RAD54B promotes gastric cancer cell migration and angiogenesis via the Wnt/β-catenin pathway

The expression level of RAD54B in the gastric cancer samples and normal samples, as well as the pan-cancer RAD54B expression were analyzed by The Cancer Genome Atlas (TCGA). In addition, the level of RAD54B in the gastric cancer samples and normal samples was also determined by the Gene Expression Profiling Interactive Analysis (GEPIA; http://gepia2.cancer-pku.cn), an online open-access RNA-seq analysis tool¹⁴, in which the cutoff mRNA/transcript value and p value were the default values with 1 and 0.01, respectively.

Human gastric cancer lines, including AGS (CL-0022), MKN45 (CL-0292), NCI-N87 (CL-0169), HGC-27 (CL-0107), human gastric epithelial cells GES-1 (CL-0563) and human umbilical vein endothelial cells (HUVECs, CL-0122) were bought from Procell (Wuhan, China). All the cell lines except for HUVECs were cultured in RPMI-1640 media (PM150110, Procell), while HUVECs were maintained in DMEM/F12 basic media (PM150312, Procell) with 10% fetal bovine serum (FBS, 1600044, Gibco, Rockville, MD, USA) and 1% penicillin/streptomycin (PB180120, Procell) at 37°C with 5% carbon dioxide (CO₂).

Two short hairpin RNAs (shRNAs) targeting RAD54B (sh-RAD54B#1 and sh-RAD54B#2) and negative controls (sh-NC) were purchased from GenePharma (Shanghai, China). Overexpression of RAD54B was achieved via the transfection of pcDNA vector plasmids containing the sequences of RAD54B (pcDNA-RAD54B). The transfection assays were conducted with Lipofectamine 3000 (L3000015, Invitrogen, Carlsbad, CA, USA). Briefly, sh-RAD54B#1, sh-RAD54B#2, sh-NC, pcDNA-RAD54B and empty pcDNA vector plasmids (pcDNA), as well as Lipofectamine 3000 reagents were diluted with Opti-MEM™ (31985070, Invitrogen), and then mixed with a ratio of 1:1. AGS and MKN45 cells were inoculated into 6-well plates with 6×10⁵ cells per well and cultured at 37°C with 5% CO₂. When the convergence reached 70%–90%, the mixtures were added into AGS and MKN45 cells for transfection. Cells were harvested for the subsequent examination after transfection for 48 h.

Total proteins from transfected AGS and MKN45 cells were isolated by RIPA buffer (R0010, Solarbio, Beijing, China) and quantified with the BCA protein quantification kit (ab102536, Abcam, Cambridge, UK) in line with the operating instructions. 20 μg protein samples were dissolved and electrically transferred onto a PVDF membrane (IPVH00010, EMD Millipore, Billerica, MA, USA). After blocking with 5% skim milk (D8340, Solarbio) at room temperature for 1 h, the membranes were incubated with primary antibodies against diverse proteins (RAD54B, 1:1000, ab168463; β-catenin, 1:500, ab16051; Axin, 1:1000, ab32197; cmyc, 1:20000, ab152146; MMP-7, 1:1000, ab216631; GAPDH, 1:2500, ab9485; all from Abcam) at 4°C overnight. Subsequently, the membranes were incubated with corresponding secondary antibodies for 2 h at room temperature and visualized by an ECL assay (P0018S, Beyotime, Shanghai, China). The band intensity was determined by ImageJ software (National Institutes of Health, USA).

FIGURE 1.

Transfected AGS and MKN45 cells with 6×10⁵ cells per well were seeded into 6-well plates and maintained at 37°C for 12 h with 5% CO₂. Following the incubation with 1 ml of EdU working solution (20 μM) for 2 h at 37°C, cells were immobilized with immunol-staining fix solution (P0098, Beyotime), permeated with 0.3% Triton X-100 (ST795, Beyotime) and incubated with the anti-EdU Click reaction solution in dark for 30 min. Hoechst 33342 (5 μg/mL, C1022, Beyotime) was utilized for the stain of cell nucleus. The stained cells were photographed under a fluorescence microscopy (Olympus, Tokyo, Japan) and five random fields were chosen for the analysis of the EdU-positive cell percentage.

FIGURE 2.

Transfected AGS and MKN45 cells with 6×10⁵ cells per well were plated into 6-well plates. Cells were hatched at 37°C for 14 days and then immobilized with 4% paraformaldehyde (P0099, Beyotime) and stained with 0.1% crystal violet (C0121, Beyotime) for 30 min, separately. The clone numbers were manually counted.

The mobility and invasion of transfected AGS and MKN45 cells were assessed by transwell assay using 24-well transwell chambers with 8.0-μm pore size polycarbonate membranes. Briefly, 200 μl of cell suspension with a total of 2 × 10⁵ cells and 600 μl of RPMI-1640 with 10% FBS were severally appended into the upper and lower chamber for the cell migration determination. Additionally, Matrigel matrix was filled in the transwell chamber with serum-free medium dilution for the cell invasion detection. The upper and lower chambers were diffused with 200 μl of cell suspension with a total of 2 × 10⁵ cells and 600 μl of RPMI-1640 with 10% FBS, respectively. After the continuous culture for 24 h, cells were immobilized with 4% paraformaldehyde and stained with 0.1% crystal violet for 30 min orderly, and then photographed under an inverted microscope (Olympus). Five random fields were selected for the analysis of the number of migrated and invasive cells by using the Image J software (National Institutes of Health, USA).

FIGURE 3.

sh-RAD54B#1, sh-RAD54B#2, sh-NC, pcDNA-RAD54B and empty pcDNA vector plasmids (pcDNA) were transfected into AGS and MKN45 cell lines. Supernatant was collected for the tube formation assay after transfection for 48 h. In brief, HUVECs were cultured in conditioned medium containing supernatant. Then, HUVECs were plated in a 6-well plate coated with matrigel (354248, Corning Company, New York, NY, USA) at a density of 6×10⁵ cells per well. Following 4 h, cells were examined with a phase-contrast microscopy, and the tube formation capacity was evaluated by the number of brunching points.

4 weeks-old BALB/c nude mice were bought from Vital River (Beijing, China). Mice were raised in a temperature-controlled SPF animal room with the 12-h cycle of light-dark. Ten mice were randomly divided into two groups, including sh-NC group and sh-RAD54B#1 group. Mice in both groups were subcutaneously injected with a total of 2 × 10⁶ MKN45 cells transfected with sh-NC and sh-RAD54B#1, severally. Tumor volume was monitored every seven days for continuous five weeks and calculated based on the formula: 1/2×length×width². After consecutive five weeks, mice were intraperitoneally injected with 120 mg/kg sodium pentobarbital for euthanasia based on the previous study¹⁵, and the tumors samples were enucleated and weighed. The sh-NC group was served as a negative control. MKN45 cells transfected with sh-NC was not affecting tumor growth compared to untreated tumors that could be supported with in vitro results showing no differences in cell proliferation (the percent EDU positive cells and number of colonies) between control untreated and sh-NC group (Supplementary Figure 1A and B). All animal experiments were authorized by the Animal Research Ethics Committee of Changzhou TCM Hospital.

FIGURE 4.

Tumor tissues were fixed in 4% formaldehyde, dehydrated with gradient concentrations ethanol, embedded into parrafin (YA0011, Solarbio) and cut into sections with a thick of 5 μm. Sections were retrieved in 10 mM sodium citrate buffer (pH 6.0, P0083, Beyotime) for 15 min at 94°C. Following cooling to room temperature, sections were blocked with 1% bovine serum albumin (BSA, ST2249, Beyotime) for 30 min and then incubated with primary antibodies against RAD54B (1:200, ab238579, Abcam), Ki-67, c-myc (1:1000, ab32072, Abcam) and MMP-7 (1:500, ab216631, Abcam) respectively. Subsequently, sections were incubated with biotinylation-labeled secondary antibody (1:1000, ab207996, Abcam), re-stained with hematoxylin, and captured under a light microscope (Olympus). The relative level of RAD54B, Ki-67, cmyc and MMP-7 was determined as the ratio of the number of positive cells to the total number of cells.

Three technical replicates with three independent experimental replicates were conducted in the cell experiments, while five technical replicates with three independent experimental replicates were performed in the animal experiments. Results were expressed as mean ± standard deviation (SD). Statistical differences were determined through the Student’s t-test between two groups followed by Post Hoc Bonferroni test by SPSS 26.0 software (IBM, Armonk, New York, USA). P < 0.05 was considered as significant difference.

FIGURE 5.

FIGURE 6.

As presented in Figure 1A, the level of RAD54B in stomach adenocarcinoma (STAD) was significantly increased compared with that in normal samples. Besides, the pan-cancer analysis revealed that RAD54B expression was also prominently enhanced in the majority of cancers, including STAD (Figure 1B). Moreover, the level of RAD54B was confirmed to be enhanced in the STAD primary tumor samples relative to that in normal samples based on the GEPIA (Figure 1C). Furthermore, a prominent increase in RAD54B expression was also observed in the gastric cancer cell lines, including AGS, MKN45, NCI-N87 and HGC-27, relative to that in human gastric epithelial cells GES-1. Among them, the expression level of RAD54B in AGS and MKN45 was significantly higher than that in NCI-N87 and HGC-27, thus the first two cell lines were chosen for subsequent assays (Figure 1D). Briefly, RAD54B expression was up-regulated in gastric cancer.

Upregulated expression of RAD54B indicated that RAD54B might exert a crucial effect during the progress of gastric cancer. Hence, the expression of RAD54B was first downregulated or upregulated in both MKN45 and AGS cells with transfection of shRAD54B or overexpression plasmid respectively (Figure 2A). The Edu positive cells and numbers of colonies were observably decreased with silencing of RAD54B, while markedly increased by overexpression of RAD54B in both AGS and MKN45 cells based on the Edu (Figure 2B and C) and colony formation assays (Figure 2D and E). Moreover, no statistical difference was found in the percent EDU positive cells and number of colonies in MKN45 cells between untreated control group and sh-NC group (Supplementary Figure 1A and B). Therefore, RAD54B increased the viability of gastric cancer cells.

In addition, the effect of RAD54B on the mobility and invasion of gastric cancer was also assessed by transwell assay. Both the numbers of migrated and invasive cells were notably reduced in both AGS and MKN45 cells transfected with shRAD54B (Figure 3). On the other hand, upregulation of RAD54B markedly enhanced the numbers of both migrated and invasive cells in both AGS and MKN45 cells (Figure 3). Besides, no statistical difference was found in the numbers of both migrated and invasive cells in MKN45 cells between untreated control group and sh-NC group (Supplementary Figure 1C and D). Thus, RAD54B expedited the mobility and invasion of gastric cancer cells.

Moreover, supernatants form cultured AGS and MKN45 cells with the knockdown of RAD54B or with the overexpression of RAD54B were used to incubate with HUVEC. The results showed that the number of brunching points of HUVECs was significantly diminished after HUVECs were cultured with supernatants form cultured AGS and MKN45 cells with the knockdown of RAD54B, while that was prominently increased after HUVECs were cultured with supernatants form cultured AGS and MKN45 cells with the overexpression of RAD54B (Figure 4), indicating that RAD54B promoted the tube formation of gastric cancer cells.

To explore the molecular mechanism of RAD54B in the progress of gastric cancer, the relative protein levels of β-catenin, Axin, c-myc and MMP-7 were examined via western blot. As displayed in Figure 5, the relative protein expressions of β-catenin, c-myc and MMP-7 were significantly decreased in both AGS and MKN45 cells with transfection of shRAD54B, while notably augmented in both AGS and MKN45 cells transfected with RAD54B overexpression plasmid. On the contrary, downregulation of RAD54B markedly enhanced the relative protein expression of Axin, whereas upregulation of RAD54B observably decreased the relative protein expression of Axin in both AGS and MKN45 cells. Therefore, these results manifested that RAD54B facilitated the activation of Wnt/β-catenin signaling axis in gastric cancer cells.

To further verify the role of RAD54B in gastric cancer, nude mice were injected with MKN45 transfected with sh-NC or sh-RAD54B#1 and then monitored for sequential five weeks. As shown in Figure 6A, results exhibited that knockdown of RAD54B significantly decreased the tumor volume and weight (Figure 6A). In addition, silencing of RAD54B also prominently reduced the expression levels of RAD54B, Ki-67, c-myc and MMP-7 relative to sh-NC group (Figure 6B). Thus, these outcomes demonstrated that RAD54B accelerated the proliferation of gastric cancer and activation of Wnt/β-catenin signaling axis in vivo.