Discussion
GC is the major cause of mortality from neoplastic disorders ( Wu et al., 2019 ), with gastric adenocarcinoma accounting for about 95% of stomach cancers ( Liu et al., 2017 ). As has been shown in recent decades, the development of stomach cancer is a complex process involving several components, and different molecular manifestations have variable clinical prognostic repercussions for patients. Tumor staging in clinical practice only evaluates tumor size, histological classification and metastasis ( Niimi et al., 2013 ), but not the patient’s gene expression level. Recent research has demonstrated that stomach cancer development is influenced by epigenetic changes ( Zhang et al., 2018 ). Non-coding RNA, histone modification, RNA methylation and DNA methylation are all examples of epigenetics ( Chen et al., 2016 ; Xie et al., 2021 ), with RNA methylation becoming a popular topic in recent years. It is a dynamic and reversible modification mechanism, with methyltransferases, demethylases and binding proteins regulating the whole process ( Mo et al., 2020 ; Romanowska et al., 2021 ). Evidence is accumulating in GC, underscoring the influence of RNA methylation on the malignancy and prognosis of tumor cells. For example, A recent investigation has suggested that METTL3 enhances the proliferation, formation of colonies, migration, and invasion of GC cells in an m6A-dependent manner via the pre-B-cell leukemia homeobox 1 (PBX1)/GTP cyclohydrolase 1 (GCH1)/tetrahydrobiopterin (BH4) axis ( Liu et al., 2022 ).

By downloading and retrieving a large number of GC samples from the TCGA database, we observed that the expression of 14 RNA methylation regulators was significantly altered in GC tissues. NOP2 , DNMT1 , YTHDF1 , NSUN2 , TRMT6 , METTL1 , WDR4 , RBM15 , KIAA1429 , NSUN5 , DNMT3B , TRMT10C , ALYREF and ZC3H13 are among the genes that have been identified. All 14 DERMGs were found to be elevated. According to our GO enrichment analysis, these DERMGs are significantly associated with RNA methylation, tRNA modification, and related processes. Previous research suggests that the expression of NOP2 primarily relies on the m5C methylation level, as it can promote cancer cell proliferation through m5C-dependent inhibition of Cyclin-Dependent Kinase Inhibitor 1B ( CDKN1B , p27Kip1 ) ( Mei et al., 2020 ; Yang et al., 2023 ). The m7G tRNA methylation facilitated by the METTL1 / WDR4 complex selectively enhances the translation of specific cyclin and oncogenic transcripts, along with downstream pathway-related mRNAs, thus governing cell proliferation and apoptosis in conjunction with the abundant homologous codons of m7G tRNAs ( Porta, Paglino & Mosca, 2014 ). Hence, we speculation that the elevated expression of these DERMGs may exert a significant impact on their target transcripts, especially those involved in cancer development, rendering them pivotal epigenetic regulatory factors. This mechanism could potentially serve as a common oncogenic driver in GC progression.

Following that, we used univariate COX analysis to find four genes, RBM15 , DNMT1 , WDR4 and ALYREF , that were linked with overall survival (OS) in STAD patients. RBM15 , DNMT1 , WDR4 and ALYREF were identified as protective factors affecting STAD patient survival. Previous research has demonstrated that silences DNMT1 gene activity and leads to the activation of a number of tumor-related target genes ( Chen et al., 2018 ). WDR4 is a methyltransferase that participates in cell cycle progression, signaling, gene control and apoptosis ( Michaud et al., 2000 ; Arrondel et al., 2019 ). WDR4 gene expression was found to be down-regulated in advanced renal papillary carcinoma but constant in advanced rectal adenocarcinoma and GC in one study ( Zeng et al., 2021 ). WDR4 expression was also found to be a protective factor in rectal adenocarcinoma in the corresponding OS outcome study. ALYREF is thought to be a preventive factor against colon cancer ( Huang et al., 2022 ). This is in line with the findings of our research. RBM15 may have a function as an oncogene in lung adenocarcinoma, according to relevant publications ( Li et al., 2020 ); Su, Huang & Hu (2019) discovered that RBM15 may contribute to the malignant evolution of GC and alter clinical prognosis. This might be due to the fact that RBM15 influences the formation of malignant tumors via a variety of molecular processes.

Furthermore, we discovered that the RNA methylation genes APOD , ACTA2 , PDK4 and SAPCD2 are linked to the prognosis of GC. Simultaneously, these four genes have been shown to reliably predict patient prognosis in predictive risk profiles. Patients who meet the criteria for being classified as high-risk have a bad prognosis. These data show that RNA methylation-associated prognostic genes may have predictive prognostic value as well, and as a result, they might be employed as a potential prognostic predictor in patients with stomach cancer.

APOD , ACTA2 , PDK4 and SAPCD2 have all been linked to the development of cancer in several studies. APOD is a protein that is generated in many different regions of the body ( Braesch-Andersen et al., 2014 ). APOD -related complexes deliver cholesteryl esters to the liver and encourage their breakdown ( Utermann et al., 1980 ). Additionally, apolipoproteins in the blood may provide lipids to cancer cells, giving them energy for expansion and invasion ( Xu et al., 2007 ). Compared to women without breast cysts, those with breast cysts had a threefold higher chance of developing BC ( Zhou & Luo, 2020 ). Because APOD is the most abundant component of cyst fluid, it is possible that APOD plays a role in the development of breast cancer ( Sánchez et al., 1992 ). APOD was substantially expressed in the high-risk group, increased in colon cancer tissues, and had potential predictive value, according to a correlation study in the TCGA database against colon cancer ( Liang, Su & Wu, 2021 ). Hu et al. (2018) discovered that APOD expression was substantially connected with the diagnosis of GC as a risk factor, and that it might be a possible prognostic biomarker for GC. ACTA2 promotes cell mechanical tension and shape maintenance, as well as tumor cell metastasis ( Lambrechts, Van Troys & Ampe, 2004 ). Patients with high ACTA2 expression in lung adenocarcinoma had considerably increased distant metastases and a poor prognosis, according to one research ( Lee et al., 2013 ). ACTA2 may hasten the formation of human tumors by acetylating late 6-phosphogluconate dehydrogenase (6PGD) at K76 and K294 ( Shan et al., 2014 ). ACTA2 inhibition causes an intracellular buildup of unesterified oxysterols, which limits the development of hepatoma cell lines and xenografts ( Lu et al., 2013 ). He et al. (2022) discovered that greater ACTA2 expression was related with a worse clinical outcome in the GC high-risk group. PDK4 belongs to a group of isozymes (PDK1-4) that help the body convert to aerobic glycolysis by transferring pyruvate metabolism from the mitochondria to the cytoplasm for glycolysis ( Woolbright et al., 2018 ). Because most cancers have some degree of hypoxia, Hypoxia-inducible factor 1 (HIF-1) is connected to a poor prognosis in bladder cancer and is assumed to be the key mediator of transcriptional control of aerobic glycolysis in cancer ( Chai et al., 2008 ; Denko, 2008 ), could be the main transcription factor driving PDK4 upregulation in cancer. PDK4 is a biomarker of GC prognosis and a prospective therapeutic target, according to Liu, Zhang & Suo (2021) . It is also linked to the quantity of invading immune cells and patients’ poor prognosis, and it is a biomarker of GC prognosis as well as a viable therapeutic target in GC. PDK4 may have predictive relevance in various malignancies, such as liver and colorectal cancer ( Leclerc et al., 2017 ; Deng et al., 2018 ; Qin et al., 2020 ). Furthermore, we discovered for the first time, based on the existing prognostic model, that SAPCD2 expression was connected with survival in STAD patients and was recognized as a protective prognostic factor. SAPCD2 is a recently discovered highly conserved mammalian gene that regulates cell cycle and chromosomal segregation ( Mao et al., 2014 ). This gene is substantially expressed in GC, glioma, liver cancer and other tumor tissues, according to relevant research, and is linked to tumor malignancy, invasion and metastasis ( Wan et al., 2014 ; Ni et al., 2015 ; Jayanthi, Das & Saxena, 2020 ). We think that SAPCD2 ’s role in the formation of STAD is complicated and dynamic, but the molecular mechanism has to be investigated further. Overall, Our study suggests that the RNA methylation-related risk signatures we constructed may serve as prognostic indicators for clinical treatment decisions in GC patients.

Several investigations have shown that genetic alterations often result in phenotypic abnormalities linked to carcinogenesis ( Binang et al., 2020 ; Chen et al., 2021 ). CNV changes have been shown to disrupt genes involved in cell cycle regulation, the complement system and antigen presentation, potentially contributing to cancer ( Brown et al., 2016 ). APOD and ACTA2 were deleted, and four prognostic genes, APOD , ACTA2 , PDK4 and SAPCD2 , were mutated and substantially related to CNV, according to our analysis. Following this study’s GSEA analysis, certain biological mechanisms have been found to have an important part in the onset and progression of cancer. Chromosome instability is a critical diagnostic sign for most malignancies ( Olson et al., 2010 ); it is caused by abnormal cell division during mitosis or by changes in gene expression levels following chromosomal separation. Defects that guide proper and accurate chromosomal separation during mitosis can cause chromosome aneuploidy and instability, which can lead to malignant tumor formation and spread ( Zhu et al., 2015 ). A cell cycle checkpoint is a crucial quality control measure that ensures that cell cycle events proceed normally. Cell growth will become uncontrollable if the regulatory method fails ( Yin et al., 2018 ). Loss of checkpoint control of cell cycle pathways occurs with cancer growth, and their dysregulation may impact the efficiency of cancer treatment ( Zhang et al., 2011 ; Ho et al., 2017 ). Furthermore, the quick transition of the G2/M phase during mitosis promotes GC cell growth ( Lim et al., 2020 ). By controlling cell cycle progression and tumor growth, the PI3K / AKT signaling pathway increases the onset and progression of GC ( Pereira et al., 2020 ). Many studies have shown that the MAPK / ERK signaling pathway is the primary molecular mechanism behind liver carcinogenesis ( Zender et al., 2010 ), and that HCC can be efficiently generated by activating oncogenes or inhibiting tumor suppressor genes in conjunction with MAPK / ERK signaling ( Zhang, Budker & Wolff, 1999 ). The inflammatory response has been linked to cytokine–cytokine receptor interactions, and cancer and inflammation have a very strong connection ( Kan et al., 2019 ). Pro-tumor inflammation accelerates the growth of tumor cells and, to some extent, reduces anti-tumor immunity ( Yu, Pardoll & Jove, 2009 ).

TME, which includes tumors, extracellular matrix, lymph, blood vessels and interstitial cells, plays a crucial part in the development, invasion and metastasis of tumors ( Palicelli et al., 2021 ). According to one research, myofibroblasts are the most common stromal cell type in most malignancies, and they have been proven to aid tumor development by secreting a range of growth factors ( Sharma, Evans & Hemers, 2016 ). At the same time, myofibroblasts can produce a variety of pro-tumor factors and directly participate in promoting metastasis ( Karnoub et al., 2007 ; Seiler et al., 2020 ). Hwang et al. (2008) discovered that pancreatic tumor fibroblasts have a key role in tumor cell proliferation, motility, invasion and treatment resistance. By stimulating epithelial cell transition into mesenchymal cells and secreting energy-rich substances to enhance cancer cell development, fibroblasts boost prostate cancer aggressiveness. Fibroblasts also promote capillary morphogenesis and endothelial cell proliferation ( Taddei et al., 2014 ). Disruptions in the quantity of mucous cells in the antrum and homeostasis have been reported to increase the probability of GC ( Yu et al., 2016 ). Glandular mucus cells release glandular mucin, help make gastric mucin, and subsequently form a mucus gel layer to protect the stomach mucosa from the outside world ( Ota & Katsuyama, 1992 ). At the same time, glandular mucin secreted by glandular mucus cells can inhibit helicobacter pylori infection to prevent GC, and can also be used as an inhibitor of differentiated gastric adenocarcinoma ( Nakayama, 2014 ). In this study, we found that ACTA2 , APOD and PDK4 were mainly expressed in myofibroblasts and fibroblasts respectively, while SAPCD2 was mainly expressed in alveolar mucous cells and glandular mucous cells of gastric adenocarcinoma. We speculated that ACTA2 , APOD and PDK4 were risk factors. It may promote the onset and progression of STAD via the aforementioned mechanism, while SAPCD2 plays a protective role.

In recent years, immune infiltration has been a research focus. PDK4 expression was shown to be favorably linked with the numbers of several invading immune cells, including CD4 + T cells, B cells and dendritic cells, as well as macrophages ( Chai et al., 2008 ). APOD was shown to be a risk factor and substantially linked with lymphoid infiltration in a biologic information investigation on colon cancer ( Liang, Su & Wu, 2021 ). Many immunosuppressive agents, such as csF1R , ADORA2A , TGFBR1 and BTLA , were discovered to be related with APOD , ACTA2 , PDK4 and SAPCD2 in this research. In addition to immunosuppressive agents, we discovered immune stimulators such as CD48 , CXCR4 , C10 and PVR . APOD , ACTA2 , PDK4 and SAPCD2 are thought to be essential in the immunomodulation of STAD. As a result, combining inhibitors and activators might possibly improve STAD patients’ anti-cancer effects.

Pharmacological chemotherapy is still often used to treat GC patients, but the development of chemoresistance during treatment is a key factor in patient fatalities. Clinically, patients with different tumor stages often respond differently to various chemotherapy treatments. We carried out chemotherapy sensitivity analysis for high and low risk categories so that GC patients might have superior antitumor chemotherapy regimens. Dasatinib, one of the most extensively studied tyrosine kinase inhibitors in clinical research, was initially developed as a dual inhibitor targeting BCR-ABL and SRC ( Li et al., 2010 ). In other cancers, Dasatinib is capable of impeding cell migration and invasion through the inhibition of SRC , which blocks the transmission of downstream signals to proteins like focal adhesion kinase ( FAK ), responsible for adhesion, as well as MAPK and p27 , which are involved in regulating the cell cycle ( Johnson et al., 2005 ; Nam et al., 2005 ). In GC, dasatinib leads to a notable rise in actin accumulation at the cell cortex, which is also indicative of an enhanced cell-cell adhesion ( Fenton, Hutchens & Denning, 2015 ; Montenegro et al., 2020 ). BIBW2992 not only directly inhibits HER2 receptor activation in GC but also suppresses its downstream signaling pathways, such as PI3K / AKT / mTOR and MAPK ( Keller et al., 2018 ). Additionally, BIBW2992 induces apoptosis and arrests the cell cycle at the G1 phase ( Chen et al., 2019 ). However, the effectiveness and applicability of these drugs still need validation through clinical trials.

We summarized four RNA methylation regulators, divided them into two subclasses according to their expression, and then analyzed them through the study of RNA methylation. We believe that more characteristic genes are often not conducive to large-scale clinical application ( Li et al., 2021 ). Therefore, the four prognostic genes related to RNA methylation that we analyzed will be conducive to the successful implementation of clinical practice. These prognostic genes express differently in the high-risk and low-risk groups, which indicates that they are important in the incidence rate and progression of STAD. We established a risk model to study the clinical relevance of prognostic genes associated with RNA methylation. The AUC value and ROC curve show that compared with previous studies ( Guan et al., 2020 ), the risk model we built has more advantages in predicting patient survival. In addition, this study found that the expression of prognostic genes is related to patient survival, which is an independent factor in the prognosis of STAD. At the same time, we also found that stage is an independent predictor, and we created a nomogram as a clear reference. It is worth noting that compared with the previous study ( Guan et al., 2020 ; Wang, Meng & Ma, 2021 ), we increased the use of real-time PCR experiments to verify the expression of characteristic genes. In conclusion, this is the first time to study the predictive significance of RNA methylation related disease subtypes and RNA methylation related genes in STAD. These genes were linked to STAD prognosis and have many research prospects and may be good biological targets.

While our findings are promising, we must acknowledge that our research has significant limitations. First, bigger samples are required in clinical investigations to confirm the clinical importance of the built characteristics as independent prognostic markers of STAD, which will be the subject of future study. Second, while we validated the differential expression of prognostic genes in clinical samples through qRT-PCR, further investigations involving the establishment of animal or cell models will facilitate our understanding of the mechanisms underlying these prognostic genes in STAD. Additionally, the findings in our study, including the applicability of the prognostic model and the efficacy of drugs with varying sensitivities, require validation in clinical samples to demonstrate their clinical relevance. These areas represent the focal points of our future research endeavors.