TALEN-induced disruption of Nanog expression results in reduced proliferation, invasiveness and migration, increased chemosensitivity and reversal of EMT in HepG2 cells

Yu,Ai  Qing; Ding,Yan; Li,Cheng  Lin; Yang,Yi; Yan,Shi  Rong; Li,Dong  Sheng

doi:10.3892/or.2015.4483

TALEN-induced disruption of Nanog expression results in reduced proliferation, invasiveness and migration, increased chemosensitivity and reversal of EMT in HepG2 cells

Authors:
- Ai Qing Yu
- Yan Ding
- Cheng Lin Li
- Yi Yang
- Shi Rong Yan
- Dong Sheng Li
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Affiliations: Hubei Key Laboratory of Embryonic Stem Cell Research, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, P.R. China
Published online on: December 10, 2015 https://doi.org/10.3892/or.2015.4483
Pages: 1657-1663

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Abstract

Accumulating evidence indicates that Nanog plays a central role in modulating the biological behaviors of human hepatocellular carcinoma (HCC). However, the underlying mechanisms remain unclear. In the present study, we employed transcription activator-like effector nucleases (TALEN) to disrupt Nanog expression in HepG2 cells and obtained subcloned cells with diallelic Nanog mutations. Significantly, we found that the expression of pluripotency factors Sox2, Oct4 and Klf4, as well as expression of cancer stem cell (CSC) marker CD133, in the Nanog-targeted HepG2 cells was markedly downregulated. This finding suggests that Nanog may play an important role in maintaining the pluripotency and malignancy of HepG2 cells. We also revealed that Nanog regulated cell proliferation by modulating the expression of cyclin D1/D3/E1 and CDK2, respectively. Additionally, the disruption of Nanog resulted in the downregulation of epithelial-mesenchymal transition (EMT) regulators Snail and Twist, which contributed to the elevated level of epithelial marker E-cadherin, and to the decreased level of mesenchymal markers N-cadherin and vimentin in the HepG2 cells. In addition, the Nanog-targeted HepG2 cells exhibited reduced ability of invasion, migration and chemoresistance in vitro. In conclusion, the disruption of Nanog expression results in less proliferation, invasiveness, migration, more chemosensitivity and reversal of EMT in HepG2 cells, by which Nanog plays crucial roles in influencing the malignant phenotype of HepG2 cells.

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1	Shan J, Shen J, Liu L, Xia F, Xu C, Duan G, Xu Y, Ma Q, Yang Z, Zhang Q, et al: Nanog regulates self-renewal of cancer stem cells through the insulin-like growth factor pathway in human hepatocellular carcinoma. Hepatology. 56:1004–1014. 2012. View Article : Google Scholar : PubMed/NCBI
2	Sun C, Sun L, Jiang K, Gao DM, Kang XN, Wang C, Zhang S, Huang S, Qin X, Li Y, et al: NANOG promotes liver cancer cell invasion by inducing epithelial-mesenchymal transition through NODAL/SMAD3 signaling pathway. Int J Biochem Cell Biol. 45:1099–1108. 2013. View Article : Google Scholar : PubMed/NCBI
3	Hashimoto N, Tsunedomi R, Yoshimura K, Watanabe Y, Hazama S and Oka M: Cancer stem-like sphere cells induced from de-differentiated hepatocellular carcinoma-derived cell lines possess the resistance to anti-cancer drugs. BMC Cancer. 14:7222014. View Article : Google Scholar : PubMed/NCBI
4	Li X, Luo Q and Song G: Novel therapeutic strategies for treatment of hepatocellular carcinoma: Targeting intervention on liver cancer stem cells. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi. 30:894–898. 2013.In Chinese. PubMed/NCBI
5	Wang J, Levasseur DN and Orkin SH: Requirement of Nanog dimerization for stem cell self-renewal and pluripotency. Proc Natl Acad Sci USA. 105:6326–6331. 2008. View Article : Google Scholar : PubMed/NCBI
6	Pan G and Thomson JA: Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res. 17:42–49. 2007. View Article : Google Scholar : PubMed/NCBI
7	Wang J, Rao S, Chu J, Shen X, Levasseur DN, Theunissen TW and Orkin SH: A protein interaction network for pluripotency of embryonic stem cells. Nature. 444:364–368. 2006. View Article : Google Scholar : PubMed/NCBI
8	Hyslop L, Stojkovic M, Armstrong L, Walter T, Stojkovic P, Przyborski S, Herbert M, Murdoch A, Strachan T and Lako M: Downregulation of NANOG induces differentiation of human embryonic stem cells to extraembryonic lineages. Stem Cells. 23:1035–1043. 2005. View Article : Google Scholar : PubMed/NCBI
9	Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M and Yamanaka S: The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell. 113:631–642. 2003. View Article : Google Scholar : PubMed/NCBI
10	Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S and Smith A: Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell. 113:643–655. 2003. View Article : Google Scholar : PubMed/NCBI
11	Wang ML, Chiou SH and Wu CW: Targeting cancer stem cells: Emerging role of Nanog transcription factor. Onco Targets Ther. 6:1207–1220. 2013.PubMed/NCBI
12	Iv Santaliz-Ruiz LE, Xie X, Old M, Teknos TN and Pan Q: Emerging role of nanog in tumorigenesis and cancer stem cells. Int J Cancer. 135:2741–2748. 2014. View Article : Google Scholar : PubMed/NCBI
13	Yang L, Zhang X, Zhang M, Zhang J, Sheng Y, Sun X, Chen Q and Wang LX: Increased Nanog expression promotes tumor development and cisplatin resistance in human esophageal cancer cells. Cell Physiol Biochem. 30:943–952. 2012. View Article : Google Scholar : PubMed/NCBI
14	Lu Y, Zhu H, Shan H, Lu J, Chang X, Li X, Lu J, Fan X, Zhu S, Wang Y, et al: Knockdown of Oct4 and Nanog expression inhibits the stemness of pancreatic cancer cells. Cancer Lett. 340:113–123. 2013. View Article : Google Scholar : PubMed/NCBI
15	Wang D, Lu P, Zhang H, Luo M, Zhang X, Wei X, Gao J, Zhao Z and Liu C: Oct-4 and Nanog promote the epithelial-mesenchymal transition of breast cancer stem cells and are associated with poor prognosis in breast cancer patients. Oncotarget. 5:10803–10815. 2014. View Article : Google Scholar : PubMed/NCBI
16	Huang CE, Yu CC, Hu FW, Chou MY and Tsai LL: Enhanced chemosensitivity by targeting Nanog in head and neck squamous cell carcinomas. Int J Mol Sci. 15:14935–14948. 2014. View Article : Google Scholar : PubMed/NCBI
17	Ding Y, Yu AQ, Li CL, Fang J, Zeng Y and Li DS: TALEN-mediated Nanog disruption results in less invasiveness, more chemosensitivity and reversal of EMT in Hela cells. Oncotarget. 5:8393–8401. 2014. View Article : Google Scholar : PubMed/NCBI
18	Chanrion M, Kuperstein I, Barrière C, El Marjou F, Cohen D, Vignjevic D, Stimmer L, Paul-Gilloteaux P, Bièche I, Tavares SR, et al: Concomitant Notch activation and p53 deletion trigger epithelial-to-mesenchymal transition and metastasis in mouse gut. Nat Commun. 5:50052014. View Article : Google Scholar : PubMed/NCBI
19	Rangel MC, Karasawa H, Castro NP, Nagaoka T, Salomon DS and Bianco C: Role of Cripto-1 during epithelial-to-mesenchymal transition in development and cancer. Am J Pathol. 180:2188–2200. 2012. View Article : Google Scholar : PubMed/NCBI
20	Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, et al: The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 133:704–715. 2008. View Article : Google Scholar : PubMed/NCBI
21	Polyak K and Weinberg RA: Transitions between epithelial and mesenchymal states: Acquisition of malignant and stem cell traits. Nat Rev Cancer. 9:265–273. 2009. View Article : Google Scholar : PubMed/NCBI
22	Thiery JP and Sleeman JP: Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol. 7:131–142. 2006. View Article : Google Scholar : PubMed/NCBI
23	Katsuyama T, Akmammedov A, Seimiya M, Hess SC, Sievers C and Paro R: An efficient strategy for TALEN-mediated genome engineering in Drosophila. Nucleic Acids Res. 41:e1632013. View Article : Google Scholar : PubMed/NCBI
24	Li T, Huang S, Zhao X, Wright DA, Carpenter S, Spalding MH, Weeks DP and Yang B: Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res. 39:6315–6325. 2011. View Article : Google Scholar : PubMed/NCBI
25	Zhu H, Lau CH, Goh SL, Liang Q, Chen C, Du S, Phang RZ, Tay FC, Tan WK, Li Z, et al: Baculoviral transduction facilitates TALEN-mediated targeted transgene integration and Cre/LoxP cassette exchange in human-induced pluripotent stem cells. Nucleic Acids Res. 41:e1802013. View Article : Google Scholar : PubMed/NCBI
26	Jiang D, Zhu W, Wang Y, Sun C, Zhang KQ and Yang J: Molecular tools for functional genomics in filamentous fungi: Recent advances and new strategies. Biotechnol Adv. 31:1562–1574. 2013. View Article : Google Scholar : PubMed/NCBI
27	Kucia M, Jankowski K, Reca R, Wysoczynski M, Bandura L, Allendorf DJ, Zhang J, Ratajczak J and Ratajczak MZ: CXCR4-SDF-1 signalling, locomotion, chemotaxis and adhesion. J Mol Histol. 35:233–245. 2004. View Article : Google Scholar : PubMed/NCBI
28	Müller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN, et al: Involvement of chemokine receptors in breast cancer metastasis. Nature. 410:50–56. 2001. View Article : Google Scholar : PubMed/NCBI
29	Egeblad M and Werb Z: New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer. 2:161–174. 2002. View Article : Google Scholar : PubMed/NCBI
30	Wang XQ, Ng RK, Ming X, Zhang W, Chen L, Chu AC, Pang R, Lo CM, Tsao SW, Liu X, et al: Epigenetic regulation of pluripotent genes mediates stem cell features in human hepatocellular carcinoma and cancer cell lines. PLoS One. 8:e724352013. View Article : Google Scholar : PubMed/NCBI
31	Han J, Zhang F, Yu M, Zhao P, Ji W, Zhang H, Wu B, Wang Y and Niu R: RNA interference-mediated silencing of NANOG reduces cell proliferation and induces G0/G1 cell cycle arrest in breast cancer cells. Cancer Lett. 321:80–88. 2012. View Article : Google Scholar : PubMed/NCBI
32	Casas E, Kim J, Bendesky A, Ohno-Machado L, Wolfe CJ and Yang J: Snail2 is an essential mediator of Twist1-induced epithelial mesenchymal transition and metastasis. Cancer Res. 71:245–254. 2011. View Article : Google Scholar : PubMed/NCBI
33	Acloque H, Adams MS, Fishwick K, Bronner-Fraser M and Nieto MA: Epithelial-mesenchymal transitions: The importance of changing cell state in development and disease. J Clin Invest. 119:1438–1449. 2009. View Article : Google Scholar : PubMed/NCBI
34	Chiou SH, Wang ML, Chou YT, Chen CJ, Hong CF, Hsieh WJ, Chang HT, Chen YS, Lin TW, Hsu HS, et al: Coexpression of Oct4 and Nanog enhances malignancy in lung adenocarcinoma by inducing cancer stem cell-like properties and epithelial-mesenchymal transdifferentiation. Cancer Res. 70:10433–10444. 2010. View Article : Google Scholar : PubMed/NCBI
35	Valastyan S and Weinberg RA: Tumor metastasis: Molecular insights and evolving paradigms. Cell. 147:275–292. 2011. View Article : Google Scholar : PubMed/NCBI
36	Meng HM, Zheng P, Wang XY, Liu C, Sui HM, Wu SJ, Zhou J, Ding YQ and Li J: Over-expression of Nanog predicts tumor progression and poor prognosis in colorectal cancer. Cancer Biol Ther. 9:295–302. 2010. View Article : Google Scholar