Chinese-German Journal of Clinical Oncology

  August 2011, Vol. 10, No. 8, P468–P471

DOI  10.1007/s10330-011-0826-3

The influence of hepatitis B virus X protein on the clock genes in liver cells and its significance Shengli Yang, Xiaoli Pan, Zhifan Xiong, Bo Wei, Hongyi Yao Department of General Surgery, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430077, China Received: 4 April 2011 / Revised: 20 May 2011 / Accepted: 25 June 2011 © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2011 Abstract  Objective: The aim of this study was to investigate the influence of hepatitis B virus X protein (HBx) on the clock genes in LO2 cells and its significance. Methods: A cell line LO2-HBx, Stably transfected with HBx gene, was established. The levels of mRNA and protein expression of CLOCK and BMAL1 were detected by real-time PCR and western blot. Results: The expression of CLOCK mRNA and protein were increased in cell line LO2-HBx (P < 0.05), while the expression of BMAL1 mRNA and protein were decreased in cell line LO2-HBx (P < 0.05). Conclusion: The expressions of core clock gene CLOCK and BMAL1 have been changed by HBx, which breaks down the previous circadian rhythm of liver cells. This maybe one of the reasons leads to the formation of liver cancer. Key words  hepatitis B virus X protein (HBx); circadian clock; CLOCK; BMAL1; hepatic carcinoma

Although the occurrence mechanism of liver cancer is not clear, but it’s explicitly that there is a close relationship between Hepatitis B virus X protein (HBx) and oncogenesis, development of liver cancer, which can result in malignant transformation of nude mice, deactivation of tumor suppressor gene p53 and interaction of multiple cell signal pathways leading to oncogenesis and development of liver cancer [1]. Circadian clock is the organism’s inner rhythm formed in the course of long-term evolution, which is important in body’s temperature, breath, sleep, eating, blood pressure, blood glucose, endocrine and many other homeostasis, and has close relationship with occurrence and development of tumor. The composition of molecular clock is based on the transcription-translation feedback loop of multiple biological clock genes such as CLOCK, BMAL1, per1, per2, per3, cry1 and cry2, by the interaction and oscillation formation of circadian rhythms [2–5]. Circadian clock disorder is also one of the common causes of the liver diseases [6, 7], but how these factors cause liver cell circadian clock disorder is still not clear at present. Would HBx lead to liver cell circadian clock disorder? We have conducted the following studies.

Correspondence to: Zhifang Xiong. Email: [email protected]

Materials and methods Reagents Mouse anti-human HBx monoclonal antibody was Chemicon International (USA). Rabbit anti-human polyclonal antibody β-actin, CLOCK and BMAL1 were from Santa Cruz Biotechnology (USA). Liposome transfection agent Lipofectamine 2000 and G418 were from Invitrogen (USA). DMEM and fetal bovine serum were from Hyclone (USA). PCR primers were produced by Shanghai Sangon Biological Engineering Technology & Services Co.Ltd. (China). Cell lines and cell culture The normal human liver cell line, LO2, was purchased from the China Center for Type Culture Collection (Wuhan University, China). Cell lines were grown in DMEM culture medium (containing 10% fetal bovine serum, 2 mmoL/L L-glutamine, 50 units/mL of penicillin, and 50 g/mL streptomycin) at 37 °C in an atmosphere of 5% CO2 in air. The expression vector pcDNA3.1-HBx was constructed by inserting HBx DNA fragments between the EcoR1 and Xho1 cloning sites of the pcDNA3.1 vector. After amplification in Escherichia coli DH-5α, we used the EZNA plasmid maxiprep kit (Omega Biotech, USA) to purify the plasmids. LO2 cells at 70%–80% confluence were transfected with plasmids, using lipofectamine 2000 ac-

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cording to the manufacturer’s protocol. Cells were seeded 48 h after transfection for the selection of G418-resistant colonies. Cells were cultured in the DMEM medium containing G418 (500 μg/mL) for 21 d. After wards, individual G418-resistant colonies were isolated. Expression of HBx in various cell lines was verified by reverse transcriptasePCR (RT-PCR) and western blot. The cell line (LO2-pcDNA3.1) with pcDNA3.1 vector alone, served as a control. After isolation of resistant colonies, the concentration of G418 was reduced to 250 μg/mL. RT-PCR Total cellular RNA of the stably transfected LO2-HBX cells was extracted with Trizol and reverse transcribed into cDNA with 2 units of Moloney murine leukemia virus reverse transcriptase (MMLV-RT; MBI Fermentas Inc., USA), 4 μL of 10 mmoL/L dNTPs (Fermentas Inc., USA), 2 μL of oligo (dT), 10 μL of 5X reaction buffer (Fermentas Inc., USA), 2 μL of ribonuclease inhibitor and 26 μL of deionized water for 90 min at 37 ℃. PCR was performed with 100 ng of cDNA in a 50 μL reaction volume containing 20 mmoL/L Tris, pH 8.0, 50 mmol/L KCl, 2 mmoL/L MgCl2, 10 mmoL/L of each dNTP, 20 pmoL of each primer, and 1.25 units of Taq DNA polymerase (Fermentas Inc., USA). The profile was 94 °C for 5 min before 25 cycles of 94 °C for 45 s, 55 ℃ for 45 s, 72 ℃ for 55 s, and a final extension at 72 ℃ for 10 min. All amplification products were separated on agarose gels and visualized by ethidium bromide staining under uv transillumination. The primers used for semi-quantitative PCR were: sense 5’-TCCTTTGTTTACGTCCCGTC-3’ and antisense 5’-TGCCTAC AGCCTCCTAATAC-3’ for HBx; sense 5’CTATCCCTGTACGCCTCTG-3’ and antisense 5’-ATGTCACGCACGATTTCC-3’ for β-actin. Protein preparation and western blot analyses Stably transfected LO2 cells were rinsed with 0.01 moL/L PBS, and then lysed in lysis buffer containing 50 mmoL/L Tris-HCl (pH 8.5), 150 mmoL/L NaCl, 0.2 g/L NaN3, 0.1 g/L SDS, 100 μg/mL phenylmethylsulfonyl fluoride (PMSF), 1 μg/mL aprotinin, 10 mL/L NP-40, and 5 g/L sodium deoxycholate. Cells were centrifuged at 14 000 g for 15 min to remove the cellular debris. Protein concentrations were determined by the Bradford method. A total of 50–100 μg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) using 6% or 10% s (according to the molecular mass of target protein), transferred to an immunoblot nitrocellulose membrane, blocked with nonfat milk for 12 h, and incubated with polyclonal antibodies to HBx, CLOCK and BMAL1 (each diluted to 1: 400) at 4 °C overnight. The membranes were then washed with TBST (10 mmoL/L Tris-Cl, pH 7.4, 150 mmoL/L NaCl, and 10 mL/ L Tween 20) 4 times (15 min incubation each time) and

hybridized with the appropriate peroxidase (HRP) conjugated secondary antibody. The antibody-specific protein bands were visualized using enhanced chemiluminescence (ECL) fluorography (Pierce Biotechnology Inc., USA). Real-time PCR Total RNA was extracted from log phase cells of the LO2-pcDNA3.1 group, LO2-HBx group, and LO2-HBx group treated with 50 μmoL PDTC for 24 h. The concentration and purity of the RNA was measured and equal amounts of the mRNA were reverse transcribed into cDNA. The cDNA of each group was quantitated in triplicate by real-time PCR using the ABI 7000 real time PCR system (Applied Biosystems, USA). Following reverse transcription, the cDNA samples were diluted 1:5 in water. The primer sequences used herein were CLOCK forward: 5’-AGGGCACCACCCATAATAGG-3’, reverse: 5’-TTGCCCCTTAGTCAG GAACC-3’; BMAL1 forward: 5’-CAAAGAGGACCCACCCCACT-3’, reverse: 5’-GGGAGGCGTACTCGTGATGT-3’; β-actin forward: 5’-GTCCACCGCAAATGCTT CTA-3’, reverse: 5’-TGCTGTCACCTTCACCGTTC-3’. Each reaction used SybrGreen mix, cDNA template, and 10 μM forward primer and 10 μM reverse primer and double distilled H2O in a total volume of 25 μL. Reaction conditions were: 94 ℃ denaturation 1 min, 94 ℃ 60 s within 40 circulations, annealing at 55 ℃ for 60 s, extension at 72 ℃ for 60 s. The program was set to automatically record average fluorescence value of the last 10% in the last cycle to which was equal to the amount of amplification at the end of each cycle. After reactions finished, baseline and threshold were adjusted in ABI 7000 software system where Ct value of each reaction hole was read. Data were analyzed according to the comparative Ct method and were normalized according to the β-actin expression in each sample. Statistical analysis Data were expressed as means ± S.D., and were analyzed using SPSS version 11.5. T-tests were used to compare between group differences, and one-factor analysis of variance was used for comparing multiple groups. P < 0.05 indicated that the difference has statistical significance.

Results Identification of HBx-expressing cells Stable transfection was indicated by the detection of HBx at mRNA level by PCR (Fig. 1) and protein level by immunoblot analysis (Fig. 2). Bands corresponding to HBx could be seen in the HBx-expressing cells but not in the control cells.

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Fig. 1 Confirmation of the successful transfection of HBx into LO2 cells by RT-PCR. M: markers (the brightest band of the marker presents the 400 bp); 1: control cells; 2: transfected cells

Fig. 3 mRNA levels of CLOCK and BMAL1 genes in different groups of cells. 1: control cells; 2: LO2-HBx cells

Fig. 2 Western blot analysis of HBx protein in samples isolated from LO2 cells with or without HBx transfection. 1: cells transfected with empty vector; 2: cells transfected with HBx gene

Fig. 4 Protein levels of CLOCK and BMAL1 genes in different groups of cells. 1: LO2-HBx cells; 2: control cells

The levels of CLOCK and BMAL1 mRNA were detected by real-time PCR Relative to expression in the control, CLOCK mRNA expression was increased 1.25 ± 0.08 times after HBx transfection, the BMAL1 mRNA expression was decreased to 0.75 ± 0.05 after HBx transfection (Fig. 3). The levels of CLOCK and BMAL1 proteins were detected by Western blot Relative to expression in the control, CLOCK protein expression was increased 1.56 ± 0.07 times after HBx transfection, the BMAL1 protein expression was decreased to 0.63 ± 0.04 after HBx transfection (Fig. 4).

Discussion HBV infection is regarded as one of the most important risk factors in the occurrence of liver cancer, and HBx, among HBV-coded products, has been proved to play an important role in viral replication and cancerous induction. HBx is a multi-functional protein which can bind to multiple transcription and gene regulation related factors in cell, widely activate promoter of viruses and cells, participate in the regulation of genes, and has close relationship with the occurrence and development of hepatitis and liver cancer [1].

Circadian clock genes are the time-sequential controller of life activities, their activities make the organ tissues and cellular lever life activity highly ordered, coordinated, and showing obvious circadian rhythm. Most of the molecular oscillation of biological circadian rhythm mainly includes two structures: iso-dimer formed by PAS domain protein and circadian clock genes contained Ebox. The former formation of iso-dimer is transcription factor, by using the upper E-box of the circadian clock genes to promote gene transcription, known as positive process. After the protein comes into the nucleus, it will suppress the activity of the transcription factor, which is called negative process to form the complete negative feedback loop. The positive process, plays a core position in the molecular oscillation of biological circadian rhythm, is composed of iso-dimer formed by CLOCK and BMAL1. The changes of CLOCK expression have close relationship with occurrence and development of many tumors [8, 9], so is BMAL1 [10, 11]. The research find out that after transfection of HBx, biological clock gene expression of CLOCK and BMAL1 have changed in the normal human liver cell LO2, with CLOCK up-regulated and BMAL1 down. As the important members in the circadian oscillation, the changes of CLOCK and BMAL1 expression can cause the disorder of LO2 cellular biological clock. One of the characteristics of malignant tumor is the cell’s lost of control and prolif-

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eration with disorder, and this disorder should embody in the disorderly time dimension. The circadian clock genes are closely related with the cell cycle regulation, especially influenced in the cell proliferation cycle and apoptosis, therefore the circadian clock genes are closely related with tumor, which are the other cause of tumor’s oncogenesis and development. A large epidemiological studies have found that circadian rhythm upset is associated with breast cancer for lighting effects. Women on the night shift and working in shifts have increasingly risk of developing breast cancer [8, 12]. Elisabeth Filipski’s research found the circadian clock disorders can speed up the development of liver cancer [13]. This phenomenon our study found maybe the other cause HBx induces normal liver cell loss of control, disorderly proliferation and consequently carcinogenesis. With the in-depth study on the biological clock, we are expected to broaden the human knowledge on occurrence and development of liver cancer, meanwhile it is also possible to provide a new idea for the treatment of liver cancer.

References 1. Wei Y, Neuveut C, Tiollais P, et al. Molecular biology of the hepatitis B virus and role of the X gene. Pathol Biol (Paris), 2010, 58: 267–272. 2. Froy O. The circadian clock and metabolism. Clin Sci (Lond), 2011, 120: 65–72.

471 3. Takeda N, Maemura K. Circadian clock and vascular disease. Hypertens Res, 2010, 33: 645–651. 4. Carey RM. Aldosterone and cardiovascular disease. Curr Opin Endocrinol Diabetes Obes, 2010, 17: 194–198. 5. Rana S, Mahmood S. Circadian rhythm and its role in malignancy. J Circadian Rhythms, 2010, 8: 3. 6. Huang MC, Ho CW, Chen CH, et al. Reduced expression of circadian clock genes in male alcoholic patients. Alcohol Clin Exp Res, 2010, 34: 1899–1904. 7. Kudo T, Tamagawa T, Shibata S. Effect of chronic ethanol exposure on the liver of Clock-mutant mice. J Circadian Rhythms, 2009, 7: 4. 8. Hoffman AE, Yi CH, Zheng T, et al. CLOCK in breast tumorigenesis: genetic, epigenetic, and transcriptional profiling analyses. Cancer Res, 2010, 70: 1459–1468. 9. Alhopuro P, Björklund M, Sammalkorpi H, et al. Mutations in the circadian gene CLOCK in colorectal cancer. Mol Cancer Res, 2010, 8: 952–960. 10. Taniguchi H, Fernández AF, Setién F, et al. Epigenetic inactivation of the circadian clock gene BMAL1 in hematologic malignancies. Cancer Res, 2009, 69: 8447–8454. 11. Zeng ZL, Wu MW, Sun J, et al. Effects of the biological clock gene Bmal1 on tumour growth and anti-cancer drug activity. J Biochem, 2010, 148: 319–326. 12. Stevens RG. Working against our endogenous circadian clock: breast cancer and electric lighting in the modern world. Mutat Res, 2009, 680: 106–108. 13. Filipski E, Subramanian P, Carrière J, et al. Circadian disruption accelerates liver carcinogenesis in mice. Mutat Res, 2009, 680: 95–105.