Human gastric tissue was collected with a protocol approved by the Ethic Committee for the Conduct of Human Research at Ningxia Medical University. Written consent was obtained from every individual according to the Ethic Committee for the Conduct of Human Research protocol. All participants were over 18 years of age and provided written informed consent for the publication of the data. The Human Research Ethic Committee at Ningxia Medical University approved this study.

Twelve tumor samples with histologic evidence of gastric adenocarcinoma and matched adjacent non-tumor tissues (close to adjacent tissues about 2 cm) were collected, ten gastric tissues diagnosed as antral or gastric inflammation and their adjacent tissues were archival samples from the Department of Medical Pathology, General Hospital of Ningxia Medical University (Table I). The pathologic tumor staging was determined according to the International Union Against Cancer (2009) (16).

The expression of SKP2 in clinic human GC and matched adjacent non-tumor tissues was evaluated by Immunohistochemistry (IHC) staining using mouse anti-human SKP2 antibody (1:100; Abgent Biotech Co., Ltd., Suzhou, China) as previously described (6,9,17,18). The expression of SKP2 protein was arbitrarily scored from -(<25% positive cells), +(25–50% positive cells), ++(50–75% positive cells) and +++(>75% positive cells), based on the intensity and number of positive cells by a single experienced pathologist (Table I).

Cell lines of human embryonic kidney 293 and human gastric cancer cell SGC-7901 were purchased from American Type Culture Collection (Mannasas, VA, USA). The cells were cultured and maintained at 37°C in a humidified atmosphere of 5% CO₂ 95% air in dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Plasmid DNA transfection was performed using TransLipid Transfection Reagent (Beijing TransGen Biotech Co., Ltd., Beijing, China) per manufacturer's instruction.

In order to validate the SKP2 mRNA was a target of miR-508-5p, a reporter plasmid containing luciferase with the 3′UTR sequence of SKP2 mRNA was generated. The following primers were designed based on GenBank database (NM_001243120), and were used for amplification of wild-type and mutated 3′UTR of SKP2 mRNA: The sequence of common forward primer was 5′-CGACGCGTCGTTTATTGCAGGATGGTGT-3′, reverse primer for the wild-type of SKP2 mRNA 3′UTR was 5′-GACTAGTCAGAATGAAGGCAAAGGGA-3′, and the reverseec primer for mutated SKP2 mRNA 3′UTR was 5′-GACTAGTCACTCACGGATAACCCGAAGGACGGATAAAA-3′. The cDNA generated from SGC7901 RNA was used as templates for amplification of SKP2 3′UTR fragment by a PCR argeting SKP2 mRNA was ascertained by co-transfection plasmid DNA of pSicoR/massay as previously described (19,20). The specificity of miR-508-5p tiR-508-5p, miR-508-5p/inhibitor or pSicoR/NC and pMIR-Report/SKP2 or pMIR-Report/Mut-SKP2 into 293T cells and determined by the relative activity of firefly luciferase unit (RLU) at 48 h post-transfection using a dual-luciferase Reporter assay kit (Promega Corp., Madison, WI, USA). A Renilla luciferase expressing plasmid pRL-TK (Promega Corp.) was always included in the transfection to normalize the efficiency of each transftion.

Total RNA from infected cells was isolated with a Miniprep kit, and small RNA was purified with an RNAiso kit per manufacturer's recommendations (Takara Bio, Dalian, China). Total RNA from archival paraffin sections was isolated using a Mag-Bind® FFPE RNA Kit (Omega Bio-Tek, Norcross, GA, USA). The sequence of primer set for amplification of miR-508-5p was 5′-ACACTCCAGCTGGGTACTCCAGAGGGCGTCACT-3′ (forward) and 5′-TGGTGTCGTGGAGTCG-3′ (reverse). The sequences of RT-PCR primer set for SKP2 was as: Forward primer 5′-TGTGACTGGTCGGTTGC-3′; reverse primer 5′-GGAGGGTGGACACTTCTAT-3′. The RT-qPCR was performed using a Bio-Rad iQ5 lightcycler using a TaKaRa SYBR RT-PCR kit (Takara Bio). Relative expression was calculated as previously described using real-time PCR efficiencies and the crossing point deviation of unknown sample vs. control using the ΔΔCq method (21).

In order to construct a lentiviral vector expressing miR-508-5p, oligonucleotides of sense strand (5′-TTACTCCAGAGGGCGTCACTCATGTTCAAGAGACATGAGTGACGCCCTCTGGAGTATTTTTTC-3) and antisense strand (5′-TCGAGAAAAAATACTCCAGAGGGCGTCACTCATCTCTCTTGAACATGAGTGACGCCCTCTGGAGTAA-3′) were synthesized, which were based on the sequence of human miR-508-5p (MIMAT0004778: 5′-UACUCCAGAGGGCGUCACUCAUG-3′) from miRBase database. Restriction endonuclease HpaI and XhoI were introduced at 5′-ends of these oligonucleotides, respectively. The miR-508-5p precursor was modified with appropriate restriction enzymes, and cloned into a miRNA expressing plasmid, pSicoR (Department of Biological Chemistry, School of Medicine, Fudan University, Shanghai, China) to generate the vector expressing miR-508-5p, which was designated as pSicoR/miR-508-5p in this study. Using the same approch, a negative control vector was also generated, the primer of miR-508 −5p/inhibitor was 5′-CAUGAGUGACGCCCUCUGGAGUA-3′.

For production of the lentiviral vector, the proviral plasmid (1.5 µg) was co-transfected with packaging plasmids pCMV-VSV-G (0.5 µg) and pCMV-dR8.91 (1 µg) (Department of Biological Chemistry) with TransLipid Transfection Reagent as suggested by the manufacturer. The medium was replaced with 2 ml of DMEM/10% FBS at 6 h post transfection. The viral particles were titrated in 293T cells by counting EGFP-positive cells. SGC7901 cells were infected with vector-containing supernatant directly after 1 day in culture, which was designated as LV-miR-508-5p, LV-NC and inhibitor.

Whole cell lysates (75 µg) were prepared in a lysis buffer, and were resolved by a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), followed by being transferred to a PVDF membrane (Millipore Corp., Billerica, MA, USA). The membranes were probed with mouse anti-SKP2 antibody and anti-GAPDH antibody (Boster, Wuhan, China) were for the interested protein SKP2 and endogenous GAPDH for loading control, respectively. The blots were developed using the enhanced chemiluminescence (ECL) reagent (Advansta Inc., Menlo Park, CA, USA) after they were incubated with the appropriate peroxidase labeled secondary antibodies.

Cell proliferation was determined by using the MTT cell proliferation kit (Solarbio, Beijing, China). 5×103 of SGC 7901 cells were seeded in each 96-well plate and allowed to adhere overnight. The cells were then infected with lentiviral vector for the indicated times prior to they were used for MTT assay per the manufacturer's instruction (Bio-Rad Laboratories, Inc., Irvine, CA, USA).

For the preparation of Hoechst staining, SGC 7901 cells were plated with 1×105 cells/ml in 6-well plates. The cells were infected with lentiviral vectors for directly stained with Hoechst kit from Beyotime Institute of Biotechnology (Shanghai, China). The nuclear morphology was observed under a fluorescent microscope (Leica, Mannheim, Germany).

The SGC-7901 cells were seeded at 80% confluent and infected with lentiviral vectors for 48 h (cells were grown to confluence) in 6-well culture plates. The cells were then scratched with 200 ml pipette tips. The resultant unattached cells were removed by washing with pre-warmed PBS for three times, the wounded monolayers were cultured for additional 48 h prior to be stained with 0.1% crystal violet solution. The closure of the wounded areas was observed under a microscope at ×40 magnification (Leica) and photographed. The distance of closure was quantified with the NIH Image J image processing program. These experiments were performed in triplicate. Each condition was tested in duplicate and each experiment was repeated at least three times.

Transwell assay was used for accessing the migration and invasion of cells. For cell migration, 2×104 lentiviral infected cells suspended in serum-free medium were seed on onto uncoated 8-µm transwell filter inserts (Corning Inc., Corning, NY, USA) of 24-well plates in duplicate. The lower chambers contained 500 µl medium containing 10% FBS as a chemoattractant and cultured for additional 24 h. For the invasion assay, the inserts were previously coated with Matrigel (BD Biosciences, Bedford, MA, USA) and cultured for 48 h. At the end of the experiments, the cells in the upper chamber were removed with a cotton swab, and the cells on the lower surface were fixed and stained with 0.1% crystal violet solution. Cells of three randomly visual fields of each insert were counted under a light microscope. Each condition was tested in duplicate and each experiment was repeated for three times.

The apoptosis and cell cycle were determined by a flow cytometry assay. SGC-7901 cells were infected with various lentiviral vectors prior to be collected and stained with Annexin V and PI using an Apoptosis Detection kit I (BD Pharmigen, San Jose, CA, USA) for flow cytometry analysis. The flow cytometry analysis was performed on a BD FACSCanto II, and data was analyzed with FlowJo 8.8.6 software (Tree Star Inc, Ashland, OR, USA). All experiments were performed with biological triplicates and data are representative of at least three independent experiments.

All data collected in this study was obtained from at least three independent experiments for each condition. SPSS 17.0 analysis software (SPSS, Inc., Chicago, IL, USA) and PRISM 5 (GraphPad Software, Inc., La Jolla, CA, USA) were used for the statistic analysis. Statistical evaluation of the data was performed by one-way ANOVA when more than two groups were compared with a single control, and t-test for comparison of differences between the two groups. Significant differences were assigned to P<0.05, P<0.01 and P<0.0001 denoted by *, ** and ***, respectively. Data was presented as the mean ± standard deviation (SD).

Since SKP2 expression is positively correlated with poor prognosis and metastasis in many cancers, including the GC (6). To further interrogate clinical relevance of SKP2 with the pathogenesis in human GC, its expression was first evaluated in GC tumor tissues and the adjacent non-tumor tissues by IHC staining against anti-SKP2 antibody. IHC staining displayed a predominantly nuclear staining of SKP2 (Fig. 1A). Notably, the expression of SKP2 was strikingly provoked with the malignant stages of tumors from the twelve examined archival GC samples, relative to the adjacent non-tumor tissues and antral or inflammatory gastric tissues (Fig. 1B), supporting the previous findings of that SKP2 was an important signaling molecule that plays an oncogenic role in the carcinogenesis of GC, and an attractive potential target for treatment of this disease (6,8,17).

Previous miRNA microarray analysis has demonstrated a downregulation of miR-508-5p in many cancers, including the GC (22–24). In order to further validate a correlation of the expression of miR-508-5p and clinicopathologic stages of GC, the relative expression of miR-508-5p in tissues of various stages of GC, antral and grastric inflammation was evaluated by a RT-qPCR assay (Fig. 1B and Table I). The results revealed an inverse correlation of miR-508-5p transcripts with the progression of GC (Table I); miR-508-5p was dramatically downregulated in GC relative to other non-cancer gastric disorders (Fig. 1B and Table I).

In order to identify the biological significances of miR-508-5p in GC, the online computational miRNA target prediction tool, TargetScan, was used to identify potential targets of miR-508-5p. The SKP2 was thus identified as a candidate target of miR-508-5p by virtue of possessing several seed sequences of miR-508-5p within the 3′UTR of their mRNA. To experimentally validate whether SKP2 is a potential target of miR-508-5p in GC. Luciferase reporter vector containing a 3′UTR of SKP2 mRNA (pMIR-Report/SKP2 3′UTR), or a mutated 3′UTR (pMIR-Report/Mut-SKP2 3′UTR) were first constructed (Fig. 2A). The 293T cells were co-transfected with pMIR-Report/SKP2 3′UTR or pMIR-Report/Mut-SKP2 3′UTR, and proviral plasmid pSicoR/NC, pSicoR/miR-508-5p, miR-508-5p/inhibitor. The results of dual luciferase assay showed a significant decrease and increase of relative luciferase activity in the cells transfected with pSicoR/miR-508-5p, miR-508-5p/inhibitor, respectively, in comparison with the pSicoR/NC transfected cells (Fig. 2B). There was no significant change of luciferase activity in the cells transfected with pSicoR/NC or pMIR-Report/Mut-SKP2 3′UTR plasmid DNA (Fig. 2B). Immunoblotting assay further confirmed that the endogenous SFP2 protein was also strikingly suppressed in the 293T cells transfected with pSicoR/miR-508-5p (Fig. 2C). This data indicated that SKP2 might be a potential target for tumor suppressor miR-508-5p in GC.

We next sought to explore the capacity of miR-508-5p to regulate SKP2 in GC. SGC-7901 cells were infected with LV-NC, LV-miR-508-5p, inhibitor vectors. Although miR-508-5p transcript was detected in GC SGC-7901 cells, a significant augmentation and inhibition of miR-508-5p transcript were observed in cells infected with LV-miR-508-5p, inhibitor vectors as determined by a qRT-PCR assay, in comparison with the cells infected with LV-NC, respectively (Fig. 3A and B). The total cell lysates were harvested for immunoblotting analysis against anti-SKP2 antibody at 48 h post infection. The result demonstrated that the expression of SKP2 was downregulated and upregulated at both mRNA (Fig. 3C and D) and protein levels (Fig. 3E) in cells infected with LV-miR-508-5p, inhibitor vectors, as compared with the LV-NC, respectively. These results suggested that miR-508-5p was capable of downregulating SKP2 expression in GC cells at both of transcriptional and post-transcriptional levels, indicative of an underlying mechanism of miR-508-5p in carcinogenesis of GC.

To better characterize the functionality of miR-508 in GC cell proliferation, the SGC-7901 cells were infected with LV-NC, LV-miR-508-5p, or inhibitor vectors, the cell proliferation and apoptosis was determined by an MTT, Hoechst staining and flow cytometric assays. The infection of LV-miR-508-5p showed a significant reduction of cell proliferation, in comparison with those infected with LV-NC and inhibitor at 72 h post-infection (P<0.05), as determined by an MTT assay (Fig. 4A). Flow cytometry analysis further demonstrated an increased frequency of apoptosis cells in the SGC-7901 cells infected with LV-miR-508-5p, in comparison with those infected with LV-NC (P<0.01) (Fig. 4B); and an increased abundance of cells with characteristics of chromosome condensation was also found in the cells infected with LV-miR-508-5p (Fig. 4C and D).

The aforementioned result of inverse relationship between miR-508-5p and clinical pathogenesis of GC led us to investigate the impact of miR-508-5p in the metastasis of GC by a cell scratch assay and a transwell assay in vitro. The migration capacity in the SGC-7901 cells infected with LV-miR-508-5p was significantly reduced, as compared with those infected with LV-NC or inhibitor, as determined by both of scratch assay (Fig. 5) and transwell assay (Fig. 6A) (P<0.05 and P<0.01, respectively). In addition, the ability of invasion through the Matrigel was also dramatically decreased in the SGC-7901 cells overexpressed miR-508-5p, in comparison with the controls (P<0.01) (Fig. 6B).

Since the SKP2 was a regulator of cell cycle, we next sought to determine whether the miR-508-5p regulated the cell cycle of GC cells in vitro. Flow cytometric assay demonstrated a significantly decreased G2/M phase cell fraction in LV-miR-508-5p cells, as compared with the controls (P<0.05) (Fig. 7). Of interest, an overexpression of miR-508-5p also showed a trend toward cell arrest at G0/G1 phase (Fig. 7). These data suggested that miR-508-5p was capable of regulating SGC-7901 cell cycle progression by inducing cell cycle arrest at the G0/G1 phase in part through a mechanism of targeting SKP2.