Mechanism of epithelial‑mesenchymal transition inhibited by miR‑203 in non‑small cell lung cancer

Huang,Weicong; Wu,Yuanbo; Cheng,Dezhi; He,Zhifeng

doi:10.3892/or.2019.7433

Mechanism of epithelial‑mesenchymal transition inhibited by miR‑203 in non‑small cell lung cancer

Authors:
- Weicong Huang
- Yuanbo Wu
- Dezhi Cheng
- Zhifeng He
View Affiliations

Affiliations: Department of Cardiothoracic Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China, Department of Thoracic Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China
Published online on: December 13, 2019 https://doi.org/10.3892/or.2019.7433
Pages: 437-446
Copyright: © Huang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The aim of the present study was to investigate whether miR‑203 can inhibit transforming growth factor‑β (TGF‑β)‑induced epithelial‑mesenchymal transition (EMT), and the migration and invasion ability of non‑small cell lung cancer (NSCLC) cells by targeting SMAD3. In the present study, the expression levels of miR‑203, SMAD3 mRNA and protein in NSCLC tissues were examined, as well as their corresponding paracancerous samples. The miR‑203 mimics and miR‑203 inhibitor were transfected into the H226 cell line. RT‑qPCR was used to assess the expression levels of E‑cadherin, Snail, N‑cadherin and vimentin mRNA, and western blotting was performed to detect the expression levels of p‑SMAD2, SMAD2, p‑SMAD3, SMAD3 and SMAD4. The cell migration and invasion abilities were detected by Transwell assays. The target site of SMAD3 was predicted by the combined action between miR‑203 and dual luciferase. The results revealed that the RNA levels of miR‑203, compared with paracancerous tissues, were decreased in NSCLC tissues, while SMAD3 mRNA and protein levels were upregulated, and miR‑203 inhibited SMAD3 expression. Induction of TGF‑β led to decreased E‑cadherin mRNA levels, upregulation of Snail, N‑cadherin and vimentin mRNA levels (P<0.05), and significant increase in cell migration and invasion, whereas transfection of miR‑203 mimics reversed the aforementioned results (P<0.05). Conversely, miR‑203 inhibitor could further aggravate the aforementioned results (P<0.05). Western blot results revealed that transfection of miR‑203 mimics significantly reduced the protein expression of SMAD3 and p‑SMAD3 (P<0.05). Furthermore, the results of the Dual‑Luciferase assay revealed that miR‑203 inhibited SMAD3 expression by interacting with specific regions of its 3'‑UTR. Overall, a novel mechanism is revealed, in which, miR‑203 can inhibit SMAD3 by interacting with specific regions of the 3'‑UTR of SMAD3, thereby restraining TGF‑β‑induced EMT progression and migration and invasion of NSCLC cells.

View Figures

View References

1	Jemal A, Bray F, Center MM, Ferlay J, Ward E and Forman D: Global cancer statistics. CA Cancer J Clin. 61:69–90. 2011. View Article : Google Scholar : PubMed/NCBI
2	Siegel RL, Miller KD, Fedewa SA, Ahnen DJ, Meester RGS, Barzi A and Jemal A: Colorectal cancer statistics, 2017. CA Cancer J Clin. 67:177–193. 2017. View Article : Google Scholar : PubMed/NCBI
3	Chen W, Zhang S and Zou X: Estimation and projection of lung cancer incidence and mortality in China. Zhongguo Fei Ai Za Zhi. 13:488–493. 2010.(In Chinese). PubMed/NCBI
4	Robinson KW and Sandler AB: The role of MET receptor tyrosine kinase in non-small cell lung cancer and clinical development of targeted anti-MET agents. Oncologist. 18:115–122. 2013. View Article : Google Scholar : PubMed/NCBI
5	Rosell R and Karachaliou N: Lung cancer: Maintenance therapy and precision medicine in NSCLC. Nat Rev Clin Oncol. 10:549–550. 2013. View Article : Google Scholar : PubMed/NCBI
6	Verdecchia A, Francisci S, Brenner H, Gatta G, Micheli A, Mangone L and Kunkler I; EUROCARE-4 Working Group, : Recent cancer survival in Europe: A 2000-02 period analysis of EUROCARE-4 data. Lancet Oncol. 8:784–796. 2007. View Article : Google Scholar : PubMed/NCBI
7	Molina JR, Yang P, Cassivi SD, Schild SE and Adjei AA: Non-small cell lung cancer: Epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc. 83:584–594. 2008. View Article : Google Scholar : PubMed/NCBI
8	Gal A, Sjöblom T, Fedorova L, Imreh S, Beug H and Moustakas A: Sustained TGF beta exposure suppresses Smad and non-Smad signalling in mammary epithelial cells, leading to EMT and inhibition of growth arrest and apoptosis. Oncogene. 27:1218–1230. 2008. View Article : Google Scholar : PubMed/NCBI
9	Ding X, Park SI, McCauley LK and Wang CY: Signaling between transforming growth factor β (TGF-β) and transcription factor SNAI2 represses expression of microRNA miR-203 to promote epithelial-mesenchymal transition and tumor metastasis. J Biol Chem. 288:10241–10253. 2013. View Article : Google Scholar : PubMed/NCBI
10	Yang J and Weinberg RA: Epithelial-mesenchymal transition: At the crossroads of development and tumor metastasis. Dev Cell. 14:818–829. 2008. View Article : Google Scholar : PubMed/NCBI
11	De Craene B and Berx G: Regulatory networks defining EMT during cancer initiation and progression. Nat Rev Cancer. 13:97–110. 2013. View Article : Google Scholar : PubMed/NCBI
12	Moustakas A and Heldin CH: Signaling networks guiding epithelial-mesenchymal transitions during embryogenesis and cancer progression. Cancer Sci. 98:1512–1520. 2007. View Article : Google Scholar : PubMed/NCBI
13	Zhang HJ, Wang HY, Zhang HT, Su JM, Zhu J, Wang HB, Zhou WY, Zhang H, Zhao MC, Zhang L and Chen XF: Transforming growth factor-β1 promotes lung adenocarcinoma invasion and metastasis by epithelial-to-mesenchymal transition. Mol Cell Biochem. 355:309–314. 2011. View Article : Google Scholar : PubMed/NCBI
14	Yang H, Wang L, Zhao J, Chen Y, Lei Z, Liu X, Xia W, Guo L and Zhang HT: TGF-β-activated SMAD3/4 complex transcriptionally upregulates N-cadherin expression in non-small cell lung cancer. Lung Cancer. 87:249–257. 2015. View Article : Google Scholar : PubMed/NCBI
15	Millet C and Zhang YE: Roles of Smad3 in TGF-beta signaling during carcinogenesis. Crit Rev Eukaryot Gene Expr. 17:281–293. 2007. View Article : Google Scholar : PubMed/NCBI
16	Attisano L and Wrana JL: Signal transduction by the TGF-beta superfamily. Science. 296:1646–1647. 2002. View Article : Google Scholar : PubMed/NCBI
17	Yang G and Yang X: Smad4-mediated TGF-beta signaling in tumorigenesis. Int J Biol Sci. 6:1–8. 2010. View Article : Google Scholar : PubMed/NCBI
18	Levy L and Hill CS: Alterations in components of the TGF-beta superfamily signaling pathways in human cancer. Cytokine Growth Factor Rev. 17:41–58. 2006. View Article : Google Scholar : PubMed/NCBI
19	Roberts AB, Tian F, Byfield SD, Stuelten C, Ooshima A, Saika S and Flanders KC: Smad3 is key to TGF-beta-mediated epithelial-to-mesenchymal transition, fibrosis, tumor suppression and metastasis. Cytokine Growth Factor Rev. 17:19–27. 2006. View Article : Google Scholar : PubMed/NCBI
20	Xue J, Lin X, Chiu WT, Chen YH, Yu G, Liu M, Feng XH, Sawaya R, Medema RH, Hung MC and Huang S: Sustained activation of SMAD3/SMAD4 by FOXM1 promotes TGF-β-dependent cancer metastasis. J Clin Invest. 124:564–579. 2014. View Article : Google Scholar : PubMed/NCBI
21	Vincent T, Neve EP, Johnson JR, Kukalev A, Rojo F, Albanell J, Pietras K, Virtanen I, Philipson L, Leopold PL, et al: A SNAIL1-SMAD3/4 transcriptional repressor complex promotes TGF-beta mediated epithelial-mesenchymal transition. Nat Cell Biol. 11:943–950. 2009. View Article : Google Scholar : PubMed/NCBI
22	Huang H, Sun P, Lei Z, Li M, Wang Y, Zhang HT and Liu J: miR-145 inhibits invasion and metastasis by directly targeting Smad3 in nasopharyngeal cancer. Tumour Biol. 36:4123–4131. 2015. View Article : Google Scholar : PubMed/NCBI
23	Zhao W, Zou J, Wang B, Fan P, Mao J, Li J, Liu H, Xiao J, Ma W, Wang M, et al: microRNA-140 suppresses the migration and invasion of colorectal cancer cells through targeting Smad3. Zhonghua Zhong Liu Za Zhi. 36:739–745. 2014.(In Chinese). PubMed/NCBI
24	Huang S, Wu S, Ding J, Lin J, Wei L, Gu J and He X: MicroRNA-181a modulates gene expression of zinc finger family members by directly targeting their coding regions. Nucleic Acids Res. 38:7211–7218. 2010. View Article : Google Scholar : PubMed/NCBI
25	Chen K and Rajewsky N: Natural selection on human microRNA binding sites inferred from SNP data. Nat Genet. 38:1452–1456. 2006. View Article : Google Scholar : PubMed/NCBI
26	Wang C, Wang X, Liang H, Wang T, Yan X, Cao M, Wang N, Zhang S, Zen K, Zhang C and Chen X: miR-203 inhibits cell proliferation and migration of lung cancer cells by targeting PKCα. PLoS One. 8:e739852013. View Article : Google Scholar : PubMed/NCBI
27	Schneider MR: MicroRNAs as novel players in skin development, homeostasis and disease. Br J Dermatol. 166:22–28. 2012. View Article : Google Scholar : PubMed/NCBI
28	Butz H, Rácz K, Hunyady L and Patócs A: Crosstalk between TGF-β signaling and the microRNA machinery. Trends Pharmacol Sci. 33:382–393. 2012. View Article : Google Scholar : PubMed/NCBI
29	Chen T, Xu C, Chen J, Ding C, Xu Z, Li C and Zhao J: MicroRNA-203 inhibits cellular proliferation and invasion by targeting Bmi1 in non-small cell lung cancer. Oncol Lett. 9:2639–2646. 2015. View Article : Google Scholar : PubMed/NCBI
30	Wang N, Liang H, Zhou Y, Wang C, Zhang S, Pan Y, Wang Y, Yan X, Zhang J, Zhang CY, et al: miR-203 suppresses the proliferation and migration and promotes the apoptosis of lung cancer cells by targeting SRC. PLoS One. 9:e1055702014. View Article : Google Scholar : PubMed/NCBI
31	Dickie LJ, Aziz AM, Savic S, Lucherini OM, Cantarini L, Geiler J, Wong CH, Coughlan R, Lane T, Lachmann HJ, et al: Involvement of X-box binding protein 1 and reactive oxygen species pathways in the pathogenesis of tumour necrosis factor receptor-associated periodic syndrome. Ann Rheum Dis. 71:2035–2043. 2012. View Article : Google Scholar : PubMed/NCBI
32	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
33	Yauch RL, Januario T, Eberhard DA, Cavet G, Zhu W, Fu L, Pham TQ, Soriano R, Stinson J, Seshagiri S, et al: Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin Cancer Res. 11:8686–8698. 2005. View Article : Google Scholar : PubMed/NCBI
34	Shintani Y, Okimura A, Sato K, Nakagiri T, Kadota Y, Inoue M, Sawabata N, Minami M, Ikeda N, Kawahara K, et al: Epithelial to mesenchymal transition is a determinant of sensitivity to chemoradiotherapy in non-small cell lung cancer. Ann Thorac Surg. 92:1794–1804. 2011. View Article : Google Scholar : PubMed/NCBI
35	Jung JW, Hwang SY, Hwang JS, Oh ES, Park S and Han IO: Ionising radiation induces changes associated with epithelial-mesenchymal transdifferentiation and increased cell motility of A549 lung epithelial cells. Eur J Cancer. 43:1214–1224. 2007. View Article : Google Scholar : PubMed/NCBI
36	Zhou YC, Liu JY, Li J, Zhang J, Xu YQ, Zhang HW, Qiu LB, Ding GR, Su XM, Mei-Shi and Guo GZ: Ionizing radiation promotes migration and invasion of cancer cells through transforming growth factor-beta-mediated epithelial-mesenchymal transition. Int J Radiat Oncol Biol Phys. 81:1530–1537. 2011. View Article : Google Scholar : PubMed/NCBI
37	Theys J, Jutten B, Habets R, Paesmans K, Groot AJ, Lambin P, Wouters BG, Lammering G and Vooijs M: E-Cadherin loss associated with EMT promotes radioresistance in human tumor cells. Radiother Oncol. 99:392–397. 2011. View Article : Google Scholar : PubMed/NCBI
38	Huber MA, Kraut N and Beug H: Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr Opin Cell Biol. 17:548–558. 2005. View Article : Google Scholar : PubMed/NCBI
39	Zeisberg M and Neilson EG: Biomarkers for epithelial-mesenchymal transitions. J Clin Invest. 119:1429–1437. 2009. View Article : Google Scholar : PubMed/NCBI
40	Derynck R and Akhurst RJ: Differentiation plasticity regulated by TGF-beta family proteins in development and disease. Nat Cell Biol. 9:1000–1004. 2007. View Article : Google Scholar : PubMed/NCBI
41	Thiery JP: Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol. 15:740–746. 2003. View Article : Google Scholar : PubMed/NCBI
42	Wang L, Yang H, Lei Z, Zhao J, Chen Y, Chen P, Li C, Zeng Y, Liu Z, Liu X and Zhang HT: Repression of TIF1γ by SOX2 promotes TGF-β-induced epithelial-mesenchymal transition in non-small-cell lung cancer. Oncogene. 35:867–877. 2016. View Article : Google Scholar : PubMed/NCBI
43	Cho HJ, Baek KE, Saika S, Jeong MJ and Yoo J: Snail is required for transforming growth factor-beta-induced epithelial-mesenchymal transition by activating PI3 kinase/Akt signal pathway. Biochem Biophys Res Commun. 353:337–343. 2007. View Article : Google Scholar : PubMed/NCBI
44	Shintani Y, Maeda M, Chaika N, Johnson KR and Wheelock MJ: Collagen I promotes epithelial-to-mesenchymal transition in lung cancer cells via transforming growth factor-beta signaling. Am J Respir Cell Mol Biol. 38:95–104. 2008. View Article : Google Scholar : PubMed/NCBI
45	Bruna A, Darken RS, Rojo F, Ocaña A, Peñuelas S, Arias A, Paris R, Tortosa A, Mora J, Baselga J and Seoane J: High TGFbeta-Smad activity confers poor prognosis in glioma patients and promotes cell proliferation depending on the methylation of the PDGF-B gene. Cancer Cell. 11:147–160. 2007. View Article : Google Scholar : PubMed/NCBI
46	Liu RY, Zeng Y, Lei Z, Wang L, Yang H, Liu Z, Zhao J and Zhang HT: JAK/STAT3 signaling is required for TGF-β-induced epithelial-mesenchymal transition in lung cancer cells. Int J Oncol. 44:1643–1651. 2014. View Article : Google Scholar : PubMed/NCBI
47	Wang C, Song X, Li Y, Han F, Gao S, Wang X, Xie S and Lv C: Low-dose paclitaxel ameliorates pulmonary fibrosis by suppressing TGF-β1/Smad3 pathway via miR-140 upregulation. PLoS One. 8:e707252013. View Article : Google Scholar : PubMed/NCBI
48	Cao H, Yang CS and Rana TM: Evolutionary emergence of microRNAs in human embryonic stem cells. PLoS One. 3:e28202008. View Article : Google Scholar : PubMed/NCBI
49	Krichevsky AM, King KS, Donahue CP, Khrapko K and Kosik KS: A microRNA array reveals extensive regulation of microRNAs during brain development. RNA. 9:1274–1281. 2003. View Article : Google Scholar : PubMed/NCBI
50	Nakayama K, Nakayama N, Katagiri H and Miyazaki K: Mechanisms of ovarian cancer metastasis: Biochemical pathways. Int J Mol Sci. 13:11705–11717. 2012. View Article : Google Scholar : PubMed/NCBI
51	Cuesta R, Martínez-Sánchez A and Gebauer F: miR-181a regulates cap-dependent translation of p27(kip1) mRNA in myeloid cells. Mol Cell Biol. 29:2841–2851. 2009. View Article : Google Scholar : PubMed/NCBI
52	Dai Y, Huang YS, Tang M, Lv TY, Hu CX, Tan YH, Xu ZM and Yin YB: Microarray analysis of microRNA expression in peripheral blood cells of systemic lupus erythematosus patients. Lupus. 16:939–946. 2007. View Article : Google Scholar : PubMed/NCBI
53	Abella V, Valladares M, Rodriguez T, Haz M, Blanco M, Tarrío N, Iglesias P, Aparicio LA and Figueroa A: miR-203 regulates cell proliferation through its influence on Hakai expression. PLoS One. 7:e525682012. View Article : Google Scholar : PubMed/NCBI
54	Zhou Y, Liang H, Liao Z, Wang Y, Hu X, Chen X, Xu L and Hu Z: miR-203 enhances let-7 biogenesis by targeting LIN28B to suppress tumor growth in lung cancer. Sci Rep. 7:426802017. View Article : Google Scholar : PubMed/NCBI
55	Yang Y, Liu L, Cai J, Wu J, Guan H, Zhu X, Yuan J, Chen S and Li M: Targeting Smad2 and Smad3 by miR-136 suppresses metastasis-associated traits of lung adenocarcinoma cells. Oncol Res. 21:345–352. 2013. View Article : Google Scholar : PubMed/NCBI
56	Wang C, Liu P, Wu H, Cui P, Li Y, Liu Y, Liu Z and Gou S: MicroRNA-323-3p inhibits cell invasion and metastasis in pancreatic ductal adenocarcinoma via direct suppression of SMAD2 and SMAD3. Oncotarget. 7:14912–14924. 2016.PubMed/NCBI