# GNAI2 Is a Risk Factor for Gastric Cancer: Study of Tumor Microenvironment (TME) and Establishment of Immune Risk Score (IRS)

Authors: Han Yu; Sha Liu; ZuGuang Wu; FenFei Gao
Journal: Oxidative Medicine and Cellular Longevity
 (2022)

---

## Abstract

Purpose. Although the G protein subunit α i2 (GNAI2) is upregulated in multiple cancers, its prognostic value and exact role in the development of gastric cancer (GC) remain largely unknown. Methods. This study evaluated the effect of GNAI2 on the tumor microenvironment (TME) in GC, constructed an immune risk score (IRS) model based on differentially-expressed immune genes, and systematically correlated GNAI2 and epigenetic factor expression patterns with TME and IRS. Also, RT-qPCR, flow cytometry, Western blotting (WB), and transwell assays were carried out to explore the regulatory mechanism of GNAI2 in GC. Results. High GNAI2 expression was associated with poor prognosis. Cytokine activation, an increase in tumor-infiltrating immune cells (TIIC), and the accumulation of regulatory T cells in the tumor immune cycle were all promoted by the TME, which was significantly associated with GNAI2 expression. Two different differentially expressed mRNA (DER) modification patterns were determined. These two DERs-clusters had significantly different TME cell infiltrations and were classified as either noninflamed or immune-inflamed phenotypes. The IRS model constructed using differentially expressed genes (DEGs) had great potential in predicting GC prognosis. The IRS model was also used in assessing clinicopathological features, such as microsatellite instability (MSI) status, epithelial-mesenchymal transition (EMT) status, clinical stages, tumor mutational burden (TMB), and tumor immune dysfunction and exclusion (TIDE) scores. Low IRS scores were associated with high immune checkpoint gene expression. Cell and animal studies confirmed that GNAI2 activated PI3K/AKT pathway and promoted the growth and migration of GC cells. Conclusion. The IRS model can be used for survival prediction and GNAI2 serves as a candidate therapeutic target for GC patients.


---

## Body

## 1. Introduction

Gastric cancer (GC) is a common malignant tumor. The interaction between GC cells and their microenvironment (tumor microenvironment, TME) causes them to continuously proliferate and resist growth inhibition, resulting in a high recurrence rate and a poor prognosis [1]. The new treatment methods and strategies partly increase the overall survival (OS) time for GC patients, but drug tolerance and tumor heterogeneity significantly worsen patient prognoses. Resultantly, it is very important to identify more effective treatment strategies to improve the OS of GC patients.Immunotherapy for cancer, including immune checkpoint blocking (ICB), has achieved some success in the field of gastric cancer treatment [2]. Immune checkpoint inhibitors (ICIs) have been shown to improve patient response and prolong survival by normalizing the immune system, whether in a single cancer type or a joint cohort of multiple cancer types [3–6]. Notably, in tumors with the inflammatory TME, tumor immunogenic genes can also be targeted by immune checkpoint blocking (ICB) to promote the recruitment of tumor-infiltrating immune cells (TIIC) and enhance tumor remission [7]. Considering the huge economic burden and adverse side effects of tumor treatment, a combined study of TME and cancer immunotherapy can significantly advance the treatment of GC [8].G protein subunitα i2 (GNAI2) belongs to the isotrimeric guanine nucleotide-binding protein (G protein) family [9]. Existing studies suggest that high GNAI2 expression can promote the occurrence and development of epithelial intestinal t-cell lymphoma, colitis-associated cancer, and ovarian cancer [10–12]. GNAI2 can also inhibit the proliferation of tongue squamous cell carcinoma [9] and hepatocellular carcinoma [13]. However, its role in the occurrence and progression of GC and its TME remains unknown.This study analyzed the expression pattern and immunological effects of GNAI2 using pan-cancer analysis and elucidated the effect of GNAI2 on GC TME inflammation, as well as GC cell growth and migration. In addition, an immune risk score (IRS) system was established to quantify patient prognostic outcomes. The findings suggest that anti-GNAI2 therapy is suitable for GC.


## 2. Material and Methods

### 2.1. Retrieval and Preprocessing of the Dataset

RNA sequencing (RNA-seq) (namely, the FPKM value) data were downloaded from UCSC Xena (https://xenabrowser.net/datapages/). Tumor diseases with fewer than 20 normal samples were excluded, and expression data, including GNAI2 expression, were selected from 21 malignant tumors. GC RNA-seq data were retrieved from the Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga), whereas several microarray data (GSE29272, GSE26901, GSE33335, GSE54129, GSE79973, GSE13861, GSE13911, and GSE26899) related to GC were retrieved from the NCBI Gene Expression Omnibus (GEO) database. Normal tissue data were retrieved from the GTEx database (Genotype-Tissue Expression, https://gtexportal.org/). GNAI2 expression data in tumor cells were retrieved from the Cancer Cell Line Encyclopedia (CCLE) project. Using the meta package in the R software, a random effects model was adopted for meta-analysis. In the Cox regression model, logarithmic rank test and Kaplan-Meier analysis were conducted to analyze OS, disease-specific survival (DSS), and progression-free survival (PFS) rates for the high and low GNAI2 expression groups.The copy number variation (CNV) data were retrieved from the UCSC Xena data portal, and the GISTIC 2.0 algorithm was used to analyze the CNV pattern of GC with differential clustering. Then, methylation data were obtained from the LinkedOmics data portal, and the methylated genome data were processed using weighted gene co-expression network analysis (WGCNA) before clustering into modules marked by different colors. In addition, a somatic mutation was downloaded from the TCGA database and analyzed with maftools R package.TCGA-stomach adenocarcinoma (STAD) cohort, two GC GEO cohorts (GSE62254 and GSE84437), and two immunotherapy-related cohorts (GSE78220 and IMvigor210) were downloaded. Detailed information about these datasets is summarized in TableS1.


### 2.2. Correlation between GNAI2 and TME Cell Infiltration

First, TCGA data on 105 immunomodulators collected in Charoentong et al.’s work [14] (including MHC, receptors, chemokines, and immune stimulators) (Table S2), we performed a pan-cancer analysis to explore the correlation between GNAI2 and PD-1, PD-L1, and CTLA-4 as well as LAG-3 within GC (Table S3). In addition, data relating to the infiltration level of T lymphocytes, M1/M2 macrophages, CD8+ T cells, and B cells were obtained, and the differences in effector genes in these five TIICs of high versus low GNAI2 expression groups were analyzed (Table S4).Next, the differential expression and activity of GNAI2 in the cancer immune cycle (CIC) were analyzed. The immune response to antitumor therapy, or CIC, involves 7 steps: (1) antigen release from dead tumor cells, (2) presentation of the cancer antigen, (3) initiation and activation of T cells, (4) migration of cytotoxic T lymphocytes (CTLs) into cancer site, (5) tumor infiltration by CTLs, (6) identification of cancer cells, and (7) immune-mediated cancer cell killing. Chen et al. [15] found that cancer cell fate was determined by the activities of the aforementioned steps.Thereafter, 20 suppressed immune checkpoints previously considered to have a potential [16] in treatment were collected for correlation analysis with GNAI2 in RNA level. Reverse phase protein array (RPPA) data in STAD, downloaded from the Cancer Proteome Atlas (TCPA), was explored to analyze the correlation between GNAI2 and the 20 suppressed immune checkpoints at the protein level. The Human Protein Atlas (HPA, http://www.proteinatlas.org/) online database was explored to validate the CD86, CD276, PD-L1, IDO1, and CD200 protein expression in GC using immunohistochemical staining.Finally, the immune and stromal scores calculated based on the ESTIMATE algorithm can predict the purity of tumor cells. A single sample gene set enrichment analysis (ssGSEA) algorithm was used to quantify the relative abundance of each cell infiltration in the GC TME. In addition, to accurately evaluate TIIC penetration in GC and calculation errors initiated by different algorithms and TIIC marker gene sets, six algorithms were used (Cibersort-ABS, MCP-counter, quanTIseq, TIMER, xCell, and TIP) to calculate TIIC penetration using the IOBR R package (TableS5) [17–22].


### 2.3. Identification of Differentially Expressed mRNAs (DERs) Associated with the GNAI2 Immune Microenvironment and Functional Annotation

Samples were divided into different groups based on the median level of GNAI2, interstitial, and immune scores. DERs were identified using the limma package, and a volcanic map was used to visualize the DERs. Using adjustedP<0.05 and logFC>0.8as the criteria for determining differential DERs, the common DERs were obtained and analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Gene Set Enrichment Analysis (GSEA). The clusterProfiler package was used to perform the GSEA, KEGG, and GO analyses for identifying possible pathways and functional annotation (Table S6).


### 2.4. Unsupervised Clustering for GNAI2-Associated Immune Microenvironment in GC and Gene Set Variation Analysis (GSVA)

ConsensusClusterPlus R packages were used to classify various cluster patterns associated with the GNAI2 immune microenvironment based on the expression of DERs, and 1000 repetitions were carried out to ensure classification stability. To explore the biological processes and final results in different cluster patterns, the gene sets of “c2.cp.kegg.v6.2.symbols” were downloaded from the MSigDB database for running GSVA analysis (TableS7).


### 2.5. Construction and Verification of IRS Model

According to the patient inclusion to the experiment, the GSE62254 dataset was classified as the verification and training sets at the 3 : 7 ratio. In the training set, common DERs were analyzed by univariate Cox analysis, and then, the optimal candidate DERs were screened using the LASSO algorithm. The best candidate DERs were selected using multivariate Cox regression coefficients, and an IRS model was developed based on the IRS RNA expression patterns and weighted using the formula:IRS=∑βi∗RNAi, where βi represents the coefficient of expression pattern for the ‘ i’th IRS RNA. Thereafter, the logarithmic rank test and Kaplan-Meier analysis were used to evaluate the prognosis prediction value of the IRS model. In addition, based on the GSE62254 verification set, TCGA external cohort, and GSE84437 dataset, the prognostic prediction efficacy of the IRS model was verified. The significance of the IRS model was evaluated using the timeROC package. The IRS model was also used to predict the survival rate of the IMvigor210 cohort subgroup.TIDE algorithm related to T cell function can accurately predict the efficacy of immunotherapy [23]. Next, the accuracy of the IRS and TIDE algorithms in predicting survival probability between the GSE78220 immunotherapy cohort and the TCGA dataset was compared. These datasets were divided into low and high IRS groups based on the cutoff point determined by the “surv-cutpoint” function in the survminer package to explore the significance of IRS in predicting clinical prognosis.


### 2.6. Clinical Specimens and Cell Culture

From 2019 to 2020, 20 GC tissues and matched paracancerous tissue specimens from patients with GC in Meizhou People’s Hospital who underwent surgical resection were collected. All patient specimens were verified by a pathologist. None of the patients had been diagnosed with other tumor diseases or had undergone other related surgery, and none had been treated with preoperative targeted therapy, biotherapy, or chemotherapy. Informed consent was obtained from all patients and their families. The study protocols were approved by the Ethics Committee of Meizhou People’s Hospital (Approval NO. 2019-C-80).The human GC MKN-7, NCI-N87, Hs-746T, and HGC27 cell lines and Genetic Suppressor Element 1 (GSE-1), provided by Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China), were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (NCM Biotech, Suzhou, China) at 37°C in a 5% CO2 incubator. The MycoAlert™ Mycoplasma Detection Kit (Lonza, Basel, Switzerland) was used to test the negative mycoplasma contamination in all cells before experimentation.


### 2.7. Construction and Transfection of Lentivirus

The full-length cDNA for GNAI2 was introduced into the pLVX-IRES-ZsGreen1 vector to construct a GNAI2-overexpressing lentivirus. Two lentivirus shRNAs targeting GNAI2 were inserted into the pLVX-shRNA-puro vector in constructing the GNAI2-silencing lentivirus. All plasmids were verified by sequencing. Thereafter, the cells (5×105/well) were inoculated into 6-well plates (NEST Biotechnology Co., Ltd, Wuxi, China) and cultured for 24 or 48 h before transfection. HGC27 and Hs-746T cells were transfected with plasmids and siRNAs using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to specific protocols. At 48-h posttransfection, gene expressions were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting (WB) assays. RNA separation, total cellular protein isolation, and cell functional analysis were also carried out 48-h posttransfection.


### 2.8. Detection of mRNA Level of GNAI2 Using RT-qPCR

Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract the total cellular RNA, which was later prepared in cDNA by reverse transcription using the PrimeScript RT Master Mix (538100; Toyobo, Osaka, Japan). Thereafter, the Applied Biosystems 7900HT real-time PCR system (Foster City, CA, USA) was used for qPCR through SYBR-Green PCR Master Mix (15153900; Roche, Basel, Switzerland). The cDNA concentration was adjusted to 30 ng/μL. Fluorescence qPCR was performed as follows: 2 min at 95°C, then 15 s at 95°C, and 30 s at 60–68°C for 40 cycles, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) being the endogenous reference gene. Fold changes (FCs) in the gene level were determined using the 2-ΔCt approach. All assays were carried out in triplicate. The primer sequences for GNAI2 were forward primer: TACCGGGCGGTTGTCTACA and reverse primer: GGGTCGGCAAAGTCGATCTG.


### 2.9. Western Blotting (WB)

Antibodies (rabbit antihuman antibodies) to GNAI2, Ki67, PCNA, AKT, P-AKT, PI3K, P-PI3K, and GAPDH were provided by Abnova (Taipei, China), Proteintech (Rosemont, IL, USA), and Abcam (Cambridge, UK). Cell lysis buffer (Pierce, Rockford, IL, USA) was employed in extracting total protein. Thereafter, 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used in separating the cell lysates. The separated lysates were then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Subsequently, 5% skimmed milk powder was utilized to block the membranes for 2 h, followed by incubation using specific primary antibodies at 4°C overnight and another 2 h of incubation with secondary antibodies after dilution. The DNR Bio Imaging System (DNR, Jerusalem, Israel) was used to detect protein bands ECL (Pierce). GAPDH was the reference gene used for protein expression normalization. The PI3K antibodies used for the study detected the PI3K enzyme catalytic subunit gamma.


### 2.10. Cell Cycle Detection Using Flow Cytometry

After trypsin digestion, the transfected cells were suspended at a density of3×104 cells/mL, followed by propidium iodide (PI) staining (BD Biosciences, San Jose, CA, USA) and 30 min of treatment with 0.5 mg/mL RNase A (Keygen Biotech, Nanjing, China) supplemented with phosphate-buffered saline (PBS) at 37°C, and subsequent inoculation into 4 plates for detection at 5 time points. After collection, washing, fixation, RNA removal, and staining, approximately 1.5×105 cells were measured by flow cytometry. Then, the FlowJo software (TreeStar, Ashland, OR, USA) was employed for cell cycle analysis. Experiments were carried out in triplicate.


### 2.11. Measurement of Invasive Ability of Cells by Transwell Assay

After 24 h of culture within the serum-free basic medium, the transfected cells were suspended at a density of1×105 cells/mL. Thereafter, 0.3 mL of the cell suspension was inoculated into the top transwell chamber (BD Biosciences, San Jose, CA, USA). Then, all wells were added with a complete culture medium of 0.7 mL that contained 10% FBS in the bottom transwell chamber. Later, the transwells were cultured at 37°C for 24 hours, fixed, washed, and stained, and then, cells remaining in the upper chamber were removed. The number of invasive cells was calculated in 8 random fields of view (FOVs) with a microscope, followed by imaging.


### 2.12. Measurement of In Vitro Cell Proliferation by Cell Counting Kit-8 (CCK-8) Assay

To determine the effect of GNAI2 on cell proliferation, cells were inoculated into four 96-well plates, followed by incubation with the CCK-8 (Dojindo, Kumamoto, Japan) reagent in a normal medium at 37°C until a color change occurred. After transfection for 0, 24, 48, and 72 h, the absorbance at 450 nm was measured using a microplate reader. The culture medium was used as the blank control for calibration. Measurements were performed in triplicate.


### 2.13. Subcutaneous Tumorigenesis in Nude Mice

The 5-6-week-old male athymic BALBC/c nude mice were provided by the Hunan Acura Biotechnology Co., Ltd. (Changsha, China). All animals were housed in a specific pathogen-free (SPF), temperature-controlled environment and fed exclusively with autoclaved water and sterilized food.Each animal experiment was carried out according to guidelines from the Ethics Committee of Meizhou People’s Hospital. The density of HGC27 cells transfected with the blank plasmid or GNAI2 plasmid was adjusted to4×107 cells/mL after trypsin digestion. Twelve BALB/c nude mice were randomized into 2 groups containing six mice each. Thereafter, the control nude mice were subcutaneously injected with HGC27+LV-NC cells (0.1 mL; 4×106 cells), whereas the experimental nude mice were subcutaneously injected with HGC27+LV-GNAI2 cells. On day 26, all mice were euthanized by cervical dislocation, followed by imaging and tumor weighing. The following formula was used to determine the tumor volume: volume=L×W2/2.


### 2.14. Statistical Analysis

Pearson or Spearman methods were applied to explore correlations between variables. A studentt-test was used to compare two groups that fit a normal distribution. Otherwise, a Wilcoxon rank-sum test was used for variables with nonnormal distribution. For comparisons of more than two groups, the Kruskal-Wallis tests or one-way ANOVA were applied. Logarithmic rank test and Kaplan-Meier analysis were applied in analyzing categorical variables. In Kaplan-Meier survival analysis, the “surv_cutpoint” function of the survminer package was used to determine the optimal risk cutoff. Logarithmic rank tests were used for univariate analysis and Cox proportional hazard regression for multivariate analysis. The area under the curve (AUC), which is calculated based on receiver operating characteristic curves or ROC curves, was calculated as well as sensitivity, specificity, and accuracy. The prognostic prediction efficacy of the IRS model was evaluated using the timeROC package. P<0.05 was considered statistically significant. All statistical analyses were performed using the R software (version 4.1).


## 2.1. Retrieval and Preprocessing of the Dataset

RNA sequencing (RNA-seq) (namely, the FPKM value) data were downloaded from UCSC Xena (https://xenabrowser.net/datapages/). Tumor diseases with fewer than 20 normal samples were excluded, and expression data, including GNAI2 expression, were selected from 21 malignant tumors. GC RNA-seq data were retrieved from the Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga), whereas several microarray data (GSE29272, GSE26901, GSE33335, GSE54129, GSE79973, GSE13861, GSE13911, and GSE26899) related to GC were retrieved from the NCBI Gene Expression Omnibus (GEO) database. Normal tissue data were retrieved from the GTEx database (Genotype-Tissue Expression, https://gtexportal.org/). GNAI2 expression data in tumor cells were retrieved from the Cancer Cell Line Encyclopedia (CCLE) project. Using the meta package in the R software, a random effects model was adopted for meta-analysis. In the Cox regression model, logarithmic rank test and Kaplan-Meier analysis were conducted to analyze OS, disease-specific survival (DSS), and progression-free survival (PFS) rates for the high and low GNAI2 expression groups.The copy number variation (CNV) data were retrieved from the UCSC Xena data portal, and the GISTIC 2.0 algorithm was used to analyze the CNV pattern of GC with differential clustering. Then, methylation data were obtained from the LinkedOmics data portal, and the methylated genome data were processed using weighted gene co-expression network analysis (WGCNA) before clustering into modules marked by different colors. In addition, a somatic mutation was downloaded from the TCGA database and analyzed with maftools R package.TCGA-stomach adenocarcinoma (STAD) cohort, two GC GEO cohorts (GSE62254 and GSE84437), and two immunotherapy-related cohorts (GSE78220 and IMvigor210) were downloaded. Detailed information about these datasets is summarized in TableS1.


## 2.2. Correlation between GNAI2 and TME Cell Infiltration

First, TCGA data on 105 immunomodulators collected in Charoentong et al.’s work [14] (including MHC, receptors, chemokines, and immune stimulators) (Table S2), we performed a pan-cancer analysis to explore the correlation between GNAI2 and PD-1, PD-L1, and CTLA-4 as well as LAG-3 within GC (Table S3). In addition, data relating to the infiltration level of T lymphocytes, M1/M2 macrophages, CD8+ T cells, and B cells were obtained, and the differences in effector genes in these five TIICs of high versus low GNAI2 expression groups were analyzed (Table S4).Next, the differential expression and activity of GNAI2 in the cancer immune cycle (CIC) were analyzed. The immune response to antitumor therapy, or CIC, involves 7 steps: (1) antigen release from dead tumor cells, (2) presentation of the cancer antigen, (3) initiation and activation of T cells, (4) migration of cytotoxic T lymphocytes (CTLs) into cancer site, (5) tumor infiltration by CTLs, (6) identification of cancer cells, and (7) immune-mediated cancer cell killing. Chen et al. [15] found that cancer cell fate was determined by the activities of the aforementioned steps.Thereafter, 20 suppressed immune checkpoints previously considered to have a potential [16] in treatment were collected for correlation analysis with GNAI2 in RNA level. Reverse phase protein array (RPPA) data in STAD, downloaded from the Cancer Proteome Atlas (TCPA), was explored to analyze the correlation between GNAI2 and the 20 suppressed immune checkpoints at the protein level. The Human Protein Atlas (HPA, http://www.proteinatlas.org/) online database was explored to validate the CD86, CD276, PD-L1, IDO1, and CD200 protein expression in GC using immunohistochemical staining.Finally, the immune and stromal scores calculated based on the ESTIMATE algorithm can predict the purity of tumor cells. A single sample gene set enrichment analysis (ssGSEA) algorithm was used to quantify the relative abundance of each cell infiltration in the GC TME. In addition, to accurately evaluate TIIC penetration in GC and calculation errors initiated by different algorithms and TIIC marker gene sets, six algorithms were used (Cibersort-ABS, MCP-counter, quanTIseq, TIMER, xCell, and TIP) to calculate TIIC penetration using the IOBR R package (TableS5) [17–22].


## 2.3. Identification of Differentially Expressed mRNAs (DERs) Associated with the GNAI2 Immune Microenvironment and Functional Annotation

Samples were divided into different groups based on the median level of GNAI2, interstitial, and immune scores. DERs were identified using the limma package, and a volcanic map was used to visualize the DERs. Using adjustedP<0.05 and logFC>0.8as the criteria for determining differential DERs, the common DERs were obtained and analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Gene Set Enrichment Analysis (GSEA). The clusterProfiler package was used to perform the GSEA, KEGG, and GO analyses for identifying possible pathways and functional annotation (Table S6).


## 2.4. Unsupervised Clustering for GNAI2-Associated Immune Microenvironment in GC and Gene Set Variation Analysis (GSVA)

ConsensusClusterPlus R packages were used to classify various cluster patterns associated with the GNAI2 immune microenvironment based on the expression of DERs, and 1000 repetitions were carried out to ensure classification stability. To explore the biological processes and final results in different cluster patterns, the gene sets of “c2.cp.kegg.v6.2.symbols” were downloaded from the MSigDB database for running GSVA analysis (TableS7).


## 2.5. Construction and Verification of IRS Model

According to the patient inclusion to the experiment, the GSE62254 dataset was classified as the verification and training sets at the 3 : 7 ratio. In the training set, common DERs were analyzed by univariate Cox analysis, and then, the optimal candidate DERs were screened using the LASSO algorithm. The best candidate DERs were selected using multivariate Cox regression coefficients, and an IRS model was developed based on the IRS RNA expression patterns and weighted using the formula:IRS=∑βi∗RNAi, where βi represents the coefficient of expression pattern for the ‘ i’th IRS RNA. Thereafter, the logarithmic rank test and Kaplan-Meier analysis were used to evaluate the prognosis prediction value of the IRS model. In addition, based on the GSE62254 verification set, TCGA external cohort, and GSE84437 dataset, the prognostic prediction efficacy of the IRS model was verified. The significance of the IRS model was evaluated using the timeROC package. The IRS model was also used to predict the survival rate of the IMvigor210 cohort subgroup.TIDE algorithm related to T cell function can accurately predict the efficacy of immunotherapy [23]. Next, the accuracy of the IRS and TIDE algorithms in predicting survival probability between the GSE78220 immunotherapy cohort and the TCGA dataset was compared. These datasets were divided into low and high IRS groups based on the cutoff point determined by the “surv-cutpoint” function in the survminer package to explore the significance of IRS in predicting clinical prognosis.


## 2.6. Clinical Specimens and Cell Culture

From 2019 to 2020, 20 GC tissues and matched paracancerous tissue specimens from patients with GC in Meizhou People’s Hospital who underwent surgical resection were collected. All patient specimens were verified by a pathologist. None of the patients had been diagnosed with other tumor diseases or had undergone other related surgery, and none had been treated with preoperative targeted therapy, biotherapy, or chemotherapy. Informed consent was obtained from all patients and their families. The study protocols were approved by the Ethics Committee of Meizhou People’s Hospital (Approval NO. 2019-C-80).The human GC MKN-7, NCI-N87, Hs-746T, and HGC27 cell lines and Genetic Suppressor Element 1 (GSE-1), provided by Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China), were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (NCM Biotech, Suzhou, China) at 37°C in a 5% CO2 incubator. The MycoAlert™ Mycoplasma Detection Kit (Lonza, Basel, Switzerland) was used to test the negative mycoplasma contamination in all cells before experimentation.


## 2.7. Construction and Transfection of Lentivirus

The full-length cDNA for GNAI2 was introduced into the pLVX-IRES-ZsGreen1 vector to construct a GNAI2-overexpressing lentivirus. Two lentivirus shRNAs targeting GNAI2 were inserted into the pLVX-shRNA-puro vector in constructing the GNAI2-silencing lentivirus. All plasmids were verified by sequencing. Thereafter, the cells (5×105/well) were inoculated into 6-well plates (NEST Biotechnology Co., Ltd, Wuxi, China) and cultured for 24 or 48 h before transfection. HGC27 and Hs-746T cells were transfected with plasmids and siRNAs using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to specific protocols. At 48-h posttransfection, gene expressions were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting (WB) assays. RNA separation, total cellular protein isolation, and cell functional analysis were also carried out 48-h posttransfection.


## 2.8. Detection of mRNA Level of GNAI2 Using RT-qPCR

Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract the total cellular RNA, which was later prepared in cDNA by reverse transcription using the PrimeScript RT Master Mix (538100; Toyobo, Osaka, Japan). Thereafter, the Applied Biosystems 7900HT real-time PCR system (Foster City, CA, USA) was used for qPCR through SYBR-Green PCR Master Mix (15153900; Roche, Basel, Switzerland). The cDNA concentration was adjusted to 30 ng/μL. Fluorescence qPCR was performed as follows: 2 min at 95°C, then 15 s at 95°C, and 30 s at 60–68°C for 40 cycles, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) being the endogenous reference gene. Fold changes (FCs) in the gene level were determined using the 2-ΔCt approach. All assays were carried out in triplicate. The primer sequences for GNAI2 were forward primer: TACCGGGCGGTTGTCTACA and reverse primer: GGGTCGGCAAAGTCGATCTG.


## 2.9. Western Blotting (WB)

Antibodies (rabbit antihuman antibodies) to GNAI2, Ki67, PCNA, AKT, P-AKT, PI3K, P-PI3K, and GAPDH were provided by Abnova (Taipei, China), Proteintech (Rosemont, IL, USA), and Abcam (Cambridge, UK). Cell lysis buffer (Pierce, Rockford, IL, USA) was employed in extracting total protein. Thereafter, 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used in separating the cell lysates. The separated lysates were then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Subsequently, 5% skimmed milk powder was utilized to block the membranes for 2 h, followed by incubation using specific primary antibodies at 4°C overnight and another 2 h of incubation with secondary antibodies after dilution. The DNR Bio Imaging System (DNR, Jerusalem, Israel) was used to detect protein bands ECL (Pierce). GAPDH was the reference gene used for protein expression normalization. The PI3K antibodies used for the study detected the PI3K enzyme catalytic subunit gamma.


## 2.10. Cell Cycle Detection Using Flow Cytometry

After trypsin digestion, the transfected cells were suspended at a density of3×104 cells/mL, followed by propidium iodide (PI) staining (BD Biosciences, San Jose, CA, USA) and 30 min of treatment with 0.5 mg/mL RNase A (Keygen Biotech, Nanjing, China) supplemented with phosphate-buffered saline (PBS) at 37°C, and subsequent inoculation into 4 plates for detection at 5 time points. After collection, washing, fixation, RNA removal, and staining, approximately 1.5×105 cells were measured by flow cytometry. Then, the FlowJo software (TreeStar, Ashland, OR, USA) was employed for cell cycle analysis. Experiments were carried out in triplicate.


## 2.11. Measurement of Invasive Ability of Cells by Transwell Assay

After 24 h of culture within the serum-free basic medium, the transfected cells were suspended at a density of1×105 cells/mL. Thereafter, 0.3 mL of the cell suspension was inoculated into the top transwell chamber (BD Biosciences, San Jose, CA, USA). Then, all wells were added with a complete culture medium of 0.7 mL that contained 10% FBS in the bottom transwell chamber. Later, the transwells were cultured at 37°C for 24 hours, fixed, washed, and stained, and then, cells remaining in the upper chamber were removed. The number of invasive cells was calculated in 8 random fields of view (FOVs) with a microscope, followed by imaging.


## 2.12. Measurement of In Vitro Cell Proliferation by Cell Counting Kit-8 (CCK-8) Assay

To determine the effect of GNAI2 on cell proliferation, cells were inoculated into four 96-well plates, followed by incubation with the CCK-8 (Dojindo, Kumamoto, Japan) reagent in a normal medium at 37°C until a color change occurred. After transfection for 0, 24, 48, and 72 h, the absorbance at 450 nm was measured using a microplate reader. The culture medium was used as the blank control for calibration. Measurements were performed in triplicate.


## 2.13. Subcutaneous Tumorigenesis in Nude Mice

The 5-6-week-old male athymic BALBC/c nude mice were provided by the Hunan Acura Biotechnology Co., Ltd. (Changsha, China). All animals were housed in a specific pathogen-free (SPF), temperature-controlled environment and fed exclusively with autoclaved water and sterilized food.Each animal experiment was carried out according to guidelines from the Ethics Committee of Meizhou People’s Hospital. The density of HGC27 cells transfected with the blank plasmid or GNAI2 plasmid was adjusted to4×107 cells/mL after trypsin digestion. Twelve BALB/c nude mice were randomized into 2 groups containing six mice each. Thereafter, the control nude mice were subcutaneously injected with HGC27+LV-NC cells (0.1 mL; 4×106 cells), whereas the experimental nude mice were subcutaneously injected with HGC27+LV-GNAI2 cells. On day 26, all mice were euthanized by cervical dislocation, followed by imaging and tumor weighing. The following formula was used to determine the tumor volume: volume=L×W2/2.


## 2.14. Statistical Analysis

Pearson or Spearman methods were applied to explore correlations between variables. A studentt-test was used to compare two groups that fit a normal distribution. Otherwise, a Wilcoxon rank-sum test was used for variables with nonnormal distribution. For comparisons of more than two groups, the Kruskal-Wallis tests or one-way ANOVA were applied. Logarithmic rank test and Kaplan-Meier analysis were applied in analyzing categorical variables. In Kaplan-Meier survival analysis, the “surv_cutpoint” function of the survminer package was used to determine the optimal risk cutoff. Logarithmic rank tests were used for univariate analysis and Cox proportional hazard regression for multivariate analysis. The area under the curve (AUC), which is calculated based on receiver operating characteristic curves or ROC curves, was calculated as well as sensitivity, specificity, and accuracy. The prognostic prediction efficacy of the IRS model was evaluated using the timeROC package. P<0.05 was considered statistically significant. All statistical analyses were performed using the R software (version 4.1).


## 3. Results

### 3.1. Pan-Cancer Expression Pattern, Prognostic Significance, and Immunological Correlation of GNAI2

GNAI2 was specifically expressed in different cancer types in the TCGA, GTEx, and CCLE databases, and was highly expressed in most cancers, including GC (FigureS1). The differential expression of GNAI2 affected the rates of OS, PFS, and DSS in patients with various cancer types (Figure S2-S4). High levels of GNAI2 in GC tumors were associated with poor OS, PFS, and DSS rates, suggesting that GNAI2 may be a prognostic factor for GC.It is speculated that GNAI2 overexpression is related to the specificity of the TME. GNAI2 levels were positively correlated with most of the immunomodulatory factors (chemokines, receptors, major histocompatibility complexes [MHC], and immunostimulators) in GC (Figure1(a)). In addition, GNAI2 levels are positively correlated with most of the TIICs in GC (Figure 1(c)). In the TCGA database, GNAI2 expression in GC was positively correlated with several immune checkpoints, including PD-1, PD-L1, LAG3, and CTLA-4 (Figure 1(b), Table S3). Notably, the expression of GNAI2 in many cancers was negatively correlated with MSI and TMB (Figure S5), suggesting that GNAI2 reflects the tumor immunogenicity of the various malignant tumors.Figure 1
Pan-cancer analysis of immunological status induced by GNAI2. (a) Correlations between GNAI2 and 105 immunomodulators. Negative correlation coefficient, blue; positive correlation coefficient, red. (b)Correlations between GNAI2 and four immune checkpoints. Cancer types are represented by the dots; y-axis:correlation coefficient; x-axis :-log10P. (c) Correlations between GNAI2 and 28 tumor-associated immune cells calculated with the ssGSEA algorithm. Correlation coefficient is indicated by the color. The asterisks represented the statisticaP value (∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001).
(a)(b)(c)


### 3.2. GNAI2 Shapes an Inflamed TME in GC

GNAI2 expression was positively correlated with many immunomodulators (chemokines, receptors, MHC, and immune stimulators) in GC (Figure2(a)). In the high GNAI2 expression group, four key chemokines (CCL1, CCL2, CCL17, and CCL22) that can induce T lymphocyte recruitment to the TME and three chemokines (CX3CL1, CXCL9, and CXCL10) that induce T lymphocyte infiltration were upregulated (Figure 2(a)). Chemokines (including CCL2-5, CCL7-8, CCL11, CCL13, CCL17, CCL19-22, CCL24, and CCL26) and matched receptors (such as CXCR3, CCR1-3, and CCR5-6) promoting effector TIIC recruitment, including antigen-presenting cells, Th17 cells, and CD8+ T cells, were positively correlated with GNAI2 expression (Figure 2(a)). Most MHC molecules were upregulated in the high GNAI2 expression group, and the content of various immune cells and mesenchymal cells increased, suggesting that antigen presentation and antitumor immune response processing were improved.Figure 2
GNAI2 shapes an inflamed TME in GC. (a) Differential expression of 105 immunomodulators between high- and low-GNAI2 groups in GC. (b) Differential levels in the 7 steps of the cancer immunity cycle between high- and low-GNAI2 groups. The asterisks represented the statisticalP value (∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001). (c) Correlations between GNAI2 and the infiltration levels of six kinds of immune cells. (d) Correlations between GNAI2 and 20 inhibitory immune checkpoints in RNA expression level.Correlation coefficient is indicated by the color (∗P<0.05). (e) Correlations between GNAI2 and 20 inhibitory immune checkpoints in protein expression level. Negative correlation coefficient, blue; positive correlation coefficient, red (∗P<0.05).
(a)(b)(c)(d)(e)Most cancer immune cycle activities were upregulated in the high GNAI2 expression group, but the initiation and activation of T cells (step 3) and CTL migration to the TME (step 4) were downregulated (Figure2(b)), suggesting the accumulation or apoptosis of regulatory T cells in the high GNAI2 expression group. Six independent algorithms were used to confirm that GNAI2 expression was positively correlated with five TIIC types—namely, natural killer (NK) cells, CD8+T cells, Th1 cells, macrophages, and dendritic cells (DCs) (Figure 2(c) and Figure S8-S14). Similarly, GNAI2 expression was positively correlated with ICIs, such as CD200, LAIR1, CD86, HAVCR2, PD-L1, and PD-1 in both RNA and protein expression levels (Figures 2(d) and 2(e)). The expressions of CD86, CD276, CD200, PD-L1, and IDO1 were consistent with that of GNAI2 in GC and normal gastric tissues (Figure S15).Previous studies have confirmed that downregulated CD200 expression induces an alteration of Th1/Th2 cytokine production, resulting in a decrease in the cellular immune response [24]. HAVCR2 inhibited the activity of T cells [25]. Downregulation of CD86 expressed on the cell membrane cannot effectively induce the activation of T cells, leading to tumor immune escape [26]. CD276 can enhance CD8+T cell-mediated antitumor immunity [27]. Evalyn E. Mulder et al. have confirmed that the PD-1/PD-L1 axis significantly promotes T cell apoptosis [28].An increase in CD80 expression by activated CD8+T cells may contribute to PD-L1-induced apoptosis of these cells [29]. Thus, it is speculated that the initiation and activation of T cells (step 3) and CTL migration to the TME (step 4) are associated with high ICI expression levels in the high GNAI2 expression group.Growing evidence suggests that cytokine activation [30], increased immune cell infiltration [31], accumulation of regulatory T cells [7], activation of the PD-1/PD-L1 pathway [32], and upregulation of inhibitory molecules [33] can lead to an immune dysfunction of the TME and produce an inflammatory TME [34]. Based on the above studies, it was confirmed that GNAI2 promotes the interaction between many inflammatory factors to influence the TME in GC. Two independent external cohorts with large sample sizes, GSE29272 and GSE26901, corroborated these findings (Figure S6-S7).To sum up, the overexpression pattern of GNAI2 in GC is TME-specific, suggesting that GNAI2 shapes an inflamed TME in GC.


### 3.3. KEGG, GO, and GSEA Analysis of DERs and TME Cell Infiltration Characteristics in Various DERs Modification Modes

Next, 570 common immune-related DERs were identified (FigureS16A-G). Interestingly, there were 411 overlapping RNAs in upregulated DERs between the high stromal/immune score and high GNAI2 expression groups and 159 overlapping RNAs in downregulated DERs between the low stromal/immune score and low GNAI2 expression groups (Figure S16J-K). However, the downregulated DERs were not intersected between high immune/stromal score and low GNAI2 expression groups, and the upregulated DERs were not intersected between low immune/stromal score and high GNAI2 expression groups (Figure S16H-I). These confirmed that the level of GNAI2 in the TME was positively correlated with the stromal and immune score (Figure 3(a)). According to GO and KEGG analyses, these 570 immune-related DERs are closely related to receptor-ligand activation, glycosaminoglycan binding, leukocyte-cell adhesion, cytokine-cytokine receptor interaction, and other functional processes (Figure S18A-B). GSEA showed that the DERs between the low and high GNAI2 expression groups were associated with PI3K/AKT, Rap1, RAS, and Wnt signaling pathways (Figure S18C).Figure 3
Different clinical and TME cell infiltration characteristics of two Cluster patterns about GNAI2-associated immune microenvironment in TCGA-STAD cohorts. (a) Differential levels of Stromalscore and Immunescore between low- and high-GNAI2 groups (Wilcox test,∗∗∗∗P<0.001). (b) Survival analysis of two clusters in TCGA-STAD cohorts. (c) GSVA analysis showing the activation states of biological pathways among two cluster patterns about GNAI2-related immune microenvironment. The two cluster patterns, TCGA-STAD subtypes, Epstein-Barr virus(EBV), Laurern, AJCC stage, Stromalscore, Immunescore, tumor purity, and survival status were used as sample annotations. Activated pathways: red; inhibited pathways: blue. (d) The abundance of each TME infiltrating cell in two cluster patterns about GNAI2-associated immune microenvironment. The upper and lower ends of the boxes represented interquartile range of values. The horizontal line within the box represented median value, and dots showed outliers. The asterisks represented the statistical P value (∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001). (e) Differences in DNA replication, mismatch repair, and stroma-activated pathways between A and B clusters. EMT: epithelial-mesenchymal transition; TGFb: transforming growth factor beta. (f) Different tumor mutation burden (TMB) levels between A and B clusters in TCGA-STAD cohort (Wilcox test, ∗∗∗P<0.001). (g) The proportion of TCGA-STAD molecular subtypes in the A and B clusters.CIN subtype, red; EBV subtype, blue; GS subtype, green; MSI subtype, purple. (h) The proportion of TCGA-STAD DNA microsatellite instability (MSI) status in the A and B clusters. MS stable (MSS), red; MSI-low (MSI-L), blue; MSI-high (MSI-H), green.
(a)(b)(c)(d)(e)(f)(g)(h)Subsequent GSVA showed that DERs-cluster-A was significantly enriched in DNA repair pathways, such as base excision repair, nucleotide excision repair, and mismatch repair (Figure3(c)). The majority of patients with MSI subtypes were clustered in the DERs-cluster-A group (Figure 3(g)), which was consistent with the aforementioned results. Notably, the MSI-H subtype in DERs-cluster-A was significantly higher than that in DERs-cluster-B (Figure 3(h)). In addition, DERs-cluster-B was significantly associated with stromal and immune activation, such as extracellular matrix (ECM) receptor interaction, transforming growth factor (TGF)-β signaling pathway, cell adhesion, chemokine signaling pathway activation, cytokine-cytokine receptor interaction, T cell receptor signaling pathway, and Toll-like receptor signaling pathway (Figure 3(c)). The activation of epithelial-mesenchymal transition (EMT) and TGF-β was significantly enhanced in DERs-cluster-B, whereas mismatch repair and cell cycle were significantly enhanced in DERs-cluster-A (Figure 3(e)).The level of TME cell infiltration was significantly higher in DERs-cluster-B than in DERs-cluster-A (Figure3(d)). Figure 3(f) shows that TMB in GC patients was significantly higher in DERs-cluster-A than in DERs-cluster-B. Based on the characteristics of TME cell infiltration, DERs-cluster-A was classified as a noninflamed phenotype and DERs-cluster-B as an inflamed phenotype. Patients with DERs-cluster modification patterns showed a matching survival advantage (Figure 3(b)).


### 3.4. Association between Immune Microenvironment DERs Modification Modes and Copy Number Variation (CNV), Methylation, and Mutational Signatures

This study used WGCNA to cluster methylated genes into diverse co-methylation modules to compare DNA methylation patterns between DERs-cluster-A and DERs-cluster-B. Generally, the type of DERs-cluster was the most significantly negatively correlated with the black module (Bonferroni threshold,P =1e-08; absolute Pearson correlation coefficient, -0.36) (Figure S17A-B). Thirty-two genes were associated with the immune microenvironment DERs modification modes (Figure S17C). The beta value of NETO1 (involved in neural development and function), GYPC (chemokine receptor), and CR1 was negatively correlated with the expression of GNAI2 (Figure S17D). Among them, CR1, an immune adhesion receptor, promoted the intracellular immunity of cancer cells.Mutations were detected in many genes, including RELN, DCLK1, ADGRB3, and PCDH10 in both DERs-cluster-A and DERs-cluster-B. Among them, SYNE1 showed the highest mutation frequency (FigureS18A).In the DERs-cluster-A and DERs-cluster-B, there were many remarkably deleted or amplified loci. There were 11 overlapping genes between the DERs-cluster-B and DERs-cluster-A copy number deletion groups, low GNAI2 expression group, and low stromal/immune score groups, while there were 2 overlapping genes between the DERs-cluster-B and DERs-cluster-A copy number amplification groups, high GNAI2 expression group, and high stromal/immune score groups (FigureS18B-C). It is suggested that CNV alterations could be the prominent factors resulting in perturbations in the expression of immune microenvironment DERs. The location of CNV alteration of DER regulators related to the immune microenvironment DERs-cluster patterns, such as TMEM89, UGT1A8, UGT1A3, UGT1A9, and AQP12B, is shown in Figure S18D.In conclusion, methylation and CNV in the GNAI2-related immune microenvironment play an important role in the occurrence and development of GC.


### 3.5. Construction of IRS Model and Its Predictive Role in Immunotherapy

The IRS model (Figures4(a)–4(c)) was based on 570 immune-related DERs. In the GSE62254 training set, the low IRS group showed a significant survival advantage compared with the high IRS group (Figure 4(d)). The AUC of IRS in 1-, 3-, and 5-year OS was 0.95, 0.91, and 0.94, respectively. The study also found that the expression patterns of IRS RNA had great potential in predicting the prognosis of all the GC cohorts (Figures 4(e)–4(g)).Figure 4
Construction of IRS using LASSO Cox regression and performance of IRS in different cohorts. (a) The variation coefficients trajectory of each gene with different lambda. (b) The standard deviation distribution of models with different lambda. (c) In multivariate COX regression, IRS RNA-expression profiles were used to construct a forest map. (d) The KM survival distribution curve, AUC, and ROC curve of IRS in the GSE62254 training set. (e–g) the KM survival distribution curve, AUC, and ROC curve of IRS in three external independent sets: GSE62254, GSE84437, and TCGA validation set.
(a)(b)(c)(d)(e)(f)(g)A nomogram was then constructed to predict 1-, 3-, and 5-year OS using 433 GC cases by incorporating distinct prognostic factors, including age, sex, T stage, N stage, M stage, and IRS prediction score (Figures5(a) and 5(b)). The AUC of 1-, 3-, and 5-year OS was 0.95, 0.88, and 0.87, respectively (Figure 5(c)). At the same time, calibration plots were also drawn, showing the possibility of overestimation or underestimation of mortality by the nomogram (combined model) (Figure 5(d)). The C-index value for the pooled model, as well as age, sex , T stage, N stage, M stage, and IRS prediction score, was 0.81.Figure 5
IRS (immune risk score) associated with a GNAI2-related immune microenvironment is a valuable prognostic model in GSE84437 cohorts. (a) A nomogram was constructed using independent prognostic factors such as age, sex, T stage, N stage, and IRS (immune risk score). (b) In multivariate COX regression, age, sex, T stage, N stage, IRS (immune risk score), and other independent prognostic factors were used to construct a forest map. (c) Time-dependent ROC curve of the line map comparing the overall survival at 1, 3, and 5 years. (d) The calibration diagrams were applied to evaluate 1, 3, and 5 years overall survival probabilities.Y axis: the actual survival probability; X axis: the predicted survival probability by the nomogram. (e) Alluvial diagram depicting the relationship of GNAI2 expression, DERs-clusters, TCGA-STAD molecular subtype, and IRS (immune risk score) group.
(a)(b)(c)(d)(e)The prognostic value of the IRS and TIDE algorithms was evaluated. The results showed that IRS had superior performance in predicting prognosis prediction to TIDE for the GSE78220 (melanoma) and TCGA-STAD cohorts (FigureS20).The relationship between GNAI2 expression, DERs-cluster, TCGA subtype, and IRS score was visualized in an alluvial diagram (Figure5(e)).Next, the role of IRS scores in predicting response to immunotherapy was explored. Due to the lack of published data on anti-PD1/PD-L1 immunotherapy in GC, published IMvigor210 (advanced urothelial cancer cohort) [35] and GSE72280 (metastatic melanoma cohort) were studied [36]. Interestingly, in the IMvigor210 cohort, the IRS score for immuno-desert phenotype was the lowest, whereas the IRS score for immuno-inflammatory phenotype was the highest (Figure 6(b)), and patients in the IMvigor210 cohort who responded to immunotherapy also showed lower IRS scores (Figure 6(c)). High IRS scores were significantly correlated with poor OS (Figure 6(a)).Figure 6
The role of IRS (immune risk score) model in IMvigor210, TCGA-STAD, and GSE62254 cohorts. (a) Kaplan-Meier curve (IMvigor210 cohort;P=0.04, log-rank test) showing the overall survival probability of patients with low and high IRS. (b) Different IRS (immune risk score) among various immune phenotypes in IMvigor210 cohort (Kruskal-Wallis test, P<0.05). (c) Different IRS (immune risk score) between CR/PR group and SD/PD group in IMvigor210 cohort (Wilcox test, ∗∗∗P<0.001). SD, stable disease; PD, progressive disease; CR, complete response; PR, partial response. (d) Differential expression of immune checkpoint genes between low and high IRS groups in GSE62254 cohort (Wilcox test, ∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001). (e) Different IRS (immune risk score) among various molecular subtypes in GSE62254 cohort (Kruskal-Wallis test, P<0.05). (f) Different IRS (immune risk score) among various AJCC stage in GSE62254 cohort (Kruskal-Wallis test, P<0.05). (g) Different TIDE levels between high and low IRS groups in GSE62254 cohort (Wilcox test, ∗∗∗P<0.001). (h) Different Tumor Mutation burden (TMB) levels between low and high IRS groups in TCGA-STAD (Wilcox test, ∗∗∗P<0.001).
(a)(b)(c)(d)(e)(f)(g)(h)In addition, differential expression of immune checkpoint genes was detected in low and high IRS groups in the GSE62254 cohort, and the low IRS group showed higher expression levels of immune checkpoint genes (Figure6(d)). Similarly, EMT subtypes that did not respond to treatment [37] also showed the highest IRS scores (Figure 6(e)), whereas MSI subtypes showed the lowest IRS scores. The IRS score for patients with stage IV was higher than for patients with early GC. This suggests that the IRS score is a potential clinical index to quantify the survival risk of GC patients (Figure 6(f)).TMB is often used to predict the effectiveness of immunotherapy. Higher TMB is often associated with better immune response [38]. The study showed that the IRS score was positively correlated with TIDE and negatively correlated with TMB (Figures 6(g) and 6(h)). These results explain the potential value of IRS scores in predicting the prognosis of GC patients and to some extent reflect the response of immunotherapy.


### 3.6. GNAI2 Levels within GC Cells and Tissues

Twenty GC and matched paracancerous samples were examined using WB assay. In comparison to adjacent healthy samples, GNAI2 protein levels increased in GC samples (P<0.01, Figure S21D). Additionally, GNAI2 mRNA and protein levels were evaluated in four human GC cell lines (Hs-746 T, HGC27, NCI-N87, and MKN7) and a normal cell line (GSE-1) using qPCR and WB. Relative to healthy cells, GNAI2 mRNA and protein levels within Hs-746T and NCI-N87 cells were increased (Figure S21C).


### 3.7. GNAI2 Promotes GC Cell Proliferation and Invasion

To explore the effect of GNAI2 expression on GC occurrence and development, two Hs-746T stable cell lines in which GNAI2 was knocked down were first established. The expression of GNAI2 was then verified using qPCR and WB. Substantial knockdown of GNAI2 mRNA was observed in Hs-746T cells (P<0.01). Compared with the negative control group, GNAI2 silencing remarkably suppressed P-AKT and P-PI3K expression (Figure 7(a)). Additionally, flow cytometry showed that GNAI2 knockdown significantly increased the cell proportion at the G1 phase (Figure 7(b)) and inhibited cell proliferation (Figure 7(h)). The transwell assay results showed that GNAI2 knockdown significantly decreased the invasion of the two GC cell lines (Figure 7(c)). To study the effect of GNAI2 overexpression on GC cells, a stable HGC27 cell line overexpressing GNAI2 was constructed. Compared with the blank transfection vector group, the flow cytometry and WB assays revealed that GNAI2 overexpression significantly enhanced p-AKT and p-PI3K levels (Figure 7(d)). The cell proportion at the G1 phase decreased (Figure 7(e)), whereas the cell proliferation rate increased (Figure 7(g)). The transwell assay results showed that GNAI2 overexpression significantly increased GC cell invasion (Figure 7(f)). These findings suggest that GNAI2 levels remarkably affect GC cell growth and migration in vitro and are related to the P-AKT and P-PI3K levels.Figure 7
Study on the function of GNAI2 in GC cells in vitro and vivo. (a, d) Q-PCR detects that the table of GNAI2 mRNA reaches the level. Western blotting was used to detect the expression of AKT, P-AKT, PI3K, P-PI3K, and GNAI2. (b, e) Cell cycle analysis by flow cytometry. (c, f) Measurement of cell invasion by transwell assay. (g, h) Cell proliferation was detected by a CCK-8 assay. (i) After 26 days, the nude mice were euthanized, and tumors were measured. Tumor size and weight significantly differed between the two groups (P<0.01). (j) After the tumor was treated, cells were extracted, and the protein expression levels of PCNA, MCM2, AKT, P-AKT, PI3K, P-PI3K, and GAPDH were detected by western blotting. (a, b, c, h) Hs-746T+SINC，Hs-746T+siGNAI2-1，and Hs-746T+siGNAI2-2. (d, e, f, g) HGC27+Vector, HGC27+GNAI2OE, numerical control, and Vector; (i, j) HGC27+Vector and HGC27+GNAI2oE; ∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.008.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)To better understand the clinical significance of GNAI2 in the occurrence and development of GC, HGC27 cells transfected with blank and GNAI2 plasmids were inoculated into nude mice. After 26 days, the GNAI2 plasmid-transfected group had a higher tumor growth rate and a larger tumor size than the blank vector control group (Figure7(i)). These findings suggest that GNAI2 overexpression promotes GC cell proliferation in vivo. The WB assay showed that PCNA, Ki67, p-PI3K, and p-AKT protein levels in the subcutaneous tumors of nude mice induced by GNAI2-overexpressed HGC27 cells were significantly increased (P<0.01, Figure 7(j)). These findings suggest that GNAI2 overexpression enhances GC cell proliferation and migration in vivo, as well as the phosphorylation of AKT and PI3K.


### 3.8. Mechanism of GNAI2-Induced GC Cell Growth and Migration

PI3K and AKT were more highly phosphorylated in GC cells with high GNAI2 expression compared to GC cells with low GNAI2 expression, suggesting that GNAI2 activates the PI3K/AKT signaling pathway. To verify this, the study assessed the direct function of GNAI2 in the PI3K/AKT signaling pathway.MK-2206 is a pan-AKT inhibitor that inhibits all three AKT isoforms–AKT1, AKT2, and AKT3. HGC27 cells transfected with the GNAI2 plasmid or blank plasmid were treated with MK2206. As revealed by the transwell assay, GNAI2 overexpression increased GC cell invasion, whereas MK-2206 decreased cell invasion (Figure8(a)).Figure 8
GNAI2 activates the PI3K/AKT pathway in GC cells. MK2206 counteracted the effect of GNAI2 on invasion and proliferation of gastric cancer cells. Comparison of HGC27 cells transfected with blank plasmid vs. HGC27 cells transfected with GNAI2 plasmid. Compared with the negative control Hs-746T cells, 740Y-P enhanced the effects of GNAI2 on proliferation and invasion of gastric cancer cells in Hs-746T cells transfected with a GNAI2-silencing plasmid. (a) Enhanced invasive ability of cells detected by a cell invasion assay in HGC27 cells. (b) Images of cell cycles after transfection with GNAI2 lentivirus were assessed by flow cytometry in HGC27 cells. (c) Proliferation of GC cells after transfection with GNAI2 lentivirus was assessed by CCK-8 assay in HGC27 cells. (d) Protein expression after transfection with GNAI2 lentivirus as assessed by western blotting in HGC27cells. (e) Data representmean±SD of relative protein expression of genes in each group. GAPDH was used as an internal control. (f) Inhibited invasive ability of cells detected by a cell invasion assay in Hs-746T cells. (g) Images of cell cycles after transfection with a GNAI2-silencing plasmid as assessed by flow cytometry in Hs-746T cells. (h) Proliferation of GC cells after transfection with a GNAI2-silencing plasmid as assessed by CCK-8 assay in Hs-746T cells. (i) Protein expression after transfection with a GNAI2-silencing plasmid was assessed by western blotting in Hs-746T cells. (j) Data represent mean±SD of relative protein expression of genes in each group. GAPDH was used as an internal control. Cell line: (a, b, c, d, e) HGC27+Vector, HGC27+GNAI2oE, HGC27+Vector+MK2206, and HGC27+GNAI2oE+MK2206. Vector, blank transfection vector: (f, g, h, i, j) Hs-746T+NC, Hs-746T+GNAI2Sh1, Hs-746T+NC+740Y-P, and Hs-746T+GNAI2Sh1+740Y-P. NC: negative control group; Sh1: GNAI2-silencing transfection vector; ∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.008.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)Flow cytometry and cell proliferation assays showed that GNAI2 overexpression reduced the cell proportion at the G1 phase (Figure8(b)) and increased the cell proliferation rate (Figure 8(c)) of the HGC27 cells. In the absence of MK-2206 treatment, the cell proportion at the G1 phase significantly increased (Figure 8(b)), whereas the cell proliferation rate decreased in the presence of MK-2206. Additionally, MK-2206 increased the proliferation rate of GC cells while inhibiting the effect of GNAI2 overexpression on cell proportion at the G1 phase (Figures 8(b) and 8(c)). GNAI2 overexpression promoted p-AKT and p-PI3K levels in HGC27 cells, whereas MK-2206 significantly inhibited p-PI3K and p-AKT levels induced by GNAI2 overexpression (Figures 8(d) and 8(e)).Hs-746T cells transfected with two GNAI2 siRNAs and blank plasmid were treated with 740Y-P, a cell-permeable PI3K phosphopeptide activator [38]. Transwell assays revealed that GNAI2 silencing significantly reduced GC cell invasion, whereas 740Y-P increased cell invasion (Figure 8(f)).Flow cytometry and cell proliferation assays showed that knockdown of GNAI2 increased the cell proportion at the G1 phase (Figure8(g)) but reduced the cell proliferation rate (Figure 8(h)) of Hs-746T cells. In the absence of 740Y-P treatment, the cell proportion at the G1 phase decreased (Figure 8(g)), whereas the cell proliferation rate increased after adding 740Y-P. 740Y-P also activated the effect of knockdown of GNAI2 on the accumulation in the G1 phase percentage (Figure 8(g)) and decrease of GC cell proliferation (Figure 8(h)). The WB assay also indicated that GNAI2 silencing decreased p-AKT and p-PI3K levels in the Hs-746T cell line (Figures 8(i) and 8(j)), whereas 740Y-P increased p-PI3K and p-AKT levels following knockdown of GNAI2.In summary, these findings indicate that PI3K/AKT signaling pathway activation contributes to GNAI12-induced GC cell growth and invasion.


## 3.1. Pan-Cancer Expression Pattern, Prognostic Significance, and Immunological Correlation of GNAI2

GNAI2 was specifically expressed in different cancer types in the TCGA, GTEx, and CCLE databases, and was highly expressed in most cancers, including GC (FigureS1). The differential expression of GNAI2 affected the rates of OS, PFS, and DSS in patients with various cancer types (Figure S2-S4). High levels of GNAI2 in GC tumors were associated with poor OS, PFS, and DSS rates, suggesting that GNAI2 may be a prognostic factor for GC.It is speculated that GNAI2 overexpression is related to the specificity of the TME. GNAI2 levels were positively correlated with most of the immunomodulatory factors (chemokines, receptors, major histocompatibility complexes [MHC], and immunostimulators) in GC (Figure1(a)). In addition, GNAI2 levels are positively correlated with most of the TIICs in GC (Figure 1(c)). In the TCGA database, GNAI2 expression in GC was positively correlated with several immune checkpoints, including PD-1, PD-L1, LAG3, and CTLA-4 (Figure 1(b), Table S3). Notably, the expression of GNAI2 in many cancers was negatively correlated with MSI and TMB (Figure S5), suggesting that GNAI2 reflects the tumor immunogenicity of the various malignant tumors.Figure 1
Pan-cancer analysis of immunological status induced by GNAI2. (a) Correlations between GNAI2 and 105 immunomodulators. Negative correlation coefficient, blue; positive correlation coefficient, red. (b)Correlations between GNAI2 and four immune checkpoints. Cancer types are represented by the dots; y-axis:correlation coefficient; x-axis :-log10P. (c) Correlations between GNAI2 and 28 tumor-associated immune cells calculated with the ssGSEA algorithm. Correlation coefficient is indicated by the color. The asterisks represented the statisticaP value (∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001).
(a)(b)(c)


## 3.2. GNAI2 Shapes an Inflamed TME in GC

GNAI2 expression was positively correlated with many immunomodulators (chemokines, receptors, MHC, and immune stimulators) in GC (Figure2(a)). In the high GNAI2 expression group, four key chemokines (CCL1, CCL2, CCL17, and CCL22) that can induce T lymphocyte recruitment to the TME and three chemokines (CX3CL1, CXCL9, and CXCL10) that induce T lymphocyte infiltration were upregulated (Figure 2(a)). Chemokines (including CCL2-5, CCL7-8, CCL11, CCL13, CCL17, CCL19-22, CCL24, and CCL26) and matched receptors (such as CXCR3, CCR1-3, and CCR5-6) promoting effector TIIC recruitment, including antigen-presenting cells, Th17 cells, and CD8+ T cells, were positively correlated with GNAI2 expression (Figure 2(a)). Most MHC molecules were upregulated in the high GNAI2 expression group, and the content of various immune cells and mesenchymal cells increased, suggesting that antigen presentation and antitumor immune response processing were improved.Figure 2
GNAI2 shapes an inflamed TME in GC. (a) Differential expression of 105 immunomodulators between high- and low-GNAI2 groups in GC. (b) Differential levels in the 7 steps of the cancer immunity cycle between high- and low-GNAI2 groups. The asterisks represented the statisticalP value (∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001). (c) Correlations between GNAI2 and the infiltration levels of six kinds of immune cells. (d) Correlations between GNAI2 and 20 inhibitory immune checkpoints in RNA expression level.Correlation coefficient is indicated by the color (∗P<0.05). (e) Correlations between GNAI2 and 20 inhibitory immune checkpoints in protein expression level. Negative correlation coefficient, blue; positive correlation coefficient, red (∗P<0.05).
(a)(b)(c)(d)(e)Most cancer immune cycle activities were upregulated in the high GNAI2 expression group, but the initiation and activation of T cells (step 3) and CTL migration to the TME (step 4) were downregulated (Figure2(b)), suggesting the accumulation or apoptosis of regulatory T cells in the high GNAI2 expression group. Six independent algorithms were used to confirm that GNAI2 expression was positively correlated with five TIIC types—namely, natural killer (NK) cells, CD8+T cells, Th1 cells, macrophages, and dendritic cells (DCs) (Figure 2(c) and Figure S8-S14). Similarly, GNAI2 expression was positively correlated with ICIs, such as CD200, LAIR1, CD86, HAVCR2, PD-L1, and PD-1 in both RNA and protein expression levels (Figures 2(d) and 2(e)). The expressions of CD86, CD276, CD200, PD-L1, and IDO1 were consistent with that of GNAI2 in GC and normal gastric tissues (Figure S15).Previous studies have confirmed that downregulated CD200 expression induces an alteration of Th1/Th2 cytokine production, resulting in a decrease in the cellular immune response [24]. HAVCR2 inhibited the activity of T cells [25]. Downregulation of CD86 expressed on the cell membrane cannot effectively induce the activation of T cells, leading to tumor immune escape [26]. CD276 can enhance CD8+T cell-mediated antitumor immunity [27]. Evalyn E. Mulder et al. have confirmed that the PD-1/PD-L1 axis significantly promotes T cell apoptosis [28].An increase in CD80 expression by activated CD8+T cells may contribute to PD-L1-induced apoptosis of these cells [29]. Thus, it is speculated that the initiation and activation of T cells (step 3) and CTL migration to the TME (step 4) are associated with high ICI expression levels in the high GNAI2 expression group.Growing evidence suggests that cytokine activation [30], increased immune cell infiltration [31], accumulation of regulatory T cells [7], activation of the PD-1/PD-L1 pathway [32], and upregulation of inhibitory molecules [33] can lead to an immune dysfunction of the TME and produce an inflammatory TME [34]. Based on the above studies, it was confirmed that GNAI2 promotes the interaction between many inflammatory factors to influence the TME in GC. Two independent external cohorts with large sample sizes, GSE29272 and GSE26901, corroborated these findings (Figure S6-S7).To sum up, the overexpression pattern of GNAI2 in GC is TME-specific, suggesting that GNAI2 shapes an inflamed TME in GC.


## 3.3. KEGG, GO, and GSEA Analysis of DERs and TME Cell Infiltration Characteristics in Various DERs Modification Modes

Next, 570 common immune-related DERs were identified (FigureS16A-G). Interestingly, there were 411 overlapping RNAs in upregulated DERs between the high stromal/immune score and high GNAI2 expression groups and 159 overlapping RNAs in downregulated DERs between the low stromal/immune score and low GNAI2 expression groups (Figure S16J-K). However, the downregulated DERs were not intersected between high immune/stromal score and low GNAI2 expression groups, and the upregulated DERs were not intersected between low immune/stromal score and high GNAI2 expression groups (Figure S16H-I). These confirmed that the level of GNAI2 in the TME was positively correlated with the stromal and immune score (Figure 3(a)). According to GO and KEGG analyses, these 570 immune-related DERs are closely related to receptor-ligand activation, glycosaminoglycan binding, leukocyte-cell adhesion, cytokine-cytokine receptor interaction, and other functional processes (Figure S18A-B). GSEA showed that the DERs between the low and high GNAI2 expression groups were associated with PI3K/AKT, Rap1, RAS, and Wnt signaling pathways (Figure S18C).Figure 3
Different clinical and TME cell infiltration characteristics of two Cluster patterns about GNAI2-associated immune microenvironment in TCGA-STAD cohorts. (a) Differential levels of Stromalscore and Immunescore between low- and high-GNAI2 groups (Wilcox test,∗∗∗∗P<0.001). (b) Survival analysis of two clusters in TCGA-STAD cohorts. (c) GSVA analysis showing the activation states of biological pathways among two cluster patterns about GNAI2-related immune microenvironment. The two cluster patterns, TCGA-STAD subtypes, Epstein-Barr virus(EBV), Laurern, AJCC stage, Stromalscore, Immunescore, tumor purity, and survival status were used as sample annotations. Activated pathways: red; inhibited pathways: blue. (d) The abundance of each TME infiltrating cell in two cluster patterns about GNAI2-associated immune microenvironment. The upper and lower ends of the boxes represented interquartile range of values. The horizontal line within the box represented median value, and dots showed outliers. The asterisks represented the statistical P value (∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001). (e) Differences in DNA replication, mismatch repair, and stroma-activated pathways between A and B clusters. EMT: epithelial-mesenchymal transition; TGFb: transforming growth factor beta. (f) Different tumor mutation burden (TMB) levels between A and B clusters in TCGA-STAD cohort (Wilcox test, ∗∗∗P<0.001). (g) The proportion of TCGA-STAD molecular subtypes in the A and B clusters.CIN subtype, red; EBV subtype, blue; GS subtype, green; MSI subtype, purple. (h) The proportion of TCGA-STAD DNA microsatellite instability (MSI) status in the A and B clusters. MS stable (MSS), red; MSI-low (MSI-L), blue; MSI-high (MSI-H), green.
(a)(b)(c)(d)(e)(f)(g)(h)Subsequent GSVA showed that DERs-cluster-A was significantly enriched in DNA repair pathways, such as base excision repair, nucleotide excision repair, and mismatch repair (Figure3(c)). The majority of patients with MSI subtypes were clustered in the DERs-cluster-A group (Figure 3(g)), which was consistent with the aforementioned results. Notably, the MSI-H subtype in DERs-cluster-A was significantly higher than that in DERs-cluster-B (Figure 3(h)). In addition, DERs-cluster-B was significantly associated with stromal and immune activation, such as extracellular matrix (ECM) receptor interaction, transforming growth factor (TGF)-β signaling pathway, cell adhesion, chemokine signaling pathway activation, cytokine-cytokine receptor interaction, T cell receptor signaling pathway, and Toll-like receptor signaling pathway (Figure 3(c)). The activation of epithelial-mesenchymal transition (EMT) and TGF-β was significantly enhanced in DERs-cluster-B, whereas mismatch repair and cell cycle were significantly enhanced in DERs-cluster-A (Figure 3(e)).The level of TME cell infiltration was significantly higher in DERs-cluster-B than in DERs-cluster-A (Figure3(d)). Figure 3(f) shows that TMB in GC patients was significantly higher in DERs-cluster-A than in DERs-cluster-B. Based on the characteristics of TME cell infiltration, DERs-cluster-A was classified as a noninflamed phenotype and DERs-cluster-B as an inflamed phenotype. Patients with DERs-cluster modification patterns showed a matching survival advantage (Figure 3(b)).


## 3.4. Association between Immune Microenvironment DERs Modification Modes and Copy Number Variation (CNV), Methylation, and Mutational Signatures

This study used WGCNA to cluster methylated genes into diverse co-methylation modules to compare DNA methylation patterns between DERs-cluster-A and DERs-cluster-B. Generally, the type of DERs-cluster was the most significantly negatively correlated with the black module (Bonferroni threshold,P =1e-08; absolute Pearson correlation coefficient, -0.36) (Figure S17A-B). Thirty-two genes were associated with the immune microenvironment DERs modification modes (Figure S17C). The beta value of NETO1 (involved in neural development and function), GYPC (chemokine receptor), and CR1 was negatively correlated with the expression of GNAI2 (Figure S17D). Among them, CR1, an immune adhesion receptor, promoted the intracellular immunity of cancer cells.Mutations were detected in many genes, including RELN, DCLK1, ADGRB3, and PCDH10 in both DERs-cluster-A and DERs-cluster-B. Among them, SYNE1 showed the highest mutation frequency (FigureS18A).In the DERs-cluster-A and DERs-cluster-B, there were many remarkably deleted or amplified loci. There were 11 overlapping genes between the DERs-cluster-B and DERs-cluster-A copy number deletion groups, low GNAI2 expression group, and low stromal/immune score groups, while there were 2 overlapping genes between the DERs-cluster-B and DERs-cluster-A copy number amplification groups, high GNAI2 expression group, and high stromal/immune score groups (FigureS18B-C). It is suggested that CNV alterations could be the prominent factors resulting in perturbations in the expression of immune microenvironment DERs. The location of CNV alteration of DER regulators related to the immune microenvironment DERs-cluster patterns, such as TMEM89, UGT1A8, UGT1A3, UGT1A9, and AQP12B, is shown in Figure S18D.In conclusion, methylation and CNV in the GNAI2-related immune microenvironment play an important role in the occurrence and development of GC.


## 3.5. Construction of IRS Model and Its Predictive Role in Immunotherapy

The IRS model (Figures4(a)–4(c)) was based on 570 immune-related DERs. In the GSE62254 training set, the low IRS group showed a significant survival advantage compared with the high IRS group (Figure 4(d)). The AUC of IRS in 1-, 3-, and 5-year OS was 0.95, 0.91, and 0.94, respectively. The study also found that the expression patterns of IRS RNA had great potential in predicting the prognosis of all the GC cohorts (Figures 4(e)–4(g)).Figure 4
Construction of IRS using LASSO Cox regression and performance of IRS in different cohorts. (a) The variation coefficients trajectory of each gene with different lambda. (b) The standard deviation distribution of models with different lambda. (c) In multivariate COX regression, IRS RNA-expression profiles were used to construct a forest map. (d) The KM survival distribution curve, AUC, and ROC curve of IRS in the GSE62254 training set. (e–g) the KM survival distribution curve, AUC, and ROC curve of IRS in three external independent sets: GSE62254, GSE84437, and TCGA validation set.
(a)(b)(c)(d)(e)(f)(g)A nomogram was then constructed to predict 1-, 3-, and 5-year OS using 433 GC cases by incorporating distinct prognostic factors, including age, sex, T stage, N stage, M stage, and IRS prediction score (Figures5(a) and 5(b)). The AUC of 1-, 3-, and 5-year OS was 0.95, 0.88, and 0.87, respectively (Figure 5(c)). At the same time, calibration plots were also drawn, showing the possibility of overestimation or underestimation of mortality by the nomogram (combined model) (Figure 5(d)). The C-index value for the pooled model, as well as age, sex , T stage, N stage, M stage, and IRS prediction score, was 0.81.Figure 5
IRS (immune risk score) associated with a GNAI2-related immune microenvironment is a valuable prognostic model in GSE84437 cohorts. (a) A nomogram was constructed using independent prognostic factors such as age, sex, T stage, N stage, and IRS (immune risk score). (b) In multivariate COX regression, age, sex, T stage, N stage, IRS (immune risk score), and other independent prognostic factors were used to construct a forest map. (c) Time-dependent ROC curve of the line map comparing the overall survival at 1, 3, and 5 years. (d) The calibration diagrams were applied to evaluate 1, 3, and 5 years overall survival probabilities.Y axis: the actual survival probability; X axis: the predicted survival probability by the nomogram. (e) Alluvial diagram depicting the relationship of GNAI2 expression, DERs-clusters, TCGA-STAD molecular subtype, and IRS (immune risk score) group.
(a)(b)(c)(d)(e)The prognostic value of the IRS and TIDE algorithms was evaluated. The results showed that IRS had superior performance in predicting prognosis prediction to TIDE for the GSE78220 (melanoma) and TCGA-STAD cohorts (FigureS20).The relationship between GNAI2 expression, DERs-cluster, TCGA subtype, and IRS score was visualized in an alluvial diagram (Figure5(e)).Next, the role of IRS scores in predicting response to immunotherapy was explored. Due to the lack of published data on anti-PD1/PD-L1 immunotherapy in GC, published IMvigor210 (advanced urothelial cancer cohort) [35] and GSE72280 (metastatic melanoma cohort) were studied [36]. Interestingly, in the IMvigor210 cohort, the IRS score for immuno-desert phenotype was the lowest, whereas the IRS score for immuno-inflammatory phenotype was the highest (Figure 6(b)), and patients in the IMvigor210 cohort who responded to immunotherapy also showed lower IRS scores (Figure 6(c)). High IRS scores were significantly correlated with poor OS (Figure 6(a)).Figure 6
The role of IRS (immune risk score) model in IMvigor210, TCGA-STAD, and GSE62254 cohorts. (a) Kaplan-Meier curve (IMvigor210 cohort;P=0.04, log-rank test) showing the overall survival probability of patients with low and high IRS. (b) Different IRS (immune risk score) among various immune phenotypes in IMvigor210 cohort (Kruskal-Wallis test, P<0.05). (c) Different IRS (immune risk score) between CR/PR group and SD/PD group in IMvigor210 cohort (Wilcox test, ∗∗∗P<0.001). SD, stable disease; PD, progressive disease; CR, complete response; PR, partial response. (d) Differential expression of immune checkpoint genes between low and high IRS groups in GSE62254 cohort (Wilcox test, ∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.001). (e) Different IRS (immune risk score) among various molecular subtypes in GSE62254 cohort (Kruskal-Wallis test, P<0.05). (f) Different IRS (immune risk score) among various AJCC stage in GSE62254 cohort (Kruskal-Wallis test, P<0.05). (g) Different TIDE levels between high and low IRS groups in GSE62254 cohort (Wilcox test, ∗∗∗P<0.001). (h) Different Tumor Mutation burden (TMB) levels between low and high IRS groups in TCGA-STAD (Wilcox test, ∗∗∗P<0.001).
(a)(b)(c)(d)(e)(f)(g)(h)In addition, differential expression of immune checkpoint genes was detected in low and high IRS groups in the GSE62254 cohort, and the low IRS group showed higher expression levels of immune checkpoint genes (Figure6(d)). Similarly, EMT subtypes that did not respond to treatment [37] also showed the highest IRS scores (Figure 6(e)), whereas MSI subtypes showed the lowest IRS scores. The IRS score for patients with stage IV was higher than for patients with early GC. This suggests that the IRS score is a potential clinical index to quantify the survival risk of GC patients (Figure 6(f)).TMB is often used to predict the effectiveness of immunotherapy. Higher TMB is often associated with better immune response [38]. The study showed that the IRS score was positively correlated with TIDE and negatively correlated with TMB (Figures 6(g) and 6(h)). These results explain the potential value of IRS scores in predicting the prognosis of GC patients and to some extent reflect the response of immunotherapy.


## 3.6. GNAI2 Levels within GC Cells and Tissues

Twenty GC and matched paracancerous samples were examined using WB assay. In comparison to adjacent healthy samples, GNAI2 protein levels increased in GC samples (P<0.01, Figure S21D). Additionally, GNAI2 mRNA and protein levels were evaluated in four human GC cell lines (Hs-746 T, HGC27, NCI-N87, and MKN7) and a normal cell line (GSE-1) using qPCR and WB. Relative to healthy cells, GNAI2 mRNA and protein levels within Hs-746T and NCI-N87 cells were increased (Figure S21C).


## 3.7. GNAI2 Promotes GC Cell Proliferation and Invasion

To explore the effect of GNAI2 expression on GC occurrence and development, two Hs-746T stable cell lines in which GNAI2 was knocked down were first established. The expression of GNAI2 was then verified using qPCR and WB. Substantial knockdown of GNAI2 mRNA was observed in Hs-746T cells (P<0.01). Compared with the negative control group, GNAI2 silencing remarkably suppressed P-AKT and P-PI3K expression (Figure 7(a)). Additionally, flow cytometry showed that GNAI2 knockdown significantly increased the cell proportion at the G1 phase (Figure 7(b)) and inhibited cell proliferation (Figure 7(h)). The transwell assay results showed that GNAI2 knockdown significantly decreased the invasion of the two GC cell lines (Figure 7(c)). To study the effect of GNAI2 overexpression on GC cells, a stable HGC27 cell line overexpressing GNAI2 was constructed. Compared with the blank transfection vector group, the flow cytometry and WB assays revealed that GNAI2 overexpression significantly enhanced p-AKT and p-PI3K levels (Figure 7(d)). The cell proportion at the G1 phase decreased (Figure 7(e)), whereas the cell proliferation rate increased (Figure 7(g)). The transwell assay results showed that GNAI2 overexpression significantly increased GC cell invasion (Figure 7(f)). These findings suggest that GNAI2 levels remarkably affect GC cell growth and migration in vitro and are related to the P-AKT and P-PI3K levels.Figure 7
Study on the function of GNAI2 in GC cells in vitro and vivo. (a, d) Q-PCR detects that the table of GNAI2 mRNA reaches the level. Western blotting was used to detect the expression of AKT, P-AKT, PI3K, P-PI3K, and GNAI2. (b, e) Cell cycle analysis by flow cytometry. (c, f) Measurement of cell invasion by transwell assay. (g, h) Cell proliferation was detected by a CCK-8 assay. (i) After 26 days, the nude mice were euthanized, and tumors were measured. Tumor size and weight significantly differed between the two groups (P<0.01). (j) After the tumor was treated, cells were extracted, and the protein expression levels of PCNA, MCM2, AKT, P-AKT, PI3K, P-PI3K, and GAPDH were detected by western blotting. (a, b, c, h) Hs-746T+SINC，Hs-746T+siGNAI2-1，and Hs-746T+siGNAI2-2. (d, e, f, g) HGC27+Vector, HGC27+GNAI2OE, numerical control, and Vector; (i, j) HGC27+Vector and HGC27+GNAI2oE; ∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.008.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)To better understand the clinical significance of GNAI2 in the occurrence and development of GC, HGC27 cells transfected with blank and GNAI2 plasmids were inoculated into nude mice. After 26 days, the GNAI2 plasmid-transfected group had a higher tumor growth rate and a larger tumor size than the blank vector control group (Figure7(i)). These findings suggest that GNAI2 overexpression promotes GC cell proliferation in vivo. The WB assay showed that PCNA, Ki67, p-PI3K, and p-AKT protein levels in the subcutaneous tumors of nude mice induced by GNAI2-overexpressed HGC27 cells were significantly increased (P<0.01, Figure 7(j)). These findings suggest that GNAI2 overexpression enhances GC cell proliferation and migration in vivo, as well as the phosphorylation of AKT and PI3K.


## 3.8. Mechanism of GNAI2-Induced GC Cell Growth and Migration

PI3K and AKT were more highly phosphorylated in GC cells with high GNAI2 expression compared to GC cells with low GNAI2 expression, suggesting that GNAI2 activates the PI3K/AKT signaling pathway. To verify this, the study assessed the direct function of GNAI2 in the PI3K/AKT signaling pathway.MK-2206 is a pan-AKT inhibitor that inhibits all three AKT isoforms–AKT1, AKT2, and AKT3. HGC27 cells transfected with the GNAI2 plasmid or blank plasmid were treated with MK2206. As revealed by the transwell assay, GNAI2 overexpression increased GC cell invasion, whereas MK-2206 decreased cell invasion (Figure8(a)).Figure 8
GNAI2 activates the PI3K/AKT pathway in GC cells. MK2206 counteracted the effect of GNAI2 on invasion and proliferation of gastric cancer cells. Comparison of HGC27 cells transfected with blank plasmid vs. HGC27 cells transfected with GNAI2 plasmid. Compared with the negative control Hs-746T cells, 740Y-P enhanced the effects of GNAI2 on proliferation and invasion of gastric cancer cells in Hs-746T cells transfected with a GNAI2-silencing plasmid. (a) Enhanced invasive ability of cells detected by a cell invasion assay in HGC27 cells. (b) Images of cell cycles after transfection with GNAI2 lentivirus were assessed by flow cytometry in HGC27 cells. (c) Proliferation of GC cells after transfection with GNAI2 lentivirus was assessed by CCK-8 assay in HGC27 cells. (d) Protein expression after transfection with GNAI2 lentivirus as assessed by western blotting in HGC27cells. (e) Data representmean±SD of relative protein expression of genes in each group. GAPDH was used as an internal control. (f) Inhibited invasive ability of cells detected by a cell invasion assay in Hs-746T cells. (g) Images of cell cycles after transfection with a GNAI2-silencing plasmid as assessed by flow cytometry in Hs-746T cells. (h) Proliferation of GC cells after transfection with a GNAI2-silencing plasmid as assessed by CCK-8 assay in Hs-746T cells. (i) Protein expression after transfection with a GNAI2-silencing plasmid was assessed by western blotting in Hs-746T cells. (j) Data represent mean±SD of relative protein expression of genes in each group. GAPDH was used as an internal control. Cell line: (a, b, c, d, e) HGC27+Vector, HGC27+GNAI2oE, HGC27+Vector+MK2206, and HGC27+GNAI2oE+MK2206. Vector, blank transfection vector: (f, g, h, i, j) Hs-746T+NC, Hs-746T+GNAI2Sh1, Hs-746T+NC+740Y-P, and Hs-746T+GNAI2Sh1+740Y-P. NC: negative control group; Sh1: GNAI2-silencing transfection vector; ∗P<0.05; ∗∗P<0.01; ∗∗∗P<0.008.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)Flow cytometry and cell proliferation assays showed that GNAI2 overexpression reduced the cell proportion at the G1 phase (Figure8(b)) and increased the cell proliferation rate (Figure 8(c)) of the HGC27 cells. In the absence of MK-2206 treatment, the cell proportion at the G1 phase significantly increased (Figure 8(b)), whereas the cell proliferation rate decreased in the presence of MK-2206. Additionally, MK-2206 increased the proliferation rate of GC cells while inhibiting the effect of GNAI2 overexpression on cell proportion at the G1 phase (Figures 8(b) and 8(c)). GNAI2 overexpression promoted p-AKT and p-PI3K levels in HGC27 cells, whereas MK-2206 significantly inhibited p-PI3K and p-AKT levels induced by GNAI2 overexpression (Figures 8(d) and 8(e)).Hs-746T cells transfected with two GNAI2 siRNAs and blank plasmid were treated with 740Y-P, a cell-permeable PI3K phosphopeptide activator [38]. Transwell assays revealed that GNAI2 silencing significantly reduced GC cell invasion, whereas 740Y-P increased cell invasion (Figure 8(f)).Flow cytometry and cell proliferation assays showed that knockdown of GNAI2 increased the cell proportion at the G1 phase (Figure8(g)) but reduced the cell proliferation rate (Figure 8(h)) of Hs-746T cells. In the absence of 740Y-P treatment, the cell proportion at the G1 phase decreased (Figure 8(g)), whereas the cell proliferation rate increased after adding 740Y-P. 740Y-P also activated the effect of knockdown of GNAI2 on the accumulation in the G1 phase percentage (Figure 8(g)) and decrease of GC cell proliferation (Figure 8(h)). The WB assay also indicated that GNAI2 silencing decreased p-AKT and p-PI3K levels in the Hs-746T cell line (Figures 8(i) and 8(j)), whereas 740Y-P increased p-PI3K and p-AKT levels following knockdown of GNAI2.In summary, these findings indicate that PI3K/AKT signaling pathway activation contributes to GNAI12-induced GC cell growth and invasion.


## 4. Discussion

GNAI2, which belongs to the G protein family, is related to cancer genesis, progression, proliferation, migration, and invasion [13]. Growing evidence suggests that GNAI2 plays an indispensable role in inflammation [39], innate immunity [40], and tumor promotion by interacting with a variety of regulatory factors. ICIs [41, 42] have been shown to cause tumor regression in patients with inflammatory tumor microenvironment (TME) [43]. Therefore, it is important to develop more effective immunotherapeutic strategies to better understand the GNAI2-mediated TME and related mechanisms of various GNAI2 phenotypes.The state of the TME reflects the interaction between immunoregulatory factors in the tumor and can predict sensitivity to immunotherapy regimens. In pancreatic ductal adenocarcinoma [44], breast cancer [45], ovarian cancer [46], and other tumors, the TME has been confirmed to be related to immune response and chemotherapy. A variety of chemokines with the characteristics of promoting cell migration in inflammation and immune response were found to be positively correlated with GNAI2. In addition, GNAI2 was positively correlated with multiple immune cell infiltration levels [47] and most immunomodulators [48]. These findings support earlier reports that the upregulation of immunosuppressive factors, such as PD-L1/PD-1 [49], is triggered by high TIIC levels and promotes GNAI2-induced inflammatory TME in GC. However, the survival analysis showed that patients with high GNAI2 expression had poor prognoses. This contradictory phenomenon suggests that GNAI2 is related to tumor immune escape.GNAI2 was positively correlated with ICIs, such as CD200, CD276, CD86, HAVCR2, PD-L1, and PD-1, in both RNA and protein expression levels. There is evidence that the overexpression and activation of immune checkpoints in tumors can lead to immune recognition disorders, resulting in immune escape and cancer-promoting effects [50–52]. Also, GNAI2 downregulated the expression of major MHC, such as HLA-G, HLA-DQA2, and HLA-DOB, which may play an immunosuppressive role by inhibiting cell-cell recognition and downregulating cancer immune response.Using 570 DERs, 2 different DERs modification patterns were revealed. These two DERs-clusters had significantly different TME cell infiltration characterization. DERs-cluster-A was significantly enriched in DNA repair pathways, such as nucleotide excision repair and mismatch repair, corresponding to a noninflamed phenotype; DERs-cluster-B showed enrichment pathways related to stromal, carcinogenesis, and immune complex activation, corresponding to an immune-inflamed phenotype.An IRS model to predict the effect of GNAI2 levels on the prognosis of GC patients was developed and successfully verified using multiple external cohorts. The IRS model could also be used in assessing patient clinicopathological features, such as MSI status, EMT status, clinical stages, TMB, and TIDE scores. Low IRS scores correlated with high expression levels of immune checkpoint genes. These findings explain the potential value of IRS scores in predicting the prognosis of GC patients and, to some extent, reflect immunotherapy response.The PI3K/AKT signaling pathway is an important intracellular signaling pathway that promotes cell growth and proliferation. Previous studies indicate that PI3K/AKT signaling pathway activation inhibits osteoblasts [53], NCI-N87 cells [54], CTC-MCC-41 cells, and lung cancer cell apoptosis [55, 56]. This pathway, as discovered by Wang et al., is activated in colon cancer cells by increased TRIM11 expression, which in turn promotes cell growth, invasion, and migration [57]. The present study used GSEA and found that GNAI2 was enriched in the PI3K/AKT signaling pathway. In vivo and in vitro experiments showed that GNAI2 upregulation contributed to PI3K/AKT signaling pathway activation and GC cell growth and migration. Following PI3K/AKT signaling pathway inhibition, GNAI2 had a reduced ability to promote GC cell proliferation. Following PI3K/AKT signaling pathway activation, si-GNAI2 had an increased ability to inhibit GC cell proliferation, suggesting that this pathway may be the main downstream pathway of GNAI2 that promotes GC cell proliferation.To sum up, the IRS model developed can be used as a biomarker to independently predict patient prognosis. Additionally, GNAI2 shapes the inflammatory TME in GC while promoting GC cell growth and migration. There were also a few limitations to this study. Firstly, an optimal cutoff value for GNAI2 was not determined. The study group was divided by the median GNAI2 expression value. Secondly, the sample size used in this study was small. Resultantly, there is a need for further studies using larger sample sizes to understand the relationship between the expression profile of GNAI2 in tumor cells and TIIC.


## 5. Conclusion

The present study established an IRS model for predicting the OS of GC patients, contributing to the personalized treatment decision-making process. The study demonstrates that GNAI2 serves as a candidate therapeutic target in GC. GNAI2 induces an inflamed TME and enhances GC cell growth and migration through the PI3K/AKT signaling pathway.



---

*Source: 1254367-2022-10-14.xml*