http / / cjbmb. bjmu. edu. cn Chinese Journal of Biochemistry and Molecular Biology HBx HBxΔ127

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1 ISSN CN / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 430 X HBxΔ127 ERK 1 * * hepatitis B virus HBV X HBx HBx HBxΔ127 1 calpain small subunit 1 Capn4 HBx HBxΔ127 HBxΔ127 Capn4 Capn4 ERK PD98059 HBxΔ127 Capn4 HBxΔ127 ERK1 /2 p-erk1 /2 Capn4 HBxΔ127 Capn4 p-erk1 /2 HBxΔ127 p-erk1 /2 Capn4 HBx HBxΔ127 X 1 Capn4 ERK1 /2 Q2 Q51 Hepatitis B Virus X Protein Mutant HBxΔ127 Promotes Hepatoma Cells Migration via ERK-induced Upregulation of Calpain Small Subunit 1 SHAN Chang-Liang ZHANG Shuai ZHANG Xiao-Dong * YE Li-Hong * Key Laboratory of Bioactive Material of Ministry of Education College of Life Sciences Nankai University Tianjin China Abstract Hepatitis B virus X protein HBx plays a crucial role in the development of hepatocellular carcinoma HCC A mutant of HBx HBxΔ127 was reported to enhance proliferation and migration of hepatoma cells In the present study we investigated whether calpain small subunit 1 Capn4 a protein known to associate with the migration proliferation and differentiation of tumor cells was involved in the HBxΔ127 promoted hepatoma cell migration Our data showed that the Capn4 promoter activities and Capn4 expression were increased in HBxΔ127 stably transfected HepG2 HepG2-XΔ127 cells The treatment with PD98059 an inhibitor of ERK blocked the HBxΔ127 upregulation of Capn4 expression suggesting that phosphorylated ERK1 /2 p-erk1 /2 was involved The wound healing assay confirmed that HBxΔ127 promoted the migration of hepatoma cells and the involvement of Capn4 and p-erk1 /2 Thus we conclude that HBxΔ127 upregulates Capn4 through p-erk1 /2 to promote the migration of hepatoma cells Key words hepatitis B virus X protein calpain small subunit 1 hepatocellular carcinoma ERK1 /2 migration No * Tel zhangxd@ nankai edu cn Tel yelihong@ nankai edu cn Received December Accepted March Supported by National Natural Science Foundation of China No * Corresponding author ZHANG Xiao-Dong Tel zhangxd@ nankai edu cn YE Li-Hong Tel yelihong@ nankai edu cn

2 5 X HBxΔ127 ERK hepatocellular carcinoma 40 1 mmol / L PMSF 1% SDS protease inhibitor HCC cocktails 20 min g 4 hepatitis B virus HBV 15 min - 70 HBV % PAGE S C P X X 17 kd PVDF 5 % 1 h 4 1 X HBx 2 Capn Chemicon HBV HBx Temecula CA HBx Abcam X Cambridge UK p-erk1 /2 T-ERK1 / HBx Cell Signaling USA β-actin nt HBxΔ127 Sigma-Aldrich USA 2 h HBx h ECL 4 HBxΔ127 3 osteopontin OPN HBxΔ127 pgl3-capn4 prl-tk psilencer 3 0 psilencer 3 0-X HBx RNAi 6 pcdna3 pcdna3-xδ127 4 ml 100 μl pgl3-capn4 prl-tk LARⅡ Capn4 5'-GCTTTTGTT 2 s 10 s CTCTCAGTAC-3' 100 μl Stop & HepG2 Glo reagent HepG2-P pcdna3 HepG2 10 s HepG2-XΔ127 pcdna3-xδ127 3 HepG HepG2 HepG2-P HepG2-XΔ h DMEM Gibco CA USA 10% 0 h U / ml 100 U / ml h 36 h 48 h 37 5 % CO HepG2 HepG2-P HepG2-XΔ h 80% 8 2 μg psilencer 3 0-X 2 μg 2 pcdna3 1 μg pcdna3-xδ127 2 μg pcdna3- XΔ127 3 μg pcdna3-xδ nmol / L sirna sictrl 80 nmol / L Capn4 sirna sicapn4 1 4 calpain small subunit 1 Capn4 PBS 3 9 HBxΔ mmol / L Tris-HCl ph mmol / L EDTA 150 mmol / L NaCl 1% NP- pcdna3-xδ h 15 min 1 5 ml r / min 10 min HepG2 HepG2-P HepG2-XΔ t 2 1 HBxΔ127 Capn4 HBxΔ127 OPN 5 1 Capn4 HBxΔ127

3 HBxΔ127 RNA HepG2-XΔ127 Capn4 Capn4 Fig 1B HepG2 HBx Fig 1A 1 μg 2 μg 3 μg pcdna3-xδ127 Capn4 Capn4 HepG2-XΔ127 HBx Fig 1C Fig 1 Examination of Capn4 expression in HepG2 HepG2-P and HepG2-XΔ127 cells A Reporter gene assay showed that the promoter activity of Capn4 was increased in HepG2-X Δ127 cells relative to HepG2 cells and HepG2-P cells Meanwhile the transient transfection with plasmid pcdna3-xδ127 was able to increase the promoter activity of Capn4 as well in a dose-dependent manner in HepG2 cell * P < 0 05 or ** P < 0 01 vs mock HepG2 cells Student's t test The results were representative of three independent experiments B Western blot analysis showed that the expression levels of Capn4 were up-regulated in HepG2-XΔ127cells relative to HepG2 cells and HepG2-P cells RNA interference RNAi targeting mrna of HBxΔ127 was able to result in the downregulation of Capn4 in HepG2-XΔ127cells Cell lysates were prepared The proteins in lysates were separated by 12% SDS-PAGE After transferring onto the membrane the blots were probed with antibodies β-actin was used as a loading control The results were representative of three independent experiments C Western blot analysis showed that the expression levels of Capn4 were increased in a dose-depended manner when HepG2 cells were transfected with 1 μg 2 μg and 3 μg pcdna3-xδ127 plasmids or 2 μg pcdna3 plasmids respectively The results were representative of three independent experiments 2 2 HBxΔ127 p-erk1 /2 Capn4 extracellular signal regulated kinase ERK HBxΔ127 Capn4 HBxΔ127 Capn4 ERK1 /2 p-erk1 /2 Capn4 HepG2- p- XΔ127 Capn4 Fig ERK1 /2 HepG2-XΔ127 3A HepG2 ERK1 /2 T-ERK1 /2 Fig 2A HBXΔ127 p-erk1 /2 Capn4 HepG2-XΔ μmol / L 50 μmol / L ERK1 /2 PD h Capn4 PD98059 Fig 2B HBxΔ127 p-erk1 / 2 Capn4 2 3 HBxΔ127 Capn4 Capn4 HepG2-XΔ127 HBxΔ127 Capn4 HepG2-XΔ127 Fig 3B 30 μmol / L ERK1 /2 PD98059 HepG2- XΔ127 Fig 3 HBxΔ127 p- ERK1 /2 Capn4

4 5 X HBxΔ127 ERK Fig 2 ERK1 /2 was responsible for the upregulation of Capn4 mediated by HBxΔ127 in HepG2-XΔ127 cells A Western blot analysis showed that the levels of p-erk1 /2 were increased in HepG2-XΔ127cells relative to HepG2 cells and HepG2-P cells B Western blot analysis showed that the expression levels of Capn4 were decreased in a dose-depended manner when HepG2-XΔ127 were treated with 30 μmol / L or 50 μmol / L PD98059 an inhibitor of ERK for 2 hours The results were representative of three independent experiments 3 HBV Capn4 were decreased when HepG2-XΔ127 cells were treated with 80 nmol / L sirna targeting Capn4 mrna sicapn4 HBV The results were representative of three independent HBV DNA experiments B Wound healing assay showed that HBXΔ127 HCC was able to promote the migration of HepG2-XΔ127 cells The HBx migration ability of HepG2-XΔ127 cells were decreased when HBx ras myc HBx HBx inhibitor of ERK Images were taken from wound healing assays at and 48 hours in a phase-contrast HBx microscope 100 Black arrows indicate the wound edge 4 closure of monolayer cells Results are representative of three calpain μ-calpains independent experiments RasGAP SerinB2 Fig 3 HBxΔ127 promotes migration ability of HepG2 cells through up-regulating Capn4 involving ERK1 /2 A Western blot analysis showed that the expression levels of the cells transfected with 80 nmol / L sirna targeting Capn4 4 mrna sicapn4 or treated with 30 μmol / L PD98059 an μ-calpains Capn4 Capn4 1 HBxΔ127 9 Capn4 5 HBXΔ127 Capn HBxΔ127 ERK MMP9 HepG2 Capn4

5 Fig 1 HBxΔ127 Capn4 HBxΔ127 Capn4 HepG2 pcdna3- XΔ127 Capn4 HBXΔ127 HBxΔ127 J Capn4 HBxΔ Capn4 18 ERK HBxΔ127 Capn4 HepG2-XΔ127 p-erk1 /2 HepG2-XΔ127 p-erk1 /2 HepG2-XΔ μmol / L 50 μmol / L ERK1 /2 PD h Capn4 PD98059 Fig 2 HBxΔ127 Capn4 p-erk1 /2 HBxΔ127 p-erk1 /2 Capn4 Fig Zhu PA Tan DM Peng ZT et al HBxΔ127 southern China J References 1 Zhang X Zhang H Ye L Effects of hepatitis B virus X protein on the development of liver cancer J J Lab Clin Med Zhang X Zhang W Ye L Pathogenesis of hepatitis B virus infection J Future Virol Liu XH Lin J Zhang SH et al COOH-terminal deletion of HBx gene is a frequent event in HBV-associated hepatocellular carcinoma J World J Gastroenterol Zhang H Shan C Li N et al Identification of a natural mutant of HBV X protein truncated 27 amino acids at COOH2terminal and its effect on liver cell proliferation J Acta Pharmacol Sin Zhang X Ye LH Zhang XD A mutant of hepatitis B virus X protein HBx Delta 127 enhances hepatoma cell migration via osteopontin involving 5-lipoxygenase J Acta Pharmacol Sin Zhang X Dong N Yin L et al Hepatitis B virus X protein upregulates survivin expression in hepatoma tissues J J Med Virol Wang Q Zhang W Liu Q et al A mutant of hepatitis B virus X protein HBxDelta127 promotes cell growth through a positive feedback loop involving 5-lipoxygenase and fatty acid syntheses Neoplasia Shan C Xu F Zhang S et al Hepatitis B virus X protein promotes liver cell proliferation via a positive cascade loop involving arachidonic acid metabolism and p-erk1 /2 J Cell Res Mellgren RL ZhangW Miyake K et al Calpain is required for the rapid calcium-dependent repair of wounded plasma membrane J J Biol Chem Roux PP Blenis J ERK and p38 MAPK-activated protein kinases a family of protein kinases with diverse biological functions J Microbiol Mol Biol Rev Lawrence MC Jivan A Shao C et al The roles of MAPKs in disease J Cell Res Murakami Y Saigo K Takashima H et al Large scaled analysis of hepatitis B virus HBV DNA integration in HBV related hepatocellular carcinomas J Gut Polymorphism analyses of hepatitis B virus X gene in hepatocellular carcinoma patients from Acta Biochim Biophys Sin Chen GG Li MY Ho RL et al Identification of hepatitis B virus X gene mutation in Hong Kong patients with hepatocellular carcinoma J J Clin Virol Pamonsinlapatham P Gril B Dufour S et al Capns1 a new binding partner of RasGAP-SH3 domain in K-RasV12 oncogenic cells Modulation of cell survival and migration J Cell Signal Tonnetti L Netzel-Arnett S Darnell G A et al SerpinB2 protection of retinoblastoma protein from calpain enhances tumor cell survival J Cancer Res Byun HJ Hong IK Kim E et al A splice variant of CD99 increases motility and MMP-9 expression of human breast cancer cells through the AKT- ERK- and JNK-dependent AP-1 activation signaling pathways J J Biol Chem Kunigal S Lakka SS Gondi CS et al RNAi-mediated down regulation of urokinase plasminogen activator receptor and matrix metalloprotease-9 in human breast cancer cells results in decreased tumor invasion angiogenesis and growth J Int J Cancer