Abstract
The long noncoding RNA growth-arrest specific 5 (GAS5) is a suppressor of many cancers. However, the role and mechanism of action of GAS5 in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) remain unclear. Here, the expression of hepatitis B virus x gene (HBx) mRNA and GAS5 was assessed by qRT–PCR, and western blot analysis was performed to determine the protein expression levels. In addition, the cell viability and invasion of cells were confirmed using MTT assay and Transwell assay, respectively. The DNA methylation level of GAS5 was measured by methylation-specific PCR. Moreover, RIP assay and RNA pull down assay were carried out to examine the combination of Y-box-binding protein 1 (YBX1) and GAS5. First, our data proved that HBx is increased, while GAS5 is decreased in HCC cell lines. Subsequently, we found that HBx facilitates HCC cell viability and invasion by inhibiting GAS5 expression. Then, we further clarified that HBx induces the DNA methylation of GAS5 by promoting methyltransferase expression, thereby suppressing GAS5 expression. Furthermore, GAS5 binds YBX1 and promotes YBX1 and p21 expression. Finally, the functional analysis revealed that the upregulation of GAS5 could attenuate cell viability and invasion by boosting p21 expression via binding YBX1. Overall, our results demonstrated that HBx promotes HCC progression by inducing GAS5 methylation to reduce its expression. The upregulation of GAS5 suppressed HBV-related HCC by activating YBX1/p21 signaling. Our data provide novel evidence supporting the potential of GAS5 as a treatment target in HBV-related HCC.
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Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
References
Cabibbo G, Celsa C, Cammà C, Craxì A (2018) Should we cure hepatitis C virus in patients with hepatocellular carcinoma while treating cancer? Liver Int 38(12):2108–2116. doi:https://doi.org/10.1111/liv.13918
Chen J, Gingold JA, Su X (2019) Immunomodulatory TGF-β signaling in hepatocellular carcinoma. Trends Mol Med 25(11):1010–1023. https://doi.org/10.1016/j.molmed.2019.06.007
Chen T, Pei J, Wang J, Luo R, Liu L, Wang L, Jia H (2019b) HBx-related long non-coding RNA 01152 promotes cell proliferation and survival by IL-23 in hepatocellular carcinoma. Biomed Pharmacother 115:108877. doi:https://doi.org/10.1016/j.biopha.2019.108877
Chen Z, Yu W, Zhou Q, Zhang J, Jiang H, Hao D, Wang J, Zhou Z, He C, Xiao Z (2019) A novel lncRNA IHS promotes tumor proliferation and metastasis in HCC by regulating the ERK- and AKT/GSK-3β-signaling pathways. Mol Ther Nucleic Acids 16:707–720. https://doi.org/10.1016/j.omtn.2019.04.021
Chen Z, Zhang Z, Zhao D, Feng W, Meng F, Han S, Lin B, Shi X (2019) Long noncoding RNA (lncRNA) FOXD2-AS1 promotes cell proliferation and metastasis in hepatocellular carcinoma by regulating MiR-185/AKT axis. Med Sci Monit 25:9618–9629. https://doi.org/10.12659/msm.918230
Fang P, Xiang L, Chen W, Li S, Huang S, Li J, Zhuge L, Jin L, Feng W, Chen Y, Pan C (2019) LncRNA GAS5 enhanced the killing effect of NK cell on liver cancer through regulating miR-544/RUNX3. Innate Immun 25(2):99–109. doi:https://doi.org/10.1177/1753425919827632
Fu WM, Zhu X, Wang WM, Lu YF, Hu BG, Wang H, Liang WC, Wang SS, Ko CH, Waye MM, Kung HF, Li G, Zhang JF (2015) Hotair mediates hepatocarcinogenesis through suppressing miRNA-218 expression and activating P14 and P16 signaling. J Hepatol 63:886–895. doi:https://doi.org/10.1016/j.jhep.2015.05.016
Gao Q, Wang K, Chen K, Liang L, Zheng Y, Zhang Y, Xiang J, Tang N (2017) HBx protein-mediated ATOH1 downregulation suppresses ARID2 expression and promotes hepatocellular carcinoma. Cancer Sci 108(7):1328–1337. doi:https://doi.org/10.1111/cas.13277
Gao ZQ, Wang JF, Chen DH, Ma XS, Yang W, Zhe T, Dang XW (2018) Long non-coding RNA GAS5 antagonizes the chemoresistance of pancreatic cancer cells through down-regulation of miR-181c-5p. Biomed Pharmacother 97:809–817. doi:https://doi.org/10.1016/j.biopha.2017.10.157
He X, Wang S, Li M, Zhong L, Zheng H, Sun Y, Lai Y, Chen X, Wei G, Si X, Han Y, Huang S, Li X, Liao W, Liao Y, Bin J (2019) Long noncoding RNA GAS5 induces abdominal aortic aneurysm formation by promoting smooth muscle apoptosis. Theranostics 9:5558–5576. doi:https://doi.org/10.7150/thno.34463
Kim EM, Jung CH, Kim J, Hwang SG, Park JK, Um HD (2017) The p53/p21 complex regulates cancer cell invasion and apoptosis by targeting Bcl-2 family proteins. Cancer Res 77:3092–3100. https://doi.org/10.1158/0008-5472.can-16-2098
Kreis NN, Friemel A, Ritter A, Roth S, Rolle U, Louwen F, Yuan J (2019) Function of p21 (Cip1/Waf1/CDKN1A) in Migration and Invasion of Cancer and Trophoblastic Cells. Cancers 11 doi:https://doi.org/10.3390/cancers11070989
Lee H, Sohn JB, Kim SS, Jang KL (2013) DNA methyltransferase inhibitor assay system based on the HBx-induced DNA methylation of E-cadherin. Biochem Biophys Res Commun 438:540–545. doi:https://doi.org/10.1016/j.bbrc.2013.07.094
Li C, Xu X (2019) Biological functions and clinical applications of exosomal non-coding RNAs in hepatocellular carcinoma. Cell Mol Life Sci 76:4203–4219. doi:https://doi.org/10.1007/s00018-019-03215-0
Liu Y, Zhao J, Zhang W, Gan J, Hu C, Huang G, Zhang Y (2015) lncRNA GAS5 enhances G1 cell cycle arrest via binding to YBX1 to regulate p21 expression in stomach cancer. Sci Rep 5:10159. doi:https://doi.org/10.1038/srep10159
Marchese FP, Raimondi I, Huarte M (2017) The multidimensional mechanisms of long noncoding RNA function. Genome Biol 18:206. doi:https://doi.org/10.1186/s13059-017-1348-2
Martens-Uzunova ES, Böttcher R, Croce CM, Jenster G, Visakorpi T, Calin GA (2014) Long noncoding RNA in prostate, bladder, and kidney cancer. Eur Urol 65:1140–1151. doi:https://doi.org/10.1016/j.eururo.2013.12.003
Park JW, Chen M, Colombo M, Roberts LR, Schwartz M, Chen PJ, Kudo M, Johnson P, Wagner S, Orsini LS, Sherman M (2015) Global patterns of hepatocellular carcinoma management from diagnosis to death: the BRIDGE study. Liver Int 35:2155–2166. https://doi.org/10.1111/liv.12818
Tu W, Yang Y, Song Y, Zhu W (2019) Hepatitis B virus x protein accelerated the proliferation of hepatocellular carcinoma cell through lncRNA SNHG20/PTEN pathway. J Biochem 165:423–431. doi:https://doi.org/10.1093/jb/mvy120
Wang Y, Su J, Wang Y, Fu D, Ideozu JE, Geng H, Cui Q, Wang C, Chen R, Yu Y, Niu Y, Yue D (2019a) The interaction of YBX1 with G3BP1 promotes renal cell carcinoma cell metastasis via YBX1/G3BP1-SPP1- NF-κB signaling axis. J Exp Clin Cancer Res 38(1):386. doi:https://doi.org/10.1186/s13046-019-1347-0
Wang YG, Wang T, Shi M, Zhai B (2019b) Long noncoding RNA EPB41L4A-AS2 inhibits hepatocellular carcinoma development by sponging miR-301a-5p and targeting FOXL1. J Exp Clin Cancer Res 38(1):153. doi:https://doi.org/10.1186/s13046-019-1128-9
Wei X, Xiang T, Ren G, Tan C, Liu R, Xu X, Wu Z (2013) miR-101 is down-regulated by the hepatitis B virus x protein and induces aberrant DNA methylation by targeting DNA methyltransferase 3A. Cell Signal 25(2):439–446. doi:https://doi.org/10.1016/j.cellsig.2012.10.013
Xiao L, Zhou Z, Li W, Peng J, Sun Q, Zhu H, Song Y, Hou JL, Sun J, Cao HC, Zhongyi D, Wu D, Liu L (2019) Chromobox homolog 8 (CBX8) Interacts with Y-Box binding protein 1 (YBX1) to promote cellular proliferation in hepatocellular carcinoma cells. Aging 11:7123–7149. doi:https://doi.org/10.18632/aging.102241
Yan J, Lu Q, Dong J, Li X, Ma K, Cai L (2012) Hepatitis B virus X protein suppresses caveolin-1 expression in hepatocellular carcinoma by regulating DNA methylation. BMC Cancer 12:353. doi:https://doi.org/10.1186/1471-2407-12-353
Yang JD, Hainaut P, Gores GJ (2019a) A global view of hepatocellular carcinoma: trends, risk, prevention and management. Nat Rev Gastroenterol Hepatol 16:589–604. doi:https://doi.org/10.1038/s41575-019-0186-y
Yang W, Xu X, Hong L, Wang Q, Huang J, Jiang L (2019b) Upregulation of lncRNA GAS5 inhibits the growth and metastasis of cervical cancer cells. J Cell Physiol 234:23571–23580. doi:https://doi.org/10.1002/jcp.28926
Yuan JH, Yang F, Wang F, Ma JZ, Guo YJ, Tao QF, Liu F, Pan W, Wang TT, Zhou CC, Wang SB, Wang YZ, Yang Y, Yang N, Zhou WP, Yang GS, Sun SH (2014) A long noncoding RNA activated by TGF-β promotes the invasion-metastasis cascade in hepatocellular carcinoma. Cancer Cell 25:666–681. doi:https://doi.org/10.1016/j.ccr.2014.03.010
Zhang N, Wang AY, Wang XK, Sun XM, Xue HZ (2016) GAS5 is downregulated in gastric cancer cells by promoter hypermethylation and regulates adriamycin sensitivity. Eur Rev Med Pharmacol Sci 20:3199–3205
Zhao H, Yu H, Zheng J, Ning N, Tang F, Yang Y, Wang Y (2018) Lowly-expressed lncRNA GAS5 facilitates progression of ovarian cancer through targeting miR-196-5p and thereby regulating HOXA5. Gynecol Oncol 151:345–355. doi:https://doi.org/10.1016/j.ygyno.2018.08.032
Zhao X, Liu Y, Yu S (2017a) Long noncoding RNA AWPPH promotes hepatocellular carcinoma progression through YBX1 and serves as a prognostic biomarker. Biochim Biophys Acta Mol Basis Dis 1863:1805–1816. doi:https://doi.org/10.1016/j.bbadis.2017.04.014
Zhao Z, Hu Y, Shen X, Lao Y, Zhang L, Qiu X, Hu J, Gong P, Cui H, Lu S, Zheng Y, Zhou M, Fan H (2017b) HBx represses RIZ1 expression by DNA methyltransferase 1 involvement in decreased miR-152 in hepatocellular carcinoma. Oncol Rep 37:2811–2818. doi:https://doi.org/10.3892/or.2017.5518
Zhou F, Shang W, Yu X (2018) Glypican-3: A promising biomarker for hepatocellular carcinoma diagnosis and treatment. Med Res Rev 38(2):741–767. doi:https://doi.org/10.1002/med.21455
Zhu K, Huang W, Wang W, Liao L, Li S, Yang S, Xu J, Li L, Meng M, Xie Y, He S, Tang W, Zhou H, Liang L, Gao H, Zhao Y, Hou Z, Tan J, Li R (2020) Up-regulation of S100A4 expression by HBx protein promotes proliferation of hepatocellular carcinoma cells and its correlation with clinical survival. Gene 749:144679. https://doi.org/10.1016/j.gene.2020.144679
Acknowledgements
We thank Xue Hong and Dai Yiwen for their help for shared routine work (short-time changing media for cells and some cleaning work etc.), which is not essential for this paper.
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This study was supported by Youth Program of National Natural Science Foundation of China (No. 81702829).
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XY designed the data and drafted the paper; XY, ZY, LH, XZ, ZL, RW, FH, GW, XG, HZ participated the experiments; XY, HZ analyzed the data; all author read and approved the paper.
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12079_2021_645_MOESM1_ESM.tif
Supplementary Figure 1. GAS5 has no effect on the YBX1 mRNA level. (A-B) The expression of YBX1 mRNA was determined using qRT-PCR. The experiments were performed in triplicate.
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Yu, X., Ye, Z., Hou, L. et al. Hepatitis B virus x gene-downregulated growth-arrest specific 5 inhibits the cell viability and invasion of hepatocellular carcinoma cell lines by activating Y-box-binding protein 1/p21 signaling. J. Cell Commun. Signal. 16, 179–190 (2022). https://doi.org/10.1007/s12079-021-00645-z
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DOI: https://doi.org/10.1007/s12079-021-00645-z