whereas downregulation of ACVR2B, CDC34, KHDRBS3 or RORA had a reduced effect on HBV propagation. Thus, ABHD2 and EREG are likely to be essential for HBV propagation, such that inhibited expression reduces HBV levels.

In summary, by utilizing the Human Whole Genome Bioarray, we performed a global analysis of gene expression induced by HBV and lamivudine. As a result, we identified 22 important HBV infection-related genes. We validated nine genes, including IGF2, TGFB1, EPC1, ABHD2, ACVR2B, CDC34, EREG, KHDRBS3 and RORA using RT-PCR technology. ASODNs targeting ABHD2 and EREG significantly inhibited HBV propagation, respectively. Thus, we would conclude that ABHD2 and EREG are essential for HBV propagation and may serve as potential targets for anti-HBV drugs.

Materials and methods

Compounds

Lamivudine was abstracted from the lamivudine tablets and was dissolved in phosphate-buffered saline with the concentration of 1 mM.

Cell culture

The HepG2.2.15 cells (clonal cells derived from HepG2 cells that were transfected with a plasmid containing HBV DNA), which secrete hepatitis B virions,^{40, 41} were kindly provided by the Beijing Medical University and was originally from the Mount Sinai Medical Center (New York, NY, USA). HepG2.2.15 cells and HepG2 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, at 37°C, 5% CO₂. 380 g/ml G418 was needed in HepG2.2.15 cells.

Treatment of HepG2.2.15 cells with lamivudine

The HepG2.2.15 cells were inoculated at a density of 3 10⁵ cells per 5 ml in 25 cm² cell culture flask. The lamivudine was added to the medium with the eventual concentration of 25 mol/l⁴² 2 days after the inoculation. Cells were grown in the presence of lamivudine for 8 days with changes of medium every 4 days. The culture medium was collected each time for HBsAg, HBeAg and HBV DNA assays.

RNA isolation and target preparation

Total RNA from HepG2 cells, HepG2.2.15 cells and HepG2.2.15 cells treated by lamivudine at 8-day point were extracted using Trizol total RNA extraction Kit (Invitrogen, Carlsbad, CA, USA) as the manufacturer's instructions, followed by purification using RNeasy Mini columns (Qiagen Inc., Valencia, CA, USA). The products were quality-controlled on formaldehyde agarose gels. The concentration was examined by using nuclear acid and protein analyzer (DU640, Beckman, Fullerton, CA, USA). Double-stranded cDNA and subsequent cRNA was synthesized from 2 g of total RNA using the CodeLink Expression Assay Kit (Amersham Biosciences, Chandler, AZ, USA) according to manufacturer's instructions. Briefly, cRNA was prepared by in vitro transcription (IVT) using a single, labeled nucleotide, biotin-11-UTP in the IVT reaction at a concentration of 1.875 mM. Unlabeled UTP was present at 5.625 mM, whereas GTP, ATP and CTP were at 7.5 mM. cRNA was synthesized at 37°C overnight for 14 h, and the target product was recovered by RNeasy MiNi Kit (Qiagen Inc.) and quantified with an UV spectrophotometer.

Arrays hybridization and image processing

A 20 g of target cRNA was fragmented in 1 fragmentation buffer (40 mM Tris-acetate (pH 7.9), 100 mM KOAc and 31.5 mM MgOAc) at 94°C for 20 min. For hybridization with CodeLink bioarrays (Amersham Biosciences), 20 g of fragmented cRNA in 260 l of hybridization solution was injected into each hybridization chamber of a CodeLink Human Whole Genome Bioarray, which targets 57 000 transcripts and ESTs, including 45 000 well-characterized human gene and transcript targets based on the NCBI/UniGene database (Amersham Biosciences). The slides were incubated at 37°C for 22 h with shaking, The four bioarrays, in this study, were processed in parallel using the CodeLink Shaker Kit and CodeLink Parallel Processing Kit (Amersham Biosciences). Bioarrays were stained with Cy5™-streptavadin (Amersham Biosciences) and scanned using a GenePix 4000B scanner (Axon Instruments, Union City, CA, USA). The scanned image files were analyzed using CodeLink Scanning and Expression Analysis software (version 2.3; Amersham Biosciences), which produced both raw and normalized hybridization signal intensities for each spot on the arrays.

Analysis of mRNA expression by semiquantitive RT-PCR

RT-PCR quantification was used to verify the microarray data. The cDNA templates were synthesized from 2 g total RNA, using SuperScript II reverse transcriptase (Invitrogen) and oligo(dT) primer. PCR was performed in a single reaction of 20 l volume. The schedule consisted of incubation for 5 min at 94°C followed by 20–30 cycles of 94°C for 20 s, 55°C for 20 s and 72°C for 20 s, then incubation for 5 min at 72°C. The PCR products were analyzed by running the reaction products on 2% agarose/ethidium bromide gels. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as inner standard. The primers for each gene validated by RT-PCR are shown in Table 3.

Table 3 - Primer sequences for the genes used in RT-PCR analysis.

Full table

ASODNs design and synthesis

The whole-length mRNA sequence of ABHD2 (GeneBank no.: NM_007011), EREG (GeneBank no.: NM_001432), ACVR2B (GeneBank no.: NM_001106), CDC34 (GeneBank no.: NM_004359), KHDRBS3 (GeneBank no.: NM_006558) and RORA (GeneBank no.: NM_134261) were chosen as target sequence. Using IDT SciTools web server (http://www.idtdna.com/Scitools/Applications/AntiSense/Antisense.aspx), one region with local structures motifs, which have been presented to be favorable for the invasion of ASODN, was chosen as target site. Blast confirmed that ASODN was specific for corresponding gene above (Table 4). All ASODNs were synthesized at ABI8909 nucleic acid synthesis system (Applied Biosystems, Foster City, CA, USA) and purified by OPC (Perkin-Elmer, Foster City, CA, USA). Considering that phosphorothiate ASODNs are very resistant to nucleases,^{43, 44} all these ASODNs had been chemically modified to phosphorithioate ODNs by substituting the oxygen molecules of the phosphate backbone with sulfur.

Table 4 - ASODNs sequence.

Full table

Transfections

For transient transfection experiments, HepG2.2.15 cells were plated at 1.5 10⁵ cells per 35 mm well or at 1 10⁴ cells per well in 96-well tissue culture plates (Nunc, Roskilde, Denmark). Transfections were carried out 3 days after plating by using lipofectin transfection reagent (8 g/ml lipofectin for 0.8 mol/l oligos; Invitrogen) as instructed by the manufacturer. After incubation of oligos with cells for 6 h, the media was replaced and cultures were incubated for 72 h before analysis of mRNA and protein levels, HBV DNA, HBsAg and HBeAg levels.

Detecting HBV DNA in cell medium by real-time PCR

To assess HBV DNA levels, HBV DNA copies in culture medium were determined by real-time PCR. Cell mediums were incubated at 94°C for 15 min. After centrifugation at 12 000 r.p.m. for 10 min, the supernatants were used as the template for real-time PCR. The real-time PCR were performed as described previously.^{45, 46} The forward primer of HBV DNA is 5'-GGA GTATGGATTCGCACTCCTC-3', the reverse primer is 5'-TTGTTGTTGTAGGGGACCTGCCT-3', the fluorescent probe is 5'-ACTTCCGGAAACTACTGTTAGACGA-3', and the quenching probe is 5'-GTAGTTTCCGGAAGT-3'. PCR amplification and analysis were performed using iCycler real-time PCR detector (Bio-Rad, Hercules, CA, USA). Assays were repeated in triplicate and average threshold cycle values were used to determine the concentration of HBV DNA. The inhibition rate was calculated as the following formula: IR (%)=(C_control-C_tester)/C_control 100%. C_control represents HBV DNA copies in HepG2.2.15 cells, which were normalized by cell number; C_tester represents HBV DNA copies in treated cells, which were normalized by cell number.

Cytotoxicity

Cell morphologic character was monitored everyday after lamivudine's treatment in the microscope. After HepG2.2.15 cells were grown in the presence of lamivudine for 8 days, the cell number was determined by using hemacytometer.

Hepatitis B surface antigen and hepatitis B e antigen assay

HBsAg and HBeAg concentrations in culture medium were determined using diagnostic kit for HBsAg and diagnostic kit for HBeAg (enzyme-linked immunosorbent assay (ELISA); Sino-American Biotechnology Co., Beijing, China) with the method described in the manufacturer's manual. The inhibition rate was calculated according to the following formula: inhibition rate (%)=(A_control-A_test)/A_control 100%, where A represented the absorbance values, which were calibrated against the cell number determined using a 1/400-mm² hemocytometer (Qiujing Biochemical Reagent & Instrument, Shanghai, China). Assays were performed in triplicate, and the average inhibitory rate was expressed as meansstandard deviation (s.d.).

Statistical analysis

The data were expressed as meanss.d., statistical analysis was performed by Student's t-test (two-tailed) and one-way ANOVA.

Duality of Interest

None declared.

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Acknowledgements

This work is a Key Program supported by grant from the national nature science foundation of China (No. 30530650), a grant from National Science Fund for Distinguished Young Scholars (No. 30625041) and the special funds for major state basic research program of China (973 program) (NO. 2005CB522902). We acknowledge Yiwei Fan for synthesizing the ASODNs.