ETV5 Silencing Produces Mesenchymal to Epithelial Transition in INS-1 (832/13) Cell Line

Abstract

ETV5 has been described to be involved in the epithelial to mesenchymal transition (EMT) mainly in cancer. It is known that EMT provokes cytoskeleton remodeling, improving cellular migratory, and invasive capabilities. Moreover, overexpression of ETV5 has been correlated to cancer development and this gene has been implicated in cell proliferation. However, little is known about the downregulation of ETV5 expression in a pancreatic cell line and the inverse mesenchymal to epithelial transition (MET). Therefore, we studied the implications of ETV5 silencing over the phenotype of the insulinoma INS-1 (832/13) cell line and described the MET by partial ETV5 silencing in the INS-1 (832/13) cell line. The downregulation of ETV5 expression was obtained by using ETV5 siRNA in the insulinoma rat cell line, INS-1 (832/13). Then, ETV5 knockdown provoked a MET phenotype observed by crystal violet staining and verified by immunohistochemistry against E-cadherin. Wound healing assay showed no migration, and F-actin stain revealed rearrangement of actin microfilaments. In addition, TGFβ1 and TGFβ3 were downregulated in the absence of ETV5. ETV5 silencing induces epithelial phenotype by downregulating TGFβ1 and TGFβ3 in INS-1 (832/13) cell line.

Keywords

ETV5 - mesenchymal to epithelial transition (MET) - β-cell - INS-1 (832/13) - TGFβ1 - TGFβ3

Publication History

Received: 08 August 2022

Accepted after revision: 11 January 2024

Article published online:
09 February 2024

• References

• 1 Alonso-Alconada L, Eritja N, Muinelo-Romay L. et al. ETV5 transcription program links BDNF and promotion of EMT at invasive front of endometrial carcinomas. Carcinogenesis 2014; 35: 2679-2686
• 2 Colas E, Muinelo-Romay L, Alonso-Alconada L. et al. ETV5 cooperates with LPP as a sensor of extracellular signals and promotes EMT in endometrial carcinomas. Oncogene 2012; 31: 4778-4788
• 3 Meng D, Li Z, Ma X. et al. ETV5 overexpression contributes to tumor growth and progression of thyroid cancer through PIK3CA. Life Sci 2020; 253: 117693
• 4 Puli OR, Danysh BP, McBeath E. et al The transcription factor ETV5 mediates BRAFV600E-induced proliferation and TWIST1 expression in papillary thyroid cancer cells. Neoplasia (United States) 20218 20: 1121-1134
• 5 Qi T, Qu Q, Li G. et al. Function and regulation of the PEA3 subfamily of ETS transcription factors in cancer. 2020; 10: 3083-3105
• 6 Beuran M, Negoi I, Paun S. et al. The epithelial to mesenchymal transition in pancreatic cancer: A systematic review. Pancreatology 2015; 15: 217-225
• 7 Kobberup S, Nyeng P, Juhl K. et al. ETS-family genes in pancreatic development. Dev Dyn 2007; 236: 3100-3110
• 8 Francis M, Ashok A, Ashwathnarayan A. et al. Advancement in understanding the concept of epithelial to mesenchymal transition in pancreatic β-cells: implication in diabetes. Curr Diabetes Rev 2023; 19: e190522205030
• 9 de Jesus DS, Mak TCS, Wang YF. et al. Dysregulation of the Pdx1/Ovol2/Zeb2 axis in dedifferentiated β-cells triggers the induction of genes associated with epithelial–mesenchymal transition in diabetes. Mol Metab 2021; 53: 101248
• 10 Title AC, Silva PN, Godbersen S. et al. The miR-200–Zeb1 axis regulates key aspects of β-cell function and survival in vivo. Mol Metab 2021; 53: 101267
• 11 Montgomery AMP, Yebra M. (2011) The epithelial-to-mesenchymal transition of human pancreatic β-cells: inductive mechanisms and implications for the cell-based therapy of type I diabetes. Curr Diabetes Rev 2011; 7: 346-355
• 12 Wang H-L, Wang L, Zhao C-Y. et al. Role of TGF-beta signaling in beta cell proliferation and function in diabetes. Biomolecules 2022; 12: 373
• 13 Jiang Y, Fischbach S, Xiao X. The role of the TGFβ receptor signaling pathway in adult beta cell proliferation. Int J Mol Sci 2018; 19: 3136
• 14 Gutierrez-Aguilar R, Kim D-H, Casimir M. et al. The role of the transcription factor ETV5 in insulin exocytosis. Diabetologia 2014; 57: 383-391
• 15 Díaz-López YE, Pérez-Figueroa GE, Cázares-Domínguez V. et al. ETV5 regulates proliferation and cell cycle genes in the INS-1 (832/13) cell line independently of the concentration of secreted insulin. FEBS Open Bio 2023; 13: 2263-2272
• 16 Ofori JK, Karagiannopoulos A, Nagao M. et al. Human islet microRNA-200c is elevated in type 2 diabetes and targets the transcription factor ETV5 to reduce insulin secretion. Diabetes 2022; 71: 275-284
• 17 Belgardt B, Ahmed K, Spranger M. et al. The microRNA-200 family regulates pancreatic beta cell survival in type 2 diabetes. Nat Med 2015; 21: 619-627
• 18 Title AC, Silva PN, Godbersen S. et al. The miR-200-Zeb1 axis regulates key aspects of b-cell function and survival in vivo. Mol Metab 2021; 53: 101267
• 19 Filios SR, Xu G, Chen J. et al. MicroRNA-200 is induced by thioredoxin-interacting protein and regulates Zeb1 protein signaling and beta cell. J Biol Chem 2014; 289: 36275-36283
• 20 Hohmeier HE, Mulder H, Chen G. et al. Isolation of INS-1-derived cell lines with robust ATP-sensitive K+channel-dependent and -independent glucose-stimulated insulin secretion. Diabetes 2000; 49: 424-430
• 21 Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest 2009; 119: 1420-1428
• 22 Jones MC, Zha J, Humphries MJ. Connections between the cell cycle, cell adhesion and the cytoskeleton. Philos Trans R Soc B Biol Sci 2019; 374: 1-10
• 23 Reshetnikova G, Barkan R, Popov B. et al. Disruption of the actin cytoskeleton leads to inhibition of mitogen-induced cyclin E expression, Cdk2 phosphorylation, and nuclear accumulation of the retinoblastoma protein-related p107 protein. Exp Cell Res 2000; 259: 35-53
• 24 Heng YW, Koh CG. Actin cytoskeleton dynamics and the cell division cycle. Int J Biochem Cell Biol 2010; 42: 1622-1633
• 25 Akagi T, Kuure S, Uranishi K. et al. ETS-related transcription factors Etv4 and Etv5 are involved in proliferation and induction of differentiationassociated genes in embryonic stem (ES) cells. J Biol Chem 2015; 290: 22460-22473
• 26 Llauradó M, Abal M, Castellví J. et al. ETV5 transcription factor is overexpressed in ovarian cancer and regulates cell adhesion in ovarian cancer cells. Int J Cancer 2012; 130: 1532-1543
• 27 Cheng X, Jin Z, Ji X. et al. ETS variant 5 promotes colorectal cancer angiogenesis by targeting platelet-derived growth factor BB. Int J Cancer 2019; 145: 179-191
• 28 Quinault A, Gausseres B, Bailbe D. et al. Disrupted dynamics of F-actin and insulin granule fusion in INS-1 832/13 beta-cells exposed to glucotoxicity: partial restoration by glucagon-like peptide 1. Biochim Biophys Acta Mol Basis Dis 2016; 1862: 1401-1411
• 29 Li D, Shen Y, Ren H. et al. ETV5 transcriptionally activates TGFβ1 and promotes cancer cell invasion and migration of NSCLC. J Mol Histol 2023; 54: 419-426
• 30 Arase M, Tamura Y, Kawasaki N. et al. Dynamics of chromatin accessibility during TGF-β-induced EMT of Ras-transformed mammary gland epithelial cells. Sci Rep 2017; 7: 1166
• 31 Moin ASM, Sathyapalan T, Atkin SL. et al. Diagnostic and prognostic protein biomarkers of β-cell function in type 2 diabetes and their modulation with glucose normalization. Metabolites 2022; 12: 196
• 32 Dahl U, Sjødin A, Semb H. Cadherins regulate aggregation of pancreatic β-cells in vivo. Development 1996; 122: 2895-2902
• 33 Wakae-Takada N, Xuan S, Watanabe K. et al. Molecular basis for the regulation of islet beta cell mass in mice: the role of E-cadherin. Diabetologia 2013; 56: 856-866
• 34 Katoch A, Jamwal VL, Faheem MM. et al. Overlapping targets exist between the Par-4 and miR-200c axis which regulate EMT and proliferation of pancreatic cancer cells. Transl Oncol 2012; 14: 100879
• 35 Aiello NM, Kang Y. Context-dependent EMT programs in cancer metastasis. J Exp Med 2019; 216: 1016-1026