Downregulation of matrix metalloproteinase 14 by the antitumor miRNA, miR-150-5p, inhibits the aggressiveness of lung squamous cell carcinoma cells
- Authors:
- Published online on: December 21, 2017 https://doi.org/10.3892/ijo.2017.4232
- Pages: 913-924
Abstract
Introduction
The improvement of the treatment efficacy of lung cancer is an important issue worldwide. In 2012, 1.8 million individuals were diagnosed with lung cancer worldwide, resulting in the death of 1.6 million individuals (1). The majority of lung cancers (approximately 80%) are classified as non-small cell lung cancer (NSCLC). NSCLC is subdivided into four histopathological subtypes as follows: adenocarcinoma (LUAD), squamous cell carcinoma (LUSQ), large cell carcinoma and neuroendocrine cancer (2). The overall survival rate of patients with NSCLC is poor as almost half of patients have metastatic disease at initial diagnosis (3). In patients with adenocarcinoma, the survival rates are markedly improved by treatment with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), inhibitor of anaplastic lymphoma kinase (ALK) and immune check point drugs (4). By contrast, recent targeted molecular therapies have little benefit to the management of patients with LUSQ (5–7). Therefore, in order to improve the prognosis of patients with LUSQ, it is important to analyze the molecular mechanisms of metastatic pathways using the latest genomic approaches.
The human genome sequencing project has shown that a large proportion of non-coding RNAs (ncRNAs) are transcribed (8). The functions of ncRNAs are varied and they can act as effective regulatory molecules in a wide range of biological progresses (9). ncRNAs are categorized into two classes based on their molecular sizes: long ncRNAs (lncRNAs) and small ncRNAs (9). MicroRNAs (miRNAs or miRs) are members of the small ncRNA family. They are typically 19 to 23 nucleotides length, and they regulate the expression of protein-coding RNAs or non-coding RNAs (10). miRNAs possess unique properties, including the ability of a single miRNA species to regulate a vast number of protein-coding or ncRNAs in human cells (10). Thus, aberrantly expressed miRNAs can disrupt systematically regulated RNA networks in cancer cells. In fact, dysregulated miRNAs are deeply involved in the pathogenesis of human cancers (11).
To elucidate the aggressive nature of LUSQ, we previously identified miRNAs that play regulatory roles in this disease (12–16). For example, our previous studies have revealed that the clustered miRNAs, miR-1 and miR-133a, markedly inhibit cancer cell aggressiveness by regulating actin binding protein CORO1C (12). All family members of miR-29 (miR-29a, miR-29b and miR-29c) act as anti-metastatic miRNAs by targeting lysyl oxidase homolog 2 (LOXL2) (13,14). The over-expression of LOXL2 has been observed in several types of cancer and the knockdown of LOXL2 interferes with cancer cell aggressiveness (17,18). Moreover, miR-206 has been shown to inhibit cancer cell malignancies by targeting two pivotal tyrosine kinase receptors, MET and EGFR, in LUSQ (16). These findings provide new knowledge into the novel molecular mechanisms underlying the pathogenesis of LUSQ.
The analysis of miRNA expression signatures of head and neck squamous cell carcinoma (HNSCC) by RNA sequencing revealed that miR-150-5p was downregulated in cancer tissues (19). In addition, the downregulation of miR-150-5p was detected in cancer signatures derived from prostate cancer and bladder cancer (20,21). However, the functional significance of miR-150-5p in LUSQ remains unknown. Thus, in this study, we focused on miR-150-5p and investigated its functional significance and the regulatory RNA networks in LUSQ cells.
Materials and methods
Clinical samples and cell lines
In this study, a total of 33 LUSQs and 24 non-cancerous lung specimens apart from the tumors were obtained from patients who underwent lobectomy at Kagoshima University Hospital from 2010 to 2013. The clinicopathological data of the patients with LUSQ (33 LUSQs and 24 non-cancerous lung specimens) are summarized in Table I. Our study was approved by the Institutional Review Board for Clinical Research of Kagoshima University Hospital. Each patient provided written informed consent and approval prior to obtaining the samples.
Two human LUSQ cell lines (SK-MES-1and EBC-1) were used in this study as previously described (15,16). The cell lines, SK-MES-1 and EBC-1, were obtained from the Japanese Cancer Research Resources Bank (JCRB) and the American Type Culture Collection (Manassas, VA, USA), respectively.
Mature miRNA and small interfering RNA transfection into LUSQ cells
The following RNA species were used in this study: mature miRNAs, Pre-miR™ miRNA Precursors (has-miR-150-5p, assay ID: PM 10070; Applied Biosystems, Foster City, CA, USA), negative control miRNA (Applied Biosystems, assay ID: AM 17111), small interfering RNA (Stealth Select RNAi siRNA, Invitrogen, si-MMP14 P/N: HSS106639 and HSS106640). RNA species were incubated with Lipofectamine RNAiMax reagent (Invitrogen, Carlsbad, CA, USA) and Opti-MEM (Invitrogen) prior to plating. Subsequently, the complex was added to suspended 1×105 cells per-well plated in 6-well plates. Mock-transfected cells were transfected only with Lipofectamine RNAiMax reagent and Opti-MEM at plating. The transfection procedures were as previously described (15,16).
Reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was isolated using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. The integrity of the RNA was checked using an RNA 6000 Nano assay kit and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
The procedure for PCR quantification was as previously described (12,15,16). In brief, we first synthesized cDNA from total RNA of each sample using the TaqMan Reverse Transcription kit (P/N N8080234 and 4366596, Applied Biosystems). Subsequently, we evaluated the expression of the gene by TaqMan Real-Time PCR Assays. TaqMan probes and primers for MMP14 (P/N: Hs1037003_g1; Applied Biosystems) were assay-on-demand gene expression products. Stem-loop RT-PCR for miR-150-5p (assay ID: 000473; Applied Biosystems) was used. Human GUSB (P/N: Hs99999908_m1; Applied Biosystems) and RNU48 (assay ID: 001006; Applied Biosystems) were used as normalized controls. All reactions were performed in triplicate and the ΔΔCt method was employed to calculate the fold change.
Cell proliferation, migration and invasion assays
To investigate the functional significance of miR-150-5p or MMP14 silencing by siRNA knockdown, we performed cell proliferation, migration and invasion assays using the SK-MES-1 and EBC-1 cells.
The cells were transfected with 10 nM miRNA or siRNA by reverse transfection and plated in 96-well plates at 5×104 cells per well. After 72 h, cell proliferation was determined by XTT assay using Cell Proliferation kit (Biological Industries, Kibbutz Beit Haemek, Israel).
Cell migration activity was evaluated by wound healing assay. The cells were seeded in 6-well plates at 1×105 cells per well. At 48 h after transfection, the cell monolayer was scraped using a P-200 micropipette tip. The initial gap length and residual gap length 24 h after wounding were calculated from photomicrographs.
Cell invasion assay was carried out using modified Boyden chambers, consisting Transwell pre-coated Matrigel membrane filter inserts with 8 µm pores in 24-well tissue culture plates (BD Biosciences, Bedford, MA, USA). At 48 h after transfection, the cells were planted in the inserts at 1×105 per well. At 72 h after transfection, the cells invaded to the lower side were fixed and stained with Diff-Quick (Sysmex Corp., Kobe, Japan). The number of cells invaded to the lower surface was determined microscopically by counting 8 areas of constant size per well. All experiments were performed in triplicate. The procedures of these functional assays were as previously described (15,16).
Western blot analysis
At 72 h after transfection, protein lysates (50 µg) were separated on NuPAGE on 4–12% Bis-Tris gels (Invitrogen) and transferred onto polyvinylidene fluoride membranes (GE Healthcare Japan, Tokyo, Japan). Immunoblotting was performed with monoclonal MMP14 antibody (1:2,000 dilution) (ab51074; Abcam, Cambridge, UK). GAPDH antibody (1:10,000 dilution) (ab8245; Abcam) was used as an internal control. The membranes were washed and then incubated with an anti-rabbit-IgG, HRP-linked anti body (#7074; Cell Signaling Technology, Danvers, MA, USA). Complexes were visualized with Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA). The procedures have been described in our previous studies (15,16).
Identification of miR-150-5p target oncogenic genes in LUSQ cells
Specific genes regulated by miR-150-5p were identified by a combination of in silico and comprehensive gene expression analyses. Our target search strategies were described as previously (15,16). First, we selected putative miR-150-5p target genes using the TargetScan 7.1 database (http://www.targetscan.org/vert_71/). We then performed comprehensive gene analysis of downregulated genes in miR-150-5p transfected EBC-1 cells and upregulated genes in NSCLC from GEO database. The microarray data were deposited into GEO (http://www.ncbi.nlm.nih.gov/geo/), with the Accession number: GSE82108. Upregulated genes in NSCLC clinical specimens were obtained from the GEO database (Accession number: GSE19188).
Plasmid construction and dual-luciferase reporter assays
The wild-type or deletion-type sequences of the 3′-untranslated region (UTR) of MMP14 in miR-150-5p target sites were inserted in the psiCHECK-2 vector (C8021; Promega, Madison, WI, USA). We used 3 sequences that were putative miR-150-5p target sites of MMP14 by the TargetScan database (position 567–573: UUGGGAG; position 804–810: UGGGAGA and position 1381–1388: UUGGGAGA). The procedure for dual luciferase reporter assays was as previously described (15,16). The synthesized DNA was cloned into the psiCHECK-2 vector. EBC-1 cells were transfected with the vector, miRNAs and Lipofectamine 2000 in Opti-MEM (both from Invitrogen). The activities of Firefly and Renilla luciferases in cell lysates were determined with a dual-luciferase assay system (E1910; Promega). Normalized data were calculated as the quotient of Renilla/Firefly luciferase activities.
Immunohistochemistry staining and scoring
A tissue microarray containing a total of 30 lung samples, 20 LUSQ specimens and 10 normal lung samples was obtained from US Biomax (Derwood, MD, USA; Cat. no. BC04002). The patient characteristics for the tissue microarray are shown in Table II. The TNM classification of cancer tissues was according to the 7th edition of the American Joint Committee on Cancer (22).
We confirmed the expression status of MMP14 in the LUSQ clinical specimens using immunohistochemical staining. The procedure for immunohistochemistry was as previously described (16,23,24). The tissue sections were incubated with the primary rabbit monoclonal antibody against MMP14 (1:5,000 dilution; ab51074; Abcam). The slide was treated with biotinylated goat anti-rabbit antibodies. Diaminobenzidine hydrogen peroxidase was the chromogen and counterstaining was done with 0.5% hematoxylin. Each tissues sample was scored on the basis of the intensity and area of staining. The procedure for score for staining tissues was as previously described (25).
Statistical analysis
Relationships between two or three variables and numerical values were analyzed using Mann-Whitney U tests or Bonferroni-adjusted Mann-Whitney U tests. Expert StatView software (version 5.0; SAS Institute Inc., Cary, NC, USA) was used for these analyses. Statistical analysis was carried out as previously described (19,23).
Results
Expression levels of miR-150-5p in LUSQ clinical specimens and cell lines
The expression levels of miR-150-5p were significantly decreased in the cancer tissues compared with non-cancerous tissues (P<0.0001; Fig. 1A). In addition, the expression levels of miR-150-5p in the cancer cell lines, SK-MES-1 and EBC-1, were markedly downregulated (Fig. 1A).
Effects of the ectopic overexpression of miR-150-5p on cell proliferation, migration and invasion in LUSQ cell lines
To validate the antitumor effects of miR-150-5p, we carried out gain-of-function assays by transfecting miRNA into two LUSQ cell lines (SK-MES-1 and EBC-1). Cell proliferation was significantly inhibited in the cells transfected with the mature miR-150-5p in comparison with the mock (transfection reagent only)- or miR-control-transfected cells (Fig. 1B). Furthermore, cell migration and invasion activities were markedly reduced in the cells transfected with the miR-150-5p expression plasmid compared to the mock- or miR-control-transfected cells (Fig. 1C and D).
Identification of putative targets of miR-150-5p regulation in LUSQ cells
Our strategy for the selection of miR-150-5p target oncogenic genes is shown in Fig. 2. First, we selected putative miR-150-5p target genes using the TargetScan 7.1 database and identified 351 genes. We then performed comprehensive gene expression analysis using miR-150-5p transfectants of EBC-1 cells, with negative control miRNA transfectants serving as controls (Accession number: GSE 82108). In this assessment, 15 genes were commonly downregulated (log2 ratio <−1.0). The gene set was then analyzed with a publicly available gene expression data set in GEO (Accession number: GSE 19188) and genes upregulated in LUSQ were selected (fold change >0). A total of 9 genes were identified as candidate targets of miR-150-5p regulation (Table III).
Among these candidate genes, we focused on MMP14 as the aberrant expression of the matrix metalloproteinase family enhances cancer cell migration and invasion (26). The ectopic overexpression of miR-150-5p significantly inhibited cancer cell migration and invasion in the LUSQ cells (Fig. 1C and D).
Direct regulation of MMP14 by miR-150-5p in LUSQ cells
We investigated whether transfection of the LUSQ cell lines with the miR-150-5p expression plasmid would reduce the expression of MMP14/MMP14. The mRNA expression levels of MMP14 were decreased by transfection with the miR-150-5p expression plasmid compared with the mock- or miR-control-transfected cells (Fig. 3A). Furthermore, the protein expression levels of MMP14 were also decreased by the overexpression of miR-150-5p compared with the mock- or miR-control-transfected cells (Fig. 3B).
We also performed luciferase reporter assays with a vector that included the 3′-UTR of MMP14 to confirm that miR-150-5p directly regulated MMP14 in a sequence-dependent manner. We used vectors encoding the partial wild-type or deletion-type sequences of the 3′-UTR of the MMP14 with miR-150-5p target sites. Three binding sites for miR-150-5p in the 3′-UTR of MMP14 (positions 567–573, 804–810 and 1381–1388) were predicted by the TargetScan Human database (Fig. 3C). We observed that the luminescence intensities of the proteins coded by vectors carrying the wild-type sequences (positions 804–810 and 1381–1388) were significantly reduced by co-transfection with miR-150-5p (Fig. 3C). By contrast, transfection with the deletion-type vector blocked the reduction of luminescence intensities (Fig. 3C). These findings indicated that miR-150-5p directly bound to specific two sites in the 3′-UTR of MMP14.
Effects of knockdown of MMP14 on the proliferation, migration and invasion of LUSQ cell lines
Loss-of-function assays using siRNA were performed to examine the function of MMP14 in LUSQ cell lines (SK-MES-1 and EBC-1). The mRNA and protein expression levels of MMP14 were decreased by transfection with si-MMP14 in the LUSQ cell lines (Fig. 4A and B).
We then investigated the effects of MMP14 knockdown on the proliferation, migration, and invasion of LUSQ cell lines. Cancer cell proliferation was significantly reduced in the si-MMP14-transfected cells (Fig. 4C). In addition, the migration and invasion activities were significantly attenuated in the si-MMP14-transfected cells (Fig. 4D and E).
Expression of MMP14 in LUSQ clinical specimens
We examined the protein expression levels of MMP14 in LUSQ clinical specimens by immunostaining. We confirmed the overexpression of MMP14 in LUSQ lesions compared with that in normal tissues (Fig. 5).
Identification of MMP14-regulated genes in LUSQ cells
To investigate downstream genes regulated by MMP14, we performed genome-wide gene expression analysis using si-MMP14 in EBC-1 cells. A total of 92 genes were identified as MMP14-regulated genes (Table IV).
Discussion
Recently, several treatment options have been approved for post-first-line therapy for LUSQ, such as chemotherapy with or without an angiogenesis inhibitor (27), or immunotherapy (4). However, treatment outcomes do not appear to have improved. Therefore, clinicians need new treatment options in their approach to LUSQ. Presumably, they should be based on the latest molecular analyses of the pathology of this disease.
In this study, we demonstrated that the restoration of miR-150-5p significantly attenuated with cancer cell aggressiveness, suggesting that this miRNA possesses antitumor activity in LUSQ cells. It has been demonstrated that miR-150-5p has multiple functions, possessing antitumor activity or oncogenic functions (28). The overexpression of miR-150-5p has been reported in several types of cancer (29,30). In contrast to the overexpression of miR-150-5p in cancer cells, the antitumor roles of miR-150-5p have been reported in several types of cancer through its targeting of oncogenic genes (19,31,32). Previous studies showed that contradiction resulted as to the expression pattern of miR-150-5p in lung cancer (28). Our preliminary data demonstrated that the expression levels of miR-150-5p were reduced in lung adenocarcinoma clinical specimens (data not shown). One such study showed that miR-150-5p expression was significantly reduced in HNSCC tissues and had antitumor roles. A low expression of miR-150-5p has been shown to be significantly associated with a poor prognosis of patients with HNSCC (19). Moreover, integrin subunit alpha 3 (ITGA3), integrin subunit alpha 6 (ITGA6) and tenascin C (TNC) have been identified as oncogenes in HNSCC that were downregulated by miR-150-5p (19). Previous studies have shown that the aberrant expression of integrin family genes and the activation of integrin-mediated oncogenic signaling promotes cancer cell metastasis and the epithelial-mesenchymal transition (EMT) phenotype (33,34). Other studies have demonstrated that SPOCK1 is directly regulated by miR-150-5p in prostate cancer and esophageal squamous cell carcinoma (31,32). The aberrant expression of SPOCK1 has been observed in several types of cancer and has been shown to play pivotal roles in cancer cell progression, metastasis and drug resistance (35,36). Of note, the ectopic expression of SPOCK1 induces EMT in lung cancer (37). These findings suggest that the downregulation of miR-150-5p induces several cancer-promoting genes and that its expression is deeply involved in cancer pathogenesis.
In this study, to identify oncogenic genes targeted by miR-150-5p, we applied gene expression analysis and in silico database searches. A total of 9 genes (MMP14, CPD, LRRC58, CDC73, ENSA, DSEL, CSNK1A1, CHD2 and CAPZB) were identified as putative targets of miR-150-5p regulation in LUSQ cells. Among these, we focused on MMP14 as the matrix metalloproteinase family contributes to cancer cell migration and invasion. Our luciferase reporter assay revealed that MMP14 was directly regulated by miR-150-5p in LUSQ cells. The overexpression of MMP14 was observed in LUSQ clinical specimens and the knockdown of MMP14 by siRNA significantly interfered in vitro with cancer cell malignancies. These findings indicate that MMP14 acts as an oncogene regulated by the antitumor miR-150-5p in LUSQ cells.
MMP14 belongs to the membrane-type matrix metalloprotease (MT-MMP) family that includes pivotal regulators of cell invasion, growth and survival in normal cells (26). The upregulation of MMP14 has been observed in many types of cancer, and the overexpression of MMP14 has been shown to correlate with a poor prognosis in patients with NSCLC, renal cell carcinoma and breast cancer (38–40). Activated MMP14 cleaves pro-MMP2 and pro-MMP13 and the activated form of MMP2 and MMP13 are involved in cancer pathogenesis (26). A major substrate of MMP14 is type I collagen in ECM components. MMP14 is localized to the leading edge of invadopodia in migrating cells, achieving spatially coordinated matrix degradation to support invasion (41). MMP14 influences cancer cells and cancer niches (ECM and fibroblast cells). MMP14 is mediated through membrane proteins, e.g., receptor tyrosine kinases (RTK) and integrins, and these events promote cancer cell aggressiveness (42,43). For instance, he interaction of MMP14 and CD44 induces the phosphorylation of the EGF receptor and enhances downstream activation of the MAPK and PI3K signaling pathways (44,45).
Proteolytic enzymes, MMPs or MT-MMPs are tightly controlled by tissue inhibitors of metalloproteases (TIMPs) (26). The expression and activation of MMPs and TIMPs are important to biological processes and essential for tissue homeostasis. Dysregulated MMPs and TIMPs have been implicated in cancer cell progression and metastasis (46). Recent studies have indicated that several downregulated miRNAs caused the aberrant expression of MMP14 in cancer cells (47,48). Our recent studies showed the antitumor functions of miR-375 and miR-139-5p/-3p that targeted MMP13 and MMP11, respectively (49,50). These MMPs were overexpressed in cancer specimens, and the restoration of these miRNAs or knockdown of MMP13 or MMP11 inhibited cancer cell migration and invasion (49,50). The identification of antitumor miRNAs that regulate novel LUSQ networks may lead to a better understanding of the aggressiveness of this disease.
Taken together, this study demonstrates miR-150-5p acts as an antitumor miRNA by targeting MMP14 in LUSQ cells. The overexpression of MMP14 may be enhanced in lung cancer oncogenesis. The exploration of antitumor miRNA-mediated regulatory networks may lead to the development of novel treatment strategies for this disease.
Acknowledgments
This study was supported by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI), 17K16893, 15K10801, 16K19458, and 17K09660.
Notes
[1] Competing interests
The authors declare that they have no competing interests.
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