HCC tissues RT-qPCR analyses indicated that the expression of HBx were positively correlated with those of HBXIP in 20 clinical HCC tissues In

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1 ISSN CN / Q http / /cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 480 DOI /j cnki cjbmb X CREB HBx * X hepatitis B virus X protein HBx HBx HBx Boyden s chamber HBx HepG2 HBx HepG2 HepG2-X HBx hepatitis B X-interacting protein HBXIP RNA HBx PCR HBXIP HBx HBx HBXIP HBx RNA HepG2-X HBXIP HBXIP camp CREB HBx HBXIP CREB RNA HBx HBXIP HBx CREB HBXIP HBXIP HBx CREB HBXIP HBx X X camp R735 7 Hepatitis B Virus X Protein Promotes Hepatoma Cell Migration via CREB-induced Up-regulation of Hepatitis B X-interacting Protein ZHANG Wei-Ying ZHANG Xiao-Dong YE Li-Hong * State Key Laboratory of Medicinal Chemical Biology 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 However the underlying mechanism by which HBx promotes the migration of hepatoma cells remains poorly understood In the present study we investigated the role of hepatitis B X- interacting protein HBXIP in the HBx-mediated migration of hepatoma cells The results from wound healing assays and Boyden chamber assays showed that HBXIP contributed to HBx-elevated migration of hepatoma cells Tissue microarrays manifested that the expression of HBXIP was up-regulated in clinical No 2015CB CB No 2014AA No No 14JCZDJC32800 * Tel yelihong@ nankai edu cn Received November Accepted January Supported by National Basic Research Program of China 973 Program No 2015CB CB National High Technology Research and Development Program of China 863 Program No 2014AA National Natural Science Foundation of China No and Tianjin Natural Science Foundation No 14JCZDJC32800 * Corresponding author Tel yelihong@ nankai edu cn

2 HCC tissues RT-qPCR analyses indicated that the expression of HBx were positively correlated with those of HBXIP in 20 clinical HCC tissues In in vitro experiments the over-expression of HBx resulted in the up-regulation of HBXIP whereas HBx RNA interference reduced HBXIP expression in stably HBxtransfected HepG2 /H7402 cell lines HepG2-X /H7402-X in a dose-dependent manner suggesting that HBx was responsible to up-regulate HBXIP in hepatoma cells We further demonstrated that HBx activated the promoter of HBXIP except when the transcription factor camp response element binding CREB site in HBXIP promoter was mutated Moreover CREB knockdown blocked the HBx-enhanced HBXIP up-regulation Chromatin immunoprecipitation assay ChIP confirmed that HBx was able to bind to the promoter of HBXIP through CREB Thus we concluded that HBx up-regulates HBXIP through the inteaction with CREB to promote the migration of hepatoma cells Key words hepatitis B virus X protein HBx hepatitis B X-interacting protein HBXIP hepatocellular carcinoma camp response element binding protein CREB cell migration hepatocellular carcinoma HCC HBx hepatitis B virus HBV 1 HBV DNA DNA 4 open reading frame ORF X hepatitis B virus X protein kb nuclear factor-kb NF-kB 5-5- CREB sirna si-creb lipoxygenase 5-LOX osteopontin OPN 1 4 HBx ORF 154 psi-hbx 5 pgl3-hbxip wt 17 kd 1 2 HBx pgl3-hbxip mut prl-tk 3 4 HBXIP RNA si- HBx yes-associated protein YAP HBXIP CGGAAGCGCAGUGAUGUU si-creb GA LIN28A /B RAB GAGAGGUCCGUCUAAUG NC AAUGGUCAUGGU CUUAUUCC 11 HBx HBV 12 HepG2 HepG2-P HBV HBx pcdna3 HepG2 HepG2-X HBx pcdna3-hbx HepG2 H7402 HBx H7402-P pcdna3 H α vascular endothelial 13 growth factor VEGF 1 2 HepG2 HepG2-P HepG2-X DMEM Gibco CA USA 8 HBx 1 H7402 H7402-P H7402-X amplified in breast cancer1 AIB1 E3 RPMI-1640 Gibco CA USA F-box and WD repeat domain containing 7α Fbw7α AIB1 Fbw7α AIB1 HBx AIB matrix metallopeptidase 9 MMP-9 HepG2 HepG2-X H7402 H7402-X MMP-9 HepG h 9 80% 14 psi-hbx 10 HBx sirna NC HBXIP sirna si-hbxip -1α hypoxia inducible factor-1α HIF- 1 calpain small subunit Capn4 1 1 psilencer3 0-HBx Hbx RNA H7402 pcdna3-hbx H % 100 U /ml 100 U /ml 37 5% CO 2 HepG2 HepG2-X 12 h

3 5 X CREB HBx PCR real-time quantitative PCR 0 h 24 h RT-qPCR boyden s chamber HepG2 HepG2-X 12 h Trizol Sigma-Aldrich USA RNA 0 2% BSA 1 ml Trizol Chamber min 0 2 ml 30 s 10% 8 μm 30 min r /min 15 min 96 Chamber 0 5 ml 10 ~ 30 min 4 50 μl r /min 10 min 1 ml 75% Boyden s Chamber CO r /min 5 min 12 h Giemsa 5 ~ 10 min DEPC-H 2 O 1 h RNA RNA M-MLV 1 6 PBS mmol /L Tris-HCl 1 9 ph8 0 1 mmol /L EDTA 150 mmol /L NaCl 1% NP-40 1 mmol /L PMSF 1% SDS HBXIP mut prl-tk pcdna3-hbx psi- 15 min h 14 15% 15 min PAGE PVDF 5% ml HBXIP Santa Cruz r /min 10 min 1 USA HBx Abcam UK β-actin Sigma-Aldrich USA LARⅡ 2 h 2 s S2P 1 10 ChIP 3% H 2 O 2 5 ~ 10 min PBS 3 10 min Immunoprecipitation Kit Epigentek Group Inc PBS 3 2 min Brooklyn NY HepG2-X 30 min 4 24 h 1% PBS 3 5 min DNA rpm 1 h PBS 3 10 min 4 5 min K min DNA 30 min PBS 3 5 min DNA DNA - 20 DAB 3 ~ 10 min 1 11 PBS RNA RNA cdna GAPDH RNA Ct 2 -ΔΔ Ct pgl3-hbxip wt pgl3- cocktails 20 min g 4 HBx NC si-creb 1 5 ml 100 μl h ECL 10 s 100 μl Stop&Glo reagent 10 s ChIP EpiQuikTM Chromatin SPSS 13 0 mean ± SD

476 31 t P < 0 05 Wilcoxon Wilcoxon signed-rank test HBXIP 1A Modified boyden s chamber Pearson s correlation coefficient HepG2 HepG2-X 2 HepG2-X HBXIP HepG2-X Fig HBXIP HepG2-X Fig 1B HepG2 HepG2-X

4 t P < 0 05 Wilcoxon Wilcoxon signed-rank test HBXIP 1A Modified boyden s chamber Pearson s correlation coefficient HepG2 HepG2-X 2 HepG2-X HBXIP HepG2-X Fig HBXIP HepG2-X Fig 1B HepG2 HepG2-X HBx 2 1 HBx HBXIP HBXIP HBx HBx HepG2-X HBXIP NF-kB /5-LOX /OPN /Capn4 Fig 1C HBx 10 HBXIP HBXIP HBx 2 2 HBXIP HBx HBXIP HBx HBXIP HBx HepG2 HepG2-X Fig 1 HBx promotes the migration of hepatoma cells through HBXIP A A wound healing assay was performed in HepG2 HepG2-X and HepG2-X cells transfected with si-hbxip HBXIP sirna One representative experiment was shown Black arrows indicated the wound edge The residual gap between the migrating cells from the opposing wound edge was expressed as a percentage of the initial scraped area B Cell migration potential of HepG2 HepG2-X and HepG2-X cells transfected with si- HBXIP or si-nc sirna control was measured by Boyden s chamber assay C The expression levels of HBXIP and HBx were tested in HepG2 HepG2-X and HepG2-X cells transfected with si-hbxip or si-nc by Western blotting analysis β-actin was used as a loading control Statistically significant differences are indicated ** P < 0 01 * P < 0 05 NS Student s t test

5 X CREB HBx 477 HBXIP P < 0 001 Fig 2B 149 HBXIP HBx 112 HBXIP Fig 2A -PCR P < 0 001 HBXIP 20 r = 0 8041 Fig 2C HBXIP HBx Fig 2 HBXIP is positively correlated with HBx in clinical HCC samples A The

5 5 X CREB HBx 477 HBXIP P < Fig 2B 149 HBXIP HBx 112 HBXIP Fig 2A -PCR P < HBXIP 20 r = Fig 2C HBXIP HBx Fig 2 HBXIP is positively correlated with HBx in clinical HCC samples A The expression of HBXIP was examined by immunohistochemistry staining IHC in clinical HCC tissues using tissue microarray The results showed that positive signals of HBXIP were localized in the nucleus and cytoplasm of tumor cells Representative image of IHC without anti-hbxip antibody as negative control Representative image of IHC observed in HCC tissues using anti-hbxip antibody 400 B Relative mrna levels of HBXIP were examined by RT-qPCR in 20 pairs of HCC clinical tissues and corresponding nontumorous tissues *** P < Student s t test C Correlation of HBXIP mrna levels with those of HBx was examined by RT-qPCR in 20 cases of HCC clinical tissues P < Pearson s correlation coefficient r = HBx HBXIP HBx HBXIP H7402-X 1 μg 2 μg psi-hbx HBx HBXIP HBXIP Fig HepG2 H μg 2 μg pcdna3-hbx HBx 3D 2 4 HBx CREB HBXIP HBXIP HBx Fig 3A HBx HBx HBXIP HepG2-X H7402-X HBXIP genomatix software Fig 3B suite HBXIP 14 HBXIP HepG2-X H7402-X Fig 3C HepG2-X HBXIP

478 31 Fig 3 HBx up-regulates HBXIP expression in HCC cells A The activities of HBXIP promoter were examined by luciferase reporter gene assays in HepG2 and H7402 cells transfected with different

6 Fig 3 HBx up-regulates HBXIP expression in HCC cells A The activities of HBXIP promoter were examined by luciferase reporter gene assays in HepG2 and H7402 cells transfected with different dose pcdna3-hbx plasmids * P < 0 05 ** P < 0 01 Student s t test B The promoter activities of HBXIP were analyzed by luciferase reporter gene assays in HepG2-X and H7402-X cells transfected with different dose psi-hbx psilencer3 0-HBx * P < 0 05 ** P < 0 01 Student s t test C The expression of HBXIP at the levels of protein was examined in H7402 or HepG2 H7402-P or HepG2-P and H7402-X or HepG2-X cells by Western blotting D The expression of HBXIP at the levels of protein was examined by Western blotting analysis in H7402-X and HepG2-X cells transfected with psi-con RNAi control vector or psi-hbx Results are representative of three independent experiments CREB 3 CREB HBx HBXIP CREB CREB HBV HBx HBXIP HBXIP CREB HBx HBx HBXIP HBx Fig 4A sirna NC HepG2-X H7402-X 50 nmol /L mitogen-activated protein kinase 100 nmol /L si-creb MAPK NF-κB Fas C protein kinase C HBXIP Fig 4B PKC 3 phosphatidylinositol 3- NC si-creb kinases PI3K 20 HBx HepG2-X Fig 4C HBx CREB HBXIP Ras Raf Wnt c-jun HBx hyaluronic acid HA CD44 HBx HBXIP 23 HBx CREB HBx HBXIP MMPs -2 cyclooxygenase-2 COX- HBx CREB HBXIP 2 Fig 4D HBx

5 X CREB HBx 479 Fig 4 HBx activates HBXIP promoter through CREB A The effect of HBx on HBXIP promoter was measured in HepG2-X and H7402-X cell lines transfected with vectors of WT wild type or MUT

7 5 X CREB HBx 479 Fig 4 HBx activates HBXIP promoter through CREB A The effect of HBx on HBXIP promoter was measured in HepG2-X and H7402-X cell lines transfected with vectors of WT wild type or MUT the mutant in CREB binding site of HBXIP promoter by luciferase reporter gene assays B Luciferase activities were measured in HepG2-X and H7402-X cell lines cotransfected with HBXIP promoter vector and NC control sirna 100 nmol /L or si-creb 50 or 100 nmol /L respectively C The expression of HBXIP and CREB were detected by Western blotting analysis in HepG2-X cells transfected with NC 100 nmol /L or si-creb 100 nmol /L D ChIP assays were performed to test the interaction of HBx with the promoter region of HBXIP Statistically significant differences are indicated * P < 0 05 ** P < 0 01 Student t test - HBx CREB 27 HBXIP HBXIP HBx NF-kB 5-LOX OPN HBx Capn4 10 HBXIP 1 X 28 HBXIP 1 References S100A4 LMO4 SKP2 LIN28B Zhang X Zhang H Ye L Effects of hepatitis B virus X protein HBXIP on the development of liver cancer J J Lab Clin Med 2006 CD46 CD55 CD HBx HBXIP 2 Zhang X Zhang W Ye L Pathogenesis of hepatitis B virus infection J Future Med HBXIP HBx HBXIP HBx HBx HBXIP HBXIP 3 Tanaka Y Kanai F Ichimura T et al The hepatitis B virus X protein enhances AP-1 activation through interaction with Jab1 J Oncogene Chen GG Li MY Ho RL et al Identification of hepatitis B virus X gene mutation in Hong Kong patients with hepatocellular

8 carcinoma J J Clin Virol Zhang T Zhang J You X et al Hepatitis B virus X protein modulates oncogene Yes-associated protein by CREB to promote growth of hepatoma cells J Hepatology You X Liu F Zhang T et al Hepatitis B virus X protein upregulates Lin28A / Lin28B through Sp-1 / c-myc to enhance the proliferation of hepatoma cells J Oncogene You X Liu F Zhang T et al Hepatitis B virus X protein upregulates oncogene Rab18 to result in the dysregulation of lipogenesis and proliferation of hepatoma cells J Carcinogenesis Yoo YG Oh SH Park ES et al Hepatitis B virus X protein enhances transcriptional activity of hypoxia-inducible factor- 1alpha through activation of mitogen-activated protein kinase pathway J J Biol Chem Liu Y Tong Z Li T et al Hepatitis B virus X protein stabilizes amplified in breast cancer 1 protein and cooperates with it to promote human hepatocellular carcinoma cell invasiveness J Hepatology Zhang X You X Wang Q et al Hepatitis B virus X protein drives multiple cross-talk cascade loops involving NF-kappaB 5- LOX OPN and Capn4 to promote cell migration J PLoS One e Shan C Zhang S Cui W et al Hepatitis B virus X protein activates CD59 involving DNA binding and let-7i in protection of hepatoma and hepatic cells from complement Carcinogenesis attack J 12 Li Y Zhang Z Zhou X et al The oncoprotein HBXIP enhances migration of breast cancer cells through increasing filopodia formation involving MEKK2 /ERK1 /2 /Capn4 Cancer Lett signaling J 13 Kong G Zhang J Zhang S et al Upregulated microrna-29a by hepatitis B virus X protein enhances hepatoma cell migration by targeting PTEN in cell culture model J PLoS One e Zhang W Lu Z Kong G et al Hepatitis B virus X protein accelerates hepatocarcinogenesis with partner survivin through modulating mir-520b and HBXIP J Mol Cancer Liu F You X Wang Y et al The oncoprotein HBXIP enhances angiogenesis and growth of breast cancer through modulating FGF8 and VEGF J Carcinogenesis Liu Q Bai X Li H et al The oncoprotein HBXIP upregulates Lin28B via activating TF II D to promote proliferation of breast cancer cells J Int J Cancer Liu S Li L Zhang Y et al The oncoprotein HBXIP uses two pathways to up-regulate S100A4 in promotion of growth and migration of breast cancer cells J J Biol Chem Xu F You X Liu F et al The oncoprotein HBXIP up-regulates Skp2 via activating transcription factor E2F1 to promote proliferation of breast cancer cells J Cancer Lett Hu N Zhang J Cui W et al mir-520b regulates migration of breast cancer cells by targeting hepatitis B X-interacting protein and interleukin-8 J J Biol Chem Feitelson MA Duan LX Hepatitis B virus X antigen in the pathogenesis of chronic infections and the development of hepatocellular carcinoma J Am J Pathol Feitelson MA Lee J Hepatitis B virus integration fragile sites and hepatocarcinogenesis J Cancer Lett Diao J Garces R Richardson C D X protein of hepatitis B virus modulates cytokine and growth factor related signal transduction pathways during the course of viral infections and hepatocarcinogenesis J Cytokine Growth Factor Rev Lara-Pezzi E Serrador J M Montoya M C et al The hepatitis B virus X protein HBx induces a migratory phenotype in a CD44- dependent manner possible role of HBx in invasion and metastasis J Hepatology Chung T W Lee Y C Kim C H Hepatitis B viral HBx induces matrix metalloproteinase-9 gene expression through activation of ERK and PI-3K /AKT pathways involvement of invasive potential J FASEB J Lara-Pezzi E Gomez-Gaviro M V Galvez B G et al The hepatitis B virus X protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression J J Clin Invest Ou D P Tao Y M Tang F Q et al The hepatitis B virus X protein promotes hepatocellular carcinoma metastasis by upregulation of matrix metalloproteinases J Int J Cancer Lara-Pezzi E Majano P L Yanez-Mo M et al Effect of the hepatitis B virus HBx protein on integrin-mediated adhesion to and migration on extracellular matrix J J Hepatol Melegari M Scaglioni P P Wands J R Cloning and characterization of a novel hepatitis B virus x binding protein that inhibits viral replication J J Virol Yue L Li L Liu F et al The oncoprotein HBXIP activates transcriptional coregulatory protein LMO4 via Sp1 to promote proliferation of breast cancer cells J Carcinogenesis

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环球中医药 2016 年 5 月第 9 卷第 5 期摇 Global Traditional Chinese Medicine, May 2016,Vol 郾 9, No 郾 5 摇 557 两种电针对脊髓损伤 14 天后大鼠运动功能神经元及 MEK2 p 鄄 ERK1 表达的影响论著宋良玉摇吕威摇景泉凯摇莫雨