Increased expression of Ribonucleic acid export 1 (RAE1) gene promotes gastric carcinogenesis and is associated with activation of Hippo signaling pathway

Introduction

Gastric cancer (GC) is one of the most common cancers in the world, with the fifth-highest morbidity rate among malignant tumors (1). GC has no obvious symptoms in the early stage, and most patients have already progressed to the advanced stage by the time of diagnosis. Early-stage GC accounts for only 20% of all GC cases in China, resulting in an overall 5-year survival rate of less than 50% (2). Therefore, understanding the mechanism in the development of GC is of great significance in reducing the risk of death of GC.

In our recent study, it was shown that Ribonucleic acid export 1 (RAE1) autoantibody may have significant potential for early detection of GC (3). RAE1 was originally discovered during a genetic screen for proteins involved in ribonucleic acid export in yeast (4). RAE1 has multiple functions in mitotic spindle formation (5) and is associated with cellular senescence, cell cycle, and tumors (6). A study reported that reduced expression of the mitotic checkpoint protein Bub3/RAE1 caused faster senescence in mice, and inhibition of RAE1 may induce cellular senescence through activation of P19, P53, P21, and P16 pathways (7). Studies also showed that aberrant RAE1 expression led to chromosome aneuploidy segregation thus causing cancer (8,9). The role of RAE1 in some tumors has been reported, which can be used as a prognostic marker of hepatocellular carcinoma, promote the growth of colorectal cancer, and promote epithelial mesenchymal transition in breast cancer (10-12), but the related mechanisms are unknown. However, few studies about the role and mechanism of RAE1 in GC are currently available.

In this study, we investigated the role and potential mechanism of RAE1 in GC. The expression of RAE1 was observed by immunohistochemistry in GC tissues and precancerous lesion (PL) tissues, and its correlation with clinical features was investigated. The effects of RAE1 on proliferation, migration, apoptosis and cell cycle of GC cells were investigated in vivo and in vitro. The signaling pathway of RAE1 on GC development was explored by transcriptome sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-1-1019/rc).

Methods

Patients

From among patients who underwent gastroscopy and from whom pathologic tissue was obtained between 2015 and 2017 at Beijing Friendship Hospital, Capital Medical University, 43 patients with well-preserved paraffin-embedded tissue blocks were selected. Clinical data were collected for all patients including age, gender, history of smoking, alcohol consumption, family history of tumor, tumor location, degree of differentiation, stages, and tumor markers such as carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 125 (CA12-5), and alpha-fetoprotein (AFP). Stages of GC were classified according to the Chinese Medical Association Guidelines for Clinical Diagnosis and Treatment of Gastric Cancer (2021 Edition). The overall survival (OS) analysis included all stages of GC patients. First progression (FP) is defined as the time from diagnosis to first documented disease progression. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Beijing Friendship Hospital, Capital Medical University (No. 2018-P2-058-01). All participants provided written informed consent.

Immunohistochemistry (IHC)

IHC analysis was performed as previously described (13). Primary antibody against RAE1 (1:300, Abclonal) was used. Staining intensities were determined by measuring the integrated optical density (IOD) / area by light microscopy using ImageJ.

Cell culture and siRNA transfection

Human GC cell lines AGS and HGC-27 were purchased from the Cell Resource Center of the Chinese Academy of Medical Science (Beijing, China), and STR authentication was performed prior to use. AGS and HGC-27 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) in a 37 ℃ incubator supplemented with 5% CO₂. RAE1-specific siRNA (sense: GCAGUAACCAAGCGAUACATT, antisense: UGUAUCGCUUGGUUACUGCTT) and negative control siRNA (sense: UUCUCCGAACGUGUCACGUTT, antisense: ACGUGACACGUUCGGAGAATT) were synthesized by Sangon Biotech. Transfection of GC cells with siRNA oligonucleotides was performed using Lipofectamine RNAiMAX (Thermo Fisher) according to the manufacturer’s instructions. Transfected cells were used for downstream analysis.

Generation of GC cells in which RAE1 was stably knocked down or overexpressed

The target sequence of sh-RAE1#1 is 5'-TGGGATACTCGATCGTCAAAT-3' (Merck, TRCN0000337192) and sh-RAE1#2 is 5'-CCTCAGCAGTAACCAAGCGAT-3'. GC cells stabling knockdown and overexpression of RAE1 were generated according to standard protocols using pLKO.1 vector and PCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector. Control cells were generated by transfecting cells with each empty vector. Transfected GC cells were selected with 2 µg/mL puromycin for 1–2 weeks.

Real-time reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was isolated from GC cells using TRI Reagent (Sigma) in accordance with the manufacturer’s instructions. cDNA was generated by Reverse Transcription Kit (Takara) and real time PCR was carried out using the SYBR green master mix (Yeasen) in applied biosystems. Each sample was examined in triplicate. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. Real-time PCR primers are listed in Table S1.

Western blotting

Protein lysates were collected with RIPA buffer with protease and phosphatase inhibitor cocktail (NCM Biotech). Equal quantities of proteins were separated by 10–12% SDS-PAG and transferred onto a PVDF membrane (GE) using a Bio-Rad wet transfer unit. After blocking with 5% non-fat milk at room temperature for 1 h, the membrane was incubated with the primary antibodies overnight at 4 ℃, followed by HRP-conjugated goat anti-mouse or goat anti-rabbit IgGs (1:5,000) for 1 h at room temperature. The blots were detected with an ECL protein chromogenic kit (Millipore) using Image Lab Software (BIO-RAD). Antibody against RAE1 (1:1,000, A6713) was purchased from Abclonal (). Antibody against GAPDH was purchased from Abcam. Antibodies against Cyclin A1/A2, Cyclin B1 were purchased from Selleck. Antibodies against cyclin D1, CDK2, CDK4, Bax, Bcl-2, β-catenin, YAP, c-Myc were purchased from Cell Signaling Technology.

Cell growth assay and colony formation assay

Cell proliferation assay was evaluated by Cell Counting Kit-8 (CCK-8) (Bioss) according to the manufacturer’s instructions. Cells were seeded onto 96-well dishes at 4,000 cells/well and the absorbance at 450 nm was measured every 24 h using a SpectraMax M3 microplate reader (Molecular Devices). For colony formation assay, GC cells with knockdown and overexpression of RAE1, along with their corresponding controls were seeded at 5,000–10,000 cells/well in 35 mm culture vessel. After 14 days, colonies were stained using crystal violet staining solution (Beyotime) according to the manufacturer’s instructions. Colony counts were determined using ImageJ software.

Cell migration assay

For these experiments, 10⁵ cells were placed in each 24 well transwell permeable supports (Costar). After 24 h incubation, migrating cells were fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature and washed 2 times with phosphate buffer saline (PBS) and then stained using crystal violet staining solution for 10 min and observed via fluorescent microscopy. Acquired images were analysed using ImageJ software.

Cell cycle assay and apoptosis assay

GC cells with knockdown and overexpression of RAE1, along with their corresponding controls were fixed with 75% ethanol overnight, and washed with PBS and stained with PI/RNase staining solution (Thermo Fisher Scientific). Cells were detected using the Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific). Apoptosis assays were performed according to the manufacturer’s instructions using the Annexin V-FITC Apoptosis Detection Kit (Bioss) and Annexin V-APC/PI Apoptosis Detection Kit (KeyGEN BioTECH). Cell cycle and apoptosis analysis was performed using FlowJo software.

Murine xenograft model

Animal studies were performed in compliance with Animal Care and Use Committee, Beijing Friendship Hospital/Capital Medical University. Four to six-week-old BALB/C nude male mice (n=5/group) were housed in a specific pathogen-free facility under standard conditions (12-h light/dark cycle, 22±2 ℃, 50%±10% humidity) with free access to autoclaved food and water. Mice were group-housed (up to 5 per cage) in individually ventilated cages with environmental enrichment (nesting material and shelters), and were injected subcutaneously in the right flank regions with 4×10⁶ HGC-27 cells expressing sh-NC or sh-RAE1 suspended in 200 µL (PBS/matrigel mixed at 1:1). Tumor sizes and mouse weights were measured weekly for a month before the mice were sacrificed. Tumor tissues were fixed in 4% fixative solution (Solarbio) for IHC and frozen for protein extraction. Tumor sizes were calculated as length × width² × 0.52.

Transcriptome sequencing and enrichment analysis

AGS cells transfected with si-NC and si-RAE1 were performed transcriptome sequencing (GENEWIZ, Suzhou, China). Differential genes with fold change (FC) of 1.5 were selected for KEGG enrichment analysis.

Statistical analysis

Non-normally distributed continuous variables were expressed as the median (25th to 75th percentiles), and normally distributed continuous variables were expressed as the mean and standard deviation (SD). P values were calculated using t-tests or the Mann-Whitney U test. Categorical variables were expressed as frequencies and percentiles, and associations between variables were tested with chi-square test or analysis of variance (ANOVA). Data of cell and animal studies are represented as means ± SD. Differences between groups were analyzed using the Student’s t-test. All statistical analyses were carried out with Graphpad Prism 8.0.2 and SPSS 22.0 software. P<0.05 was considered to indicate statistical significance.

Results

RAE1 was highly expressed in GC and was associated with poor prognosis

We first examined the expression of RAE1 in GC by The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interactive Analysis (GEPIA) databases. Both the results showed the expression of RAE1 was significantly higher in tumor tissue than the normal tissue (Figure 1A,1B). Then, we used Kaplan-Meier online tool (http://kmplot.com) to examine whether high expression of RAE1 associated with poor prognosis in GC. Among all patients (N=875), higher RAE1 expression was associated with worse OS (P=0.003; Figure 1C) and FP survival (P=1.7×10⁻⁶; Figure 1D).

Figure 1 RAE1 is highly expressed in gastric cancer. RAE1 expression in GC tissue and normal tissue in TCGA (A) and GEPIA (B) dataset. Kaplan-Meier overall survival curves (C) and first progression survival curves (D) of GC patients according to RAE1 expression. (E) Immunohistochemical staining for RAE1 in normal tissues, PL, and early GC tissues. Original magnification, ×20. The OS analysis included all stages of gastric cancer patients. FP is defined as the time from diagnosis to first documented disease progression. *, P<0.05; ****, P<0.0001. AOD, average optical density; CI, confidence interval; FP, first progression; GC, gastric cancer; GEPIA, Gene Expression Profiling Interactive Analysis; OS, overall survival; PL, precancerous lesion; RAE1, Ribonucleic acid export 1; STAD, stomach adenocarcinoma; TCGA, The Cancer Genome Atlas.

A total of 43 patients with well-preserved paraffin-embedded tissue blocks were enrolled, including 20 of PL and 23 of early GC, and from which 18 cases of normal tissue were selected. IHC showed that patients with early GC had higher levels of RAE1 expression, and the average optical density (AOD) was significantly higher than that of patients with PL and normal tissues (Figure 1E). The mean AOD of the 43 patients was 0.1208±0.0221, and based on the AOD value we categorized the patients into RAE1 high expression group (N=21) and low expression group (N=22). Correlation analysis between RAE1 and clinicopathological features was shown in Table 1. We observed a positive correlation between RAE1 expression and clinical staging (P<0.001).

RAE1 affected proliferation in GC cell lines

Affections in the proliferation of GC cells by RAE1 knockdown or overexpression were performed using both cell growth assay and colony formation assays. Knockdown of RAE1 by siRNA obviously decreased the expression of RAE1 in the protein and mRNA level (Figure 2A). Due to low efficiency of transducing RAE1 overexpression plasmid, we established GC cells stably overexpressing RAE1 via lentiviral vectors (Figure 2B). Our results showed RAE1 knockdown suppressed cell proliferation and overexpression of RAE1 increased cell proliferation (Figure 2C,2D). Overexpression of RAE1 did not significantly promote cell proliferation in colony formation assays, it may be related to the endogenous expression of RAE1.

Figure 2 RAE1 affected proliferation in GC cell lines. (A) RAE1 mRNA expression and protein expression in control and RAE1 knockdown cells using siRNA. (B) RAE1 mRNA expression and protein expression in control and RAE1 overexpression cells using lentiviral. (C) CCK-8 assays using RAE1 knockdown and overexpression cells. (D) Colony formation assays using RAE1 knockdown and overexpression cells (staining method: magnification). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, gastric cancer; NC, negative control; OD, optical density; RAE1, Ribonucleic acid export 1.

RAE1 is negatively correlated with apoptosis and may promote GC cell migration

Apoptosis was assessed by flow cytometry analysis using Annexin V-FITC/PI staining. Both early apoptotic (Annexin V-FITC positive) and late apoptotic (Annexin V-FITC/PI double positive) cells were included in the quantification. Knockdown of RAE1 significantly increased the percentage of apoptotic cells, whereas overexpression of RAE1 reduced apoptosis (Figure 3A). Changes in the proportion of apoptosis were statistically significant in both two GC cell lines. Western blot also showed RAE1-knockdown cells increased levels of Bax, a molecular marker of apoptosis, compared with control cells (Figure 3B). Bcl-2 is a molecular marker of apoptotic inhibitors. Knockdown of RAE1 showed decreased levels of Bcl-2. The results of RAE1-overexpression cells were in the opposite.

Figure 3 RAE1 is negatively correlated with apoptosis and promote GC cell migration. (A) Apoptosis assays of GC cell lines transfected with siRNA and lentiviruses. The percentages shown represent the sum of early apoptotic cells and late apoptotic cells. The red box in each plot indicates the gating strategy used for quantification. (B) Protein expression for Bax and Bcl-2 in RAE1 knockdown and overexpression GC cells. GAPDH was used as the loading control. (C) A migration assay were performed to measure the migratory abilities of GC cells (staining method; magnification). ns, not significant; **, P<0.01; ****, P<0.0001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, gastric cancer; NC, negative control; PI, propidium iodide; RAE1, Ribonucleic acid export 1.

Next, cell migration assay with RAE1 knockdown or overexpression cells was conducted. We found that RAE1 promoted migration in AGS cell line but had no significant effect in HGC-27 cell lines (Figure 3C). It may be related to the different cytogenetic background and the endogenous expression of RAE1. These findings indicated that RAE1 was negatively correlated with apoptosis and may promote GC cell migration and invasion.

Knockdown of RAE1 prevented cell cycle progression from the S to G2/M phase in vitro and inhibited tumor growth in vivo

To explore whether knockdown of RAE1 affects cell cycle progression, we conducted cell cycle assay on GC cells with RAE1 knockdown using siRNA. Compared with controls, the proportion of HGC-27 cells in S phase was increased, while the proportion of G2/M phase was decreased significantly, suggesting that the cell cycle was blocked in S phase after knocking down RAE1 (Figure 4A). However, there was no significant difference in the percentage of AGS cells in each phase compared to the control group. Further, we performed western blot to verify whether RAE1 knockdown inhibits S to G2/M phase transition. Both HGC-27 and AGS cells showed decreased expression of CDK2, which can bind to cyclinA in the S phase, thus blocking cell cycle progression. Meanwhile, knockdown of RAE1 resulted a slight decrease in cyclinA (Figure 4B). Taken together, these results revealed that RAE1 knockdown prevented cell cycle progression from the S to G2/M phase in GC cells. We also observed the effect of overexpression of RAE1 on cell cycle. After overexpression of RAE1, HGC-27 cells showed a decrease in the percentage of G1 phase and S phase, and the percentage of G2/M phase was significantly increased, suggesting that the expression of RAE1 facilitates the cells from the S phase to G2/M phase. In AGS cells, similar to the results of RAE1 knockdown, the percentage of cell cycle phases did not show significant differences compared with the control group (Figure S1A). Meanwhile, CDK2 was increased after overexpression of RAE1, which promoted the cells to enter the G2/M phase, and Cyclin B1, a protein of the G2/M phase of the cell cycle, was also increased slightly (Figure S1B).

Figure 4 RAE1 knockdown prevents cell cycle progression and inhibits tumor growth. (A) Cell cycle analysis by flow cytometry in controls and RAE1 knockdown cells using siRNA. (B) Western blot analysis of cell cycle-related proteins in control and RAE1 knockdown cells. (C) RAE1 mRNA expression and protein expression in control and RAE1 knockdown cells using shRNA. (D) In vivo analysis using a xenograft model. Tumor size and tumor weight in RAE1 knockdown cells and control cells. (E) Immunohistochemical staining and protein expression for RAE1 in tumor tissues from control and RAE1 knockdown cells. Original magnification, ×20. *, P<0.05; **, P<0.01; ****, P<0.0001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, gastric cancer; NC, negative control; RAE1, Ribonucleic acid export 1.

To validate the effect of RAE1 on tumor growth in vivo, we constructed HGC-27 cells with RAE1 stable knockdown using shRNA. We verified the knockdown efficiency of the two shRNA through western blot and selected sh-RAE1#2 for subsequent experiments, and the knockdown efficiency of sh-RAE1#2 was also confirmed to be more than 50% at mRNA level (Figure 4C). Compared with controls, RAE1 knockdown decreased the volume and weight of GC tumors in xenograft mouse models (Figure 4D). IHC analysis of xenograft tumors demonstrated that tumor tissues with stable knockdown of RAE1 showed weaker RAE1 staining compared with controls (Figure 4E). The results of western blot also showed that the expression of RAE1 in the tumors of the RAE1 knockdown group was lower than that in the control group, consistent with the IHC results (Figure 4E). Taken together, these results revealed that knockdown of RAE1 prevented cell cycle progression and inhibited GC tumor growth.

RAE1 may affect GC development by regulating Hippo signaling pathway

Transcriptome sequencing analysis showed a total of 331 differentially expressed genes between the RAE1 knockdown group and the control group, of which 176 genes were up-regulated and 155 genes were down-regulated (Figure 5A). KEGG results showed that the down-regulated genes after knockdown of RAE1 were mainly enriched in Hippo signaling pathways, in addition, genes in other pathways such as metabolic , biological processes, as well as GC and basal cell carcinoma were also identified (Figure 5B). We detected the five genes TEAD1, TEAD2, FZD4, FZD5, GLI2 enriched in Hippo signaling pathway and three other random genes CCNE2, CASP7 and RYR2 by q-PCR. The results showed that the expression of TEAD1, TEAD2, FZD4, FZD5, GLI2, and CCNE2 was significantly decreased after knockdown of RAE1, while CASP7 and RYR2 expression was increased, which was consistent with the transcriptome sequencing results (Figure 5C).

Figure 5 RAE1 is associated with GC development by regulating Hippo signaling pathway. (A) Volcano map of the differential genes after knockdown of RAE1 compared to controls. (B) Top 10 pathways analyzed for KEGG enrichment of down-regulated genes after RAE1 knockdown. (C) mRNA expression of individual genes after RAE1 knockdown using q-PCR. (D,E) Western blot analysis of YAP, β-catenin, and c-Myc expression after RAE1 knockdown and treatment with the Hippo agonist GA-017. *, P<0.05; **, P<0.01. DEG, differentially expressed gene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, gastric cancer; KEGG, Kyoto Encyclopedia of Genes and Genomes; q-PCR; RAE1, Ribonucleic acid export 1.

Since FZD is also a Wnt signaling pathway gene, and it can also affect TEAD expression and regulate the transcription of downstream genes through the key molecule β-catenin. Therefore, we verified the expression of YAP, β-catenin, and c-Myc by Western blots analysis. The results showed that the expression of β-catenin, YAP, and c-Myc decreased after knockdown of RAE1, while increased after overexpression of RAE1 (Figure 5D). To further test whether RAE1 acts through the Hippo pathway, we treated RAE1 knockdown cells with the Hippo agonist GA-017. Western blot analysis showed that the agonist enhanced the protein levels of β-catenin, YAP, and c-Myc (Figure 5E). Taken together, these results suggest that RAE1 may affect GC development associated with activation of the Hippo signaling pathway, at least in part.

Discussion

GC is a common tumor with a poor prognosis at an advanced stage. Therefore, it is of great significance to understand the potential mechanism for the development of GC and find new therapeutic targets to provide a reference for clinical therapy. RAE1 is required for mRNA export (14) and is associated with the viral infection (15), cell cycle, cellular senescence, and tumors. TCGA database shows that RAE1 expression is up-regulated in several cancers, including GC. However, the role and mechanism of RAE1 in GC are rarely studied. In this study, we found the expression of RAE1 is significantly higher in GC tissues than in PLs and normal tissues. RAE1 promotes the proliferation and migration of GC cells, affects the cell cycle, inhibits apoptosis, and may affect GC development by regulating signaling pathways such as Hippo.

Immunohistochemistry of GC tissue showed that RAE1 was expressed in both the nucleus and cytoplasm, and the expression of RAE1 in early GC tissues was significantly higher than in PL and normal tissues. Further, the expression of RAE1 was positively correlated with the clinical stage. Currently, there are limited studies on RAE1 immunohistochemical staining in tumor tissues. One study on breast cancer (12) included 98 patients whose tumor tissues were subjected to RAE1 immunohistochemical staining, a positive correlation between RAE1 expression and histological grade was found. RAE1 in gastric carcinogenesis and epithelial mesenchymal transition (EMT) was also reported in GC (16). In addition, since overexpression of antigen is one of the main mechanisms of autoantibody production (17), we also found a positive correlation between RAE1 overexpression and its autoantibody level. However, due to the small sample size, there was no statistical significance and the data were not shown in the paper, further research is needed.

To explore the effects of RAE1 on the behaviour of GC cells, we applied siRNA transfection and lentiviral infection to construct GC cells with knockdown and overexpression of RAE1. Both in vivo and in vitro experiments demonstrated that knockdown of RAE1 inhibited the proliferation of GC, whereas overexpression of RAE1 promoted the proliferation of GC cells. Apoptosis assay suggested that knockdown of RAE1 promoted apoptosis in GC cells, whereas overexpression of RAE1 showed the opposite trend. Transwell assay showed that knockdown of RAE1 inhibited the migration of AGS cells, while overexpression of RAE1 increased the migration of AGS cells. The migration of HGC-27 cells was not statistically significant, which may be related to the different genetic backgrounds. Our results are consistent with previous studies indicating RAE1 promotes nitrosamine-induced malignant transformation of human esophageal epithelial cells (18), and promotes the invasion and migration of breast cancer by inducing EMT (12). EMT plays a key role in the metastasis of cancers, contributing to the development of epithelial cells with the ability of mesenchymal cells, thereby enhancing cell migration. It is characterized by the absence of markers for epithelial cells, such as E-cadherin, and upregulation of markers for mesenchymal cells, such as N-cadherin, vimentin, and Snail (19).

It has been shown that the absence of RAE1 in Drosophila results in decrease of the cell cycle proteins such as Cyclin A and Cyclin B, whereas overexpression of RAE1 increases the levels of Cyclin A and Cyclin B (20). Another study reported that knockdown of RAE1 inhibits the transition from G1 to S phase in colorectal cancer cells (10). In this study, we found that the percentage of S phase in HGC-27 cells was increased significantly after knockdown of RAE1, and the percentage of G2/M phase was decreased significantly, suggesting that the cell cycle was blocked in S phase after knockdown of RAE1. Cyclin and CDK interact for cell cycle progression, including Cyclin A/CDK2 in the S phase, Cyclin D/CDK4, Cyclin D/CDK6, and Cyclin E/CDK2 in the G1 to S phase transition, and Cyclin B/CDK1 in the G2 to M phase transition (21). Our results showed that knockdown of RAE1 reduced the level of Cyclin A1/A2 and CDK2, which blocked the cell cycle in S phase.

In this study, we also explored the pathways involved in the development of GC by transcriptome sequencing and KEGG enrichment analysis, and found that the down-regulated genes were mainly enriched in the Hippo signaling pathway after knocking down RAE1. In mammals, the core components of the Hippo signalling pathway include MST1/2, SAV1, LATS1/2 and MOB1. SAV1 and MOB1 are able to bind MST1/2 and LATS1/2, respectively, thereby enhancing their phosphorylation activity (22). YAP is a key effector molecule at a downstream position in the signalling pathway and can be phosphorylated by LATS1/2. The phosphorylated YAP binds to proteins in the cytoplasm, and then remains in the cytoplasm to be ubiquitinated and degraded, which inhibits the pro-growth and anti-apoptotic functions of YAP, thus negatively regulating the activity of YAP by the Hippo pathway (23). When the Hippo pathway is inhibited, YAP enters the nucleus and binds to transcription factors such as TEADs to promote the expression of downstream genes, thus contributing to cell proliferation. Therefore, TEADs are important transcription factors for YAP to regulate biological processes such as cell proliferation and EMT (24). Deficiency of Hippo signalling leads to over-activation of YAP and TAZ functions, which promotes the proliferation of tumour cells and is associated with a variety of cancers. In addition, YAP plays an important role in cell migration and invasion, and highly active YAP/TAZ helps cells escape cell-to-cell contact inhibition and apoptosis, thus providing favourable conditions for tumour metastasis (25,26).

Our results showed that knockdown of RAE1 in AGS cells led to a significant decrease in the expression of TEAD1, TEAD2, and YAP, while YAP was significantly increased in GC cells after overexpression of RAE1. c-Myc is one of the downstream target genes, which is associated with cell proliferation. We verified c-Myc by Western blot and found that the trend was consistent with YAP. A study has reported that there is a mutual regulatory relationship between the Hippo signalling pathway and the Wnt signalling pathway (27). Specifically, YAP/TAZ in the cytoplasm can bind to FZD, a positive regulator upstream of the Wnt signalling pathway, thus inhibiting its activity. In the nucleus, YAP/TAZ can help to promote the binding of β-catenin to transcription factors and enhance the expression of its downstream target genes. On the other hand, the Wnt signalling pathway may also activate YAP/TAZ. Wierzbicki et al. (28) indicated that YAP/TAZ can be regarded as a target of the Wnt signalling pathway, which can be negative feedback regulated, thus preventing over-activation of the Wnt signalling pathway. Our study showed that knockdown of RAE1 decreased the expression of FZD4, FZD5, and β-catenin, while β-catenin expression was increased after overexpression of RAE1. In addition, qPCR also showed that knockdown of RAE1 decreased the expression of CCNE2 and increased CASP7, which was associated with cell cycle and apoptosis, respectively. The results also demonstrated knockdown RAE1 inhibited proliferation and promoted apoptosis of GC cells, which was corroborated by the behavioral experiments. It was report that RAE1 promotes gastric carcinogenesis through the ERK/MAPK pathway and EMT (16), suggesting that RAE1 may exert its oncogenic functions via multiple signaling cascades.

There are some limitations in this study. Firstly, the sample size was small, we only detected the immunohistochemical expression of RAE1 in the tissue of 43 patients with early GC and PL. Secondly, this study did not knock down or overexpress RAE1 in normal gastric epithelial cell GES-1, which limited to investigate whether RAE1 promotes the malignant transformation of GES-1, and it is essential to explore whether RAE1 affects tumor initiation or progression, using depletion of RAE1 in established tumors in vivo. Thirdly, our current cohort did not include patients with metastatic GC. Future studies with a larger cohort encompassing all stages, including stage IV patients, are warranted to fully characterize the correlation between RAE1 expression and GC progression. Finally, although we explored the possibility that RAE1 may affect tumor proliferation through signaling pathways such as Hippo, further exploration of the mechanism of RAE1 in the development and progression of GC is still needed by using multiomics methods and molecular interaction analysis to illustrate how mechanistically RAE1 intersects with the Hippo pathway or Wnt signaling.

In conclusion, the expression of RAE1 in early GC tissues was significantly higher than that in PL and normal tissues, and RAE1 promotes the proliferation and migration of GC cells, affects the cell cycle, inhibits apoptosis, and may affect GC development by regulating signaling pathways such as Hippo.

Conclusions

RAE1 promotes GC cells proliferation and migration and is associated with the inhibition of Hippo signaling pathway, and may be a potential biomarker for early diagnosis and treatment of GC.

Acknowledgments

We thank the patients for providing their samples for this study. We acknowledge the Beijing Friendship Hospital, Capital Medical University for sample collection.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-1-1019/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-1-1019/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-1-1019/prf

Funding: This work was funded by Grants from the National Major Science and Technology Projects of China (No. 2017ZX10201201) and the Digestive Medical Coordinated Development Center of Beijing Hospitals Authority (No. XXZ0103).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-1-1019/coif). All authors report research grants from the National Major Science and Technology Projects of China (No. 2017ZX10201201) and the Digestive Medical Coordinated Development Center of Beijing Hospitals Authority (No. XXZ0103); and report a patent (Patent No. ZL202310422419.5) related to gastric cancer autoantibody detection. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Beijing Friendship Hospital, Capital Medical University (No. 2018-P2-058-01). All participants provided written informed consent. All animal experiments complied with the ARRIVE guidelines and were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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