GPR176 enhances the epithelial-mesenchymal transition in gastric cancer cells by activating the PI3K/AKT/mTOR signaling pathway

GPR176 enhances the epithelial-mesenchymal transition in gastric cancer cells by activating the PI3K/AKT/mTOR signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article GPR176 enhances the epithelial-mesenchymal transition in gastric cancer cells by activating the PI3K/AKT/mTOR signaling pathway Guangchuan Mu, Kaiyan Li, Chaozhen Hu, Jintao Cai, Huichao Ruan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5713551/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Gastric cancer is characterized by a high incidence and unfavorable prognosis. The exploration of novel molecular markers and a deeper understanding of their mechanisms of action hold the potential to offer fresh insights into gastric cancer treatment. Leveraging the TCGA-STAD and GSE66254 datasets, this study conducted an analysis on the relationship between GPR176 and clinical pathological features. Furthermore, it was validated in patients from The First Affiliated Hospital of Guangxi Medical University. Cell migration and invasion capabilities were evaluated through Transwell and scratch assays. Western blot was performed to detect the impact of GPR176 on PI3K/AKT/mTOR signaling pathway. Nude mouse tumorigenesis experiments were conducted to validate the impact of GPR176 on tumor growth in vivo . GPR176 exhibited higher expression levels in gastric cancer tissues, and was associated with a poor prognosis in patients with gastric cancer. Significant downregulation of GPR176 suppressed the invasive and migratory capabilities of gastric cancer cells, concomitant with the inhibition of the PI3K and EMT signaling pathways. However, the phenotypic changes induced by GPR176 downregulation and its inhibitory effects could be reversed by the overexpression of PIP5K1A. Nude mouse tumorigenesis experiments validated the findings from cell experiments, demonstrating that GPR176 downregulation suppressed tumor growth, while GPR176 overexpression promoted tumor growth. Similarly, after GPR176 downregulation, the EMT and PI3K/AKT/mTOR signaling pathways in tumor cells were significantly inhibited, whereas GPR176 upregulation led to their substantial activation. In conclusion, GPR176 emerged as a newly identified prognostic marker in this study. GPR176 may promote the EMT of gastric cancer cells by activating the PI3K/AKT/mTOR signaling pathway. Gastric cancer GPR176 EMT PI3K signaling pathway PIP5K1A Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction In 2020, the incidence of gastric cancer reached approximately 1.09 million cases, ranking fifth among malignant tumors and accounting for 5.6% of all malignant diseases. The number of deaths caused by gastric cancer totaled around 770,000, ranking fourth and accounting for 7.7% of overall malignant tumor-related fatalities 1 , 2 . The high prevalence and grim prognosis of gastric cancer have significantly impacted the well-being of the population, especially in China 3 . In 2022, China reported approximately 397,000 new cases of gastric cancer, accounting for 37% of the global total, with both incidence and mortality rates ranking third among malignant tumors in China 4 , 5 . East Asia bears the brunt of the global burden, with around 60% of gastric cancer cases occurring in this region 6 – 8 . Along with the health burden comes a significant economic loss to residents and the government due to the diagnosis and treatment of gastric cancer 9 , 10 . Early-stage gastric cancer often presents with inconspicuous symptoms, which leads to late detection, suboptimal treatment outcomes, high recurrence rates, and low survival rates 6 , 10 – 12 . The 5-year survival rate for advanced gastric cancer remains as low as 5% 13–15 . Currently, the predominant treatment strategy for gastric cancer is comprehensive, with a primary emphasis on surgical intervention 16 – 18 . Surgical procedures dominate the treatment landscape for early-stage gastric cancer, while chemotherapy improves survival and quality of life for locally advanced or metastatic cases (stage Ib to IIIb) 8 , 17 , 19 , 20 . Despite the mature theory and practice of abdominal anatomy, coupled with inherent shortcomings of chemotherapy, advances in surgical and chemotherapeutic approaches to the treatment of gastric cancer have been limited in recent decades 8 . Overall, the efficacy of gastric cancer treatment remains unsatisfactory, with only modest improvements in prognosis 21 . New therapeutic approaches, such as targeted drugs and immunotherapy, offer new hope for gastric cancer patients 22 , 23 . Targeting central mechanisms of tumor development, developing drugs that prevent uncontrolled cell proliferation, or directly inducing apoptosis represents a promising path to a successful transformation and even a possible cure of advanced gastric cancer 24 . GPR176, a G protein-coupled receptor located on 15q14-q15.1, belongs to the G protein-coupled receptor family and functions as a cell surface receptor that responds to hormones, growth factors, and neurotransmitters 25 . GPR176 is primarily expressed in the brain, followed by the gallbladder and testis 25 . While earlier studies focused primarily on the role and mechanisms of GPR176 in circadian rhythms, more recent research recognizes its significance in tumors. For example, Tang et al. have revealed that GPR176 recruits GNAS, activates the cAMP/PKA/BNIP3L signaling pathway, and inhibits mitochondrial autophagy, which promotes stem cell formation and proliferation of colorectal adenocarcinoma (CRC) 26 . Zheng HC and others pointed out a correlation between the expression of GPR176 and the prognosis of breast adenocarcinoma 27 . Interfering with GPR176 suppresses the PI3K/AKT/mTOR signaling pathway, glycolysis, epithelial-mesenchymal transition, and proliferation of breast adenocarcinoma cells 27 . The prognostic value of GPR176 in esophageal adenocarcinoma has also been established, indicating a correlation with prognosis and resistance of esophageal adenocarcinoma 28 . Public data analyses have hinted at a link between GPR176 and gastric cancer prognosis, warranting further exploration of underlying mechanisms 29 , 30 . Our research team has found a correlation between GPR176 and the prognosis of gastric cancer patients. In-depth studies were conducted on the impact mechanisms of GPR176 on the occurrence and development of gastric cancer, with results validated by animal experiments. PIP5K1A, which encodes the protein phosphatidylinositol-4-phosphate 5-kinase type 1 alpha, plays a role in several processes, including activation of GTPase activity 31 , 32 . It serves as an upstream regulator of the PI3K/AKT/mTOR signaling pathway, and genetic or pharmacological inhibition of PIP5K1A significantly inhibits AKT phosphorylation 33 , 34 . In our study, we discovered an interaction between GPR176 and PIP5K1A, confirming through extensive experiments that GPR176 activates the PI3K/AKT/mTOR signaling pathway by upregulating PIP5K1A. This activation promotes epithelial-mesenchymal transition and invasive capabilities of gastric cancer cells. This study innovatively explores the association of GPR176 with the prognosis and clinicopathologic factors of gastric adenocarcinomas, shedding light on its potential as a significant biomarker. Specifically, in gastric cancer tissues, GPR176 expression is significantly elevated, particularly in advanced tumor stages, suggesting its role in disease progression. Importantly, high GPR176 expression correlates with poorer overall survival and recurrence-free survival in gastric adenocarcinoma patients, indicating its prognostic significance. Furthermore, exploration of GPR176's mechanisms of action reveals its involvement in key signaling pathways such as PI3K/AKT/mTOR and EMT, highlighting its potential as a therapeutic target. Mechanistic studies further demonstrate that GPR176 enhances tumor migration, invasion, and EMT, and its effects are mediated, at least in part, through the PI3K/AKT/mTOR pathway. Additionally, the study uncovers a positive correlation between GPR176 and PIP5K1A, further elucidating the regulatory network underlying GPR176's oncogenic functions. Collectively, these findings underscore the innovative contributions of this study, positioning GPR176 as a promising prognostic biomarker and therapeutic target in gastric adenocarcinoma. Considering the potential of GPR176/PIP5K1A as intervention targets for future gastric cancer metastasis, our results provide a novel theoretical basis for precision treatment of gastric cancer and point to new research directions. Materials and Methods Data Acquisition RNA-seq data from 448 gastric cancer (STAD) samples, including 410 tumor and 38 normal samples, were meticulously collected, along with clinical data from 383 STAD patients, sourced from the TCGA database ( https://adenocarcinomagenome.nih.gov/ ) 35 . The Limma package in R ( https://bioconductor.org/packages/release/bioc/html/limma.html ) facilitated the standardization of RNA-seq information 36 . The transcriptomic details for all samples were preserved to investigate the nuanced differences between adenocarcinomatous and adjacent tissues. However, 17 samples with incomplete clinical information were excluded from subsequent survival analysis. The RNA-seq data and associated clinical/prognostic information for 300 patients diagnosed with gastric cancer were methodically extracted from the GSE66254 dataset in the GEO database ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE66254) 37 . The original chip data from GSE66254 underwent a rigorous annotation and standardization process in R, utilizing the Limma package. Cell Culture The cell lines HGC-27 and NCI-N87 were procured from the ATCC (The Global Bioresource Center, https://www.atcc.org/ ) cell repository. Culturing these cells involved a comprehensive medium consisting of DMEM (Dulbecco's Modified Eagle Medium) (Gibco, 10566016, New York), supplemented with 10% FBS (Fetal Bovine Serum) (Gibco, 10099141C, Australia), 1% streptomycin, and 1% penicillin (Solarbio, P1410, Beijing). The controlled environment for these cell cultures was maintained incubators with 5% CO 2 and 37°C. RNA Extraction and PCR The meticulous procedure of RNA extraction from each sample followed the strict TRI Reagent™ protocol (Invitrogen, AM9738, Canada). Subsequently, reverse transcription into cDNA was carefully performed according to the manufacturer’s instructions for the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, RR047B, China). The primers designed through Primer3 ( https://primer3.ut.ee/ ), had specific sequences as listed in Table S1 . The application of FastStart Universal SYBR® Green Master Mix (Roche, 06402712001, Germany) in qRT-PCR allowed precise calculation of gene expressions using the 2-∆∆CT method. Construction of Lentivirus and Stable Cell Lines The design and packaging of OE (Overexpression)/RNAi (RNA interfering) lentiviruses was entrusted to SHANGHAI GENECHEM CO.,LTD ( https://www.genechem.com.cn/ ). Lentiviruses for GPR176 overexpression, GPR176 knockdown (sh-GPR176), PIP5K1A overexpression, and PIP5K1A knockdown (sh-PIP5K1A) were constructed using the wild-type sequences of GPR176 and PIP5K1A, respectively. Lentiviruses containing empty vectors were used as the control group for infection. The infection concentration of all lentiviruses in this study was set to 5 moi, with an infection duration of 12 hours. Puromycin was added at a concentration of 10 ng/ml to select cells with off-target effects, and the selection period was 5 days. The GPR176 overexpression lentivirus contained the wild-type sequence of the GPR176 gene, while the PIP5K1A overexpression lentivirus contained the wild-type sequence of the PIP5K1A gene. The functional sequences of sh-GPR176 and sh-PIP5K1A are provided in Table S2 . The efficacy of these lentiviruses was unequivocally verified through a comprehensive investigation using qRT-PCR assay and Western Blot Assay. Western Blot Assay (WB) The extraction of proteins entailed a combination of RIPA reagent (Solarbio, R0010, Beijing) and 1% PMSF (Solarbio, P0100, Beijing). Quantification of protein concentration was carefully performed using the BCA protein assay kit (Beyotime, P0009, Shanghai). The proteins underwent a separation via SDS-PAGE electrophoresis, and then transferred to polyvinylidene difluoride (PVDF) membranes. Following a meticulous blocking with 5% skim milk, the PVDF membranes were incubated overnight at 4℃ with the diluent for the primary antibody. Post dual washes with PBST, the membranes were incubated with secondary antibody diluent at 23℃. The visualization of protein bands was performed using the Bio-Rad ChemiDoc MP Imaging System. The information and usage concentrations of the corresponding antibodies are listed in Table S3 . Cell Invasion Assays Before adding the cell suspension in 6.5 mm Transwell® with 8.0 µm Pore Polycarbonate Membrane Inserts (Corning, 3422, Shanghai), the Matrigel was carefully prepared. The addition of 200µL of serum-free suspension containing 100,000 cells to the Transwell® Inserts, placed on a 24-well plate filled with 600 ul of 10% FBS DMEM medium, marked the start of a 48-hour incubation at 37°C and 5% CO2. After incubation, the insert was fixed with 4% paraformaldehyde for 30 minutes and stained with a crystal violet solution for 20 minutes. Subsequent actions included the erasure of cells on the upper layer were then removed, carefully washed PBS, and the invaded cells were closely observed under an inverted microscope. Cell Wound Healing Assays Matched to cell proliferation, a precise wound was induced when the cells covered 90–100% of the culture plate area, facilitated by a 200µL suction tip. After washing twice with PBS, serum-free medium was added to the plate. Subsequently, the width of the wound was then carefully observed and photographed consecutively under the microscope after 0h and 48h. Nude-mouse Transplanted Tumor Model Construction The cells, which were carefully maintained in a stable growth state, were diligently diluted to 10 6 cells/mL with pre-cooled PBS. A 200 ul cell suspension containing 10^7 cells was aseptically injected under the skin of nude mice. Since transplantation, the weight, tumor volume, and general health status of the nude mice were regularly monitored and recorded. Tumor volumes were calculated using the exact formula: V = length × width^2 × π/6. Statistical Analysis Each experiment was carefully replicated, with each group containing at least three replicates. The analysis of experimental data was impeccably executed using IBM SPSS Statistics software (version 26.0). The results of experiments were judiciously presented as mean ± standard error. Comparison of differences between two groups was performed using Student's t-test, and multiple comparisons were performed using two-way analysis of variance (ANOVA). A significance level of P < 0.05 was unequivocally deemed statistically significant. Results GPR176 is associated with the prognosis and clinicopathologic factors of gastric adenocarcinomas. The expression matrices and corresponding clinical data were respectively obtained from TCGA-STAD dataset and GSE66254 dataset, and comprehensively evaluated the expression of GPR176, its prognostic significance, and its correlation with clinicopathologic factors. Analysis of the pan-cancer data from TCGA revealed significant differences in GPR176 expression between cancer and adjacent tissues in CHOL, COAD, ESCA, HNSC, KICH, KIRC, LIHC, LUAD, STAD and UCEC (P < 0.05) (Fig. 1 a). In the TCGA-STAD dataset, the expression of GPR176 was significantly higher in gastric cancer tissues than in adjacent tissues. Additionally, GPR176 expression was lower in TNM stage 1 gastric adenocarcinoma patients than in stage 2/3/4 patients, and GPR176 expression was significantly lower in stage T1 patients compared with T2/T3/T4 (Fig. 1 b-d). Statistical analysis confirmed the association of GPR176 with tissue type and tumor stage, suggesting its importance in the occurrence and progression of gastric adenocarcinoma. Subsequently, a significant correlation was observed between GPR176 expression and overall survival (OS)/recurrence-free survival (RFS) in gastric adenocarcinoma, with high GPR176 expression associated with poorer prognosis (Fig. 1 e-g). Using the median expression of GPR176 as a cut-off point, patients were categorized into GPR176-High and GPR176-Low groups. We observed a higher proportion of T3/T4 patients in the GPR176-High group (Fig. 1 h). The area under the ROC curve reached 0.8315, indicating GPR176 as a good biomarker to distinguish gastric adenocarcinoma from normal gastric tissue (Fig. 1 i). Correlation analysis of GPR176 with PIP5K1A expression in gastric adenocarcinoma tissues using the TCGA-STAD dataset confirmed a linear positive correlation (Fig. 1 j). Nomogram construction The bar charts were created based on GPR176 expression and clinicopathologic parameters. Univariate Cox regression analysis and multivariate Cox regression analysis were performed using clinical case characteristics such as age, sex, TNM stage, T stage, N stage, M stage, histologic grade, and GPR176 expression ( Table S4 ). The results of univariate Cox regression analysis results indicated that age, sex, TNM stage, T stage, N stage, M stage, and histological grade were associated with the overall survival of gastric adenocarcinoma patients. In Multivariate Cox regression analysis, only age, histologic grade, and GPR176 expression were associated with overall survival of gastric adenocarcinoma patients. Bar charts based on age, gender, TNM stage, T stage, N stage, M stage, histological grade, and GPR176 expression were constructed to assess the risk of death for specific patients (Fig. 2 a). The predictive power of the histogram was evaluated by comparing the grade between the training group and the validation group. The nomogram showed a high degree of overlap between the self-validation cohort and the training group in predicting the 1-, 3-, or 5-year prognosis (Fig. 2 b-d). Exploration of the mechanisms of GPR176 action using bioinformatics tools. Based on the expression levels of GPR176, patients in the TCGA-STAD dataset were categorized into high and low expression groups. Differential expression analysis was performed using the Limma package for RNA sequencing data (RNA-seq), and a volcano plot was generated (Fig. 3 a). The corresponding pie charts showed that 326 genes were upregulated, and 581 genes were downregulated. Functional enrichment analysis of differentially expressed genes associated with GPR176 revealed enrichment in signaling pathways such as PI3K/AKT/mTOR (Fig. 3 b) and cellular functions such as cell adhesion and G-protein-coupled receptor signaling (Fig. 3 c). Gene Set Enrichment Analysis (GSEA) indicated associations between GPR176 and tumor-related signaling pathways such as cell adhesion, Hedgehog signaling pathway, Jak-stat signaling pathway, and MAPK signaling pathway (Fig. 3 d). CDK2, FOXO1, FOXO3, and VEGFA were identified as downstream target genes of the PI3K/AKT/mTOR signaling pathway, reflecting the activation/inhibition status of the pathway. Linear regression analysis using RNA-seq data from gastric adenocarcinoma tissues in TCGA-STAD revealed a strong correlation between GPR176 and CDK2, FOXO1, FOXO3, and VEGFA (Fig. 3 e). GPR176 enhances the migration and invasion capabilities of gastric cancer cells and induces EMT. To confirm the effect of GPR176 on the biological behavior of gastric cancer cells and explore the corresponding mechanisms, we used lentiviral vectors to overexpress and knockdown GPR176. PCR and immunoblotting confirmed the satisfactory efficiency of OE lentivirus and RNAi lentivirus in HGC-27 and NCI-N87 cells ( Fig S1 a-d ). Subsequently, scratch healing and trans-well assays were performed to evaluate the effects of modulating GPR176 expression on cell migration and invasion. After upregulation of GPR176, the migration and invasion ability of cells increased significantly, while downregulation of GPR176 resulted in a significant decrease in this ability (Fig. 4 a, b). Subsequent PCR analysis revealed a significant increase in the mRNA levels of EMT-activating genes, such as CDH2, VIM, and SNAI1, following upregulation of GPR176. In contrast, the expression of the gene CDH1, associated with the inhibition of the EMT pathway, significantly decreased. Conversely, downregulation of GPR176 led to a significant decrease in the mRNA levels of EMT-activating genes, accompanied by a significant increase in the expression of the gene CDH1, associated with the inhibition of the EMT pathway. (Fig. 4 c). The WB assay results confirmed the PCR results and showed a significant increase in the protein concentration of N-cadherin, vimentin, and Snai1, and a decrease in the protein concentration of E-cadherin after upregulation of GPR176 (Fig. 4 d), with the corresponding bar chart shown in Fig S2 a-b . The above PCR and WB experiments indicated that GPR176 promotes the activation of the EMT signaling pathway and enhances the migration and invasion ability of gastric adenocarcinoma cells. Downregulation of PIP5K1A reverses the effects of GPR176 on cell migration/invasion, EMT, and PI3K/AKT/mTOR. Previous studies have confirmed that PIP5K1A is an upstream molecule in the PI3K/AKT/mTOR signaling pathway. Analysis of RNA-seq data from the TCGA-STAD dataset in this study revealed a significant positive correlation between GPR176 and PIP5K1A expression. Therefore, we hypothesized that GPR176 might activate the PI3K/AKT/mTOR signaling pathway by inducing upregulation of PIP5K1A expression. We separately performed interference and overexpression of PIP5K1A based on the overexpression of GPR176 and the repression of GPR176. We found that the interference of PIP5K1A expression significantly reversed the migration and invasion induced by GPR176 overexpression (Fig. 5 a-b). Conversely, downregulation of PIP5K1A also significantly reversed the slowed migration and invasion of gastric cancer cells caused by GPR176 knockdown (Fig. 5 a-b). Following that, we conducted WB assay to evaluate the phosphorylation levels of molecules within the PI3K/AKT/mTOR pathway and the EMT signaling pathway, along with assessing the expression levels of PIP5K1A. The activation of the PI3K/AKT/mTOR pathway and EMT signaling pathway after GPR176 upregulation was attenuated by the downregulation of PIP5K1A. Conversely, the inhibition of the PI3K/AKT/mTOR pathway and EMT signaling pathway after GPR176 downregulation was counteracted by the overexpression of PIP5K1A (Fig. 6 a-b). Corresponding bar graphs illustrating protein expression levels and phosphorylation levels are depicted in Fig S3 a-b . The results of the PCR assay were in concordance with the aforementioned experiments, indicating a reversal in the mRNA expression levels of molecules in the EMT signaling pathway after GPR176 upregulation, which was suppressed by the downregulation of PIP5K1A in HGC-27 and NCI-N87 cells; Similarly, in HGC-27 and NCI-N87 cells, the downregulation of GPR176 resulted in a reversal of the mRNA expression levels of molecules in the EMT signaling pathway, under the upregulating influence of PIP5K1A. (Fig. 7 a-l). The status of EMT and PI3K/AKT/mTOR between para-cancer and cancer tissues, as well as the expression of PIP5K1A and GPR176. Firstly, gastric cancer patients were categorized into two groups based on the presence or absence of lymph node metastasis: those with lymph node metastasis and those without. Further classification was made based on histological type, distinguishing between the para-cancer group and the cancer group. Initially, Western Blot analysis was employed to assess the protein levels of E-cadherin, N-cadherin, PIP5K1A, GPR176, AKT, p-AKT, mTOR, and p-mTOR in each group. Our findings revealed that irrespective of lymph node metastasis, the expression of E-cad in the cancer group was significantly lower compared to the para-cancer group. Conversely, levels of N-cad, PIP5K1A, GPR176, p-AKT, p-mTOR, p-AKT/AKT, and p-mTOR/mTOR were notably higher in the cancer group compared to the para-cancer group (Fig. 8 a-j). The representative blots of WB experiments are referenced in Fig S4 a . Subsequently, validation of the expression levels of key genes in the EMT pathway and PI3K/AKT/mTOR signaling pathway, as well as PIP5K1A and GPR186, was conducted using qPCR. Among patients without lymph node metastasis, no significant differences were observed in GPR186 and CDH2 expression between the para-cancer and cancer groups (Fig. 8 l, n). However, CDH1 expression was significantly lower in the cancer group compared to the para-cancer group (Fig. 8 k). Conversely, PIP5K1A expression was markedly higher in the cancer group (Fig. 8 m). In patients with lymph node metastasis, there were no significant differences in CDH1 expression between the para-cancer and cancer groups (Fig. 8 o). Nevertheless, GPR186, CDH2, and PIP5K1A expression levels were significantly higher in the cancer group compared to the para-cancer group (Fig. 8 p, q, r). In summary, the expression levels of PIP5K1A and GPR176 were higher in the cancer group compared to the para-cancer group. Additionally, activation of the EMT pathway and PI3K/AKT/mTOR pathway was evident in the cancer group. GPR176 promotes the growth of transplanted tumors by activating the PI3K/AKT/mTOR pathway. The cellular experiments have provided evidence that GPR176 could activate the PI3K/AKT/mTOR pathway, facilitating EMT, and promoting cell proliferation by inducing the overexpression of PIP5K1A. To further validate the role of GPR176 in gastric cancer, a nude mouse subcutaneous tumor experiment was designed in this study to assess the influence of GPR176 expression on the PI3K/AKT/mTOR pathway and EMT. In comparison to the Lenti-NC group, the GPR176 upregulation group exhibited a significantly accelerated tumor growth rate. Conversely, relative to the sh-NC group, the sh-GPR176 group displayed a substantial reduction in tumor growth rate (Fig. 9 a). Subsequent examination of the mRNA expression levels of EMT pathway molecules, consistent with the in vitro results, revealed activation of the EMT pathway following GPR176 upregulation, while the EMT signaling pathway was inhibited after GPR176 downregulation (Fig. 9 b). Western blot results consistently indicated activation of the EMT pathway and the PI3K/AKT/mTOR pathway after GPR176 upregulation, whereas inhibition of the EMT pathway and PI3K/AKT/mTOR pathway occurred after GPR176 downregulation (Fig. 9 c). The corresponding bar graphs illustrating protein expression levels and phosphorylation levels are presented in Fig S5 a-k . Discussion Gastric cancer is a highly malignant tumor characterized by the absence of typical early symptoms, often leading to late-stage diagnosis and missing the optimal treatment window. This poses several challenges for the treatment of gastric cancer 38 . In the advanced stages, gastric cancer has usually spread to lymph nodes or other organs, making treatment considerably more difficult 39 – 41 . Due to the different subtypes of gastric cancer, each with different biological behaviors and treatment responses, the development of universal treatment plans becomes complex and requires more individualized and precise treatment strategies 42 , 43 . Some patients develop resistance to conventional chemotherapeutic agents, leading to a decrease in treatment efficacy and requiring constant adjustment of drug combinations during treatment to overcome the tumor’s adaptability 44 , 45 . In cases of advanced or inoperable gastric cancer, the effectiveness of surgical treatment is limited. Although radiation and chemotherapy are conventional treatment methods, their control over the tumor is also limited 8 . Despite the remarkable success of immunotherapy in certain adenocarcinomas, its use in gastric cancer still faces challenges 22 . The use of immunotherapy is hampered by issues such as immune tolerance and immune escape of the tumor, which hinders its widespread use in the treatment of gastric cancer 46 . Some patients have difficulty accessing high-quality medical services due to economic or societal factors, which exacerbates inequality in treatment and put certain patients in a more difficult treatment situation. Giving these challenges, there is an urgent need for further research in the field of gastric cancer treatment to develop more effective and personalized treatment strategies. In addition, improving early detection and diagnosis is crucial to improving patient survival rates and overall quality of life. GPR176 is generally thought to be associated with circadian rhythms 47 . However, in recent years, some researchers have begun to recognize its significant role in tumors. For example, Tang et al. found that GPR176 interacts with the G protein GNAS to inhibit mitochondrial autophagy in colorectal cancer cells, thereby promoting cancer progression 48 . In this study, a significant upregulation of GPR176 expression in gastric cancer was found, with significant differences in expression between different TNM stages and tissue types. This heterogeneous expression suggests that GPR176 may play different biological roles in different subtypes of gastric cancer. Through integrated analysis of data from TCGA and GEO (Gene Expression Omnibus) databases, it was clarified that high GPR176 expression is significantly associated with shorter overall survival and disease-free survival in patients with gastric cancer. This strongly implies that GPR176 could serve as an independent prognostic marker, providing a new molecular standard for the assessment of patient survival. Similarly, researchers such as Wen-Jing Yun have previously suggested that GPR176 may also serve as a prognostic biomarker in breast cancer 49 . Bioinformatic analysis revealed that the gene expression profile regulated by the accumulation of GPR176 is associated with signaling pathways such as PI3K/AKT/mTOR. This suggests that GPR176 may be involved in the development of gastric cancer via this signaling pathway. Further experimental verification demonstrated the link between GPR176 and the PI3K/AKT/mTOR signaling pathway and provided an experimental basis for understanding the specific role of GPR176 in cell proliferation, survival, and metastasis. The PI3K signaling pathway plays a crucial role in the proliferation and progression of various cancer cells, including gastric cancer 50 . Numerous previous studies have indicated that the PI3K signaling pathway promotes the progression of gastric cancer through various mechanisms, including inhibiting cell apoptosis, inducing drug resistance, facilitating metastasis, and promoting angiogenesis 51 , 52 . After activation by PI3K and PIP2, AKT kinase relocates downstream to the cell membrane, triggering its conformational activation 53 . AKT plays a pivotal role in activating the PI3K axis. Elevated expression of AKT and p-AKT has been detected in over 74% of gastric cancer cases 54 . Aberrant expression of p-AKT is strongly correlated with the overexpression of PI3K and HER2, and high levels of p-AKT are regarded as indicators of tumor progression, metastasis, and poor prognosis in GC 55 . Analysis of the TCGA molecular subtypes reveals that most gastric cancer cases studied display varying degrees of PIK3CA gene mutations, along with amplifications of RTK genes such as EGFR and HER2 56 . Genomic amplifications significantly contribute to tumor progression. Amplification of PIK3CA is closely linked to tumor progression, prognosis, and the development of GC resistance 57 . The substantial involvement of the PI3K/AKT/mTOR signaling pathway in GC progression suggests that targeting this signaling axis holds promise for cancer therapy 58 . Our experimental results indicate that high expression of GPR176 significantly promotes epithelial-mesenchymal transition (EMT) in gastric cancer cells, a critical step in the invasion and metastasis of adenocarcinoma cells. Future research could explore the detailed mechanisms of GPR176 in EMT regulation, including its interaction with other EMT-related factors and regulatory networks. We observed a positive correlation between GPR176 expression and PIP5K1A, and downregulation of PIP5K1A reversed the effects of GPR176 on cell migration/invasion, EMT, and the PI3K/AKT/mTOR signaling pathway. This suggests that GPR176 may exert a more precise regulatory role in the PI3K/AKT/mTOR signaling pathway through synergistic regulation with PIP5K1A. Future studies could use RNA interference and gene knock-in experiments to verify the interaction between GPR176 and PIP5K1A and elucidate their specific mechanisms in regulating the pathway. Through experiments in a xenograft model, it was confirmed that the overexpression of GPR176 significantly promotes tumor growth and activates EMT and the PI3K/AKT/mTOR signaling pathway. This not only supports the experimental in vitro results but also provides an experimental basis for further investigation of the role of GPR176 in the tumor microenvironment. The study of the effects of GPR176 on gastric cancer cells and the corresponding mechanisms holds significant scientific and clinical value. As a member of the G protein-coupled receptor family, the mechanisms of action of GPR176 in normal physiology and pathology are not yet fully understood. In-depth exploration of GPR176 expression and function in gastric cancer contributes to a more complete understanding of its role in adenocarcinoma biology, including its effects on cell signal transduction, proliferation, and metastasis. If GPR176 plays a crucial role in gastric cancer, it could prove to be a potential therapeutic target. Understanding the mechanisms of action of GPR176 may provide a theoretical basis for the development of drugs targeting this receptor and offer new strategies for gastric cancer treatment. Gastric cancer is a highly heterogeneous tumor, and patients respond differently to treatment. The study of GPR176 may lead to a better understanding of its expression and function in different subtypes of tumors and thus contribute to the development of more individualized treatment plans. The correlation between GPR176 expression and overall survival and disease-free survival in gastric cancer patients suggests that GPR176 may be a potential biomarker for assessing patient prognosis and formulating personalized treatment plans. Investigating the mechanisms of action of GPR176 helps to uncover the signaling pathways it regulates and the biological processes in which it is involved. This is not only crucial for understanding the specific role of GPR176 in the occurrence and development of gastric cancer but also contributes to the discovery of new therapeutic targets and strategies. In summary, the in-depth study of the effect of GPR176 on gastric cancer cells and its mechanisms offers a new perspective for our understanding of this disease and provides essential scientific insights for the treatment and prognosis assessment of gastric cancer. In summary, this study comprehensively elucidates the intricate regulatory mechanisms of GPR176 in gastric adenocarcinoma. This includes its diverse expression in the different subtypes, its correlation with patient prognosis, its influence on the PI3K/AKT/mTOR signaling pathway, the mechanism of EMT induction, and its interplay with PIP5K1A. These findings provide a solid theoretical basis for considering GPR176 as a potential target in the treatment of gastric adenocarcinoma and offer a new perspective for a deeper understanding of the mechanisms of cancer development. Subsequent research efforts should aim to further substantiate these findings and investigate the interconnections between GPR176 and additional signaling pathways to advance its in-depth exploration in the field of tumor biology. Abbreviations GPR176 G protein-coupled receptor 176 GC gastric cancer PIP5K1A phosphatidylinositol-4-phosphate 5-kinase type 1 alpha GSEA Gene Set Enrichment Analysis DAVID Database for Annotation, Visualization and Integrated Discovery GO Gene ontology ROC Receiver operating characteristic curve AUC The Area Under Curve qPCR Quantitative polymerase chain reaction WB Western Blot EMT Epithelial-Mesenchymal Transition Declarations Ethics approval and consent to participate The investigation had been approved by the ethics committee of Guangxi Medical University the first affiliated hospital (Approval number: 2022 [KY‐E‐251]). All methods in this research were carried out in accordance with Declaration of Helsinki. Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request Declaration of No Conflict of Interest: I declare that there are no relevant financial or non-financial conflicts of interest in the preparation of this manuscript. Funding This work was supported in part by the Medical Excellence Award Funded by the Creative Research Development Grant from the First Affiliated Hospital of Guangxi Medical University (NO.202204), Self-funded Research Project of Health Commission of Guangxi Zhuang Autonomous Region (Z-A20220397) and Hubei Chen Xiaoping Science and Technology Development Foundation (CXPJJH122002-027) Authors' contributions gc M conceived and designed the research program; gc M and KY L made acquisition of data; ZC H, HC R, DL, GY Z and JT C performed data analysis. gc M, ZC H, HC R, and KY L performed the experiments. KY L wrote the manuscript. gc M guided and supervised the manuscript. All authors read and approved the final manuscript. All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. Acknowledgements The authors thank the contributors of GSE66254 and TCGA database for sharing the data on open access. References Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. Cancer J Clin. 2021;71(3):209–49. 10.3322/caac.21660 . Guo HX, Wang Q, Wang C, Yin QC, Huo HZ, Yin BH. 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Significance of Akt phosphorylation on tumor growth and vascular endothelial growth factor expression in human gastric carcinoma. Pathobiology. 2006;73(1):8–17. 10.1159/000093087 . Additional Declarations No competing interests reported. Supplementary Files Figurelegendsofsupplemantary.docx TableS1.docx TableS2.docx TableS3.docx TableS4.docx FigureS1.jpg FigureS2.jpg FigureS3.jpg FigureS4.jpg FigureS5.jpg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5713551","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":394520275,"identity":"44c5b487-b2e6-40f7-ada1-75e330175f01","order_by":0,"name":"Guangchuan Mu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYDADAxDxAcqRIFoL4wyStTDzEKNFd0b6NYmfO2rlzSWSnz22+XU42uAA88HbPAx2ebi0mN3IKZPsPXPccOeMNHPj3L603A0H2JKteRiSi/FoSZPgbTuWYHAjwUw6t8cGqIXHTJqH4UBiAx4tkn/BWtK/SVv2SAC18H8joCX9mDRvWw1QS46ZNMMPsC1s+LWcecNsLdt2wHDDmTdATzWk5c48zGZsOccgGbeW4+kPb75tq5M3OJ6+TeLHn8O5fcebH954U2GHUwsDAw8oRg5D2IxtQIIZxDLAqR4I2B8AiToo5w8+laNgFIyCUTBSAQDNXVzyvgtXKAAAAABJRU5ErkJggg==","orcid":"","institution":"The First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Guangchuan","middleName":"","lastName":"Mu","suffix":""},{"id":394520276,"identity":"c3626e52-8e5b-4b49-bb45-5817709b2d90","order_by":1,"name":"Kaiyan Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kaiyan","middleName":"","lastName":"Li","suffix":""},{"id":394520277,"identity":"b147f4a4-47f5-4f46-8fdc-dd8ee35d1797","order_by":2,"name":"Chaozhen Hu","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chaozhen","middleName":"","lastName":"Hu","suffix":""},{"id":394520278,"identity":"f761f57a-ca7b-4865-a469-d5dc2fac69fd","order_by":3,"name":"Jintao Cai","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jintao","middleName":"","lastName":"Cai","suffix":""},{"id":394520279,"identity":"ab9e951b-fb77-4f18-a753-9db7942e3e7d","order_by":4,"name":"Huichao Ruan","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Huichao","middleName":"","lastName":"Ruan","suffix":""},{"id":394520280,"identity":"bd64c02a-9c87-4533-ba5e-a9e783c0d750","order_by":5,"name":"Guanyu Zhu","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guanyu","middleName":"","lastName":"Zhu","suffix":""},{"id":394520281,"identity":"ab8c2370-d035-4f95-bcf3-60dce3c3ee53","order_by":6,"name":"Dang Liu","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dang","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-12-26 04:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5713551/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5713551/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76483968,"identity":"ff590017-f7ce-46c2-8d3c-b7d175492e54","added_by":"auto","created_at":"2025-02-17 15:13:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1561999,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between GPR176 expression levels and clinical-pathological features, as well as prognosis, in STAD patients. a. GPR176 expression levels in common malignant tumors and adjacent tissues; b. Comparison of GPR176 expression levels in STAD tumor tissues and adjacent stomach tissues; c. Expression levels of GPR176 mRNA in STAD tumors after TNM staging (I, II, III, IV); d. Expression levels of GPR176 mRNA in STAD tumors at T1, T2, T3, and T4 stages; e. Tn the TCGA-STAD dataset, Overall survival of STAD patients of GPR176 high and low expression groups using the median value as a cutoff; f. In the GSE66254 dataset, Overall survival of STAD patients of GPR176 high and low expression groups using the 75th percentile value as a cutoff; g. In the GSE66254 dataset, Disease-free survival of STAD patients of GPR176 high and low expression groups using the 75th percentile as a cutoff; h. Association of GPR176 expression with T staging in the TCGA-STAD dataset; i. The ROC curve based on GPR176 plotted for distinguishing between gastric cancer and adjacent tissues; j. Significant positive correlation between GPR176 and PIP5K1A expression in the gastric cancer samples in TCGA-STAD dataset.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/bee5038c27b0d913967f39de.jpg"},{"id":76483979,"identity":"89029e2c-a553-4cff-84e8-63027f4d25f0","added_by":"auto","created_at":"2025-02-17 15:14:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1068990,"visible":true,"origin":"","legend":"\u003cp\u003eNomogram constructed based on GPR176 and clinical-pathological features in the TCGA-STAD dataset. a. Nomogram; b. Fitted curve evaluating the accuracy of one-year survival prediction; c. Fitted curve evaluating the accuracy of three-year survival prediction; d. Fitted curve evaluating the accuracy of five-year survival prediction.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/4932b3cca7fcd82fffc31e7b.jpg"},{"id":76483953,"identity":"5dc69e98-b8cc-4399-acfd-8e82363f10e9","added_by":"auto","created_at":"2025-02-17 15:13:58","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1562771,"visible":true,"origin":"","legend":"\u003cp\u003eBioinformatics analysis of the mechanism of action of GPR176. a. Volcano plot of differentially expressed genes between GPR176 high and low expression groups in the TCGA-STAD dataset; b. KEGG signaling pathways enriched based on differentially expressed genes; c. GO terms enriched based on differentially expressed genes; d. GSEA analysis results based on differentially expressed genes; e. Correlation between GPR176 expression and the expression of target genes of PI3K/AKT/mTOR signaling pathway.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/e762fec7dfc99dbf43ebfa12.jpg"},{"id":76483982,"identity":"07e15b79-039f-4d69-9e9e-0cdf45cafdbf","added_by":"auto","created_at":"2025-02-17 15:14:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5495275,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic regulation of GPR176 influences cell invasion/migration and EMT signaling pathways in gastric cancer cells. a. Upregulation and downregulation of GPR176 promote and inhibit migration of gastric cancer cells, respectively; b. Upregulation and downregulation of GPR176 promote and inhibit invasion of gastric cancer cells, respectively; c. Effects of upregulation and downregulation of GPR176 on expression of EMT pathway genes in mRNA level in HGC-27 and NCI-N87 cells; d. Effects of upregulation and downregulation of GPR176 on protein expression in the EMT pathway in HGC-27 and NCI-N87 cells, with the bar chart displayed in Figure S1.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/071657f1fd4589063082ba94.jpg"},{"id":76484232,"identity":"83dbef16-ad96-4f27-ad84-c97057df93b8","added_by":"auto","created_at":"2025-02-17 15:21:59","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5176263,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic regulation of PIP5K1A counteracts the effects of GPR176 on invasion, migration. a. Combined genetic regulation of GPR176 and PIP5K1A on cell invasion; b. Combined genetic regulation of GPR176 and PIP5K1A on cell migration.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/f38f29b5a460830c3f3339ec.jpg"},{"id":76483983,"identity":"fef6e38f-a2d4-4d60-93f7-38060e302f2f","added_by":"auto","created_at":"2025-02-17 15:14:00","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1493931,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic regulation of PIP5K1A counteracts the effects of GPR176 on EMT pathway, and PI3K/AKT/mTOR pathway. a. Combined genetic regulation of GPR176 and PIP5K1A on protein expression and phosphorylation in the EMT pathway and PI3K/AKT/mTOR pathway in HGC-27 cells, with the bar chart displayed in Figure S2; b. Combined genetic regulation of GPR176 and PIP5K1A on protein expression and phosphorylation in the EMT pathway and PI3K/AKT/mTOR pathway in HGC-27 cells, with the bar chart displayed in Figure S2.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/4e469da3ced7878c8361cfcd.jpg"},{"id":76484239,"identity":"ce4f19cb-4cee-4a5b-bd66-d3c0e74a365a","added_by":"auto","created_at":"2025-02-17 15:22:00","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1506580,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic regulation of PIP5K1A counteracts the effects of GPR176 on the expression of EMT pathway genes in mRNA level. a-f. Combined genetic regulation of GPR176 and PIP5K1A on expression levels of EMT pathway genes in HGC-27 cells; g-i. Combined genetic regulation of GPR176 and PIP5K1A on expression levels of EMT pathway genes in NCI-N87 cells.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/9b429d6ca4a9883dfef1bece.jpg"},{"id":76484233,"identity":"e345ef39-57ce-414c-b243-19a168c6dc70","added_by":"auto","created_at":"2025-02-17 15:21:59","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1169130,"visible":true,"origin":"","legend":"\u003cp\u003eExpression level of key molecules EMT signaling pathway and PI3K/AKT/mTOR pathway, as well as the expression of GPR176 and PIP5K1A. a, The relative expression of E-cadherin at the protein level in the specific group; b, Relative expression of GPR176 at the protein level in the specific group; c, Relative expression of N-cadherin at the protein level in the specific group; d, Relative expression of PIP5K1A at the protein level in the specific group; e, Relative expression of p-AKT at the protein level in the specific group; f, Relative expression of AKT at the protein level in the specific group; g, relative expression of p-AKT/AKT at the protein level in the specific group; h, Relative protein expression of p-mTOR at the protein level in the specific group; i, Relative expression of mTOR at the protein level in the specific group; j, Relative expression of p-mTOR/mTOR at the protein level in the specific group; k, Relative expression of CDH1(E-cadherin) at the mRNA level in the specific group; i, Relative expression of GPR176 at the mRNA level in the specific group; m, Relative expression of PIK5K1A at the mRNA level in the specific group; n, Relative expression of CDH2(N-cadherin) at the mRNA level in the specific group; o, Relative expression of CDH1(E-cadherin) at the mRNA level in the specific group; p, Relative expression of GPR176 at the mRNA level in the specific group; r, Relative expression of PIK5K1A at the mRNA level in the specific group; s, Relative expression of CDH2(N-cadherin) at the mRNA level in the specific group.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/55259b89c96a90a1a9050759.jpg"},{"id":76483977,"identity":"8aa5995f-1564-424c-b176-ffc29d3fce60","added_by":"auto","created_at":"2025-02-17 15:14:00","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1543284,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic regulation of GPR176 expression affects mouse subcutaneous graft tumor growth, PI3K/AKT/mTOR pathway and the EMT pathway. a. Effects of upregulation and knockdown of GPR176 expression on tumor growth; b. Effects of GPR176 expression regulation on the expression of PIP5K1A and EMT signaling pathway genes; c. Effects of GPR176 expression regulation on the EMT signaling pathway and PI3K/AKT/mTOR pathway, with the bar chart displayed in Figure S3.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/1794b2fdc429747bab0d9956.jpg"},{"id":78078342,"identity":"7d10e01f-2608-43cb-9c1e-abf4f61dd106","added_by":"auto","created_at":"2025-03-09 10:46:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21476811,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/98d74d64-10ba-4641-825a-9191bf20b938.pdf"},{"id":76485154,"identity":"9f7e2d94-4ce1-4eb2-9876-fd1a43289f48","added_by":"auto","created_at":"2025-02-17 15:30:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25622,"visible":true,"origin":"","legend":"","description":"","filename":"Figurelegendsofsupplemantary.docx","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/a115f88b97d74a17ae88ae63.docx"},{"id":76483974,"identity":"ec1c26f0-4b32-4030-9a93-5e2e5efdc6c8","added_by":"auto","created_at":"2025-02-17 15:13:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":23480,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/f07ac2c8e031334e0eff4d8e.docx"},{"id":76483956,"identity":"0e4eaf1f-14ac-4f8d-abd9-0433c35db9ca","added_by":"auto","created_at":"2025-02-17 15:13:59","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":23715,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/262db6138f8a6bf37c306f20.docx"},{"id":76483969,"identity":"5c1c7929-ae0b-4bc9-8674-579ac281ab5b","added_by":"auto","created_at":"2025-02-17 15:13:59","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":25501,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/c4a4139d7aee68e300e933fb.docx"},{"id":76483960,"identity":"b9fb8a04-a9c8-41f1-a5a7-bd11ca8f7494","added_by":"auto","created_at":"2025-02-17 15:13:59","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":35023,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/6836e6ee1f90f560fd00cfa7.docx"},{"id":76484234,"identity":"f1dd5cd8-99f1-4da6-9d9b-1904585eb092","added_by":"auto","created_at":"2025-02-17 15:21:59","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":752121,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/2304785605f15c75cfcb375b.jpg"},{"id":76483958,"identity":"2dfe62fe-3044-4628-8fcb-480dd716ee1a","added_by":"auto","created_at":"2025-02-17 15:13:59","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":509609,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/b0f879f8072e7932426dfbcf.jpg"},{"id":76483995,"identity":"c8c67881-9f0a-44d5-8441-6745f4609b39","added_by":"auto","created_at":"2025-02-17 15:14:01","extension":"jpg","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":960307,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/f2cf371edd21685428eb39e8.jpg"},{"id":76483965,"identity":"2d6b32eb-f298-4dc7-9955-c1def26044f9","added_by":"auto","created_at":"2025-02-17 15:13:59","extension":"jpg","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":409985,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/a95461603b60fddabb7953b4.jpg"},{"id":76484006,"identity":"aa03300c-2847-4796-8504-b6e958b3cada","added_by":"auto","created_at":"2025-02-17 15:14:02","extension":"jpg","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":972924,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5713551/v1/80f5295cfe0785ad5576c3a5.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"GPR176 enhances the epithelial-mesenchymal transition in gastric cancer cells by activating the PI3K/AKT/mTOR signaling pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn 2020, the incidence of gastric cancer reached approximately 1.09\u0026nbsp;million cases, ranking fifth among malignant tumors and accounting for 5.6% of all malignant diseases. The number of deaths caused by gastric cancer totaled around 770,000, ranking fourth and accounting for 7.7% of overall malignant tumor-related fatalities\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The high prevalence and grim prognosis of gastric cancer have significantly impacted the well-being of the population, especially in China\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In 2022, China reported approximately 397,000 new cases of gastric cancer, accounting for 37% of the global total, with both incidence and mortality rates ranking third among malignant tumors in China\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. East Asia bears the brunt of the global burden, with around 60% of gastric cancer cases occurring in this region\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Along with the health burden comes a significant economic loss to residents and the government due to the diagnosis and treatment of gastric cancer\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEarly-stage gastric cancer often presents with inconspicuous symptoms, which leads to late detection, suboptimal treatment outcomes, high recurrence rates, and low survival rates\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The 5-year survival rate for advanced gastric cancer remains as low as 5%\u003csup\u003e13\u0026ndash;15\u003c/sup\u003e. Currently, the predominant treatment strategy for gastric cancer is comprehensive, with a primary emphasis on surgical intervention\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Surgical procedures dominate the treatment landscape for early-stage gastric cancer, while chemotherapy improves survival and quality of life for locally advanced or metastatic cases (stage Ib to IIIb)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Despite the mature theory and practice of abdominal anatomy, coupled with inherent shortcomings of chemotherapy, advances in surgical and chemotherapeutic approaches to the treatment of gastric cancer have been limited in recent decades\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Overall, the efficacy of gastric cancer treatment remains unsatisfactory, with only modest improvements in prognosis\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNew therapeutic approaches, such as targeted drugs and immunotherapy, offer new hope for gastric cancer patients\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Targeting central mechanisms of tumor development, developing drugs that prevent uncontrolled cell proliferation, or directly inducing apoptosis represents a promising path to a successful transformation and even a possible cure of advanced gastric cancer\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGPR176, a G protein-coupled receptor located on 15q14-q15.1, belongs to the G protein-coupled receptor family and functions as a cell surface receptor that responds to hormones, growth factors, and neurotransmitters\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. GPR176 is primarily expressed in the brain, followed by the gallbladder and testis\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. While earlier studies focused primarily on the role and mechanisms of GPR176 in circadian rhythms, more recent research recognizes its significance in tumors. For example, Tang et al. have revealed that GPR176 recruits GNAS, activates the cAMP/PKA/BNIP3L signaling pathway, and inhibits mitochondrial autophagy, which promotes stem cell formation and proliferation of colorectal adenocarcinoma (CRC)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Zheng HC and others pointed out a correlation between the expression of GPR176 and the prognosis of breast adenocarcinoma \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Interfering with GPR176 suppresses the PI3K/AKT/mTOR signaling pathway, glycolysis, epithelial-mesenchymal transition, and proliferation of breast adenocarcinoma cells\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The prognostic value of GPR176 in esophageal adenocarcinoma has also been established, indicating a correlation with prognosis and resistance of esophageal adenocarcinoma\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Public data analyses have hinted at a link between GPR176 and gastric cancer prognosis, warranting further exploration of underlying mechanisms\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur research team has found a correlation between GPR176 and the prognosis of gastric cancer patients. In-depth studies were conducted on the impact mechanisms of GPR176 on the occurrence and development of gastric cancer, with results validated by animal experiments.\u003c/p\u003e \u003cp\u003ePIP5K1A, which encodes the protein phosphatidylinositol-4-phosphate 5-kinase type 1 alpha, plays a role in several processes, including activation of GTPase activity\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. It serves as an upstream regulator of the PI3K/AKT/mTOR signaling pathway, and genetic or pharmacological inhibition of PIP5K1A significantly inhibits AKT phosphorylation\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In our study, we discovered an interaction between GPR176 and PIP5K1A, confirming through extensive experiments that GPR176 activates the PI3K/AKT/mTOR signaling pathway by upregulating PIP5K1A. This activation promotes epithelial-mesenchymal transition and invasive capabilities of gastric cancer cells.\u003c/p\u003e \u003cp\u003eThis study innovatively explores the association of GPR176 with the prognosis and clinicopathologic factors of gastric adenocarcinomas, shedding light on its potential as a significant biomarker. Specifically, in gastric cancer tissues, GPR176 expression is significantly elevated, particularly in advanced tumor stages, suggesting its role in disease progression. Importantly, high GPR176 expression correlates with poorer overall survival and recurrence-free survival in gastric adenocarcinoma patients, indicating its prognostic significance. Furthermore, exploration of GPR176's mechanisms of action reveals its involvement in key signaling pathways such as PI3K/AKT/mTOR and EMT, highlighting its potential as a therapeutic target. Mechanistic studies further demonstrate that GPR176 enhances tumor migration, invasion, and EMT, and its effects are mediated, at least in part, through the PI3K/AKT/mTOR pathway. Additionally, the study uncovers a positive correlation between GPR176 and PIP5K1A, further elucidating the regulatory network underlying GPR176's oncogenic functions. Collectively, these findings underscore the innovative contributions of this study, positioning GPR176 as a promising prognostic biomarker and therapeutic target in gastric adenocarcinoma. Considering the potential of GPR176/PIP5K1A as intervention targets for future gastric cancer metastasis, our results provide a novel theoretical basis for precision treatment of gastric cancer and point to new research directions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData Acquisition\u003c/h2\u003e \u003cp\u003eRNA-seq data from 448 gastric cancer (STAD) samples, including 410 tumor and 38 normal samples, were meticulously collected, along with clinical data from 383 STAD patients, sourced from the TCGA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://adenocarcinomagenome.nih.gov/\u003c/span\u003e\u003cspan address=\"https://adenocarcinomagenome.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e35\u003c/sup\u003e. The Limma package in R (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioconductor.org/packages/release/bioc/html/limma.html\u003c/span\u003e\u003cspan address=\"https://bioconductor.org/packages/release/bioc/html/limma.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) facilitated the standardization of RNA-seq information\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The transcriptomic details for all samples were preserved to investigate the nuanced differences between adenocarcinomatous and adjacent tissues. However, 17 samples with incomplete clinical information were excluded from subsequent survival analysis.\u003c/p\u003e \u003cp\u003eThe RNA-seq data and associated clinical/prognostic information for 300 patients diagnosed with gastric cancer were methodically extracted from the GSE66254 dataset in the GEO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u0026thinsp;=\u0026thinsp;GSE66254)\u003csup\u003e37\u003c/sup\u003e. The original chip data from GSE66254 underwent a rigorous annotation and standardization process in R, utilizing the Limma package.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Culture\u003c/h3\u003e\n\u003cp\u003eThe cell lines HGC-27 and NCI-N87 were procured from the ATCC (The Global Bioresource Center, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.atcc.org/\u003c/span\u003e\u003cspan address=\"https://www.atcc.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) cell repository. Culturing these cells involved a comprehensive medium consisting of DMEM (Dulbecco's Modified Eagle Medium) (Gibco, 10566016, New York), supplemented with 10% FBS (Fetal Bovine Serum) (Gibco, 10099141C, Australia), 1% streptomycin, and 1% penicillin (Solarbio, P1410, Beijing). The controlled environment for these cell cultures was maintained incubators with 5% CO\u003csub\u003e2\u003c/sub\u003e and 37\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eRNA Extraction and PCR\u003c/h3\u003e\n\u003cp\u003eThe meticulous procedure of RNA extraction from each sample followed the strict TRI Reagent\u0026trade; protocol (Invitrogen, AM9738, Canada). Subsequently, reverse transcription into cDNA was carefully performed according to the manufacturer\u0026rsquo;s instructions for the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, RR047B, China). The primers designed through Primer3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://primer3.ut.ee/\u003c/span\u003e\u003cspan address=\"https://primer3.ut.ee/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), had specific sequences as listed in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. The application of FastStart Universal SYBR\u0026reg; Green Master Mix (Roche, 06402712001, Germany) in qRT-PCR allowed precise calculation of gene expressions using the 2-∆∆CT method.\u003c/p\u003e\n\u003ch3\u003eConstruction of Lentivirus and Stable Cell Lines\u003c/h3\u003e\n\u003cp\u003eThe design and packaging of OE (Overexpression)/RNAi (RNA interfering) lentiviruses was entrusted to SHANGHAI GENECHEM CO.,LTD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genechem.com.cn/\u003c/span\u003e\u003cspan address=\"https://www.genechem.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Lentiviruses for GPR176 overexpression, GPR176 knockdown (sh-GPR176), PIP5K1A overexpression, and PIP5K1A knockdown (sh-PIP5K1A) were constructed using the wild-type sequences of GPR176 and PIP5K1A, respectively. Lentiviruses containing empty vectors were used as the control group for infection. The infection concentration of all lentiviruses in this study was set to 5 moi, with an infection duration of 12 hours. Puromycin was added at a concentration of 10 ng/ml to select cells with off-target effects, and the selection period was 5 days. The GPR176 overexpression lentivirus contained the wild-type sequence of the GPR176 gene, while the PIP5K1A overexpression lentivirus contained the wild-type sequence of the PIP5K1A gene. The functional sequences of sh-GPR176 and sh-PIP5K1A are provided in \u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e. The efficacy of these lentiviruses was unequivocally verified through a comprehensive investigation using qRT-PCR assay and Western Blot Assay.\u003c/p\u003e\n\u003ch3\u003eWestern Blot Assay (WB)\u003c/h3\u003e\n\u003cp\u003eThe extraction of proteins entailed a combination of RIPA reagent (Solarbio, R0010, Beijing) and 1% PMSF (Solarbio, P0100, Beijing). Quantification of protein concentration was carefully performed using the BCA protein assay kit (Beyotime, P0009, Shanghai). The proteins underwent a separation via SDS-PAGE electrophoresis, and then transferred to polyvinylidene difluoride (PVDF) membranes. Following a meticulous blocking with 5% skim milk, the PVDF membranes were incubated overnight at 4℃ with the diluent for the primary antibody. Post dual washes with PBST, the membranes were incubated with secondary antibody diluent at 23℃. The visualization of protein bands was performed using the Bio-Rad ChemiDoc MP Imaging System. The information and usage concentrations of the corresponding antibodies are listed in \u003cb\u003eTable \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell Invasion Assays\u003c/h2\u003e \u003cp\u003eBefore adding the cell suspension in 6.5 mm Transwell\u0026reg; with 8.0 \u0026micro;m Pore Polycarbonate Membrane Inserts (Corning, 3422, Shanghai), the Matrigel was carefully prepared. The addition of 200\u0026micro;L of serum-free suspension containing 100,000 cells to the Transwell\u0026reg; Inserts, placed on a 24-well plate filled with 600 ul of 10% FBS DMEM medium, marked the start of a 48-hour incubation at 37\u0026deg;C and 5% CO2. After incubation, the insert was fixed with 4% paraformaldehyde for 30 minutes and stained with a crystal violet solution for 20 minutes. Subsequent actions included the erasure of cells on the upper layer were then removed, carefully washed PBS, and the invaded cells were closely observed under an inverted microscope.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Wound Healing Assays\u003c/h3\u003e\n\u003cp\u003eMatched to cell proliferation, a precise wound was induced when the cells covered 90\u0026ndash;100% of the culture plate area, facilitated by a 200\u0026micro;L suction tip. After washing twice with PBS, serum-free medium was added to the plate. Subsequently, the width of the wound was then carefully observed and photographed consecutively under the microscope after 0h and 48h.\u003c/p\u003e\n\u003ch3\u003eNude-mouse Transplanted Tumor Model Construction\u003c/h3\u003e\n\u003cp\u003eThe cells, which were carefully maintained in a stable growth state, were diligently diluted to 10\u003csup\u003e6\u003c/sup\u003e cells/mL with pre-cooled PBS. A 200 ul cell suspension containing 10^7 cells was aseptically injected under the skin of nude mice. Since transplantation, the weight, tumor volume, and general health status of the nude mice were regularly monitored and recorded. Tumor volumes were calculated using the exact formula: V\u0026thinsp;=\u0026thinsp;length \u0026times; width^2\u0026thinsp;\u0026times;\u0026thinsp;π/6.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eEach experiment was carefully replicated, with each group containing at least three replicates. The analysis of experimental data was impeccably executed using IBM SPSS Statistics software (version 26.0). The results of experiments were judiciously presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Comparison of differences between two groups was performed using Student's t-test, and multiple comparisons were performed using two-way analysis of variance (ANOVA). A significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was unequivocally deemed statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":" \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003cp\u003e \u003cb\u003eGPR176 is associated with the prognosis and clinicopathologic factors of gastric adenocarcinomas.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe expression matrices and corresponding clinical data were respectively obtained from TCGA-STAD dataset and GSE66254 dataset, and comprehensively evaluated the expression of GPR176, its prognostic significance, and its correlation with clinicopathologic factors. Analysis of the pan-cancer data from TCGA revealed significant differences in GPR176 expression between cancer and adjacent tissues in CHOL, COAD, ESCA, HNSC, KICH, KIRC, LIHC, LUAD, STAD and UCEC (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In the TCGA-STAD dataset, the expression of GPR176 was significantly higher in gastric cancer tissues than in adjacent tissues. Additionally, GPR176 expression was lower in TNM stage 1 gastric adenocarcinoma patients than in stage 2/3/4 patients, and GPR176 expression was significantly lower in stage T1 patients compared with T2/T3/T4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-d). Statistical analysis confirmed the association of GPR176 with tissue type and tumor stage, suggesting its importance in the occurrence and progression of gastric adenocarcinoma. Subsequently, a significant correlation was observed between GPR176 expression and overall survival (OS)/recurrence-free survival (RFS) in gastric adenocarcinoma, with high GPR176 expression associated with poorer prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-g). Using the median expression of GPR176 as a cut-off point, patients were categorized into GPR176-High and GPR176-Low groups. We observed a higher proportion of T3/T4 patients in the GPR176-High group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). The area under the ROC curve reached 0.8315, indicating GPR176 as a good biomarker to distinguish gastric adenocarcinoma from normal gastric tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). Correlation analysis of GPR176 with PIP5K1A expression in gastric adenocarcinoma tissues using the TCGA-STAD dataset confirmed a linear positive correlation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eNomogram construction\u003c/h2\u003e \u003cp\u003eThe bar charts were created based on GPR176 expression and clinicopathologic parameters. Univariate Cox regression analysis and multivariate Cox regression analysis were performed using clinical case characteristics such as age, sex, TNM stage, T stage, N stage, M stage, histologic grade, and GPR176 expression (\u003cb\u003eTable \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e). The results of univariate Cox regression analysis results indicated that age, sex, TNM stage, T stage, N stage, M stage, and histological grade were associated with the overall survival of gastric adenocarcinoma patients. In Multivariate Cox regression analysis, only age, histologic grade, and GPR176 expression were associated with overall survival of gastric adenocarcinoma patients. Bar charts based on age, gender, TNM stage, T stage, N stage, M stage, histological grade, and GPR176 expression were constructed to assess the risk of death for specific patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The predictive power of the histogram was evaluated by comparing the grade between the training group and the validation group. The nomogram showed a high degree of overlap between the self-validation cohort and the training group in predicting the 1-, 3-, or 5-year prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExploration of the mechanisms of GPR176 action using bioinformatics tools.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on the expression levels of GPR176, patients in the TCGA-STAD dataset were categorized into high and low expression groups. Differential expression analysis was performed using the Limma package for RNA sequencing data (RNA-seq), and a volcano plot was generated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The corresponding pie charts showed that 326 genes were upregulated, and 581 genes were downregulated. Functional enrichment analysis of differentially expressed genes associated with GPR176 revealed enrichment in signaling pathways such as PI3K/AKT/mTOR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and cellular functions such as cell adhesion and G-protein-coupled receptor signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Gene Set Enrichment Analysis (GSEA) indicated associations between GPR176 and tumor-related signaling pathways such as cell adhesion, Hedgehog signaling pathway, Jak-stat signaling pathway, and MAPK signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). CDK2, FOXO1, FOXO3, and VEGFA were identified as downstream target genes of the PI3K/AKT/mTOR signaling pathway, reflecting the activation/inhibition status of the pathway. Linear regression analysis using RNA-seq data from gastric adenocarcinoma tissues in TCGA-STAD revealed a strong correlation between GPR176 and CDK2, FOXO1, FOXO3, and VEGFA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGPR176 enhances the migration and invasion capabilities of gastric cancer cells and induces EMT.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo confirm the effect of GPR176 on the biological behavior of gastric cancer cells and explore the corresponding mechanisms, we used lentiviral vectors to overexpress and knockdown GPR176. PCR and immunoblotting confirmed the satisfactory efficiency of OE lentivirus and RNAi lentivirus in HGC-27 and NCI-N87 cells (\u003cb\u003eFig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea-d\u003c/b\u003e). Subsequently, scratch healing and trans-well assays were performed to evaluate the effects of modulating GPR176 expression on cell migration and invasion. After upregulation of GPR176, the migration and invasion ability of cells increased significantly, while downregulation of GPR176 resulted in a significant decrease in this ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Subsequent PCR analysis revealed a significant increase in the mRNA levels of EMT-activating genes, such as CDH2, VIM, and SNAI1, following upregulation of GPR176. In contrast, the expression of the gene CDH1, associated with the inhibition of the EMT pathway, significantly decreased. Conversely, downregulation of GPR176 led to a significant decrease in the mRNA levels of EMT-activating genes, accompanied by a significant increase in the expression of the gene CDH1, associated with the inhibition of the EMT pathway. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The WB assay results confirmed the PCR results and showed a significant increase in the protein concentration of N-cadherin, vimentin, and Snai1, and a decrease in the protein concentration of E-cadherin after upregulation of GPR176 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), with the corresponding bar chart shown in \u003cb\u003eFig \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea-b\u003c/b\u003e. The above PCR and WB experiments indicated that GPR176 promotes the activation of the EMT signaling pathway and enhances the migration and invasion ability of gastric adenocarcinoma cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDownregulation of PIP5K1A reverses the effects of GPR176 on cell migration/invasion, EMT, and PI3K/AKT/mTOR.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have confirmed that PIP5K1A is an upstream molecule in the PI3K/AKT/mTOR signaling pathway. Analysis of RNA-seq data from the TCGA-STAD dataset in this study revealed a significant positive correlation between GPR176 and PIP5K1A expression. Therefore, we hypothesized that GPR176 might activate the PI3K/AKT/mTOR signaling pathway by inducing upregulation of PIP5K1A expression. We separately performed interference and overexpression of PIP5K1A based on the overexpression of GPR176 and the repression of GPR176. We found that the interference of PIP5K1A expression significantly reversed the migration and invasion induced by GPR176 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). Conversely, downregulation of PIP5K1A also significantly reversed the slowed migration and invasion of gastric cancer cells caused by GPR176 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). Following that, we conducted WB assay to evaluate the phosphorylation levels of molecules within the PI3K/AKT/mTOR pathway and the EMT signaling pathway, along with assessing the expression levels of PIP5K1A. The activation of the PI3K/AKT/mTOR pathway and EMT signaling pathway after GPR176 upregulation was attenuated by the downregulation of PIP5K1A. Conversely, the inhibition of the PI3K/AKT/mTOR pathway and EMT signaling pathway after GPR176 downregulation was counteracted by the overexpression of PIP5K1A (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-b). Corresponding bar graphs illustrating protein expression levels and phosphorylation levels are depicted in \u003cb\u003eFig \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ea-b\u003c/b\u003e. The results of the PCR assay were in concordance with the aforementioned experiments, indicating a reversal in the mRNA expression levels of molecules in the EMT signaling pathway after GPR176 upregulation, which was suppressed by the downregulation of PIP5K1A in HGC-27 and NCI-N87 cells; Similarly, in HGC-27 and NCI-N87 cells, the downregulation of GPR176 resulted in a reversal of the mRNA expression levels of molecules in the EMT signaling pathway, under the upregulating influence of PIP5K1A. (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-l).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThe status of EMT and PI3K/AKT/mTOR between para-cancer and cancer tissues, as well as the expression of PIP5K1A and GPR176.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFirstly, gastric cancer patients were categorized into two groups based on the presence or absence of lymph node metastasis: those with lymph node metastasis and those without. Further classification was made based on histological type, distinguishing between the para-cancer group and the cancer group. Initially, Western Blot analysis was employed to assess the protein levels of E-cadherin, N-cadherin, PIP5K1A, GPR176, AKT, p-AKT, mTOR, and p-mTOR in each group. Our findings revealed that irrespective of lymph node metastasis, the expression of E-cad in the cancer group was significantly lower compared to the para-cancer group. Conversely, levels of N-cad, PIP5K1A, GPR176, p-AKT, p-mTOR, p-AKT/AKT, and p-mTOR/mTOR were notably higher in the cancer group compared to the para-cancer group (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003ea-j). The representative blots of WB experiments are referenced in \u003cb\u003eFig \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003ea\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, validation of the expression levels of key genes in the EMT pathway and PI3K/AKT/mTOR signaling pathway, as well as PIP5K1A and GPR186, was conducted using qPCR. Among patients without lymph node metastasis, no significant differences were observed in GPR186 and CDH2 expression between the para-cancer and cancer groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003el, n). However, CDH1 expression was significantly lower in the cancer group compared to the para-cancer group (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003ek). Conversely, PIP5K1A expression was markedly higher in the cancer group (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003em). In patients with lymph node metastasis, there were no significant differences in CDH1 expression between the para-cancer and cancer groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003eo). Nevertheless, GPR186, CDH2, and PIP5K1A expression levels were significantly higher in the cancer group compared to the para-cancer group (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003ep, q, r).\u003c/p\u003e \u003cp\u003eIn summary, the expression levels of PIP5K1A and GPR176 were higher in the cancer group compared to the para-cancer group. Additionally, activation of the EMT pathway and PI3K/AKT/mTOR pathway was evident in the cancer group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGPR176 promotes the growth of transplanted tumors by activating the PI3K/AKT/mTOR pathway.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe cellular experiments have provided evidence that GPR176 could activate the PI3K/AKT/mTOR pathway, facilitating EMT, and promoting cell proliferation by inducing the overexpression of PIP5K1A. To further validate the role of GPR176 in gastric cancer, a nude mouse subcutaneous tumor experiment was designed in this study to assess the influence of GPR176 expression on the PI3K/AKT/mTOR pathway and EMT. In comparison to the Lenti-NC group, the GPR176 upregulation group exhibited a significantly accelerated tumor growth rate. Conversely, relative to the sh-NC group, the sh-GPR176 group displayed a substantial reduction in tumor growth rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). Subsequent examination of the mRNA expression levels of EMT pathway molecules, consistent with the in vitro results, revealed activation of the EMT pathway following GPR176 upregulation, while the EMT signaling pathway was inhibited after GPR176 downregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e9\u003c/span\u003eb). Western blot results consistently indicated activation of the EMT pathway and the PI3K/AKT/mTOR pathway after GPR176 upregulation, whereas inhibition of the EMT pathway and PI3K/AKT/mTOR pathway occurred after GPR176 downregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e9\u003c/span\u003ec). The corresponding bar graphs illustrating protein expression levels and phosphorylation levels are presented in \u003cb\u003eFig \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003ea-k\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eGastric cancer is a highly malignant tumor characterized by the absence of typical early symptoms, often leading to late-stage diagnosis and missing the optimal treatment window. This poses several challenges for the treatment of gastric cancer\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. In the advanced stages, gastric cancer has usually spread to lymph nodes or other organs, making treatment considerably more difficult\u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Due to the different subtypes of gastric cancer, each with different biological behaviors and treatment responses, the development of universal treatment plans becomes complex and requires more individualized and precise treatment strategies\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Some patients develop resistance to conventional chemotherapeutic agents, leading to a decrease in treatment efficacy and requiring constant adjustment of drug combinations during treatment to overcome the tumor\u0026rsquo;s adaptability\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn cases of advanced or inoperable gastric cancer, the effectiveness of surgical treatment is limited. Although radiation and chemotherapy are conventional treatment methods, their control over the tumor is also limited\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Despite the remarkable success of immunotherapy in certain adenocarcinomas, its use in gastric cancer still faces challenges\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The use of immunotherapy is hampered by issues such as immune tolerance and immune escape of the tumor, which hinders its widespread use in the treatment of gastric cancer\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Some patients have difficulty accessing high-quality medical services due to economic or societal factors, which exacerbates inequality in treatment and put certain patients in a more difficult treatment situation.\u003c/p\u003e \u003cp\u003eGiving these challenges, there is an urgent need for further research in the field of gastric cancer treatment to develop more effective and personalized treatment strategies. In addition, improving early detection and diagnosis is crucial to improving patient survival rates and overall quality of life.\u003c/p\u003e \u003cp\u003eGPR176 is generally thought to be associated with circadian rhythms\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. However, in recent years, some researchers have begun to recognize its significant role in tumors. For example, Tang et al. found that GPR176 interacts with the G protein GNAS to inhibit mitochondrial autophagy in colorectal cancer cells, thereby promoting cancer progression\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In this study, a significant upregulation of GPR176 expression in gastric cancer was found, with significant differences in expression between different TNM stages and tissue types. This heterogeneous expression suggests that GPR176 may play different biological roles in different subtypes of gastric cancer.\u003c/p\u003e \u003cp\u003eThrough integrated analysis of data from TCGA and GEO (Gene Expression Omnibus) databases, it was clarified that high GPR176 expression is significantly associated with shorter overall survival and disease-free survival in patients with gastric cancer. This strongly implies that GPR176 could serve as an independent prognostic marker, providing a new molecular standard for the assessment of patient survival. Similarly, researchers such as Wen-Jing Yun have previously suggested that GPR176 may also serve as a prognostic biomarker in breast cancer\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBioinformatic analysis revealed that the gene expression profile regulated by the accumulation of GPR176 is associated with signaling pathways such as PI3K/AKT/mTOR. This suggests that GPR176 may be involved in the development of gastric cancer via this signaling pathway. Further experimental verification demonstrated the link between GPR176 and the PI3K/AKT/mTOR signaling pathway and provided an experimental basis for understanding the specific role of GPR176 in cell proliferation, survival, and metastasis.\u003c/p\u003e \u003cp\u003eThe PI3K signaling pathway plays a crucial role in the proliferation and progression of various cancer cells, including gastric cancer\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Numerous previous studies have indicated that the PI3K signaling pathway promotes the progression of gastric cancer through various mechanisms, including inhibiting cell apoptosis, inducing drug resistance, facilitating metastasis, and promoting angiogenesis\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfter activation by PI3K and PIP2, AKT kinase relocates downstream to the cell membrane, triggering its conformational activation\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. AKT plays a pivotal role in activating the PI3K axis. Elevated expression of AKT and p-AKT has been detected in over 74% of gastric cancer cases\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Aberrant expression of p-AKT is strongly correlated with the overexpression of PI3K and HER2, and high levels of p-AKT are regarded as indicators of tumor progression, metastasis, and poor prognosis in GC\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAnalysis of the TCGA molecular subtypes reveals that most gastric cancer cases studied display varying degrees of PIK3CA gene mutations, along with amplifications of RTK genes such as EGFR and HER2\u003csup\u003e56\u003c/sup\u003e. Genomic amplifications significantly contribute to tumor progression. Amplification of PIK3CA is closely linked to tumor progression, prognosis, and the development of GC resistance\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The substantial involvement of the PI3K/AKT/mTOR signaling pathway in GC progression suggests that targeting this signaling axis holds promise for cancer therapy\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur experimental results indicate that high expression of GPR176 significantly promotes epithelial-mesenchymal transition (EMT) in gastric cancer cells, a critical step in the invasion and metastasis of adenocarcinoma cells. Future research could explore the detailed mechanisms of GPR176 in EMT regulation, including its interaction with other EMT-related factors and regulatory networks.\u003c/p\u003e \u003cp\u003eWe observed a positive correlation between GPR176 expression and PIP5K1A, and downregulation of PIP5K1A reversed the effects of GPR176 on cell migration/invasion, EMT, and the PI3K/AKT/mTOR signaling pathway. This suggests that GPR176 may exert a more precise regulatory role in the PI3K/AKT/mTOR signaling pathway through synergistic regulation with PIP5K1A. Future studies could use RNA interference and gene knock-in experiments to verify the interaction between GPR176 and PIP5K1A and elucidate their specific mechanisms in regulating the pathway.\u003c/p\u003e \u003cp\u003eThrough experiments in a xenograft model, it was confirmed that the overexpression of GPR176 significantly promotes tumor growth and activates EMT and the PI3K/AKT/mTOR signaling pathway. This not only supports the experimental in vitro results but also provides an experimental basis for further investigation of the role of GPR176 in the tumor microenvironment.\u003c/p\u003e \u003cp\u003eThe study of the effects of GPR176 on gastric cancer cells and the corresponding mechanisms holds significant scientific and clinical value. As a member of the G protein-coupled receptor family, the mechanisms of action of GPR176 in normal physiology and pathology are not yet fully understood. In-depth exploration of GPR176 expression and function in gastric cancer contributes to a more complete understanding of its role in adenocarcinoma biology, including its effects on cell signal transduction, proliferation, and metastasis. If GPR176 plays a crucial role in gastric cancer, it could prove to be a potential therapeutic target. Understanding the mechanisms of action of GPR176 may provide a theoretical basis for the development of drugs targeting this receptor and offer new strategies for gastric cancer treatment. Gastric cancer is a highly heterogeneous tumor, and patients respond differently to treatment. The study of GPR176 may lead to a better understanding of its expression and function in different subtypes of tumors and thus contribute to the development of more individualized treatment plans. The correlation between GPR176 expression and overall survival and disease-free survival in gastric cancer patients suggests that GPR176 may be a potential biomarker for assessing patient prognosis and formulating personalized treatment plans. Investigating the mechanisms of action of GPR176 helps to uncover the signaling pathways it regulates and the biological processes in which it is involved. This is not only crucial for understanding the specific role of GPR176 in the occurrence and development of gastric cancer but also contributes to the discovery of new therapeutic targets and strategies. In summary, the in-depth study of the effect of GPR176 on gastric cancer cells and its mechanisms offers a new perspective for our understanding of this disease and provides essential scientific insights for the treatment and prognosis assessment of gastric cancer.\u003c/p\u003e \u003cp\u003eIn summary, this study comprehensively elucidates the intricate regulatory mechanisms of GPR176 in gastric adenocarcinoma. This includes its diverse expression in the different subtypes, its correlation with patient prognosis, its influence on the PI3K/AKT/mTOR signaling pathway, the mechanism of EMT induction, and its interplay with PIP5K1A. These findings provide a solid theoretical basis for considering GPR176 as a potential target in the treatment of gastric adenocarcinoma and offer a new perspective for a deeper understanding of the mechanisms of cancer development. Subsequent research efforts should aim to further substantiate these findings and investigate the interconnections between GPR176 and additional signaling pathways to advance its in-depth exploration in the field of tumor biology.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGPR176\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eG protein-coupled receptor 176\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egastric cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePIP5K1A\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphatidylinositol-4-phosphate 5-kinase type 1 alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGSEA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Set Enrichment Analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAVID\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDatabase for Annotation, Visualization and Integrated Discovery\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReceiver operating characteristic curve\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAUC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThe Area Under Curve\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eqPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eQuantitative polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWestern Blot\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpithelial-Mesenchymal Transition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe investigation had been approved by the ethics committee of Guangxi Medical University the first affiliated hospital (Approval number: 2022 [KY‐E‐251]). All methods in this research were carried out in accordance with Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003eAvailability of data and material\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003eDeclaration of No Conflict of Interest:\u003c/p\u003e\n\u003cp\u003eI declare that there are no relevant financial or non-financial conflicts of interest in the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by the Medical Excellence Award Funded by the Creative Research Development Grant from the First Affiliated Hospital of Guangxi Medical University (NO.202204), Self-funded Research Project of Health Commission of Guangxi Zhuang Autonomous Region (Z-A20220397) and Hubei Chen Xiaoping Science and Technology Development Foundation (CXPJJH122002-027)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthors' contributions\u003c/p\u003e\n\u003cp\u003egc M\u0026nbsp;conceived and designed the research program; gc M and KY L made acquisition of data; ZC H, HC R, DL, GY Z and JT C performed data analysis. gc M, ZC H, HC R, and KY L performed the experiments. KY L wrote the manuscript. gc M guided and supervised the manuscript. All authors read and approved the final manuscript. All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThe authors thank the contributors of GSE66254 and TCGA database for sharing the data on open access.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 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Pathobiology. 2006;73(1):8\u0026ndash;17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1159/000093087\u003c/span\u003e\u003cspan address=\"10.1159/000093087\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Gastric cancer, GPR176, EMT, PI3K signaling pathway; PIP5K1A","lastPublishedDoi":"10.21203/rs.3.rs-5713551/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5713551/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGastric cancer is characterized by a high incidence and unfavorable prognosis. The exploration of novel molecular markers and a deeper understanding of their mechanisms of action hold the potential to offer fresh insights into gastric cancer treatment.\u003c/p\u003e \u003cp\u003eLeveraging the TCGA-STAD and GSE66254 datasets, this study conducted an analysis on the relationship between GPR176 and clinical pathological features. Furthermore, it was validated in patients from The First Affiliated Hospital of Guangxi Medical University. Cell migration and invasion capabilities were evaluated through Transwell and scratch assays. Western blot was performed to detect the impact of GPR176 on PI3K/AKT/mTOR signaling pathway. Nude mouse tumorigenesis experiments were conducted to validate the impact of GPR176 on tumor growth \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eGPR176 exhibited higher expression levels in gastric cancer tissues, and was associated with a poor prognosis in patients with gastric cancer. Significant downregulation of GPR176 suppressed the invasive and migratory capabilities of gastric cancer cells, concomitant with the inhibition of the PI3K and EMT signaling pathways. However, the phenotypic changes induced by GPR176 downregulation and its inhibitory effects could be reversed by the overexpression of PIP5K1A. Nude mouse tumorigenesis experiments validated the findings from cell experiments, demonstrating that GPR176 downregulation suppressed tumor growth, while GPR176 overexpression promoted tumor growth. Similarly, after GPR176 downregulation, the EMT and PI3K/AKT/mTOR signaling pathways in tumor cells were significantly inhibited, whereas GPR176 upregulation led to their substantial activation.\u003c/p\u003e \u003cp\u003eIn conclusion, GPR176 emerged as a newly identified prognostic marker in this study. GPR176 may promote the EMT of gastric cancer cells by activating the PI3K/AKT/mTOR signaling pathway.\u003c/p\u003e","manuscriptTitle":"GPR176 enhances the epithelial-mesenchymal transition in gastric cancer cells by activating the PI3K/AKT/mTOR signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-17 15:13:52","doi":"10.21203/rs.3.rs-5713551/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"24aa8b96-cf54-4260-908c-230a2606800e","owner":[],"postedDate":"February 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-09T10:38:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-17 15:13:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5713551","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5713551","identity":"rs-5713551","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}