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miR-206 regulates cisplatin resistance and EMT in human lung adenocarcinoma cells partly by targeting MET

miR-206 regulates cisplatin resistance and EMT in human lung adenocarcinoma cells partly by... www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 17 miR-206 regulates cisplatin resistance and EMT in human lung adenocarcinoma cells partly by targeting MET 1,* 1,* 1,* 1,* 1 Qing-yong Chen , De-min Jiao , Jian Wang , Huizhen Hu , Xiali Tang , 1 1 2 Jun Chen , Hao Mou , Wei Lu Department of Respiratory Disease, The 117th Hospital of PLA, Hangzhou, Zhejiang, 310013, P.R. China Department of Oncology, The 117th Hospital of PLA, Hangzhou, Zhejiang, 310013, P.R. China These authors have contributed equally to this work Correspondence to: Qingyong Chen, e-mail: [email protected] Wei Lu, e-mail: [email protected] Keywords: miR-206, cisplatin resistance, epithelial-mesenchymal transition (EMT), MET, lung adenocarcinoma Received: April 27, 2015 Accepted: March 04, 2016 Published: March 21, 2016 ABSTRACT MicroRNAs (miRNAs) play a critical role in drug resistance and epithelial- mesenchymal transition (EMT). The aims of this study were to explore the potential role of miR-206 in governing cisplatin resistance and EMT in lung cancer cells. We found that both lung adenocarcinoma A549 cisplatin-resistant cells (A549/DDP) and H1299 cisplatin-resistant cells (H1299/DDP) acquired mesenchymal features and were along with low expression of miR-206 and high migration and invasion abilities. Ectopic expression of miR-206 mimics inhibited cisplatin resistance, reversed the EMT phenotype, decreased the migration and invasion in these DDP-resistant cells. In contrast, miR-206 inhibitors increased cisplatin resistance, EMT, cell migration and invasion in non-DDP-resistant cells. Furthermore, we found that MET is the direct target of miR-206 in lung cancer cells. Knockdown of MET exhibited an EMT and DDP resistant inhibitory effect on DDP-resistant cells. Conversely, overexpression of MET in non-DDP- resistant cells produced a promoting effect on cell EMT and DDP resistance. In lung adenocarcinoma tissues, we demonstrated that low expression of miR-206 were also correlated with increased cisplatin resistance and MET expression. In addition, we revealed that miR-206 overexpression reduced cisplatin resistance and EMT in DDP-resistant cells, partly due to inactivation of MET/PI3K/AKT/mTOR signaling pathway, and subsequent downregulation of MDR1, ZEB1 and Snail expression. Finally, we found that miR-206 could also sensitize A549/DDP cells to cisplatin in mice model. Taken together, our study implied that activation of miR-206 or inactivation of its target gene pathway could serve as a novel approach to reverse cisplatin resistance in lung adenocarcinomas cells. such as high efficiency, mild side effects and easy INTRODUCTION administration. However, cisplatin resistance often occurs in clinical practice [2]. Thus, adjuvant therapy Lung cancer, predominantly non-small cell lung to enhance cisplatin efficiency becomes an important cancer (NSCLC), is the leading cause of cancer-related chemotherapeutic strategy. mortality worldwide. Patients with NSCLC are mostly Accumulating studies indicate that there are several treated with platinum-based chemotherapy in combination major mechanisms of drug resistance in cancer cells, with radiation therapy. However, the development of such as increased detoxification of anticancer drugs by chemoresistance, either intrinsic or acquired, is a major glutathione system, defective apoptosis pathway, increased obstacle limiting successful treatment [1]. Cisplatin (DDP) levels of DNA repair or DNA tolerance, decreased uptake is still one of the commonly used chemotherapeutic agents of water-soluble drugs and enhanced drug efflux from against lung cancer due to its therapeutic advantages, www.impactjournals.com/oncotarget 24510 Oncotarget cancer cells mediated by ATP-binding cassette (ABC) In this study, we found miR-206 was down- transporters[3–5]. Several studies showed that the drug- regulated in both A549/DDP cells and H1299/DDP cells. resistant cancer cells display features of epithelial- Overexpression of miR-206 or knockdown of its target mesenchymal transition (EMT), which is defined by the MET reversed the mesenchymal features and sensitized loss of intracellular links along with the gain of migratory DDP-resistant cells to cisplatin. More importantly, we and invasive abilities [6, 7]. Specifically, cells with demonstrated that decreased miR-206 levels induced decreased expression of the epithelial marker E-cadherin cisplatin resistance and EMT phenotype due to activation and increased expression of mesenchymal molecules of MET/PI3K/AKT/mTOR axis, upregulation of MDR1, including Snail/Snai1, Slug/Snai2, Vimentin, zinc-finger ZEB1 and Snail expression in DDP-resistant cells. E-box binding homeobox 1 (ZEB1), lead to enhanced These results provide new insights into the molecular motility, invasion and drug resistance[8]. In addition, mechanisms of cisplatin resistance induced by decreased PI3K/AKT/mammalian target of rapamycin (mTOR) miR-206 levels in lung adenocarcinoma cells and suggest signaling has also found to confer resistance to DDP-based miR-206 and its target gene pathway could be novel treatment in many cancers [9, 10]. Furthermore, it has therapeutic targets to reverse cisplatin resistance of lung been reported that there exist a cross-talk between EMT adenocarcinoma cells. programming and PI3K/AKT/mTOR pathway [11]. The microRNAs (miRNAs) are a group of small RESULTS non-coding RNAs. The basic mechanism of miRNA action is that miRNA could bind to the 3'UTR of target Cisplatin resistant lung adenocarcinoma cells mRNAs, resulting in translational repression or target exhibit EMT features and have enhanced MDR1 mRNA cleavage[12]. Recent studies suggested that the expression acquisition of drug resistance by cancer cells might be modulated via the changes in miRNA levels[13, 14]. Previous studies have demonstrated that For instance, miR-135a/b are downregulated in cisplatin chemotherapeutic drug can induce EMT, enhance resistance (A549/DDP) cells, and the overexpression of invasive ability, resulting in drug resistance [31, 32]. miR-135a/b sensitizes A549/DDP cells to cisplatin by Snail and ZEB1 are two crucial EMT inducers [33]. targeting MCL1 (myeloid cell leukemia 1) [15]. Up- Multi-drug resistance gene 1 (MDR1, ABCB1), encoding regulation of miR-27a can suppress RKIP (Raf kinase P-glycoprotein (P-gp), is one of pharmaceutical inhibitory protein) expression and in turn contribute carriers that can decrease the effective intracellular to chemoresistance of lung adenocarcinoma cells to concentration of the drug, leading to drug resistance cisplatin[16]. Upregulation of miR-451 expression [34]. To determine the mechanism of cisplatin resistance inactivates the AKT signaling pathway and enhanced in lung adenocarcinoma cells, we first compared A549/ cisplatin induced apoptosis in A549 cells[17]. MiR- DDP cells and H1299/DDP cells with its parental cells 513a-3p can sensitize human lung adenocarcinoma in cisplatin sensitivity, MDR1 expression levels, EMT cells to cisplatin by targeting GSTP1 (Glutathione morphology and related markers expression. MTT S-transferase P1)[18]. MiR-92b is significantly up- assay showed that A549/DDP and H1299/DDP cells regulated in lung cancer cells and knockdown of miR- exhibited significantly higher resistance to cisplatin 92b inhibits cell growth and sensitizes the A549/DDP than non-DDP-resistant cells (Figure 1A). IC50 of cells to DDP by target PTEN (phosphatase and tensin cisplatin in A549/DDP cells was 2.60 fold higher than homolog) [19]. that in A549 cells and IC50 of cisplatin in H1299/DDP MiR-206 is one of the most studied and best cells was 2.69 fold higher than that in H1299 cells. characterized miRNAs to date, which specifically Western blotting showed that DDP-resistant cells had expressed in skeletal muscle[20]. Recently, there has higher levels of MDR1 protein expression than their been increasing interest in understanding the role of miR- non-DDP-resistant cells (Figure 1B). Furthermore, 206 in cancer, and down-regulation of miR-206 has been A549 cells and H1299 lung adenocarcinoma cells observed in different types of cancers [21–27]. Decreased display epithelial characteristics, whereas A549/DDP expression of miR-206 in gastric cancer is associated and H1299/DDP cells exhibited elongated, fibroblastoid with tumor progression and poor survival [24]. miR-206 morphology and separated from one another (Figure suppresses breast cancer cell migration and invasion by 1C). Morphological conversion DDP-resistant cells targeting Cdc42 [28]. miR-206 can inhibit the expression associated with EMT were also reflected by changes of VEGF and regulate the apoptosis and migration of in protein levels. Western blotting showed that A549/ laryngeal cancer cells[29]. We and others have also DDP exhibited downregulation of E-cadherin levels reported miR-206 overexpression could inhibit invasion and upregulation of N-cadherin, Vimentin, Snail of lung cancers [22, 26, 30]. However, whether miR-206 and ZEB1 (Figure 1D). H1299/DDP cells also have is involved in regulating cisplatin resistance and EMT in upregulation of N-cadherin, Vimentin, Snail and ZEB1 human lung adenocarcinomas remains unclear. www.impactjournals.com/oncotarget 24511 Oncotarget than H1299 cells, but E-cadherin expression could not miR-206 overexpression reverses cisplatin be detected (Supplementary Figure 1A). Additionally, resistance, EMT, migration and invasion in wound healing assay and transwell invasion assay DDP-resistant cells demonstrated that the migration and invasion abilities were significantly stronger in A549/DDP cells and miR-206 has been found to be down-regulated in H1299/DDP cells (Figure 1E-1F, Supplementary Figure many types of cancers including lung cancer[21-27, 30]. 1B-1C). To determine whether miR-206 plays a pivotal role in drug Figure 1: Differences between DDP-resistant cells and non-DDP-resistant cells. A. Two DDP-resistant cells and their parental cells were treated with indicated concentrations of cisplatin for 48 h and then were subjected to MTT assay (n = 5). The results showed that A549/DDP and H1299/DDP cells were more resistant to cisplatin than their parental cells in vitro. B. Western blotting illustrated increased expression of MDR1 in A549/DDP and H1299/DDP cells. C. A549 and H1299 cells displayed epithelial morphology, but A549/DDP and H1299/DDP cells exhibited fibroblastic morphology (original magnification, ×200). D. Western blotting showed increased expression of N-cadherin, Vimentin, ZEB1, Snail and reduced expression of E-cadherin in A549/DDP cells. E. Wound healing assay and F. transwell invasion assay revealed significant enhancement of migration and invasion ability in A549/ DDP cells. Data are means of three separated experiments ± SD, * P <0.05, ** P <0.01 1. compared with A549 cell group. www.impactjournals.com/oncotarget 24512 Oncotarget resistance in lung cancer cells, we measured the expression miR-206 target site in the 3′-UTR of MET mRNA was of miR-206 in the A549/DDP cells, H1299/DDP cells and responsible for its regulation, we cloned MET 3′-UTR their parental cells. Real-time PCR assay revealed that wild type (MET-wt) or 3′-UTR mutant type (MET-mut) miR-206 was significantly lowered in both A549/DDP into downstream of the luciferase reporter gene and cells and H1299/DDP cells (Figure 2A, Supplementary cotransfected with miR-206 mimics into A549 cells. A Figure 2A) compared with their parental cells. To further luciferase reporter containing miR-206 inhibitor sequence validate the role of miR-206 in cisplatin resistance, we was used as a positive control (PC). As indicated in Figure transfected miR-206 mimics into A549/DDP cells and 3A. Luciferase activity from a construct harboring miR- H1299/DDP cells, transfected miR-206 inhibitors into 206 inhibitor sequence (PC group) was significantly A549 cells and H1299 cells. MTT assay revealed that decreased in A549 cells expressing either miR-206 or its miR-206 mimics treatment led to significantly decreased negative control form. Luciferase activity from A549 cells resistance of A549/DDP cells and H1299/DDP cells cotransfected with miR-206 and the construct containing to cisplatin, whereas miR-206 inhibitors transfection MET-mut form did not induce any significant change enhanced the resistance of A549 cells and H1299 cells in luciferase activity, whereas luciferase activity from to cisplatin (Figure 2B, Supplementary Figure 2B-2C). A549 cells cotransfected with miR-206 and the construct Furthermore, western blotting showed that miR-206 containing MET-wt was decreased by more than 95% mimics significantly decreased the expression of MDR1 when compared to negative control cells. These results in A549/DDP cells and H1299/DDP cells, while miR-206 indicate that miR-206 regulates MET protein in A549 cells inhibitors increased the expression of MDR1 in A549 cells by directly targeting MET 3′-UTR. and H1299 cells (Figure 2C, Supplementary Figure 2D). On the other hand, we found that A549/DDP cells Previous studies have shown that the drug-resistant and H1299/DDP cells expressed higher levels of MET cancer cells display features of epithelial-mesenchymal protein than A549 cells and H1299 cells (Figure 3B), transition (EMT)[32, 35, 36]. Here, we observed that but basal expression of p-MET is undetectable in these miR-206 mimics transfection led to a change from cell lines (data not shown). Western blotting showed that elongated, fibroblastoid morphology to a rounded shap miR-206 overexpression could significantly decrease in both A549/DDP cells and H1299/DDP cells, whereas MET expression in A549/DDP and H1299/DDP cells. miR-206 inhibitors transfection resulted in an elongated Meanwhile, down-regulated miR-206 could increase MET fibroblast-like morphology of A549 cells and H1299 cells expression level in A549 cells and H1299 cells (Figure (Figure 2D, Supplementary Figure 2E). Furthermore, 3C). These findings further confirmed the existence of an miR-206 mimics treatment caused the higher expression inverse correlation between the expression of miR-206 of E-cadherin and lower expression of mesenchymal and MET expression in these cell lines. markers including Vimentin, Snail and ZEB1 in A549/ DDP cells. Also, miR-206 mimics decreased the Low expression of miR-206 in lung expression of N-cadherin, Vimentin, Snail and ZEB1 in adenocarcinoma tissues correlates with increased H1299/DDP cells (Figure 2E, Supplementary Figure 2F). cisplatin resistance and MET expression On the contrary, miR-206 inhibitors reduced E-cadherin expression, induced the expression of Vimentin, ZEB1 To better understand the association between and Snail in A549 cells, while induced N-cadherin, miR-206 and cisplatin resistance, a total of 34 clinical Snail and ZEB1expression in H1299 cells (Figure 2F, lung tumor tissue samples were collected from patients Supplementary Figure 2G). In addition, invasion and with advanced lung adenocarcinoma and divided into migration assay further demonstrated that miR-206 mimics “sensitive” and “insensitive” groups according to the suppressed the invasion and migration of A549/DDP patient’s response to cisplatin-based chemotherapy. cells and H1299/DDP cells (Figure 2G, Supplementary As shown in Figure 4A, miR-206 was significantly Figure 3A-3B), whereas miR-206 inhibitors enhanced down-regulated in the “insensitive” group tissues (n the invasion and migration of A549 cells and H1299 cells = 17) compared with that in the “sensitive” group (n = (Figure 2H, Supplementary Figure 3C-3D). These results 17). Importantly, immunohistochemistry assay showed indicated that miR-206 could reverses cisplatin resistance, that 16 of 17 “insensitive” group tissues (94.12%) had EMT, migration and invasion of cisplatin resistant cells. positive immunostaining of MET protein but it was 4 of 17 (23.52%) in “sensitive” group (Figure 4B). Moreover, the stronger immunoreactivity of MET was significantly MET gene is a target of miR-206 in lung associated with lower miR-206 expression (r = 0.4086, cancer cells P = 0.0165, Figure 4C), suggesting that miR-206-MET Identification of miRNA-regulated gene targets is interaction might be biologically significant in cisplatin a necessary step to understand miRNA functions. Based resistance. In addition, IHC staining for MET, MDR1, on target prediction programs, we found that MET is a E-cadherin and Vimentin were performed on 5 samples tentative target of miR-206. To test whether the predicted of each group. We found that except Vimentin, all five www.impactjournals.com/oncotarget 24513 Oncotarget Figure 2: miR-206 decreased cisplatin resistance, EMT, migration and invasion of A549/DDP cells. A. qRT-PCR assay showed a significant down-regulation of miR-206 in A549/DDP cells compared with in A549 cells. B. A549/DDP cells were transfected with miR-206 mimics, and A549 cells were transfected with miR-206 inhibitors. After 24 hrs of transfection, 5×10 cells/well were seeded in 96-well cell culture plates. The next day, cells were incubated with or without the indicated concentration of cisplatin for 48 h and subsequently subjected to an MTT assay. (C-F) A549/DDP cells or A549 cells were transfected with the indicated plasmid. After 48 h, C. the expression of MDR1 was determined by Western blotting analysis. D. Cell morphology was observed by microscopy (Original magnification, ×200). E-F. Western blotting analysis was used to detect the expression of E-cadherin, N-cadherin, Vimentin, ZEB1 and Snail (Left panel), Quantitative results are illustrated for left panel. (G-H) Wound healing assays (Left panel) and invasion assay (Right panel) were used to detect the migration and invasion ability in G. miR-206 mimics transfected A549/DDP cells or H. miR-206 inhibitors transfected A549 cells. Data are means of three separated experiments ± SD, * P <0.05, ** P <0.01 compared with their control. www.impactjournals.com/oncotarget 24514 Oncotarget cisplatin “insensitive” tissues have higher MET and the expression of MET was significantly suppressed by MDR1 expression, and lower E-cadherin expression. MET-shRNA, and activated by MET expression vector In contrast, 3 out of 5 cisplatin “sensitive” tissues have (ex-MET) (Figure 5A, Supplementary Figure 5A). lower MET and MDR1 expression, and higher E-cadherin MET inhibitor SU11274 treatment partially reversed expression (Supplementary figure 4). the mesenchymal phenotype of A549/DDP cells. MET silence also reversed the mesenchymal phenotype in both A549/DDP cells and H1299/DDP cells, while MET MET mediated EMT and cisplatin resistance overexpression induced a mesenchymal phenotype in To determine whether the anti-EMT and anti- A549 cells and H1299 cells (Figure 5B, Supplementary cisplatin resistance effects of miR-206 on DDP-resistant Figure 5B). Western blotting analysis showed that both cells could be partly explained by its targeting of MET, MET inhibitor SU11274 and MET-shRNA increased we first analyzed how MET inhibitors, MET silence and the E-cadherin protein expression, but decreased the MET overexpression affected EMT. Our data showed that expression of mesenchymal markers including N-cadherin, Figure 3: MET is a direct target of miR-206. A. Luciferase assay was performed in A549 cells that were cotransfected with miRNA mimics and reporter vectors carrying MET 3'UTR wild type (MET-wt), MET 3'UTR mutated type (MET-mut), and miR-206 inhibitor sequences (positive control) element. B. Variable MET expression in A549 and A549/DDP was obtained by Western blotting analysis. C. A549/DDP cells were transfected with miR-206 mimics and A549 cells were transfected with miR-206 inhibotors for 48 h respectively. Western blotting was used to detect MET expression. Data are means of three separated experiments ± SD, ** P <0.01, compared with negative control (NC). www.impactjournals.com/oncotarget 24515 Oncotarget Vimentin, ZEB1 and Snail (Figure 5C-5D, Supplementary silence decreased the MDR1 expression(Supplementary Figure 5D). In contrast, MET overexpression decreased Figure 5E), suppressed cell migration and invasion the E-cadherin protein expression, but increased the (Supplementary Figure 6A-6B). While MET expression of N-cadherin, Vimentin, ZEB1 and Snail overexpression increased MDR1 protein expression in A549 cells (Figure 5E). Similarly, MET-shRNA (Supplementary Figure 5F), enhanced the capability of decreased the expression of N-cadherin, Vimentin, ZEB1 H1299 cell migration and invasion(Supplementary Figure and Snail (Supplementary Figure 5B). In contrast, MET 6C-6D). overexpression increased the expression of N-cadherin, Vimentin, ZEB1 and Snail in A549 cells (Supplementary miR-206 inhibits EMT and cisplatin resistance Figure 5C). However, E-cadherin expression could not be via MET dependent PI3K/AKT /mTOR detected in both conditions. signaling pathways We next analyzed the effect of MET inhibitors, MET silence and MET overexpression on cell cisplatin PI3K/AKT/mTOR is one of the definite downstream sensitivity, migratory and invasive capability. As shown targets of MET receptor. Inhibition of PI3K/AKT in Figure 6, SU11274 significantly promoted cell growth signaling has proven to be an efficient way to attenuate inhibition induced by cisplatin (Figure 6A) and decreased the resistance of chemotherapy[37]. In present study, we the MDR1 expression in A549/DDP cells (Figure 6B). found that AKT/mTOR pathway is activated in A549/ Similarly, MET shNA transfection also increased the DDP cells and H1299/DDP cells compared with the sensitivity of A549/DDP cells to cisplatin (Figure 6D) parental cells (Figure 7A, Supplementary Figure 7A). and decreased the MDR1 expression (Figure 6E). IC50 To determine whether miR-206 inhibits MET dependent in SU11274 (0.5μM) group and MET shRNA group were PI3K/AKT/mTOR signaling pathways, we analyzed the 10.58μM and 3.53μM, significantly lower than their DDP effects of miR-206 or MET expression changes on PI3K/ control groups (12.82μM and 10.06μM, respectively. AKT/mTOR signaling. The results showed that both Supplementary Table 1). Furthermore, SU11274 miR-206 mimics transfection and MET-shRNA treatment treatment and MET shRNA transfection showed similar significantly decreased AKT, p-AKT, mTOR and p-mTOR effects on suppressing migration and invasion of A549/ protein levels in A549/DDP cells (Figure 7B-7C,), and DDP cells (Figure 6C and Figure 6F). In contrast, MET MET inhibitor SU11274 also resulted in a decrease in overexpression increased the cisplatin resistance and p-AKT, p-mTOR protein expression(Figure 7D). In MDR1 protein expression (Figure 6G-6H, Supplementary contrast, miR-206 inhibitors and MET overexpression Table 1), enhanced the capability of cell migration increased p-AKT, mTOR and p-mTOR protein levels in and invasion (Figure 6I). In H1299/DDP cells, MET A549 cells (Figure 7E-7F). Although the degree of gene Figure 4: Low expression of miR-206 in lung adenocarcinoma tissues correlates with increased cisplatin resistance and MET expression. A. Expression levels of miR-206 and B. MET protein were detected in cisplatin “sensitive” (a,n =17) and “insensitive” (b,n = 17) lung adenocarcinoma tissues via qRT-PCR (normalized to U6 RNA) and immunostaining (Origninal magnification, ×100), respectively. C. The immunoreactivity of MET protein in cisplatin “sensitive” and “insensitive” tissues showed a statistically significant inverse correlation with the relative expression level of miR-206. Data are means of three separated experiments ± SD, * P <0.05. www.impactjournals.com/oncotarget 24516 Oncotarget Figure 5: MET is involved in miR-206 inhibited EMT. A. Western blotting analysis confirmed that the expression of MET was suppressed by MET-shRNA, and activated by MET expression vector (ex-MET). (B-E) A549/DDP cells were treated with MET inhibitor SU11274, or transfected with MET shRNA for 48h, and the A549 cells were transfected with MET expression vector (ex-MET) for 48h, B. cell morphological changes associated with EMT are shown in the phase contrast image (Original magnification, ×200). C-E.The expression of E-cadherin, N-cadherin, Vimentin, ZEB1, Snail were examined by western blotting. Data are means of three separated experiments ± SD, * P <0.05, ** P <0.01 compared with their control. www.impactjournals.com/oncotarget 24517 Oncotarget expression and protein levels varied between different cell (Figure 7G-7H). Furthermore, both LY294002 (0.5μM) lines, similar results were also found in H1299/DDP and and rapamycin (10nM) enhanced cisplatin sensitivity, H1299 cell lines (Supplementary Figure 7B-7C). decreased the MDR1 expression in A549/DDP cells To further support the role of PI3K/AKT/mTOR (Figure 8A–8B). IC50 decreased from 11.12 μM to 7.78 signaling in suppression of EMT and cisplatin resistance μM for LY294002, and decreased from 11.12 μM to 8.31 by miR-206, PI3K selective inhibitor LY294002 and μM for rapamycin (Supplimentary Table 1). In addition, mTOR inhibitor rapamycin was utilized in A549/ both LY294002 and rapamycin reversed mesenchymal DDP cells. LY294002 (10μM, 20μM) were observed to characteristics (Figure 8C), decreased the expression of remarkably reduce the protein of p-AKT and p-mTOR, mesenchymal markers (Figure 8D-8E), and inhibited the and rapamycin to reduce p-mTOR in A549/DDP cells migration and invasion of A549/DDP cells (Figure 8F). Figure 6: MET is involved in miR-206 inhibited cisplatin resistance. A549/DDP cells were treated with MET inhibitor SU11274 (indicated concentration), or were transfected with MET shRNA for 48h, and A549 cells were transfected with MET expression vector (ex-MET) for 48h, A, D and G. the changes of cisplatin sensitivity, B, E and H. MDR1 expression, C, F and I. migration and invasion ability in each groups were detected. Data are means of three separated experiments ± SD, * P <0.05, ** P <0.01 compared with their control. www.impactjournals.com/oncotarget 24518 Oncotarget Figure 7: miR-206/MET regulated PI3K/AKT/mTOR pathway. Western blotting analysis was performed to detect the protein expression of AKT, p-AKT, mTOR, p-mTOR in A. A549/DDP cells and A549 cells, B. miR-206 mimics transfected A549/DDP cells, C. MET shRNA transfected A549/DDP cells, D. MET inhibitors SU11274 treated A549/DDP cells, E. miR-206 inhibitors transfected A549 cells, F. MET overexpression vectors (ex-MET) transfected A549 cells, G. PI3K inhibitor LY294002 treated A549/DDP cells, H. mTOR inhibitor rapamycin treated A549/DDP cells. Cell lysates were collected 48 h after transfection or 2h after treatment with MET, PI3K and mTOR inhibitors. Data are means of three separated experiments ± SD, * P <0.05, ** P <0.01 compared with their control. www.impactjournals.com/oncotarget 24519 Oncotarget Figure 8: PI3K/AKT/mTOR pathway is involved in miR-206/MET regulated cisplatin resistance, EMT, migration and invasion. A549/DDP cells were treated with LY294002 (indicated concentration) or rapamycin (indicated concentration) respectively. After 48h, A. The changes of cisplatin sensitivity, B. MDR1 expression, C. cell morphology (Original magnification, ×200), D-E. E-cadherin, N-cadherin, Vimentin, ZEB1 and Snail expression, F. migration and invasion ability in each group of cells were detected. Data are means of three separated experiments ± SD, * P <0.05, ** P <0.01 compared with their control. www.impactjournals.com/oncotarget 24520 Oncotarget These data indicated the involvement PI3K/AKT/mTOR decreased in the miRNA-206 plus cisplatin group pathway in suppression of EMT and cisplatin resistance compared with DDP combined mimic NC group or DDP by miR-206. group. (Figure 9C-9D). These results further suggest that miR-206 and its downstream MET/AKT/mTOR pathway play important roles in controlling A549/DDP cells miR-206 enhances A549/DDP cells to cisplatin cisplatin sensitivity. sensitivity in vivo To further investigate the effect of miR-206 DISCUSSION expression on cisplatin sensitivity, we evaluated the in vivo antitumor activity of miR-206 in xenograft model. Previous studies have reported that miR-206 could As shown in Figure 9A-9B, The in vivo results parallel the act as a tumor-suppressor in various cancers including in vitro results and show that miR-206 resulted in dramatic lung cancer[21–30]. We and others have demonstrated tumor regressions compared with both negative control or that miR-206 overexpression inhibited invasion and DDP control group. Furthermore, the expression of MET, metastasis in lung cancer cells[26, 30]. In the current AKT, p-AKT, mTOR and p-mTOR were significantly study, we demonstrated that miR-206 could suppress EMT Figure 9: miR-206 enhances A549/DDP cells to cisplatin sensitivity in vivo. A. The gross morphology of tumor samples B. The final xenograft tumor weights were measured after 30 days of treatment. C. MET/AKT/ mTOR pathway proteins expression in excised xenograft tumor were detected by western blotting assay. D. Relatve expression levels of MET/AKT/mTOR pathway proteins. sample1, 2, 3 stand for mouse tumor tissues from each group. Data are means of three separated experiments ± SD, (* P < 0.05, ** P < 0.01 compared with indicated control). www.impactjournals.com/oncotarget 24521 Oncotarget process and cisplatin resistance of lung adenocarcinoma MET is also one of the targets of miR-206. Despite MET cells, partly through targeting MET and its downstream has recently been identified as a target of miR-206 in PI3K/AKT/ mTOR pathway both in vitro and in vivo. Our rhabdomyosarcoma cells [44]. We further demonstrated data provide a first insight into the function of miR-206 that miR-206 directly target MET in lung cancer A549 in regulating cisplatin resistance and EMT in cisplatin cells. In addition, our results in clinical lung cancer tissue resistant lung adenocarcinoma cells. samples show that the decreased expression of miR-206 The EMT is a molecular process through which closely correlated with increased MET expression and an epithelial cell undergoes transdifferentiation into poor cisplatin sensitivity. These data suggest that miR- a mesenchymal phenotype. Recent evidences have 206-MET play important roles in regulating EMT and suggested that EMT processes may play an important cisplatin resistance in lung adenocarcinoma cells. role in the development of chemoresistance. It has PI3K/AKT/mTOR is a crucial downstream been reported knockdown of snail and slug reverses pathway of MET and can regulate many of the biological the EMT phenotype and reduces ovarian cancer phenomena, such as cell proliferation and survival, cell resistance to cisplatin[17]. Inhibiting EMT by motility and migration, and tumor cell invasion. In the overexpressing the microRNA miR-200 could abrogate present study, we found miR-206-MET axis regualted cyclophosphamide resistance in spontaneous breast-to- PI3K/AKT/mTOR pathway in lung cancer cells. Both lung metastasis models[38]. Furthermore, in pancreatic miR-206 mimics and MET-shRNA suppresses the ductal adenocarcinoma (PDAC) mouse models, EMT phosphorylation of AKT/mTOR in A549/DDP cells. In suppression by Snail or Twist deletion leads to an increase contrast, miR-206 inhibitors or MET overexpression in cancer cell proliferation with enhanced expression enhanced phosphorylation of AKT/ mTOR in A549 cells. of nucleoside transporters in tumours, contributing To further investigate the molecular mechanisms linking to enhanced sensitivity to gemcitabine treatment and the PI3K/AKT/mTOR pathway and DDP resistance, increased overall survival of mice[39]. In our models of we detected the expression of MDR1, a multispecific cisplatin resistant lung cancers, we found that EMT gene efflux transporter of drugs, after PI3K selective inhibitor signatures were also significantly correlated with the LY294002 or mTOR inhibitor rapamycin treatmnet in presence of cisplatin resistance and increasing expression A549/DDP cells. The results showed that both these two of MDR1. The results further suggested a close correlation inhibitors reduced the expression of MDR1. In contrast, between EMT and cisplatin resistance in lung cancer cells. miR-206 inhibitors and MET overexpression activated It is becoming increasingly evident that miRNAs the PI3K/AKT/mTOR pathway and increased MDR1 are key modulators of cisplatin resistance and EMT in expression in A549 cells. These results provides a possible lung cancer. For instance, miR-451 is downregulated in mechanism linking miR-206, MET/PI3K/AKT/ mTOR NSCLC tissues and is capable of conferring resistance to pathway, cisplatin resistance, by which downregulated cisplatin in non-small cell lung cancer cell line (A549) expression of multi-drug resistance genes leads to [40]. miR-224 can promote both in vitro and in vivo cisplatin resistance in A549 cells. In addition, We further cisplatin resistance of A549 cells via targeting gene p21 detected the expression of two transcription factors ZEB1 (WAF1/CIP1) and regulating G1/S cell cycle transition and Snail, which are two crucial EMT activators. We and apoptosis [16]. Moreover, it has been reported that demonstrated that decrease of EMT related transcription downregulation of miRNA-27a is responsible for EMT factors, such as ZEB1 and Snail expression is one of the and cisplatin resistance in A549 cells by directly targeting molecular mechanisms from the deregulated miR-206 Raf Kinase Inhibitory Protein (RKIP)[41]. In support of levels to the EMT. the role of miRNAs in cisplatin resistance and EMT in In the present study, we also assessed the anti- lung cancer, our study identified that miR-206 is down- tumour effect of miR-206 in a cisplatin-resistant in vivo regulated and could confer cisplatin resistance and EMT mice model. We found that miR-206 inhibited the MET/ in A549/DDP cells and H1299/DDP cells. AKT/ mTOR pathway and enhanced the A549/DDP cell Targeting MET might be an effective way to sensitivity to cisplatin in vivo. Therefore, these results enhance cisplatin sensitivity in certain tumors. It has been further demonstrated in vivo that miR-206 inhibiting MET reported that MET inhibition in osteosarcoma cancer is and its downstream PI3K/AKT/mTOR pathways is one associated with higher tumor aggressive behavior and potential mechanism to overcome cisplatin resistance in resistance to cisplatin therapy[42], and overexpression lung cancer. To the best of our knowledge, we provided of MET enhances survival of ovarian cancer cells and a first insight into the roles and possible mechanisms of increased resistance to cisplatin[43]. Moreover, HGF miR-206 upregulation in chemosensitivity of A549 cells increases cisplatin resistance via activation of MET in to cisplatin. However, because only two pairs of cisplatin- lung cancer cells[27]. Consistent with these reports, our resistance cell lines were used in our study, further data showed that downregulation of MET reduced EMT investigation in other lung adenocarcinoma cell lines is process and reversed cisplatin resistance in A549/DDP necessary to explore the function and mechanisms of miR- cells and H1299/DDP cells. Interestingly, we found that 206 in cisplatin resistance. www.impactjournals.com/oncotarget 24522 Oncotarget Taken together, our study demonstrated that miR- stage IIIB-IV; first-line chemotherapy either with cisplatin 2 2 206 overexpression in human lung adenocarcinoma 100 mg/m and pemetrexed 500 mg/m or cisplatin 100 2 2 cisplatin resistant cells inhibited the EMT and cisplatin mg/m and gemcitabine 1000 mg/m administered every 3 resistance by targeting MET and suppressing its weeks for a maximum of 5 cycles. Samples were divided downstream PI3K/AKT/mTOR signaling pathway. Low into “sensitive” (complete response or partial response) expression of miR-206 and high levels of MET were and “insensitive” (stable disease or progressive disease) strongly associated with the poor cisplatin sensitivity of groups according to the patient’s responses assessed lung adenocarcinoma patients. Therefore, activation of via medical image analysis and detection of serum miR-206 or inactivation of its target gene pathway may be tumor markers after 4 or 5 cycles of cisplatin-based a potential strategy to reverse cisplatin resistance in human chemotherapy. lung adenocarcinoma cisplatin resistant cells. Transient transfection MATERIALS AND METHODS FAM-labled mimic negative control (mimic NC), miR-206 mimics (mimics), inhibitor NC, miR-206 Cell culture inhibitors, MET silence vectors p-GPU6-MET-shRNA (MET-shRNA), shRNA control, MET (Accession NO: Human lung cancer cell line A549, H1299 and NM_000245) overexpression vector pEZ/M98/neo- A549/DDP were obtained from China Center for Type MET (ex-MET) and the ex-control were purchased Culture Collection (CCTCC, Shanghai, China). All the from GenePharma (Shanghai, China). The MET shRNA cells were cultured in RPMI-1640 medium supplemented sequences were designed as showed in Supplimentary with 10% fetal bovine serum (FBS, Gibco, USA) in a Table 2. The cells were seeded into 6-well plates and humidified atmosphere containing 5% CO at 37 °C. To transfected with 75 pmol oligonucleotides or 2.5μg establish cisplatin-resistant H1299 cell lines, H1299 cells shRNA vectors using Lipofectamine 2000 (Invitrogen, were first treated with 0.6 μM of cisplatin (DDP, Sigma, USA) according to the instructions provided by the St. Louis, MO), and then were treated with increased manufacturer. The cells were used for further analysis 48h concentrations of DDP in a stepwise manner during each after transfection. passage. To maintain the drug-resistant phenotype, DDP (with final concentration of 2μM) was added to the culture Quantitative real-time PCR analysis media for A549/DDP cells and H1299/DDP cells. Total RNA was isolated with Trizol reagent Reagents and antibodies (Invitrogen, USA). The concentration and purity of the RNA samples were determined spectroscopically. MET inhibitor (SU11274) and PI3 kinase inhibitor Expression of mature miRNA was assayed using (LY294002) were purchased from Selleck Chemicals stem-loop RT followed by real-time PCR analysis. (Houston,TX). Primary antibodies phospho-MET The SYBR and U6 gene were used for detecting the (Y1234/35), phospho-AKT (S473), phospho-mTOR gene amplification and normalizing the each sample, (S2448), MET, AKT, mTOR, E-cadherin, N-cadherin, respectively. The primers for RT-PCR were designed Vimentin, ZEB1, Snail and GAPDH were purchased based on the miR-206 sequences provided by the Sanger from Cell Signaling Technology (Beverley, MA). MDR1 Center miRNA Registry. The RT primers were designed antibody was purchased from Santa Cruz Biotechnology as showed in Supplimentary Table 3. qRT-PCR was (Santa Cruz, CA). Secondary antibodies, HRP-conjugated performed on the ABI (Applied Biosystems) 7900 HT goat anti-mouse IgG and goat anti-rabbit IgG, were Thermal cycler in standard mode for 40 cycles. The fold obtained from Jackson (West Grove, PA). -ΔΔCt change was calculated using the 2 Method. PCR was performed in triplicate. Tissue samples A total of 34 lung adenocarcinoma tissues Luciferase reporter assay were collected from patients with advanced lung Based on the miRNA databases (microRNA.org, adenocarcinoma who received chemotherapy at The miRDB and TargetScan database), MET is a predicted 117th Hospital of PLA (Hangzhou, China) between June target of miR-206 in humans. According to the results of 2013 and June 2014. Informed consent was obtained from prediction, we cloned MET 3'UTR fragment containing all subjects and thisstudy was approved by the Clinical the predicted site into pGL3 luciferase reporter vector Research Ethics Committee of The 117th Hospital of (pmirGLO3, Promega, Madison, USA) and named as PLA. Patients met all of the following criteria: primary MET-wt. We cloned MET 3'UTR fragment with mutant lung adenocarcinoma; histological diagnosis of lung sequence into pmirGLO3 luciferase reporter vector and adenocarcinoma with at least 1 measurable lesion; clinical www.impactjournals.com/oncotarget 24523 Oncotarget named as MET-mut. In addition, miR-206 inhibitor by SDS-polyacrylamide gel electrophoresis(PAGE) sequence was also cloned into pmirGLO3 luciferase and transferred to a polyvinylidene dfluoride (PVDF) reporter vector as a positive control (PC). For luciferase membrane (Millipore, Germany). The blots were blocked assay, the reporter plasmid was cotransfected with miR- in BSA (5% w/v in PBS + 0.1% Tween 20) for 1 h at 206 mimics or mimic NC in A549 cells. After 48 h, cells room temperature and immunostained with antibodies at were harvested, and the luciferase activity was measured 4 °C overnight. Immunoreactive bands were visualized using the Dual-Luciferase Reporter Assay System by enhanced chemiluminescence (Millipore, Germany) (Promega,Madison, USA). according to the manufacturer’s instructions. Data were normalized to GAPDH. Migration and invasion assay Immunohistochemistry Wound healing experiment and Transwell insert (24- well insert; pore size 8μm, Corning, USA) assays were Tissue slides were incubated for 2 h at 56 °C and de- used to determine the migration and invasion abilities paraffinized. Antigen retrieval was obtained by microwave of the cells, respectively. Briefly, for the wound healing treatment in citrate buffer for 15 min to retrieve experiment, cells were grown to confuence wounded antigenicity. After peroxidase activity was blocked with using a pipette tip and photographed at 0 h and subsequent 3% H O /methanol for 10 min, sections were incubated 2 2 time points. Cell migration was evaluated by measuring with normal goat serum for 20 min to block non-specific the width of the wound at the identical position. For the antibody binding sites. Sections were incubated with the invasion assay, the lower chambers of matrigel-coated primary antibodies for 1 h at 25°C followed by incubations invasion plates were used. Cells (50,000) were added to with biotinylated anti-rabbit/mouse IgG and peroxidase- the upper chamber in serum-free media and invasion at labelled streptavidin for 10 min each. The percentage of 37 °C towards 10% FBS-containing growth media was the cells with cytoplasmic labeling was recorded from determined after 24 h. Cells that invaded through the two areas of each specimen, and the labeling intensity was membrane were fixed, stained with crystal violet and estimated as 1+, 2+ or 3+. The immunohistochemistry photographed. The invaded cells were counted by Image results were categorized into two groups: the samples J software. All experiments were carried out in triplicate. without any labeling, 1+ labeling in <25% cells, and 2+ labeling in <5% cells were considered negative; all the remaining samples were defined as positive. In vitro drug sensitivity assay The cells were plated in 6-well plates (3×10 cells/ Animal studies well) and 75 pmol of the miR-206 mimic or negative control were transfected into the A549/DDP cells, while a miR-206 All experimental procedures used in this study inhibitors or inhibitor negative control were transfected into had been approved by the ethics committee in the 117th the A549 cells, using Lipofectamine-2000 (Invitrogen, USA) Hospital of PLA. Male nude mice (BALB/c, 4-6wk) were according to the manufacturer's instructions. Twenty-four purchased from Shanghai Laboratory Animal Center hours after transfection, the cells were seeded in 96-well (Shanghai, China). For preparation of subcutaneous plates (5×10 cells/well) for the following experiment. After xenograft model, 0.2 ml A549/DDP lung cancer cells (2.0 cell adhesion, freshly prepared anticancer drug (cisplatin; × 10 ) in phosphate buffered saline/100 μl were injected Qilu Pharmaceutical Co., Ltd., Jinan, China) was added at subcutaneously into the right flank of the nude mice. 15 a final concentration of 2, 4, 8, 16, 32 μM for A549/DDP, days after cell inoculation, total of 20 mice were divided and 1, 2, 4, 8, 16 μM for A549 cells. Forty-eight hours after randomly into four groups (five mice per group). miR-206 the addition of the drug, cell viability was assessed by the agomirs and miR-206 agomir negative control (NC) (2 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium nmol; Genepharma, Shanghai, China) were given locally bromide (MTT) assay. The absorbance at 490 nm (A490) by direct injection into the xenografts every two days. of each well was read on a spectrophotometer. The Meanwhile, cisplatin was administered via intraperitoneal concentration at which the drug produced 50% inhibition of injection at a dose of 5 mg/kg every other day. After 30 growth (IC50) was estimated by the relative survival curve. days of treatment, all mice were sacrificed. Transplanted Three independent experiments were performed in duplicate. tumors were excised, and the wet weight was recorded. Protein expression in tumors was detected by western blotting assay. Western blot analysis Cells were washed in PBS and lysed in RIPA lysis Statistical analysis buffer supplemented with protease inhibitor cocktail (Roche, Germany). Total protein was quantfied by All statistical analyses were performed using SPSS BCA Protein Assay Kit (Beyotime, Nanjing, China), 13.0. Numerical data were presented as mean ± SD. 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Zhang J, Liang Q, Lei Y, Yao M, Li L, Gao X, Feng J, Zhang Y, Gao H, Liu DX, Lu J, Huang B. SOX4 induces www.impactjournals.com/oncotarget 24526 Oncotarget http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Oncotarget Unpaywall

miR-206 regulates cisplatin resistance and EMT in human lung adenocarcinoma cells partly by targeting MET

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www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 17 miR-206 regulates cisplatin resistance and EMT in human lung adenocarcinoma cells partly by targeting MET 1,* 1,* 1,* 1,* 1 Qing-yong Chen , De-min Jiao , Jian Wang , Huizhen Hu , Xiali Tang , 1 1 2 Jun Chen , Hao Mou , Wei Lu Department of Respiratory Disease, The 117th Hospital of PLA, Hangzhou, Zhejiang, 310013, P.R. China Department of Oncology, The 117th Hospital of PLA, Hangzhou, Zhejiang, 310013, P.R. China These authors have contributed equally to this work Correspondence to: Qingyong Chen, e-mail: [email protected] Wei Lu, e-mail: [email protected] Keywords: miR-206, cisplatin resistance, epithelial-mesenchymal transition (EMT), MET, lung adenocarcinoma Received: April 27, 2015 Accepted: March 04, 2016 Published: March 21, 2016 ABSTRACT MicroRNAs (miRNAs) play a critical role in drug resistance and epithelial- mesenchymal transition (EMT). The aims of this study were to explore the potential role of miR-206 in governing cisplatin resistance and EMT in lung cancer cells. We found that both lung adenocarcinoma A549 cisplatin-resistant cells (A549/DDP) and H1299 cisplatin-resistant cells (H1299/DDP) acquired mesenchymal features and were along with low expression of miR-206 and high migration and invasion abilities. Ectopic expression of miR-206 mimics inhibited cisplatin resistance, reversed the EMT phenotype, decreased the migration and invasion in these DDP-resistant cells. In contrast, miR-206 inhibitors increased cisplatin resistance, EMT, cell migration and invasion in non-DDP-resistant cells. Furthermore, we found that MET is the direct target of miR-206 in lung cancer cells. Knockdown of MET exhibited an EMT and DDP resistant inhibitory effect on DDP-resistant cells. Conversely, overexpression of MET in non-DDP- resistant cells produced a promoting effect on cell EMT and DDP resistance. In lung adenocarcinoma tissues, we demonstrated that low expression of miR-206 were also correlated with increased cisplatin resistance and MET expression. In addition, we revealed that miR-206 overexpression reduced cisplatin resistance and EMT in DDP-resistant cells, partly due to inactivation of MET/PI3K/AKT/mTOR signaling pathway, and subsequent downregulation of MDR1, ZEB1 and Snail expression. Finally, we found that miR-206 could also sensitize A549/DDP cells to cisplatin in mice model. Taken together, our study implied that activation of miR-206 or inactivation of its target gene pathway could serve as a novel approach to reverse cisplatin resistance in lung adenocarcinomas cells. such as high efficiency, mild side effects and easy INTRODUCTION administration. However, cisplatin resistance often occurs in clinical practice [2]. Thus, adjuvant therapy Lung cancer, predominantly non-small cell lung to enhance cisplatin efficiency becomes an important cancer (NSCLC), is the leading cause of cancer-related chemotherapeutic strategy. mortality worldwide. Patients with NSCLC are mostly Accumulating studies indicate that there are several treated with platinum-based chemotherapy in combination major mechanisms of drug resistance in cancer cells, with radiation therapy. However, the development of such as increased detoxification of anticancer drugs by chemoresistance, either intrinsic or acquired, is a major glutathione system, defective apoptosis pathway, increased obstacle limiting successful treatment [1]. Cisplatin (DDP) levels of DNA repair or DNA tolerance, decreased uptake is still one of the commonly used chemotherapeutic agents of water-soluble drugs and enhanced drug efflux from against lung cancer due to its therapeutic advantages, www.impactjournals.com/oncotarget 24510 Oncotarget cancer cells mediated by ATP-binding cassette (ABC) In this study, we found miR-206 was down- transporters[3–5]. Several studies showed that the drug- regulated in both A549/DDP cells and H1299/DDP cells. resistant cancer cells display features of epithelial- Overexpression of miR-206 or knockdown of its target mesenchymal transition (EMT), which is defined by the MET reversed the mesenchymal features and sensitized loss of intracellular links along with the gain of migratory DDP-resistant cells to cisplatin. More importantly, we and invasive abilities [6, 7]. Specifically, cells with demonstrated that decreased miR-206 levels induced decreased expression of the epithelial marker E-cadherin cisplatin resistance and EMT phenotype due to activation and increased expression of mesenchymal molecules of MET/PI3K/AKT/mTOR axis, upregulation of MDR1, including Snail/Snai1, Slug/Snai2, Vimentin, zinc-finger ZEB1 and Snail expression in DDP-resistant cells. E-box binding homeobox 1 (ZEB1), lead to enhanced These results provide new insights into the molecular motility, invasion and drug resistance[8]. In addition, mechanisms of cisplatin resistance induced by decreased PI3K/AKT/mammalian target of rapamycin (mTOR) miR-206 levels in lung adenocarcinoma cells and suggest signaling has also found to confer resistance to DDP-based miR-206 and its target gene pathway could be novel treatment in many cancers [9, 10]. Furthermore, it has therapeutic targets to reverse cisplatin resistance of lung been reported that there exist a cross-talk between EMT adenocarcinoma cells. programming and PI3K/AKT/mTOR pathway [11]. The microRNAs (miRNAs) are a group of small RESULTS non-coding RNAs. The basic mechanism of miRNA action is that miRNA could bind to the 3'UTR of target Cisplatin resistant lung adenocarcinoma cells mRNAs, resulting in translational repression or target exhibit EMT features and have enhanced MDR1 mRNA cleavage[12]. Recent studies suggested that the expression acquisition of drug resistance by cancer cells might be modulated via the changes in miRNA levels[13, 14]. Previous studies have demonstrated that For instance, miR-135a/b are downregulated in cisplatin chemotherapeutic drug can induce EMT, enhance resistance (A549/DDP) cells, and the overexpression of invasive ability, resulting in drug resistance [31, 32]. miR-135a/b sensitizes A549/DDP cells to cisplatin by Snail and ZEB1 are two crucial EMT inducers [33]. targeting MCL1 (myeloid cell leukemia 1) [15]. Up- Multi-drug resistance gene 1 (MDR1, ABCB1), encoding regulation of miR-27a can suppress RKIP (Raf kinase P-glycoprotein (P-gp), is one of pharmaceutical inhibitory protein) expression and in turn contribute carriers that can decrease the effective intracellular to chemoresistance of lung adenocarcinoma cells to concentration of the drug, leading to drug resistance cisplatin[16]. Upregulation of miR-451 expression [34]. To determine the mechanism of cisplatin resistance inactivates the AKT signaling pathway and enhanced in lung adenocarcinoma cells, we first compared A549/ cisplatin induced apoptosis in A549 cells[17]. MiR- DDP cells and H1299/DDP cells with its parental cells 513a-3p can sensitize human lung adenocarcinoma in cisplatin sensitivity, MDR1 expression levels, EMT cells to cisplatin by targeting GSTP1 (Glutathione morphology and related markers expression. MTT S-transferase P1)[18]. MiR-92b is significantly up- assay showed that A549/DDP and H1299/DDP cells regulated in lung cancer cells and knockdown of miR- exhibited significantly higher resistance to cisplatin 92b inhibits cell growth and sensitizes the A549/DDP than non-DDP-resistant cells (Figure 1A). IC50 of cells to DDP by target PTEN (phosphatase and tensin cisplatin in A549/DDP cells was 2.60 fold higher than homolog) [19]. that in A549 cells and IC50 of cisplatin in H1299/DDP MiR-206 is one of the most studied and best cells was 2.69 fold higher than that in H1299 cells. characterized miRNAs to date, which specifically Western blotting showed that DDP-resistant cells had expressed in skeletal muscle[20]. Recently, there has higher levels of MDR1 protein expression than their been increasing interest in understanding the role of miR- non-DDP-resistant cells (Figure 1B). Furthermore, 206 in cancer, and down-regulation of miR-206 has been A549 cells and H1299 lung adenocarcinoma cells observed in different types of cancers [21–27]. Decreased display epithelial characteristics, whereas A549/DDP expression of miR-206 in gastric cancer is associated and H1299/DDP cells exhibited elongated, fibroblastoid with tumor progression and poor survival [24]. miR-206 morphology and separated from one another (Figure suppresses breast cancer cell migration and invasion by 1C). Morphological conversion DDP-resistant cells targeting Cdc42 [28]. miR-206 can inhibit the expression associated with EMT were also reflected by changes of VEGF and regulate the apoptosis and migration of in protein levels. Western blotting showed that A549/ laryngeal cancer cells[29]. We and others have also DDP exhibited downregulation of E-cadherin levels reported miR-206 overexpression could inhibit invasion and upregulation of N-cadherin, Vimentin, Snail of lung cancers [22, 26, 30]. However, whether miR-206 and ZEB1 (Figure 1D). H1299/DDP cells also have is involved in regulating cisplatin resistance and EMT in upregulation of N-cadherin, Vimentin, Snail and ZEB1 human lung adenocarcinomas remains unclear. www.impactjournals.com/oncotarget 24511 Oncotarget than H1299 cells, but E-cadherin expression could not miR-206 overexpression reverses cisplatin be detected (Supplementary Figure 1A). Additionally, resistance, EMT, migration and invasion in wound healing assay and transwell invasion assay DDP-resistant cells demonstrated that the migration and invasion abilities were significantly stronger in A549/DDP cells and miR-206 has been found to be down-regulated in H1299/DDP cells (Figure 1E-1F, Supplementary Figure many types of cancers including lung cancer[21-27, 30]. 1B-1C). To determine whether miR-206 plays a pivotal role in drug Figure 1: Differences between DDP-resistant cells and non-DDP-resistant cells. A. Two DDP-resistant cells and their parental cells were treated with indicated concentrations of cisplatin for 48 h and then were subjected to MTT assay (n = 5). The results showed that A549/DDP and H1299/DDP cells were more resistant to cisplatin than their parental cells in vitro. B. Western blotting illustrated increased expression of MDR1 in A549/DDP and H1299/DDP cells. C. A549 and H1299 cells displayed epithelial morphology, but A549/DDP and H1299/DDP cells exhibited fibroblastic morphology (original magnification, ×200). D. Western blotting showed increased expression of N-cadherin, Vimentin, ZEB1, Snail and reduced expression of E-cadherin in A549/DDP cells. E. Wound healing assay and F. transwell invasion assay revealed significant enhancement of migration and invasion ability in A549/ DDP cells. Data are means of three separated experiments ± SD, * P <0.05, ** P <0.01 1. compared with A549 cell group. www.impactjournals.com/oncotarget 24512 Oncotarget resistance in lung cancer cells, we measured the expression miR-206 target site in the 3′-UTR of MET mRNA was of miR-206 in the A549/DDP cells, H1299/DDP cells and responsible for its regulation, we cloned MET 3′-UTR their parental cells. Real-time PCR assay revealed that wild type (MET-wt) or 3′-UTR mutant type (MET-mut) miR-206 was significantly lowered in both A549/DDP into downstream of the luciferase reporter gene and cells and H1299/DDP cells (Figure 2A, Supplementary cotransfected with miR-206 mimics into A549 cells. A Figure 2A) compared with their parental cells. To further luciferase reporter containing miR-206 inhibitor sequence validate the role of miR-206 in cisplatin resistance, we was used as a positive control (PC). As indicated in Figure transfected miR-206 mimics into A549/DDP cells and 3A. Luciferase activity from a construct harboring miR- H1299/DDP cells, transfected miR-206 inhibitors into 206 inhibitor sequence (PC group) was significantly A549 cells and H1299 cells. MTT assay revealed that decreased in A549 cells expressing either miR-206 or its miR-206 mimics treatment led to significantly decreased negative control form. Luciferase activity from A549 cells resistance of A549/DDP cells and H1299/DDP cells cotransfected with miR-206 and the construct containing to cisplatin, whereas miR-206 inhibitors transfection MET-mut form did not induce any significant change enhanced the resistance of A549 cells and H1299 cells in luciferase activity, whereas luciferase activity from to cisplatin (Figure 2B, Supplementary Figure 2B-2C). A549 cells cotransfected with miR-206 and the construct Furthermore, western blotting showed that miR-206 containing MET-wt was decreased by more than 95% mimics significantly decreased the expression of MDR1 when compared to negative control cells. These results in A549/DDP cells and H1299/DDP cells, while miR-206 indicate that miR-206 regulates MET protein in A549 cells inhibitors increased the expression of MDR1 in A549 cells by directly targeting MET 3′-UTR. and H1299 cells (Figure 2C, Supplementary Figure 2D). On the other hand, we found that A549/DDP cells Previous studies have shown that the drug-resistant and H1299/DDP cells expressed higher levels of MET cancer cells display features of epithelial-mesenchymal protein than A549 cells and H1299 cells (Figure 3B), transition (EMT)[32, 35, 36]. Here, we observed that but basal expression of p-MET is undetectable in these miR-206 mimics transfection led to a change from cell lines (data not shown). Western blotting showed that elongated, fibroblastoid morphology to a rounded shap miR-206 overexpression could significantly decrease in both A549/DDP cells and H1299/DDP cells, whereas MET expression in A549/DDP and H1299/DDP cells. miR-206 inhibitors transfection resulted in an elongated Meanwhile, down-regulated miR-206 could increase MET fibroblast-like morphology of A549 cells and H1299 cells expression level in A549 cells and H1299 cells (Figure (Figure 2D, Supplementary Figure 2E). Furthermore, 3C). These findings further confirmed the existence of an miR-206 mimics treatment caused the higher expression inverse correlation between the expression of miR-206 of E-cadherin and lower expression of mesenchymal and MET expression in these cell lines. markers including Vimentin, Snail and ZEB1 in A549/ DDP cells. Also, miR-206 mimics decreased the Low expression of miR-206 in lung expression of N-cadherin, Vimentin, Snail and ZEB1 in adenocarcinoma tissues correlates with increased H1299/DDP cells (Figure 2E, Supplementary Figure 2F). cisplatin resistance and MET expression On the contrary, miR-206 inhibitors reduced E-cadherin expression, induced the expression of Vimentin, ZEB1 To better understand the association between and Snail in A549 cells, while induced N-cadherin, miR-206 and cisplatin resistance, a total of 34 clinical Snail and ZEB1expression in H1299 cells (Figure 2F, lung tumor tissue samples were collected from patients Supplementary Figure 2G). In addition, invasion and with advanced lung adenocarcinoma and divided into migration assay further demonstrated that miR-206 mimics “sensitive” and “insensitive” groups according to the suppressed the invasion and migration of A549/DDP patient’s response to cisplatin-based chemotherapy. cells and H1299/DDP cells (Figure 2G, Supplementary As shown in Figure 4A, miR-206 was significantly Figure 3A-3B), whereas miR-206 inhibitors enhanced down-regulated in the “insensitive” group tissues (n the invasion and migration of A549 cells and H1299 cells = 17) compared with that in the “sensitive” group (n = (Figure 2H, Supplementary Figure 3C-3D). These results 17). Importantly, immunohistochemistry assay showed indicated that miR-206 could reverses cisplatin resistance, that 16 of 17 “insensitive” group tissues (94.12%) had EMT, migration and invasion of cisplatin resistant cells. positive immunostaining of MET protein but it was 4 of 17 (23.52%) in “sensitive” group (Figure 4B). Moreover, the stronger immunoreactivity of MET was significantly MET gene is a target of miR-206 in lung associated with lower miR-206 expression (r = 0.4086, cancer cells P = 0.0165, Figure 4C), suggesting that miR-206-MET Identification of miRNA-regulated gene targets is interaction might be biologically significant in cisplatin a necessary step to understand miRNA functions. Based resistance. In addition, IHC staining for MET, MDR1, on target prediction programs, we found that MET is a E-cadherin and Vimentin were performed on 5 samples tentative target of miR-206. To test whether the predicted of each group. We found that except Vimentin, all five www.impactjournals.com/oncotarget 24513 Oncotarget Figure 2: miR-206 decreased cisplatin resistance, EMT, migration and invasion of A549/DDP cells. A. qRT-PCR assay showed a significant down-regulation of miR-206 in A549/DDP cells compared with in A549 cells. B. A549/DDP cells were transfected with miR-206 mimics, and A549 cells were transfected with miR-206 inhibitors. After 24 hrs of transfection, 5×10 cells/well were seeded in 96-well cell culture plates. The next day, cells were incubated with or without the indicated concentration of cisplatin for 48 h and subsequently subjected to an MTT assay. (C-F) A549/DDP cells or A549 cells were transfected with the indicated plasmid. After 48 h, C. the expression of MDR1 was determined by Western blotting analysis. D. Cell morphology was observed by microscopy (Original magnification, ×200). E-F. Western blotting analysis was used to detect the expression of E-cadherin, N-cadherin, Vimentin, ZEB1 and Snail (Left panel), Quantitative results are illustrated for left panel. (G-H) Wound healing assays (Left panel) and invasion assay (Right panel) were used to detect the migration and invasion ability in G. miR-206 mimics transfected A549/DDP cells or H. miR-206 inhibitors transfected A549 cells. Data are means of three separated experiments ± SD, * P <0.05, ** P <0.01 compared with their control. www.impactjournals.com/oncotarget 24514 Oncotarget cisplatin “insensitive” tissues have higher MET and the expression of MET was significantly suppressed by MDR1 expression, and lower E-cadherin expression. MET-shRNA, and activated by MET expression vector In contrast, 3 out of 5 cisplatin “sensitive” tissues have (ex-MET) (Figure 5A, Supplementary Figure 5A). lower MET and MDR1 expression, and higher E-cadherin MET inhibitor SU11274 treatment partially reversed expression (Supplementary figure 4). the mesenchymal phenotype of A549/DDP cells. MET silence also reversed the mesenchymal phenotype in both A549/DDP cells and H1299/DDP cells, while MET MET mediated EMT and cisplatin resistance overexpression induced a mesenchymal phenotype in To determine whether the anti-EMT and anti- A549 cells and H1299 cells (Figure 5B, Supplementary cisplatin resistance effects of miR-206 on DDP-resistant Figure 5B). Western blotting analysis showed that both cells could be partly explained by its targeting of MET, MET inhibitor SU11274 and MET-shRNA increased we first analyzed how MET inhibitors, MET silence and the E-cadherin protein expression, but decreased the MET overexpression affected EMT. Our data showed that expression of mesenchymal markers including N-cadherin, Figure 3: MET is a direct target of miR-206. A. Luciferase assay was performed in A549 cells that were cotransfected with miRNA mimics and reporter vectors carrying MET 3'UTR wild type (MET-wt), MET 3'UTR mutated type (MET-mut), and miR-206 inhibitor sequences (positive control) element. B. Variable MET expression in A549 and A549/DDP was obtained by Western blotting analysis. C. A549/DDP cells were transfected with miR-206 mimics and A549 cells were transfected with miR-206 inhibotors for 48 h respectively. Western blotting was used to detect MET expression. Data are means of three separated experiments ± SD, ** P <0.01, compared with negative control (NC). www.impactjournals.com/oncotarget 24515 Oncotarget Vimentin, ZEB1 and Snail (Figure 5C-5D, Supplementary silence decreased the MDR1 expression(Supplementary Figure 5D). In contrast, MET overexpression decreased Figure 5E), suppressed cell migration and invasion the E-cadherin protein expression, but increased the (Supplementary Figure 6A-6B). While MET expression of N-cadherin, Vimentin, ZEB1 and Snail overexpression increased MDR1 protein expression in A549 cells (Figure 5E). Similarly, MET-shRNA (Supplementary Figure 5F), enhanced the capability of decreased the expression of N-cadherin, Vimentin, ZEB1 H1299 cell migration and invasion(Supplementary Figure and Snail (Supplementary Figure 5B). In contrast, MET 6C-6D). overexpression increased the expression of N-cadherin, Vimentin, ZEB1 and Snail in A549 cells (Supplementary miR-206 inhibits EMT and cisplatin resistance Figure 5C). However, E-cadherin expression could not be via MET dependent PI3K/AKT /mTOR detected in both conditions. signaling pathways We next analyzed the effect of MET inhibitors, MET silence and MET overexpression on cell cisplatin PI3K/AKT/mTOR is one of the definite downstream sensitivity, migratory and invasive capability. As shown targets of MET receptor. Inhibition of PI3K/AKT in Figure 6, SU11274 significantly promoted cell growth signaling has proven to be an efficient way to attenuate inhibition induced by cisplatin (Figure 6A) and decreased the resistance of chemotherapy[37]. In present study, we the MDR1 expression in A549/DDP cells (Figure 6B). found that AKT/mTOR pathway is activated in A549/ Similarly, MET shNA transfection also increased the DDP cells and H1299/DDP cells compared with the sensitivity of A549/DDP cells to cisplatin (Figure 6D) parental cells (Figure 7A, Supplementary Figure 7A). and decreased the MDR1 expression (Figure 6E). IC50 To determine whether miR-206 inhibits MET dependent in SU11274 (0.5μM) group and MET shRNA group were PI3K/AKT/mTOR signaling pathways, we analyzed the 10.58μM and 3.53μM, significantly lower than their DDP effects of miR-206 or MET expression changes on PI3K/ control groups (12.82μM and 10.06μM, respectively. AKT/mTOR signaling. The results showed that both Supplementary Table 1). Furthermore, SU11274 miR-206 mimics transfection and MET-shRNA treatment treatment and MET shRNA transfection showed similar significantly decreased AKT, p-AKT, mTOR and p-mTOR effects on suppressing migration and invasion of A549/ protein levels in A549/DDP cells (Figure 7B-7C,), and DDP cells (Figure 6C and Figure 6F). In contrast, MET MET inhibitor SU11274 also resulted in a decrease in overexpression increased the cisplatin resistance and p-AKT, p-mTOR protein expression(Figure 7D). In MDR1 protein expression (Figure 6G-6H, Supplementary contrast, miR-206 inhibitors and MET overexpression Table 1), enhanced the capability of cell migration increased p-AKT, mTOR and p-mTOR protein levels in and invasion (Figure 6I). In H1299/DDP cells, MET A549 cells (Figure 7E-7F). Although the degree of gene Figure 4: Low expression of miR-206 in lung adenocarcinoma tissues correlates with increased cisplatin resistance and MET expression. A. Expression levels of miR-206 and B. MET protein were detected in cisplatin “sensitive” (a,n =17) and “insensitive” (b,n = 17) lung adenocarcinoma tissues via qRT-PCR (normalized to U6 RNA) and immunostaining (Origninal magnification, ×100), respectively. C. The immunoreactivity of MET protein in cisplatin “sensitive” and “insensitive” tissues showed a statistically significant inverse correlation with the relative expression level of miR-206. Data are means of three separated experiments ± SD, * P <0.05. www.impactjournals.com/oncotarget 24516 Oncotarget Figure 5: MET is involved in miR-206 inhibited EMT. A. Western blotting analysis confirmed that the expression of MET was suppressed by MET-shRNA, and activated by MET expression vector (ex-MET). (B-E) A549/DDP cells were treated with MET inhibitor SU11274, or transfected with MET shRNA for 48h, and the A549 cells were transfected with MET expression vector (ex-MET) for 48h, B. cell morphological changes associated with EMT are shown in the phase contrast image (Original magnification, ×200). C-E.The expression of E-cadherin, N-cadherin, Vimentin, ZEB1, Snail were examined by western blotting. Data are means of three separated experiments ± SD, * P <0.05, ** P <0.01 compared with their control. www.impactjournals.com/oncotarget 24517 Oncotarget expression and protein levels varied between different cell (Figure 7G-7H). Furthermore, both LY294002 (0.5μM) lines, similar results were also found in H1299/DDP and and rapamycin (10nM) enhanced cisplatin sensitivity, H1299 cell lines (Supplementary Figure 7B-7C). decreased the MDR1 expression in A549/DDP cells To further support the role of PI3K/AKT/mTOR (Figure 8A–8B). IC50 decreased from 11.12 μM to 7.78 signaling in suppression of EMT and cisplatin resistance μM for LY294002, and decreased from 11.12 μM to 8.31 by miR-206, PI3K selective inhibitor LY294002 and μM for rapamycin (Supplimentary Table 1). In addition, mTOR inhibitor rapamycin was utilized in A549/ both LY294002 and rapamycin reversed mesenchymal DDP cells. LY294002 (10μM, 20μM) were observed to characteristics (Figure 8C), decreased the expression of remarkably reduce the protein of p-AKT and p-mTOR, mesenchymal markers (Figure 8D-8E), and inhibited the and rapamycin to reduce p-mTOR in A549/DDP cells migration and invasion of A549/DDP cells (Figure 8F). Figure 6: MET is involved in miR-206 inhibited cisplatin resistance. A549/DDP cells were treated with MET inhibitor SU11274 (indicated concentration), or were transfected with MET shRNA for 48h, and A549 cells were transfected with MET expression vector (ex-MET) for 48h, A, D and G. the changes of cisplatin sensitivity, B, E and H. MDR1 expression, C, F and I. migration and invasion ability in each groups were detected. Data are means of three separated experiments ± SD, * P <0.05, ** P <0.01 compared with their control. www.impactjournals.com/oncotarget 24518 Oncotarget Figure 7: miR-206/MET regulated PI3K/AKT/mTOR pathway. Western blotting analysis was performed to detect the protein expression of AKT, p-AKT, mTOR, p-mTOR in A. A549/DDP cells and A549 cells, B. miR-206 mimics transfected A549/DDP cells, C. MET shRNA transfected A549/DDP cells, D. MET inhibitors SU11274 treated A549/DDP cells, E. miR-206 inhibitors transfected A549 cells, F. MET overexpression vectors (ex-MET) transfected A549 cells, G. PI3K inhibitor LY294002 treated A549/DDP cells, H. mTOR inhibitor rapamycin treated A549/DDP cells. Cell lysates were collected 48 h after transfection or 2h after treatment with MET, PI3K and mTOR inhibitors. Data are means of three separated experiments ± SD, * P <0.05, ** P <0.01 compared with their control. www.impactjournals.com/oncotarget 24519 Oncotarget Figure 8: PI3K/AKT/mTOR pathway is involved in miR-206/MET regulated cisplatin resistance, EMT, migration and invasion. A549/DDP cells were treated with LY294002 (indicated concentration) or rapamycin (indicated concentration) respectively. After 48h, A. The changes of cisplatin sensitivity, B. MDR1 expression, C. cell morphology (Original magnification, ×200), D-E. E-cadherin, N-cadherin, Vimentin, ZEB1 and Snail expression, F. migration and invasion ability in each group of cells were detected. Data are means of three separated experiments ± SD, * P <0.05, ** P <0.01 compared with their control. www.impactjournals.com/oncotarget 24520 Oncotarget These data indicated the involvement PI3K/AKT/mTOR decreased in the miRNA-206 plus cisplatin group pathway in suppression of EMT and cisplatin resistance compared with DDP combined mimic NC group or DDP by miR-206. group. (Figure 9C-9D). These results further suggest that miR-206 and its downstream MET/AKT/mTOR pathway play important roles in controlling A549/DDP cells miR-206 enhances A549/DDP cells to cisplatin cisplatin sensitivity. sensitivity in vivo To further investigate the effect of miR-206 DISCUSSION expression on cisplatin sensitivity, we evaluated the in vivo antitumor activity of miR-206 in xenograft model. Previous studies have reported that miR-206 could As shown in Figure 9A-9B, The in vivo results parallel the act as a tumor-suppressor in various cancers including in vitro results and show that miR-206 resulted in dramatic lung cancer[21–30]. We and others have demonstrated tumor regressions compared with both negative control or that miR-206 overexpression inhibited invasion and DDP control group. Furthermore, the expression of MET, metastasis in lung cancer cells[26, 30]. In the current AKT, p-AKT, mTOR and p-mTOR were significantly study, we demonstrated that miR-206 could suppress EMT Figure 9: miR-206 enhances A549/DDP cells to cisplatin sensitivity in vivo. A. The gross morphology of tumor samples B. The final xenograft tumor weights were measured after 30 days of treatment. C. MET/AKT/ mTOR pathway proteins expression in excised xenograft tumor were detected by western blotting assay. D. Relatve expression levels of MET/AKT/mTOR pathway proteins. sample1, 2, 3 stand for mouse tumor tissues from each group. Data are means of three separated experiments ± SD, (* P < 0.05, ** P < 0.01 compared with indicated control). www.impactjournals.com/oncotarget 24521 Oncotarget process and cisplatin resistance of lung adenocarcinoma MET is also one of the targets of miR-206. Despite MET cells, partly through targeting MET and its downstream has recently been identified as a target of miR-206 in PI3K/AKT/ mTOR pathway both in vitro and in vivo. Our rhabdomyosarcoma cells [44]. We further demonstrated data provide a first insight into the function of miR-206 that miR-206 directly target MET in lung cancer A549 in regulating cisplatin resistance and EMT in cisplatin cells. In addition, our results in clinical lung cancer tissue resistant lung adenocarcinoma cells. samples show that the decreased expression of miR-206 The EMT is a molecular process through which closely correlated with increased MET expression and an epithelial cell undergoes transdifferentiation into poor cisplatin sensitivity. These data suggest that miR- a mesenchymal phenotype. Recent evidences have 206-MET play important roles in regulating EMT and suggested that EMT processes may play an important cisplatin resistance in lung adenocarcinoma cells. role in the development of chemoresistance. It has PI3K/AKT/mTOR is a crucial downstream been reported knockdown of snail and slug reverses pathway of MET and can regulate many of the biological the EMT phenotype and reduces ovarian cancer phenomena, such as cell proliferation and survival, cell resistance to cisplatin[17]. Inhibiting EMT by motility and migration, and tumor cell invasion. In the overexpressing the microRNA miR-200 could abrogate present study, we found miR-206-MET axis regualted cyclophosphamide resistance in spontaneous breast-to- PI3K/AKT/mTOR pathway in lung cancer cells. Both lung metastasis models[38]. Furthermore, in pancreatic miR-206 mimics and MET-shRNA suppresses the ductal adenocarcinoma (PDAC) mouse models, EMT phosphorylation of AKT/mTOR in A549/DDP cells. In suppression by Snail or Twist deletion leads to an increase contrast, miR-206 inhibitors or MET overexpression in cancer cell proliferation with enhanced expression enhanced phosphorylation of AKT/ mTOR in A549 cells. of nucleoside transporters in tumours, contributing To further investigate the molecular mechanisms linking to enhanced sensitivity to gemcitabine treatment and the PI3K/AKT/mTOR pathway and DDP resistance, increased overall survival of mice[39]. In our models of we detected the expression of MDR1, a multispecific cisplatin resistant lung cancers, we found that EMT gene efflux transporter of drugs, after PI3K selective inhibitor signatures were also significantly correlated with the LY294002 or mTOR inhibitor rapamycin treatmnet in presence of cisplatin resistance and increasing expression A549/DDP cells. The results showed that both these two of MDR1. The results further suggested a close correlation inhibitors reduced the expression of MDR1. In contrast, between EMT and cisplatin resistance in lung cancer cells. miR-206 inhibitors and MET overexpression activated It is becoming increasingly evident that miRNAs the PI3K/AKT/mTOR pathway and increased MDR1 are key modulators of cisplatin resistance and EMT in expression in A549 cells. These results provides a possible lung cancer. For instance, miR-451 is downregulated in mechanism linking miR-206, MET/PI3K/AKT/ mTOR NSCLC tissues and is capable of conferring resistance to pathway, cisplatin resistance, by which downregulated cisplatin in non-small cell lung cancer cell line (A549) expression of multi-drug resistance genes leads to [40]. miR-224 can promote both in vitro and in vivo cisplatin resistance in A549 cells. In addition, We further cisplatin resistance of A549 cells via targeting gene p21 detected the expression of two transcription factors ZEB1 (WAF1/CIP1) and regulating G1/S cell cycle transition and Snail, which are two crucial EMT activators. We and apoptosis [16]. Moreover, it has been reported that demonstrated that decrease of EMT related transcription downregulation of miRNA-27a is responsible for EMT factors, such as ZEB1 and Snail expression is one of the and cisplatin resistance in A549 cells by directly targeting molecular mechanisms from the deregulated miR-206 Raf Kinase Inhibitory Protein (RKIP)[41]. In support of levels to the EMT. the role of miRNAs in cisplatin resistance and EMT in In the present study, we also assessed the anti- lung cancer, our study identified that miR-206 is down- tumour effect of miR-206 in a cisplatin-resistant in vivo regulated and could confer cisplatin resistance and EMT mice model. We found that miR-206 inhibited the MET/ in A549/DDP cells and H1299/DDP cells. AKT/ mTOR pathway and enhanced the A549/DDP cell Targeting MET might be an effective way to sensitivity to cisplatin in vivo. Therefore, these results enhance cisplatin sensitivity in certain tumors. It has been further demonstrated in vivo that miR-206 inhibiting MET reported that MET inhibition in osteosarcoma cancer is and its downstream PI3K/AKT/mTOR pathways is one associated with higher tumor aggressive behavior and potential mechanism to overcome cisplatin resistance in resistance to cisplatin therapy[42], and overexpression lung cancer. To the best of our knowledge, we provided of MET enhances survival of ovarian cancer cells and a first insight into the roles and possible mechanisms of increased resistance to cisplatin[43]. Moreover, HGF miR-206 upregulation in chemosensitivity of A549 cells increases cisplatin resistance via activation of MET in to cisplatin. However, because only two pairs of cisplatin- lung cancer cells[27]. Consistent with these reports, our resistance cell lines were used in our study, further data showed that downregulation of MET reduced EMT investigation in other lung adenocarcinoma cell lines is process and reversed cisplatin resistance in A549/DDP necessary to explore the function and mechanisms of miR- cells and H1299/DDP cells. Interestingly, we found that 206 in cisplatin resistance. www.impactjournals.com/oncotarget 24522 Oncotarget Taken together, our study demonstrated that miR- stage IIIB-IV; first-line chemotherapy either with cisplatin 2 2 206 overexpression in human lung adenocarcinoma 100 mg/m and pemetrexed 500 mg/m or cisplatin 100 2 2 cisplatin resistant cells inhibited the EMT and cisplatin mg/m and gemcitabine 1000 mg/m administered every 3 resistance by targeting MET and suppressing its weeks for a maximum of 5 cycles. Samples were divided downstream PI3K/AKT/mTOR signaling pathway. Low into “sensitive” (complete response or partial response) expression of miR-206 and high levels of MET were and “insensitive” (stable disease or progressive disease) strongly associated with the poor cisplatin sensitivity of groups according to the patient’s responses assessed lung adenocarcinoma patients. Therefore, activation of via medical image analysis and detection of serum miR-206 or inactivation of its target gene pathway may be tumor markers after 4 or 5 cycles of cisplatin-based a potential strategy to reverse cisplatin resistance in human chemotherapy. lung adenocarcinoma cisplatin resistant cells. Transient transfection MATERIALS AND METHODS FAM-labled mimic negative control (mimic NC), miR-206 mimics (mimics), inhibitor NC, miR-206 Cell culture inhibitors, MET silence vectors p-GPU6-MET-shRNA (MET-shRNA), shRNA control, MET (Accession NO: Human lung cancer cell line A549, H1299 and NM_000245) overexpression vector pEZ/M98/neo- A549/DDP were obtained from China Center for Type MET (ex-MET) and the ex-control were purchased Culture Collection (CCTCC, Shanghai, China). All the from GenePharma (Shanghai, China). The MET shRNA cells were cultured in RPMI-1640 medium supplemented sequences were designed as showed in Supplimentary with 10% fetal bovine serum (FBS, Gibco, USA) in a Table 2. The cells were seeded into 6-well plates and humidified atmosphere containing 5% CO at 37 °C. To transfected with 75 pmol oligonucleotides or 2.5μg establish cisplatin-resistant H1299 cell lines, H1299 cells shRNA vectors using Lipofectamine 2000 (Invitrogen, were first treated with 0.6 μM of cisplatin (DDP, Sigma, USA) according to the instructions provided by the St. Louis, MO), and then were treated with increased manufacturer. The cells were used for further analysis 48h concentrations of DDP in a stepwise manner during each after transfection. passage. To maintain the drug-resistant phenotype, DDP (with final concentration of 2μM) was added to the culture Quantitative real-time PCR analysis media for A549/DDP cells and H1299/DDP cells. Total RNA was isolated with Trizol reagent Reagents and antibodies (Invitrogen, USA). The concentration and purity of the RNA samples were determined spectroscopically. MET inhibitor (SU11274) and PI3 kinase inhibitor Expression of mature miRNA was assayed using (LY294002) were purchased from Selleck Chemicals stem-loop RT followed by real-time PCR analysis. (Houston,TX). Primary antibodies phospho-MET The SYBR and U6 gene were used for detecting the (Y1234/35), phospho-AKT (S473), phospho-mTOR gene amplification and normalizing the each sample, (S2448), MET, AKT, mTOR, E-cadherin, N-cadherin, respectively. The primers for RT-PCR were designed Vimentin, ZEB1, Snail and GAPDH were purchased based on the miR-206 sequences provided by the Sanger from Cell Signaling Technology (Beverley, MA). MDR1 Center miRNA Registry. The RT primers were designed antibody was purchased from Santa Cruz Biotechnology as showed in Supplimentary Table 3. qRT-PCR was (Santa Cruz, CA). Secondary antibodies, HRP-conjugated performed on the ABI (Applied Biosystems) 7900 HT goat anti-mouse IgG and goat anti-rabbit IgG, were Thermal cycler in standard mode for 40 cycles. The fold obtained from Jackson (West Grove, PA). -ΔΔCt change was calculated using the 2 Method. PCR was performed in triplicate. Tissue samples A total of 34 lung adenocarcinoma tissues Luciferase reporter assay were collected from patients with advanced lung Based on the miRNA databases (microRNA.org, adenocarcinoma who received chemotherapy at The miRDB and TargetScan database), MET is a predicted 117th Hospital of PLA (Hangzhou, China) between June target of miR-206 in humans. According to the results of 2013 and June 2014. Informed consent was obtained from prediction, we cloned MET 3'UTR fragment containing all subjects and thisstudy was approved by the Clinical the predicted site into pGL3 luciferase reporter vector Research Ethics Committee of The 117th Hospital of (pmirGLO3, Promega, Madison, USA) and named as PLA. Patients met all of the following criteria: primary MET-wt. We cloned MET 3'UTR fragment with mutant lung adenocarcinoma; histological diagnosis of lung sequence into pmirGLO3 luciferase reporter vector and adenocarcinoma with at least 1 measurable lesion; clinical www.impactjournals.com/oncotarget 24523 Oncotarget named as MET-mut. In addition, miR-206 inhibitor by SDS-polyacrylamide gel electrophoresis(PAGE) sequence was also cloned into pmirGLO3 luciferase and transferred to a polyvinylidene dfluoride (PVDF) reporter vector as a positive control (PC). For luciferase membrane (Millipore, Germany). The blots were blocked assay, the reporter plasmid was cotransfected with miR- in BSA (5% w/v in PBS + 0.1% Tween 20) for 1 h at 206 mimics or mimic NC in A549 cells. After 48 h, cells room temperature and immunostained with antibodies at were harvested, and the luciferase activity was measured 4 °C overnight. Immunoreactive bands were visualized using the Dual-Luciferase Reporter Assay System by enhanced chemiluminescence (Millipore, Germany) (Promega,Madison, USA). according to the manufacturer’s instructions. Data were normalized to GAPDH. Migration and invasion assay Immunohistochemistry Wound healing experiment and Transwell insert (24- well insert; pore size 8μm, Corning, USA) assays were Tissue slides were incubated for 2 h at 56 °C and de- used to determine the migration and invasion abilities paraffinized. Antigen retrieval was obtained by microwave of the cells, respectively. Briefly, for the wound healing treatment in citrate buffer for 15 min to retrieve experiment, cells were grown to confuence wounded antigenicity. After peroxidase activity was blocked with using a pipette tip and photographed at 0 h and subsequent 3% H O /methanol for 10 min, sections were incubated 2 2 time points. Cell migration was evaluated by measuring with normal goat serum for 20 min to block non-specific the width of the wound at the identical position. For the antibody binding sites. Sections were incubated with the invasion assay, the lower chambers of matrigel-coated primary antibodies for 1 h at 25°C followed by incubations invasion plates were used. Cells (50,000) were added to with biotinylated anti-rabbit/mouse IgG and peroxidase- the upper chamber in serum-free media and invasion at labelled streptavidin for 10 min each. The percentage of 37 °C towards 10% FBS-containing growth media was the cells with cytoplasmic labeling was recorded from determined after 24 h. Cells that invaded through the two areas of each specimen, and the labeling intensity was membrane were fixed, stained with crystal violet and estimated as 1+, 2+ or 3+. The immunohistochemistry photographed. The invaded cells were counted by Image results were categorized into two groups: the samples J software. All experiments were carried out in triplicate. without any labeling, 1+ labeling in <25% cells, and 2+ labeling in <5% cells were considered negative; all the remaining samples were defined as positive. In vitro drug sensitivity assay The cells were plated in 6-well plates (3×10 cells/ Animal studies well) and 75 pmol of the miR-206 mimic or negative control were transfected into the A549/DDP cells, while a miR-206 All experimental procedures used in this study inhibitors or inhibitor negative control were transfected into had been approved by the ethics committee in the 117th the A549 cells, using Lipofectamine-2000 (Invitrogen, USA) Hospital of PLA. Male nude mice (BALB/c, 4-6wk) were according to the manufacturer's instructions. Twenty-four purchased from Shanghai Laboratory Animal Center hours after transfection, the cells were seeded in 96-well (Shanghai, China). For preparation of subcutaneous plates (5×10 cells/well) for the following experiment. After xenograft model, 0.2 ml A549/DDP lung cancer cells (2.0 cell adhesion, freshly prepared anticancer drug (cisplatin; × 10 ) in phosphate buffered saline/100 μl were injected Qilu Pharmaceutical Co., Ltd., Jinan, China) was added at subcutaneously into the right flank of the nude mice. 15 a final concentration of 2, 4, 8, 16, 32 μM for A549/DDP, days after cell inoculation, total of 20 mice were divided and 1, 2, 4, 8, 16 μM for A549 cells. Forty-eight hours after randomly into four groups (five mice per group). miR-206 the addition of the drug, cell viability was assessed by the agomirs and miR-206 agomir negative control (NC) (2 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium nmol; Genepharma, Shanghai, China) were given locally bromide (MTT) assay. The absorbance at 490 nm (A490) by direct injection into the xenografts every two days. of each well was read on a spectrophotometer. The Meanwhile, cisplatin was administered via intraperitoneal concentration at which the drug produced 50% inhibition of injection at a dose of 5 mg/kg every other day. After 30 growth (IC50) was estimated by the relative survival curve. days of treatment, all mice were sacrificed. Transplanted Three independent experiments were performed in duplicate. tumors were excised, and the wet weight was recorded. Protein expression in tumors was detected by western blotting assay. Western blot analysis Cells were washed in PBS and lysed in RIPA lysis Statistical analysis buffer supplemented with protease inhibitor cocktail (Roche, Germany). Total protein was quantfied by All statistical analyses were performed using SPSS BCA Protein Assay Kit (Beyotime, Nanjing, China), 13.0. Numerical data were presented as mean ± SD. 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