Gastric cancer cell lines SGC-7901, MKN-45, MGC-803 and normal gastric epithelial GES-1 cells were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA) and the Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI-1640 medium (Life Technologies, Inc., Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), penicillin (100 U/ml) and streptomycin (100 mg/ml). All cell cultures were kept at 37°C in a humidified incubator with 5% CO₂.

The approval to conduct the experiments involving human tissue samples was obtained from the Institutional Research Ethics Committee of Tongren Hospital, Shanghai Jiao Tong University School of Medicine. One hundred pairs of gastric cancer tissues and adjacent non-tumor tissues were collected from Tongren Hospital, Shanghai Jiao Tong University School of Medicine. All these clinical tissues were stored at −80°C before the RNA extraction. The informed consents were obtained from all enrolled patients in this study. Demographic and clinicopathological information of the included patients are presented in Table I.

The mimic and the inhibitor of miR-187 were obtained from Ruibobio, Guangzhou, China. FOXA2 siRNA and FOXA2 expression vector was purchased from Sigma-Aldrich (St. Louis, MO, USA). The day before the transfection, GC cells were seeded in 6-well plates. Then, miR-187 mimic or inhibitor/FOXA2 siRNA or plasmid were transfected into GC cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manusfacturers protocol.

Total RNA from GC tissues and cells were extracted with TRIzol reagent (Invitrogen). Reverse transcription reactions and real-time PCR were performed with Transcriptor First Strand cDNA Synthesis kit (Roche, Indianapolis, IN, USA) and SYBR-Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Primers for miR-187 and U6 were purchased from GeneCopoeia (Guangzhou, China). U6 was used as the control gene for the relative expression level of miR-187.

Western blot analysis was performed following standard protocols. Generally, cellular protein was extracted with the RIPA lysis buffer, and protein concentration was measured using the BCA kit (Pierce, Rockford, IL, USA). A total of 20–40 µg cellular proteins were separated by SDS-PAGE and transferred to PVDF membrane. The following primary antibodies including FOXA2 (1:1,000; Cell Signaling Technology, Danvers, MA, USA) and GAPDH (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were incubated with the membrane overnight at 4°C. The blots were then incubated with secondary antibodies (1:3,000; Santa Cruz Biotechnology) at room temperature for 2 h. The signals were visualized with ECL reagents (Amersham Biosciences Corp., Piscataway, NJ, USA).

For MTT assay, 5,000 GC cells transfected with miR-187 mimic or inhibitor were plated into 96-well plates. At 24, 28 and 72 h after cell seeding, the cells were stained with MTT (Sigma-Aldrich) for 4 h at 37°C, and then the absorbance at 490 nm was examined.

GC cells were cultured to confluence in 6-well plates. A 100-µl pipette tip was used to make the wounds on the confluent cells. The width of wounds was measured at 0 and 24 h after scratching and then the percentage of wound healing was calculated.

The invasive ability of GC cells was evaluated by Transwell assays. Generally, upper chamber of Transwell device was coated with 80 µl mixture of RPMI-1640 and Matrigel with a ratio of 1:8. Then, GC cells suspended in serum-free RPMI-1640 medium were plated in the upper chamber while 700 µl serum-containing medium RPMI-1640 were placed in the lower chamber. Twenty-four hours later, GC cells invaded into the lower surface were stained with crystal violet. Cell number for the invaded cells was counted under a microscope.

Wild-type 3-UTR sequence of FOXA2 containing the miR-187 predicted target site or the mutated sequence within the predicted target sites was cloned into the pGL3 control vector (Promega, Madison, WI, USA), designated as the wild-type FOXA2-3-UTR or mutant FOXA2-3-UTR, respectively. Then, GC cells seeded into 12-well plates were maintained in Opti-MEM reduced serum media (Life Technologies) the day before the transfection, and were cotransfected with wild-type or mutant 3′-UTR of FOXA2 along with miR-187 mimic or inhibitor. Forty-eight hours after the transfection, luciferase activity was measured by a single luciferase reporter assay (Promega).

All animal experiments were approved by the Animal Care Committee of Tongren Hospital, Shanghai Jiao Tong University School of Medicine. For tumor growth studies, nude mice were injected subcutaneously with 1×10⁶ cancer cells transfected with control vector or miR-187 mimic. Tumor sizes were measured every three days after cancer cell injection. Three weeks later, tumors were removed and the volumes were measured. For metastasis assay, GC cells were injected through tail vein into nude mice. Eight weeks later, the lungs of nude mice were subjected to H&E staining for potential lung metastasis of GC cells.

All quantatively data are presented as mean ± standard error of the mean (SEM). Statistical analysis including Student's t-test, Chi-square, correlation analysis and Kaplan-Meier analysis was performed with SPSS software (SPSS, Inc., Chicago, IL, USA) and GraphPad software. P<0.05 was considered statistically significant.

We performed qRT-PCR to determine the expression status of miR-187 in GC tissues. Compared with adjacent normal tissues, the expression of miR-187 in GC tissues was increased significantly (Fig. 1A; P<0.01). In addition, compared to those of small size, tumors of large size had significantly increased level of miR-187 (Fig. 1B; P<0.05). Furthermore, compared to those without metastasis, the expression level of miR-187 in patients with metastasis was significantly elevated (Fig. 1C; P<0.05). Moreover, we examined the expression of miR-187 in GC cells and the normal gastric epithelial GES-1 cells. Compared to GES-1 cells, miR-187 in GC cells lines was significantly increased (Fig. 1D; P<0.05). Besides, among the four GC cell lines, miR-187 level was lowest in SGC-7901 cells while was highest in AGS cells (Fig. 1D), indicating that miR-187, which is elevated in GC tissues and cells, probably plays an oncogenic role in GC.

After confirming the increased expression of miR-187 in GC, we further determined whether miR-187 expression level was associated with clinical features and the survival of GC patients. Association analysis (Table II) showed that high expression level was associated with large tumor size (P<0.001), lymphatic metastasis (P<0.001) and advanced TNM stage (P<0.001). Furthermore, survival analysis showed the patients with high level of miR-187 had significantly decreased overall survival (Fig. 2A; P<0.001) and disease-free survival (Fig. 2B; P<0.001). These data indicate that miR-187 is actively involved in the pathogenesis of GC, and can potentially serve as a novel biomarker for the prognosis of GC patients.

Aberrantly increased expression of miR-187 in GC prompted us to explore the biological function of miR-187 in GC cells. Transfection of miR-187 mimic in SGC-7901 cells significantly increased miR-187 expression in SGC-7901 cells (Fig. 3A; P<0.01). Functionally, overexpression of miR-187 obviously increased cell proliferation of SGC-7901 cells (Fig. 3B; P<0.05) as suggested by MTT assay. Moreover, the wound healing assay and Transwell assay showed that the migration and invasion of SGC-7901 was obviously increased after overexpression of miR-187 (Fig. 3C and D; P<0.05 for wound healing assay, P<0.01 for invasion assay). On the other hand, miR-187 inhibitor significantly decreased the expression of miR-187 in AGS cells (Fig. 4A; P<0.01). Subsequently, inhibition of miR-187 resulted in decreased cell proliferation (Fig. 4B; P<0.05), migration (Fig. 4C; P<0.05) and invasion (Fig. 4D; P<0.01) of AGS cells. These data indicate that miR-187 promotes the proliferation, migration and invasion of GC cells in vitro.

To further confirm the in vitro influence of miR-187 on GC cells, we further carried out nude mouse experiments to determine whether miR-187 could promote the in vivo growth and metastasis of GC cells. The result of subcutaneous tumor formation assay showed that the growth of SGC-7901 cells was significantly increased after overexpression of miR-187 (Fig. 5A and B; P<0.01). Moreover, the tail vein injection experiments in nude mice showed that overexpression of miR-187 significantly increased the lung metastasis of SGC-7901 cells (Fig. 5C and D; P<0.01). These data suggest that miR-187 potentiate the growth and metastasis of GC cells in vivo.

After confirming the functional influence of miR-187 in GC cells, we further investigated the underlying mechanism mediating the function of miR-187. The data from the database of TargetScan and miRNA showed that FOXA2 contained the potential binding sites for miR-187 (Fig. 6A), suggesting FOXA2 is a potential downstream target of miR-187. Then, we performed luciferase assay and found that overexpression of miR-187 significantly inhibited the luciferase activity of wild-type 3-UTR of FOXA2 (Fig. 6B; P<0.01) while inhibiting miR-187 significantly increased the luciferase activity of wild-type FOXA2 3-UTR (Fig. 6B; P<0.05). The influence of miR-187 overexpression and inhibition on the luciferase activity of wild-type 3-UTR of FOXA2 was absent in the mutant 3-UTR of FOXA2 (Fig. 6B). Furthermore, we performed qRT-PCR and western blot analysis to confirm that miR-187 could affect the expression of FOXA2 in GC cells. As suggested by Fig. 6C and D, overexpression of miR-187 significantly decreased the mRNA (P<0.01) and protein (P<0.01) level of FOXA2 in SGC-7901 cells. On the other hand, inhibition of miR-187 significantly increased the expression of FOXA2 mRNA (Fig. 6E; P<0.05) and protein (Fig. 6F; P<0.01) in AGS cells.

Lastly, we examined whether FOXA2 could affect the biological function of miR-187 in gastric cancer cells. FOXA2 expression vector significantly increased the protein level of FOXA2 in SGC-7901 cells overexpressing miR-187 (Fig. 7A; P<0.01). Functionally, overexpression of FOXA2 abrogated the promoting effects of miR-187 mimic on the proliferation (Fig. 7B; P<0.05), migration (Fig. 7C; P<0.05) and invasion (Fig. 7D; P<0.01) of SGC-7901 cells. On the contrary, FOXA2 siRNA significantly decreased the expression of FOXA2 in AGS cells with miR-187 inhibition (Fig. 8A; P<0.05). Inhibition of FOXA2 prevented the inhibitory effects of miR-187 inhibitor on the proliferation (Fig. 8B; P<0.01 for MTT), migration (Fig. 8C; P<0.05) and invasion (Fig. 8D; P<0.01) of AGS cells. These data indicate that FOXA2 is a functional mediator for miR-187 in GC cells.