The present study was approved by the Ethics Committee of The First Affiliated Hospital of Guangxi Medical University (Nanning, China). Written informed consent was obtained from all participants. A total of 49 GC and matched adjacent normal gastric tissue samples were collected from patients (28 males, 21 females; age range, 52–79 years) who underwent surgery resection at The First Affiliated Hospital of Guangxi Medical University between July 2014 and November 2016. No patients received chemotherapy, radiotherapy or other treatments prior to surgery. Upon resection, all tissues were immediately frozen in liquid nitrogen and subsequently stored at −80°C.

GC cell lines (AGS, SGC-7901, BGC-823 and MGC-803) and the human gastric epithelial immortalized GES-1 cell line were purchased from American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution at 37°C in a humidified incubator with 5% CO₂. DMEM, FBS and antibiotic solution were all purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

miR-18b inhibitor, miRNA inhibitor negative control (NC inhibitor), KLF6-specific small interfering RNA (siRNA) and negative control siRNA (NC siRNA) were synthesized by Shanghai GenePharma Co., Ltd., (Shanghai, China). The miR-18b inhibitor sequence was 5′-CUAACUGCACUAGAUGCACCUUA-3′ and the NC inhibitor sequence was 5′-CAGUACUUUUGUGUAGUACAAA-3′.

The KLF6 siRNA sequence was 5′-GCAGGAAAGUUUACACCAATT-3′ and the NC siRNA sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. Cells were plated into 6-well plates at a density of 8×10⁵ cells/well. Following incubation overnight, cells were transfected with miR-18b inhibitor (100 pmol), NC inhibitor (100 pmol), KLF6 siRNA (100 pmol) or NC siRNA (100 pmol) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 48 h following transfection, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine miR-18b expression. Cell Counting kit-8 (CCK8) and cell invasion assays were performed at 24 and 48 h post transfection. Western blot analysis was carried out at 72 h following transfection.

Total tissue or cell RNA was isolated using TRIzol^® reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. To quantify miR-18b expression, total RNA was converted into first-strand complementary DNA (cDNA) using a TaqMan microRNA reverse transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The temperature protocol for reverse transcription was as follows: 16°C for 30 min, 42°C for 30 min and 85°C for 5 min. Subsequently, PCR amplification was performed to detect miR-18b expression with the TaqMan microRNA PCR kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The cycling conditions 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 KLF6 mRNA expression, cDNA was synthesized from total RNA using a Moloney Murine Leukemia Virus reverse transcriptase kit (Promega Corporation, Madison, WI, USA). The temperature protocol for reverse transcription was as follows: 95°C for 2 min; 20 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2 min; and 72°C for 5 min. cDNA was subsequently subjected to qPCR using SYBR^® Premix Ex TaqTM II (Takara Biotechnology Co., Ltd., Dalian, China). The cycling conditions were as follows: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 65°C for 45 sec. U6 snRNA and GAPDH were used as internal reference for miR-18b and KLF6 mRNA, respectively. The primers were designed as follows: miR-18b, 5′-GGGTAAGGTGCATCTAGTGC-3′ (forward) and 5′-CAGTGCGTGTCGTGGAGT-3′ (reverse); U6, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ (forward) and 5′-CGCTTCACGAATTTGCGTGTCAT-3′ (reverse); KLF6, 5′-CGGACGCACACAGGAGAAAA-3′ (forward) and 5′-CGGTGTGCTTTCGGAAGTG-3′ (reverse); and GAPDH, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ (forward) and 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′ (reverse). Data were analyzed using the 2^−ΔΔCq method (18).

A CCK8 assay was performed to detect cell proliferation. Cells were plated into 96-well plates at a density of 3×10³ cells/well. Following transfection, cells were incubated at 37°C in a humidified incubator with 5% CO₂ for 0, 24, 48 and 72 h. At each time point, the CCK8 assay was conducted according to the manufacturer's protocol. Briefly, 10 µl CCK8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well and incubated at 37°C with 5% CO₂ for further 2 h. Optical density values were measured at a wavelength of 450 nm using a microplate spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT, USA).

Matrigel coated Transwell chambers (pore size, 8-µm; BD Biosciences, Franklin Lakes, NJ, USA) were used to examine cell invasive ability. Transfected cells were collected 48 h post-transfection. A total of 1×10⁵ cells in 200 µl FBS-free DMEM medium were added to the upper chamber. The lower chambers were filled with 500 µl DMEM supplemented with 10% FBS. Following incubation for 24 h, cells remaining on the upper surface were removed using cotton swabs. The invaded cells were fixed with 100% methanol at room temperature for 15 min, stained with 0.1% crystal violet at room temperature for 15 min and washed with PBS. The number of invasive cells was counted under an inverted light microscope (Olympus Corporation, Tokyo, Japan) in five randomly selected fields.

The putative target genes of miR-18b were predicted using miRanda (August 2010 Release, Last Update: 2010-11-01; www.microrna.org) and TargetScan (version 7.1; www.targetscan.org). KLF6 was predicted as a potential target of miR-18b. A luciferase reporter assay was utilized to investigate if KLF6 is a direct target of miR-18b. Luciferase plasmids pmirGLO-KLF6-3′-UTR wild type (Wt) and pmirGLO-KLF6-3′-UTR mutant (Mut), were chemically synthesized by Shanghai GenePharma Co., Ltd. Cells were seeded in 24-well plates at an initial density of 1.5×10⁵ cells/well. Following an overnight incubation, cells were transfected with miR-18b inhibitor or NC inhibitor, in addition to pmirGLO-KLF6-3′-UTR Wt or pmirGLO-KLF6-3′-UTR Mut using Lipofectamine^® 2000. Following incubation at 37°C with 5% CO₂ for 48 h, cells were harvested and the luciferase activity was detected using the Dual-Luciferase Reporter assay system (Promega Corporation) according to the manufacturer's protocol. Firefly luciferase activity was normalized Renilla luciferase activity.

The primary antibodies used in the present study were mouse anti-human KLF6 primary antibody (1:1,000; cat. no. sc-134374) and mouse anti-human GAPDH primary antibody (1:1,000; cat. no. sc-32233), purchased from Santa Cruz Biotechnology, Inc., Dallas, TX, USA. Total protein was extracted from tissues or cells using a radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). The concentration of total protein was quantified with a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein (30 µg) were loaded into each lane and separated by 10% SDS-PAGE, followed by transfer onto polyvinylidene difluoride membranes. Membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 1 h, followed by incubation with primary antibodies at 4°C overnight. Membranes were subsequently washed with TBST three times and incubated with goat anti-mouse horseradish peroxidase-conjugated secondary IgG goat anti-mouse (1:5,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) at room temperature for 2 h. Protein bands were visualized using an enhanced chemiluminescence plus reagent (GE Healthcare Life Sciences, Little Chalfont, UK). Protein expression was quantified using Quantity One software version 4.62 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Data is expressed as the mean ± standard deviation of at least 3 independent experiments and was analyzed using the Student's t-test or one-way analysis of variance followed by the Student-Newman-Keuls multiple comparisons test. The association between miR-18b and clinicopathological characteristics of patients with GC was analyzed using a univariate χ² test. Spearman's correlation analysis was performed to evaluate the association between miR-18b and KLF6 mRNA levels in GC tissues. SPSS software, version 13.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

miR-18b expression was detected in 49 GC and matched adjacent normal gastric tissue samples. RT-qPCR analysis revealed that miR-18b expression was significantly upregulated in GC tissues compared with the adjacent normal gastric tissues (P<0.05; Fig. 1A). Additionally, the expression levels of miR-18b in GC cell lines and the human gastric epithelial immortalized cell line GES-1 were also determined using RT-qPCR. Compared with GES-1, higher expression levels of miR-18b were detected in all four GC cell lines (P<0.05; Fig. 1B). As AGS and BGC-823 cells expressed relatively higher miR-18b expression amongst the four GC cell lines, these two cell lines were selected for the subsequent experiments. These results indicated that miR-18b is upregulated in both GC tissues and cell lines.

To explore the clinical value of miR-18b in GC, patients were divided into miR-18b high-expression group (n=25) and low-expression groups (n=24) based on the median expression of miR-18b (1.86). As presented in Table I, high miR-18b expression was associated with lymph node metastasis (P=0.032), invasive depth (P=0.015) and Tumor Node Metastasis (TNM) stage (P=0.015) in patients with GC. However, no significant difference was observed between miR-18b expression and other clinicopathological factors, including age, sex, tumor size and differentiation (P>0.05). These results suggested that miR-18b may be associated with the malignant progression of GC.

To investigate the role of miR-18b in GC, AGS and BGC-823 cells were transfected with miR-18b inhibitor to decrease its expression. Following transfection, RT-qPCR revealed that miR-18b was significantly downregulated in cells transfected with miR-18b inhibitor compared with the cells transfected with NC inhibitor (Fig. 2A; P<0.05). A CCK-8 assay was subsequently conducted to examine the effect of miR-18b knockdown on GC cell proliferation. The downregulation of miR-18b significantly suppressed the proliferation of AGS and BGC-823 cells (Fig. 2B; P<0.05). Additionally, a cell invasion assay was performed to evaluate cell invasion ability in cells transfected with miR-18b inhibitor or NC inhibitor. Transfection with miR-18b inhibitor markedly attenuated the invasion capacities of AGS and BGC-823 cells compared with the NC inhibitor group (Fig. 2C; P<0.05). Taken together, this indicated that miR-18b may have an oncogenic role in GC progression.

To elucidate the mechanism underlying the oncogenic role of miR-18b in GC, bioinformatics analysis was used to predict the potential targets of miR-18b. KLF6, which has previously been reported to affect GC initiation and progression (19–21), was predicted as a putative target of miR-18b. As presented in Fig. 3A, the 3′-UTR of KLF6 contains two miR-18b binding sites (1 and 2). A luciferase reporter assay was performed to investigate if miR-18b interacted with the 3′-UTR of KLF6. miR-18b downregulation increased the luciferase activity of pmirGLO-KLF6-3′-UTR Wt (1 and 2) in AGS and BGC-823 cells (Fig. 3B; P<0.05). However, no significant changes in luciferase activity were detected in cells transfected with pmirGLO-KLF6-3′-UTR Mut (1 and 2) in the presence or absence of miR-18b inhibitor. Furthermore, RT-qPCR and western blot analysis were performed to investigate if miR-18b regulated KLF6 expression in GC cell lines. Transfection of miR-18b inhibitor increased the expression of KLF6 expression at both mRNA (P<0.05; Fig. 3C) and protein (P<0.05; Fig. 3D) levels in AGS and BGC-823 cells. Collectively, this data indicated that KLF6 may be a direct target of miR-18b in GC.

To further explore the association between miR-18b and KLF6 in GC, the expression of KLF6 in 49 pairs of GC and matched adjacent normal gastric tissues was detected. RT-qPCR and western blot analysis revealed that KLF6 expression was downregulated in GC tissues compared with adjacent normal gastric tissues at the mRNA (Fig. 4A; P<0.05) and protein (Fig. 4B) level. Additionally, a negative association between miR-18b and KLF6 mRNA expression in GC tissues was confirmed by Spearman's correlation analysis (Fig. 4C; r=−0.6030; P<0.0001). These results suggested that the downregulation of KLF6 in GC may be attributed at least in part to the upregulation of miR-18b.

Rescue experiments were performed to further confirm that the effect of miR-18b knockdown on GC cell proliferation and invasion is mediated by the regulation of KLF6 expression. AGS and BGC-823 cells were transfected with miR-18b inhibitor in combination with KLF6 siRNA or NC siRNA. Western blot analysis indicated that co-transfection of KLF6 siRNA partially abrogated the miR-18b inhibitor-mediated upregulation of KLF6 (Fig. 5A; P<0.05). Additionally, functional experiments demonstrated that KLF6 restoration rescued the inhibition of cell proliferation (Fig. 5B; P<0.05) and invasion (Fig. 5C; P<0.05) caused by miR-18b knockdown in AGS and BGC-823 cells. Collectively, these results suggested that the oncogenic roles of miR-18b in GC are achieved, at least in part, by regulation of KLF6 expression.