Prognostic significance and immune characteristics of GPR27 in gastric cancer

Xena Shiny

Xena Shiny is an easy-to-use database for quickly searching, analyzing and visualizing data from UCSC Xena data hubs [10]. We used Xena Shiny to explore the expression of GPR27 in pan-cancer and its association with TMB and MSI.

GEPIA 2

GEPIA 2 is a web-based tool for in-depth analysis of transcriptome data in TCGA database [11]. It was used to investigate GPR27 expression in GC.

MethSurv

MethSurv [12], is a network tool for multivariate survival analysis using DNA methylation data, which we used to explore survival and correlation between 12CpG and GPR27 gene methylation. We defined patients as high groups and low groups according to methylation medians.

cBioPortal

cBioPortal contains large-scale cancer genomics datasets and provides capabilities including visualization, downloading, and analysis [13]. cBioPortal was used to explore mutation of GPR27 in GC and survival analysis of GC patients between GPR27 mutation group and unaltered group.

Kaplan–Meier plotter

KM plotter was applied to investigate the prognostic worth of GPR27 in GC [14]. GC patients were defined as high and low groups according to median GPR27 level, then the HRs, 95% CIs, and logarithmic rank P values of overall survival (OS) and disease-free survival (DFS) were examined.

TIMER

TIMER was applied for investigation of various immune cells’ infiltration levels [15]. Firstly, we applied the “Diff Exp” module to examine the expression level of GPR27 in all human malignancies. Then we used the “Gene” module to examine the correlation between GPR27 mRNA level and infiltration levels of immune cells using the dataset we gained from TCGA. Finally, we applied the “Correlation” module to examine the correlation between GPR27 mRNA level and immune cells’ markers.

TISIDB

TISIDB is a website for gene-immune and tumor-immune interaction analysis [16]. It was applied to investigate GPR27 mRNA level in different molecular subtypes and immune subtypes.

ImmuneCellAI

The Immune Cells Abundance Identifier (ImmuCellAI) is a novel network tool for assessing the number of immune cells, focusing on T cell subsets associated with tumor progression and elicitation [17]. Using transcription data from GC in TCGA, the distinction in abundance of 24 immune cells in the low group and high group of GPR27 was estimated.

The human protein atlas

HPA is a public database launched in 2003 that uses a variety of holographic techniques to detect various human proteins. This database consists of 10 separate sections, and we explored the Single Cell section to identify which immune cells express GPR27 via single-cell sequencing analysis.

Collection of GC tissues

We purchased a total of 180 paraffin-embedded tissue arrays (HStmA180Su19-M-066) from Shanghai Outdo Biotech Co, Ltd, including 97 gastric tumors and 83 normal tissues, with 5-year follow-up information. All clinical samples were clustered under patients’ informed consent. Our study plan was checked and accepted by the Ethics Committee of Taihe Hospital of Hubei University of Medicine (2022KS44) and performed based on the Declaration of Helsinki.

Immunohistochemical

Immunohistochemistry (IHC) staining was conducted to explore GPR27 protein levels in gastric cancerous tissues and adjacent gastric normal tissue as reported [18]. Firstly, we deparaffinized the tissue sections by immersing them in xylene and rehydrating them using a series of graded alcohols. Then, we performed antigen retrieval to enhance antigen accessibility. We heated the sections in a 10mM citrate buffer (pH 6.0) for 20 mins. Subsequently, we blocked the sections with 3% H₂O₂ solution to prevent nonspecific binding. We incubated the tissue sections with anti-GPR27 protein antibody (1:300 dilution, No. bs-13528R, Bioss, Beijing, China) in 4° C refrigerators overnight. Then we washed the sections multiple times with PBS buffer to remove any unbound antibodies. Next, we incubated the tissue section with the secondary antibody. We developed the sections using 3, 3’- diaminobenzidine (DAB) and counterstained the sections with hematoxylin.

Immunohistochemical scoring

IHC scoring is a method used to quantify the staining intensity and distribution of a target protein in tissue samples. We applied a semi-quantitative scale in our study that takes into account staining intensity and staining scope. Positive staining of GPR27 protein refers to cytoplasmic staining of GC cells. Staining intensity may be categorized as 0 point (absent), 1 point (weak), 2 points (moderate), and 3 points (strong), while the staining scope can be categorized as 1 point (1%–25%), 2 points (26%–50%), 3 points (51%–75%), and 4 points (76%–100%). The final IHC scores of each tissue were obtained by the multiplication of the scores for staining intensity and staining scope. Then we defined GC patients as low GPR27 protein and high GPR27 protein groups according to the median value of GPR27 protein level in all those GC patients.

Statistical analysis

Our analysis was conducted using SPSS software (version 21) and R software (version 3.5.1). Student’s t-tests were applied to examine the statistical significance of the GPR27 immunohistochemical (IHC) scores between GC tissues and adjacent tissues. To assess the correlations between GPR27 mRNA expression and clinical characteristics of GC, we utilized either Chi-square or Fisher’s exact test, depending on the specific circumstances. To present the associations between survival outcomes and GPR27 mRNA, DNA methylation, and protein expression, we utilized Kaplan-Meier survival analysis. A P value of less than 0.05 was considered statistically significant.

Data availability statement

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

GPR27 has low expression in several cancerous tissues

Firstly, we investigated GPR27 mRNA expression level in various human cancers through TIMER (Figure 1A) and Xena Shiny (Figure 1B). Results show that GPR27 is lowly expressed in several cancerous tissues including STAD, COAD, READ, LUAD, GBM, KIRC, KICH, KIRP, HNSC and UCEC. By contrast, GPR27 mRNA was highly expressed in CHOL and PCPG. Then we investigated the GPR27 mRNA levels between GC tissues and normal gastric mucosa basing on data gained from TCGA dataset, and results show that GPR27 mRNA was down-regulated in GC tissues than in normal gastric mucosa (Figure 1C). GPR27 mRNA level in different molecular subtypes of STAD, ESCA, KIRP, LGG, LUSC, BRCA, OV and PCPG were significantly different (Figure 1D).

Figure 1. The transcription levels of GPR27 in human cancers. GPR27 mRNA expression in pan-cancer from TIMER (https://cistrome.shinyapps.io/timer/) (A) and Xena Shiny (https://shiny.hiplot.com.cn/ucsc-xena-shiny/) (B) and gastric cancer (C). The expression of GPR27 in different molecular subtypes of cancers via TISIDB (http://cis.hku.hk/TISIDB/index.php) (D).

GPR27 mutation correlates with GPR27 expression and GC patients’ survival

cBioPortal was applied to analyze genetic alteration of GPR27 in GC patients. The results show that missense mutation of GPR27 was found in 4% of GC patients (Supplementary Figure 1A–1C). The mutation of GPR27 was well correlated with some clinical indices, such as TMB and gender (Supplementary Table 1). Next, we explored the correlation between GPR27 mutation and GC patients’ survival. We found that GC individuals with GPR27 alteration showed worse OS (log rank P=0.0103, Supplementary Figure 1D) and DFS (log rank P=0.0116, Supplementary Figure 1E) than those without GPR27 alteration.

GPR27 mRNA expression level correlates with GC patients’ survival

We examined the prognostic worth of GPR27 in GC via K-M Plotter. Firstly, we analyzed TCGA-STAD cohort and found that GC patients with lower level of GPR27 had a longer OS (HR=1.63, 95%CI:1.13-2.34, P=0.0083, Figure 2A) and DFS (HR=3.12, 95%CI:1.47-6.63, P=0.0018, Figure 2B). To further authenticate our conclusion, we also conducted survival analysis in GEO cohort. Based on 875 GC patients, we gained a similar conclusion: GC patients with lower expression of GPR27 exhibited longer OS (HR=1.61, 95%CI:1.34-1.95, P=5.7e-07, Figure 2C) and DFS (HR=1.42, 95%CI:1.16-1.74, P=0.00072, Figure 2D). Furthermore, we examined the association between GPR27 mRNA level and survival in GC patients with various clinical metrics using Kaplan-Meier plotter (Table 1).

Figure 2. Survival analysis of GPR27 mRNA in gastric cancer. Low levels of GPR27 are correlated with longer overall time (A) and disease-free survival time (B) based on TCGA dataset. Low levels of GPR27 are related to longer overall time (C) and disease-free survival time (D) based on KM Plotter (http://kmplot.com).

Subgroup survival analysis stratified by tumor mutation burden (TMB) in GC

Xena Shiny was used to calculate TMB for each sample. Then we analyzed correlations between GPR27 mRNA level and TMB, and GPR27 methylation level and TMB. The results show that the GPR27 mRNA level is inversely linked to TMB (r=-0.267, P<0.0001, Supplementary Figure 2A), while GPR27 methylation level is positively linked to TMB (r=0.327, P<0.0001, Supplementary Figure 2B). In survival analysis of low-TMB group, GC patients with lower expression of GPR27 displayed longer OS (HR=1.34, 95%CI:0.85-2,13, P=0.21, Supplementary Figure 2C) and DFS (HR=2.52, 95%CI:1.02-6.21, P=0.037, Supplementary Figure 2D). While high TMB group survival analysis showed the same results: GC patients with lower expression of GPR27 exhibited longer OS (HR=1.76, 95%CI:1.08-2.85, P=0.02, Supplementary Figure 2E) and DFS (HR=3.5, 95%CI:1.27-9.61, P=0.01, Supplementary Figure 2F).

Epigenetic regulation of GPR27 in GC

We applied the UCXC Xena to investigate the epigenetic regulation of GPR27 in GC patients and found that the methylation level of the GPR27 promoter was inversely linked to the transcription volume of GPR27 (Figure 3A). Therefore, Spearman correlation analysis was employed to examine the linkage between GPR27 promoter methylation level and GPR27 transcription level. There was a negative correlation (r=-0.6178 P<0.0001) between GPR27 promoter methylation and GPR27 transcription in GC (Figure 3B). Moreover, survival analysis was performed and results show that hypermethylation of GPR27 is correlated with relatively superior OS and DFS in GC patients (Figure 3C). Spearman correlation analysis was employed to evaluate the linkage between GPR27 mRNA level and 12CpG sites of GPR27 DNA promoter in GC. As shown in the correlation plots (Supplementary Figure 3), the cg22823146CpG site exhibited the strongest association with GPR27 mRNA level in GC (r=-0.6639, P<0.0001). We also explored the associations of the 6 CpG sites with clinical characteristics, including ethnicity, sex, age and prognosis (Supplementary Figure 4A). We conducted survival analysis and results show that GC patients with hypermethylation of cg10172415, cg03024619 and cg13562542 exhibit better survival outcome than those with hypermethylation of GPR27 (Supplementary Figure 4B–4G).

Figure 3. Prognostic significance of GPR27 methylation in gastric cancer. The heat map unveils that high expression of GPR27 corresponds to low DNA methylation in gastric cancer (A). A strong correlation between GPR27 expression and DNA methylation is noticed in gastric cancer (B). Hypermethylation of GPR27 is correlated with relatively superior overall survival (C) and disease-free survival (D) in sufferers with gastric cancer.

Correlation between GPR27 mRNA level and tumor-immune microenvironment

Using TISIDB, we analyzed the expression pattern of GPR27 mRNA levels in several immune subtypes. We found a significant difference in GPR27 expression pattern across immune subtypes in LGG, LUSC, BLCA, PCPG, STAD, SARC and UVM (Figure 4A). While in other cancers, we did not observe clear differences (Supplementary Figure 5).

Figure 4. GPR27 is associated with immune infiltration in several human cancers. (A) GPR27 mRNA expression in different immune subtypes in BLCA, LGG, LUSC, PCPG, STAD, SARC, UVM from TISIDB (http://cis.hku.hk/TISIDB/index.php), (B) The fraction of tumor infiltrating immune cells in GPR27 high and low subgroups.

To examine the association between GPR27 and immune cells, we applied ImmuCellAI to investigate the levels of 24 types of tumor infiltrating immune cells in GC. We found the proportion of B cells, CD4 naïve cells, CD4 T cells, central memory cells, iTreg cells, MAIT cells, nature killer cells, nTreg cells, Tfh cells, and TH17, Th2, and Tr1 cells was increased in the GPR27-high subgroup, whereas the proportion of neutrophils, macrophages, gamma delta cells, and cytotoxic cells was increased in the GPR27-low subgroup (Figure 4B).

We examine the association between GPR27 mRNA levels and six types of immune cells, and found that GPR27 mRNA level is positively linked to the infiltration of all those six types of immune cells in GC (Figure 5A). We examined the linkage between GPR27 mRNA level and various immune characteristics in GC. The genes listed in Tables 2, 3 were the markers of corresponding immune cells. Results show that GPR27 mRNA level was remarkably correlated with majority markers of immune cells in GC (Figure 5B). Finally, for the purpose of which cells express GPR27, we mined the human protein atlas database. Single-cell sequencing analysis reveals that GPR27 is mainly expressed in macrophages, followed by gastric mucus secreting cells (Supplementary Figure 6).

Figure 5. Correlation between GPR27 and immune cells from TIMER (https://cistrome.shinyapps.io/timer/). (A) The correlation of the expression of GPR27 with the infiltration of different immune cells. (B) The correlation of the expression of GPR27 and gene markers of immune cells.

Clinical validation with 97 GC cases

We utilized 97 GC patients to explore GPR27 protein level in GC patients. We performed semi-quantitative analysis and results showed a significant reduction in GPR27 staining intensity in GC tissues (Figure 6A, 6B). Furthermore, we utilized the Chi-square test to examine the clinical features between the GPR27 protein low and high group. As shown in Supplementary Table 2, significant differences were observed in tumor stage (P=0.0016) and distant metastasis (P=0.0335) between low-GPR27 and high-GPR27 groups.

Figure 6. GPR27 is downregulated in gastric cancer clinical samples and correlated with longer survival in gastric cancer patients. (A) Immunohistochemical staining of normal and gastric cancer tissues with anti-GPR27 antibody. (B) Quantitative analysis of GPR27 staining shows significantly H-score in gastric tumor samples compared with adjacent normal tissues (83 normal tissues and 97 tumor samples). (C) Gastric cancer patients with GPR27 over-expression displayed less favorable overall survival than those with low GPR27 expression.

Survival analysis showed that GC patients with lower GPR27 protein level had a longer OS times (HR=0.6354, 95%CI:03919-1.030, P=0.0087, Figure 6C). In conclusion, we conclude that high levels of GPR27 protein are a risk factor for poor prognosis in GC patients, which is consistent with our conclusions from the TCGA and GEO databases.