microRNA-9 Targets Matrix Metalloproteinase 14 to Inhibit Invasion, Metastasis, and Angiogenesis of Neuroblastoma Cells

Neuroblastoma, an embryonic malignancy derived from the neural crest, is characterized by heterogeneous biologic behaviors, including spontaneous regression or aggressive progression (1). Because metastasis is the leading cause of death in this disease, better elucidation of the underlying mechanisms is important for improving the therapeutic efficiencies (1). The metastatic process of tumor cells is highly complex and consists of multiple steps, such as local invasion, intravasation into the circulatory system, migration to a distant site, and colonization in a different microenvironment (2). Matrix metalloproteinase (MMP)-14, also named as membrane type-1 MMP (MT1-MMP), plays a critical role in facilitating the tumor cells to remodel and penetrate extracellular matrix (ECM; ref. 3). It has been established that MMP-14 promotes tumor invasion by functioning as a pericellular collagenase and an activator of proMMP-2, and is directly linked to tumorigenesis, metastasis, and angiogenesis (4, 5). However, the expression of MMP-14 and underlying mechanisms in neuroblastoma still remain largely unknown.

In recent years, emerging evidence has indicated that miRNAs, highly conserved and small noncoding RNA molecules, participate in the metastasis processes by interfering with the expression of tumor- and metastasis-associated genes through posttranscriptional repression or mRNA degradation (2). For example, miR-21 has been established as one of the most intensively studied metastasis-promoting miRNAs by targeting multiple tumor suppressor genes, such as tissue inhibitor of metalloproteinase 3 (6), phosphatase, and tensin homolog deleted on chromosome 10 (7), tropomyosin 1 (8), and programmed cell death 4 (9). The let-7 family, which comprises 13 miRNA members located on 9 different chromosomes, is downregulated in many cancer types, such as lung cancer, ovarian cancer, head and neck squamous cell carcinoma, and regulates the metastasis process of cancer cells (10). miR-146a inhibits cancer cell migration and invasion by targeting Rho-associated coiled-coil containing protein kinase 1 (11). Thus, it is currently urgent to investigate the roles of miRNAs and target genes in tumor metastasis by experimental models.

miR-9 is a kind of miRNA selectively expressed in neuron tissues that plays essential roles in developing neurons, neural carcinogenesis, and other diseases of the nervous system (12–15). In brain cancers, miR-9 is elevated and used to distinguish primary or metastatic brain tumors with very high accuracy (16). Meanwhile, miR-9 is downregulated in lung and ovarian cancer (17, 18), and is regarded as a biomarker in recurrent ovarian cancer (18). These findings indicate that the miR-9 expression profile in cancer relies on tissue distribution. Recent evidence shows that miR-9 is closely correlated with metastasis of several kinds of cancer (19–21). It has been indicated that miR-9 is downregulated in 50% of primary neuroblastoma tumors, suggesting its potential function as an oncosuppressor gene (22). However, the exact roles of miR-9 and target gene in neuroblastoma still remain elusive. In this study, we showed, for the first time, that miR-9 attenuated the expression of MMP-14 through directly targeting the 3′-untranslated region (3′-UTR), and suppressed the invasion, metastasis, and angiogenesis of neuroblastoma cells in vitro and in vivo.

Approval to conduct this study was obtained from the Institutional Review Board of Tongji Medical College. Paraffin-embedded specimens from 42 well-established primary neuroblastoma cases were obtained from the Department of Pediatric Surgery, Union Hospital of Tongji Medical College (23). The pathologic diagnosis of neuroblastoma was confirmed by at least 2 pathologists. On the basis of Shimada classification system, including the mitosis karyorrhexis index (MKI), degree of neuroblastic differentiation and stromal maturation, and patient's age, 19 patients were classified as favorable histology and 23 as unfavorable histology. According to the International Neuroblastoma Staging System (INSS), 7 patients were classified as stage 1, 7 as stage 2, 9 as stage 3, 11 as stage 4, and 8 as stage 4. In subtotal 20 neuroblastoma patients, fresh tumor specimens were collected at surgery and stored at −80°C until use. Protein and RNAs of normal human dorsal ganglia were obtained from Clontech.

Immunohistochemical staining was done as previously described (23), with antibodies specific for MMP-14 (Abcam Inc.; Santa Cruz Biotechnology; 1:200 dilutions), VEGF and CD31 (Santa Cruz Biotechnology; 1:200 dilutions). The negative controls included parallel sections treated with omission of the primary antibody, in addition to an adjacent section of the same block in which the primary antibody was replaced by rabbit polyclonal IgG (Abcam Inc.) as an isotype control. The immunoreactivity in each tissue section was assessed by at least 2 pathologists without knowledge of the clinicopathologic features of tumors or patients' survival. The degree of positivity was initially classified according to the percentage of positive tumor cells as the following: (−) <5% cells positive, (1+) 6% to 25% cells positive, (2+) 26% to 50% cells positive, and (3+) >50% cells positive.

Tissue or cellular protein was extracted with 1× cell lysis buffer (Promega). Western blotting was done as previously described (24), with antibodies specific for MMP-14, VEGF, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology). Enhanced chemiluminescence substrate kit (Amersham) was used for the detection of signals with autoradiography film (Amersham).

Total RNA was isolated with RNeasy Mini Kit (Qiagen Inc.). The reverse transcription reactions were conducted with Transcriptor First Strand cDNA Synthesis Kit (Roche). The PCR primers for MMP-14, VEGF, and GAPDH were designed by Premier Primer 5.0 software (Supplementary Table S1). RT-PCR was done as previously described (24). Real-time quantitative RT-PCR with SYBR Green PCR Master Mix (Applied Biosystems) was done using ABI Prism 7700 Sequence Detector (Applied Biosystems). The fluorescent signals were collected during extension phase, C_t values of the sample were calculated, and the transcript levels were analyzed by |$2^{- \Delta \Delta C_t}$| method.

The levels of mature miR-9 in primary tissues and cell lines were determined using Bulge-Loop miRNAs qPCR Primer Set (RiboBio Co. Ltd.). After cDNA was synthesized with a miRNA-specific stem-loop primer, the quantitative PCR was conducted with the specific primers. The miR-9 levels were normalized to those of U6 snRNA.

miRNA targets were predicted using the algorithms, including miRanda Human miRNA Targets (25), miRDB (26), RNA22 (27), and TargetScan (28). To identify the genes commonly predicted by these 4 different algorithms, results of predicted targets were intersected using miRWalk (29).

Human neuroblastoma cell lines IMR32 (CCL-127), SK-N-AS (CRL-2137), SH-SY5Y (CRL-2266), and SK-N-SH (HTB-11), and human endothelial cell line HUVEC (human umbilical vein endothelial cell; CRL-1730) were purchased from American Type Culture Collection. Cells were grown in RPMI-1640 medium (Life Technologies, Inc.) supplemented with 10% FBS (Life Technologies, Inc.), penicillin (100 U/mL) and streptomycin (100 μg/mL). Cells were maintained at 37°C in a humidified atmosphere of 5% CO₂. Anti-miR-9 or negative control inhibitors (RiboBio Co. Ltd.) were transfected into confluent cells with Lipofectamine 2000 (Life Technologies, Inc.).

According to the pre-miR-9 (5′-TCTTTGGTTATCTAGCTGTATG-3′) sequence documented in miRNA Registry database (30), oligonucleotides encoding miR-9 precursor (Supplementary Table S2) were subcloned into the BamHI and XhoI restrictive sites of pPG/miR/EGFP (GenePharma Co., Ltd.), and verified by DNA sequencing. The plasmids pPG/miR/EGFP and pPG-miR-9-EGFP were transfected into tumor cells, and stable cell lines were screened by administration of Blasticidin (Invitrogen).

Human MMP-14 3′-UTR (1,555 bp) containing the putative binding site of miR-9, and its identical sequence with a mutation of the miR-9 seed sequence (mutant) were amplified by PCR (Supplementary Table S2), inserted between the restrictive sites XhoI and NotI of firefly/Renilla luciferase reporter vector pmiR-RB-REPOR (RiboBio Co. Ltd.), and validated by sequencing. Tumor cells were plated at 1 × 10⁵ cells/well on 24-well plates, and transfected with pmiR-RB-MMP-14 3′-UTR (30 ng) or its mutant construct. Twenty-four hours posttransfection, firefly and Renilla luciferase activities were consecutively measured, according to the dual-luciferase assay manual (Promega). The Renilla luciferase signal was normalized to the firefly luciferase signal for each individual analysis.

Human MMP-14 cDNA (1,749 bp) was amplified from neuroblastoma tissue (Supplementary Table S2), subcloned into the HindIII and XhaI restrictive sites of pcDNA3.1/Zeo(+) (Invitrogen), and validated by sequencing. To restore the miR-9–induced downregulation of MMP-14, stable cell lines were transfected with the recombinant vector pcDNA3.1-MMP14. The 21-nucleotide small interfering RNA (siRNA) targeting the encoding region of MMP-14 (31) was chemically synthesized (RiboBio Co. Ltd.) and transfected with Genesilencer Transfection Reagent (Genlantis). The scramble siRNA (si-Scb) was applied as controls (Supplementary Table S2).

Homogeneous single-cell suspensions (2 × 10⁴ tumor cells) were inoculated into each well of 96-well plates that were precoated with 100 μL of 20 μg/mL Matrigel (BD Biosciences) or 50 μL of 10 mg/L fibronectin (BD Biosciences), and incubated at 37°C in serum-free complete medium (pH 7.2) for 1 hour. After incubation, the wells were washed 3 times with PBS and the remaining cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature. The cells were stained with 0.1% crystal violet and washed 3 times with PBS to remove free dye. After extraction with 10% acetic acid, absorbance of the samples was measured at 570 nm.

Tumor cells were cultured in 24-well plates and scraped with the fine end of 1-mL pipette tips (time 0). Plates were washed twice with PBS to remove detached cells, and incubated with the complete growth medium. Cell migration was photographed using 10 high-power fields, at 0 and 24 hours postinduction of injury. Remodeling was measured as diminishing distance across the induced injury, normalized to the 0 hour control, and expressed as outgrowth (μm).

The Boyden chamber technique (Transwell analysis) was done as previously described (24). Homogeneous single-cell suspensions (1 × 10⁵ cells/well) were added to the upper chambers and allowed to invade for 24 hours at 37°C in a CO₂ incubator. Migrated cells were stained with 0.1% crystal violet for 10 minutes at room temperature and examined by light microscopy. Quantification of migrated cells was done according to published criteria (24).

Fifty microliters of growth factor-reduced Matrigel were polymerized on 96-well plates. HUVECs were serum starved in RPMI-1640 medium for 24 hours, suspended in RPMI-1640 medium preconditioned with tumor cells, added to the Matrigel-coated wells at the density of 5 × 10⁴ cells/well, and incubated at 37°C for 18 hours. Quantification of antiangiogenic activity was calculated by measuring the length of tube walls formed between discrete endothelial cells in each well relative to the control (24).

All animal experiments were approved by the Animal Care Committee of Tongji Medical College (approval number: Y20080290). For the in vivo tumor growth studies, 2-month-old male nude mice (n = 6 per group) were injected subcutaneously in the upper back with 1 × 10⁶ tumor cells stably transfected with empty vector or pPG-miR-9-EGFP. Six weeks later, mice were sacrificed and examined for tumor weight, gene expression, and angiogenesis. The experimental metastasis (0.4 × 10⁶ tumor cells per mouse) studies were conducted with 2-month-old male nude mice as previously described (32).

Unless otherwise stated, all data were shown as mean ± SEM. The SPSS 12.0 statistical software (SPSS Inc.) was applied for statistical analysis. The χ² analysis and Fisher exact probability analysis were applied for comparison among the expression of MMP-14, miR-9, and individual clinicopathologic features. Pearson's coefficient correlation was applied for analyzing the relationship between miR-9 and MMP-14 transcripts. The Kaplan–Meier method was used to estimate survival rates, and the log-rank test was used to assess survival difference. Difference of tumor cells was determined by t test or ANOVA.

To investigate the expression of MMP-14 in neuroblastoma, paraffin-embedded sections from 42 well-established primary cases were collected (23). Immunohistochemical staining with antibodies from different companies revealed that MMP-14 was expressed in the cytoplasm or at the membrane of tumor cells within the neuroblastic nests (Fig. 1A). MMP-14 was also expressed in some ganglionic differentiated tumor cells (Fig. 1A). MMP-14 was detected in 29/42 cases (69.0%) and the staining was weak in 8, moderate in 8, and intense in 13 (Supplementary Table S3). The MMP-14 immunoreactivity was significantly higher in neuroblastoma cases with age more than 1 year (P = 0.005), poorer differentiation (P = 0.01), higher MKI (P = 0.03), and higher INSS stages (P = 0.035; Supplementary Table S3). The median survival time (20.6 months) of MMP-14–positive patients (n = 29) was significantly shorter than that (35.4 months) of MMP-14–negative patients (n = 13, P = 0.005; Fig. 1B). In addition, Western blotting and real-time quantitative RT-PCR were applied to measure the expression levels of MMP-14 and mature miR-9 in subtotal 20 neuroblastoma specimens, normal dorsal ganglia, and cultured IMR32, SK-N-AS, SH-SY5Y, and SK-N-SH cell lines. As shown in Fig. 1C and D, higher levels of MMP-14 were observed in neuroblastoma tissues and cell lines than those in normal dorsal ganglia. In contrast, mature miR-9 was downregulated in the neuroblastoma tissues and cell lines compared with normal dorsal ganglia (Fig. 1E). There was an inverse correlation between miR-9 expression and MMP-14 mRNA levels in neuroblastoma tissues (Fig. 1F). In situ hybridization further revealed that the miR-9 expression mainly located at the cytoplasm of tumor cells in the neuroblastoma tissues (Supplementary Fig. S1). These results indicated high MMP-14 expression in primary neuroblastoma tissues and cell lines, which was inversely correlated with endogenous miR-9 levels.

To investigate the hypothesis that miR-9 may influence the MMP-14 expression in neuroblastoma, computational prediction was done by miRNA databases. In the MMP-14 3′-UTR, there was one potential binding site of miR-9 with high complementarity (Fig. 2A), which was coincidentally predicted by at least 3 independent sources. The miR-9–binding site was located at bases 1,336 to 1,349 of the MMP-14 3′-UTR (Fig. 2A).

To investigate the direct effects of miR-9 on MMP-14 expression in neuroblastoma cell lines, we conducted the miRNA overexpression experiments. Stable transfection of miR-9 precursor into neuroblastoma cells resulted in increase of miR-9 levels (Fig. 2B). Western blotting, RT-PCR, and real-time quantitative RT-PCR showed that stable transfection of miR-9 precursor resulted in decreased protein and transcriptional levels of MMP-14 in neuroblastoma cells, when compared with untransfected parental cells and those stably transfected with empty vector (mock; Fig. 2C–E). The expression levels of VEGF, an MMP-14 downstream target gene (33), were significantly downregulated in miR-9–overexpressing neuroblastoma cells, consistent with the MMP-14 reduction (Fig. 2C–E). Because the analysis of miRNA databases from at least 3 independent sources revealed no potential binding site of miR-9 within 3′-UTR of VEGF, and combining the evidence that overexpression or knockdown of MMP-14 promoted or suppressed the VEGF expression in neuroblastoma cells, respectively (Supplementary Fig. S2), we ruled out the possibility that miR-9 may directly regulate the VEGF expression. To further examine the suppressive role of miR-9 on MMP-14 expression, we conducted the miR-9 knockdown experiments by transfection of anti-miR-9 or negative control (anti-NC) inhibitors into SH-SY5Y and SK-N-SH cells. Transfection of anti-miR-9 obviously downregulated the miR-9 expression (Supplementary Fig. S3A) and upregulated MMP-14 and VEGF protein levels than those of anti-NC (Fig. 2F). Both RT-PCR and real-time quantitative RT-PCR analyses showed the enhanced transcriptional levels of MMP-14 and VEGF in neuroblastoma cells transfected with anti-miR-9, when compared with those transfected with anti-NC (Fig. 2G). Overall, these results showed that miR-9 considerably inhibited MMP-14 expression through posttranscriptional repression.

To determine whether or not miR-9 could repress MMP-14 expression by targeting its binding sites in the MMP-14 3′-UTR, the PCR products containing intact target sites or a mutation of miR-9 seed recognition sequence (Fig. 3A) were inserted into the luciferase reporter vector. The plasmids were transfected into neuroblastoma cells stably transfected with empty vector (mock) or miR-9 precursor. The Renilla luciferase activity normalized to that of firefly was significantly reduced in the tumor cells stably transfected with miR-9 precursor (Fig. 3B), and the effect was abolished by mutating the putative miR-9–binding site within the 3′-UTR of MMP-14 (Fig. 3B). Moreover, knockdown of miR-9 with anti-miR-9 inhibitor increased the luciferase activity in SH-SY5Y and SK-N-SH cells (Fig. 3C), whereas mutation of miR-9 recognition site abolished these effects (Fig. 3C). These results indicated that miR-9 directly and specifically interacted with the target site in the MMP-14 3′-UTR.

Because previous studies indicate that MMP-14 promotes the invasion and angiogenesis of tumor cells (4, 5), we further investigated the effects of miR-9 overexpression and MMP-14 restoration on cultured neuroblastoma cells. Western blotting indicated that transfection of MMP-14 rescued the miR-9–induced downregulation of MMP-14 (Fig. 4A). In adhesion assay, tumor cells stably transfected with miR-9 precursor possessed the decreased ability in adhesion to the precoated Matrigel or fibronectin, when compared with those stably transfected with empty vector (mock; Fig. 4B). In scratch migration assay, miR-9 overexpression attenuated the migration capabilities of SH-SY5Y and SK-N-SH cells (Fig. 4C; Supplementary Fig. S4). Transwell analysis showed that neuroblastoma cells stably transfected with miR-9 precursor presented an impaired invasion capacity than mock cells (Fig. 4D). The tube formation of endothelial cells was suppressed by treatment with the medium preconditioned by stable transfection of neuroblastoma cells with miR-9 precursor (Fig. 4E). In addition, transfection of MMP-14 into SH-SY5Y and SK-N-SH cell lines restored the decrease in adhesion, migration, invasion, and angiogenesis induced by stable overexpression of miR-9 (Fig. 4B–E; Supplementary Fig. S4). On the other hand, we examined the effects of miR-9 knockdown on neuroblastoma cells. Introduction of anti-miR-9 inhibitor into neuroblastoma cells resulted in enhanced abilities in adhesion (Supplementary Fig. S3B), migration (Supplementary Fig. S3C), invasion (Supplementary Fig. S3D), and angiogenic capabilities (Supplementary Fig. S3E). These results indicated that miR-9 remarkably decreased the adhesion, migration, invasion, and angiogenesis of neuroblastoma cells in vitro.

We next investigated the efficacy of miR-9 against tumor growth, metastasis, and angiogenesis in vivo. Stable transfection of miR-9 precursor into SH-SY5Y cells resulted in decreased growth and tumor weight of subcutaneous xenograft tumors in athymic nude mice, when compared with those stably transfected with empty vector (mock; Fig. 5A and B). In addition, the expression of MMP-14 and downstream VEGF was also reduced by stable transfection of miR-9 precursor (Fig. 5C). Moreover, stable transfection of miR-9 precursor resulted in a decrease in CD31-positive mean vessel density within tumors (Fig. 5C). In the experimental metastasis studies, SH-SY5Y cells stably transfected with miR-9 precursor established statistically fewer lung metastatic colonies than mock group (Fig. 5D). These results suggested that miR-9 could inhibit the growth, metastasis, and angiogenesis of neuroblastoma cells in vivo.

Because above results indicated the negative regulation of MMP-14 expression by miR-9, we hypothesized that knockdown of MMP-14 should have a similar effect on cultured neuroblastoma cells. siRNAs targeting the encoding region of MMP-14 were designed and transfected into SH-SY5Y and SK-N-SH cells. Transfection of si-MMP-14, but not of si-Scb, resulted in decreased MMP-14 expression in neuroblastoma cells (Fig. 6A). Knockdown of MMP-14 suppressed the adhesion, migration, invasion, and angiogenesis of SH-SY5Y and SK-N-SH cells (Fig. 6B–E). These results were consistent with the findings that overexpression of miR-9 suppressed the adhesion, migration, invasion, and angiogenesis of neuroblastoma cells in vitro, providing further evidence that MMP-14 was involved in miR-9-mediated suppression of neuroblastoma. Accordingly, identification of MMP-14 as a miR-9 target gene may explain, at least in part, why overexpression of miR-9 suppressed the migration, invasion, and angiogenesis of neuroblastoma cells.

The mature human miR-9 transcript is encoded by 3 independent genes, miR-9-1, miR-9-2, and miR-9-3, which locate on chromosomes 1, 5, and 15, respectively (34). miR-9 is one of the crucial regulators of neuronal development, neuronal stem cell fate determination, and migration of neural progenitors (12), and is upregulated in oligodendroglioma (13), glioblastoma (14), and gliomas (15). However, in colon cancer, lung cancer, breast cancer, and melanoma, the promoters of 2 miR-9 genes are aberrantly hypermethylated, resulting in undetectable miR-9 transcripts (21, 35). Downregulation of miR-9 is also noted in pancreatic cancer (36), gastric cancer (37), and ovarian cancer (18). Altered miR-9 expression is associated with the malignant progression and metastasis in liver cancer (19), breast cancer (20), and colorectal cancer (21). In this study, we showed the downregulation of miR-9 in primary neuroblastoma tissues and cell lines, which was in line with previous studies (22). It is proposed that the downregulated miR-9 expression may be not because of chromosome aberrations, because the miR-9 gene family (miR-9-1, miR-9-2, and miR-9-3) is not positioned within the chromosomal regions rearranged in neuroblastoma (38). Importantly, we noted the inverse correlation between miR-9 levels and MMP-14 expression in neuroblastoma tissues, suggesting that MMP-14 expression may be negatively regulated by miR-9.

On the basis of the base pairing between miRNA and 3′-UTR of target gene, computational algorithms have been the major methods in predicting miRNA targets (39). Although bioinformatics reveal over 1,000 target genes with predicted miR-9 seed sites in their 3′-UTR, the presence of a conserved seed is not sufficient to indicate a true biologic interaction between miR-9 and putative target genes (40). In this study, our experimental evidence showed that MMP-14 was a target of miR-9. First, the ability of miR-9 to regulate MMP-14 expression was likely direct because of its high complementarity to the 3′-UTR of MMP-14 mRNA. Second, the activities of MMP-14 3′-UTR luciferase reporter were responsive to miR-9 overexpression. Third, mutation of the miR-9–binding site abolished the regulatory effects of miR-9 on the MMP-14 3′-UTR luciferase reporter. Fourth, knockdown of miR-9 with anti-miR-9 inhibitor increased the activities of MMP-14 3′-UTR luciferase reporter. Finally, endogenous MMP-14 expression, both mRNA and protein, was decreased in miR-9 precursor-transfected neuroblastoma cells, suggesting that miR-9 may regulate MMP-14 expression by inducing mRNA degradation and/or translational suppression.

The adhesion of tumor cells to ECM is a key step in the initial process of migration and invasion. MMP-14 enhances cell attachment to matrix, suggesting that MMP-14 can facilitate cell attachment at a new site for metastasis, aside from its leading role in matrix degradation (41). Matrigel, a solubilized basement membrane preparation extracted from EHS mouse sarcoma, resembles the biologically active matrix, and its major components include laminin, collagen IV, heparan sulfate proteoglycans, and entactin (42). In this study, we showed that miR-9 inhibited the MMP-14 expression and reduced the adhesion of neuroblastoma cells to Matrigel or fibronectin. It has been established that MMP-14–mediated ECM degradation at cell–matrix adhesion facilitates the focal adhesion turnover, which regulates integrin-generated signal transduction and subsequent cell migration (43). MMP-14 also regulates cell–ECM interaction by processing cell adhesion molecule CD44, and eventually promotes cell migration (44). We believe that the dynamic attachment and detachment of neuroblastoma cells to ECM is intricately regulated for cell migration, and the underlying mechanisms for MMP-14–promoted cell–ECM adhesion warrant our further investigation. MMP-14, but not other collagenases, can promote the invasion of epithelial cells, fibroblasts, and cancer cells (5). MMP-14–mediated degradation of ECM occurs throughout the angiogenic process and contributes to vascular regression (4). Furthermore, MMP-14 promotes tumor growth and angiogenesis through upregulating the protein and mRNA expression of VEGF (33). Multiple lines of preclinical evidence have shown the linkage between high MMP-14 expression and cancer progression, such as lymph node metastases, invasion, poor clinical stage, larger tumor size, and increasing tumor stage (45). In this study, we showed that high MMP-14 expression in neuroblastoma was correlated with clinicopathologic features and shorter patients' survival time. Thus, in light of the emerging pivotal role of MMP-14 in cancer progression, the miR-9–mediated MMP-14 inhibition seems attractive as a strategy to suppress the tumor growth, invasion, and metastases of neuroblastoma.

Previous studies suggest that the function of miR-9 is tumor-type specific. Overexpression of miR-9 suppresses the in vitro and in vivo growth of ovarian cancer cells through downregulating the expression of nuclear factor κB1 (46). However, miR-9 can directly target its binding site in the caudal-type homeobox 2 (CDX2) 3′-UTR, resulting in blockage of CDX2 protein translation in gastric cancer cells (47), although knockdown of miR-9 inhibits the proliferation of gastric cancer cells, which is similar to the effects of CDX2 overexpression (47). In addition, miR-9 increases the invasion and epithelial mesenchymal transition through targeting E-cadherin in hepatic cancer (48) and breast cancer cell lines (32). When breast cancer cells overexpressing miR-9 are implanted in mice, the tumors show enhanced angiogenesis and growth than those formed by miR-9 low-expressing cells (32). In colorectal cancer cells, overexpression of miR-9 promotes cell migration and cytoskeleton reorganization through downregulating the α-catenin expression (49). Thus, miR-9 seems to be a useful marker for tumor metastasis, but its role in this process is also dependent on the type of cancer. In this study, we showed that overexpression of miR-9 attenuated the invasion, metastasis, and angiogenesis of neuroblastoma cells, which was similar to that of MMP-14 knockdown, suggesting the potential application of miR-9 as a target for the therapeutics of neuroblastoma.

In summary, we have shown that miR-9 expression is downregulated in human neuroblastoma, and overexpression of miR-9 inhibits the invasion, metastasis, and angiogenesis of neuroblastoma cells in vitro and in vivo. Furthermore, miR-9 suppresses the MMP-14 expression via the binding site in the 3′-UTR in neuroblastoma cell lines. This study extends our knowledge about the regulation of MMP-14 at the posttranscriptional level by miRNA, and suggests that miR-9 may be of potential values as novel therapeutic target for human neuroblastoma.

No potential conflicts of interest were disclosed.

Conception and design: L. Zheng, Q. Tong

Development of methodology: H. Zhang, M. Qi, S. Li, T. Qi, H. Mei, K. Huang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Zhang, M. Qi, S. Li

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Zhang, M. Qi, S. Li, L. Zheng, Q. Tong

Writing, review, and/or revision of the manuscript: L. Zheng, Q. Tong

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Qi, H. Mei, K. Huang

Study supervision: L. Zheng, Q. Tong

L. Zheng is supported by the National Natural Science Foundation of China (30600278, 81071997). Q. Tong is supported by the National Natural Science Foundation of China (30200284, 30772359, 81072073), Program for New Century Excellent Talents in University (NCET-06-0641), Scientific Research Foundation for the Returned Overseas Chinese Scholars (2008-889), and Fundamental Research Funds for the Central Universities (2010JC025).

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