Gastric adenocarcinoma MKN45 and gastric carcinoma HGC27 cells (Ningbo Mingzhou Biotechnology Co., Ltd.) were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 15% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin solution (Ningbo Mingzhou Biotechnology Co., Ltd.). The culture environment was maintained at 37°C with 5% CO₂.

The cells were seeded into a 6-well plate at a density of 2×10⁵ cells/well and were transfected with 2 µg pcDNA3.1 PDGFRB overexpression vector or an empty vector (NovoPro Bioscience, Inc.) using RFect reagent (Baidai; Changzhou EMI Biotechnology Co., Ltd.) at 37°C. After transfection for 48 h at 37°C, the cells were treated with lenvatinib (40 µM; Selleck Chemicals) for 24 h at 37°C.

MKN45 and HGC27 cells were seeded into 96-well plates at a density of 3×10³ cells/well, and were incubated in the presence of gradient concentrations of lenvatinib (0–100 µM) for 24 h at 37°C. Subsequently, 10 µl CCK-8 reagent was added to the wells. The optical density was measured at a wavelength of 450 nm using a microplate reader (Tecan Group, Ltd.) after 1 h of incubation. The percentage cell inhibition rate (%) was calculated using the following formula: Cell inhibition=(OD value of control group-OD value of experimental group)/(OD value of control group-OD value of blank group) ×100.

MKN45 and HGC27 cells were seeded into 96-well plates, and cell proliferation was assessed using the EdU staining kit (Beyotime Institute of Biotechnology). As aforementioned, MKN45 and HGC27 cells were treated with lenvatinib, transfected with Ov-PDGFRB or Ov-NC, and were then incubated with 10 µM EdU reagent for 2 h at 37°C. The cells were washed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 15 min at room temperature and the nuclei were stained with DAPI. Images of the stained cells were captured under a fluorescence microscope (Olympus Corporation).

MKN45 and HGC27 cells treated with lenvatinib and transfected with Ov-PDGFRB or Ov-NC were inoculated into culture dishes (500 cells/dish) and evenly dispersed. After culturing the cells for 14 days, they were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 0.1% crystal violet (Selleck Chemicals) for 30 min at room temperature. Images of visible colonies (≥50 cells) were captured under a light microscope and colonies were counted using ImageJ software (version 1.8; National Institutes of Health).

Cell apoptosis was analyzed using an Annexin V-FITC Apoptosis Detection kit (BD Biosciences). After being treated with lenvatinib and transfected with Ov-PDGFRB or Ov-NC, the MKN45 and HGC27 cells were digested with 0.25% trypsin and washed twice with PBS. The cells were then suspended in binding buffer, and were incubated with 5 µl Annexin V-FITC for 30 min, followed by incubation with 5 µl propidium iodide for 5 min at room temperature in the dark. Apoptosis was analyzed using a FACSCalibur flow cytometer (BD Biosciences) and FlowJo software (version 10.7.2; FlowJo LLC).

After MKN45 and HGC27 cells were treated with lenvatinib and transfected with Ov-PDGFRB or Ov-NC, total proteins were extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology) and were quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Inc.). Protein samples (30 µg) were separated by SDS-PAGE (10% gel; Bio-Rad Laboratories, Inc.). PVDF membranes carrying transferred proteins were then blocked with 5% skimmed milk for 1 h at room temperature, followed by an overnight incubation with primary antibodies against Bax (cat. no. 50599-2-Ig; 1:8,000 dilution; Wuhan Sanying Biotechnology), Bcl-2 (cat. no. 12789-1-AP; 1:9,000 dilution; Wuhan Sanying Biotechnology), PDGFRB (cat. no. 3169S; 1,000 dilution; Cell Signaling Technology, Inc.) and GAPDH (cat. no. 10494-1-AP; 1:20,000 dilution; Wuhan Sanying Biotechnology) at 4°C. Thereafter, the membranes were incubated with an HRP-conjugated goat anti-rabbit antibody (cat. no. SA00001-2; 1:5,000 dilution; Wuhan Sanying Biotechnology) at 37°C for 1 h. Signals were visualized using an ECL Western Blotting substrate kit (BioVision, Inc.) and were analyzed using ImageJ software (version 1.8; National Institutes of Health).

The structure of Lenvatinib was drawn in the ChemDraw software (version 18.0) and then imported into OpenBabel software (version 2.3.1) for hydrogenation, and converted into a mol2 format file. Subsequently, the structure of PDGFRB (PDB ID: AF-P09619-F1) was obtained from the RCSB PDB (https://www.rcsb.org/). Thereafter, the protein PDGFRB file was opened in PyMOL software (version 2.2.0) to remove the excess water molecules, delete any irrelevant small ligands originally carried and to keep only the protein structure. As the downloaded protein structure had ligands, the original ligands were deleted and the original ligand positions were set as the docking sites. AutoDock (version 1.5.6) (14) was used to display the specific docking energy value after running. Finally, the results were analyzed with the adoption of Protein-Ligand Interaction Profiler (PLIP; http://plip-tool.biotec.tu-dresden.de/plip-web).

After being treated with lenvatinib and transfected with Ov-PDGFRB or Ov-NC, serum-starved MKN45 and HGC27 cells were grown until cells reached 90% confluence, and the central cells on the monolayer were scraped away using a 200-µl pipette tip. The distance of migration within 24 h was analyzed using ImageJ software (version 1.8) to calculate the migration rate under a light microscope.

After being treated with lenvatinib and transfected with Ov-PDGFRB or Ov-NC, 1×10⁵ MKN45 or HGC27 cells suspended in fresh serum-free RPMI 1640 medium were seeded into the upper chamber of Transwell plates (8-µm pore size; Costar; Corning, Inc.) precoated with Matrigel at 37°C for 30 min, and RPMI 1640 medium containing 20% FBS was added to the lower chamber. After a 24-h incubation at 37°C, cells that passed through the Matrigel were stained with 0.5% crystal violet at room temperature for 10 min and were captured under a light microscope. The results were analyzed using ImageJ software (version 1.8).

Differentially expressed genes between gastric cancer tissue and paired normal tissue obtained from the GSE79973 (19) and GSE118916 (20) datasets from the GEO database (ncbi.nlm.nih.gov/gds) were determined using the Limma package in R software (version 4.1.2; http://www.bioconductor.org/packages/release/bioc/html/limma.html). The differentially expressed genes in gastric cancer were acquired from The Cancer Genome Atlas (https://www.cancer.gov/ccg/research/genome-sequencing/tcga). The target genes of lenvatinib were analyzed through TargetNet (http://targetnet.scbdd.com/) and SuperPred (https://prediction.charite.de/) databases. A Venn diagram was generated to display intersection genes, and the Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn/) was used to display the association between intersection genes and overall survival.

Statistical analysis was performed using GraphPad Prism software (version 9.5; Dotmatics) and quantitative data are presented as the mean ± SD of three independent experiments. One-way ANOVA and Tukey's post hoc test were used to determine statistical differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Gradient concentrations of lenvatinib (0–100 µM) were used to treat MKN45 and HGC27 cells, and their cell viability was measured at 24 h. According to the results of the CCK-8 assay, lenvatinib inhibited the viability of both cells in a concentration-dependent manner (Fig. 1A and B). Given that the IC₅₀ values of lenvatinib in MKN45 and HGC27 cells were 30.75 and 29.03 µM at the 24-h time point, concentrations of lenvatinib at 0, 10, 30 and 40 µM were selected for subsequent assays. The effects of lenvatinib on cell proliferation were determined by EdU staining. Proliferation levels were quantified by assessing the proportion of EdU-positive cells. It was discovered that Lenvatinib markedly suppressed the proliferation of MKN45 and HGC27 cells in a concentration-dependent manner (Fig. 1C and D). In addition, lenvatinib inhibited colony formation and the number of colonies visible to the naked eye was reduced (Fig. 1E and F).

The effect of lenvatinib on MKN45 and HGC27 cell apoptosis was evaluated by flow cytometry. Lenvatinib increased the early and late apoptosis of both cells in a concentration-dependent manner (Fig. 2A and B). According to the results of western blot analysis, lenvatinib reduced Bcl-2 and increased Bax protein expression levels in cells, supporting the effect of lenvatinib on apoptosis (Fig. 2C and D). Scratch and Transwell assays were applied to evaluate the migration and invasion of the two cell lines, respectively. Lenvatinib significantly reduced the migration rate and the number of cells that invaded the matrix membrane, in both MKN45 (Fig. 3A and B) and HGC27 cells (Fig. 3C and D).

Volcano plots displaying the differentially expressed genes in gastric cancer are shown in Fig. 4A and B. The Venn diagrams exhibit the intersection of the predicted targets of lenvatinib and the differentially upregulated (Fig. 4C) or downregulated (Fig. 4D) genes in gastric cancer, suggesting that only 8 upregulated genes in gastric cancer can act as potential targets of lenvatinib. As depicted in Fig. 4E, the hazard ratios of these eight intersection genes were exhibited in the heatmap, and PDGFRB, which had the highest hazard ratio in gastric cancer, was screened out. Based on GEPIA, low- and high-expression PDGFRB groups were classified based on the median expression of PDGFRB, and it was shown that high PDGFRB expression was associated with poor survival (Fig. 4F); therefore, the effect of lenvatinib on PDGFRB was subsequently studied. Molecular docking analysis revealed that lenvatinib formed multiple hydrogen bonds with PDGFRB (Fig. 4G), indicating that lenvatinib bound well to amino acids in the protein pocket. Western blot analysis indicated that the expression levels of PDGFRB in MKN45 and HGC27 cells were reduced in a concentration-dependent manner upon lenvatinib treatment (Fig. 4H). Furthermore, PDGFRB was successfully overexpressed by transfection of MKN45 and HGC27 cells with a pcDNA3.1 PDGFRB overexpression vector, which was confirmed by western blotting (Fig. 4I).

To explore the regulatory effects of lenvatinib on PDGFRB, cells were induced to overexpress PDGFRB, and the proliferation and colony formation of the cells were evaluated. The results demonstrated that PDGFRB overexpression promoted MKN45 and HGC27 cell proliferation (Fig. 5A and B) and colony formation (Fig. 5C and D), and reversed the inhibitory effects of lenvatinib. Flow cytometry (Fig. 6A and B) and apoptosis-related protein analysis (Fig. 6C and D) also revealed that PDGFRB overexpression reduced the proportion of apoptotic cells and the protein expression levels of Bax, and increased the protein expression levels of Bcl-2 compared with the lenvatinib 40 µM + Ov-NC group. The migration and invasion of MKN45 (Fig. 7A and B) and HGC27 cells (Fig. 7C and D) were also enhanced in response to PDGFRB overexpression, compared with the negative control group, upon treatment with lenvatinib.