Four human gastric cancer cell lines were used in this study: MKN45, NUGC4, MKN7 (all three cell lines from JCRB Cell Bank), and KE39 (from RIKEN Cell Bank). All cell lines were cultured in RPMI-1640 medium (Nissui Pharmaceutical Co., Ltd.) supplemented with 10% fetal bovine serum and 100 U/ml penicillin at 37°C in a humidified atmosphere of 5% CO₂. 5-FU was purchased from FUJIFILM Wako Pure Chemical Corporation. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium Bromide Thiazolyl Blue (MTT) was purchased from Nakalai Tesque.

Cell transfection was performed using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol for knockdown experiments. All siRNAs were purchased from FASMAC. siRNA sequences were as follows: siRPL11#1, 5′-GGUGCGGGAGUAUGAGUUA-3′; siRPL11#2, 5′-AAGGUGCGGGAGUAUGAGUUA-3′; siControl, 5′-UUCUCCGAACGUGUCACGU-3′. siP53, 5′-CGGCGCACAGAGGAAGAGAAT-3 [Knockdown of siRNA-mediated P53 expression is previously described (21,22)].

Cells were seeded at 7,000 cells per well in a 96-well plate and transfected with the indicated siRNAs for 24 h. After transfection, the cells were exposed to different concentrations of 5-FU for 3 days. Subsequently, the MTT solution was added to each well, and the cells were cultured for an additional 4 h. After removing the media, 100 µl of DMSO was added to each well to dissolve the formazan crystals. The absorbance values at 570A of each well were measured with a microplate reader (Sunrise Remote; Tecan Japan Co. Ltd.) and applied to the following calculation: Relative cell viability=A value of cells treated with drugs/A value of cells treated with vehicle.

For protein analysis, cells were washed twice with phosphate-buffered saline (PBS) and lysed with lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Sodium Vanadate, 1 mM EDTA, 50 mM NaF, 1% Triton X-100) supplemented with Protease Inhibitor Cocktail (Nakalai Tesque), followed by sonication to reduce viscosity. Protein concentration of each sample was determined by the Protein Assay CBB Solution, according to the manufacturer's protocol (Nakalai Tesque). Lysates containing proteins (20 µg) were resolved on an SDS-polyacrylamide gel and transferred to an Immobilon-P membrane (Millipore). The membranes were blocked with blocking solution (4% BSA in TBST; 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 0.1% Tween-20) for 1 h at 37°C prior to subsequent incubation with the following primary antibodies: Anti-P53 antibody (1:500 dilution; Santa Cruz Biotechnology), anti-P21 antibody (1:400 dilution; Santa Cruz), anti-RPL11 antibody (1:1,000 dilution; Invitrogen), and anti-Actin (1:3,000 dilution; Bio Matrix Research) primary antibodies for 1 h at 37°C. Following this, the membranes were washed three times for 10 min in TBST and incubated with horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG secondary antibodies (1:3,000 dilution; Cell Signaling Technology) for 1 h at 37°C. Immunoreactive bands were visualized with enhanced chemiluminescence using Clarity Western ECL Substrate (Bio-Rad). Representative images from repeated experiments are presented in each figure.

Total RNA from cultured cells was isolated using the TRIzol reagent (Molecular Research Center) according to the manufacturer's instructions. RNA (1 µg) was reverse-transcribed using the ReverTra Ace kit (Toyobo). The mRNA expression levels of P21, FAS, and RPL11 were determined by real-time RT-PCR (StepOnePlus Real-Time PCR System; Applied Biosystems) using GoTaq qPCR Master Mix (Promega) according to the manufacturer's instructions. Human GAPDH was used for normalization. The expression of the target gene was quantified using the comparative cycle threshold method. The primer sequences used were as follows: RPL11 forward, 5′-GAAAAGGAGAACCCCATGC-3′ and reverse, 5′-CATTTCTCCGGATGCCAA-3′; P21 forward, 5′-CTGGACTGTTTTCTCTCGGCTC-3′ and reverse, 5′-TGTATATTCAGCATTGTGGGAGGA-3′; FAS forward, 5′-TCTGCCATAAGCCCTGT-3′ and reverse, 5′-GTCTGTGTACTCCTTCCCT-3′; GAPDH forward, 5′-TGCACCACCAACTGCTTAG-3′ and reverse, 5′-GAGGCAGGGATGATGTTC-3′.

The prognostic significance of the mRNA expression of RPL11 genes in gastric cancer was evaluated using the Kaplan-Meier plotter online database, including gene expression data and clinical data (GSE14210, GSE15459, GSE22377, GSE29272, GSE51105 and GSE62254). The database automatically divided RPL11 mRNA expression into high and low expression groups. The survival curve, log-rank P-value, and hazard ratio with 95% confidence intervals were calculated and displayed in the results by the computer. The datasets analyzed during the present study are available in the Kaplan-Meier plotter online database (http://kmplot.com/analysis/index.php?p=service&cancer=gastric).

Data are presented as the mean ± standard deviation. Differences between multiple groups were determined by one-way analysis of variance followed by Dunnett's post-hoc test. The analyses were performed using GraphPad Prism software (version 8.1.1; GraphPad software Inc.). P<0.05 was considered to indicate a statistically significant difference.

To study the relationship between RPL11 expression and the effect of 5-FU on gastric cancers, we first performed survival analysis using a Kaplan-Meier plotter (www.kmplot.com). Fig. 1A indicates that 5-FU based adjuvant therapy-treated gastric cancer patients in the RPL11-high expression group showed better prognosis than those in RPL11-low expression group (P=0.041). In contrast, gastric cancer patients treated by surgery alone showed no significant difference between the RPL11-high and RPL11-low expression groups (P=0.11; Fig. 1B). Similarly, survival analysis in only stage IV gastric cancer patients with surgery alone showed no significant difference between the RPL11-high and RPL11-low expression groups (P=0.19; Fig. 1C). The results suggest that RPL11 expression alters the sensitivity of gastric cancer against 5-FU.

RPL11 is known to regulate the P53 pathway (7). To investigate whether the growth inhibitory effect of 5-FU in gastric cancer involves regulation of P53 via RPL11, we performed an MTT assay using gastric cancer cell lines, MKN45 (wild-type TP53), NUGC4 (wild-type), MKN7 (mutated), and KE39 (mutated) cells. siRPL11-transfected TP53 wild-type cells, MKN45 and NUGC4 cells, showed more resistant to 5-FU than their corresponding non-specific control siRNA-transfected cells. Further, in TP53-mutant cell lines, MKN7 and KE39 cells, there was no significant difference in growth suppressive effect of 5-FU between siRPL11-transfected cells and non-specific control siRNA-transfected cells (Fig. 2A), Further, MKN45 and NUGC4 cells with siRNA-mediated P53 knockdown showed more resistant to 5-FU than those with scramble siRNA as a control (Fig. 2B); however, double knockdown of P53 and RPL11 did not show further resistance to 5-FU in MKN45 and NUGC4 cells in comparison with either P53 or RPL11 single knockdown (Fig. 2B). These observations suggested that cell growth suppression by RPL11-mediated P53 activation in 5-FU treated gastric cancer cell lines is dependent on normal P53 function.

To investigate whether RPL11 is involved in regulation of the P53 pathway in 5-FU treated gastric cancer cells, we examined protein expression levels of P53 and a P53 downstream target, P21. As a result, 5-FU increased protein expression levels of P53 and P21 in MKN45 and NUGC4 cells, and they were markedly reduced by siRNA knockdown of RPL11. Conversely, MKN7 and KE39 cells did not have any significant effects on P53 and P21 protein expression (Fig. 3A). Because the extrinsic apoptosis factor FAS is crucial for P53 mediation of apoptosis in 5-FU treated cancer cells (23), 5-FU treatment increased mRNA levels of P53 target genes, P21 and FAS, which were determined using the quantitative real-time PCR assay. In MKN45 and NUGC4 cells, knockdown of RPL11 markedly reduced the increased mRNA levels of P21 and FAS by 5-FU treatment. However, in MKN7 and KE39 cells treated with 5-FU, there was little difference in mRNA levels of P21 and FAS between siRPL11-transfected cell lines and non-specific control siRNA-transfected cell lines (Fig. 3B). These results demonstrate that RPL11 expression is involved in regulating the P53 pathway in 5-FU-treated gastric cancer cell lines.