A total of 57 gastric cancer and matched, non-tumorous gastric tissue samples were collected from patients of the The Affiliated Huai'an Hospital of Xuzhou Medical University (Huai'an, China) and The Second People's Hospital of Huai'an (Huai'an, China) between 2011 and 2014. None of the gastric cancer patients received chemotherapy or radiotherapy treatments prior to surgery. All tissue samples were immediately frozen in liquid nitrogen and stored at −80°C. The seventh edition of UICC tumor node metastasis (TNM) classification system was used as clinical staging criteria (18). The present study was approved by the Ethics Committee of The Affiliated Huai'an Hospital of Xuzhou Medical University and The Second People's Hospital of Huai'an, and written informed consent was obtained from all gastric cancer patients.

Five gastric cancer cell lines (SGC-7901, BGC-823, AGS, MKN-1 and MKN-45) and a normal gastric epithelium cell line (GES-1) were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China) and cultured in Gibco Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with Gibco 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) at 37°C in an atmosphere consisting of 5% CO₂.

Total RNA was isolated from tissue samples and cells using Invitrogen TRIzol (Thermo Fisher Scientific, Inc.). A SYBR PrimeScript miRNA RT PCR kit (Takara Biotechnology Co., Ltd., Dalian, China) was used to determine the miR-455 expression level and U6 served as an internal control. To quantify IGF-1R mRNA expression, total RNA was used to synthesize cDNA using a PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd.), followed by qPCR using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.). The amplification reaction was performed using the following cycling conditions: An initial predenaturation step for 5 min at 95°C, followed by 40 cycles of denaturation at 95°C for 30 sec and annealing at 65°C for 45 sec. The primer sequences were as follows: Forward, 5′-ACACTCCAGCTGGGTATGTGCCTT-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′ for miR-455; forward, 5′-CTCGCTTCGGCAGCACATATACT-3′ and reverse, 5′-ACGCTTCACGAATTTGCGTGTC-3′ for U6; forward, 5′-CCGCTGCCAGAAAATGTGCCCA-3′ and reverse, 5′-TGTCGTTGTCAGGCGCGCTG-3′ for IGF-1R; and forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′ for GAPDH. RT-qPCR was performed in triplicate on an AB7300 thermo-recycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The relative expression of miR-455 and IGF-1R mRNA were determined using the 2^−ΔΔCq cycle threshold method (19).

miR-455 mimics and negative control miRNA mimics (NC) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). Small interfering RNAs (siRNAs) specifically for IGF-1R and the control siRNA (IGF-1R siRNA and NC siRNA) were obtained from the Chinese Academy of Sciences (Changchun, China). Cells were transfected with these oligonucleotides using Invitrogen Lipofectamine 2000 Thermo Fisher Scientific, Inc.). Following transfection for 6–8 h, the culture medium containing Lipofectamine 2000 and oligonucleotides was discarded and replaced with fresh culture medium.

Following the 24-h transfection, the transfected cells were collected and re-seeded in 96-well plates at a density of 2,000 cells per well. Cells were then incubated at 37°C in 5% CO₂ atmosphere. At different time points (24, 48, 72 and 96 h), an MTT assay (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was performed according to the manufacturer's instructions. Briefly, 20 µl MTT solution (5 mg/ml) was added to each well and subsequently incubated at 37°C for 4 h. The culture medium containing MTT solution was discarded and 200 µl DMSO solution (Sigma-Aldrich; Merck KGaA) was added to dissolve the formazan precipitates. The absorbance at a wavelength of 490 nm was measured using an automatic multi-well spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA).

Following a 48-h transfection, the transfected cells were harvested, washed with Gibco phosphate-buffered saline (PBS; Thermo Fisher Scientific, Inc.) three times and resuspended in FBS-free culture medium. For the Transwell migration assay, 5×10⁴ cells were plated into the top side of polycarbonate Transwell chambers (8 µm; Costar; Thermo Fisher Scientific, Inc.). For the Transwelll invasion assay, 5×10⁴ cells were plated into the upper chambers coated with Matrigel (BD Biosciences, San Jose, CA, USA). The lower chambers were filled with DMEM containing 20% FBS. The chambers were incubated at 37°C in an atmosphere of 5% CO₂ for 48 h. Migratory and invasive cells on the lower membranes were fixed, stained with 0.5% crystal violet, washed with PBS three times and counted in five random fields (magnification, ×200) per Transwell chamber using an IX71 inverted microscope (Olympus Corporation, Tokyo, Japan).

Following transfection for 72 h, cells were washed with ice-cold PBS and lysed in RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) in the presence of protease inhibitor cocktail. The concentration of total protein was quantified using the BCA Protein Assay kit (Beyotime Institute of Biotechnology). Proteins (30 µg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membrane at 100 V for 1.5 h. Subsequent to blocking in 5% non-fat milk in Tris-buffered saline containing Tween-20 for 1 h at room temperature, the membranes were probed with mouse anti-human monoclonal IGF-1R (1:1,000 dilution; cat. no. sc-81464; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and mouse anti-human monoclonal GAPDH (1:1,000 dilution; cat. no. sc-137179; Santa Cruz Biotechnology, Inc.) at 4°C overnight, followed by incubation with the goat anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:5,000 dilution; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The signals were visualized using the ECL Plus Detection kit (Pierce; Thermo Fisher Scientific, Inc.).

Bioinformatic analysis was performed to investigate the potential target genes of miR-455 using three algorithm databases: TargetScan (http://www.targetscan.org/), PicTar (http://pictar.bio.nyu.edu) and miRanda (http://www.sanger.ac.uk).

Luciferase reporter assay was performed to investigate whether IGF-1R was a direct target gene of miR-455. Luciferase report vectors, PGL3-IGF-1R-3′UTR Wt and PGL3-IGF-1R-3′UTR Mut, were synthesized by Shanghai GenePharma Co., Ltd. HEK293T cells, purchased from Shanghai Institute of Biochemistry and Cell Biology, were seeded into 24-well plates at a density of 30–40%, and transfected with PGL3-IGF-1R-3′UTR Wt or PGL3-IGF-1R-3′UTR Mut, along with miR-455 mimics or NC using Lipofectamine 2000. At 48 h post-transfection, transfected cells were collected, washed with PBS, and subjected to luciferase reporter assay using a Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. All samples were run in triplicate.

Data are presented as the mean ± standard deviation, and were compared using a Student's t-test or one-way analysis of variance followed by the Student-Newman-Keuls post hoc test using SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA). Spearman's correlation analysis was used to analyze the association between miR-455 and IGF-1R mRNA expression levels. Two-tailed P<0.05 was considered to indicate a statistically significant difference.

miR-455 expression was detected in gastric cancer tissue samples and their matched non-tumorous gastric tissue samples using RT-qPCR. The expression level of miR-455 in the gastric cancer tissue samples was significantly lower than that in the matched non-tumorous gastric tissue samples (Fig. 1A; P<0.05).

In addition, the association between miR-455 expression and the clinicopathological features of gastric cancer were evaluated. As shown in Table I, reduced miR-455 expression was significantly associated with the clinical stage (P=0.003), lymph node metastasis (P=0.008) and tumor invasion (P=0.032). However, there was no correlation between miR-455 expression and age (P=0.962), sex (P=0.607), tumor size (P=0.314) or age (P=0.578).

Subsequently, miR-455 expression levels in the gastric cancer cell lines were compared with the normal gastric epithelium cell line. As presented in Fig. 1B, miR-455 was downregulated in all five gastric cancer cell lines (SGC-7901, BGC-823, AGS, MKN-1 and MKN-45) compared with that in GES-1 (P<0.05).

Given the downregulation of miR-455 in gastric cancer, it was hypothesized that miR-455 may be involved in the progression of cervical cancer. Firstly, BGC-823 and MKN-45 cells were transfected with miR-455 mimics or NC. At 48 h post-transfection, results of RT-qPCR indicated that miR-455 was markedly upregulated in miR-455 mimic-transfected BGC-823 and MKN-45 cells (Fig. 2A; P<0.05). Subsequently, MTT assay, and Transwell migration and invasion assays were used to analyzed the effects of miR-455 on cell proliferation, migration and invasion in gastric cancer. BGC-823 and MKN-45 cells transfected with miR-455 mimics exhibited decreased proliferation (Fig. 2B; P<0.05), migration and invasion (Fig. 2C; P<0.05) compared with NC.

Bioinformatic analysis with three algorithm databases indicated that IGF-1R was a potential target of miR-455 (Fig. 3A). A luciferase reporter assay was used to confirm whether the 3′-UTR of IGF-1R was directly targeted by miR-455. HEK293T cells were co-transfected with luciferase reporter vectors and miR-455 mimics or NC. As presented in Fig. 3B, miR-455 overexpression significantly decreased the luciferase activities of the PGL3-IGF-1R-3′UTR Wt. However, there was no significant difference between the miR-455 mimics and NC groups co-transfected with PGL3-IGF-1R-3′UTR Mut.

The expression levels of IGF-1R mRNA in gastric cancer tissue samples and their matched non-tumorous gastric tissue samples were observed using qRT-PCR. As demonstrated in Fig. 3C, IGF-1R mRNA was expressed at higher levels in gastric cancer tissue samples when compared with the matched non-tumorous gastric tissue samples (P<0.05). Spearman's correlation analysis revealed a significant inverse correlation between miR-455 and IGF-1R mRNA (r=−0.8408) in gastric cancer tissue samples (Fig. 3D; P<0.001).

To investigate whether miR-455 regulates the IGF-1R mRNA and protein expression levels in gastric cancer cells, RT-qPCR and western blotting were performed. The results demonstrated that IGF-1R mRNA (Fig. 3E; P<0.05) and protein (Fig. 3F; P<0.05) expression levels were suppressed by upregulation of miR-455 in the BGC-823 and MKN-45 cells. These findings indicate that miR-455 may directly target IGF-1R in gastric cancer cells via interaction with the binding sites in the 3′UTR of IGF-1R.

To evaluate the effects of IGF-1R on gastric cancer, IGF-1R siRNA or NC siRNA was injected into BGC-823 and MKN-45 cells. Following transfection, RT-qPCR and western blotting were conducted and IGF-1R was demonstrated to be downregulated at the mRNA (Fig. 4A; P<0.05) and protein (Fig. 4B; P<0.05) levels in the BGC-823 and MKN-45 cells transfected with IGF-1R siRNA. Consistent with the results of the current study, downregulation of IGF-1R mimics the effects of miR-455 overexpression on BGC-823 and MKN-45 cell proliferation (Fig. 4C; P<0.05), migration and invasion (Fig. 4D; P<0.05).