Short hairpin RNA silencing of TGF-βRII and FZD-7 synergistically suppresses proliferation and metastasis of hepatocellular carcinoma cells

Chen,Cong; Xue,Yuyang; Zhang,Dejun; Xu,Wei; Xu,Hao; Yao,Hong; Pei,Dongsheng; Gu,Yuming

doi:10.3892/ol.2016.4208

Short hairpin RNA silencing of TGF-βRII and FZD-7 synergistically suppresses proliferation and metastasis of hepatocellular carcinoma cells

Authors:
- Cong Chen
- Yuyang Xue
- Dejun Zhang
- Wei Xu
- Hao Xu
- Hong Yao
- Dongsheng Pei
- Yuming Gu
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Affiliations: Department of Interventional Radiology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu 221002, P.R. China, Department of Oncology, Huazhong University of Science and Technology, Wuhan, Hubei 430030, P.R. China, Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, Xuzhou, Jiangsu 221002, P.R. China
Published online on: February 9, 2016 https://doi.org/10.3892/ol.2016.4208
Pages: 2039-2046
Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Transforming growth factor-β (TGF-β) is a multifunctional regulator of cell growth, apoptosis, differentiation and migration. The Wnt/β-catenin signaling pathway has been implicated in a wide spectrum of diseases, including numerous cancers and degenerative disease. The aim of the present study was to investigate if simultaneous blocking of TGF‑β and Wnt/β-catenin signaling pathways exerts synergistic anti‑tumor effects on hepatocellular carcinoma (HCC) cells. Short hairpin (sh) RNA eukaryotic expression vectors, specific to TGF‑β receptor II (RII) and Frizzled receptor (FZD)‑7, were constructed by gene recombination. The expression vectors were transfected into human HCC HepG2 and Huh‑7 cells using Lipofectamine 2000 to investigate the synergistic effects between TGF‑β and Wnt/β‑catenin signaling pathways on HCC cell proliferation, invasion and migration and the cell‑cycle distribution. Western blot analysis was used to identify the expression of β‑catenin, c‑Myc and cyclin D1 in transfected cells to investigate the underlying mechanisms that cause TGF‑β and Wnt/β‑catenin signaling in HCC cells. shTGF‑βRII‑c and shFZD‑7‑2 were selected as the most efficient plasmids. A cell growth assay and colony‑forming assay consistently demonstrated that the proliferative activity of the co‑transfected group was significantly decreased compared to the single‑transfected group. A wound healing invasion and migration assay demonstrated that co‑transfection of shTGF‑βRII‑c and shFZD‑7‑2 decreased the invasion and migration abilities of the cells compared with either single‑transfected group. In addition, the present study demonstrated that the observed reduction in cell proliferation was due to the cells arresting at the G1 phase of the cell cycle, and the downregulation of β‑catenin, c‑Myc and cyclin D1 impaired the proliferative and invasive abilities of the HCC cells. The present results demonstrate that simultaneous blocking of TGF‑β and Wnt/β‑catenin signaling by targeting TGF‑βRII and FZD‑7 may inhibit the proliferation and metastasis of HCC cells more effectively compared with blocking either the TGF-β or Wnt/β-catenin pathway.

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1	Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J and Jemal A: Global cancer statistics, 2012. CA Cancer J Clin. 65:87–108. 2015. View Article : Google Scholar : PubMed/NCBI
2	Eskens FA, van Erpecum KJ, de Jong KP, van Delden OM, Klumpen HJ, Verhoef C, Jansen PL, van den Bosch MA, Méndez Romero A, Verheij J, et al: Hepatocellular carcinoma: Dutch guideline for surveillance, diagnosis and therapy. Neth J Med. 72:299–304. 2014.PubMed/NCBI
3	Lee HS, Park MH, Yang SJ, Park KC, Kim NS, Kim YS, Kim DI, Yoo HS, Choi EJ and Yeom YI: Novel candidate targets of Wnt/beta-catenin signaling in hepatoma cells. Life Sci. 80:690–698. 2007. View Article : Google Scholar : PubMed/NCBI
4	Ogasawara N, Tsukamoto T, Mizoshita T, Inada K, Cao X, Takenaka Y, Joh T and Tatematsu M: Mutations and nuclear accumulation of beta-catenin correlate with intestinal phenotypic expression in human gastric cancer. Histopathology. 49:612–621. 2006. View Article : Google Scholar : PubMed/NCBI
5	Ozaki S, Ikeda S, Ishizaki Y, Kurihara T, Tokumoto N, Iseki M, Arihiro K, Kataoka T, Okajima M and Asahara T: Alterations and correlations of the components in the Wnt signaling pathway and its target genes in breast cancer. Oncol Rep. 14:1437–1443. 2005.PubMed/NCBI
6	Mårtensson A, Öberg A, Jung A, Cederquist K, Stenling R and Palmqvist R: Beta-catenin expression in relation to genetic instability and prognosis in colorectal cancer. Oncol Rep. 17:447–452. 2007.PubMed/NCBI
7	Fuchs SY, Ougolkov AV, Spiegelman VS and Minamoto T: Oncogenic beta-catenin signaling networks in colorectal cancer. Cell Cycle. 4:1522–1539. 2005. View Article : Google Scholar : PubMed/NCBI
8	Willert K and Jones KA: Wnt signaling: Is the party in the nucleus? Genes Dev. 20:1394–1404. 2006. View Article : Google Scholar : PubMed/NCBI
9	Holland JD, Klaus A, Garratt AN and Birchmeier W: Wnt signaling in stem and cancer stem cells. Curr Opin Cell Biol. 25:254–264. 2013. View Article : Google Scholar : PubMed/NCBI
10	Ye N, Wang B, Quan ZF, Pan HB, Zhang ML and Yan QG: The research progress of the interactions between miRNA and Wnt/beta-catenin signaling pathway in breast cancer of human and mice. Asian Pac J Cancer Prev. 15:1075–1079. 2014. View Article : Google Scholar : PubMed/NCBI
11	Roberts AB and Wakefield LM: The two faces of transforming growth factor beta in carcinogenesis. Proc Natl Acad Sci USA. 100:8621–8623. 2003. View Article : Google Scholar : PubMed/NCBI
12	Tan Y, Xu Q, Li Y, Mao X and Zhang K: Crosstalk between the p38 and TGF-β signaling pathways through TβRI, TβRII and Smad3 expression in plancental choriocarcinoma JEG-3 cells. Oncol Lett. 8:1307–1311. 2014.PubMed/NCBI
13	Miyazono K, Suzuki H and Imamura T: Regulation of TGF-beta signaling and its roles in progression of tumors. Cancer Sci. 94:230–234. 2003. View Article : Google Scholar : PubMed/NCBI
14	Labbé E, Letamendia A and Attisano L: Association of Smads with lymphoid enhancer binding factor 1/T cell-specific factor mediates cooperative signaling by the transforming growth factor-beta and wnt pathways. Proc Natl Acad Sci USA. 97:8358–8363. 2000. View Article : Google Scholar : PubMed/NCBI
15	Li Z, Zhang LJ, Zhang HR, Tian GF, Tian J, Mao XL, Jia ZH, Meng ZY, Zhao LQ, Yin ZN and Wu ZZ: Tumor-derived transforming growth factor-β is critical for tumor progression and evasion from immune surveillance. Asian Pac J Cancer Prev. 15:5181–5186. 2014. View Article : Google Scholar : PubMed/NCBI
16	Nishita M, Hashimoto MK, Ogata S, Laurent MN, Ueno N, Shibuya H and Cho KW: Interaction between Wnt and TGF-beta signalling pathways during formation of Spemann's organizer. Nature. 403:781–785. 2000. View Article : Google Scholar : PubMed/NCBI
17	Labbé E, Lock L, Letamendia A, Gorska AE, Gryfe R, Gallinger S, Moses HL and Attisano L: Transcriptional cooperation between the transforming growth factor-β and Wnt pathways in mammary and intestinal tumorigenesis. Cancer Res. 67:75–84. 2007. View Article : Google Scholar : PubMed/NCBI
18	Akhurst RJ and Derynck R: TGF-beta signaling in cancer - a double-edged sword. Trends Cell Biol. 11:S44–S51. 2001. View Article : Google Scholar : PubMed/NCBI
19	Howard S, Deroo T, Fujita Y and Itasaki N: A positive role of cadherin in Wnt/β-catenin signalling during epithelial-mesenchymal transition. PLoS One. 6:e238992011. View Article : Google Scholar : PubMed/NCBI
20	Merle P, de la Monte S, Kim M, Herrmann M, Tanaka S, Von Dem Bussche A, Kew MC, Trepo C and Wands JR: Functional consequences of frizzled-7 receptor overexpression in human hepatocellular carcinoma. Gastroenterology. 127:1110–1122. 2004. View Article : Google Scholar : PubMed/NCBI
21	Miyazono K, Ehata S and Koinuma D: Tumor-promoting functions of transforming growth factor-β in progression of cancer. Ups J Med Sci. 117:143–152. 2012. View Article : Google Scholar : PubMed/NCBI
22	Singh AM and Dalton S: The cell cycle and Myc intersect with mechanisms that regulate pluripotency and reprogramming. Cell Stem Cell. 5:141–149. 2009. View Article : Google Scholar : PubMed/NCBI
23	Ferraz C, Lorenz S, Wojtas B, Bornstein SR, Paschke R and Eszlinger M: Inverse correlation of miRNA and cell cycle-associated genes suggests influence of miRNA on benign thyroid nodule tumorigenesis. J Clin Endocrinol Metab. 98:E8–E16. 2013. View Article : Google Scholar : PubMed/NCBI
24	Shi QQ, Zuo GW, Feng ZQ, Zhao LC, Luo L, You ZM, Li DY, Xia J, Li J and Chen DL: Effect of trichostatin A on anti HepG2 liver carcinoma cells: Inhibition of HDAC activity and activation of Wnt/β-Catenin signaling. Asian Pac J Cancer Prev. 15:7849–7855. 2014. View Article : Google Scholar : PubMed/NCBI
25	Lan FF, Wang H, Chen YC, Chan CY, Ng SS, Li K, Xie D, He ML, Lin MC and Kung HF: Hsa-let-7g inhibits proliferation of hepatocellular carcinoma cells by downregulation of c-Myc and upregulation of p16(INK4A). Int J Cancer. 128:319–331. 2011. View Article : Google Scholar : PubMed/NCBI
26	Liao DJ, Thakur A, Wu J, Biliran H and Sarkar FH: Perspectives on c-Myc, Cyclin D1, and their interaction in cancer formation, progression, and response to chemotherapy. Crit Rev Oncog. 13:93–158. 2007. View Article : Google Scholar : PubMed/NCBI
27	Shachaf CM, Kopelman AM, Arvanitis C, Karlsson A, Beer S, Mandl S, Bachmann MH, Borowsky AD, Ruebner B, Cardiff RD, et al: MYC inactivation uncovers pluripotent differentiation and tumour dormancy in hepatocellular cancer. Nature. 431:1112–1117. 2004. View Article : Google Scholar : PubMed/NCBI
28	Giuriato S, Ryeom S, Fan AC, Bachireddy P, Lynch RC, Rioth MJ, van Riggelen J, Kopelman AM, Passegué E, Tang F, et al: Sustained regression of tumors upon MYC inactivation requires p53 or thrombospondin-1 to reverse the angiogenic switch. Proc Natl Acad Sci USA. 103:16266–16271. 2006. View Article : Google Scholar : PubMed/NCBI
29	Jemal A, Siegel R, Xu J and Ward E: Cancer statistics, 2010. CA Cancer J Clin. 60:277–300. 2010. View Article : Google Scholar : PubMed/NCBI
30	Brabletz T, Hlubek F, Spaderna S, Schmalhofer O, Hiendlmeyer E, Jung A and Kirchner T: Invasion and metastasis in colorectal cancer: Epithelial-mesenchymal transition, mesenchymal-epithelial transition, stem cells and β-catenin. Cells Tissues Organs. 179:56–65. 2005. View Article : Google Scholar : PubMed/NCBI
31	Kim H, Yoo SB, Sun P, Jin Y, Jheon S, Lee CT and Chung JH: Alteration of the E-Cadherin/β-Catenin complex is an independent poor prognostic factor in lung adenocarcinoma. Korean J Pathol. 47:44–51. 2013. View Article : Google Scholar : PubMed/NCBI
32	Nelson WJ and Nusse R: Convergence of Wnt, beta-catenin, and cadherin pathways. Science. 303:1483–1487. 2004. View Article : Google Scholar : PubMed/NCBI