MicroRNA-610 inhibits the migration and invasion of gastric cancer cells by suppressing the expression of vasodilator-stimulated phosphoprotein

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MicroRNA-610 inhibits the migration and invasion of gastric cancer cells by suppressing the expression of vasodilator-stimulated phosphoprotein Jing Wang a,e, Jingwei Zhang Yihao Tian a, Lei Wei a,*

b,e

, Junzhu Wu c, Daji Luo d, Ke Su a, Wentao Shi a, Jian Liu a,

a

Department of Pathology and Pathophysiology, School of Basic Medical Sciences, Hubei Provincial Key Laboratory of Allergy and ImmuneRelated Diseases and Center for Medical Research, Research Center of Food and Drug Evaluation, Wuhan University, Wuhan, PR China b Department of Oncology, Zhongnan Hospital of Wuhan University, Hubei Key Laboratory of Tumor Biological Behaviors, Hubei Cancer Clinical Study Center, Wuhan, PR China c Department of Biochemistry, School of Basic Medical Sciences, Wuhan University, Wuhan, PR China d Department of Medical Genetics, School of Basic Medical Sciences, Wuhan University, Wuhan, PR China

A R T I C L E I N F O

A B S T R A C T

Article history:

Vasodilator-stimulated phosphoprotein (VASP) has been implicated in the establishment of

Available online 19 December 2011

cancerous phenotypes. However, the role of VASP in gastric cancer progression and metastasis remains poorly understood. Here, we demonstrated that VASP was upregulated by

Keywords:

epidermal growth factor (EGF) and promoted the migration and invasion of gastric cancer

Gastric cancer

cells. Then we explored the regulatory mechanisms responsible for high expression of

miR-610

VASP in gastric cancer. Based on miRNA expression profiling of the paired gastric cancer

VASP

tissues and their adjacent non-tumour gastric tissues 18 miRNAs were identified including

Epidermal growth factor

microRNA-610 (miR-610) which were down-regulated in gastric cancer. Next, we observed

Cell migration

an inverse correlation between VASP and miR-610 expression levels in gastric cancer cells

Invasion

after EGF stimulation. Then we performed bioinformatics analysis, Western blot and

Metastasis

reverse transcription polymerase chain reaction (RT-PCR) analysis and luciferase assay to establish that miR-610 directly targets VASP 3 0 -UTR and inhibits its expression. Functionally, we demonstrated that miR610-mediated inhibition of VASP expression resulted in a significant reduction in the migration and invasion properties of gastric cancer cells. The identification of miR-610 as a novel miRNA regulated by EGF that targets VASP in gastric cancer cells suggests that EGF-miR610-VASP axis may be exploited for therapeutic intervention to inhibit gastric cancer progression and metastasis.  2011 Elsevier Ltd. All rights reserved.

1.

Introduction

Gastric cancer is one of the most aggressive and lethal malignancies in Asia.1,2 Over the last two decades a number of oncogenic and tumour-suppressor proteins have been identified that are associated with the development of gastric

cancer.3 However, the mechanism underlying gastric cancer progression remains poorly understood. Binding of epidermal growth factor (EGF) to cell surface receptor tyrosine kinases (RTKs) including epidermal growth factor receptor (EGFR) stimulates a series of cellular events that drive cell fate decisions.4,5 EGFR is often mutated or

* Corresponding author: Tel.: +86 15327396969; fax: +86 27 6875 9171. E-mail addresses: [email protected], [email protected] (L. Wei). e These authors contributed equally to this work. 0959-8049/$ - see front matter  2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ejca.2011.11.026

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overexpressed in gastric cancer, and its overexpression is correlated with poor survival.6 Deregulation of signalling cascades downstream of EGFR has been implicated in diverse types of human cancer.7,8 These cascades consist of small GTPases of the Rho-family, polyphosphoinositide (PPI) modulating enzymes and the actin-binding proteins (ABPs).9,10 The expression levels of different actin-binding proteins such as WAVE, Arp2/3 complex subunits, cofilin and profilin are altered in cancer cells.11–14 Especially, Ena/vasodilatorstimulated phosphoprotein (Ena/VASP) family has been implicated in the establishment of the cancerous cell phenotype.15–18 However, few studies have addressed the potential role of VASP in gastric cancer progression and metastasis. Our previous studies showed that VASP is critical for the migration and invasion properties of breast cancer cells.15,18 Therefore, in this study we examined whether VASP contributes to the migration and invasion of gastric cancer. Then we explored the potential regulatory mechanisms responsible for the high expression of VASP in gastric cancer. Given that miRNAs emerge as powerful regulators of gene expression and are involved in cancer progression,19,20 we decided to screen miRNAs that regulate VASP expression and function in gastric cancer metastasis. We performed miRNA expression profiling of the paired gastric cancer tissues and their adjacent non-tumour gastric tissues and identified 18 miRNAs including miR610 which were down-regulated in gastric cancer. Next, we demonstrated an inverse correlation between VASP and miR-610 expression levels in cultured gastric cancer cells after EGF stimulation, prompting us to hypothesise that VASP expression might be regulated by miR-610. Then we performed bioinformatics analysis, Western blot and RT-PCR analysis and luciferase assay to establish that miR-610 directly targets the 3 0 -UTR of the VASP and inhibits its expression. Functionally, we demonstrated that miR610-mediated inhibition of VASP expression resulted in a significant reduction in the migration and invasion properties of gastric cancer cells. This is the first report to identify VASP as a novel target of miR-610 and demonstrate the antagonistic effects of VASP and miR-610 on the regulation of gastric cancer cell migration and invasion.

2.

Materials and methods

2.1.

Clinical gastric cancer specimens and cell culture

Three pairs of primary gastric cancer tissues and corresponding adjacent non-tumour gastric tissues were obtained from untreated patients undergoing resection of gastric cancer in Zhongnan Hospital of Wuhan University. Non-tumour gastric tissues were obtained far from the centre of the cancer in surgical specimens. Informed consent was obtained from each patient and the protocol was approved by the Ethics Committee of Zhongnan Hospital. The specimens were immediately snap frozen in liquid nitrogen and stored at –80 C until used. The human gastric cancer cell lines BGC-823 and MKN-28 were cultured in Dulbecco 0 s modified Eagle 0 s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM Lglutamine (Invitrogen, Carlsbad, CA) at 37 C in 5% CO2.

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2.2.

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VASP knockdown

Three different pairs of oligos encoding the pre-miRNA were synthesised, annealed and cloned into pcDNA6.2-GW/ EmGFP-miR (Invitrogen, Carlsbad, CA, United States of America (USA)). These sequences are shown in Supplementary Table S1. The negative control miRNA vector with no sequence homology to human genes was provided by the manufacturer (Invitrogen). A total of 3 · 105 BGC-823 or MKN-28 cells were seeded in 6-well plates in DMEM containing 10% FBS without antibiotics for 24 h, then transfected with 4 lg purified pcDNA6.2-GW/EmGFP-miR expression vector containing either the VASP miRNA insert (miRVASP-514, miRVASP-1023 and miRVASP-1050) or the negative-control mismatch sequence (miR-control) using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. The cells were harvested 24 h later for the evaluation of VASP knockdown or functional assays.

2.3.

MicroRNA microarray

MicroRNA expression profiles of gastric cancer and adjacent non-tumour gastric tissues were generated by applying the miRCURY Locked Nucleic Acid (LNA) microarray platform (Exiqon, Denmark). All procedures were carried out according to manufacturer’s protocol. Briefly, total RNA was isolated from the tissues using Trizol (Invitrogen). One micro gram total RNA was dual-labelled with Hy3 or Hy5 fluorophore. Labelled miRNAs were hybridised on a microarray containing Tm-normalised capture probes for 471 human miRNAs. Slides were scanned using a GenePix 4000B laser scanner and the images were analysed using GenePix Pro 6.0 software (Axon Instruments, USA). The samples of gastric cancer tissues and paired adjacent non-tumour tissues were pooled to represent the study and the control group. Differentially expressed miRNAs were defined as miRNAs whose expression in the study group is consistently altered above 1.5-fold compared to the control group.

2.4. Quantitative reverse transcription-polymerase chain reaction Total RNA containing miRNAs was extracted from the cells using Trizol (Invitrogen). For miR-610 detection, 2 lg total RNA was reverse transcribed by Moloney murine leukaemia virus reverse transcriptase with 1 lg RT primer (Supplementary Table S1) according to the manufacturer’s instructions (Fermentas, Vilnius, Lithuania). Quantitative real-time polymerase chain reaction was performed by using SYBR-green PCR Master Mix (Tiangen, China) on the iCycler iQ Real-Time PCR Detection System (Bio-Rad). The mature miR-610 DNA sequence was used as the forward primer, and the 3 0 universal primer as listed in Supplementary Table S1. The human RNU6-2 (NR_002752) RNA was amplified in parallel as an internal control. PCR reactions were performed at 95 C for 10 min, followed by 40 cycles of 95 C for 15 s and 60 C for 1 min. DCt was calculated by subtracting the Ct of U6 from the Ct of miR610. DDCt was then calculated by subtracting the DCt of the control from the DCt of the treatment group. Fold change of miRNA was calculated by the equation 2–DDCt.

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2.5. MiR-610 expression plasmid construction and transfection Expression plasmid of miR-610 was constructed by PCR using human genomic DNA as a template. The primers are listed in Supplementary Table S1. PCR product (240 bp containing pre-miRNA) was digested with BamHI/XhoI and ligated into pcDNA 6.2-GW/EmGFP-miR vector (Invitrogen) to yield pcDNA6.2-miR-610 which was confirmed by DNA sequencing. PcDNA6.2-miR-610 vector was transfected to BGC-823 or MKN-28 cells plated in 6-well or 24-well plates using lipofectamine 2000 (Invitrogen). The cells were harvested 24 h later for the evaluation of gene expression or functional assays.

2.6.

Semi-quantitative reverse transcription-PCR

For the detection of VASP mRNA expression, 2 lg total RNA was reverse transcribed with oligo-dT primer according to the manufacturer’s instructions (Fermentas). The VASP primers are listed in Supplementary Table S1. Glyceraldehyde 3phosphate dehydrogenase (GAPDH) was amplified in parallel as the internal control. PCR reactions were performed at 95 C for 5 min, followed by 30 cycles of 94 C for 30 s and 60 C or 55 C for 30 s, 72 C for 30 s. PCR products were then separated on 2% agarose gels containing ethidium bromide and visualised under UV transillumination. Quantification of each product was done using GeneTools software (version 3.03; SynGene).

2.7.

Western blot analysis

Cells were washed with PBS and lysed in RIPA buffer containing 50 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM sodium fluoride, 2 mM Na3VO4, 1 mM EDTA, 1 mM EGTA and 1 · protease inhibitor cocktail. Cell lysate was quantified for protein content by BCA method. 40 lg of protein was resolved in 12% SDS–PAGE and transferred onto nitrocellulose membrane. Membranes were blocked in 5% BSA in Tris-Buffered Saline Tween-20 (TBST) for 2 h, then probed with primary antibody against VASP (Alexis Biochemicals) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 C overnight, washed extensively with TBST and incubated with secondary antibody conjugated with horseradish peroxidase at 1:2000 dilution. The signals were visualised with DAB system Western blotting detection reagents (Pierce).

2.8.

Luciferase activity assay

A 477 bp fragment of the VASP mRNA-3 0 UTR was amplified from a pCMV-SPORT vector containing VASP cDNA (Image clone 5210128, Proteintech Group) by PCR and cloned into pRL-TK reporter vector (Promega) to make pRL-TK-VASP3 0 UTR reporter construct. The primer sequences used for PCR amplification are listed in Supplementary Table S1. Site-directed mutagenesis was performed to disrupt the sequence within VASP mRNA-3 0 UTR which is complementary to the miR-610 seed region with the primers listed in Supplementary Table S1, and the mutated vector was named pRL-TK-mutVASP 3 0 UTR. The reporter constructs were confirmed by DNA sequencing. A total of 5 · 104 BGC-823 or MKN-28 cells

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were seeded in 24-well plates for 24 h and then co-transfected with 100 ng pRL-TK constructs using Lipofectamine 2000 with or without pcDNA6.2-miR-610. 100 ng of pGL3 plasmid was co-transfected to monitor the transfection efficiency. At 24 h post-transfection, the luciferase activities were measured by the dual-luciferase reporter assay system using the GloMax luminometer (Promega).

2.9.

Cell migration and invasion assay

For wound healing assay, gastric cancer cells BGC-823 and MKN-28 were grown to confluence on 6-well plates. Then, linear scratch wounds (in triplicate) were created on the confluent monolayer using a pipette tip and cells were transfected with pcDNA6.2-miR-610 or pcDNA6.2-VASP-miRNA. Immediately after wounding (time 0) and at 6 h intervals for 48 h, images of the untreated, miR610-treated, VASP-miRNA treated BGC-823 and MKN-28 cells were taken using digital camera mounted on light microscope. The width of the wound gaps were measured using NIH Image J analysis and normalised to the time 0 wounds for four independent experiments. Transwell migration assay was performed in 6.5-mm diameter Boyden chambers with pore size of 8.0 lm (Corning, NY, USA). Twenty-four hours after transfection with miR-610 or VASP-miRNA vector, 5 · 104 cells were resuspended in the migration medium (DMEM medium with 0.5% FBS), and placed in the upper compartment of transwell chambers coated with Fibronectin on the lower surface. The lower compartment was filled with 600 ll DMEM medium containing 10% FBS. After incubation for 24 h at 37 C, cells on the lower surface of the filter were fixed in 4% formaldehyde for 20 min and stained with 0.1% crystal violet and five random fields of each filter were counted at 200· magnifications. For invasion assays, cells were measured in 24-well matrigel-coated invasion chambers. The lower chambers were filled with 600 ll of DMEM medium containing 10% FBS as a chemoattractant. A cell suspension of 5 · 104 in 100 ll DMEM medium with 0.5% FBS was added to the upper chamber. After the cells were incubated for 24 h at 37 C in a humidified incubator with 5% CO2, the invasive cells attached to the lower surface of the membrane insert were fixed by 4% formaldehyde and stained with crystal violet. The number of cells was then counted under a microscope.

2.10.

Statistical analysis

The data were expressed as the mean ± standard error of the mean (SEM) from at least three independent experiments and analysed using Prism GraphPad Software. The difference between two groups was analysed by Student 0 s t-test. The difference was significant for P < 0.05.

3.

Results

3.1. VASP knockdown inhibits gastric cancer cell migration and invasion in vitro By immunochemical staining we previously observed that the expression level of VASP is positively correlated with the advanced stages of gastric cancer.21 Therefore, we employed loss

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Fig. 1 – Vasodilator-stimulated phosphoprotein (VASP) knockdown inhibits gastric cancer cell migration and invasion in vitro. (A) BGC-823 cells were transfected with three miRVASP vectors (miRVASP-514, miRVASP-1023, miRVASP-1050), then subjected to Western blot analysis using VASP and GAPDH antibodies. (B) BGC-823 and (C) MKN-28 cell monolayers were transfected as indicated and scratched, then the migration of the cells towards the wound was visualised. Images were taken at various time points and Image J was used to determine the migration distance. (D) BGC-823 and (E) MKN-28 cells were transfected as indicated and subjected to transwell migration and invasion assay. Data were presented as mean ± standard error of the mean (SEM), *P < 0.05, **P < 0.01.

of function approach to confirm that VASP is crucial for the migration and invasion of gastric cancer cells. The results showed that VASP protein level was decreased by 69% (P < 0.01) and 53% (P < 0.01) in miRVASP-514 and miRVASP-1023 transfected BGC-823 cells, respectively, compared to control cells (Fig. 1A). Similar knockdown results were observed in MKN-28 cells (data not shown). We therefore chose miRVASP514 to effectively knockdown VASP for the following functional analysis. Wound healing assay showed that miRVASP-514

mediated VASP knockdown reduced the motility of both BGC823 and MKN-28 cells (Fig. 1B and C). Transwell migration and invasion assay also demonstrated that the numbers of migratory and invasive BGC-823 and MKN-28 cells transfected with miRVASP-514 were significantly reduced compared to controls (Fig. 1D and E). Taken together, these results demonstrate that VASP is crucial for the migratory and invasive capability of BGC-823 and MKN-28 cells in vitro, consistent with our previous studies on MDA-MB-231 breast cancer cells.15,18

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3.2. MiR-610 is downregulated in human gastric cancer tissues and EGF treated gastric cancer cells Next we aimed to screen miRNAs that regulate VASP expression in gastric cancer. MicroCosm Targets predicted that VASP is targeted by 31 human miRNAs (Supplementary Table S2). We performed miRNA expression profiling of primary gastric cancer and paired adjacent non-tumour gastric tissues samples and identified 18 miRNAs significantly downregulated in primary gastric cancer compared with adjacent non-tumour gastric tissues (Supplementary Table S3). Notably, miR-610 and miR-638, which were predicted to target VASP, were downregulated in gastric cancer. Among them, miR610 attracted our attention because it was also reported to be downregulated in gastric cancer by Yao et al.22 and its function remains elusive. Therefore, miR-610 was chosen for further characterisation. Analysis of the genomic regions of DNA 2000 bp upstream of miR-610 revealed that miR-610 contained three p53 binding sites, indicating that it is a candidate target of EGF signalling. To determine whether EGF regulates miR-610 expression, we performed qRT-PCR to evaluate the mature miR-610 level in MKN-28 cells after EGF stimulation. MiR-610 expression was decreased by 60% (P < 0.001) and 80% (P < 0.001) after 24 h treatment with 15 and 50 ng/ml EGF, respectively (Fig. 2A). Interestingly, we found that VASP expression was induced by 1.6-fold in response to 50 ng/ml EGF treatment in MKN-28 cells (P < 0.05; Fig. 2B). Similar patterns of miR-610 and VASP expression in response to

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EGF were observed in BGC-823 cells (data not shown). These data demonstrate that miR-610 is downregulated in human gastric cancer tissues and there is an inverse correlation between VASP and miR-610 expression levels in cultured gastric cancer cells after EGF stimulation.

3.3. MiR-610 directly targets the 3 0 -UTR of VASP and represses its expression As shown in Fig. 3A, miR-610 targets the 741-762 nt of VASP 3 0 -UTR, indicating that VASP is a target of miR-610. Western blot analysis demonstrated that ectopic miR-610 expression significantly decreased VASP protein level in BGC-823 cells (P < 0.01; Fig. 3B) although VASP mRNA level remained unchanged (Fig. 3C). Next we made a reporter construct harbouring the 477 bp fragment of VASP 3 0 -UTR flanking the entire putative target sequence and performed luciferase assay. The results showed that ectopic miR-610 expression resulted in a significant reduction of luciferase activity in BGC-823 cells (P < 0.01; Fig. 3D). Furthermore, the mutation of the seed sequence of miR-610 within the 3 0 -UTR of VASP abrogated the inhibition of luciferase activity by exogenous miR-610 in BGC-823 cells (Fig. 3D). Similar results were obtained in MKN-28 cells (Fig. 3E), demonstrating that the effect of miR610 is not restricted to a single cell line. Taken together, these data provide strong evidence that miR-610 specifically targets VASP and represses its expression in gastric cancer cells.

3.4. MiR-610 inhibits gastric cancer cell migration and invasion in vitro by repressing VASP expression Having established that VASP is a direct target of miR-610, we decided to explore the functional significance of the regulation of VASP by miR-610 in gastric cancer. Wound healing assay demonstrated that ectopic miR-610 expression reduced the motility of BGC-823 and MKN-28 cells (Fig. 4A and B). Furthermore, transwell migration and invasion assay showed that the numbers of BGC-823 and MKN-28 cells transfected with miR-610 expression vector that passed the membrane were significantly reduced compared to the cells transfected with the control vector (Fig. 4C and D). To confirm that miR-610 modulates the biological behaviours of gastric cancer cells by repressing VASP expression, we performed rescue experiments. Upon the co-transfection of VASP expression vector without 3 0 -UTR (pEGFP-VASP) into BGC-823 and MKN-28 cells, we observed that overexpression of VASP could rescue miR-610 mediated inhibition of cell migration and invasion (Fig. 5A and B). These results indicate that miR-610 inhibits gastric cancer cell migration and invasion in vitro by repressing VASP expression.

Fig. 2 – Epidermal growth factor (EGF) regulates miR-610 and vasodilator-stimulated phosphoprotein (VASP) expression in an opposite manner. (A) qRT-PCR analysis of mature miR610 in MKN-28 cells untreated or treated with EGF (15 and 50 ng/ml) for 24 h. Expression was normalised to untreated MKN-28 cells. B. Western blot analysis of VASP expression in MKN-28 cells untreated or treated with EGF 50 ng/ml for 24 h. GAPDH served as loading control. Data were presented as mean ± standard error of the mean (SEM), *P < 0.05, ***P < 0.001.

4.

Discussion

Five-year survival rate of patients with resectable gastric cancer ranges from 10% to 30%.23 The cause of gastric cancer-related death is more often due to metastasis of the primary tumour. An understanding of the molecular pathways regulating gastric cancer metastasis could help improve the diagnosis and therapy of the disease.

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Fig. 3 – MiR-610 represses vasodilator-stimulated phosphoprotein (VASP) expression through binding to 3 0 -UTR of VASP. (A) Sequence alignment of human miR-610 with 3 0 -UTR of VASP. The seed sequence of miR-610 (top) matches 3 0 -UTR of VASP (middle). Bottom, mutation of the 3 0 -UTR of VASP in mutant luciferase reporter construct. BGC-823 cells were transfected with pcDNA6.2-miR-610 and pcDNA6.2-miR-control vector, VASP protein level was detected by Western blot with GAPDH as loading control (B), VASP mRNA level was measured by RT-PCR with GAPDH as internal control (C). BGC-823 (D) and MKN-28 (E) cells were transiently transfected with indicated plasmids and collected 24 h later for luciferase assay. Data presented were the normalised Renilla/firefly luciferase ratio. MiR-610 inhibited wild-type but not mutant VASP-3 0 -UTR reporter activity in BGC-823 and MKN-28 cells. Data were presented as mean ± standard error of the mean (SEM), **P < 0.01, ***P < 0.001.

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Fig. 4 – MiR-610 inhibits gastric cancer cell migration and invasion in vitro. BGC-823 and MKN-28 cells were transfected with pcDNA6.2-miR-610 or control vector, and then collected for wound healing assay (A, B) or transwell migration and invasion assays (C, D). Shown were pictures of representative fields for each experiment. Data were expressed as mean ± standard error of the mean (SEM) from three independent experiments. *P < 0.05, ***P < 0.001, versus miR-control.

The actin cytoskeleton mediates numerous cellular processes including adhesion, motility and shape change. Dysfunctional actin cytoskeleton is associated with diseases such as metastatic cancers.12,24 As a key regulator of actin cytoskeleton, VASP is a member of the Ena/VASP protein family which is recruited downstream of plasma membrane receptors25,26 and plays crucial roles in the regulation of cell adhesion, migration and cell-cell interaction.27 Deregulation of EGFR in gastric cancer correlates with poor disease outcome and induces tumour metastasis.6 EGF stimulation is known to activate several signalling pathways that drive cell motility and migration, processes in which VASP has been implicated.28–30 Recently, miRNAs emerge as novel regulators of the actin cytoskeleton. For example, miR-146a targets ROCK1, a Rho target that affects cell movement in androgen-independent prostate cancer.31 MiR-155 is associated with TGF-induced RhoA suppression, leading to the dissolution of cell-cell tight junctions.32 MiR-200 and miR-31 suppress WAVE3 and function to inhibit cancer cell invasion.20,33 In addition, miR-10b directly targets Tiam1, which is implicated in Rac activation and carcinoma migration.34

In this study we demonstrated that VASP contributes to gastric cancer invasion. Next we hypothesised that VASP expression and function might be regulated by miRNA in gastric cancer. We used multiple assays to confirm that VASP expression is regulated by miR-610. First, miRNA expression profiles of the paired gastric cancer and their adjacent nontumour gastric tissues identified the downregulation of miR-610 in gastric cancer. Second, qRT-PCR and Western blot analysis showed an inverse correlation between the expression levels of VASP and miR-610 in gastric cancer cells after EGF stimulation. Third, overexpression of miR-610 precursor vector in gastric cancer cells resulted in a significant downregulation of VASP expression at protein but not mRNA level. Fourth, mutation of the seed sequence for miR-610 in the 3 0 UTR of VASP abrogated miR-610 mediated inhibition of luciferase activity, clearly demonstrating that miR-610 directly targets VASP and represses its expression. Based on these results, we finally characterised the functional significance of the regulation of VASP expression by miR-610 and found that miR-610 mediated inhibition of VASP expression resulted in significant loss of the migratory and invasive phenotype of gastric cancer cells.

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Fig. 5 – Vasodilator-stimulated phosphoprotein (VASP) overexpression rescues MiR-610 mediated inhibition of gastric cancer cell migration and invasion in vitro. BGC-823 (A) and MKN-28 (B) cells were transfected with the indicated plasmids and then collected for wound healing assay or transwell migration and invasion assays. Shown were pictures of representative fields for each experiment. Data were expressed as mean ± standard error of the mean (SEM) from three independent experiments.

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In addition to VASP, many genes relevant for the metastatic phenotype were predicted to be targeted by miR-610 (Supplementary Table S4). For example, WASF1 (Wiskott-Aldrich syndrome protein family member 1), a downstream effector of Rho GTPases, might be involved in miR-610 mediated regulation of cell migration and invasion. In this study our data only show that miR-610 is an EGF-regulated miRNA that exerts tumour suppression activities in gastric cancer. Further study is necessary to identify and characterise other targets of miR-610 to fully elucidate the functional role of miR-610 in gastric cancer progression and metastasis. In conclusion, our data demonstrate that the expression of miR-610 is decreased in gastric cancer. Furthermore, the activation of EGF signalling is associated with the upregulation of VASP and the downregulation of miR-610. MiR-610 directly targets VASP and represses its expression, leading to the inhibition of VASP mediated cell migration and invasion in gastric cancer. The identification of miR-610 as a novel miRNA regulated by EGF that targets VASP in gastric cancer cells suggests that EGF-miR610-VASP axis may be exploited for therapeutic intervention to inhibit gastric cancer progression and metastasis.

Conflict of interest statement None declared.

Acknowledgements We thank Yun Wei for technical assistance. This work was supported by the Natural Science Foundation of China (No. 30971132 and No. 81172043), the Hubei Science Foundation (No. 2009CDA074), and the Fundamental Research Funds for the Central Universities (No. 3081006 and No. 20083010101000064).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejca.2011.11.026.

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