The present study was approved by the Ethics Committee of Renhe Hospital (Shanghai, China), and written informed consent was obtained from all patients between June 2015 and December 2016. A total of 45 paired gastric cancer tissues and adjacent non-tumoural tissues were obtained from patients who received surgery resection at Department of General Surgery, Renhe Hospital (Shanghai, China). None of the patients were treated with chemotherapy or radiotherapy prior to surgery. Tissue samples were immediately frozen in liquid nitrogen following surgical resection, and stored at −80°C until use.

Human gastric cancer cell lines (SGC-7901, MGC-803, AGS and BGC-823), an immortalized gastric epithelial cell line (GES-1) and the 293T cell line were acquired form the American Type Culture Collection (Manassas, VA, USA). All cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 50 U/ml penicillin and 50 µg/ml streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C.

Total RNA was extracted from tissues (1 g) or cells (1.5×10⁶) using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), following to the manufacturer's protocol. RNA concentrations were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). To detect miR-197 expression, reverse transcription was conducted using a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the TaqMan miRNA assay (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to examine miR-197 expression, with U6 as an internal control. The cycling conditions for qPCR were as follows: 50°C for 2 min, 95°C for 10 min; 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 60 sec. To quantify MTDH mRNA expression levels, total RNA was reverse transcribed to cDNA using a PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). The relative expression levels of MTDH were determined with SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.), and normalized to GAPDH expression. The amplification was performed with cycling conditions as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. The primers were designed as follows: miR-197, 5′-ATTACTTTGCCCATATTCATTTTGA-3′ (forward) and 5′-ATTCTAGAGGCCGAGGCGGCCGACATGT-3′ (reverse); U6, 5′-CGCTTCGGCAGCACATATAC-3′ (forward), and 5′-TTCACGAATTTGCGTGTCAT-3′ (reverse); MTDH, 5′-AAATAGCCAGCCTATCAAGACTC-3′ (forward) and 5′-TTCAGACTTGGTCTGTGAAGGAG-3′ (reverse); and GAPDH, 5′-GCACCGTCAAGGCTGAGAAC-3′ (forward) and 5′-TGGTGAAGACGCCAGTGGA-3′ (reverse). The relative quantification was calculated using the 2^−ΔΔCq method (25).

The miR-197 mimics and corresponding negative control miRNA (miR-NC) were synthesized and purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The miR-197 mimic sequence was 5′-UUCACCACCUUCUCCACCCCAGC-3′ and the miR-NC sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. The MTDH overexpression plasmid (pcDNA3.1-MTDH) and empty control plasmid (pcDNA3.1) were obtained from Origene Technologies, Inc. (Rockville, MD, USA). Cells (7×10⁵ cells/well) were seeded in 6-well plates and transfected with oligonucleotides (100 pmol) and/or plasmids (2 µg) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Following a 6 h incubation at 37°C, the culture medium was replaced with fresh DMEM containing 10% FBS. Cell Counting Kit-8 assay, Matrigel invasion assay, RT-qCPR and western blot analysis were performed 24, 48, 48 and 72 h post-transfection, respectively.

Cell proliferation was determined by a CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), according to the manufacturer's protocol. Briefly, transfected cells were collected at 24 h post-transfection, and seeded (3×10³ cells/well) into 96-well plates. Proliferation was monitored everyday for 4 days. A total of 10 µl CCK8 solution was added into each well and cultured for 2 h in a 37°C incubator with 5% CO₂. The absorbance at a wavelength of 450 nm was measured using a SpectraMAX Plus microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). Each assay was performed in triplicate and repeated three times.

Transwell invasion assays were performed to examine the cell invasive abilities using Transwell chambers (8 µm; BD Biosciences, San Jose, CA, USA) coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). Transfected cells were collected and resuspended in FBS-free DMEM medium. A total of 5×10⁴ transfected cells in FBS-free DMEM medium were seeded in the top chamber. DMEM supplemented with 10% FBS was added to the lower chamber and served as a chemoattractant. Following 48 h incubation at 37°C in 5% CO₂, the cells remaining on the upper side were wiped away with cotton swabs and the Transwell chambers were washed with PBS, fixed in 95% methanol at room temperature for 15 min, stained with 0.1% crystal violet at room temperature for 15 min and air-dried. The invasive cells were imaged and five independent fields were counted with an Olympus IX53 microscope (Olympus Corporation, Tokyo, Japan). The assay was repeated at least three times.

Bioinformatics analysis was conducted to predict the candidate genes of miR-197 using TargetScan (http://targetscan.org) and miRanda (http://www.microrna.org/microrna/home.do).

MTDH was predicted to be a potential target of miR-197 by bioinformatics analysis. The luciferase reporter vectors, pMiR-MTDH-3′UTR wild-type (Wt) and pMiR-MTDH-3′UTR mutant-type (Mut) were synthesized and confirmed by Shanghai GenePharma, Co., Ltd. 293T cells were seeded in 24-well plates until 60–70% confluent. Subsequently, cells were transfected with either pMiR-MTDH-3′UTR Wt (0.2 µg) or pMiR-MTDH-3′UTR Mut (0.2 µg), and co-transfected with either miR-197 mimics (50 pmol) or miR-NC (50 pmol) were transfected into 293T cells using Lipofectamine 2000, following the manufacturer's protocol. Following 48 h transfection, luciferase activities were detected using the Dual Luciferase Reporter assay system (Promega Corporation, Madison, WI, USA). Renilla luciferase activities were used to normalize firefly luciferase activities. The experiment was repeated three times, and each was performed in triplicate.

Cells (1.5×10⁶) or tissue specimens (1 g) were lysed in radioimmunoprecipitation lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Protein quantification was performed using a Bicinchoninic Acid Protein assay (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein (30 µg) were separated by 10% SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) at room temperature for 1 h, the membranes were incubated overnight at 4°C with primary antibodies against: MTDH (1:1,000 dilution; cat. no. sc-517220; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), PTEN (sc-133197; 1:1,000 dilution), p-AKT (sc-271966; 1:1,000 dilution), AKT (sc-81434; 1:1,000 dilution) and GAPDH (sc-32233; 1:1,000 dilution) (all from Santa Cruz Biotechnology, Inc.). Membranes were washed with TBST and probed with horseradish peroxidase-conjugated goat-anti mouse secondary antibody (1:5,000 dilution; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) at room temperature for 2 h. Immunoreactive bands were visualized using an Enhanced Chemiluminescence Detection kit (Thermo Fisher Scientific, Inc.). Quantification of band intensity was performed with Quantity One software (version 4.62; Bio-Rad Laboratories, Inc., Hercules, CA, USA). GAPDH served as a loading control.

Data are expressed as the mean ± standard deviation, and all statistical analyses were performed with a two-tailed Student's t test or one-way analysis of variance followed by a Student-Newman-Keuls test, using SPSS version 16.0 software (SPSS, Inc., Chicago, IL, USA). Correlation of miR-197 expression with that of MTDH mRNA expression was conducted with the Spearman's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

miR-197 expression was measured in 45 pairs of gastric cancer tissues and adjacent non-tumoural tissues by RT-qPCR to evaluate the significance of miR-197 in gastric cancer. Expression of miR-197 was significantly lower in gastric cancer tissues compared with expression in adjacent non-tumoural tissues (P<0.05; Fig. 1A). In addition, associations between miR-197 expression and clinicopathological factors of patients with gastric cancer were evaluated. Patients were divided into the following groups according to their median miR-197 values (Table I): Low-miR-197 group (n=23) and high-miR-197 group (n=22). Low miR-197 expression was significantly associated with tumour size (P=0.011), invasive depth (P=0.005), tumour nodes metastasis (TNM) staging (P=0.011) and lymph node metastasis (P=0.004). However, no significant association was observed between miR-197 expression and age (P=0.626), sex (P=0.672) or differentiation (P=0.668).

miR-197 expression levels were also examined in a panel of gastric cancer cell lines (SGC-7901, MGC-803, AGS and BGC-823) and an immortalised normal gastric epithelial cell line (GES-1). miR-197 expression levels in all of the gastric cancer cell lines were significantly decreased compared with that of GES-1 (P<0.05; Fig. 1B). These results suggested that miR-197 may serve important roles in gastric cancer progression. Notably, SGC-7901 and BGC-823 cells expressed the lowest levels of miR-197; thus, these two cell lines were selected as models for subsequent experiments.

miR-197 mimics were transfected into SGC-7901 and BGC-823 cells to overexpress the total miR-197 levels and to investigate the biological roles of miR-197 in gastric cancer progression. RT-qPCR analysis demonstrated that miR-197 was significantly upregulated in SGC-7901 and BGC-823 cells following transfection with miR-197 mimics compared with cells transfected with miR-NC (P<0.05; Fig. 2A and B, respectively). Subsequently, the effects of miR-197 overexpression on the proliferation and invasion of SGC-7901 and BGC-823 cells were investigated. CCK-8 assays demonstrated that miR-197 overexpression suppressed SGC-7901 and BGC-823 cell proliferation compared with the miR-NC (P<0.05; Fig. 2C and D, respectively). Results from the Matrigel invasion assays revealed that overexpression of miR-197 decreased the invasive capacities of the SGC-7901 and BGC-823 cells compared with the miR-NC (P<0.05; Fig. 2E and F). These results suggested that miR-197 may act as a tumour suppressor in gastric cancer.

Bioinformatics analysis was performed to predict candidate target genes and to determine the molecular mechanism underlying miR-197-mediated suppression of gastric cancer cell proliferation and invasion. Among the predicted targets, MTDH was selected for further analysis, as it has been reported to be highly expressed in gastric cancer and may serve oncogenic roles in gastric cancer initiation and progression (26,27). Two putative target sites of miR-197 in the 3′-UTR of MTDH were identified (Fig. 3A).

Luciferase reporter assays were performed to determine whether MTDH is a direct target of miR-197. 293T cells were transfected with MTDH-3′-UTR Wt or MTDH-3′-UTR Mut and co-transfected with miR-197 mimics or miR-NC. Overexpression of miR-197 significantly decreased the luciferase activities of MTDH-3′-UTR Wt site 1 and MTDH-3′-UTR Wt site 2 in 293T cells compared with cells transfected with the miR-NC (P<0.05; Fig. 3B). By contrast, no significant suppression of luciferase activity was observed on the MTDH-3′-UTR Mut reporter plasmid following miR-197 mimics transfection. In addition, the influence of miR-197 overexpression on the levels of MTDH mRNA and protein expression were determined in SGC-7901 and BGC-823 cells transfected with miR-197 mimics or miR-NC. RT-qPCR and western blot analysis results indicated that miR-197 overexpression resulted in decreased MTDH expression in the SGC-7901 and BGC-823 cells at both the mRNA (P<0.05; Fig. 3C) and the protein (P<0.05; Fig. 3D) level. These results suggested that MTDH may be a direct target gene of miR-197 in gastric cancer.

MTDH mRNA and protein expression levels were measured in gastric cancer tissues and adjacent non-tumoural tissues. MTDH mRNA expression levels were significantly higher in gastric cancer tissues compared with adjacent non-tumoural tissues (P<0.05; Fig. 4A). Similarly, a notable increase in the protein expression levels of MTDH were observed in gastric cancer tissues compared with normal tissue (P<0.05; Fig. 4B and C). In addition, an inverse correlation between MTDH mRNA and miR-197 expression levels was observed by Spearman's correlation analysis in gastric cancer tissues (P<0.0001; r=−0.6351; Fig. 4D).

Following the validation of MTDH as a direct target of miR-197, the present study examined whether MTDH upregulation may prevent the suppressive effects of miR-197 on gastric cancer cell proliferation and invasion. miR-197 mimics were transfected into SGC-7901 and BGC-823 cells with or without pcDNA3.1-MTDH. Co-transfection of pCDNA3.1-MTDH and miR-197 mimics enabled the recovery of MTDH expression (P<0.05; Fig. 5A). Furthermore, MTDH restoration rescued the proliferation (Fig. 5B and C; P<0.05) and invasion (Fig. 5D and E; P<0.05) inhibition induced by miR-197 overexpression in SGC-7901 and BGC-823 cells. These results suggested that MTDH inhibition by miR-197 may partially contribute to tumour-suppression in human gastric cancer.

MTDH negatively regulates PTEN expression by blocking PTEN transcription (28,29). Western blot analysis was conducted to detect PTEN, AKT and phosphorylated (p)-AKT protein expression levels in SGC-7901 and BGC-823 cells following transfection with miR-197 mimics or miR-NC. The results revealed that PTEN protein levels were notably increased in cells transfected with miR-197 mimics, compared with miR-NC-transfected cells (Fig. 6). p-AKT expression was reduced by overexpression of miR-197, and AKT expression levels were unaltered by miR-197 overexpression in SGC-7901 and BGC-823 cells (Fig. 6; P<0.05). These results suggested that miR-197 may indirectly regulate the PTEN/AKT signalling pathway in gastric cancer by regulating MTDH expression.