Mol Cell Biochem (2014) 396:295–305 DOI 10.1007/s11010-014-2164-6

MicroRNA 520d-3p inhibits gastric cancer cell proliferation, migration, and invasion by downregulating EphA2 expression Ruixin Li • Weijie Yuan • Wenjuan Mei Keda Yang • Zihua Chen

Received: 13 April 2014 / Accepted: 14 July 2014 / Published online: 26 July 2014 Ó Springer Science+Business Media New York 2014

Abstract Aberrant expression of microRNAs (miRNAs) has been shown to play important roles in cancer progression as a result of changes in expression of their target genes. In this study, we investigated the roles of miR-520d3p on gastric cancer (GC) cell proliferation, migration, and invasion, and confirmed that this miRNA regulates EphA2 expression. The mRNA expression levels of miR-520d-3p and EphA2 in GC tissues and cell lines were evaluated. The clinical and prognostic significance of miR-520d-3p was assessed. The biological function of miR-520d-3p in GC cells was investigated using a methylthiazolyldiphenyltetrazolium bromide assay, cell cycle assay, transwell invasion assay, and wound-healing assay. miR-520d-3p expression was down-regulated and inversely correlated with the expression of EphA2 in GC tissues and cell lines. Lower expression of miR-520d-3p was associated with tumor invasion (P = 0.0357), lymph nodes metastasis (P = 0.0272), a higher clinical stage (P = 0.0041), and poorer overall survival (P = 0.0105). Luciferase assays revealed that miR-520d-3p inhibited EphA2 expression by targeting the 30 -untranslated region of EphA2 mRNA.

R. Li  W. Yuan  Z. Chen (&) Department of Gastrointestinal Surgery, Xiangya Hospital, Central South University, Xiangya Road, Changsha 410008, Hunan, People’s Republic of China e-mail: [email protected] W. Mei Department of Nephrology, Xiangya Hospital, Central South University, Xiangya Road, Changsha 410008, Hunan, People’s Republic of China K. Yang Department of Pathology, Xiangya Hospital, Central South University, Xiangya Road, Changsha 410008, Hunan, People’s Republic of China

Overexpression of miR-520d-3p dramatically inhibited the proliferation, cell cycle progression, invasion, and migration of GC cells, while down-regulation substantially promoted these properties. Moreover, c-Myc, CyclinD1, and matrix metalloproteinase-9 expression levels were downregulated in miR-520d-3p mimic-transfected cells and upregulated in miR-520d-3p inhibitor-transfected cells. Taken together, our data showed that miR-520d-3p appears to contribute to GC progression via the regulation of EphA2 and could serve as a novel prognostic and potential therapeutic marker. Keywords miR-520d-3p  EphA2  Gastric cancer  Proliferation  Invasion

Introduction Gastric cancer (GC) is the most lethal type of gastrointestinal tract malignancy, and the third leading cause of cancer-related deaths worldwide [1]. More than 40 % of all GC cases occur in China [2]. Post-surgical recurrence, metastasis, and multidrug resistance all contribute to maintain the 5-year survival rate below 30 % [3]. Therefore, a better understanding of the pathogenesis and molecular mechanisms involved in GC progression is essential to achieve early diagnosis and positive responses to therapy. MicroRNAs (miRNAs), a class of 18–25-nucleotidelong noncoding RNAs, have been identified to be aberrantly expressed in several human malignancies [4]. These highly conserved noncoding RNAs incompletely bind to the 30 -untranslated regions (UTRs) of their target mRNAs, thereby resulting in mRNA degradation or translation inhibition [5]. Previous studies have identified a number of



miRNAs that show aberrant expression in GC [6–9]. For instance, miR-146a has been shown to be down-regulated in GC tissues, and lower levels of miR-146a are associated with lymph node metastasis and venous invasion [10]. Furthermore, miR-135a is down-regulated in early GC and suppresses GC cell migration and invasion through its target gene ROCK1 [11]. Therefore, further exploration of the expression and function of miRNAs will provide insight into the pathogenesis and progression of GC. EphA2, a member of the erythropoietin-producing hepatocellular (Eph) receptor family, is a receptor tyrosine kinase that has been shown to have oncogenic properties in several human malignancies, including GC [12–15], glioblastoma [16], ovarian cancer [17–19], cholangiocarcinoma [20], lung cancer [21, 22], breast cancer [23], colorectal cancer [24], and prostate cancer [25]. Our previous work indicated that EphA2 is overexpressed in approximately 60 % of GC tissues, and its expression has been associated with invasion, lymph node metastasis, a higher clinical stage, and poor prognosis, and was shown to promote GC cell proliferation, migration, and invasion in vitro and in vivo [12–15]. However, the molecular mechanisms underlying EphA2 overexpression in GC are still largely unknown. Recently, a number of miRNAs that have been reported to be aberrantly expressed in cancer were found to exert their biological functions through targeting EphA2, including miR-141 [26], miR-520d-3p [17], miR-200a [27], and miR-26b [28]. This evidence suggests that investigation into the precise molecular mechanisms underlying the interaction between miRNAs that target EphA2 and GC progression could provide valuable information for the development of a novel therapeutic strategy. In the current study, we evaluated the clinical significance of miR-520d-3p as a tumor suppressor in GC tissues and cell lines. Furthermore, we demonstrate the effect and mechanism of miR-520d-3p overexpression on GC cell proliferation, invasion, and migration.

Materials and methods Patient tissues and preparation A total of 120 GC tissues and their corresponding nontumorous (NT) gastric tissues were obtained from the Department of Gastrointestinal Surgery of Xiangya Hospital of Central South University between 2010 and 2012. Before surgical resection, GC was confirmed by a pathologist in all patients. No patients received neoadjuvant chemotherapy or radiotherapy before radical gastrectomy. All patients were closely followed up after surgery, and the follow-up was terminated on December 30, 2013. The follow-up periods ranged from 3 to 44.9 months, with a


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mean of 16.5 months. Resected tissues were immediately frozen in liquid nitrogen and stored at -80 °C for RNA extraction or were fixed in 37 % formaldehyde solution for paraffin embedding. All samples were collected and used after obtaining informed consent. The study was approved by the Research Ethics Committee of Xiangya Hospital, Central South University. Cell culture and transfection The gastric adenocarcinoma cell lines (AGS, SGC-7901, BGC-823, and MKN-45) and normal gastric epithelial cell line (GES) were purchased from the Cell Resource Center of Xiangya Medical School, Central South University. Cells were maintained in RPMI 1640 medium (AGS, SGC-7901, BGC-823, MKN-45) or Dulbecco’s modified Eagle medium (GES) supplemented with 10 % fetal bovine serum (Hyclone) at 37 °C in a 5 % CO2 incubator. Transfection of the cells with 2 lM of miRNA mimics or miRNA inhibitors (Genepharma) and their negative controls was performed using Lipofectamine 2000 (Invitrogen). The sequences used were as follows: forward: 50 -AAAGUGCUUCUCUUUGGUGGGU-3 0 , reverse: 50 -CCACCAAAGAGAAGCACUUUUU-30 (miR520d-3p mimics), 50 -ACCCACCAAAGAGAAGCACUU U-30 (miR-520d-3p inhibitor oligonucleotide), and 50 -CAGU ACUUUUGUGUAGUACAA-30 (control oligonucleotide). RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was isolated from tissues and cell lines using the miRNeasy Mini Kit (Qiagen). The miRNA Q-PCR Detection Kit (GeneCopoeia) was used for quantification of miRNA levels according to the manufacturer’s protocol. For quantification of EphA2 mRNA levels, the RT reactions were conducted with the RevertAidTM H Minus First Strand cDNA Synthesis Kit (Fermentas). qRT-PCR was performed using an ABI 7500 System (Bio-Rad). RNU6B and b-actin were used as normalizing controls for miRNA and mRNA quantification, respectively. The 2-DDCt method was employed to calculate the relative expression levels. The primers were as follows: miR-520d-3p, forward primer: 50 -GGTCTACAAAGGGAAGC-30 and reverse primer: 50 -TTTGGCACTAGCACATT-30 ; EphA2, forward primer: 50 -TGGCTCACACACCCGTATG-30 and reverse primer: 50 -GTCGCCAGACATCACGTTG-30 . Western blot analysis Cells were lysed using a mammalian protein extraction reagent, RIPA (Beyotime), supplemented with a protease inhibitor cocktail (Roche). Total proteins (40 lg) were separated by 10 % sodium dodecyl sulfate-polyacrylamide

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gel electrophoresis and then transferred to nitrocellulose membranes (Millipore). Membranes were then incubated with specific primary antibodies [EphA2, c-Myc, matrix metalloproteinase, MMP7 (Abcam), MMP9, GAPDH (Santa Cruz), CyclinD1 (Cell Signaling Technology)] overnight at 4 °C. Primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies. The bands were visualized using an enhanced chemiluminescence system (Millipore) according to the manufacturer’s protocol. Immunohistochemical staining Paraffin blocks were sectioned at a 4-lm thickness. Immunohistochemical staining was performed with the ElivisionTM plus Kit (Fuzhou Maxim Biotech). The slides were dewaxed in xylene and then immersed in 2.5 % hydrogen peroxide for 30 min at room temperature, followed by citrate buffer treatment at 100 °C for antigen retrieval. After washing with phosphate-buffered saline (PBS), the slides were incubated with EphA2 primary antibody overnight at 4 °C. Incubation with solution B was conducted for 30 min at room temperature, after which the slides were incubated with solution A for 20 min at room temperature. Finally, the slides were visualized in diaminobenzidine solution. The reactivity degree was assessed by two pathologists independently. The degree of positivity was classified as previously described [13, 19]. The percentage of positive cells was evaluated as follows: 0 points, 0–5 %; 2 points, 6–50 %; and 3 points, [50 %. The staining intensity was rated as follows: 1 point, weak intensity; 2 points, moderate intensity; and 3 points, strong intensity. Scores for expression and percentage of positive cells were summed. Slides were categorized into four groups: negative, 0 points; weak, 1–2 points; moderate, 3–4 points; and strong, 5–6 points.


ethanol. The cells were re-suspended in 1,000 lL PBS containing 1 mg/mL RNase and 50 mg/mL propidium iodide (Sigma) after washing with PBS. Samples were incubated for 30 min at 37 °C in the dark and cell cycle analysis was performed using a flow cytometer (Beckman Counter). Transwell invasion assays Matrigel-coated invasion chambers (8 lm; BD Biosciences) were used according to the manufacturer’s instructions. The cells were suspended in serum-free medium at 5 9 104 cells/mL. A 2-mL cell suspension was pipetted into the upper chamber of the Matrigel-coated inserts. RPMI 1640 medium with 10 % fetal bovine serum was added to the lower chamber as a chemoattractant. The cells were incubated for 24 h at 37 °C, and then the cells on the upper side of the membrane were removed with a cotton swab. The cells that had invaded the lower surface were fixed with methanol and stained with 0.1 % crystal violet. The number of cells that had invaded through the Matrigel was counted in five fields for triplicate membranes at 9200 magnification using an inverted microscope (Olympus). Wound-healing assay Transfected SGC-7901 and MKN-45 cells were cultured in six-well plates and grown to confluence overnight. Linear wounds were made with a sterile 200-lL pipette tip. Cells were washed to remove the debris using PBS and incubated at 37 °C for 24 h. The serial images of cell migration at 0 and 24 h after scratching were obtained using an inverted microscope (Olympus). Luciferase assay

Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay GC cells transfected with miR-520d-3p mimics or inhibitors and their negative normal controls (NCs) were plated in 96-well plates at 1 9 104 cells per well. After incubation for 24 h and 48 h, the culture medium was replaced with fresh RPMI 1640. Twenty microliters of MTT (Sigma) was then added to each well. After 4 h of additional incubation, the medium was replaced with 150 lL dimethyl sulfoxide (Sigma) and mixed for 10 min at room temperature. The absorbance was measured at 570 nm on a microplate reader (Beckman Counter). Cell cycle assays The GC cells transfected with miRNA mimics or inhibitors and their negative NCs were harvested and fixed in 75 %

The 30 -UTR sequence of EphA2 was amplified from normal human genomic DNA (NM_004431) and subcloned into the pmirGLO luciferase reporter vector (Promega). SGC-7901 or MKN-45 cells at 70–80 % confluence in 48-well plates were cotransfected with wild-type (WT) or mutant (Mut) 30 -UTR vectors and miR-520d-3p mimics or inhibitors using Lipofectamine 2000. At 48 h post-transfection, the cells were assayed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) by following the manufacturer’s instructions. The firefly luciferase activities were normalized to Renilla luciferase activity. The firefly luciferase activity of the cells that were transfected with miRNA mimics or inhibitors is represented as the percentage of activity relative to that of cells that were transfected with negative controls. All experiments were performed in triplicate.



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Fig. 1 The expression and correlation of EphA2 and miR-520d-3p in gastric cancer tissues. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-520d-3p and EphA2 in 120 gastric cancer (GC) tissues (a) and nontumor tissues (NT, b). c Pearson’s coefficient correlation analysis was employed to evaluate the correlation between the expression of miR-520d-3p and EphA2. d One hundred and twenty

gastric cancer tissues were graded into two groups (negative/weak vs. moderate/strong) according to the immunohistochemical staining intensity of EphA2. The miR-520d-3p expression levels were analyzed between these two groups. e Kaplan–Meier overall survival curves according to high and low miR-520d-3p expression levels. *P \ 0.05, ***P \ 0.0001

Statistical analysis

that the average expression level of miR-520d-3p was down-regulated in GC tissues and up-regulated in NT tissues (Fig. 1a, b). Inversely, the average expression level of EphA2 was up-regulated in GC tissues and down-regulated in NT tissues (Fig. 1a, b). Pearson’s correlation coefficient analysis revealed a significant inverse correlation between miR-520d-3p expression and EphA2 transcript levels, as shown in Fig. 1c (P = 0.0178). To further analyze this relationship, immunohistochemical staining was performed to observe the expression of EphA2 in these cancerous tissues. Immunostaining confirmed positive membrane and cytoplasm staining of EphA2 in 73/120 (60.83 %) of samples, and tumors with high miR-520d-3p expression showed negative or weak EphA2 staining, whereas tumors with low miR-520d-3p expression showed moderate or strong EphA2 staining (Fig. 1d). We further analyzed the relationship between miR-520d3p levels and clinicopathological factors. As shown in Table 1, low miR-520d-3p expression was correlated with increased tumor invasion (P = 0.0357), lymph node metastasis (P = 0.0272), and clinical stage (P = 0.0041). However, miR-520d-3p expression was not correlated with age, gender, tumor size, tumor position, or tumor cell

All experiments were performed three times. Continuous variables are shown as the mean ± SD, and differences between groups were evaluated using unpaired Student’s ttests. Pearson’s coefficient correlation analysis was used to determine the correlation between miR-520d-3p and EphA2 expression. Overall survival was evaluated using the Kaplan–Meier method and the log-rank test. Statistical analyses were performed with SPSS 18.0 software (SPSS, Chicago, IL). P \ 0.05 was considered statistically significant.

Results miR-520d-3p is down-regulated and inversely associated with the expression of EphA2 in clinical specimens and GC cell lines We first employed qRT-PCR to detect miR-520d-3p and EphA2 levels in 120 gastric cancerous (GC) and corresponding noncancerous tissues (NT). The results showed


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Table 1 Relationship between miR-520d-3p expression and clinicopathological factors in 120 primary gastric cancer tissues

miR-520d-3p directly targets the 30 -UTR of EphA2

Clinicopathological factors

To elucidate whether EphA2 is a potential downstream target gene of miR-520d-3p, the miRNA target prediction websites and TargetScan were used to identify a conserved miR-520d-3p-binding site in the 30 UTR of EphA2 mRNA. We then cloned WT or Mut target region sequence of the EphA2 30 -UTR, which was inserted into a luciferase reporter vector (Fig. 2a). Subsequently, these reporter vectors were cotransfected with miR-520d3p mimics or inhibitors and their corresponding NCs (miRNC and Anti-miR-NC) into the SGC-7901 and MKN-45 cell lines. The data showed that ectopic expression of miR520d-3p potently reduced the activity of the luciferase reporter fused to the WT EphA2 30 -UTR, but not of that fused to the Mut 30 -UTR (Fig. 2b). Inversely, luciferase activity was significantly increased in the cells transfected with miR-520d-3p inhibitors, but did not change in the Mut 30 -UTR cells (Fig. 2c). qRT-PCR and Western blot analysis were used to determine whether miR-520d-3p could regulate EphA2 at both the mRNA and protein levels, respectively. The ectopic expression of miR-520d-3p inhibited EphA2 at both the mRNA and protein levels, while EphA2 expression was up-regulated by miR-520d-3p inhibitors (Fig. 3). These data suggest that EphA2 is a direct functional target of miR-520d-3p.

Number of cases

Expression of miR520d-3p (mean ± SD)




1.7510 ± 0.0818


[60 Gender


1.5509 ± 0.0828

Age (years)



1.6587 ± 0.0732



1.6699 ± 0.1007



1.6373 ± 0.0715



1.7172 ± 0.1052


Tumor size (cm) 0.5305

Location Distal third


1.6814 ± 0.0632

Middle or proximal


1.5977 ± 0.1482

Well and Moderate


1.7052 ± 0.0876

Poor and Signet


1.6322 ± 0.0798


1.8865 ± 0.1411

T3 ? T4 92 Lymph node metastasis

1.5944 ± 0.0626


Differentiationa 0.5442

Invasion T1 ? T2



1.8873 ± 0.1292



1.5877 ± 0.0643

I ? II


1.8948 ± 0.1092



1.5421 ± 0.0659



Clinical stages 0.0041c

miR-520d-3p inhibits GC cell proliferation, invasion, and migration


Well-differentiated adenocarcinoma (Well), moderately differentiated adenocarcinoma (Moderate), poorly differentiated adenocarcinoma (Poor), signet ring cell carcinoma (Signet)


P \ 0.05; c P \ 0.01

differentiation. Next, we divided the 120 GC patients into two groups, the miR-520d-3p high-expression group (GC/ NT expression[0.5, n = 53) and the low-expression group (GC/NT expression \0.5, n = 67), according to the GC/ NT tissue ratio of miR-520d-3p expression, as previously described [10]. In the overall survival curve, patients in the miR-520d-3p low-expression group (median survival time 11.97 months) had a significantly poorer prognosis than those in the miR-520d-3p high-expression group (median survival time 19.17 months, P = 0.0105; Fig. 1e). A panel of GC cell lines was also used to analyze the expression of miR-520d-3p and EphA2. Compared with a normal GES, the miR-520d-3p expression level was downregulated in GC cell lines (SGC-7901, MKN-45), while the EphA2 expression level was up-regulated in these cell lines (data not shown). Taken together, these results indicated that miR-520d-3p was down-regulated and inversely associated with the expression of EphA2 in clinical specimens and GC cell lines.

To further characterize the functional importance of miR520d-3p in GC progression, we examined its effect on the proliferation of GC cells using an MTT assay. The data showed that the MTT value (reflecting cell viability) of cells transfected with miR-520d-3p mimics was significantly lower than that of cells transfected with control miRNA. However, miR-520d-3p inhibitors significantly promoted the proliferation of GC cells (Fig. 4). In addition, flow cytometry indicated that miR-520d-3p overexpression increased the percentage of SGC-7901 and MKN-45 cells in the G0/G1 phase, whereas miR-520d-3p inhibitors significantly decreased the percentage of cells in the G0/G1 phase (data not shown). To evaluate the impact of miR-520d-3p on cell invasion and migration, Matrigel invasion and wound-healing assays were employed. The results showed that miR-520d3p mimics decreased the invasion of SGC-7901 and MKN45 cells, whereas miR-520d-3p inhibitors substantially increased their invasion (Fig. 5a). Similar results were observed in migration assays of SGC-7901 and MKN-45 cells (Fig. 5b). Together, these findings demonstrate that miR-520d-3p inhibits GC cell proliferation, invasion, and migration in vitro.



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Fig. 2 miR-520d-3p directly targets the 30 -UTR of EphA2. a Representative diagram of the predicted wild-type (WT) or mutant (Mut) binding site of miR-520d-3p in the 30 untranslated region (UTR) of EphA2 mRNA. b, c The luciferase reporter plasmid containing the WT or Mut EphA2 30 -UTR was cotransfected into SGC-7901 and MKN-45 cells with miR520d-3p mimics or inhibitors. Luciferase activity of the cells was assayed at 48 h after transfection, and the values were normalized to the normal control values. *P \ 0.05, **P \ 0.01, ***P \ 0.0001 (compared with the control)

miR-520d-3p downregulates c-Myc, CyclinD1, and MMP9 expression through targeting EphA2 Our previous study indicated that silencing of EphA2 results in decreased protein levels of c-Myc, CyclinD1, and MMP9, which are oncoproteins that play an important role in cell proliferation, cell cycle regulation, and invasion, respectively [12, 29]. In the present study, Western blotting was used to investigate whether miR-520d-3p regulates the expression of c-Myc, CyclinD1, and MMP9 through


targeting EphA2. The results revealed that the expression levels of EphA2, c-Myc, CyclinD1, and MMP9 in cells transfected with miR-520d-3p mimics were significantly lower than those of cells transfected with NC miRNA. By contrast, miR-520d-3p inhibitors increased the expression of these proteins, except for MMP7, whose expression was not significantly changed (Fig. 6). These results provide further support of the inhibitory role that miR-520d-3p plays in the proliferation, invasion, and migration of GC cells through targeting EphA2.

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Fig. 3 miR-520d-3p downregulates EphA2 both at the mRNA and protein levels. miR-520d-3p mimics (miR520d-3p) or inhibitors (AntimiR-520d-3p) and their normal controls (miR-NC and AntimiR-NC, respectively) were cotransfected into SGC-7901 and MKN-45 cells using Lipofectamine 2000. Fortyeight hours later, RNA and proteins were harvested for qRT-PCR (a, b) and Western blotting (c), respectively. ***P \ 0.0001 (compared with the control)

Fig. 4 Ectopic expression of miR-520d-3p suppresses gastric cancer cell proliferation. miR-520d-3p mimics (miR-520d-3p) or inhibitors (Anti-miR-520d-3p) and their normal controls (miR-NC and AntimiR-NC, respectively) were cotransfected into SGC-7901 and MKN-

45 cells. Cells were plated in 96-well plates, and absorbance at 570 nm was measured at 0, 4, 12, 24, 48, and 72 h. **P \ 0.01, ***P \ 0.0001 (compared with miR-NC), ##P \ 0.01 (compared with Anti-miR-NC)


with recurrence and lymph node metastasis of ovarian cancer, and that patients with high miR-520d-3p expression have a significantly longer survival time compared with those with low miR-520d-3p expression [17]. Moreover, targeting of EphA2 using a combination of short interfering RNA (siRNA) and the tumor suppressor miR-520d-3p was shown to exhibit remarkable therapeutic synergy and enhance tumor suppression in vitro and in vivo [17]. This study therefore demonstrated that miR-520d-3p is a promising marker for cancer prognosis and targeted therapy.

This study showed that miR-520d-3p expression levels in cancerous tissues were significantly lower than those in noncancerous tissues in GC patients. Moreover, low levels of miR-520d-3p were associated with invasion, lymph node metastasis, higher clinical stages, and poorer prognosis, and were inversely correlated with EphA2 expression. These findings indicate that miR-520d-3p is a favorable factor for preventing GC progression. A previous report demonstrated that lower miR-520d-3p levels are associated



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Fig. 5 Ectopic expression of miR-520d-3p suppresses gastric cancer cell invasion and migration. miR-520d-3p mimics or inhibitors and their normal controls (miR-NC and AntimiR-NC, respectively) were cotransfected into SGC-7901 and MKN-45 cells. a The invasive properties of the cells were analyzed by an invasion assay using a Matrigel-coated plate. b The capacities of migration were evaluated using a scratch wound-healing assay. The rate of wound healing was measured with the following formula: (0 h width of wound - 24 h width of wound)/(0 h width of wound). *P \ 0.05, **P \ 0.01 (compared with miR-NC), # P \ 0.05, ##P \ 0.01 (compared with Anti-miR-NC)

Next, we confirmed that miR-520d-3p binds to the EphA2 30 -UTR and suppresses its expression both at the mRNA and protein levels. Recent research has revealed that the regulation of target genes by miRNAs is more complicated than previously considered. Target site recognition is primarily determined by multiple instances of seven-nucleotide core complementarity, which implies that each miRNA can influence the expression of a remarkably large number of different mRNAs [5]. Indeed, Nishimura et al. [17] confirmed that miR-520d-3p could also directly target the 30 -UTR of EphB2 in ovarian cancer. Although we did not confirm this directly, our results do not rule out


the possibility that miR-520d-3p might also target EphB2 or other genes involved in the GC phenotype, and that these targets might augment its antitumor efficiency. Therefore, further identification of other targets of miR520d-3p or other miRNAs that target EphA2 in GC is essential. Local invasion and lymph node metastasis are critical steps of GC progression and recurrence [30]. Therefore, elucidating the pathological mechanisms underlying GC invasion and metastasis is critical for development of effective therapies. As an essential tyrosine kinase receptor, EphA2 participates in the regulation of cancer cell

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Fig. 6 Overexpression of miR520d-3p downregulates the expression of c-Myc, CyclinD1, and MMP9 by targeting EphA2. miR-520d-3p mimics (miR520d-3p) or inhibitors (AntimiR-520d-3p) and their normal controls (miR-NC and AntimiR-NC, respectively) were cotransfected into SGC-7901 and MKN-45 cells. Forty-eight hours later, proteins were harvested for Western blotting; GAPDH served as an internal control

proliferation, invasion, and metastasis [18, 31–33]. Our previous studies indicated that EphA2 promotes GC cell proliferation, invasion, and migration through the canonical Wnt/b-catenin signaling pathway [12]. In the present study, we showed that overexpression of miR-520d-3p potently suppressed the GC cells’ proliferative, invasive, and migration behaviors, whereas silencing of miR-520d3p, using its specific single-stranded nucleic acid inhibitors, substantially enhanced these behaviors. Moreover, the overexpression and silencing of miR-520d-3p were accompanied by down-regulated and up-regulated expression of EphA2, respectively. Several studies have reported that the canonical Wnt/bcatenin signaling pathway target genes c-myc [34], cyclinD1 [35], MMP7 [36], and MMP9 [37] are essential for cancer cell proliferation and invasion. Ectopic expression of EphA2 could induce changes in the expression levels of c-Myc and CyclinD1 through the canonical Wnt/b-catenin signaling pathway [12]. In addition, silencing EphA2 expression with siRNA results in down-regulated MMP9 expression in vitro and in vivo [29]. These results imply that EphA2 promotes GC cell proliferation, migration, and invasion through inducing changes in c-Myc, CyclinD1, and MMP9. In the current study, we observed that expression levels of c-Myc, CyclinD1, and MMP9 were down-regulated when EphA2 was inhibited by miR-520d3p, whereas the MMP7 expression level was not significantly attenuated. It is possible that the distinct functions of EphA2 in different types of cancer reflect differences in the

intercellular Wnt signaling pathway. Although our data could not confirm whether the down-regulation of c-Myc, CyclinD1, and MMP9 resulted from direct interaction of miR-520d-3p with these genes, these results nevertheless suggest that miR-520d-3p inhibits the proliferation, invasion, and migration of GC cells through the EphA2-mediated-Wnt/b-catenin signaling pathway. In summary, our study showed that miR-520d-3p is downregulated in GC and its lower expression is associated with invasion, lymph node metastasis, a higher clinical stage, and poorer prognosis. Moreover, ectopic expression of miR-520d3p inhibited GC proliferation, invasion, and migration through targeting EphA2. Further investigation into the functional and clinical implications of miR-520d-3p may contribute to the development of a novel targeted therapy for GC. Acknowledgments This study was supported by the National Natural Scientific Foundation of China (No. 81172297), and the Fundamental Research Funds for the Central Universities of Central South University (No. 2013zzts082). Conflict of interest


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MicroRNA 520d-3p inhibits gastric cancer cell proliferation, migration, and invasion by downregulating EphA2 expression.

Aberrant expression of microRNAs (miRNAs) has been shown to play important roles in cancer progression as a result of changes in expression of their t...
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