The human gastric adenocarcinoma cell lines SGC7901/DDP and SGC7901 were obtained from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). The cisplatin-resistant SGC7901/DDP cell line was developed from the parental SGC7901 line by exposure to increasing concentrations of cisplatin over ~12 months. The cells acquired resistance at 1 µg/ml cisplatin. We have also previously confirmed that SGC7901/DDP cells are highly resistant to cisplatin using cell viability assays (13). All cells were maintained in RPMI-1640 containing 10% fetal calf serum, 100 U/ml penicillin, and 100 U/ml streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified atmosphere with 5% CO₂ at 37°C. Cells were cultured in the absence (SGC7901) or presence (SGC7901/DDP) of 800 ng/ml cisplatin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany).

Total RNA was extracted from the gastric cancer cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. RNA concentrations were measured using a NanoDrop-2000 spectrophotometer with the absorbance set at 260 nm. Aliquots of total RNA were used for the microarray and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses.

miR-138-5p-depleted or -overexpressing cell lines were constructed using lentiviral-mediated short hairpin (sh)RNA targeting or overexpression of miR-138-5p. Vectors encoding miR-138-5p (pLV-miR-138-5p) or a negative control sequence (pLV-miR-138-5p-NC) and the corresponding viruses (1×10⁸ plaque-forming units) were all purchased from GeneCopoeia, Inc. (Rockville, MD, USA). Lentiviral infection was performed according to the manufacturer's protocol. Briefly, SGC7901/DDP cells were cultured overnight in 12-well plates at a density of 1×10⁵ cells/well, and then infected with the appropriate lentivirus at a multiplicity of infection of 10 plaque-forming units/cell. A total of 72 h after the transduction, SGC7901/DDP cells were transferred to RMPI-1640 containing 2 µg/ml puromycin and cultured for 3 days. Cells that survived were selected and used for subsequent experiments.

A similar approach was used to knock down miR-138-5p in SGC7901 cells, except that the cells were infected with lentiviral vectors encoding a control sequence or short hairpin (sh)RNA specific for miR-138-5p and then cultured for 5 days in RPMI-1640 containing 150 µg/ml hygromycin. Cells that survived were selected and used for subsequent experiments.

The lentiviral vectors co-expressed green fluorescent protein (GFP) for overexpression or red fluorescent protein (RFP) for silencing to allow verification of transduction efficacy by fluorescence microscopy.

RT-qPCR was performed on an Mx3000P qPCR system with a SYBR Green single-step kit RT-qPCR kit (Biomics Biopharma, Nantong, China) according to the manufacturer's protocol. Aliquots of cDNA were amplified under the following conditions: 42°C for 30 min followed by 40 cycles of 20 sec at 95°C, 30 sec at 60°C and 30 sec at 72°C; and an extension step for 2 min at 72°C to obtain the fluorescent signals. All miRNA levels were normalized to U6 expression. Primers used in this study were: miR-138-5p forward, 5′-AGCTGGTGTTGTGAATCAGGCCG-3′ and reverse, 5′-TGGTGTCGTGGAGTCG-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′. The relative differences were quantified using the 2^−ΔΔCq method, where ΔΔCq is calculated as the: Experimental (Ct_miR-138-5p-Ct_u6)-control (Ct_miR-138-5p-Ct_u6). The procedure was performed in triplicate (14).

Total RNA was extracted from triplicate biological samples of SGC7901/DDP and SGC7901 cells as aforementioned and sent to Beijing CapitalBio Technology Co., Ltd. (Beijing, China) for microarray analysis.

Expression profiling was performed using an Affymetrix miRNA 4.0 Array, which contains 775 mature human miRNA probe sets. The output data, which contained normalized miRNA expression profiles, were analyzed in Excel spreadsheets. The two groups of data were analyzed by t-tests to obtain P-values, and false discovery rate adjustment was performed to generate corrected P-values. The fold change in differentially expressed miRNAs between SGC7901/DDP and SGC7901 cells was calculated, and Cluster 3.0 software (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm) was used to display the differential expression patterns. Genes potentially targeted by the differentially expressed miRNAs were identified using the online tools DIANAmT http://diana.imis.athena-innovation.gr/), miRanda (http://www.microrna.org/microrna/home.do), miRDB (http://www.mirdb.org/), miRWalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/index.html), RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/), PICTAR4 (http://pictar.mdc-berlin.de), PICTAR5 (http://pictar.mdc-berlin.de), PITA (http://genie.weizmann.ac.il/pubs/mir07/mir07_data.html), RNA22 (https://cm.jefferson.edu/rna22/) and TargetScan (http://www.targetscan.org/).

Total proteins were extracted from cultured SCG7901 cells and SGC7901/DDP cells with or without lentivirus infection by cell lysis in radioimmunoprecipitation assay buffer containing protease inhibitors (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentrations were quantitatively determined using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). Lysate samples containing 60 µg total protein were electrophoresed on a 10 or 8% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Beyotime Institute of Biotechnology). The PVDF membrane was blocked with 5% non-fat milk for 1.5 h at room temperature and probed overnight at 4°C with the following primary antibodies: Rabbit anti-ERCC1 (1:200 dilution; cat. no. ab129267); rabbit anti-ERCC4 (XPF) (1:300 dilution; cat. no. ab76948); rabbit anti-β-actin (1:1,000 dilution; cat. no. ab8227); and rabbit anti-GAPDH (1:1,000 dilution; cat. no. ab9485) (all from Abcam, Cambridge, UK). Following washing with Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution; cat. no. 7074S; Shanghai Youningwei Biotechnology Co. Ltd., Shanghai, China) for 4 h at 4°C. Protein bands were visualized with an ECL Plus chemiluminescent kit (Thermo Scientific Fisher, Inc.). Protein levels were quantified by densitometry (Tanon2500 Fully Automatic Digital Gel Image Analysis system; Tanon Science and Technology Co., Ltd., Shanghai, China) and are presented as the fold change in expression after normalization to GAPDH and β-actin.

Cisplatin sensitivity was determined using the colorimetric MTT assay. SGC7901/DDP and SGC7901 cells, their miR-138-5p-overexpressing or -silenced counterparts, and control cells were resuspended at 1×10⁵ cells/ml. Aliquots of 100 µl were distributed into 96-well plates, and the volumes were made up to 200 µl/well with RPMI-1640. The cells were incubated for 24 h, and 180 µl of supernatant/well was then removed and replaced with freshly prepared DDP at final concentrations of 40, 20, 2, 0.2 and 0.02 µg/ml for DDP-resistant groups; and at final concentrations of 4, 2, 0.2, 0.02 and 0.002 µg/ml for DDP-sensitive groups. These concentrations were selected because the peak concentration of DDP in human plasma is 2.0 µg/ml (15). A total of 48 h later, 180 µl/well of supernatant was removed and replaced with 180 µl fresh medium plus 20 µl of MTT/well (5 mg/ml, Sigma-Aldrich; Merck KgaA). The plate was incubated for 4 h in a humidified atmosphere at 37°C, the medium was removed, and 150 µl/well DMSO (Sigma-Aldrich; Merck KGaA) was added. The plates were mixed for 10 min at 72°C to dissolve the formazan crystals and the absorbance at 490 nm was then measured using a spectrophotometer. The 50% inhibitory concentration (IC₅₀) of DDP was calculated based on the relative viability of control wells lacking DDP. The assay was performed in triplicate.

Statistical analysis was performed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). All data are presented as the mean ± standard deviation. A two-tailed Student's t-test was used to compare differences between two groups. One-way analysis of variance and the Bonferroni post hoc test was used to compare differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

To identify miRNAs potentially involved in cisplatin resistance, miRNA microarray analysis was performed on triplicate samples of SGC7901 and SGC7901/DDP cells. As shown in Fig. 1 and Table I, 41 miRNAs were significantly differentially expressed between SGC7901 and SGC7901/DDP cells, using a threshold of ≥2-fold or ≤0.5-fold change and P<0.05. For further analysis, miR-138-5p was selected, which exhibited the largest difference in expression between the cisplatin-sensitive and -resistant cell lines. This miRNA was of particular interest as it has previously been reported to modulate drug resistance in lung cancer by acting on the NER pathway (9,16). The expression of miR-138-5p was ~3-fold lower in SGC7901/DDP cells compared with SGC7901 cells (P<0.05; Fig. 1).

To confirm these findings, the expression of miR-138-5p was examined by RT-qPCR. The analysis demonstrated that miR-138-5p expression in SGC7901/DDP cells was ~5-fold lower compared with that in SGC7901 cells (P<0.0001; Fig. 2). Thus, the PCR results were consistent with the microarray analysis, confirming that miR-138-5p was downregulated in cisplatin-resistant SGC7901/DDP cells compared with the cisplatin-susceptible parental cells.

To investigate potential target genes of miR-138-5p, the predictive algorithms miRanda, miRWalk and PicTar, which screen for complementary binding sequences between miRNAs and known gene transcripts were used. These analysis identified the excision repair proteins ERCC1 and ERCC4 as possible target genes of miR-138-5p (Fig. 3).

Western blot analysis was performed to determine the expression levels of ERCC1 and ERCC4 in SGC7901/DDP and SGC7901 cells. As shown in Fig. 4, ERCC1 and ERCC4 proteins were expressed at significantly higher levels in SGC7901/DDP cells compared with that in SGC7901 cells (P<0.01).

To suppress miR-138-5p expression, SGC7901 cells were infected with lentiviruses encoding RFP and a negative control sequence or an shRNA targeting miR-138-5p, named SGC7901-LV-NC-knockdown (KD) and SGC7901-LV-miR138-5p-KD cells, respectively. Similarly, the effects of miR-138-5p overexpression were investigated by SGC7901/DDP cell infection with lentiviruses encoding GFP and a control sequence or miR-138-5p, named SGC7901/DDP-LV-NC-overexpression (OE) and SGC7901/DDP-LV-miR-138-5p-OE cells, respectively. Fluorescence microscopy of the infected cells revealed successful transduction, as demonstrated by abundant RFP and GFP expression in the corresponding SGC7901 and SGC7901/DDP cells (Fig. 5).

RT-qPCR was used to verify the miR-138-5p expression in SGC7901 and SGC7901/DDP cells. As shown in Fig. 6A, the levels of miR-138-5p in uninfected SGC7901 cells and control SGC7901-LV-NC-KD cells were similar. However, miR-138-5p was significantly reduced (~5-fold lower) in the SGC7901-LV-miR-138-5p-KD cells compared with both control cell groups (both P<0.0001; Fig. 6A). Similarly, miR-138-5p was significantly upregulated (~4-fold increase) in SGC7901/DDP-LV-miR-138-5p-OE cells compared with either SGC7901/DDP and SGC7901/DDP-LV-NC-OE cells (both P<0.0001; Fig. 6B). Thus, miR-138-5p was successfully knocked down or overexpressed in the transduced gastric cancer cells.

As ERCC1 and ERCC4 were identified as potential target genes of miR-138-5p, their expression in SGC7901 or SGC7901/DDP cells was evaluated by western blot analysis. Notably, downregulation of miR-138-5p significantly increased the expression of ERCC1 and ERCC4 compared with uninfected SGC7901 cells or SGC7901-LV-NC-KD cells (P<0.01; Fig. 7).

Conversely, the expression of ERCC1 and ERCC4 was significantly diminished in SGC7901/DDP-LV-miR138-5p-OE cells compared with control SGC7901/DDP and SGC7901/DDP-LV-NC-OE cells (P<0.01; Fig. 8).

To evaluate the effects of miR-138-5p downregulation or upregulation on the sensitivity of SGC7901/DDP or SGC7901 cells to cisplatin, cells were treated with varying concentrations (40, 20, 2, 0.2 and 0.02 µg/ml in SGC7901/DDP, SGC7901/DDP-LV-miR138-5p-OE and SGC7901/DDP-LV-NC-OE groups; and with varying concentrations of 4, 2, 0.2, 0.02 and 0.002 µg/ml in SGC7901, SGC7901-LV-NC-KD and SGC7901-LV-miR138-5p-KD groups) of cisplatin for 48 h, and then cell viability was determined using an MTT assay. Drug resistance was evaluated by calculating the IC₅₀ of DDP relative to untreated cells. It was demonstrated that miR138-5p-overexpressing SGC7901/DDP cells were significantly more sensitive to cisplatin compared with SGC7901/DDP or SGC7901/DDP-LV-NC-OE cells, as evidenced by the IC50 values of 3.14±0.32, 6.81±0.12, and 6.89±0.14 µg/ml, respectively (P<0.01; Fig. 9A). Conversely, the cisplatin resistance of SGC7901-LV-miR138-5p-KD cells was significantly increased following depletion of miR-138-5p compared with SGC7901 and SGC7901-LV-NC-KD cells (IC50 values 0.39±0.04, 0.20±0.01, and 0.25±0.05 µg/ml, P<0.01; Fig. 9B). Collectively, these data demonstrated that low miR-138-5p levels and high ERCC1 and ERCC4 levels were associated with cisplatin resistance in gastric cancer cells.