A total of 4 GC cell lines, including SGC-7901, AGS, BGC-823 and MGC-803 were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China), maintained in our laboratory and used in the present study. A normal gastric mucosal epithelial cell line GES-1 (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) was also used. All cells were cultured in RPMI-1640 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin and 100 U/ml penicillin, in a culture incubator under the atmosphere of 5% CO₂ and 37°C.

Tissue samples of GC and adjacent normal tissues were obtained from 40 patients (male, n=24; female, n=16) who were diagnosed at the 401 Hospital of People's Liberation Army between January 2009 and December 2012. Participants were between 42 and 76 years of age and had no history of tumors. Written informed consent was obtained from patients and the protocols were approved by the ethics committee of the hospital. Non-cancerous tissues from the macroscopic tumor margin were isolated simultaneously and used as control. All tissues were snap frozen and stored in −80°C until use.

miRNAs were extracted from cells and tissues and purified using a miRNA isolation system (Omega Bio-Tek, Inc., Norcross, GA, USA) and processed with the miScript II RT kit (Qiagen GmbH, Hilden, Germany) to generate cDNA. The template cDNA was used for the qPCR assay with the miScript SYBR-Green PCR kit (Qiagen GmbH) following the manufacturer's protocol. The PCR thermal parameters were: 15 min at 95°C, 40 cycles of 15 sec at 94°C followed by 30 sec at 55°C, and 30 sec at 72°C. The expression threshold cycle values of miRNAs were calculated by normalizing with internal reference U6 and the relative quantity of miRNAs were calculated using the 2^−ΔΔCq method (29). For mRNA analysis, total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and followed by RT reaction using the Prime-script RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) and the RT reaction was 37°C for 60 min, followed by 85°C for 5 sec. RT-qPCR was conducted using SYBR Premix Ex Taq (Qiagen GmbH) and β-actin was used as internal control. The PCR reaction was 95°C for 5 min, followed by 45 cycles of 95°C for 15 sec and 58°C for 30 sec. The sequences of the PCR primers were as follows: miR-320a forward, 5′-AAAAGCTGGGTTGAGAGGGCG-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and U6 reverse, 5′-AACGCTTCACGAATTTGCGT-3′; ADAM10 forward, 5′-CTGCCCAGCATCTGACCCTAA-3′ and reverse, 5′-TTGCCATCAGAACTGGCACAC-3′; GAPDH forward, 5′-GACCCAGATCATGTTTGAGACC-3′, and reverse 5′-ATCTCCTTCTGCATCCTGTCG-3′. Relative quantification of ADAM10 expression was calculated using the 2^−ΔΔCq method (29). Each sample was analyzed in triplicate.

miR-320a mimics, mimic control, inhibitor and inhibitor control were synthesized by GenePharma (Shanghai, China) and were transfected into cells (1×10⁵) with a final concentration of 20 nmol/l using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). miR-320a mimics, 5′-AAAAGCUGGGUUGAGAGGGCGA-3′; mimics control, 5′-UUCUCCGAACGUGUCACGUTT-3′; inhibitor, 5′-UCGCCCUCUCAACCCAGCUUUU3′; inhibitor control, 5′-CAGUACUUUUGUGUAGUACAA-3′. The specific anti-ADAM10 small interfering (si)RNA (siADAM10) and its non-specific scramble siRNA (control) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and then transfected into cells using Lipofectamine^® 2000 according to the manufacturer's protocol. Briefly, cells were cultured in RPMI-1640 medium without antibiotics. Transfection procedure was performed once the cultured cells are at a confluency of 80%. SiRNA (5 µl) and 4.5 µl RNAi-MAX complexes (Thermo Fisher Scientific, Inc.) were diluted in 250 µl Opti-MEM medium and incubated for 20 min. The mixture was then added to the cells and incubated at 37°C. The efficiency of siADAM10 was confirmed by western blot analysis. ADAM10 cDNA with 3′-UTR were inserted into pcDNA3.1(+) vector (Invitrogen; Thermo Fisher Scientific, Inc.) to build the plasmid pcDNA3.1(+)-ADAM10 (pcADAM10), and the empty vector (Invitrogen; Thermo Fisher Scientific, Inc.) was used as control. The samples were collected for gene and protein detection 48 h post-transfection.

Transfected cells were seeded into 6-well culture dishes (400 cells/well) and cultured for 14 days. The colonies were fixed with 75% ethanol and stained with 0.2% crystal violet (Sangon Biotech Co., Ltd., Shanghai, China) for 20 min at room temperature and counted under a microscope (SZ61-ILST; Olympus Corporation, Tokyo, Japan). The number of formed colonies was counted in 6 different fields (magnification, ×100).

MMT was used for the viability assay identify the effect of miR-320a. The transfected GC cells were seeded in 96-well plates at 2,000 cells/well and maintained at 37°C and 5% CO₂ for 4 days. Then, 150 µl DMSO was added to block the reaction and the cell proliferation was determined by measuring the optical density at 570 nm using a spectrophotometric reader (Thermo Fisher Scientific, Inc.). All tests were performed in triplicate.

The wild-type 3′-UTR and the mutated 3′-UTR sequences of ADAM10 were inserted into the SpeI and HindIII sites of the pMiR-REPORT (Ambion; Thermo Fisher Scientific, Inc.) to produce constructs of wild-type 3′-UTR segment of ADAM10 (ADAM10-WT) and mutated 3′-UTR segment of ADAM10 (ADAM10-Mut), respectively. The following primers were used: forward 5′-AGTCCTTATGTGGCATGCCCCCTATG-3′ and reverse 5′-ATTGCGATGATACAAGTCCATCGGATTATTTCA-3′. SGC-7901 cells were seeded in 24-well plates (3×10⁵ cells/well). After 1 day, ADAM10-WT, ADAM10-Mut, and pMiR-REPORT control vectors were co-transfected with miR-320a and β-galactosidase into SGC-7901 cells. Following incubation at 37°C for 48 h, the luciferase activity was quantified using Dual-Light Combined Reporter Gene Assay system (Thermo Fisher Scientific, Inc.) 48 h after transfection.

The sensitivity of cells to cisplatin was determined using the Cell Titer 96 AQueous One Solution Cell Proliferation Assay kit (Promega Corporation, Madison, WI, USA). Cells were cultured in 96-well plates seeded at 3,000 cells/well and different concentrations of cisplatin were added. The RPMI-1640 medium was replaced with fresh medium (containing cisplatin) every 24 h. After 3 days, MTS (0.02 ml/well) was added. The absorbance was recorded at 490 nm for each well on the BioTek Synergy 2 (BioTek, Winooski, VT, USA).

Cells were collected and treated with cell lysis buffer (Beyotime Institute of Biotechnology, Haimen, China), followed by centrifugation for 30 min at 4°C and 13,400 × g, and the protein samples were collected. Protein quantity was determined using BCA Protein Assay kit (Beyotime Institute of Biotechnology) and then separated on a 10% SDS-PAGE gel and transferred onto PVDF membranes. The membranes were subsequently incubated at 4°C overnight with primary antibody ADAM10 (sc-28358; 1:500; Santa Cruz Biotechnology, Inc.) or β-actin (sc-8432; 1:2,000; Santa Cruz Biotechnology, Inc.), followed by incubated with horseradish peroxidase-conjugated secondary goat anti-mouse antibodies (STAR117P, 1:1,000; Bio-Rad Laboratories, Hercules, CA, USA) at room temperature for 30 min. The western blots were visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific, Inc.).

The miR-320a precursor sequences were cloned into pCDH-CMV-MCS-EF1-Puro vector (System Biosciences, Palo Alto, CA, USA) to construct stably miR-320a-expressing cells used in xenograft models. A total of 5 male BALB/c nu/nu mice (5-weeks old; weight, 18–20 g) were provided by the Animal Center of the Chinese Academy of Science (Shanghai, China) and used for the in vivo xenograft tumor model. Animals were housed in a specific pathogen-free room with a 12-h light/dark cycle and 40–70% humidity at 26–28°C. SGC-7901 cells (3×10⁶) with miR-320a stable expression vector or empty vector (control) were subcutaneously injected into the left and right flank. Tumor volumes were calculated using the formula: Tumor volume=(length × width²)/2. After 35 days, all mice were sacrificed following the standard procedure and harvested tumors were weighed.

Each experiment was repeated at least three times. Data are reported as mean ± standard deviation and statistical tests were performed using SPSS version 14.0 (SPSS, Inc., Chicago, IL, USA) and Prism version 5.0 (GraphPad, La Jolla, CA, USA). Statistical significance was determined using a two-sided Student's t-test. Paired-sample t-test was used to compare the expression levels of miR-320a and ADAM10 in clinical samples, and Pearson correlation was used to determine if there was a relationship between. Multiple group comparisons were analyzed using one-way analysis of variance. Tukey post hoc tests were used when comparing multiple parameters. P<0.05 was considered to indicate a statistically significant difference.

In order to determine the endogenous miR-320a level in GC cells, the present study used RT-qPCR to detect miR-320a expression in four GC cell lines (AGS, SGC-7901, BGC-823 and MGC-803 cells) and compared them with miR-320a expression in a normal gastric mucosal epithelial cell line GES-1 cells. The data revealed that compared with GES-1, all GC cells had significantly reduced expression levels of miR-320a (Fig. 1A), suggesting miR-320a may contribute to GC development.

The present study identified the role of miR-320a in GC cells by gain- or loss-of-function analysis (Fig. 1A). SGC-7901 cells were selected as they have relatively low endogenous miR-320a expression and BGC-823 cells were selected due to their relatively high endogenous miR-320a and were transfected with mimics or inhibitor. The expression of miR-320a in transfected cells was confirmed by RT-qPCR (Fig. 1B).

Colony formation and MTT assays were used to identify the effect of miR-320a on GC cell growth. As presented in Fig. 1C, the proliferation rate of SGC-7901 cells was significantly inhibited by miR-320a mimics, whereas the proliferation of BGC-823 cells was promoted by miR-320a inhibitor, in comparison with their corresponding control cells. For colony formation, the number of formed colonies was markedly reduced in miR-320a overexpressing cells, whereas it was increased in cells where miR-320a was inhibited (Fig. 1D). These findings indicated that miR-320a suppressed cell growth in vitro.

The present study determined the effects of ectopic miR-320a expression on cell chemosensitivity. It was revealed that miR-320a mimics effectively sensitized SGC-7901 and BGC-823 cells to cisplatin, compared with control-transfected cells. By contrast, the sensitivity of SGC-7901 and BGC-823 cells to cisplatin was reduced by the miR-320a inhibitor, when compared with the control cells (Fig. 2A and B). These findings collectively indicated that miR-320a sensitized GC cells to cisplatin.

As it was evident that up or downregulating miR-320a expression had a functional role in regulating GC cell growth in vitro, the present study investigated whether overexpressing miR-320a may have a similar antitumor role in inhibition of tumor growth in vivo. SGC-7901 cells were transfected with stably expressing miR-320a vector or control vector. Subsequently, cells were subcutaneously injected into five null mice. The tumors were harvested 5 weeks after injection. The findings revealed that tumors were significantly smaller when miR-320a expression was upregulated (Fig. 3A and B). Quantification of tumor weight confirmed that miR-320a markedly suppressed the ability of SGC-7901 cells to form tumors in vivo (Fig. 3C).

Three publically available databases (miRDB, microRNA, TargetScan) were used to predict the potential targets of miR-320a in GC cells and ADAM10 with conserved binding site in its 3′-UTR was selected for further analysis (Fig. 4A). To assess whether ADAM10 is a direct target of miR-320a, the luciferase reporter vectors with the putative ADAM10 3′-UTR target site for miR-320a (ADAM10-WT) and mutant type (ADAM10-Mut) were constructed. The present study confirmed that miR-320a significantly decreased luciferase activity of the ADAM10-WT plasmid, but not the ADAM10-Mut plasmid (Fig. 4B). Additionally, western blot assay also revealed that the protein level of ADAM10 was reduced in GC cells with miR-320a mimic, compared with control-transfected cells (Fig. 4C). These findings indicated that miR-320a directly targets to the 3′-UTR of ADAM10 and then suppresses its protein expression.

To clarify whether miR-320a regulates cell growth and drug sensitivity by targeting ADAM10, the current study induced knockdown or overexpression of ADAM10 in SGC-7901 cells. As presented in Fig. 5A, the protein level of ADAM10 was inhibited after siADAM10 transfection and ADAM 10 level was upregulated following cDNA transfection. The proliferation ability of SGC-7901 cells was impaired by knockdown of ADAM10 and promoted by overexpression of ADAM10 (Fig. 5B and C). Finally, the cisplatin-sensitivity of SGC-7901 cells was enhanced by siADAM10, whereas it was attenuated by overexpression of ADAM10 (Fig. 5D). These findings suggested that ADAM10 is a functional target of miR-320a in GC development and chemotherapy.

The present study determined the expression level of miR-320a in primary GC tissues and corresponding normal tissues. As presented in Fig. 6A, the expression of miR-320a was significantly downregulated in tumors when compared with adjacent normal tissues (0.1766±0.0085 vs. 0.2230±0.0134; P=0.008). The mRNA level of ADAM10 was higher in tumors compared with normal tissues (4.4278±1.6846 vs. 3.5373±1.6943; P=0.00; Fig. 6B). Pearson correlation coefficient analysis revealed that the expression of miR-320a was negatively correlated with the mRNA levels of ADAM10 (r=-0.452; P=0.003; Fig. 6C) in tumor tissues. These findings verified the negative correlation between miR-320a and ADAM10 in tumors.