ISSN 1007-7626 CN 11-3870 / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology 2011 5 27 5 426 ~ 430 X HBxΔ127 ERK 1 * * 300071 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 300071 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 2010-12-23 2011-03-18 No 81071624 * Tel 022-23506830 E-mail zhangxd@ nankai edu cn Tel 022-23501385 yelihong@ nankai edu cn Received December 23 2010 Accepted March 18 2011 Supported by National Natural Science Foundation of China No 81071624 * Corresponding author ZHANG Xiao-Dong Tel 022-23506830 E-mail zhangxd@ nankai edu cn YE Li-Hong Tel 022-23501385 yelihong@ nankai edu cn
5 X HBxΔ127 ERK 1 427 hepatocellular carcinoma 40 1 mmol / L PMSF 1% SDS protease inhibitor HCC cocktails 20 min 12 000 g 4 hepatitis B virus HBV 15 min - 70 HBV 4 6 12 % PAGE S C P X X 17 kd PVDF 5 % 1 h 4 1 X HBx 2 Capn4 1 1 000 Chemicon HBV HBx Temecula CA HBx 1 1 000 Abcam X Cambridge UK p-erk1 /2 T-ERK1 /2 1 1 000 3 HBx Cell Signaling USA β-actin 1 20 000 382-465 nt HBxΔ127 Sigma-Aldrich USA 2 h HBx 1 50 1 h ECL 4 HBxΔ127 3 osteopontin OPN 1 5 5 HBxΔ127 pgl3-capn4 prl-tk 1 1 1 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 HepG2 7 1 2 HepG2 HepG2-P HepG2-XΔ127 12 h DMEM Gibco CA USA 10% 0 h 24 100 U / ml 100 U / ml h 36 h 48 h 37 5 % CO 2 1 3 HepG2 HepG2-P HepG2-XΔ127 6 24 h 80% 8 2 μg psilencer 3 0-X 2 μg 2 pcdna3 1 μg pcdna3-xδ127 2 μg pcdna3- XΔ127 3 μg pcdna3-xδ127 80 nmol / L sirna sictrl 80 nmol / L Capn4 sirna sicapn4 1 4 calpain small subunit 1 Capn4 PBS 3 9 HBxΔ127 10 mmol / L Tris-HCl ph 8 0 1 mmol / L EDTA 150 mmol / L NaCl 1% NP- pcdna3-xδ127 48 h 15 min 1 5 ml 12 000 r / min 10 min 1 1 5 1 6 HepG2 HepG2-P HepG2-XΔ127 1 7 t 2 1 HBxΔ127 Capn4 HBxΔ127 OPN 5 1 Capn4 HBxΔ127
428 27 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 9 10 11 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-XΔ127 30 μmol / L 50 μmol / L ERK1 /2 PD98059 2 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
5 X HBxΔ127 ERK 1 429 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 12 13 HBx ras myc HBx HBx inhibitor of ERK Images were taken from wound healing assays at 0 12 24 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 17 18 Capn4 Capn4 1 HBxΔ127 9 Capn4 5 HBXΔ127 Capn4 15 16 HBxΔ127 ERK MMP9 HepG2 Capn4
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