Forty-four gastric cancer and adjacent normal tissues were obtained from patients undergoing surgery for gastric cancer at The First and Second Affiliated Hospital of Medical College of Xi’an Jiao Tong University. The samples were placed in liquid nitrogen immediately after excision from the patients and subsequently frozen at −80°C until RNA extraction. Informed consent was obtained from each patient and was approved by the Institute Research Ethics Committee at the Cancer Center, Xi’an Jiaotong University.

RNA from gastric cancer tissue samples was extracted using TRIzol (Invitrogen, Life Technologies) according to the manufacturer’s recommendations. miR-126 expression was assessed using qRT-PCR. Human U6 small nuclear RNA (snRNA) was used as an internal standard. Primer sequences are listed in Table I. In order to verify that PLK2 was regulated by miR-126, we collected cells and extracted RNA using TRIzol, according to manufacturer recommendations, after transfecting the miR-126, miR-control and anti-miR-126 individually at 24 h. The β-actin mRNA level was used as an internal standard. The PLK2 and β-actin qRT-PCR primers are shown in Table I. Reverse transcription was performed using PrimerScript™ RT reagent kit perfect Real-Time and qRT-PCR using SYBR^® Premix Ex Taq™ II (Takara). The qRT-PCR program used for amplification was: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec.

The pcDNA6.2-GW/EmGFP-miR vector was purchased from Invitrogen. The linear structure was converted to a double link-shaped structure by cloning multiple cloning sites (MCS) (using EcoRI, PstI, NheI, KpnI and HindIII) into pcDNA6.2-GW/EmGFP-miR via the vector’s joint. The Hsa-miR-126 clone vector was constructed by Jie Rui Biology Ltd. (Shanghai, China). Hsa-miR-126 mature sequences were cloned into pcDNA6.2-GW/EmGFP-miR, using EcoRI and HindIII, and named pcDNA6.2-GW/EmGFP-miR-126 (miR-126). Finally, for miR-control vector, the miR-126 mature sequence was transformed to clone MCS (short miR-control).

SGC-7901 cells were obtained from The Central Laboratory of Biomedical Research of Xi’an Jiaotong University Medical School. The cells were grown in Dulbecco’s modified Eagle medium (Gibco) with 10% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen), and incubated at 37°C in 5% CO₂. There were four experimental groups (control, anti-miR-126, miR-control and miR-126). Anti-miR-126 sequence: 5′-GCATTATTACTCACGGTACGA-3′ (Sangon Biotech). Anti-126, miR-control and miR-126 were transfected at a final concentration of 100 nmol/l by Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions, in all experiments.

SGC-7901 cells were seeded at 1×10⁴ cells/well in 96-well plates and transfected the following day with miR-126, miR-control and anti-miR-126 using Lipofectamine 2000; the control group was transfected with Lipofectamine 2000 only. MTT was added to cells (Sigma, USA) at 24, 48, 72 and 96 h, respectively, and these continued incubating for 3 h at 37°C in 5% CO₂, then the supernatant was discarded and dimethyl sulfoxide was added to the cells. Sample absorbance was measured at 490 nm by a high-throughput universal microplate assay (BMG Lab Technologies).

SGC-7901 cells were seeded at 1×10⁵ cells/well in a 6-well plate and transfected with miR-126, miR-control and anti-miR-126 using Lipofectamine 2000. Cells were harvested at 24 and 48 h post-transfection, stained for DNA content using propidium iodide (PI) and analyzed on a FACSCalibur flow cytometer (Becton-Dickinson). Briefly, cells were harvested by trypsinization and washed once with phosphate-buffered saline (PBS) before fixing overnight in 70% EtOH. For DNA staining, cells were pelleted and stained for 30 min with 300 μl PI solution (0.05 mg/ml PI, 20 μg/ml RNAse A in 0.1% bovine serum albumin).

SGC-7901 cells were seeded at 1×10⁵ cells/well in a 6-well plate and transfected with miR-126, miR-control and anti-miR-126 using Lipofectamine 2000. Cells were harvested 24 and 48 h post-transfection and washed twice with ice-cold PBS before being stained with 7-AAD/PI, and later analyzed on a FACSCalibur flow cytometer (Becton-Dickinson).

One thousand cells, which were post-transfected with miR-126, miR-control or anti-miR-126 at 24 h, were seeded into 6-well plates and cultured in common media. Approximately 2 weeks later, colonies that appeared were fixed with pre-chilled methanol and stained with 2% Giemsa solution (Sigma, St. Louis, MO, USA). Experiments were conducted in triplicate.

SGC-7901 cells (1×10⁶ cells/mouse) stably transfected with miR-126 or miR-control lentivirus vector were subcutaneously injected into 6-week-old male nude mice. Tumor size was measured every 3 days. For end-point experiments, the bioluminescence images captured in vivo were obtained using a photobiology system (Xenogen). The mice were euthanized 22 days after injection, and the tumors were removed and weighed. The tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 5 μm and stained with hematoxylin and eosin (H&E).

SGC-7901 cells were seeded at 1×10⁶ cells/6-cm culture dish, and transfected with miR-126, miR-control or anti-miR-126, using Lipofectamine 2000 according to the manufacturer instructions (Invitrogen). Cells were harvested by trypsinization, washed once with ice-cold PBS and lysed in RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% Igepal, 50 mM Tris-HCl pH 8.0, 2 mM ethylenediamine tetraacetic acid) supplemented with 1X complete mini protease inhibitor cocktail tablets. Then, 25 μg of protein/lane was resolved on 10% polyacrylamide gel electrophoresis gels and transferred to a nitrocellulose membrane (Roche Diagnostics GmbH). Primary antibodies used were PI3KR2 (Cell Signal 9402), Crk (Santa Cruz Sc-53923), Bcl-2 (Sigma V9131), Bax (Santa Cruz Sc-126), PLK2 (sc-25421; Santa Cruz Biotechnology) and β-actin (Bioworld, AP0060). The membranes were further probed with horseradish peroxidase-conjugated secondary antibodies (ZSGB-BIO). Working solutions of the Pierce ECL Substrate were prepared and added to polyvinyldifluoride membranes for 1 min. The membranes were removed from the substrates and exposed to ChemiDoc-It 510 (UVP; LLC).

The wild-type 3′ untranslated regions (UTRs) or mutant 3′UTRs of PLK2 carrying miR-126-binding sites were cloned downstream of the luciferase reporter in a pmir-GOL vector system (Promega; E1330) through SacI and XhoI digestion. The wild-type PLK2 3′UTR PCR primer was as follows: sense primer, 5′-GAGCTCGACCCTATGGGACTCCTCTTT-3′ and antisense primer, 5′-CTCGAGGTATGCCTTAGCCTGTTCTGG-3′. The PLK2 mutant sequence was chemically synthesized by Beijing AuGCT DNA-SYN Biotechnology Co., Ltd., as follows: PLK2 Mut Top: 5′-cgagtatgttgaagaagatggacatgtggtgatcctaaaacaattccccc-3′ and PLK2 Mut Bottom: 5′-tcgagggggaattgttttaggatcaccacatgtccatcttcttcaacatactcgagct-3′. The wild-type PLK2 reporter vector and mutation reporter vector were named pmir-GOL-PLK2 and pmir-GOL-mutPLK2, respectively. SGC-7901 cells were seeded in 96-well plates and transfected with miR-126 or miR-control, and pmir-GOL-PLK2 or pmir-GOL-mutPK2, using Lipofectamine 2000. Cells were harvested 24 h post-transfection and luciferase activity was measured using a Dual-Glo Luciferase Assay System (Promega; E2920) according to the manufacturer’s recommendations.

Quantitative results are presented as the mean ± standard deviation (SD). Statistical analysis was performed by using a t-test, and P<0.05 was taken as the level of significance. All results were analyzed using the statistical software SPSS10.0.

To explore miR-126 expression levels in gastric cancer, we collected 44 paired gastric cancer and adjacent normal tissue samples. We used qRT-PCR to determine the miR-126 expression level in gastric cancer. In the 44 paired clinical gastric samples, there were 29 samples with downregulation of miR-126 compared with the matched non-tumor tissue samples (relative expression ratio <1.0), and 18 gastric cancer tissues showed miR-126 expression with significant downregulation (relative expression ratio <0.5) (Fig. 1A). Furthermore, analysis of miR-126 expression in 4 gastric cancer cell lines (GES-1, BGC-823, SGC-7901 and MKN-45) revealed that miR-126 was downregulated in the BGC-823 and SGC-7901 cell lines (Fig. 1B). These data suggest that miR-126 might act as a tumor suppressor in gastric cancer.

To explore the tumor suppressor role of miR-126 in gastric cancer, SGC-7901 cells were transfected with miR-126 or miR-control. qRT-PCR was performed to examine the expression levels of miR-126 after transfection of the miR-126 or miR-control. As depicted in Fig. 2A, an approximate 10-fold increase in the expression of miR-126 was observed in SGC-7901 cells that were transfected with miR-126 relative to the cells transfected with miR-control. To study the role of miR-126 in SGC-7901 cell proliferation, an MTT assay was used. The results showed that transient overexpression of miR-126 inhibited the proliferation of SGC-7901 cells at 24, 48 and 72 h after transfection (Fig. 2B). However, the cell cycle of SGC-7901 cells was not obviously affected by miR-126 at 24 h (Fig. 2C). In order to further examine the inhibitory role of miR-126 in SGC-7901 cells, a colony formation assay was conducted after similar transient transfection. miR-126-transfected cells displayed fewer colonies compared with the control (Fig. 2D). To further investigate the mechanisms by which miR-126 inhibits the growth of SGC-7901 cells, we tested the apoptosis of SGC-7901 cells induced by miR-126. We found that cells in early apoptosis and late apoptosis (at 24 h) were increased in the miR-126 group. Thus, the inhibitory effect of miR-126 on cell growth may occur through the induction of apoptosis in SGC-7901 cells (Fig. 2E). Altogether, these results indicate that miR-126 was able to inhibit the proliferation of SGC-7901 cells in vitro.

We further confirmed the growth inhibitory role of miR-126 in vivo in a xenograft model. First, we tested the effects of miR-126 on tumor growth in the in vivo xenograft model. miR-126 and miR-control-transfected SGC-7901 cells were injected subcutaneously into either posterior flank of nude mice, and xenograft growth was observed for 22 days. As shown in Fig. 3A and C, the tumors from mice that were injected with miR-126 were significantly smaller than those in control mice on the 13th day after the first injection. On the 22nd day, the tumors were tested using IVIS Spectrum before the mice were euthanized (Fig. 3D), and the tumors were removed and weighed (Fig. 3B). The tumor tissue was fixed in 4% paraformaldehyde, embedded in paraffin and stained with H&E (Fig. 3E). Although, both tissues of miR-126 and control grew tumors, the degree of malignancy in the miR-126 groups was lower than in the miR-control group. These data indicate that miR-126 could inhibit tumorigenicity of SGC-7901 cells in the nude mouse xenograft model.

The preceding observations demonstrated that miR-126 expression inhibited the growth and increased apoptosis of SGC-7901 cells. We next asked whether downregulated expression miR-126 promotes the growth of SGC-7901 cells. To accomplish this, we transiently inhibited miR-126 in SGC-7901 cells with antisense oligonucleotides. qRT-PCR was performed to examine the expression levels of miR-126 after transfection of anti-miR-126 and control. As shown in Fig. 4A, the expression of miR-126 decreased by ~12% compared with the control. The MTT assay results showed that anti-miR-126 slightly improved the ability of growth at 48 and 72 h (Fig. 4B), but the cell cycle and colony forming ability were unaffected by the inhibitor (Fig. 4C and D). Interestingly, inhibitor-induced apoptosis of SGC-7901 cells was higher than in the control (Fig. 4E), but there was no significant difference, and the percent of apoptosis was very low in both groups. This phenomenon is worth exploring further in the future.

Feng et al reported that miR-126 functions as a tumor suppressor in gastric cancer, with Crk as a direct target (12). We wanted to know whether any other target genes of miR-126 might also contribute to the function of miR-126 in gastric cancer. Crk and PI3KR2 protein (p85) levels, targets of miR-126, were assayed in miR-126-expressing SGC-7901 cells. As shown in Fig. 5, miR-126 repressed levels of PI3KR2 and Crk protein expression. P13KR2 was upregulated, while the expression of Crk was not affected by the inhibitor of miR-126. These data indicated that miR-126 directly regulates Crk and PI3K2 in SGC-7901 cells. In addition, in overexpressing miR-126 SGC-7901 cells, the Bcl-2 and Bax protein levels were changed; Bax was upregulated and Bcl-2 was downregulated in the miR-126 group. However, protein levels of Bcl-2 and Bax were unchanged by the inhibitor of miR-126. These data were compatible with the results of the apoptosis analysis.

Through the miRBase (http://www.mirbase.org) and TargetScan (http://www.targetscan.org/) databases, we found matching bases between the 3′UTR of PLK2 and miR-126 (top of Fig. 6A). In order to determine whether miR-126 regulates PLK2 in gastric cancer SGC-7901 cells, we performed a luciferase assay to evaluate the relationship between miR-126 and PLK2. SGC-7901 cells were co-transfected with miR-126 together with pmir-GOL-PLK2 or pmir-GOL-mut-PLK2. miR-126 strongly reduced the expression of PLK2 (27.73%), but showed almost no effect on the expression of pmir-GOL-mut-PLK2 (81.81%) (bottom of Fig. 6A). As shown in Fig. 6B and C, qRT-PCR revealed that PLK2 mRNA was decreased in SGC-7901 cells overexpressing miR-126, while it was upregulated in cells treated with anti-miR-126. These results were also confirmed in SGC-7901 cells transfected with miR-126, which showed a lower amount of PLK2 protein when compared to miR-control-treated cells (right of Fig. 6D). The PLK2 protein level did not change significantly between the control and anti-miR-126 groups (Fig. 6D, left). Together, these data indicate that PLK2 is regulated by miR-126 in SGC-7901 cells.