Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), TRIzol agent, Lipofectamine 2000, SYBR Green Quantitative Polymerase Chain Reaction (qPCR) Assay kit, miRNA Reverse Transcription kit and all miRNA mimics and inhibitors were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The Bicinchoninic Acid (BCA) Protein Assay kit and Enhanced Chemiluminescence (ECL) kit were purchased from Pierce Biotechnology, Inc. (Rockford, IL, USA). 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Biosharp (Hefei, China). The Hairpin-it™ miRNAs qPCR Quantitation kit and U6 small nuclear RNA were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). PsiCHECK™-2 vector was purchased from Promega Corporation (Madison, WI, USA). A Stratagene QuikChange Site-Directed Mutagenesis kit was purchased from Agilent Technologies, Inc. (La Jolla, CA, USA). Mouse anti-sprouty2 (SPRY2), mouse anti-phosphorylated extracellular signal-related kinase (p-ERK), mouse anti-ERK and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primary antibodies, and rat anti-mouse secondary antibodies were purchased from Abcam (Cambridge, MA, USA). The Chemicon Cell Invasion Assay kit was purchased from EMD Millipore (Temecula, CA, USA).

The current study was approved by the Ethical Committee of Central South University (Changsha, China). Informed consent was obtained from the patients. A total of 15 gastric cancer tissues and matched normal adjacent gastric tissues were obtained at the Department of General Surgery, The Third Xiangya Hospital of Central South University (Changsha, China). Tissue samples were immediately frozen in liquid nitrogen subsequent to surgical removal.

Human gastric cancer HGC-27, GC7901 and AGS cells, and normal gastric mucosa epithelial GES-1 cells were obtained from the Cell Bank of Central South University (Changsha, China), and cultured in DMEM with 10% FBS at 37°C in a humidified incubator containing 5% CO₂.

According to the manufacturer's instructions, total RNA was extracted using TRIzol agent. Subsequent to that, the miRNA Reverse Transcription kit was used to convert RNA into cDNA (500 ng). The expression levels of miRNAs were then evaluated according to the manufacturer's instructions using the Hairpin-it™ miRNAs qPCR Quantitation kit. The relative expression of miRNA was analyzed by the 2^−ΔΔCt method (18). The U6 small nuclear RNA was used for normalization. Expression of SPRY2 mRNA was detected by the SYBR Green qPCR Assay kit. Expression of GAPDH was used as an endogenous control. The specific primers used are as follows: SPRY2, forward 5′-CCTACTGTCGTCCCAAGACCT-3′ and reverse 5′-GGGGCTCGTGCAGAAGAAT-3′; GAPDH, forward 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′.

Tissues or cells were lysed in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Wuhan, China). Protein was quantified using the BCA Protein Assay kit. Proteins (60 µg) were separated with 10% sodium dodecyl sulfate-polyacrylimide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF; Thermo Fisher Scientific, Inc.) membrane, which was then incubated with Tris-buffered saline with Tween-20 (Beyotime Institute of Biotechnology) containing 5% milk at room temperature for 3 h. The PVDF membrane was then incubated with mouse anti-SPRY2 (1:50; ab50317), mouse anti-p-ERK (1:100; ab50011), mouse anti-ERK (1:100; ab119933) and mouse anti-GAPDH (1:100; ab8245) primary antibodies, respectively, at room temperature for 3 h. Subsequent to washing with phosphate-buffered saline (PBS) with Tween-20 3 times, the membrane was incubated with the rat anti-mouse secondary antibody (1:1,000; ab187851) at room temperature for 40 min. Chemiluminescent detection was performed using the ECL kit. The relative protein expression was analyzed by Image-Pro Plus software, version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), represented as the density ratio versus GAPDH.

Transfection was performed using Lipofectamine 2000 according to the manufacturer's instructions. For miR-27b functional analysis, GC7901 cells were transfected with the scrambled miRNA as the negative control (NC), miR-27b mimics, or miR-27b inhibitor. For SPRY2 functional analysis, GC7901 cells were transfected with the pcDNA3.1-SPRY2 plasmid.

A QuikChange Site-Directed Mutagenesis kit was used to generate a mutant type 3′-untranslated region (UTR) of SPRY2, according to the manufacturer's instructions. The wild type or mutant 3′-UTRs of SPRY2 were inserted into the psiCHECK™-2 vector, respectively. Subsequent to culture of the GC7901 cells to approximately 70% confluence, the cells were transfected with psiCHECK™-2-SPRY2-3′-UTR or psiCHECK™-2-mutant SPRY2-3′-UTR vector, with or without 100 nM miR-27b mimics, respectively. Subsequent to transfection for 48 h, the luciferase activities were determined using the LD400 luminometer (Beckman Coulter, Inc., Brea, CA, USA). Renilla luciferase activity was normalized to firefly luciferase activity.

The wound healing assay was used to determine the cell migration. Cells were cultured to full confluence. A wound of approximately 1 mm in width was created using a plastic scriber. Cells were washed using PBS, and then cultured at 37°C with 5% CO₂ for 48 h. Subsequently, the cells were fixed with absolute ethanol (Beyotime Institute of Biotechnology) and observed under a microscope (CX41; Olympus Corporation, Tokyo, Japan).

The cell invasion assay was performed using the Cell Invasion Assay kit according to the manufacturer's instructions. In brief, the cell suspension containing 500,000 cells/ml was prepared in serum-free media (DMEM). Subsequently, 300 µl cell suspension was placed in the upper compartment of the chambers and DMEM containing 10% FBS was added into the lower chambers. Following 24 h of incubation at 37°C, cells on the upper face of the membrane were scraped using a cotton swab, and cells on the lower face were fixed with absolute ethanol, stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) and observed under a microscope. Then, the dye on the membrane was dissolved with 10% acetic acid (Beyotime Institute of Biotechnology), plated into 96-well plates (150 µl/well), then the optical density was measured at 570 nm (OD570) with an ELISA reader (ELx800; BioTek Instruments, Inc., Winooski, VT, USA).

The results are expressed as the mean ± standard deviation of three independent experiments. Statistical analysis of the differences was performed with one-way analysis of variance using SPSS software, version 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The expression levels of miR-27b were measured in gastric cancer tissues and their matched normal adjacent tissues. As presented in Fig. 1A, the expression levels of miR-27b in gastric cancer tissues were observed to be frequently increased when compared with their matched normal adjacent tissues. In addition, it was expression was observed to be upregulated in gastric cancer cell lines, when compared with normal gastric epithelial cells (Fig. 1B). Accordingly, the data suggests that miR-27b is downregulated while SPRY2 is upregulated in gastric cancer.

The putative seed sequences for miR-27b at the 3′UTR of SPRY2 were indicated in Fig. 2A based on bioinformatical analysis. To further confirm the association between SPRY2 and miR-27b, the wild type (WT) and mutant (MUT) of SPRY2 3′-UTR was generated (Fig. 2A), then the luciferase reporter assay was conducted in gastric cancer GC7901 cells. As presented in Fig. 2B, the luciferase activity was notably reduced in gastric cancer GC7901 cells co-transfected with miR-27b mimics and the WT 3′UTR of SPRY2, however was unaltered in GC7901 cells co-transfected with miR-27b mimics and MUT SPRY2 3′UTR, indicating that miR-27b is able to directly bind to the 3′-UTR of SPRY2 mRNA in gastric cancer GC7901 cells, and thus SPRY2 may be a target of miR-27b.

The GC7901 cells were then transfected with miR-27b mimics or inhibitor, in order to upregulate or inhibit the expression levels of miR-27b, respectively. As presented in Fig. 3A, the expression levels of miR-27b were increased subsequent to transfection with miR-27b mimics, however were reduced following transfection with the miR-27b inhibitor, thus the transfection efficiency was deemed to be satisfactory. In addition, it was demonstrated that the protein levels of SPRY2 were reduced following upregulation of miR-27b, however were increased subsequent to inhibition of miR-27b (Fig. 3B), indicating that miR-27b negatively regulates the protein expression of SPRY2 in gastric cancer GC7901 cells.

At present, the roles of miR-27b and SPRY2 in the mediation of gastric cancer cell migration and invasion, in addition to the association between them, remain to be elucidated. To investigate this, GC7901 gastric cancer cells were transfected with miR-27b inhibitor or the pcDNA3.1-SPRY2 plasmid, respectively. Subsequent to transfection with the pcDNA3.1-SPRY2 plasmid, the protein level of SPRY2 was significantly increased compared with the control group (Fig. 4A). As presented in Fig. 4B and C, knockdown of miR-27b inhibited migration and invasion of GC7901 gastric cancer cells, while upregulation of SPRY2 additionally suppressed GC7901 cell migration and invasion. As miR-27b negatively mediated the expression of SPRY2 in GC7901 cells, it is suggested that the role of miR-27b in the regulation of cell migration and invasion is at least partly mediated through targeting of SPRY2.

It has been demonstrated that SPRY2 inhibits the activity of ERK signaling by binding to growth factor receptor bound protein 2 (GRB2) during fibroblast growth factor receptor (FGFR) activation, disrupting the GRB2-son of sevenless complex that transduces signals from FGFR to RAS (19). Accordingly, the activity of ERK signaling in each group was determined. As presented in Fig. 5, upregulation of miR-27b increased the phosphorylation level of the ERK protein, indicating that the activity of ERK signaling was upregulated. On the contrary, knockdown of miR-27b reduced the phosphorylation level of ERK protein, exhibiting a similar effect to that of SPRY2 overexpression in GC7901 gastric cancer cells (Fig. 5). As miR-27b negatively mediated the expression of SPRY2 in GC7901 cells, it is suggested that miR-27b promotes the activity of ERK signaling via directly targeting SPRY2.

The expression levels of SPRY2 were determined by performing RT-qPCR in gastric cancer tissues in addition to in their matched normal adjacent tissues. As presented in Fig. 6, the mRNA level of SPRY2 was frequently reduced in gastric cancer tissues, compared with the matched normal adjacent tissues.