Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C

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Cancer Letters xxx (2017) 1e13

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Original Article

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Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C Tzu-Hua Chang a, 1, Meng-Feng Tsai b, 1, Chien-Hung Gow a, c, d, 1, Shang-Gin Wu a, e, Yi-Nan Liu a, Yih-Leong Chang f, Sung-Liang Yu g, Hsing-Chen Tsai a, h, Shih-Wen Lin i, Yen-Wei Chen g, Po-Yen Kuo j, Pan-Chyr Yang a, Jin-Yuan Shih a, * a

Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University, Taipei 10002, Taiwan Department of Molecular Biotechnology, Da-Yeh University, Changhua 51591, Taiwan c Department of Internal Medicine, Far Eastern Memorial Hospital, New Taipei City 22060, Taiwan d Department of Healthcare Information and Management, Ming-Chuan University, Taoyuan 33348, Taiwan e Graduate Institute of Clinical Medicine, National Taiwan University, Taipei 10002, Taiwan f Department of Pathology, National Taiwan University Hospital, National Taiwan University, Taipei 10002, Taiwan g Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University, Taipei 10002, Taiwan h Graduate Institute of Toxicology, National Taiwan University, Taipei 10002, Taiwan i Graduate Institute of Oncology, National Taiwan University, Taipei 10002, Taiwan j Department of Pathology, National Taiwan University Hospital Yun-Lin Branch, Yun-Lin 64041, Taiwan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 August 2016 Received in revised form 29 April 2017 Accepted 3 June 2017

The epithelial-mesenchymal transition (EMT) regulator, Slug, plays multifaceted roles in controlling lung cancer progression, but its downstream targets and mechanisms in promoting lung cancer progression have not been well defined. In particular, the miRNAs downstream of Slug in non-small cell lung cancer (NSCLC) remain undetermined. Here, we report that miR-137 is downstream of the EMT regulator, Slug, in lung cancer cells. Slug binds directly to the E-box of the miR-137 promoter and up-regulates its expression in lung cancer cells. Knockdown of miR-137 abolished Slug-induced cancer invasion and migration, whereas upregulation of miR-137 was found to trigger lung cancer cell invasion and progression by direct suppressing TFAP2C (transcription factor AP-2 gamma). Clinical data showed that lung adenocarcinoma patients with low-level expression of Slug and miR-137 but high-level expression of TFAP2C experienced significantly better survival. miR-137 is a Slug-induced miRNA that relays the prometastatic effects of Slug by targeting TFAP2C. Our findings add new components to the Slug-mediated regulatory network in lung cancer, and suggest that Slug, miR-137, and TFAP2C may be useful prognostic markers in lung adenocarcinoma. © 2017 Elsevier B.V. All rights reserved.

Keywords: Non-small cell lung cancer (NSCLC) Epithelial-mesenchymal transition (EMT) Slug miR-137 TFAP2C

Introduction The epithelial-mesenchymal transition (EMT) is an essential developmental process whereby epithelial cells lose their epithelial characteristics and acquire a migratory, mesenchymal-like

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* Corresponding author. Department of Internal Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, 7 Chung-Shan South Road, Taipei 10002, Taiwan. E-mail address: [email protected] (J.-Y. Shih). 1 These authors contributed equally to this work.

phenotype [1]. Accumulating evidence shows that EMT plays important roles not only in embryonic development, but also in tissue regeneration and cancer progression [1e3]. The EMT phenotype is tightly regulated by EMT transcriptional regulators, such as Slug (also known as Snail2), Snail, Twist, Zeb1, and Zeb2 [4], most of which have been suggested as potential diagnostic markers and therapeutic targets for human cancers [5e9]. Slug is a member of the SNAIL family of zinc finger transcription factors. It was initially identified as mediating the formation of the mesoderm during gastrulation and the migration of neural crest cells during embryonic development [10]. Subsequently identified as an EMT regulator, Slug has recently been shown to be associated

http://dx.doi.org/10.1016/j.canlet.2017.06.002 0304-3835/© 2017 Elsevier B.V. All rights reserved.

Please cite this article in press as: T.-H. Chang, et al., Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.06.002

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with tumor metastasis and recurrence in a variety of cancers [5,11e13]. In lung cancer cells, elevated expression of Slug has been reported to enhance angiogenesis and cause the cells to acquire traits reminiscent of those expressed by stem cells [5,14e16]. Slug also contributes to the ability of lung cancer cells to resist chemotherapeutic drugs and tyrosine kinase inhibitors [17,18]. However, the downstream targets and action mechanisms through which Slug promotes lung cancer progression remain largely unknown. MicroRNAs (miRNAs) are small noncoding single-stranded RNAs (~22 nt) that can function as an endogenous means of RNA interference [19]. These actions play critical roles in various essential biological processes, including metabolism, development, proliferation, differentiation, and apoptosis [20,21]. Accumulating evidence indicates that miRNAs are grossly dysregulated in lung cancer, and may serve as oncogenes or tumor suppressors. miRNAs can be used to sub-classify non-small cell lung cancers (NSCLCs), and specific miRNA profiles may predict the prognosis and/or recurrence of the disease [22e25]. Similar to the case of proteinencoding genes, the transcription of miRNAs can be activated or repressed by transcription factors, which interact with their conventional binding sites in polymerase II- or III-dependent manners [26]. However, although Slug is a transcription factor, and it is known to have multiple functions in lung cancer progression, no previous study has examined a possible the link between Slug and downstream miRNAs in lung cancer cells. In the present study, we used Illumina bead arrays and TaqMan low-density arrays (TLDAs) to identify miRNAs downstream of Slug in lung cancer cells. We demonstrate that miR-137 is a Sluginduced miRNA, and that the Slug-induced upregulation of miR137 plays important roles in controlling lung cancer progression by targeting and downregulating TFAP2C (transcription factor AP-2 gamma). Our novel findings suggest that the Slug-miR-137-TFAP2C axis may offer new candidate target molecules for lung cancer therapeutics.

We first used Illumina bead arrays and TLDAs to identify miRNAs that exhibited differential expression in Slugoverexpressing versus control lung cancer cells (Supplementary Methods, Table S1 and Table S2). The Illumina bead array data are available from Gene Expression Omnibus (GEO) under accession number GSE84040. These analyses identified 14 wellannotated miRNAs that were significantly altered (13 upregulated and 1 downregulated) in Slug-overexpressing cells (Supplementary Fig. S1). Of them, miR-137 was increased by ~10.0- and 49.2-fold according to the Illumina bead arrays and TLDAs, respectively (Supplementary Fig. S1). This was confirmed by poly-A tailing real-time RT-PCR, which showed that miR-137 was expressed at significantly higher levels in CL1-0-Slug-4 (14.6 fold) and PC9-Slug (23.0 fold) cells compared to controls (Fig. 1A and B). In addition to miR-137, the expression levels of miR-335, miR-181b, miR-30a-3p, miR-31, and miR-376a were all significantly increased more than 2-fold in CL1-0-Slug4 and PC9-Slug cells compared with mock cells.

Materials and methods

Slug induces miR-137 expression in lung cancer cells

Patients and tissue procurement We collected samples from 143 lung adenocarcinomas surgically resected at the National Taiwan University Hospital (NTUH, Taipei, Taiwan). Prior to surgery, all patients signed informed consent regarding the use of specimens for future molecular research. This study was approved by the Institutional Review Board (IRB) of the NTUH Research Ethics Committee. Refer to the Supplementary Methods. Immunohistochemical (IHC) staining of TFAP2C Refer to the Supplementary Methods. Statistical analysis Refer to the Supplementary Methods.

Results miRNAs downstream of Slug in lung cancer cells

Cell lines Human lung cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the Center of Genomic Medicine, National Taiwan University (Supplementary Methods). Cell lines authentication, cell culture and stable transfections were performed as described in the Supplementary Methods. The cell lines were authenticated using short tandem repeat profiling. Cell culture and stable transfections were performed as described in the Supplementary Methods. Luciferase reporter assays Luciferase activity was measured using a dual-luciferase assay (Promega, Madison, WI, USA), according to the manufacturer's instructions. Refer to the Supplementary Methods. Chromatin immunoprecipitation (ChIP) ChIP assay reagents were obtained from Millipore (Billerica, MA, USA), and assays were performed in accordance with the manufacturer's protocol. The detail procedures and promoter-specific primers are described in the Supplementary Methods. TFAP2C 30 UTR luciferase reporter assay Refer to the Supplementary Methods. Functional in vitro assays Invasion, wound healing, and single-cell mobility were assessed in vitro, as described in the Supplementary Methods. Experimental metastasis in vivo All animal experiments were approved by the Institutional Animal Care and Use Committee of the National Taiwan University. The methods were carried out in accordance with the approved guidelines. Refer to the Supplementary Methods.

Quantitative RT-PCR was used to analyze the expression levels of Slug, E-cadherin, Vimentin, N-cadherin, pri-miR-137 (the long primary miR-137 transcript) and pre-miR-137 (the shorter hairpin precursor miR-137) in HCC827-Slug and PC9/gefsi-Slug cells. As shown in Fig. 1C and D, Vimentin, N-cadherin, pri-miR-137 and pre-miR-137 were upregulated in HCC827-Slug cells compared to controls, whereas E-cadherin was downregulated. In contrast, Vimentin, N-cadherin, pri-miR-137 and pre-miRNA137 were downregulated and E-cadherin was upregulated in PC9/gef-si-Slug cells versus scramble control (PC9/gefsi-Ctrl) cells. We also indicated that Slug expression showed a significant positive correlation with miR-137 expression in various lung cancer cell lines (Pearson correlation, P < 0.05; Supplementary Fig. S2B). Slug is a direct regulator of the miR-137 promoter The gene encoding miR-137 is located at chromosome 1p22, and its promoter contains four putative Slug-binding sites (E-boxes, CANNTG and CACTGT) (Fig. 2A). The first binding site (miR-137-Ebox-1: CATTTG) is located at 207 to 202 region, while the second (miR-137-E-box-2: CACTGT) is located 321 to 316 region upstream of the transcriptional start site (TSS). We generated four miR137-luc reporter constructs with different miR-137 promoter fragments and mutations, including pGL3-miR-137-1129bp, pGL3miR-137-527bp, pGL3-miR-137-527bp-mutE1, and pGL3-miR137-527bp-mutE2 (Fig. 2B and C). Our results indicated that pGL3miR-137-1129bp, pGL3-miR-137-527bp, and pGL3-miR-137-

Please cite this article in press as: T.-H. Chang, et al., Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.06.002

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Fig. 1. The expression of miR-137 is up-regulated by Slug in human lung cancer cells. Slug-transfected lung cancer cells (CL1-0-Slug-4 and PC9-Slug) expressed high levels of Slug protein, while the corresponding control clones (CL1-0-mock and PC9-mock) exhibited low Slug expression, as established previously (Supplementary Methods). (A) Poly-A tailing real-time RTPCR confirmed that the six identified Slug-regulated miRNAs (miR-137, miR-335, miR-181b, miR-30a-3p, miR-31 and miR376a) were up-regulated in CL1-0-Slug-4 cells. (B) The expression of miR-137 in PC9-Slug cells was confirmed by poly-A tailing real-time RT-PCR. (C) The expression levels of Slug, E-cadherin, Vimentin, N-cadherin, pre-miR-137, and pri-miR-137 in HCC827-Slug cells (constitutively overexpressing Slug) and HCC827-mock cells were quantified by real-time RT-PCR (*P < 0.01). (D) The expressions of Slug, E-cadherin, Vimentin, Ncadherin, pre-miR-137, and pri-miR-137 in PC9/gef-si-Slug (with siRNA-mediated knockdown of Slug) and PC9/gef-si-Ctrl (scrambled control siRNA) cells were quantified by real-time RTPCR (*P < 0.01). Data are presented as the means ± SD of three independent experiments. P-values were determined using the Student's t-test.

Please cite this article in press as: T.-H. Chang, et al., Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.06.002

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Fig. 2. Slug increases miR-137 promoter activity in lung cancer cells. To determine whether the upregulation of miR-137 in Slug-overexpressing lung cancer cells is transcriptionally dependent on Slug, we analyzed the miR-137 promoter region spanning nucleotides 1090 to þ39. (A) Schematic representation of the potential Slug binding sites within the putative promoter region of miR-137. The transcription start site (TSS) is indicated by an arrow. Four potential Slug binding sites are represented: E-box-1 (CATTTG, 207 to 202 nt); E-box-2 (CACTGT, 321 to 316 nt); E-box-3 (CAGCTG, 901 to 896 nt); and E-box-4 (CAAATG, 987 to 982 nt). (B) The activity of the miR-137 promoter was examined using a dual-luciferase reporter assay system (*P < 0.01). PC9 cells were transiently co-transfected with mock or Slug expression plasmids (pCIneo or pCIneo-Slug, respectively) plus luciferase reporter constructs containing miR-137 promoter fragments of different lengths, as follows: pGL3-miR-137-1129bp, which contained the miR137 promoter region from þ39 to 1090; and pGL3-miR-137-527bp, which contained the miR137 promoter region from þ39 to 448. (C) Schematic representation of the pGL3-miR137-527bp reporter plasmid, which contained the potential Slug-binding sites, E-box-1 and E-box-2. The cross indicates the location of disrupting mutations introduced in E-box-1 (pGL3-miR-137-527bp-mutE1; CATTTG to TGCGCA) and E-box-2 (pGL3-miR-137-527bp-mutE2; CACTGT to TGTGCA). At 24 h post-co-transfection, luciferase activity was measured with a dual-luciferase reporter assay system (*P < 0.01). (D) Slug induces miR-137 promoter activity by binding to the E-box. ChIP was performed as described in the Materials and methods section. PC9-Slug (left panel) and H1299-Slug (right panel) cells were subjected to formaldehyde crosslinking, and the crosslinked chromatin was sonicated to an average length of 300 bp and immunoprecipitated with a Slug-specific antibody or control IgG. The miR-137 promoter regions E-box-1 to E-box-2 (202 to 321 nt) and E-box-3 to E-box-4 (896 to 987 nt) were PCR amplified using specific primer sets. The ChIP experiment was repeated three times, and similar results were obtained. Luciferase activity is presented as the means ± SD of three independent experiments. P-values were determined using the Student's t-test.

Please cite this article in press as: T.-H. Chang, et al., Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.06.002

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527bp-mutE1 (all of which possess intact miR137 E-box-2 sequences) were efficiently activated by Slug overexpression, which increased their luciferase activities by 2.3-, 2.1-, and 2.4-fold, respectively, compared to that in mock control cells (Fig. 2B and C). In contrast, pGL3-miR137-528bp-mutE2, which harbored an intact E-box-1 but a mutated E-box-2 (CACTGT to TGTGCA), did not significantly respond to the overexpression of Slug in PC9 cells. ChIP experiments performed with an antibody against Slug suggested that Slug can directly bind to the E-box-2 region of the miR-137 promoter in PC9-Slug and H1299-Slug cells (Fig. 2D). These results indicated that Slug can activate the transcription of miR-137 in

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lung cancer cells, primarily through binding to E-box-2 of the miR-137 promoter. miR-137 knockdown abolishes the Slug-induced in vitro invasion and migration abilities of lung cancer cells As shown in Fig. 3, knockdown of miR-137 significantly inhibited the migration and invasion of HCC827-Slug cells. This confirms that miR-137 is a downstream target of Slug, and suggests that this miRNA may play an important role in Slug-induced cancer cell invasion and migration (Figs. 2 and 3).

Fig. 3. Knockdown of miR-137 significantly blocks the Slug-induced migration and invasion of lung cancer cells. (A) Expression of miR-137 in HCC827-Slug cells (infected with pLVhsa-miR-137-locker for knockdown of miR-137) and HCC827-mock cells (infected with pLV-miR-locker as a control) was quantified by real-time RT-PCR (*P < 0.01). Data are presented as the means ± SD of three independent experiments. (B) The invasion ability of HCC827-Slug and HCC827-mock cells was determined using in vitro cell invasion assay (*P < 0.01). Data are presented as the means ± SD of three independent experiments. (C) The rate of cancer cell motility was determined using a single-cell motility assay based on time-lapse imaging (n ¼ 10 cells for each stable cell line). The box and whisker plots display significant differences in the average migration speeds of HCC827-Slug and HCC827mock cells (*P < 0.01). P-values were determined using the Student's t-test.

Please cite this article in press as: T.-H. Chang, et al., Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.06.002

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Fig. 4. miR-137 promotes lung cancer cell invasion and migration, and is positively correlated with poor survival in NSCLC patients. (A) The miR-137 expression levels in 427 lung adenocarcinomas (stage I-III) and 46 adjacent non-tumor lung tissue samples were downloaded from The Cancer Genome Atlas (TCGA) data portal and statistically compared using the Mann-Whitney test (*P < 0.01). We obtained only the data derived from Illumina HiSeq, and used the normalized expression results (represented as reads per million miRNA mapped) for our analyses. (B) miR-137 expression and overall survival in a cohort of 405 lung cancer patients was obtained from TCGA, and survival analysis was performed using Kaplan-Meier survival analyses and log-rank tests (P ¼ 0.0457). (C) miR-137 expression and overall survival was assessed in an independent cohort of 143 lung cancer patients

Please cite this article in press as: T.-H. Chang, et al., Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.06.002

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High-level expression of miR-137 is correlated with shorter overall survival of lung adenocarcinoma patients We next questioned whether Slug-induced miR-137 expression might promote lung cancer progression. To test this hypothesis, we analyzed the correlation between miR-137 expression and lung cancers in the relevant TCGA (The Cancer Genome Atlas) dataset. The level 3 miRNA-seq (473 samples) and the corresponding clinical data were downloaded from the TCGA data portal. Statistically significant differences in miR-137 expression levels between lung adenocarcinoma and adjacent non-tumor lung tissues were calculated by the Mann-Whitney test (P < 0.001; Fig. 4A). The lung cancer samples were then divided into high miR-137 (above the median) and low miR-137 (below the median) groups. Survival data for all subjects were censored at 100 months to exclude potential noncancer related deaths, and the survival differences between the high miR-137 and low miR-137 groups were calculated using the log-rank test (P ¼ 0.0457; Fig. 4B). The clinicopathological characteristics of each group are summarized in Supplementary Table S3. To validate these in silica results, we collected samples from 143 consecutive surgically resected lung adenocarcinomas, and used real-time quantitative RT-PCR to detect miR-137. The clinical and pathological features of the patients and tissues are summarized in Supplementary Table S4. According to the median level of miR-137 expression, we classified the lung cancer patients into high (n ¼ 72) and low (n ¼ 71) miR-137 groups. High-level miR-137 expression was significantly associated with tumor status (P ¼ 0.039; Supplementary Table S4), but miR-137 expression was not associated with other clinicopathological characteristics, including age (P ¼ 0.510), gender (P ¼ 0.054), disease stage (P ¼ 0.243), or lymph node metastasis (P ¼ 0.347). Kaplan-Meier survival analysis and log-rank tests demonstrated that patients with high-level expression of miR-137 had a significantly shorter overall survival (P ¼ 0.005; Fig. 4C). Multivariate Cox proportional hazards regression analysis indicated that the significant prognostic factors for overall survival of lung adenocarcinoma patients included miR-137 expression (hazards ratio, 1.77; 95% confidence interval, 1.13e2.78; P ¼ 0.013) and stage III disease (hazards ratio, 2.31; 95% confidence interval, 1.39e3.85; P ¼ 0.001; Supplementary Table S5). miR-137 expression affects lung cancer cell invasion and migration As shown in Fig. 4D, the expression of miR-137 varied among the tested lung cancer cell lines. We subjected these lower and higher expression groups (three cell lines per group) to overexpression and knockdown of miR-137, respectively (Fig. 4E and I). The invasive activities of miR-137-overexpressing transfectants were significantly higher than those of the mock-transfected controls (P < 0.01; Fig. 4F). The migration ability of HCC827-

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pMIRH137 cells was markedly increased over that of control cells, as assessed using a standard scratch wound-healing assay and a single-cell motility assay (Fig. 4G and H). Cell growth was similar between HCC827-pMIRH137 cells and mock-transfected controls (data not shown). Conversely, the invasion and migration capabilities of miR-137-knockdown (H1299-miR-137-locker) cells were lower than those of the corresponding control cells (Fig. 4I and J). To determine whether knockdown of miR-137 can enhance the cisplatin-mediated inhibition of cell growth and migration ability in H1299 cells. MiR-137- knockdown and control cells (H1299-miR-137-locker and H1299-Ctrl-locker) were treated with various concentrations of cisplatin, and then MTT and transwell migration assays were performed to detect the cell viability and migration ability. These results showed that knockdown of miR-137 can significantly enhance the cisplatin-mediated inhibition of cell growth and migration in lung cancer cells (Fig. 4K and L). miR-137 represses TFAP2C expression by directly targeting the 30 -UTR of its mRNA The putative miR-137 target site within the TFAP2C mRNA was found to be conserved across multiple species (Fig. 5A). TFAP2C protein expression showed a significant negative correlation with miR-137 expression in various lung cancer cell lines (Pearson correlation, P < 0.05; Fig. 5B). In addition, we indicated that TFAP2C protein expression showed a significant negative correlation with Slug expression in these lung cancer cell lines (Person correlation, P < 0.05; Supplementary Fig. S2C). Moreover, TFAP2C protein expression was significantly inhibited and enhanced in miR-137overexpressing and -knockdown cells, respectively (Fig. 5C and D). We cloned a TFAP2C 30 -UTR fragment containing the putative miR-137 targeting site into a luciferase reporter plasmid. The luciferase activity of the generated pMir-TFAP2C-30 UTR-WT was significantly reduced (~45%) in 293T cells co-transfected with an expression vector for miR-137 (P < 0.01; Fig. 5E), whereas miR-137 overexpression did not affect the luciferase activity of pMirTFAP2C-30 -UTR-Mut, in which the miR-137 targeting site was mutated (Fig. 5E). There was no significant difference in TFAP2C mRNA expression in miR137 transfectants compared with control cells (Fig. 5F). Collectively, miR-137 suppresses TFAP2C expression by specifically interacting with a target site in the 30 UTR of its mRNA and triggering translational regulation. miR-137 promotes lung cancer cell invasion and migration by downregulating TFAP2C To further explore the functional relationship between miR-137 and TFAP2C, a lentivirus-based vector (pLKO-TFAP2C) encoding

collected in-house. miR-137 expression was determined by real-time RT-PCR (range from 0.0001 to 2.0002 with a median of miR-137/RNU6B ratio ¼ 0.0294), and defined as “high” (above the median) or “low” (below the median). Kaplan-Meier survival analysis and log-rank tests indicated that patients with high-level expression of miR-137 had a significantly shorter overall survival (median, 40.4 months; 95% confidence interval, 25.3e55.5 months) compared to patients who had cancers with low-level miR-137 expression (median survival, 92.6 months; 95% confidence interval, 78.3e106.9 months; P ¼ 0.005, log-rank test). (D) The expression levels of miR-137 in lung cancer cell lines (PC9, HCC827, CL1-0, PC9/ gef, HCC827/gef, and H1299 cells) were detected by real-time RT-PCR. (E) HCC827, CL1-0, and PC9 cells were infected with a lentivector-based miR-137 overexpression system (pMIRH137-PA-1 plasmid) or mock control, and constitutively miR-137-overexpressing (HCC827-pMIRH137, CL1-0-pMIRH137, and PC9-pMIRH137) and control (HCC827-Ctrl, CL10-Ctrl, and PC9-Ctrl) stable cell lines were established. miR-137 expression was detected using real-time RT-PCR (*P < 0.01). P-values were determined using the Student's t-test. (F) The invasiveness of the cells described in (E) were evaluated by in vitro cell invasion assay. (G) The in vitro wound-healing abilities of the cells described in (E) were assessed as a measure of cancer cell migration ability. Each track was photographed upon wounding, and then at 16 h post-wounding. (H) The motility rates of the cells described in (E) were determined using a single-cell motility assay based on time-lapse imaging (n ¼ 10 cells for each stable cell line). The box and whisker plots display significant differences in the average migration speeds of HCC827-pMIRH137 and HCC827-Ctrl cells (*P < 0.01). P-values were determined using the Student's t-test. (I) H1299 lung cancer cells subjected to lentivector-mediated knockdown of miR-137 (H1299-miR-137-locker) and control cells (H1299-Ctrl-locker) (left panel) were assessed for their invasion properties using in vitro cell invasion assay (right panel). (J) The motility rates of the cells described in (I) were assessed using a single-cell motility assay (n ¼ 10 cells for each cell line). The box and whisker plots display significant differences in the average migration speeds of H1299-miR-137-locker and H1299-Ctrl-locker cells (*P < 0.01). P-values were determined using the Student's t-test. (K) H1299-miR-137-locker and H1299-Ctrl-locker cells were treated with various concentrations of cisplatin for 72 h. MTT assay was used for detection of cellular viability. (L) H1299-miR-137-locker and H1299-Ctrl-locker cells were treated with cisplatin, and transwell migration assay was used for detection of cellular migration ability. (*P < 0.01). P-values were determined using the Student's t-test.

Please cite this article in press as: T.-H. Chang, et al., Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.06.002

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Fig. 5. miR-137 targets the 30 -UTR of the TFAP2C mRNA to repress TFAP2C expression. (A) The target genes of miR-137 were predicted using Target Scan and miRWalk. Schematic representation of the putative miR-137 binding site in the 30 -UTR of the human TFAP2C mRNA (1098e1104 nt). Multiple sequence alignment of the 30 -UTRs of the TFAP2C mRNA

Please cite this article in press as: T.-H. Chang, et al., Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.06.002

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TFAP2C was infected into miR-137-overexpressing transfectants (HCC827-pMIRH137). Our results revealed that the restoration of TFAP2C expression in these cells significantly inhibited their migration and invasion (Fig. 6AeC). These results further confirm that miR-137 enhances lung cancer progression by suppressing TFAP2C. miR-137 promotes lung cancer cell metastasis in vivo We next investigated the effect of miR-137 expression on cancer metastasis in vivo. A mouse tail vein injection metastasis model was performed. H1299 cells with miR-137 locker or control locker were injected into tail veins of SCID mice. Seven weeks after the injection, lung metastases were quantified. The results showed that H1299 cells with miR-137 locker (H1299-miR-137 locker cells) significantly decreased the number of lung metastases by 4.8-fold compared with control transfectants (H1299-Ctrl locker; mean number of lung tumor nodules, 3.2 ± 1.2 for H1299-miR137-locker and 15.2 ± 2.8 for H1299-Ctrl-locker; P < 0.001; Fig. 6D). Hematoxylin and eosin staining of lung samples dissected from the mice were shown in Fig. 6D. As shown in Fig. 6E, the expression level of miR-137 was inversely correlated with the protein level of TFAP2C in lung tumor sections. The signature of low Slug, low miR-137, and high TFAP2C expression in lung adenocarcinoma predicts longer overall survival To further verify the clinical significance of Slug, miR-137, and TFAP2C expression in lung cancer progression, we collected 143 tumor specimens, quantified Slug and miR-137 transcripts with real-time quantitative RT-PCR, and assessed TFAP2C protein expression by immunohistochemistry (Fig. 6F). Kaplan-Meier survival analysis and log-rank tests demonstrated that patients in the SlugLow-miR-137Low-TFAP2CHigh group had longer overall survival than those in the SlugHigh-miR-137High-TFAP2CLow or “other” groups (P ¼ 0.029; Fig. 6G). Multivariate Cox proportional hazards regression analysis performed using a stepwise selection model revealed that the SlugLow, miR-137Low, and TFAP2CHigh profile was an independent predictor in lung adenocarcinoma (hazards ratio, 0.25; 95% confidence interval, 0.08e0.73; P ¼ 0.011) after we controlled for all other prognostic factors. Disease stage was also found to be an independent prognostic factor. Our results therefore suggest that lung adenocarcinoma patients whose tumors were SlugLow, miR-137Low, and TFAP2CHigh exhibited significantly better survival. Discussion We previously reported that elevated mRNA expression of the transcription factor and EMT regulator, Slug, in NSCLC tumor specimens was significantly associated with early post-operative relapse and shorter survival [5]. Consistent with our report,

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another investigation found poorer survival among lung adenocarcinoma patients whose tumors showed positive IHC staining for Slug [27]. In addition to regulating EMT and cancer metastasis, Slug is also temporally regulated during the cell cycle, where it contributes to ensuring accurate cell proliferation and genomic integrity. Slug was recently suggested to promote the invasivity of lung cancer cells by increasing the activity of matrix metalloproteinase2 and suppressing E-cadherin [5]. Silencing of Slug in gefitinibresistant lung cancer cells was shown to restore gefitinib-induced apoptosis, mainly via the upregulation of Bim and the activation of caspase-9 [18]. In addition, Slug has also been shown to antagonize P53-mediated apoptosis in hematopoietic progenitors by repressing Puma [28]. In this study, we show that miR-137 is a downstream target of Slug and plays an important role in lung cancer progression. Recent studies have shown that transcription factors can mediate miRNA expression by interacting with conventional binding sites in the promoters of miRNAs [26,29], and that the transcription factor-mediated dysregulation of miRNAs can cause phenotypic variations and diseases. For example, the liver-enriched transcription factors, C/EBPa, HNF1a, HNF3b, and HNF4a, contribute to regulating miR-122, which directly affects target genes that are involved in liver development [29]. The transcription factor, c-Myc, upregulates the miR-17e92 and miR-106a~363 clusters to inhibit human trophoblast differentiation by repressing hGCM1 and hCYP19A1, and aberrant regulation of these miRNAs is thought to be associated with the pathogenesis of preeclampsia [30]. The transcription factor, GATA6, regulates the transcription of the miR-302e367 cluster during lung development, and alterations of miR302~367 expression can disrupt the balance of proliferation and differentiation among lung endoderm progenitors [31]. As a transcription factor, Slug is known to play multifaceted roles in lung cancer progression. However, no previous study has examined whether Slug can modulate miRNAs, and whether this is related to its ability to induce EMT and/or lung cancer progression. In this study, Illumina bead arrays and TLDAs were used to identify Slugregulated miRNAs in lung cancer cells, and the Slug-regulated miRNA expression was confirmed by Poly-A tailing real-time RTPCR. The expression levels of six identified miRNAs (miR-137, miR30a-3p, miR-335, miR-31, and miR-181b) were up-regulated to varying degrees in Slug overexpression cells (Fig. 1A and B). Our results indicated that only miR-137 expression increased more than 10-fold in Slug overexpression cells (CL1-0-Slug-4 and PC9-Slug) compared to controls. Therefore, we focus on miR-137 for further study. Our further experiments demonstrated for the first time that Slug, can bind and activate the miR-137 promoter (Figs. 1 and 2), and that knockdown of miR-137 abolished Slug-induced cancer cell invasion and migration (Fig. 3). To investigate whether Slug can affect TFAP2C expression in lung cancer cells, and it expression can be rescued by manipulating miR137 expression. We detected miR-137 transcripts with realtime quantitative RT-PCR, and assessed Slug and TFAP2C protein

indicates that the putative miR-137 binding site is highly conserved across multiple species. (B) miR-137 expression is negatively correlated with the protein expression of TFAP2C in various lung cancer cell lines (H1437, H460, PE089, H1975, PC9, Hop62, HCC827, and H1299), as assessed by real-time RT-PCR and Western blot analysis, respectively. The protein bands were quantified using the NIH-ImageJ program, and are presented as the TFAP2C/a-tubulin density ratio. The correlation of TFAP2C and miR-137 was assessed using Pearson correlation (P < 0.05). (C) miR-137 represses TFAP2C expression in lung cancer cells. The TFAP2C protein expression levels in miR-137-overexpressing transfectants (HCC827pMIRH137 and PC9-pMIRH137) and mock control cells (HCC827-Ctrl and PC9-Ctrl) were detected using Western blot analysis (right panel), while miR-137 expression levels were determined by real-time RT-PCR (left panel). (D) The protein expression levels of TFAP2C in miR-137-knockdown (PC9-miR-137-locker) and mock control (PC9-Ctrl-locker) cells were detected using Western blot analysis (right panel), while miR-137 expression was determined by real-time RT-PCR (left panel). (E) miR-137 suppresses TFAP2C protein expression through a direct interaction with its 30 -UTR. Sequences encoding wild-type and mutated fragments of the TFAP2C 30 -UTR were inserted into a luciferase reporter plasmid (pMir target vector) to yield pMir-TFAP2C-30 UTR-WT (wild-type containing the putative miR-137 targeting site) and pMir-TFAP2C-30 UTR-Mut (harboring a mutation in the miR-137 targeting site), as presented schematically in the left panel. 293T cells were co-transfected with pMir-TFAP2C-30 UTR-WT or pMir-TFAP2C-30 UTR-Mut, plus pMIRH137-PA-1 (expressing miR-137) or pMIRNA1 (control). The relative luciferase activities are presented as the means ± SD from three independent experiments (*P < 0.01). P-values were determined using the Student's t-test. (F) miR-137 expression does not affect the mRNA level of TFAP2C. The mRNA expression of TFAP2C was detected by real-time RT-PCR in HCC827-pMIRH137, CL1-0-pMIRH137, PC9-pMIRH137 (all of which constitutively express miR-137), HCC827-Ctrl, CL1-0-Ctrl, and PC9-Ctrl (which are the corresponding mock transfectants) cells. NS, no significant difference.

Please cite this article in press as: T.-H. Chang, et al., Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.06.002

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Fig. 6. miR-137 promotes lung cancer cell progression by downregulating TFAP2C. (A) Restoration of TFAP2C expression inhibits the miR-137-induced enhancements of cancer cell migration and invasion. HCC827-pMIRH137 (miR-137-knockdown) and mock-control cells were transfected with pLKO-TFAP2C (containing the TFAP2C-encoding sequence but lacking the 30 -UTR), and TFAP2C expression was detected by Western blot analysis. (B) The invasion abilities of the cells described in (A) were determined using in vitro cell invasion assay (*P < 0.01). Data are presented as the means ± SD of three independent experiments. (C) The motility rates of the cells described in (A) were determined using a single-cell motility assay based on time-lapse imaging (n ¼ 10 cells for each stable cell line). The box and whisker plots display significant differences in the average migration speeds of HCC827-pMIR137 cells transfected with pLKO-TFAP2C versus control pLKO-Ctrl (*P < 0.01). P-values were determined using the Student's t-test. (D) A mouse tail vein injection metastasis model was performed. H1299 cells with miR-137 locker or control locker were injected into tail veins of SCID mice. Seven weeks after the injection, lung nodules were counted under gross and microscopic examination (n ¼ 6 mice per group). Hematoxylin and eosin staining of lung samples dissected from the mice were shown (T: tumor; Scale bar:200 mm). (E) miR-137 expression and TFAP2C protein levels were assessed in mouse lung tumor sections, using real-time RT-PCR (left panel) and Western blot analysis (right panel), respectively. (F) TFAP2C protein expression was examined by immunohistochemical (IHC) staining of lung tumor specimens obtained from 143 NSCLC patients who underwent surgical resections. The intensity of IHC staining in cell nuclei was scored as follows: 0, no staining; 1þ, weak staining; 2þ, moderate staining; and 3þ, strong staining. Representative images of high- (3þ; left panel) and low- (0; right panel) scoring sections are shown. (G) The expression levels of Slug, miR-137, and TFAP2C in 143 tumor specimens were examined and used to divide the patients into three groups (Supplementary Table S6): the SlugHigh-miR-137High-TFAP2CLow group (n ¼ 21); the SlugLow-miR-137Low-TFAP2CHigh group (n ¼ 23); and the “other” group (n ¼ 99). Survival analysis was performed using Kaplan-Meier survival analyses and log-rank tests.

expression using Western blot analysis in lung cancer cell lines (HCC827-mock, HCC827-Slug, HCC827-Slug-Ctrl-locker, and HCC827-Slug-miR-137-locker cells described in Fig. 3). The results showed that overexpression of Slug in HCC827 cells (HCC827-Slug)

can suppress TFAP2C protein expression. Moreover, we indicated that knockdown of miR-137 in HCC827-Slug cells (HCC827-SlugmiR-137-locker) can rescue TFAP2C expression (Supplementary Fig. S3A). In this investigation, we also found that knockdown

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and overexpression of miR-137 does not affect the Slug expression in lung cancer cells. No feedback regulation of Slug expression were observed by manipulating miR137 expression (Supplementary Fig. S3). Accumulating evidence demonstrates that miRNAs play significant roles in fundamental biological processes (e.g., cell proliferation, differentiation, development, apoptosis, and metabolism) through their ability to regulate critical signaling molecules [19e21]. Several miRNAs have been shown to function as oncogenes or tumor suppressor genes by regulating mRNA targets in a variety of cancers, including glioblastoma, osteosarcoma, breast cancer, colon cancer, gastric cancer, and lung cancer [22,32]. Among the miRNAs that have been implicated in tumorigenesis, miR-137 has been widely studied. However, its precise role during tumor progression remains controversial. Various studies have suggested that miR-137 acts as a tumor suppressor. For example, miR-137 expression was significantly downregulated and negatively correlated with lymph node metastasis in human thyroid carcinoma, where it was found to target CXCL12 [33]. Another investigation found that miR-137 inhibited the malignant progression of thyroid cancer by directly repressing EGFR [34]. In colorectal cancer, miR137 was shown to target Cdc42 and inhibit cancer cell invasion and cell cycle progression [35]. miR-137 is significantly downregulated in both osteosarcoma cell lines and osteosarcoma tumors, and lentivirus-mediated expression of a miR-137-mimic inhibited osteosarcoma cell proliferation and migration by targeting FXYD6 [36]. However, whereas miR-137 appears to act as a tumor suppressor in human thyroid carcinoma, colorectal cancer, and osteosarcoma [34e36], it has been suggested to promote bladder cancer. For example, miR-137 is upregulated in human bladder cancer tissues and cell lines, and overexpressed miR-137 was found to downregulate PAQR3 by binding to its 30 -UTR, thereby increasing bladder cancer cell proliferation and invasion [37]. miR-137 has been reported as tumor suppressor in various types of cancers, including NSCLC [33e36,38]. However, we report that the expression levels of miR-137 in lung adenocarcinoma tissues (stage I to III) were significantly higher than those in adjacent tissues. Moreover, our analysis of the TCGA lung cancer dataset revealed that lung adenocarcinoma patients with high-level expression of miRNA-137 had significantly shorter overall survival than patients with low-level miRNA-137 expression. We further obtained similar results in an independent cohort of 143 NTUH lung cancer patients (Fig. 4). These findings, together with our in vitro observations that miR-137 overexpression promoted the invasion

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and migration of lung cancer cells, whereas miR-137 knockdown inhibited lung cancer progression (Figs. 4 and 6), strongly suggest that Slug-induced miR-137 functions as an oncogene in lung cancer. A recently investigation has supported our results and also indicated miR-137, a risky microRNA, is associated with poor prognosis in NSCLC patients. Furthermore, oncogenic miR-137 contributes to cisplatin resistance through targeting and repressing caspase-3 (CASP3) in lung adenocarcinoma [39]. Using theTarget Scan and miRWalk computational algorithms, we identified TFAP2C as a potential target gene of miR-137, and found that the putative miR-137 target site within the TFAP2C mRNA are conserved across multiple species. TFAP2C is a member of the activating protein 2 (AP2) family of transcription factors; it can regulate gene transcription by interacting with viral and cellular enhancing components and binding to the sequence, 50 GCCNNNGGC-30 . Interestingly, the TFAP2C-mediated activation or repression of various genes has been implicated in tumorigenesis and early development [40]. TP53, which is a well-known tumor suppressor involved in diverse cellular processes, was shown to target and transcriptionally regulate TFAP2C, and forced TP53 expression in human breast carcinoma cells was found to upregulate TFAP2C at the mRNA and protein levels and inhibit cancer cell growth [41]. A recently study found that miR-124 expression enhances melanoma progression by suppressing TFAP2C through the direct and specific targeting of the 30 -UTR of its mRNA [42]. Finally, TFAP2C was shown to function as a tumor suppressor in melanoma, and silencing of TFAP2C in melanoma cells was shown to increase their invasive and metastatic behaviors [42]. Our present results strongly suggest that miR-137 downregulates TFAP2C in lung cancer cells by specifically interacting with a target site in the 30 -UTR of its mRNA to suppress translation. Moreover, ectopic expression of TFAP2C significantly inhibited the migration and invasion of miR137 overexpressing lung cancer cells in vitro (Fig. 6). In this study, we collected samples from 143 lung adenocarcinomas surgically resected at the National Taiwan University Hospital (described in Supplementary Methods). Statistically significant differences in Slug expression levels between high and low miR-137 groups were calculated by the Mann-Whitney test (P ¼ 0.008; Supplementary Fig. S4), and no statistically significant differences in TFAP2C expression levels between high and low miR137 groups were calculated (Pearson Chi-square test; P ¼ 0.663; Supplementary Fig. S4). Since there are many other genes controlled by Slug and miR-137 have been reported in lung cancer cells [5,18,28,34e37]. The functions of Slug and miR137 may be cell context dependent in regulating lung cancer malignancy. In clinical

Fig. 7. Schematic diagram of our proposal for how the EMT regulator, Slug, promotes lung cancer progression through the miR-137-mediated manipulation of TFAP2C. Our results suggest that the Slug-miR-137-TFAP2C pathway should be considered a novel regulatory network responsible for controlling lung cancer progression.

Please cite this article in press as: T.-H. Chang, et al., Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.06.002

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studies, we indicated that lung adenocarcinoma patients with lowlevel expression of Slug and miR-137 but high-level expression of TFAP2C experienced significantly better survival (Fig. 6G). Notably, 69% of lung adenocarcinomas patients (99/143; classified as other group) were detected with altered gene expression patterns (neither SlugLow-miR-137Low-TFAP2CHigh nor SlugHigh-miR-137HighTFAP2CLow; Supplemental Table S6). These results also point out that Slug-miR-137-TFAP2C axis in the regulation of lung cancer malignancy may influenced by cell context. In sum, and as diagrammed in Fig. 7, our results collectively demonstrate that the transcription factor, Slug, upregulates miR137 to enhance lung cancer progression-related phenotypes by downregulating TFAP2C. We further report that, in lung adenocarcinoma patients, low-level expression of Slug and miR-137 but high-level expression of TFAP2C is an independent predictor and was associated with a significantly better survival rate. Thus, we herein show that miR-137 relays the pro-metastatic effects of Slug via TFAP2C in lung cancer. These findings add new components to the Slug-controlled regulatory network in NSCLC cells, and suggest that the Slug-miR-137-TFAP2C axis may provide future target molecules for lung cancer therapeutics. Funding This work was financially supported by grant from National Taiwan University (103R7601-3) and the Far Eastern Memorial Hospital-National Taiwan University Hospital Joint Research Program (105-FTN02). Acknowledgments The authors thank the NTU Microarray Core Facility of the National Research Program for Genomic Medicine of Taiwan for technical assistance, and the Department of Medical Research in National Taiwan University Hospital for facility support. Conflict of interest The authors declare no conflicts of interest. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.canlet.2017.06.002. References [1] L. Larue, A. Bellacosa, Epithelial-mesenchymal transition in development and cancer: role of phosphatidylinositol 3' kinase/AKT pathways, Oncogene 20 (2005) 7443e7454. [2] J.P. Thiery, H. Acloque, R.Y. Huang, M.A. Nieto, Epithelialemesenchymal transitions in development and disease, Cell 139 (2009) 871e890. [3] J.Y. Shih, P.C. Yang, The EMT regulator slug and lung carcinogenesis, Carcinogenesis 32 (2011) 1299e1304. [4] N. Tiwari, A. Gheldof, M. Tatari, G. Christofori, EMT as the ultimate survival mechanism of cancer cells, Semin. Cancer Biol. 22 (2012) 194e207. [5] J.Y. Shih, M.F. Tsai, T.H. Chang, Y.L. Chang, A. Yuan, C.J. Yu, et al., Transcription repressor slug promotes carcinoma invasion and predicts outcome of patients with lung adenocarcinoma, Clin. Cancer Res. 11 (2005) 8070e8078. [6] T. Kosaka, E. Kikuchi, S. Mikami, A. Miyajima, S. Shirotake, M. Ishida, et al., Expression of snail in upper urinary tract urothelial carcinoma: prognostic significance and implications for tumor invasion, Clin. Cancer Res. 16 (2010) 5814e5823. [7] G.J. Zhang, T. Zhou, H.P. Tian, Z.L. Liu, S.S. Xia, High expression of ZEB1 correlates with liver metastasis and poor prognosis in colorectal cancer, Oncol. Lett. 5 (2013) 564e568. [8] S. Prislei, E. Martinelli, G.F. Zannoni, M. Petrillo, F. Filippetti, M. Mariani, et al., Role and prognostic significance of the epithelial-mesenchymal transition factor ZEB2 in ovarian cancer, Oncotarget 6 (2015) 18966e18979.

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Please cite this article in press as: T.-H. Chang, et al., Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.06.002

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Please cite this article in press as: T.-H. Chang, et al., Upregulation of microRNA-137 expression by Slug promotes tumor invasion and metastasis of non-small cell lung cancer cells through suppression of TFAP2C, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.06.002

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