Patients admitted to the Affiliated Hospital of Jining Medical University between February 2012 and October 2016 were evaluated. These patients with gastric cancer included 41 males and 22 females aged between 32–86 years, with a mean age of 60.6 years. Clinical stages were classified according to the International Union against Cancer TNM classification system (20). The Research Ethics Committee of the Affiliated Hospital of Jining Medical University approved the present study (JN2017015), and all patients provided written informed consent. All tissue samples were stored at −80°C before use.

Human gastric cancer cell lines, such as CTC-141 (Laboratory of Stem Cell Biology of Sichuan University, Sichuan, China) and MKN45 [American Type Culture Collection (ATCC) Manassas, VA, USA], as well as normal human gastric epithelium cell line GES-1 (Bogu Biotechnology, Shanghai, China) were maintained in the Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 U/ml penicillin, and 100 µg/ml streptomycin as well as 10% fetal bovine serum (FBS) at 37°C with 5% CO₂.

Mimic control and miR-95 mimics (miR-95 mimic), as well as inhibitor control and miR-95 inhibitors (miR-95 inhibitor) were purchased from Qiagen (Duesseldorf, Germany). Cells were transfected using Lipofectamine 3000 according to the manufacturer's instructions (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). After transfection for 48 h, the transfected cells were used for further experiments.

Total RNA was extracted from the gastric cancer tissue samples and cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol and was quantified using NanoDrop 2000 (Thermo Fisher Scientific, Inc.). An RNA sample (2 µg) was used to synthesize cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc.). A SYBR-Green (Roche Molecular Diagnostics, Pleasanton, CA, USA) was used to determine relative mRNA expression with an ABI PRISM 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) following the instructions of the manufacturer. Primers for miR-95 were purchased from GeneCopoeia Co. (Guangzhou, China). The cycling parameters were as follows: 95°C for 5 min and then 40 cycles of 95°C for 15 sec and annealing/extension at 60°C for 1 min. E-cadherin: 5′-ACCTGGTTCAGATCAAATCC-3′ (forward) and 5′-TCATTCTGATCGGTTACCGT-3′ (reverse); N-cadherin: 5′-CAGAGTTTACTGCCATGACG-3′ (forward) and 5′-AAAGTCGATTGGTTTGACCA-3′ (reverse); vimentin: 5′-ATTGAGATTGCCACCTACAG-3′ (forward) and 5′-ATCCAGATTAGTTTCCCTCAG-3′ (reverse); Slug: 5′-AGATGCATATTCGGACCCACA-3′ (forward) and 5′-CCTCATGTTTGTGCAGGAGAG-3′ (reverse); GAPDH: 5′-GAGAAGTATGACAACAGCCTC-3′ (forward) and 5′-ATGGACTGTGGTCATGAGTC-3′ (reverse); miR-95: 5′-CTGGTGGAGGGATGGATGAA-3′ (forward) and 5′-GGCCCGATCACAAACTCATC-3′ (reverse); U6: 5′-CTCGCTTCGGCAGCACA-3′ (forward) and 5′-AACGCTTCACGAATTTGCGT-3′ (reverse) GAPDH mRNA or small nuclear RNA U6 were used as internal controls. The relative mRNA expression was calculated via the 2^−∆∆Cq method (21).

Whole protein was isolated from the transfected cells using RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China), and protein concentrations were assessed using Pierce BCA Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. Protein ~45 µg was separated via 10% SDS-PAGE. After being transferred to nitrocellulose filter membranes (EMD Millipore, Bedford, MA, USA), the membranes were blocked with 5% skimmed milk at room temperature for 1 h. Subsequently, the membranes were incubated with indicated primary antibodies at 4°C overnight. After being washed with phosphate-buffered saline Tween-20 (PBST) three times at room temperature, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h and washed with PBST three times at room temperature. Finally, the blots were visualized by ECL kit (Pierce; Thermo Fisher Scientific, Inc.). Each independent experiment was performed three times. The anbodies as follow: EMT kit (1:1,000; cat. no. 9782; Cell Signaling Technology, Danvers, MA, USA), β-actin (1:2,000; cat. no. ab6276; Abcam, Cambridge, UK), goat anti-rabbit (HRP) (1:5,000; cat. no. ab205718; Abcam), goat anti-mouse (HRP) (1:3,000; cat. no. ab205719, Abcam). The densitometry of blots was determined using ImageJ software (version 4.1; National Institutes of Health, Bethesda, MD, USA).

CCK-8 assay was used to detect the effect of miR-95 on cell proliferation. In brief, 3,000 transfected CTC-141 or MKN45 cells with 200 µl media were seeded in 96-well plates. After transfection for 48 h, 20 µl CCK-8 solution (Beyotime Institute of Biotechnology) was added to the culture medium at 0, 24, 48 and 72 h, and incubated for 30 min at 37°C. The absorbance of 450 nm was measured using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each independent experiment was performed three times.

A wound healing assay was used to determine the effect of miR-95 on cell migration. Briefly, 5×10⁵ transfected CTC-141 or MKN45 cells were seeded in 6-well plates. When cell density was almost 90–100%, a linear wound was generated using a 20-µl pipette tip, and the detached cells were washed with PBS three times. The distance of wound healing was assessed at 0 and 24 h with a light microscope. Each independent experiment was performed three times.

A Transwell assay was used to determine the effect of miR-95 on cell invasion. In brief, 80 µl Matrigel was coated on the Transwell chambers (BD Biosciences, Bedford, MA, USA) and maintained at 37°C for 30 min. Approximately 4×10⁴ transfected CTC-141 or MKN45 cells in 400 µl serum-free medium were placed in the upper chamber in 24-well culture plates, and 500 µl RPMI-1640 medium containing 10% FBS was added to the lower chamber. Cells were maintained at 37°C with 5% CO₂ for 16 h. Subsequently, the cells were stained with 0.5% crystal violet at room temperature for 10 min. The cells on the surface of the upper membranes were removed by cotton swab, and the number of invading cells was counted under a light microscope. Each independent experiment was performed three times.

When cell density was almost 60%, cells were cultured with serum-free media overnight. Recombinant human TGF-β1 (10 ng/ml) was added into media for 72 h.

TargetScan Human version 7.0 (www.targetscan.org) predicted that Slug was a potential target of miR-95. The 3′-UTR of Slug was cloned into the pGL3 luciferase vector (Invitrogen; Thermo Fisher Scientific, Inc.). For the luciferase assay, CTC-141 and MKN-45 cells were co-transfected with Renilla, pGL3-Slug 3′-UTR or and miR-95 mimics or miR-95 inhibitor. After transfection for 24 h, a luciferase reporter assay was performed using a Dual-Luciferase Reporter Assay kit according to the manufacturer's instructions (Promega Corp., Madison, Wisconsin, USA). The Renilla luciferase activity was used to normalize firefly luciferase activity. Each independent experiment was performed three times.

All data were analyzed using SPSS 18.0 statistical software (SPSS, Inc., Chicago, IL, USA) and presented as the mean ± SD. A Student's t-test or one-way ANOVA followed by Tukey's were used to analyze the differences between groups. P<0.05 was considered to indicate a statistically significant difference.

To investigate the functions of miR-95 in gastric cancer, we first detected the expression of miR-95 in gastric cancer tissues. We performed qRT-PCR to analyze the miR-95 expression levels in 63 gastric tumor tissue samples and adjacent normal tissues samples. As shown in Fig. 1A, our findings revealed that miR-95 expression was significantly downregulated in gastric cancer tissues compared with adjacent normal tissues (Fig. 1A). In addition, we detected the expression of miR-95 in gastric cancer cell lines, including CTC-141 and MKN45. GES-1 was used as a control. The expression of miR-95 was lower in CTC-141 and MKN45 cells compared to GES-1 (Fig. 1B). Subsequently, clinicopathological analyses of 63 gastric cancer patients demonstrated that the expression of miR-95 was closely associated with tumor size, lymph node metastasis as well as TNM stage (Table I).

To explore the function of miR-95 in gastric cancer, we first overexpressed or knocked down miR-95 in CTC-141 and MKN45 cells with miR-95 mimics or miR-95 inhibitor. The expression of miR-95 was determined by qRT-PCR (Fig. 2A). As shown in Table I, since the expression of miR-95 was closely associated with tumor size, we assumed that miR-95 may regulate cell proliferation. To verify our hypothesis, CCK-8 and colony formation assays were performed. The results of the CCK-8 assay revealed that compared with that of the mimic control or inhibitor control groups, the proliferation of CTC-141 and MKN45 cells in the miR-95-mimic group were significantly decreased and were clearly increased in the miR-95-inhibitor group (Fig. 2B). The colony formation assay also confirmed that ectopic expression of miR-95 led to a decrease in the number of colonies, and an inhibition of miR-95 led to an increase in the number of colonies (Fig. 2C). These results revealed that miR-95 suppressed gastric cancer cell proliferation.

Our experiments revealed that miR-95 expression was negatively associated with lymph node metastasis. To determine whether miR-95 regulated migration and invasion in gastric cancer cells, we performed wound healing and Transwell assays. The results of the wound healing assays revealed that ectopic expression of miR-95 significantly decreased the distance of cell migration (Fig. 3A). In contrast, downregulation of miR-95 significantly increased the distance of cell migration (Fig. 3A). Similar results were observed in the Transwell assay, revealing that the ectopic expression of miR-95 resulted in less invading cells than that in the mimic control group (Fig. 3B). In contrast, the downregulation of miR-95 resulted in more invading cells than those in the inhibitor control group (Fig. 3B). Since MMP9 is an invasion-related factor, we next determined whether miR-95 regulated MPP9 secretion using an ELISA assay, which revealed that the secretion of MMP9 was decreased when cells were transfected with miR-95 mimics, and increased when cells were transfected with the miR-95 inhibitor (Fig. 3C). Our experiments revealed that downregulation of miR-95 promoted gastric cancer cell migration and invasion.

To further decipher the detailed mechanisms of miR-95 in gastric cancer metastasis, we aimed to explore the effects of miR-95 on EMT, which was recognized as a main cause for cell migration and invasion. CTC-141 and MKN45 cells were transfected with miR-95 mimics and mimic control. After inducing 10 ng/ml of TGF-β1, the expression of EMT-associated proteins was determined by RT-qPCR and western blotting, respectively. The results revealed that ectopic expression of miR-95 led to an increased expression of E-cadherin both at the mRNA level and protein level, and a decreased expression of N-cadherin and vimentin both at the mRNA level and protein level (Fig. 4A and B). A suppression of miR-95 reversed these results (Fig. 4C and D). The mRNA and protein levels of E-cadherin were decreased, and the levels of N-cadherin as well as vimentin were increased (Fig. 4C and D). Overall, the results implied that miR-95 inhibits the TGF-β1-induced EMT process of gastric cancer cells.

miRNAs have been found to participate in multiple physiological and pathological processes by regulating gene expression. To further decipher the detailed mechanism of miR-95 on EMT, we searched for potential targets of miR-95 by bioinformatics search using TargetScan (www.targetscan.org). We found that Slug, a key transcription factor of EMT, was a direct target of miR-95. To verify whether slug was regulated by miR-95, we overexpressed or knocked down miR-95 in CTC-141 and MKN45 and detected the expression of Slug. As shown in Fig. 5A, we found that Slug expression was decreased in CTC-141 and MKN45 cells treated with miR-95 mimics, while Slug expression was increased in CTC-141 and MKN45 cells treated with miR-95 inhibitors (Fig. 5A and B). To examine whether miR-95 directly interacted with the 3′-UTR of Slug, we performed a luciferase reporter assay. The luciferase activity was significantly decreased when cells were co-transfected with Slug 3′-UTR and either miR-95 mimics or mimic control (Fig. 5C). These findings indicated that Slug is a target of miR-95 in gastric cancer.