MiR-145 and miR-203 represses TGF-β-induced epithelial-mesenchymal transition and invasion by inhibiting SMAD3 in non-small cell lung cancer cells
Introduction
Non-small cell lung cancer (NSCLC) accounts for about 80% of all types of lung cancer, which is the primary cause of cancer-related deaths worldwide [1]. Despite the advance in the medical conditions, the prognosis and therapy of NSCLC have not yet been remarkably improved over several decades [2]. Most NSCLC patients have been in an advanced stage when diagnosed, that is to say, tumor cells have already owned invasive and metastatic character.
Epithelial-mesenchymal transition (EMT) has bcecome the focus of attention in recent years, which plays an important role not only in embryonic development but also in tumor cell invasion and metastasis [3]. In the process of EMT, epithelial cells lose cell–cell junctions, polarity and epithelial markers, along with gain of mesenchymal markers and motility and invasive phenotype, which may trigger tumor metastasis [4]. Extracellular stimulus, such as TGF-β1, may induce EMT through activating kinase-dependent signaling cascades [5]. Moreover, inhibition of TGF-β1 or its receptors may reduce the capacity of tumor metastasis and/or invasion [5]. TGF-β-mediated EMT is characterized by marker changes, such as repression of E-cadherin and induction of N-cadherin [6].
Excessive TGF-β has been reported to be related to poor prognosis in cancer patients [7]. As a key intracellular mediator of TGF-β signaling, SMAD3 is closely associated with tumorigenesis and prognosis of multiple human cancers [8]. Moreover, SMAD3 is very important for TGF-β-mediated EMT, tumor suppression and metastasis [9]. Several studies showed that SMAD3 can accelerate tumor cell invasion and metastasis via SMAD3-mediated EMT [10], [11]. In our previous study, knockdown of SMAD3 inhibited tumor cell migration and invasion in nasopharyngeal carcinoma [12] and reduced expression of N-cadherin was observed in SMAD3-silenced NSCLC cells when treated with TGF-β [6]. These data demonstrate that SMAD3 is required for TGF-β-mediated EMT. However, the miRNA-based mechanism underlying SMAD3 regulation in TGF-β-induced EMT is poorly understood.
MicroRNAs (miRNAs) are a family of 19–24 nucleotides small non-coding RNA molecules, which post-transcriptionally regulate gene expression by targeting mRNAs and lead to translational repression and/or degradation of target mRNAs [13], [14], [15]. Thus, miRNAs have a broad impact on several fundamental biological processes such as cell development, differentiation and proliferation [16]. Of more importance, abnormal expression of miRNAs is involved in tumorigenesis, progression and metastasis [17], [18], [19], [20]. Our previous studies showed that miR-203 and miR-145 inhibited cellular invasion in NSCLC and nasopharyngeal cancer, respectively [12], [21]. Ding et al. reported that miR-203 played an important role in TGF-β-induced EMT and miR-203 was down-regulated in high-metastatic breast cancer cells [22]. Hu et al. found that miR-145 repressed the EMT process of lung adenocarcinoma-initiating cells by targeting Oct4 [23]. Moreover, miR-203 and miR-145 were frequently reduced in NSCLC [21], [24], [25]. These findings suggest that miR-203 or miR-145 may act as a metastasis suppressor in NSCLC.
Taken together, we hypothesized that miR-203 and/or miR-145 could affect TGF-β-induced EMT and invasion by regulating SMAD3 in NSCLC cells. We are encouraged to test this because the 3′-untranslated region (3′-UTR) of SMAD3 is in silico predicted as a target of miR-203/or miR-145. In this study, we provided the first evidence that miR-145 and miR-203 inhibited TGF-β-induced EMT and invasion through repression of SMAD3 in NSCLC cells.
Section snippets
Cell culture
HBE (Human bronchial epithelial) cells from ScienCell Research Laboratories (Carlsbad, CA, USA) and human NSCLC cell lines A549, H460, H1299 (lung adenocarcinoma cell lines), 95C (low-metastatic giant-cell lung carcinoma cell line), and 95D (high-metastatic giant-cell lung cell line) from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were cultured in RPMI-1640 (Gibco, Grand Island, NY, USA) medium containing 10% fetal bovine serum (FBS) (Gibco) in a humidified sterile
SMAD3 expression is up-regulated in NSCLC tissues and cell lines
To examine the expression of SMAD3 in NSCLC tissues and cells, a qRT-PCR assay was conducted. As shown in Fig. 1A, the expression of SMAD3 mRNA was significantly higher in NSCLC tissues than matched noncancerous lung tissues. The comparable results were obtained in five NSCLC cell lines (H460, 95D, A549, 95C and H1299), which showed remarkably higher expression of SMAD3 mRNA and protein compared with HBE cells (Fig. 1B and C).
Expression of miR-145 and miR-203 is decreased in NSCLC tissues and cell lines
MiR-203 and miR-145 were suggested to act as tumor suppressors in
Discussion
Metastasis is a common cause of tumor deterioration in many solid tumors, including NSCLC [29]. TGF-β-induced EMT plays an important role in NSCLC cell invasion [27]. Moreover, SMAD3 is crucial to TGF-β-mediated EMT and tumor metastasis [9]. In the present study, we investigate the miRNA-based mechanism underlying SMAD3 regulation in TGF-β-induced EMT and invasion in NSCLC. Our findings indicate that miR-145 and miR-203 repress TGF-β-induced EMT and invasion by targeting SMAD3 in NSCLC cells.
Conflict of interest
No declared.
Acknowledgments
We want to thank all the NSCLC patients for their participation and cooperation. This work was supported in part by the grants from the National Natural Science Foundation of China (81372277 to Hong-Tao Zhang, and 81502498 to Zhe Lei), the Jiangsu Province’s Key Provincial Talents Program (RC2011106 to Jun Zhao), the Science and Technology Committee of Jiangsu Province (BK20131159 to Jun Zhao), Natural Science Research Foundation of the Jiangsu Higher Education Institutions of China (14KJB320012
References (37)
- et al.
Recent cancer survival in Europe: a 2000-02 period analysis of EUROCARE-4 data
Lancet Oncol.
(2007) - et al.
Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis
Dev. Cell
(2008) - et al.
TGF-beta-activated SMAD3/4 complex transcriptionally upregulates N-cadherin expression in non-small cell lung cancer
Lung Cancer
(2015) - et al.
Alterations in components of the TGF-beta superfamily signaling pathways in human cancer
Cytokine Growth Factor Rev.
(2006) - et al.
Smad3 is key to TGF-beta-mediated epithelial-to-mesenchymal transition, fibrosis, tumor suppression and metastasis
Cytokine Growth Factor Rev.
(2006) - et al.
Signaling between transforming growth factor beta (TGF-beta) and transcription factor SNAI2 represses expression of microRNA miR-203 to promote epithelial-mesenchymal transition and tumor metastasis
J. Biol. Chem.
(2013) - et al.
Cancer metastasis: building a framework
Cell
(2006) Epithelial-mesenchymal transitions in development and pathologies
Curr. Opin. Cell Biol.
(2003)- et al.
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
(2007) - et al.
MicroRNA-486 as a biomarker for early diagnosis and recurrence of non-Small cell lung cancer
PLoS One
(2015)
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
TGF-beta and epithelial-to-mesenchymal transitions
Oncogene
Roles of Smad3 in TGF-beta signaling during carcinogenesis
Crit. Rev. Eukaryot. Gene Expr.
A SNAIL1-SMAD3/4 transcriptional repressor complex promotes TGF-beta mediated epithelial-mesenchymal transition
Nat. Cell Biol.
Sustained activation of SMAD3/SMAD4 by FOXM1 promotes TGF-beta-dependent cancer metastasis
J. Clin. Invest.
miR-145 inhibits invasion and metastasis by directly targeting Smad3 in nasopharyngeal cancer
Tumour Biol.
Mammalian microRNAs predominantly act to decrease target mRNA levels
Nature
Molecular mechanisms of RNA interference
Annu. Rev. Biophys.
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These authors contributed equally to this work.