Hepatitis B spliced protein triggers TGFβ1-induced epithelial-mesenchymal transition via interaction with transforming growth factor beta-1-induced transcript 1 protein
CHEN Wan-nan, HUANG Jun-gao, LIANG Fei-fei, YAN Xiao-li, XUAN Dan-dan, LIN Xu
Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Key Laboratory of Fujian Province for Tumor Microbiology, School of Basic Medical Sciences, Fujian Medical University, Fuzhou 350122, China
Abstract：To investigate the TGFβ1-induced epithelial-mesenchymal transition (EMT) of Huh7 hepatoma cells caused by interaction of hepatitis B spliced protein (HBSP) with transforming growth factor beta-1-induced transcript 1 protein (TGFβ1I1), coding region of HBSP was cloned into lentiviral expression vector. Huh7 hepatoma cells were infected by recombinant lentivirus packaged in 293T cells. Stable cell lines expressing HBSP or control cells were selected by puromycin. Cells were incubated with 5 ng/mL TGFβ1 for 24 h, and observed under contrast-phase microspcope. Then the whole cell lysates were collected for western blot analysis using specific antibodies against EMT markers including E-cadherin, N-cadherin, Claudin-1 and β-catenin. To evaluate the effects of HBSP-TGFβ1I1 interaction on EMT, TGFβ1-induced EMT marker transition, as well as cell invasion and migration were explored after knocking down of TGFβ1I1 by siRNA. Results showed that Huh7 cell lines expressing HBSP (Huh7-HBSP-flag-HIV) and control cell lines (Huh7-flag-HIV) were successfully established. Huh7-HBSP-flag-HIV cells lost their pebble-like shape and tight cell-cell adhesion and transformed into the mesenchymal-like cells in the presence of TGFβ1. Decreased expression level of epithelial marker of E-cadherin, Claudin-1, β-catenin, increased expression level of mesenchymal marker of N-cadherin, and enhanced migration and invasion abilities were observed in Huh7-HBSP-flag-HIV cells as compared to the control cells. Moreover, the changes of EMT markers and metastasis abilities of Huh7-HBSP-flag-HIV cells could be reversed when TGFβ1I1 was knocked down by siRNA. In conclusion, HBSP could promote hepatoma cell migration and invasion by triggering EMT via interaction with TGFβ1I1. Our findings highlight new insights for HBSP-induced HCC progression.
[1] Wu HL, Chen PJ, Tu SJ, et al. Characterization and genetic analysis of alternatively spliced transcripts of hepatitis B virus in infected human liver tissues and transfected HepG2 cells[J]. J Virol, 1991, 65(4):1680-1686. [2] Soussan P, Tuveri R, Nalpas B, et al. The expression of hepatitis B spliced protein (HBSP) encoded by a spliced hepatitis B virus RNA is associated with viral replication and liver fibrosis[J]. J Hepatol, 2003, 38(3):343-348. doi:10.1016/S0168-8278(02)00422-1 [3] Mancini-Bourgine M, Bayard F, Soussan P, et al. Hepatitis B virus splice-generated protein induces T-cell responses in HLA-transgenic mice and hepatitis B virus-infected patients[J]. J Virol, 2007, 81(10):4963-4972. doi:10.1128/JVI.02619-06 [4] Chen WN, Chen JY, Jiao BY, et al. Interaction of hepatitis B spliced protein (HBSP) with cathepsin B promotes hepatoma cell migration and invasion[J]. J Virol, 2012, 86(24):13533-13541. doi:10.1128/JVI.02095-12 [5] Chen JY, Chen WN, Liu LL, et al. Hepatitis B spliced protein (HBSP) generated by a spliced hepatitis B virus RNA participates in abnormality of fibrin formation and functions by binding to fibrinogen γ chain[J]. J Med Virol, 2010, 82(12):2019-2026. doi:10.1002/jmv.21918 [6] Sciencepaper Online.Interaction of the hepatitis B spliced protein with transforming growth factor beta 1 induced transcript 1[EB/OL].[2015-7-13].http://www.paper.edu.cn/html/releasepaper/201507-118. [7] Tumbarello DA, Turner CE. Hic-5 contributes to epithelial-mesenchymal transformation through a RhoA/ROCK-dependent pathway[J]. J Cellular Physiol, 2007, 211(3):736-47. doi:10.1002/jcp.20991 [8] Soussan P, Garreau F, Zylberberg H, et al. In vivo expression of a new hepatitis B virus protein encoded by a spliced RNA[J]. J Clin Investigat, 2000, 105(1):55-60. doi:10.1172/JCI8098 [9] Chen JY, Chen WN, Jiao BY, et al. Hepatitis B spliced protein (HBSP) promotes the carcinogenic effects of benzo [alpha] pyrene by interacting with microsomal epoxide hydrolase and enhancing its hydrolysis activity[J]. BMC Cancer, 2014, 14:282. doi:10.1186/1471-2407-14-282 [10] Shibanuma M, Mashimo J, Mita A, et al. Cloning from a mouse osteoblastic cell line of a set of transforming-growth-factor-beta 1-regulated genes, one of which seems to encode a follistatin-related polypeptide[J]. Eur J Biochem, 1993, 217(1):13-19. doi:10.1111/j.1432-1033.1993.tb18212.x [11] Shibanuma M, Mashimo J, Kuroki T, et al. Characterization of the TGF beta 1-inducible hic-5 gene that encodes a putative novel zinc finger protein and its possible involvement in cellular senescence[J]. J Biol Chem, 1994, 269(43):26767-26774. doi:10.1007/s00109-010-0608-3 [12] Dabiri G, Tumbarello DA, Turner CE, et al. Hic-5 promotes the hypertrophic scar myofibroblast phenotype by regulating the TGF-beta1 autocrine loop[J]. J Invest Dermatol, 2008, 128(10):2518-2525. doi:10.1038/jid.2008.90 [13] Tumbarello DA, Turner CE. Hic-5 contributes to epithelial-mesenchymal transformation through a RhoA/ROCK-dependent pathway[J]. J Cellular Physiol, 2007, 211(3):736-747. doi:10.1002/jcp.20991 [14] Pignatelli J, Tumbarello DA,Schmidt RP, et al. Hic-5 promotes invadopodia formation and invasion during TGF-β-induced epithelial-mesenchymal transition[J]. J Cell Biol, 2012, 197(3):421-437. doi:10.1083/jcb.201108143 [15] Xu Z, Shen MX, Ma DZ, et al. TGF-beta1-promoted epithelial-to-mesenchymal transformation and cell adhesion contribute to TGF-beta1-enhanced cell migration in SMMC-7721 cells [J]. Cell Res, 2003, 13(5):343-350. doi:10.1038/sj.cr.7290179 [16] Calvisi DF, Pascale RM, Feo F. Epidermal growth factor-like repeats and discoidin I-like domains 3:a multifaceted oncoprotein at the crossroad of MAPK and TGF-beta pathways in human hepatocellular carcinoma[J]. Transl Cancer Res, 2016, 5(2):103-109. doi:10.21037/tcr.2016.03.09 [17] Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states:acquisition of malignant and stem cell traits[J]. Nat Rev Cancer, 2009, 9(4):265-273. doi:10.1038/nrc2620 [18] Tepass U, Truong K, Godt D, et al. Cadherins in embryonic and neural morphogenesis[J]. Nat Rev Mol Cell Biol, 2000, 1(2):91-100. doi:10.1038/35040042 [19] An HT, Yoo S, Ko J.α-Actinin-4 induces the epithelial-to-mesenchymal transition and tumorigenesis via regulation of Snail expression and β-catenin stabilization in cervical cancer[J]. Oncogene, 2016, 35(45):5893-5904.doi:10.1038/onc.2016.117
2017, Vol. 33 
