The GC cell lines, MKN-28, MGC-803 and SGC-7901 were purchased from the Laboratory Animal Center of Sun Yat-sen University Cell Bank (Guangzhou, China). MKN-28 is derived from MKN-74 (http://cellbank.nibiohn.go.jp/~cellbank/en/search_res_det.cgi?ID=340). Cells were grown in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; cat. no. 04-001-1A; Biological Industries, Beit-Haemek, Israel) at 37°C in a humidified atmosphere containing 5% CO₂. The primary antibodies used in western blot analysis were as follows: Mouse anti-ID1 (1:1,000; cat. no. ab168256; Abcam, Cambridge, UK); mouse anti-Nanog (1:500; cat. no. sc-376915; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); mouse anti-octamer-binding protein 4 (1:1,000; cat. no. 611203; BD Biosciences, San Jose, CA, USA); rabbit anti-cyclin D1 (1:1,000; cat. no. 2261-1; Epitomics, Burlingame, CA, USA); mouse anti-transcription factor SOX2 (Sox2; 1:500; cat. no. 561469; BD Biosciences); mouse anti-β-actin (1:1,000; cat. no. sc-47778; Santa Cruz Biotechnology, Inc., CA, USA); and mouse anti-GAPDH (1:3,000; cat. no. 60004-1-lg; Proteintech Group, Inc., Chicago, IL, USA). Mouse (cat. no. SA00001-1) or rabbit (cat. no. SA00001-2) immunoglobulin G horseradish peroxidase-conjugated secondary antibodies (both 1:10,000; Proteintech Group, Inc., Chicago, IL, USA) were obtained from the Proteintech Group, Inc. (both 1:3,000). DDP was purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany; cat. no. p4394).

ID1 knockdown was achieved by RNA interference using siRNA in the GC cell line MGC-803. The full-length ID1 mRNA sequence was retrieved from GenBank (NM_002165.3; www.ncbi.nlm.nih.gov/nuccore/NM_002165.3). The siRNA sequences were designed by Takara Biotechnology Co., Ltd., (Dalian, China) and synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). The sequences of the ID1 and negative control (NC) siRNAs are shown in Table I. A total of 1 day prior to transfection, cells were seeded into a 6-well plate at a confluency of between 50 and 60%. Cells in the logarithmic phase of growth were transfected with 100 nM siRNA-ID1 or siRNA-NC in Opti-MEM™I Reduced Serum media using Lipofectamine™2000 (both Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

The efficacy of siRNA transfection was evaluated using RT-PCR. A total of 48 h following transfection, total cellular RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. First-strand cDNA was subsequently synthesized by denaturing 1 µg RNA at 65°C for 10 min with 0.1 µg oligo-dT (Takara Biotechnology Co., Ltd.) and the denatured product was immediately incubated in an ice bath for 5 min. The denatured product was made up to a total volume of 25 µl with the addition of 1 µl dNTP (10 mM/base; cat. no. U1205), 100 units RNasin^® Ribonuclease inhibitor (cat. no. N2111), 10 units Moloney murine leukemia virus reverse transcriptase (cat. no. M1701) (all Promega Corporation, Madison, WI, USA) and 0.01% diethylpyrocarbonate (DEPC; cat. no. 472565; Sigma-Aldrich; Merck Millipore) in H₂O. The mix was incubated at 42°C for 60 min, 95°C for 5 min and then on ice for 3 min prior to being stored at −20°C until the cDNA template was required for PCR. Primers targeting ID1 and β-actin were designed using Premier Primer software (version 5.0; Premier Biosoft International, Palo Alto, CA, USA) and synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). Primer sequences are shown in Table II. PCR was performed in a total reaction volume of 10 µl, containing 5 µl 2X Taq PCR Master Mix (cat. no. K0171; Thermo Fisher Scientific Inc.), 0.25 µl forward primer (10 µM), 0.25 µl reverse primer (10 µM), 0.5 µl of template cDNA (200 ng/µl) and DEPC-treated water (cat. no. R0021; Beyotime Institute of Biotechnology, Haimen, China). PCR thermocycling conditions were as follows: 94°C for 5 min; 30 cycles of 94°C for 30 sec, 62°C for 30 sec and 72°C for 1 min; and 72°C for 7 min. Agarose gel electrophoresis was performed on the final PCR products using a 2% agarose gel containing 0.5 µg/µl ethidium bromide, and images were captured using the Tanon-4100 Gel Imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China). The expression of ID1 mRNA normalized to β-actin was determined using Image-Pro^® Plus software (version 5.1; Rockville, MD, USA).

The Cell Counting Kit-8 (CCK-8; cat. no. BB-4202; BestBio Co., Shanghai, China) assay was used to evaluate the viability of MKN-28 and MGC-803 cells. Between 2,000 and 3,000 GC cells/well were seeded into a 96-well plate and subsequently transfected with siRNA-ID1 or siRNA-NC for 24, 48 and 72 h, as described above. A total of 10 µl CCK-8 reagent was added to each well and plates were incubated for 1 h at 37°C prior to measuring the absorbance at 450 nm using the ELx800™ Absorbance Reader (BioTek Instruments, Inc., Winooski, VT, USA). The Cell-Light™ EdU Apollo^®567 In Vitro Imaging kit (cat. no. C10310; RiboBio Co., Ltd., Guangzhou, China) was used to label cells in the S phase based on EdU labeling as previously described (25). According to the manufacturer's protocol, siRNA-transfected cells were incubated with EdU solution for 3 h at 37°C. Cells were subsequently washed with PBS, fixed with 4% paraformaldehyde for 30 min, and permeated using 0.5% Triton™ X-100. Apollo567 from the Imaging kit and DAPI (Sigma-Aldrich; Merck Millipore) were used for EdU and nuclear staining, respectively. Images were captured using a fluorescence microscope (Eclipse Ti-U inverted microscope; Nikon Corporation, Tokyo, Japan). EdU-positive cells were counted using Image Pro Plus software (version 6.0; Media Cybernetics, Rockville, MD, USA).

Following transfection with si-ID1 or si-NC, MKN-28 and MGC-803 cells (2×10⁶-5×10⁶) were harvested using trypsin and resuspended in 300 µl PBS. The cell suspension was subsequently incubated in 700 µl ice-cold absolute ethanol overnight at 4°C. Cells were pelleted through centrifugation at 13,400 × g at 4°C for 5 min, and then washed with PBS, prior to resuspension in PBS containing 100 µg/ml RNase inhibitor and 25 µg/ml propidium iodide (PI). The mixture was incubated in an ice bath for 30 min prior to flow cytometric analysis of cell cycle distribution using the BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The fractions of cells in G₀/G₁, S, and G₂/M phases were analyzed using FlowJo software (version 7.6.2; Tree Star, Inc., Ashland, OR, USA).

The apoptotic rates of MKN-28 and MGC-803 cells were analyzed using the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), according to the manufacturers' protocol. Briefly, between 2×10⁶ and 5×10⁶ transfected cells were harvested using trypsin, and resuspended in 500 µl binding buffer containing 5 µl Annexin V-FITC from the Apoptosis Detection kit, and 5 µl PI. The mix was incubated for 15 min at 4°C prior to flow cytometric analysis using the BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

A total of 48 h following siRNA transfection, MKN-28 and MGC-803 cells were harvested and lysed in 1X radioimmunoprecipitation assay buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) containing phenylmethylsulfonyl fluoride (cat. no. ST506; Beyotime Institute of Biotechnology) and a phosphatase inhibitor cocktail (cat. no. CW2383; CW Biotech, Beijing, China). Proteins (100 ng/lane) were separated on a 10% (for protein with a mass of 40–170 kDa) or 12% (for protein with a mass of 15–70 kDa) gel through SDS-PAGE. Proteins were subsequently transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, US) and blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology). The membrane was subsequently incubated overnight at 4°C with the following primary antibodies: Anti-ID1; anti-Nanog; anti-Sox2; anti-Oct-4; anti-cyclin D1; and anti-GAPDH. The membrane was washed 4 times by TBS-Tween 20 buffer (6 min/wash), followed by treatment with secondary antibodies for 1 h at room temperature. Protein bands were visualized using the Enhanced Chemiluminescence Western Blot kit (cat. no. P90720; EMD Millipore). Relative protein expression analysis using Image Lab software (version 3.0.1 beta 1; Bio-Rad Laboratories, California, USA) was normalized to GAPDH or β-actin.

MKN-28 and MGC-803 cells were seeded into a 6-well plate at a density of 500 cells/well and transfected with siRNA-ID1 or siRNA-NC the following day, as described above. The cells were subsequently cultured in RPMI-1640 medium containing 10% FBS and re-transfected every 4 days for 2 weeks. In addition, certain cell groups were treated with 1 µg/ml DDP. Cell colonies were subsequently fixed with methanol and stained with crystal violet (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Visible colonies of >50 cells were counted by eye for each sample and colony formation rates were subsequently calculated as follows: Number of colonies/the number of cells seeded. Colony formation assays were performed in triplicate.

A total of 10 h following transfection, GC cells were trypsinized, washed with PBS and seeded into a 6-well ultra-low attachment plate (Corning Life Sciences, Acton, MA, USA) at a density of 1×10⁵ cells/well. Cells were cultured in serum-free Dulbecco's modified Eagle's medium: Nutrient mixture F-12 (Thermo Fisher Scientific, Inc.) supplemented with EGF, basic fibroblast growth factor and B27 (all 20 ng/ml; all Gibco; Thermo Fisher Scientific, Inc.). To study the effect of ID1 knockdown on tumor spheroid formation, transfections were repeated on the fourth day. Following 8 days of incubation at 37°C, images were captured using a fluorescence microscope (Eclipse Ti-U inverted microscope). Tumor spheroids with a diameter of >20 µm were counted using ImageJ software (version 1.37; National Institutes of Health, Bethesda, MD, USA). Data are presented as the number of spheroids in 5 randomly selected fields and are the result of triplicate experiments (26).

Cell migration assays were performed using a 24-well Transwell^® Chamber (Corning Life Sciences), according to the manufacturer's protocol. MKN-28 and MGC-803 cells were harvested 24 h following siRNA transfection and resuspended in serum-free RPMI-1640 medium. Cells were seeded into the upper chamber at a density of 200 cells/wells. RPMI-1640 medium containing 10% FBS was added into the lower chambers and cells were incubated at 37°C for 12 (MKN-28) and 20 (MGC-803) h. Migratory cells were immobilized in methanol for 10 min and stained with Giemsa (Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's protocol. Photomicrographs of 5 randomly selected fields were captured using a Nikon Eclipse Ti-U inverted microscope.

Data analysis was conducted using SPSS Statistics software (version 20.0; IBM SPSS, Armonk, NY, USA). All values are presented as the mean ± standard error of the mean. A two-tailed Student's t-test was used to analyze differences between treatment groups in the cell proliferation, colony formation, cell migration, tumor spheroid formation and EdU assays. One-way analysis of variance was performed to compare differences between multiple groups for cell viability and apoptosis analysis. P<0.05 was considered to indicate a statistically significant difference. All experiments were repeated between 3 and 5 times.

To study the effect of endogenous ID1 depletion in GC cells, four ID1-specific siRNAs, ID1-201, ID1-252, ID1-266 and ID1-316, were synthesized and transiently transfected into MGC-803 cells. The effect of the four siRNAs on ID1 mRNA and protein levels was determined in order to identify the most efficient siRNA. RT-PCR analysis was performed 48 h following transfection, as illustrated in Fig. 1. ID1 mRNA expression in si-ID1-transfected and si-NC-transfected cells was determined following normalization to β-actin. The ratio of ID1 to β-actin in the siRNA-NC-transfected group was arbitrarily set to 1.00. Relative ID1 mRNA expression was decreased by 0.65, 0.86 and 0.95 times, following transfection with ID1-201, ID1-266 and ID1-316, respectively (Fig. 1C). By contrast, transfection with ID1-252 resulted in an increased relative ID1 mRNA expression of 1.73 times (Fig. 1C). These results indicate that ID-201 is the most effective siRNA, resulting in a significant decrease in ID1 mRNA expression (P=0.008 vs. siRNA-NC; Fig. 1C). ID1 protein expression was detected using western blotting 72 h following siRNA transfection, thus allowing sufficient time for transcription. Transfection with ID1-201 and ID1-266 decreased ID1 protein expression, while transfection with ID1-252 and ID1-316 increased ID1 protein expression (Fig. 1D). Transfection with ID1-201 significantly decreased ID1 protein expression (47%; P=0.027 vs. siRNA-NC; Fig. 1D). Transfection with ID1-201 produced a 1.53- and 1.88-fold reduction in ID1 mRNA and protein expression, respectively, and was therefore used for further investigation.

ID1 has been reported to increase cell proliferation in several types of cancer, including glial, liver, colorectal and gastric (27–29); therefore, the effect of ID1 knockdown on proliferation and cell cycle distribution was investigated. The MGC-803 (poorly-differentiated) and MKN-28 (well-differentiated) gastric adenocarcinoma cell lines were used to evaluate the effect of ID1 knockdown on the proliferation of GC cells. From 2 days following transfection, MGC-803 and MKN-28 cells transfected with the siRNA-NC demonstrated significantly increased proliferation compared with the siRNA-ID1-transfected cells (MKN-28, P=0.009; MGC-803, P=0.003; Fig. 2A). EdU assays were used to analyze the proliferative ability of GC cells, as shown in Fig. 2B. A total of 48 h following transfection, siRNA-ID1-transfected MKN-28 cells exhibited a significant reduction in S phase EdU-labeling (21 to 13%) compared with the siRNA-NC-transfected cells (P=0.028). In addition, siRNA-ID1-transfected MGC-803 cells exhibited a significant reduction in S phase EdU-labeling (37 to 19%) compared with the siRNA-NC-transfected cells (P<0.001). Consistent with published data (29), these results indicate that siRNA-mediated ID1 knockdown inhibits GC cell growth.

Cell cycle dysfunction serves an essential role in the development of GC, therefore the effect of ID1 knockdown on cell cycle distribution in the MKN-28, MGC-803 and SGC-7901 cell lines was evaluated. As shown in Fig. 2C and D, the proportion of cells in the G₀/G₁ phases was significantly increased in the siRNA-ID1-transfected cells compared with the siRNA-NC-transfected cells, for all three cell lines (P=0.017). By contrast, the mean proportion of all three cell types in the S phase was significantly decreased in the siRNA-D1-transfected cells compared with the siRNA-NC-transfected cells (P=0.018; Fig. 2D). These results demonstrate that ID1 knockdown in GC cells results in G₁ cell cycle arrest. Cyclin D1 is known to be the primary cyclin that couples to cyclin-dependent kinases 4/6 and drives G₁ to S phase cell cycle progression (30), therefore cyclin D1 protein expression was evaluated using western blotting. A decrease in cyclin D1 protein expression was observed in the ID1 knockdown cells (Fig. 2E). These results indicate that ID1 knockdown attenuates aberrant cell proliferation and leads to G₁ cell cycle arrest, suggesting a role in GC cell growth and cell cycle progression.

CSCs form a rare cell population within cancer tissue, and exhibit self-renewal, differentiation potential and tumorigenic capacity (31). Nanog, Oct-4, Sox2 and Krueppel-like factor 4 (KLF4) are key factors in stem cell reprogramming, and their interactions are involved in the maintenance of stem cell pluripotency and self-renewal. As ID1 is a known inhibitor of differentiation, the effect of ID1 knockdown on the stem cell like-properties of GC was examined. Colony formation is a distinct ability of CSCs in malignant tumors; therefore colony formation was analyzed in MGC-803 and MKN-28 cell lines following siRNA transfection. As shown in Fig. 3A, colony formation rates following ID1 knockdown were significantly reduced in MKN-28 (28 to 11%) and MGC-803 (45 to 23%) cells compared with the control cells (MKN-28, P=0.005; MGC-803, P=0.001). These results demonstrate that ID1 knockdown leads to a significant reduction in the ability of GC cells to form colonies.

Tumor spheroid formation assays were used to evaluate the self-renewal ability of CSCs in vitro. MGC-803 and MKN-28 cells were transfected with siRNA-ID1 or siRNA-NC on days 1 and 4, respectively. Following an 8-day incubation, the total number of tumor spheroids with a diameter of >20 µm were counted. As shown in Fig. 3B, ID1 knockdown in MKN-28 and MGC-803 cells significantly decreased the number of tumor spheroids (2.70- and 3.33-fold decrease, respectively) compared with the siRNA-NC-transfected cells (MKN-28, P=0.021; MGC-803, P=0.037).

CSCs exhibit EMT, which enables metastasis and invasion (32). The results of the cell migration assays performed on siRNA-ID1- and siRNA-NC-transfected cells are shown in Fig. 3C. Significantly decreased migration was observed in the ID1 knockdown MKN-28 (P=0.002) and MGC-803 (P=0.015) cells compared with the siRNA-NC-transfected cells. N-cadherin protein expression is a key marker of EMT (33). Western blotting demonstrated decreased N-cadherin protein expression in ID1 knockdown cells compared with siRNA-NC-transfected cells (Fig. 3D; left panel), quantification of the mean gray value from MKN-28 and MGC-803 demonstrated the same results (Fig. 3D; right panel). These results indicated that ID1 knockdown inhibits the migratory ability of GC cells.

The effect of ID1 knockdown on CSC-associated proteins in MGC-803 and MKN-28 cells was analyzed using western blotting 72 h following siRNA transfection. As shown in Fig. 3D, Nanog and Oct-4 protein expression was decreased in ID1 knockdown cells compared with siRNA-NC-transfected cells. Sox2 protein expression remained unchanged following transfection. These results indicate that ID1 regulates CSC-like properties by targeting Nanog and Oct-4.

Previous studies have confirmed the role of ID1 in the development of resistance to chemotherapy in non-small cell lung cancer cells (21). In addition, ID1 has been demonstrated to contribute to radioresistance in glioblastoma through inhibition of DNA repair pathways (34). The effect of ID1 knockdown on resistance to DDP in the GC cell lines MKN-28 and MGC-803 was examined. Following siRNA transfection, MKN-28 and MGC-803 cells were treated with 1 and 2 µg/ml DDP. siRNA-ID1-transfected MKN-28 cell viability decreased markedly following treatment with 1 (62% decrease; P<0.05) and 2 (75% decrease; P<0.001) µg/ml DDP compared with the siRNA-NC-transfected cells (Fig. 4A). Similarly, treatment with 1 and 2 µg/ml DDP decreased MKN-28 cell viability compared with the untreated cells (Fig. 4A). siRNA-ID1-transfected MGC-803 cell viability decreased markedly following treatment with 1 (44% decrease; P<0.05) and 2 (49% decrease; P<0.05) µg/ml DDP compared with the siRNA-NC-transfected cells (Fig. 4A). In addition, treatment with 1 and 2 µg/ml DDP decreased MGC-803 cell viability compared with the untreated cells (Fig. 4A).

Colony formation assays were performed to further evaluate the effect of ID1 knockdown on GC cell sensitivity to DDP. As shown in Fig. 4B and C, MKN-28 colony formation was markedly decreased in the ID1 knockdown cells compared with the siRNA-NC-transfected cells following treatment with 1 µg/ml DDP (P<0.01). In addition, colony formation was decreased in the DDP treatment groups compared with the untreated groups. Similarly, MGC-803 colony formation was markedly decreased in the ID1 knockdown cells compared with the siRNA-NC-transfected cells, following treatment with 1 µg/ml DDP (P<0.05). Furthermore, colony formation was decreased in the DDP treatment groups compared with the untreated groups.

To further investigate the role of ID1 in DDP resistance, the effect of ID1 knockdown and treatment with DDP on apoptosis was analyzed. As shown in Fig. 4D and E, the percentage of apoptotic MKN-28 (P<0.001) and MGC-803 (P<0.01) cells was markedly increased following ID1 knockdown compared with the siRNA-NC-transfected cells. In addition, the percentage of apoptotic MKN-28 and MGC-803 cells was markedly increased following combined ID1 knockdown and treatment with DDP compared with ID1 knockdown alone (both P<0.01; Fig. 4E). These results indicate that ID1 knockdown reduces DDP resistance in MKN-28 and MGC-803 cells through the promotion of apoptosis.