For construction of eukaryotic expression vector pcDNA3.1(−)-MHBs^t167, the MHBs^t167 fragment was PCR-amplified from pCP10 containing two copies of HBV DNA subtype ayw (GenBank accession: U95551), with the forward primer (5′-GCCGGGCCCATGCAGTGGAATTCCACAAC) containing an ApaI site and reverse primer (5′-GGAAAGCTTCTATCCTGGAATTAGAGGACAAAC) containing a HindIII site. The fragment was inserted into the cloning vector pGEM-T (Promega, Madison, WI, USA), resulting in pGEM-T-MHBs^t167. An ApaI-HindIII fragment was isolated from the vector and inserted into ApaI-HindIII-digested pcDNA3.1(−) (Invitrogen Life Technologies, Carlsbad, CA, USA), resulting in pcDNA3.1(−)-MHBs^t167. The pcDNA3.1(−)-MHBs coding for intact HBV middle surface protein was constructed previously in the laboratory (8). To construct the reporter vector pCAT3-c-Myc, the promoter of human c-Myc was amplified from the genomic DNA of the HepG2 human HCC cell line (HBsAg-negative) by PCR with a pair of forward (5′-GGTACCATCCTCTCTCGCTAATCTCC) and reverse (5′-AGATCTATGGG CAGAATAGCCTCCCC) primers containing a KpnI and a BglII site, respectively. The promoter fragment was inserted into pGEM-T, yielding pGEM-T-c-Myc. A KpnI-BglII fragment was isolated from the plasmid and inserted at the KpnI-BglII sites of pCAT3-basic (promoterless; Promega), resulting in the pCAT3-c-Myc reporter vector. DL2000 DNA marker (Takara Biotechnology (Dalian) Co., Ltd, Dalian, China) was used as a DNA molecular weight marker. All the vectors were sequenced and digested with corresponding restriction enzymes to confirm the sequence accuracy.

HepG2 cells were purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and were cultivated in Dulbecco’s modified Eagle’s medium (Invitrogen Life Technologies) containing 100 IU penicillin and 100 μg streptomycin per milliliter, supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT, USA), at 37°C in 5% CO₂ and 90% relative humidity. The cells were seeded on the day before transfection at a density of 8×10⁵ cells per 35-mm dish and reached 50% confluence at the time of transfection. All transfections were performed with FuGene^® 6 Transfection Reagent (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer’s instructions. The medium was changed 5 h after transfection and cells were harvested 40–48 h following transfection. All transfections and assays were repeated independently three times in triplicate.

mRNA from HepG2 cells transfected with pcDNA3.1(−)-MHBs^t167 and pcDNA3.1(−) was isolated using a QuickPrep Micro mRNA purification kit (Amersham Biosciences, Little Chalfont, UK), and cDNA was reverse-transcribed from the mRNA. MHBs^t167 expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) with MHBs^t167-specific primers by using 35 amplification cycles, and by western blotting using lysates of the HepG2 cells. The extracts were boiled for 5 min and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes (Pierce Biotechnology, Inc., Rockford, IL, USA). The membranes were reacted with anti-pre-S2 mouse anti-human monoclonal antibody and HRP-labeled goat anti-mouse polyclonal IgG as the primary and secondary antibodies, respectively and then with a SuperSignal West Pico Chemiluminescent Substrate working solution (Pierce Protein Biology Products, Thermo Fisher Scientific, Inc., Rockford, IL, USA) according to the manufacturer’s instructions. The immunoreactive bands were visualized after exposure to X-ray film.

To detect the effect of MHBs^t167 on the SV40 promoter, various quantities of reporter vector pCAT3-promoter (Promega) containing CAT reporter gene controlled by the SV40 immediate early promoter element were transiently transfected into HepG2 cells. Co-transfections were made with pcDNA3.1(−)-MHBs^t167 (2.0 μg) + pCAT3-promoter (0.4 μg) as the test group, and pcDNA3.1(−) (2.0 μg) + pCAT3-promoter (0.4 μg) and pcDNA3.1(−)-MHBs (2.0 μg) + pCAT3-promoter (0.4 μg) as the control groups, respectively. To detect the effect of MHBs^t167 on the c-Myc promoter, various quantities of pCAT3-c-Myc were transiently transfected into HepG2 cells. The co-transfections were made with 1.0 μg reporter vector pCAT3-c-Myc and 0.5, 1.0, 1.5 and 2.0 μg effector vector pcDNA3.1(−)-MHBs^t167. The relative CAT activity was measured using enzyme-linked immunosorbent assay according to the manufacturer’s instructions (CAT ELISA kit; Roche Applied Science).

SSH was performed with the PCR-Select™ cDNA subtraction kit (Clontech Laboratories, Inc., Mountain View, CA, USA) according to the manufacturer’s instructions. In brief, 2.0 μg of poly A+ mRNA, each from the pcDNA3.1(−)-MHBs^t167 tester group and the pcDNA3.1(−) driver group was subjected to cDNA synthesis, respectively. Following restriction with RsaI, small sizes of cDNAs were obtained. The tester cDNAs were then subdivided into two parts, ligated with the specific adaptor 1 and adaptor 2, respectively. After two subtractive hybridization reactions and two suppression PCR amplifications, differentially expressed cDNAs were selectively amplified. Subsequently, the second PCR products were used as templates for PCR amplification of G3PDH (a housekeeping gene) at 18, 23, 28, 33 cycles, respectively, to analyze subtraction efficiency. The second PCR products were directly purified using the Wizard^® PCR-Preps DNA Purification system (Promega), and inserted into pGEM-T Easy (Promega) to construct the subtracted library. Colony PCRs were conducted to confirm that the size of the cDNA inserts ranged between 200 and 1,000 bp by using T7/SP6 specific primers localized in pGEM-T Easy. Following DNA sequencing of the positive colonies, nucleotide homology searches were performed using the BLAST program at NCBI (http://blast.st-va.ncbi.nlm.nih.gov/Blast.cgi).

To detect the effect of MHBs^t167 on c-Myc mRNA, total RNA was extracted from HepG2 cells transiently transfected by pcDNA3.1(−) and pcDNA3.1(−)-MHBs^t167 using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions, and was used for RT-PCR. The following primers (sense, 5′-TTCGGGTAGTGGAAAACCAG and antisense, 5′-CAGCAGCTCGAATTTCTTC) were used to amplify the c-Myc cDNA. β-actin specific primers (sense, 5′-TGACGGGGTCACCCACACTGTGCCCATCTA and antisense, 5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG) was used as internal reference. To detect the effect of MHBs^t167 on c-Myc protein, total soluble proteins were extracted in radioimmunoprecipitation assay buffer (Pierce Biotechnology, Inc.) from the transfected HepG2 cells and separated on 12.5% SDS-PAGE gels for immunoblotting assay (prepared in house, Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China). The expression of c-Myc was probed by mouse monoclonal antibody against human c-Myc derived from a cell line from American Type Culture Collection, Manassas, VA, USA. Mouse anti-human monoclonal β-actin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used as internal reference.

Digestion of recombinant vector pcDNA3.1(−)-MHBs^t167 with ApaI/HindIII, EcoRI, XbaI and XhoI yielded the expected bands (data not shown). DNA sequencing results indicated that the recombinant vector contained HBV DNA fragment encoding the truncated middle surface protein in-frame and the sequence was completely correct. MHBs^t167 mRNA and protein expression in HepG2 cells was successfully detected by RT-PCR (Fig. 1A) and western blotting (Fig. 1B), respectively.

To determine the sensitivity of the kit for CAT measurement, we first evaluated the CAT activities in total cell lysates of HepG2 cells transfected with different quantities of pCAT3-promoter. The results showed that CAT gene expression exhibited an approximately linear association with the quantity of reporter vector used in transfection (Fig. 2A). Subsequently, 0.4 μg of reporter vector was selected to be used in the transfection experiments, so that it was easy to detect the reporter activity with room for further increase if a greater quantity of reporter vector was used. In the transient co-transfection assays, CAT gene expression from the pCAT3-promoter was ~4.5-fold higher following co-transfection with pcDNA3.1(−)-MHBs^t167 compared with that after co-transfection with pcDNA3.1(−) or pcDNA3.1(−)-MHBs. The marked increase in CAT gene expression may be attributed to the transactivating effect of the truncated HBV MHBs^t167 on the SV40 early promoter element, leading to the observed increase in CAT expression, whereas the intact MHBs was not transactive (Fig. 2B).

To gain a general view of the genes which may be involved in the pathogenesis of HBV, genes that were upregulated in HepG2 cells expressing MHBs^t167 were identified by the generation of a subtracted cDNA library. Subtraction efficiency analysis showed that PCR products of the housekeeping gene G3PDH in the unsubtracted library were obviously visible after 18 cycles; however, 28 cycles were required in the subtracted library (Fig. 3), indicating that the abundance of non-differentially expressed genes was effectively reduced and the subtraction method had a high subtraction efficiency. Using SSH, a total of 94 positive clones were obtained. These clones were prescreened by using PCR amplification to ensure that they had different inserts before sequencing. Among these clones, 77 contained inserts of 200–1,000 bp. A total of 50 clones from the cDNA library were randomly chosen and sequenced, and their nucleotide sequence homology searches were performed using the BLAST program at NCBI. The analysis results showed that there were 22 coding sequences, of which 18 were known and 4 were unknown genes. Some of the proteins coded by these genes have been shown to be involved in cell cycle regulation, cell apoptosis, signal transduction pathways and tumor development. Notably, the cell proto-oncogene c-Myc was up-regulated by MHBs^t167. A summary of the data is presented in Table I.

To examine the relationship between MHBs^t167 and c-Myc, the effect of MHBs^t167 on the promoter activity of c-Myc was investigated. The promoter activity was evaluated using a CAT assay in HepG2 cells transfected with pcDNA3.1(−)-MHBs^t167. As shown in Fig. 4, transiently expressed MHBs^t167 was found to markedly increase the promoter activity of c-Myc in HepG2 cells in a dose-dependent manner. The results suggested that MHBs^t167 protein could transactivate c-Myc expression by transcriptionally activating its promoter element.

To further elucidate the mechanisms of MHBs^t167 on c-Myc expression at the transcription and translation levels, the effect of MHBs^t167 on expression of the gene was investigated. As shown in Fig. 5A, level of mRNA of c-Myc markedly increased following transient transfection with pcDNA3.1(−)-MHBs^t167. The western blot analysis indicated that the expression of the gene was low in the control groups, whereas in the experiment group, its expression was markedly enhanced (Fig. 5B). These results indicated that MHBs^t167 could transactivate the expression of c-Myc at both the transcription and translation levels.