The human GC cell line MKN-45 and 293T cells were purchased from the Beijing Beina Chuanglian Biotechnology Institute. MKN-45 cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.), 293T cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% foetal bovine serum (Biological Industries), 100 µg/ml streptomycin, and 100 U/ml penicillin, and incubated at 37°C in 5% CO₂ in a humidified chamber. Fluorouracil (5-FU) and 0 nmol/l TSN were used as positive control and negative control, respectively. The cells were treated with different concentrations of TSN (0, 60, 80 and 100 nmol/l) and 5-FU (80 nmol/l) for 48 h, or transfected with pcDNA3.1(+) (2 µg/ml), pcDNA3.1(+)-miR-23a-3p (2 µg/ml), miR-23a-3p inhibitor (miR-23a-3p-I; 1.0×10⁻⁴ mmol/l) or miR-23a-3p inhibitor-NC (miR-23a-3p-I-NC; 1.0×10⁻⁴ mmol/l) (Guangzhou RiboBio Co., Ltd.) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions, then cultured for 48 h. The sequences used were as follows: miR-23a-3p-I 5′-GGAAAAUCCCUGGCAAUGUGAU-3′; miR-23a-3p-I-negative control (NC) 5′-CAGUACUUUUGUGUAGUACAAA-3′. miR-23a-3p-I and miR-23a-3p-I-NC were 2′-O-methyl modified.

Cell survival rate and chemosensitivity were determined using a CCK-8 assay (Beyotime Institute of Biotechnology). A total of 1×10⁵ MKN-45 cells were seeded in a 96-well plate. Three duplicated wells were set for each group, and the total volume of each well was 100 µl. The plate was then placed in a humidified incubator at 37°C with 5% CO₂, and the cells were cultured for 24, 48 and 72 h with different concentrations of TSN (0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 nmol/l). After the indicated incubation times, 10 µl of CCK-8 was added to the plates and incubated at 37°C for an additional 2 h. The absorbance at 450 nm was measured using a Spark 10 M multi-well plate reader (Tecan Group, Ltd.).

Total DNA was extracted from the MKN-45 cells using a Genomic DNA Extraction kit (centrifugal column) according to the manufacturer's instructions (BioTeke Corporation). Pre-miR-23a DNA fragment (5′-ggc cgg cug ggg uuc cug ggg aug gga uuu gcu ucc ugu cac aaA UCACAUUGCCAGGGAUUUCCa acc gac c-3′) was amplified by PCR from the total DNA using Rapid PCR amplification kit according to the manufacturer's instructions (BioTeke Corporation). The PCR thermocycling conditions were as follows: Initial denaturation at 94°C for 5 min; followed by 35 cycles of denaturation at 94°C for 30 sec, primer annealing at 61°C for 30 sec, extension at 72°C for 1 min, and a final extension at 72°C for 10 min. Then pre-miR-23a DNA fragment cloned into pcDNA3.1(+) plasmids (Promega Corporation) to construct the overexpression vector of miR-23a-3p, which was termed pcDNA3.1(+)-miR-23a-3p. The 3′-UTR of BCL2 was PCR-amplified from MKN-45 cell genomic DNA using the same PCR protocol as the pre-miR-23a-3p and cloned into psiCHECK-2 dual-luciferase reporter plasmids (Promega Corporation) immediately downstream of the stop codon of the luciferase gene to generate psiCHECK-2-BCL2-wt. The mutant BCL2 3′-UTR reporter, designated as psiCHECK-2-BCL2-mu, was created by mutating the seed region of the predicted miR-23a-3p site (TAT ATG T to TAC ACA A) using PCR and the T-BCL primers was that used to amplify the sequence contained the mutant seed region of the predicted miR-23a-3p site (18). The primers used in plasmid construction are shown in Table I.

Cells were cultured on glass cover slides and treated with either different concentrations of TSN (0, 60, 80 and 100 nmol/l) or transfected with pcDNA3.1(+) (2 µg/ml), pcDNA3.1(+)-miR-23a-3p (2 µg/ml), miR-23a-3p-I-NC (1.0×10⁻⁴ mmol/l) or miR-23a-3p-I (1.0×10⁻⁴ mmol/l) for 48 h. Subsequently, cells were rinsed twice with PBS and fixed with 4% paraformaldehyde in PBS for 10 min. Next, the cells were stained with 0.1 mg/ml acridine orange (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) for 1–2 min. The images were captured using a Leica TCS SP8 confocal laser-scanning microscope (Leica Microsystems GmbH) at 488 nm excitation and 515 nm emission wavelengths (magnification, ×63).

Cells were cultured to the logarithmic phase of growth in a cell culture flask and treated with different concentrations of TSN (0, 60, 80 and 100 nmol/l) or transfected with pcDNA3.1(+), pcDNA3.1(+)-miR-23a-3p (2 µg/ml), miR-23a-3p-I-NC or miR-23a-3p-I (1.0×10⁻⁴ mmol/l) for 48 h. The cells were then collected and centrifuged at 300 × g at 4°C for 5 min. The pellet was resuspended in PBS and centrifuged again using the same conditions. Next, the pellet was resuspended in ice-cold 70% ethanol and stored at 4°C for 18 h. Fixed cells were then washed with PBS and incubated in 0.5 ml staining solution [0.1 mg/ml RNase A and 0.05 mg/ml propidium iodide (PI)] at 37°C in the dark for 30 min. The cell cycles were determined on a Cytomics FC 500 flow cytometer and CXP software 2.3 (Beckman Coulter, Inc.).

Cells were cultured to the logarithmic phase in a cell culture flask and treated with different concentrations of TSN (0, 60, 80 and 100 nM) or transfected with pcDNA3.1(+), pcDNA3.1(+)-miR-23a-3p (2 µg/ml), miR-23a-3p-I-NC or miR-23a-3p-I (1.0×10⁻⁴ mmol/l) for 48 h. The cells were collected, washed twice with cold PBS and 500 µl binding buffer was added to each sample. The cells were then stained with 5 µl annexin V-FITC and 5 µl PI for 15 min. The cells were immediately analysed by FC 500 flow cytometer and CXP software 2.3 (Beckman Coulter, Inc.) to determine the rate of apoptosis.

MMP was measured by flow cytometry using JC-1 (Nanjing KGI Biological Technology Development Co., Ltd.,) staining. Briefly, transfected cells were incubated with 500 µl diluted JC-1 (10 µg/ml) reagent at 37°C in the dark for 20 min. After incubation, cells were washed with PBS and analysed within 30 min using FC 500 flow cytometer and CXP software 2.3 (Beckman Coulter, Inc.). If the mitochondrial membrane potential is high, JC-1 is gathered in the mitochondrial matrix and the formation of polymer (J-aggregates) produces red fluorescence; When the mitochondrial membrane potential is low, JC-1 cannot be concentrated in the mitochondrial matrix. Then JC-1 is a monomer and produces green fluorescence. Thus it is convenient to detect the change of mitochondrial membrane potential by the change of fluorescent color.

MKN-45 cells were seeded in 6-well plates (1.0×10⁵ cells/well) and treated with different concentrations of TSN (0, 60, 80 and 100 nmol/l) or transfected with pcDNA3.1(+), pcDNA3.1(+)-miR-23a-3p (2 µg/ml), miR-23a-3p-I-NC, or miR-23a-3p-I (1.0×10⁻⁴ mmol/l) for 48 h. Total RNA was extracted based on the TRIzol^® method (Invitrogen; Thermo Fisher Scientific, Inc.). The concentrations of total RNA were determined spectrophotometrically using a NanoDrop 2000C Spectrophotometer (Thermo Fisher Scientific, Inc.). A first-strand cDNA was synthesized with 1µg of total RNA from each sample using a TransScriptROne-Step gDNA Removal and cDNA Synthesis Super Mix (Beijing TransGen Biotech Co., Ltd) according to the manufacturer's instructions. 2X Plus SYBR Real-time PCR mixture (BioTeke Corporation) was used for qPCR. The thermocycling conditions were as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec, and 60°C for 30 sec. miRNAs are short and have no ploy (A) structure, so stem-loop RT-PCR was used to detect the miRNA expression (19). The expression of human βactin or U6 was used as an internal control for mRNA or miRNA expression levels, respectively, and the relative expression level was measured by the 2^−∆∆Cq method (20). Primers used are detailed in Table II.

MKN-45 cells were seeded in 6-well plates (1.0×10⁵ cells/well) and treated with different concentrations of TSN (0, 60, 80 and 100 nmol/l) or transfected with pcDNA3.1(+) (2 µg/ml), pcDNA3.1(+)-miR-23a-3p (2 µg/ml), miR-23a-3p-I-NC (1.0×10⁻⁴ mmol/l), or miR-23a-3p-I (1.0×10⁻⁴ mmol/l) for 48 h. Cells were harvested and lysed in RIPA buffer containing a protease inhibitor cocktail (Beyotime Institute of Biotechnology). The lysate was centrifuged at 12,000 × g for 10 min at 4°C. The supernatant was then collected, and the protein concentration was determined using the BCA Protein Assay Kit (Beyotime Institute of Biotechnology). The same protein amounts (10 µg in each lane) were loaded and separated by 10% SDS-PAGE, followed by transfer to polyvinylidene fluoride membranes (EMD Millipore) using the western blotting apparatus. The membranes were blocked with 5% (w/v) fat-free milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and then incubated with primary antibodies at 4°C overnight. Membranes were probed with the following primary antibodies (all 1:300): Anti-BCL2 polyclonal (cat. no. ab196495; Abcam), anti-Bax polyclonal (cat. no. ab53154; Abcam), anti-active caspase-3 monoclonal (cat. no. bsm-33284M; BIOSS), anti-caspase-8 polyclonal (cat. no. bs-0052R; BIOSS), anti-caspase-9 polyclonal (cat. no. bs-0049R; BIOSS), anti-cytochrome c polyclonal (cat. no. bs-0013R; BIOSS), anti-Fas polyclonal (cat. no. bs-6477R; BIOSS), anti-apoptotic protease activating factor 1 polyclonal (cat. no. ab2000; Abcam) and anti-β actin polyclonal (cat. no. ab8227; Abcam). The membranes were subsequently incubated with IRDye 800CW^® goat anti-rabbit (926–32211) and IRDye 800CW^® goat anti-mouse (32210) secondary antibody (1:15,000; LI-COR Biosciences) for 90 min at 20–25°C and washed with PBS. The bands were visualized and quantified using the Li-COR Odyssey infrared imaging system (Li-COR Biosciences); each band was normalised to its respective β-actin band.

To explore the mechanisms of miR-23a-3p-induced apoptosis, the target genes of mir-23a-3p were predicted by TargetScan websites (TargetScan: https://www.targetscan.org, version 7.1) (21) and examined by luciferase reporter assay. 293T cells (2×10⁴ cells/well) were plated in a 24-well plate (Corning, Inc.) 24 h before transfection. Cells were co-transfected with 0.5 µg of either the psiCHECK-2-BCL2-wt, psiCHECK-2-BCL2-mu or empty vector psiCHECK-2, and either pcDNA3.1(+) (2 µg/ml), pcDNA3.1(+)-miR-23a-3p (2 µg/ml), miR-23a-3p-I-NC (1×10⁻⁴ mmol/l) or miR-23a-3p-I (1×10⁻⁴ mmol/l) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 48 h of transfection, cells were lysed in Passive Lysis Buffer (Promega Corporation), and the activities of the firefly and Renilla luciferase were measured with a GloMax 20/20 Luminometer (Promega Corporation) using the Dual-Luciferase Reporter Assay System according to the manufacturer's protocols. Transfections were performed in duplicate and were repeated three times.

All the results were expressed as mean ± SEM. All statistical analyses and graphing were performed using SPSS 17.0 software (SPSS, Inc.) and GraphPad Prism 5.0 software (GraphPad Software, Inc.), respectively. Statistical differences were determined using one-way ANOVA followed by a Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

CCK-8 and cell cycle assays were carried out to evaluate the effect of TSN on MKN-45 cell proliferation. The results showed that inhibitory rates on MKN-45 cells treated with TSN significantly increased as the rise of TSN concentration and prolongation of time, (Fig. 1A), the IC₅₀ value was 81.06 nmol/l for 48 h, and the cell cycle was arrested at the G1 phase with increasing concentrations of TSN compared with the negative control (Fig. 1B and C). Compared with those in the negative control, the TSN-treated cells showed the cell nucleus cleavage and chromatin morphological changes. Acridine orange staining revealed a higher number of apoptotic bodies following the addition of TSN (Fig. 2A), and flow cytometry demonstrated that the early apoptotic rates gradually increased (Fig. 2B and C). Moreover, compared with the negative control, the mRNA and protein expression levels of Bax, cytochrome c, Fas, caspase-3, caspase-8, caspase-9 and APAF-1 were significantly upregulated, reaching the maximum at 80 nmol/l concentration of TSN, and decreasing following the addition of 100 nmol/l (Fig. 2D-F). However, the expression of BCL2 was significantly downregulated at 80 nmol/l TSN, and increased following the addition of 100 nmol/l, which suggested that TSN may also activate other mechanisms to inhibit the proliferation of gastric cancer cells at 100 nmol/l. The effect in the 80 nmol/l TSN treatment group was more evident compared with the 5-FU treatment group.

BCL2 is an integral mitochondrial membrane protein and acts as a switch to control the process of mitochondrial pathway apoptosis (22,23). As shown in Fig. 2D-F, the expression of BCL2 was significantly downregulated in MKN-45 cells following treatment with TSN. To explore the apoptotic mechanism employed by TSN, miRNAs targeting BCL2 were predicted (Fig. 3A), and the expression of these miRNAs was evaluated using RT-qPCR in MKN-45 cells following treatment with different concentrations of TSN for 48 h. After TSN treatment, the expression levels of miR-23a-3p, miR-34a-5p and miR-365a-3p were markedly increased compared with the negative control; however, a similar change was not observed in miR-148a-5p expression (Fig. 3B). It was found that the expression of miR-23a-3p increased significantly upon increasing TSN concentration and reached a maximum when the TSN concentration was increased to 80 nmol/l. Since the expression level of miR-23a-3p was the more significant, the study focused on miR-23a-3p for the subsequent experiments.

To validate whether BCL2 was the direct target genes of miR-23a-3p, a dual-luciferase reporter system was employed. 3′UTR sequences containing the predicted target site of miR-23a-3p or mutated sequences were cloned into the psiCHECK-2, respectively. Co-transfection of pcDNA3.1 (+)-miR-23a-3p with psiCHECK-2-BCL2-wt in 293T cells showed that luciferase activity was significantly lower than those of controls (P<0.001) (Fig. 3C). Co-transfection pcDNA3.1 (+)-miR-23a-3p with psiCHECK-2- BCL2-mu did not change the luciferase activities (P>0.05) (Fig. 3C). Compared with miR-23a-3p-I-NC, miR-23a-3p-I abolished the targeting 3′UTR of BCL2 (Fig. 3D). This finding suggested that miR-23a-3p may directly target the 3′-UTR of the BCL2 mRNA. Based on this evidence, whether miR-23a-3p regulated the expression of BCL2 in MKN-45 cells was examined. Endogenous mRNA and protein expression levels of BCL2 were markedly downregulated following overexpression of miR-23a-3p and upregulated following the silencing of miR-23a-3p with the inhibitor (Fig. 3E-H).

To identify the effects of miR-23a-3p on apoptosis in MKN-45 cells, a series of cell apoptosis experiments were performed. Compared with those in the pcDNA3.1(+) group, cells transfected with pcDNA3.1(+)-miR-23a-3p exhibited more pronounced nuclear morphology changes, which included the appearance of concentrated, agglomerated and marginalised chromatin as well as a small number of nuclei becoming ruptured (Fig. 4A). By contrast, compared with cells in the miR-23a-3p-I-NC group, the nuclei transfected with miR-23a-3p-I were intact. Subsequently, annexin V-FITC/PI double staining assay was performed to detect apoptosis in transfected MKN-45 cells (Fig. 4B and C). The results indicated that the early apoptotic rate of cells transfected with pcDNA3.1(+)-miR-23a-3p was significantly higher compared with that of cells transfected with pcDNA3.1(+) for 48 h. By contrast, the rate of apoptosis of cells transfected with miR-23a-3p-I was lower compared with that of cells transfected with miR-23a-3p-I-NC.

The changes in MMP were determined by flow cytometry (Fig. 4D and E). Following transfection with pcDNA3.1(+)-miR-23a-3p, MMP was decreased in MKN-45 cells compared with its level in cells transfected with pcDNA3.1(+). However, the MMP was increased in cells transfected with miR-23a-3p-I compared with the NC cells transfected with miR-23a-3p-I-NC. These findings indicated that miR-23a-3p may induce apoptosis in MKN-45 cells.

To identify the effects of miR-23a-3p on cell cycle distribution, a cell cycle study was conducted in MKN-45 cells by PI single staining (Fig. 5A and B). Compared with pcDNA3.1(+), the number of cells in the G₁/G₀ phase of MKN-45 cells increased significantly after transfection with pcDNA3.1(+)-miR-23a-3p, and the number of cells in the S phase decreased significantly, resulting in inhibition of cell proliferation in the G₁ phase after transfection. Conversely, compared with that in the miR-23a-3p-I-NC group, the number of cells in the G₁/G₀ phase of MKN-45 cells decreased, whereas the number in the S phase increased after transfection with miR-23a-3p-I. These results indicated that the cycle became arrested in the G₁ phase following overexpression of miR-23a-3p in MKN-45 cells.