microrna-9 targets matrix metalloproteinase 14 to inhibit ... · 5/5/2012 · 1 microrna-9 targets...

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microRNA-9 targets matrix metalloproteinase 14 to inhibit invasion, metastasis

and angiogenesis of neuroblastoma cells

Huanyu Zhang 1, #, Meng Qi 1, #, Shiwang Li 1, #, Teng Qi 1, Hong Mei 1, Kai Huang 2, 3,

Liduan Zheng 2, 4,*, and Qiangsong Tong 1, 2, *

1 Department of Pediatric Surgery, 2 Clinical Center of Human Genomic Research,

3 Department of Cardiology, 4 Department of Pathology, Union Hospital of Tongji Medical College,

Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, P. R. China

Running title: miR-9 targets MMP-14 in neuroblastoma

Key words: neuroblastoma, microRNA-9, matrix metalloproteinase 14

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); L. Zheng is supported by the National

Natural Science Foundation of China (30600278, 81071997).

*Corresponding author:

Qiangsong Tong, Department of Pediatric Surgery, Union Hospital of Tongji Medical College,

Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, P. R. China. Tel:

+86-27-85726005; Fax: +86-27-85726821; Email: qs_tong@hotmail. com

Liduan Zheng, Department of Pathology, Union Hospital of Tongji Medical College, Huazhong

University of Science and Technology, Wuhan 430022, Hubei Province, P. R. China. Tel:

+86-27-85627129; Fax: +86-27-85726821; Email: [email protected]

# Zhang H., Qi M., and Li S. contributed equally to this work. No potential conflicts of interest

were disclosed.

Word count: 5853; Total number of figures and tables: 6

on November 22, 2020. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 7, 2012; DOI: 10.1158/1535-7163.MCT-12-0001

http://mct.aacrjournals.org/

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Abstract

Matrix metalloproteinase 14 (MMP-14) is the only membrane anchored MMP that plays a

critical role in tumor metastasis and angiogenesis. However, the mechanisms underlying MMP-14

expression in tumors still remain largely unknown. In this study, MMP-14 immunostaining was

identified in 29/42 neuroblastoma (NB) tissues, which was correlated with clinicopathological

features and shorter patients’ survival. In subtotal twenty NB cases, microRNA 9 (miR-9) was

down-regulated and inversely correlated with MMP-14 expression. Bioinformatics analysis

revealed a putative miR-9 binding site in the 3′-untranslated region (3′-UTR) of MMP-14 mRNA.

Over-expression or knockdown of miR-9 responsively altered both the mRNA and protein levels

of MMP-14 and its downstream gene, vascular endothelial growth factor (VEGF), in cultured NB

cell lines SH-SY5Y and SK-N-SH. In a MMP-14 3′-UTR luciferase reporter system, miR-9

down-regulated the luciferase activity, and these effects were abolished by a mutation in the

putative miR-9 binding site. Over-expression of miR-9 suppressed the invasion, metastasis and

angiogenesis of SH-SY5Y and SK-N-SH cells in vitro and in vivo. In addition, the effects of miR-9

on MMP-14 expression, adhesion, migration, invasion and angiogenesis were rescued by

over-expression of MMP-14 in these cells. Furthermore, anti-miR-9 inhibitor or knockdown of

MMP-14 respectively increased or inhibited the migration, invasion and angiogenesis of NB cells.

These data indicate that miR-9 suppresses MMP-14 expression via the binding site in the 3′-UTR,

thus inhibiting the invasion, metastasis and angiogenesis of NB.




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Introduction

Neuroblastoma (NB), an embryonic malignancy derived from the neural crest, is

characterized by heterogeneous biological behaviors, including spontaneous regression or

aggressive progression [1]. Since 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 14 (MMP-14), also

named as membrane type-1 matrix metalloproteinase (MT1-MMP), plays a critical role in

facilitating the tumor cells to remodel and penetrate extracellular matrix (ECM) [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 NB still remain largely

unknown.

In recent years, emerging evidence has indicated that microRNAs (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

post-transcriptional 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 nine different




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chromosomes, is down-regulated 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.

MicroRNA 9 (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 down-regulated 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 down-regulated in 50% of primary NB tumors,

suggesting its potential function as an oncosuppressor gene [22]. However, the exact roles of

miR-9 and target gene in NB still remain elusive. In the current study, we demonstrated, 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 NB

cells in vitro and in vivo.

Materials and methods

Patient tissue samples

Approval to conduct this study was obtained from the Institutional Review Board of Tongji

Medical College. Paraffin-embedded specimens from 42 well-established primary NB cases were




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obtained from the Department of Pediatric Surgery, Union Hospital of Tongji Medical College [23].

The pathological diagnosis of NB was confirmed by at least two pathologists. Based on 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 4S. In subtotal 20 NB 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 (Mountain View, CA).

Immunohistochemistry

Immunohistochemical staining was performed as previously described [23], with antibodies

specific for MMP-14 (Abcam Inc, Cambridge, MA; Santa Cruz Biotechnology, Santa Cruz, CA;

1:200 dilutions), vascular endothelial growth factor (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 two pathologists without

knowledge of the clinicopathological 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+) 6–25% cells positive, (2+) 26–50% cells positive, and

(3+) >50% cells positive.

Western blot




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Tissue or cellular protein was extracted with 1× cell lysis buffer (Promega, Madison, WI).

Western blot was performed 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, Piscataway, NJ) was used for the

detection of signals with autoradiography film (Amersham).

RT-PCR and real-time quantitative RT-PCR

Total RNA was isolated with RNeasy Mini Kit (Qiagen Inc., Valencia, CA). The reverse

transcription reactions were conducted with Transcriptor First Strand cDNA Synthesis Kit (Roche,

Indianapolis, IN). The PCR primers for MMP-14, VEGF and GAPDH were designed by Premier

Primer 5.0 software (Supplementary Table S1). RT-PCR was performed as previously described

[24]. Real-time quantitative RT-PCR with SYBR Green PCR Master Mix (Applied Biosystems,

Foster City, CA) was performed using ABI Prism 7700 Sequence Detector (Applied Biosystems).

The fluorescent signals were collected during extension phase, Ct values of the sample were

calculated, and the transcript levels were analyzed by 2-△△Ct method.

Quantification of miR-9 expression

The levels of mature miR-9 in primary tissues and cell lines were determined using

Bulge-LoopTM miRNAs qPCR Primer Set (RiboBio Co. Ltd, Guangzhou, China). After cDNA was

synthesized with a miRNA-specific stem-loop primer, the quantitative PCR was performed with

the specific primers. The miR-9 levels were normalized as to those of U6 snRNA.

miRNA target prediction

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




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predicted by these four different algorithms, results of predicted targets were intersected using

miRWalk [29].

Cell culture and transfection

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 (CRL-1730) were

purchased from American Type Culture Collection (Rockville, MD). Cells were grown in

RPMI1640 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal

bovine serum (Life Technologies, Inc.), penicillin (100 U/ml) and streptomycin (100 g/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.).

pre-miR-9 construct and stable transfection

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 BamH I and Xho I restrictive sites of pPG/miR/EGFP

(GenePharma Co., Ltd, Shanghai, China), and verified by DNA sequencing. The plasmids

pPG/miR/EGFP and pPG-miR9-EGFP were transfected into tumor cells, and stable cell lines were

screened by administration of Blasticidin (Invitrogen, Carlsbad, CA).

Luciferase reporter assay

Human MMP-14 3′-UTR (1555 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 Xho I and Not I of firefly/Renilla




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luciferase reporter vector pmiR-RB-REPORT™ (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-MMP143′-UTR (30 ng) or its mutant construct. Twenty-four hrs post-transfection,

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.

MMP-14 over-expression and knockdown

Human MMP-14 cDNA (1749 bp) was amplified from NB tissue (Supplementary Table S2),

subcloned into the Hind III and Xha I restrictive sites of pcDNA3.1/Zeo(+) (Invitrogen), and

validated by sequencing. To restore the miR-9-induced down-regulation 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,

San Diego, CA). The scramble siRNA (si-Scb) was applied as controls (Supplementary Table S2).

Cell adhesion assay

Homogeneous single cell suspensions (2 × 104 tumor cells) were inoculated into each well of

96-well plates that were precoated with 100 l of 20 g/ml matrigel (BD Biosciences, Franklin

Lakes, NJ) 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 hr. After incubation, the wells were washed three times with

phosphate buffered saline (PBS) and the remaining cells were fixed in 4% paraformaldehyde for

20 min at room temperature. The cells were stained with 0.1% crystal violet and washed three

times with PBS to remove free dye. After extraction with 10% acetic acid, absorbance of the




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samples was measured at 570 nm.

Scratch migration assay

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 hrs post-induction of injury. Remodeling was measured as diminishing distance across the

induced injury, normalized to the 0 hr control, and expressed as outgrowth (μm).

Matrigel invasion assay

The Boyden chamber technique (transwell analysis) was performed as previously described

[24]. Homogeneous single cell suspensions (1 × 105 cells/well) were added to the upper chambers

and allowed to invade for 24 hrs at 37°C in a CO2 incubator. Migrated cells were stained with

0.1% crystal violet for 10 min at room temperature and examined by light microscopy.

Quantification of migrated cells was performed according to published criteria [24].

Tube formation assay

Fifty microliters of growth factor-reduced matrigel were polymerized on 96-well plates.

HUVECs were serum starved in RPMI1640 medium for 24 hrs, suspended in RPMI1640 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 hrs. Quantification of anti-angiogenic activity was

calculated by measuring the length of tube walls formed between discrete endothelial cells in each

well relative to the control [24].

In vivo growth and metastasis assay

All animal experiments were approved by the Animal Care Committee of Tongji Medical




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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-miR9-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 performed with 2-month-old male nude mice as

previously described [32].

Statistical analysis

Unless otherwise stated, all data were shown as mean ± standard error of the mean (SEM).

The SPSS 12.0 statistical software (SPSS Inc., Chicago, IL) 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 clinicopathological 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 analysis of

variance (ANOVA).

Results

High levels of MMP-14 were inversely correlated with endogenous miR-9 expression in NB

tissues and cell lines

To investigate the expression of MMP-14 in NB, 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




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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 NB 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 blot and real-time

quantitative RT-PCR were applied to measure the expression levels of MMP-14 and mature miR-9

in subtotal 20 NB specimens, normal dorsal ganglia, and cultured IMR32, SK-N-AS, SH-SY5Y

and SK-N-SH cell lines. As shown in Fig. 1C and Fig. 1D, higher levels of MMP-14 were

observed in NB tissues and cell lines than those in normal dorsal ganglia. In contrast, mature

miR-9 was down-regulated in the NB 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 NB tissues (Fig. 1F). In situ hybridization further revealed that the miR-9 expression mainly

located at the cytoplasm of tumor cells in the NB tissues (Supplementary Fig. S1). These results

indicated high MMP-14 expression in primary NB tissues and cell lines, which was inversely

correlated with endogenous miR-9 levels.

MMP-14 was a candidate target of miR-9

To investigate the hypothesis that miR-9 may influence the MMP-14 expression in NB,

computational prediction was performed 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 three independent sources. The miR-9 binding site was located at bases




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1336–1349 of the MMP-14 3′-UTR (Fig. 2A).

miR-9 down-regulated MMP-14 expression through post-transcriptional repression

To investigate the direct effects of miR-9 on MMP-14 expression in NB cell lines, we

performed the miRNA over-expression experiments. Stable transfection of miR-9 precursor into

NB cells, resulted in increase of miR-9 levels (Fig. 2B). Western blot, RT-PCR and real-time

quantitative RT-PCR demonstrated that stable transfection of miR-9 precursor resulted in

decreased protein and transcriptional levels of MMP-14 in NB cells, when compared to

untransfected parental cells and those stably transfected with empty vector (mock) (Fig. 2C, Fig.

2D, and Fig. 2E). The expression levels of VEGF, a MMP-14 downstream target gene [33], were

significantly down-regulated in miR-9 over-expressing NB cells, consistent with the MMP-14

reduction (Fig. 2C, Fig. 2D, and Fig. 2E). Since the analysis of miRNA databases from at least

three independent sources revealed no potential binding site of miR-9 within 3′-UTR of VEGF,

and combining the evidence that over-expression or knockdown of MMP-14 promoted or

suppressed the VEGF expression in NB 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 performed 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 down-regulated 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 NB cells transfected with anti-miR-9, when

compared with those transfected with anti-NC (Fig. 2G). Overall, these results demonstrated that




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miR-9 considerably inhibited MMP-14 expression through post-transcriptional repression.

miR-9 interacted with a putative binding site in the MMP-14 3'-UTR

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 NB 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), while 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.

miR-9 suppressed the adhesion, migration, invasion, and angiogenesis of NB cells in vitro

Since previous studies indicate that MMP-14 promotes the invasion and angiogenesis of

tumor cells [4, 5], we further investigated the effects of miR-9 over-expression and MMP-14

restoration on cultured NB cells. Western blot indicated that transfection of MMP-14 rescued the

miR-9-induced down-regulation 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 to those stably transfected with empty vector (mock) (Fig.

4B). In scratch migration assay, miR-9 over-expression attenuated the migration capabilities of

SH-SY5Y and SK-N-SH cells (Fig. 4C and Supplementary Fig. S4). Transwell analysis showed




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that NB 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 NB 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 over-expression of miR-9 (Fig.

4B, Fig. 4C, Fig. 4D, Fig. 4E, and Supplementary Fig. S4). On the other hand, we examined the

effects of miR-9 knockdown on NB cells. Introduction of anti-miR-9 inhibitor into NB 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 NB cells in vitro.

Over-expression of miR-9 attenuated the growth, metastasis, and angiogenesis of NB cells in

vivo

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 to

those stably transfected with empty vector (mock) (Fig. 5A and Fig. 5B). 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




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the growth, metastasis and angiogenesis of NB cells in vivo.

Knockdown of MMP-14 suppressed the adhesion, migration, invasion, and angiogenesis of

NB cells

Since 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 NB cells.

siRNAs targeting the encoding region of MMP-14 were designed and transfected into SH-SY5Y

and SK-N-SH cells. Transfection of si-MMP14, but not of si-Scb, resulted in decreased MMP-14

expression in NB cells (Fig. 6A). Knockdown of MMP-14 suppressed the adhesion, migration,

invasion and angiogenesis of SH-SY5Y and SK-N-SH cells (Fig. 6B, Fig. 6C, Fig. 6D and Fig. 6E).

These results were consistent with the findings that over-expression of miR-9 suppressed the

adhesion, migration, invasion and angiogenesis of NB cells in vitro, providing further evidence

that MMP-14 was involved in miR-9-mediated suppression of NB. Accordingly, identification of

MMP-14 as a miR-9 target gene may explain, at least in part, why over-expression of miR-9

suppressed the migration, invasion and angiogenesis of NB cells.

Discussion

The mature human miR-9 transcript is encoded by three 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 up-regulated in oligodendroglioma [13], glioblastoma

[14], and gliomas [15]. However, in colon cancer, lung cancer, breast cancer and melanoma, the

promoters of two miR-9 genes are aberrantly hypermethylated, resulting in undetectable miR-9

transcripts [21, 35]. Down-regulation of miR-9 is also noted in pancreatic cancer [36], gastric




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cancer [37] and ovarian cancer [38]. 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 demonstrated the down-regulation of miR-9 in primary NB tissues and cell lines,

which was in line with previous studies [22]. It is proposed that the down-regulated miR-9

expression may be not due to chromosome aberrations, since the miR-9 gene family (miR-9-1,

miR-9-2 and miR-9-3) is not positioned within the chromosomal regions rearranged in NB [39].

Importantly, we noted the inverse correlation between miR-9 levels and MMP-14 expression in

NB tissues, suggesting that MMP-14 expression may be negatively regulated by miR-9.

Based on the base pairing between miRNA and 3′-UTR of target gene, computational

algorithms have been the major methods in predicting miRNA targets [40]. Although

bioinformatics reveal over 1000 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 biological interaction between

miR-9 and putative target genes [41]. In the current study, our experimental evidence demonstrated

that MMP-14 was a target of miR-9. First, the ability of miR-9 to regulate MMP-14 expression

was likely direct due to its high complementarity to the 3′-UTR of MMP-14 mRNA. Second, the

activities of MMP-143′-UTR luciferase reporter were responsive to miR-9 over-expression. 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 NB cells, suggesting that miR-9

may regulate MMP-14 expression by inducing mRNA degradation and/or translational

suppression.




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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 [42].

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 [43]. In this study, we demonstrated that miR-9

inhibited the MMP-14 expression and reduced the adhesion of NB 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 [44]. MMP-14 also regulates cell-ECM interaction by processing cell

adhesion molecule CD44, and eventually promotes cell migration [45]. We believe that the

dynamic attachment and detachment of NB 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 up-regulating 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 [46]. In this study, we demonstrated that high

MMP-14 expression in NB was correlated with clinicopathological features and shorter patients’

survival time. Thus, in light of the emerging pivotal role of MMP-14 in cancer progression, the




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miR-9-mediated MMP-14 inhibition appears attractive as a strategy to suppress the tumor growth,

invasion, and metastases of NB.

Previous studies suggest that the function of miR-9 is tumor-type specific. Over-expression of

miR-9 suppresses the in vitro and in vivo growth of ovarian cancer cells through down-regulating

the expression of nuclear factor B1 [47]. 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 [48], while knockdown of miR-9 inhibits the proliferation of gastric cancer

cells, which is similar to the effects of CDX2 over-expression [48]. In addition, miR-9 increases

the invasion and epithelial mesenchymal transition through targeting E-cadherin in hepatic cancer

[49] and breast cancer cell lines [32]. When breast cancer cells over-expressing 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, over-expression of miR-9 promotes

cell migration and cytoskeleton reorganization through down-regulating the -catenin expression

[50]. 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 demonstrated that over-expression of miR-9

attenuated the invasion, metastasis and angiogenesis of NB cells, which was similar to that of

MMP-14 knockdown, suggesting the potential application of miR-9 as a target for the therapeutics

of NB.

In summary, we have shown that miR-9 expression is down-regulated in human NB, and

over-expression of miR-9 inhibits the invasion, metastasis and angiogenesis of NB cells in vitro

and in vivo. Furthermore, miR-9 suppresses the MMP-14 expression via the binding site in the

3′-UTR in NB cell lines. This study extends our knowledge about the regulation of MMP-14 at the




19

post-transcriptional level by miRNA, and suggests that miR-9 may be of potential values as novel

therapeutic target for human NB.

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Figure legends

Figure 1 MMP-14 was highly expressed and inversely correlated with endogenous levels of

miR-9 in NB tissues and cell lines. A, haematoxylin/eosin (HE) and immunohistochemical

staining revealed that MMP-14 was expressed in the cytoplasm or at the membrane of tumor cells

within the neuroblastic nests, and was also expressed in some ganglionic differentiated tumor cells.

Scale bars: 100 m. B, the Kaplan-Meier method was used to estimate survival rates, and indicated

that the median survival time (20.6 months) of MMP-14-positive NB patients was significantly

shorter than that (35.4 months) of MMP-14-negative patients. C, western blot indicated higher

protein levels of MMP-14 in NB tissues (n=20) and cultured cell lines (IMR32, SK-N-AS,

SH-SY5Y and SK-N-SH) than those in normal dorsal ganglia (DG). D, real-time quantitative

RT-PCR revealed higher transcription levels of MMP-14 in NB tissues (n=20) and cultured cell

lines (IMR32, SK-N-AS, SH-SY5Y and SK-N-SH) than those in DG. E, real-time quantitative

RT-PCR indicated lower miR-9 levels in NB tissues (n=20) and cultured cell lines (IMR32,

SK-N-AS, SH-SY5Y and SK-N-SH) than those in DG. F, There was an inverse correlation

between miR-9 levels and MMP-14 transcription in NB tissues. The symbols (* and △) indicate a

significant decrease and a significant increase from DG, respectively.

Figure 2 miR-9 down-regulated MMP-14 expression through post-transcriptional

repression in NB cells. A, scheme of the potential binding site of miR-9 in the MMP-14 3′-UTR,

locating at bases 1336–1349. B, real-time quantitative RT-PCR revealed that stable transfection of




26

miR-9 precursor into cultured SH-SY5Y and SK-N-SH cells, resulted in enhanced miR-9 levels,

when compared to untransfected parental cells and those stably transfected with empty vector

(mock). C, western blot indicated that stable transfection of miR-9 precursor resulted in decreased

MMP-14 and VEGF protein levels in SH-SY5Y and SK-N-SH cells. D and E, RT-PCR and

real-time quantitative RT-PCR revealed the decreased MMP-14 and VEGF transcription levels in

miR-9 precursor-transfected SH-SY5Y and SK-N-SH cells, but not in control and mock cells. F,

western blot indicated that transfection of anti-miR-9 inhibitor (100 nmol/L), but not of negative

control inhibitor (anti-NC, 100 nmol/L), resulted in increased MMP-14 and VEGF protein levels in

SH-SY5Y and SK-N-SH cells. G, RT-PCR and real-time quantitative RT-PCR revealed the

increased MMP-14 and VEGF expression in SH-SY5Y and SK-N-SH cells transfected with

anti-miR-9 inhibitor (100 nmol/L), but not in those transfected with anti-NC. The symbols (* and

△) indicate a significant decrease and a significant increase from control or anti-NC, respectively.

Figure 3 miR-9 directly interacted with a putative binding site in the MMP-14 3′-UTR. A,

scheme and sequence of the intact miR-9 binding site (WT) and its mutation (Mut) within the

luciferase reporter vector. B, stable transfection of miR-9 precursor into SH-SY5Y and SK-N-SH

cells resulted in decreased luciferase activities of MMP-14 3′-UTR reporter, when compared with

those stably transfected with empty vector (mock). These effects were abolished by a mutation in

the putative miR-9 binding site within the 3′-UTR of MMP-14. C, transfection of anti-miR-9 (100

nmol/L) inhibitor into SH-SY5Y and SK-N-SH increased the luciferase activity when compared

with those transfected with negative control inhibitor (anti-NC, 100 nmol/L), while mutation of

miR-9 recognition site abolished these effects. The symbols (* and △) indicate a significant

decrease and a significant increase from mock or anti-NC, respectively.




27

Figure 4 Over-expression of miR-9 abolished the adhesion, migration, invasion, and

angiogenesis of NB cells in vitro. A, western blot indicated that transfection of MMP-14 restored

the down-regulation of MMP-14 induced by stable miR-9 over-expression. B, stable transfection

of miR-9 precursor into SH-SY5Y and SK-N-SH cells resulted in decreased adhesion to matrigel

or fibronectin, when compared to those stably transfected with empty vector (mock). In addition,

transfection of MMP-14 restored the cell adhesion in miR-9 over-expressing tumor cells. C, in

scratch migration assay, the migration of miR-9 over-expressing SH-SY5Y and SK-N-SH cells

was significantly reduced when compared to mock. Transfection of MMP-14 rescued the migration

of miR-9 over-expressing cells. D, matrigel invasion assay indicated the decreased invasion

capabilities of miR-9 over-expressing SH-SY5Y and SK-N-SH cells than those of mock cells.

However, transfection of MMP-14 restored the invasion of miR-9 over-expressing cells. E, the

tube formation of endothelial HUVEC cells was suppressed by treatment with the medium

preconditioned by miR-9 over-expressing SH-SY5Y and SK-N-SH cells, when compared to that of

mock cells. Transfection of MMP-14 rescued the angiogenic capabilities of miR-9 over-expressing

cells. The symbols (* and △) indicate a significant decrease and a significant increase from mock,

respectively.

Figure 5 Over-expression of miR-9 attenuated the growth, metastasis, and angiogenesis of

NB cells in vivo. A and B, hypodermic injection of SH-SY5Y cells into athymic nude mice

established subcutaneous xenograft tumors. Six weeks later, mice (n=6) from each group were

sacrificed. Stable transfection of tumor cells with miR-9 precursor resulted in decreased tumor size,

and the mean tumor weight formed from miR-9 over-expressing cells was significantly decreased.




28

C, HE and immunohistochemical staining revealed that stable transfection of miR-9 precursor

resulted in decreased expression of MMP-14, VEGF and CD31 within tumors. The mean vessel

density within tumors decreased after stable transfection of miR-9 precursor. Scale bars: 100 m.

D, SH-SY5Y cells were injected into the tail vein of athymic nude mice (0.4× 106 cells per mouse,

n=6 for each group). Tumor cells stably transfected with miR-9 precursor established significantly

fewer metastatic colonies. Scale bars: 100 m. The symbol (*) indicates a significant decrease

from empty vector (mock).

Figure 6 Knockdown of MMP-14 suppressed the adhesion, migration, invasion, and

angiogenesis of NB cells. A, western blot indicated that transfection of si-MMP14 (100 nmol/L),

but not of si-Scb (100 nmol/L), into SH-SY5Y and SK-N-SH cells, resulted in decreased MMP-14

expression when compared to untransfected cells (control). B, the adhesion of SH-SY5Y and

SK-N-SH cells to matrigel or fibronectin was significantly attenuated by transfection of si-MMP14,

but not of si-Scb, when compared to control cells. C, in scratch migration assay, the migration of

si-MMP14-transfected SH-SY5Y and SK-N-SH cells was significantly reduced when compared to

control and those transfected with si-Scb. D, matrigel invasion assay indicated the decreased

invasion capabilities of MMP-14-knockdown SH-SY5Y and SK-N-SH cells than those of control

cells. E, the tube formation of endothelial HUVEC cells was suppressed by treatment with the

medium preconditioned by MMP-14-knockdown SH-SY5Y and SK-N-SH cells, when compared

to that of control cells. The symbol (*) indicates a significant decrease from control.




Published OnlineFirst May 7, 2012.Mol Cancer Ther Huanyu Zhang, Meng Qi, Shiwang Li, et al. invasion, metastasis and angiogenesis of neuroblastoma cellsmicroRNA-9 targets matrix metalloproteinase 14 to inhibit

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