The pathogenesis of WT is still unclear, and the related epigenetic mechanism remains to be explored. As a post-transcriptional gene expression regulation model, m6A RNA methylation regulates gene expression by affecting multiple aspects of mRNA metabolism, including mRNA pre-processing, nuclear export, decay, and translation [28]. Therefore, the effect of m6A on gene expression is extensive. In other tumor studies, it was found that m6A in the peripheral blood of patients with gastric cancer increased with the progression and metastasis of gastric cancer but decreased significantly after surgery, suggesting that m6A level in peripheral blood is a promising noninvasive diagnostic biomarker for gastric cancer patients [29]. In the study of m6A RNA methylation in glioma, it was found that high expression of m6A was associated with poor prognosis and tumor grade [30]. In the study of urological malignancies, high levels of m6A RNA methylation in mRNA were found to promote prostate cancer progression by regulating subcellular protein localization, and patients with high m6A RNA methylation had poor survival benefits [31]. Our study found that m6A is up-regulated in nephroblastoma, and increased expression of m6A is associated with poor prognosis and is associated with the grade of nephroblastoma because the expression of m6A in grade III and IV is more robust than that in grade I and II. These findings suggest that abnormal methylation of m6A may show specific diagnostic biomarkers and prognostic value in nephroblastoma. In addition, m6A is reversible, and reversing high m6A expression may contribute to cancer treatment [32]. However, the fundamental question of the factors causing m6A deposition remains unanswered [33], suggesting that the observed overexpression of m6A RNA methylation in WT patients requires further investigation.

The dysregulation or mutation of m6A “writer” and “eraser” proteins is closely associated with m6A deposition in cancer cells. According to reports, the m6A “writer” proteins methyltransferase-like 3 (METTL3) and METTL14, as well as the “eraser” protein obesity-associated protein (FTO), are intricately involved in the modulation of m6A methylation and the development of various cancer types [34]. Dysregulated transcriptional regulation in cancer cells can also impact m6A deposition. Transcriptional regulators associated with cancer can potentially enhance the expression of m6A “writer” proteins, consequently leading to elevated m6A levels [35]. Furthermore, accumulating evidence indicates that various environmental factors can influence m6A deposition. For example, it has been demonstrated that UV irradiation can induce the formation of m6A methylation [33]. However, the fundamental question regarding the underlying factors leading to m6A deposition remains inadequately addressed, lacking a comprehensive and definitive elucidation [33], suggesting that the intricate mechanisms underlying the interplay of these transcriptional, mutational, and environmental factors with altered m6A RNA methylation in WT patients requires further investigation.

In recent years, more and more studies have confirmed that m6A-related genes are closely related to malignant tumors. For example, ZC3H13, CBLL1, ELAVL1, and YTHDF1 are differentially expressed in lung cancer tissues, which is significant for predicting and treating lung cancer [36]. Nine m6A-related genes were identified in glioma. The mRNA levels of these genes were highly correlated with the clinicopathological features of glioma and may be involved in glioma progression [37]. In the head and neck squamous cell carcinoma study, m6A-related gene changes were significantly correlated with tumor grade and stage [38]. Although the abnormal expression of m6A-related genes is involved in tumorigenesis in many solid tumors, the prognostic value of m6A-related genes in nephroblastoma and its correlation with clinicopathological features still need further study. We screened four highly expressed m6A-related genes (ADGRG2, CPD, CTHRC1, LRTM2) in nephroblastoma and constructed an effective diagnostic model based on these genes. In addition, we used qRT-PCR to verify the expression of m6A-related genes in different WT cell lines and normal 293T cell lines. The results showed that the expression of these four genes in normal 293T cell lines was lower than in different WT cell lines. It is suggested that these four m6A-related genes can be used as biomarkers for diagnosing WT and potential therapeutic targets. Although our study focused on m6A-related genes, we found in many results of our investigations that the DEGs between WT tissues and normal tissues partially overlapped with another study. A study by Li et al. compared the DEGs between WT and normal tissues from the GEO (Gene Expression Omnibus) database by bioinformatics methods, and 10 DEGs (ALB, CDH1, EGF, AQP2, REN, SLC2A2, SPP1, UMOD, NPHS2, and FOXM1) were screened [39]. The AQP2, SPP1, and UMOD in the results of this study coincided with the DEGs we screened. Differences in AQP2, SPP1, and UMOD between WT and normal tissues were found in two different databases, and our study provides additional evidence to support AQP2, SPP1, and UMOD as DEGs of WT.

We reviewed the research status of these four highly expressed m6A-related genes. The correlation between CTHRC1 and tumor is the most studied, followed by ADGRG2, while the correlation between CPD and LRTM2 and tumor is less studied. CTHRC1 is a vital oncogene; its expression of collagen triple helix repeat containing protein 1 is a cancer-related protein. CTHRC1 is the most widely studied and has been consistently shown to be upregulated across multiple cancer types (e.g., gastric cancer, pancreatic cancer, hepatocellular carcinoma, breast cancer, colorectal cancer, epithelial ovarian cancer, esophageal squamous cell carcinoma, cervical cancer, non-small cell lung cancer, melanoma), aligning with our observation of its elevated expression in WT [40,41,42,43]. Increased CTHRC1 expression has consistently been associated with tumor development, and its expression level significantly correlates with the prognosis of cancer patients [41]. Qi et al. first confirmed the high expression of CTHRC1 in WT tumors, and further studies have found that the survival time of patients with increased expression of CTHRC1 is shorter than that of patients with low expression of CTHRC1, and CTHRC1 can be regarded as an independent prognostic factor for WT [42]. However, a discrepancy is noted in the findings by Huang et al. that despite the high expression of CTHRC1 in WT compared to normal kidney tissue, it acts as a protective factor (HR = 0.489) associated with WT prognosis [43]. This difference could be attributed to patient cohort characteristics, highlighting the need for further validation. Our study also confirmed the high expression of CTHRC1 in WT and found that CTHRC1 significantly differed in different stages of WT. The expression of CTHRC1 in grades I and II was higher than in grades III and IV. Next, we performed a survival analysis of CTHRC1, and the results showed that patients with increased expression of CTHRC1 in WT patients had a better prognosis than patients with low expression. These results suggest that the difference in CTHRC1 expression may be related to the prognosis of WT patients. CTHRC1 is a protective or risk factor for WT; further research is needed.

ADGRG2 is considered a new pathogenic gene, and most studies have shown that it is closely related to the congenital absence of vas deferens, and there are currently no studies on WT [44, 45]. The research on CPD is limited to plant research, and there is no research involving CPD and WT [46]. Furthermore, studies investigating the correlation between LRTM2 and tumors are scarce, necessitating further research to elucidate its function in this context. Overall, this study reinforces prior knowledge of dysregulated m6A-gene expression in malignancies and reveals new WT-specific prognostic biomarkers that contribute uniquely to the current understanding of m6A biology in pediatric cancer. While our analysis was focused on m6A-related genes, the identified DEGs between tumor and normal samples also provide valuable insights. The abnormal m6A modifications can directly impact target RNA structure and function, potentially contributing to the altered gene expression patterns observed.

Conversely, the proteins encoded by DEGs may have feedback to affect the m6A process. Exploring the interactions between m6A dysregulation and downstream DEG targets could shed light on the collective molecular changes driving nephroblastoma pathogenesis. Connecting the DEGs-enriched pathways to the upstream effects of m6A on modulating gene expression represents an important future direction to dissect the complex interplay underlying Wilms tumor biology.

Understanding the immune status of the tumor microenvironment will help us deepen the understanding of anti-tumor immune responses and develop more effective immunotherapy methods. This study found that APC_co_stimulation, CCR, Macrophages, Parainflammation, Th1_cells, Treg, and Type_ II_IFN_response were significantly decreased in WT compared with normal tissues. This suggests that overall immunosuppression in the WT tumor microenvironment is consistent with many current findings [47]. Some studies have compared the differences of immune cells in tumor microenvironment between the WT high-risk and low-risk groups. In the high-risk group, it was found that except for B cells and macrophage M1 type, other types of cells were lower than those in the low-risk group, including CD8+ naive T cells, CD8+ T cells, macrophage M2 type, mast cells, neutrophils, NKT cells and Treg cells [48]. This suggests that the high-risk WT tumor microenvironment has immune inactivation and a lack of T cells.

Furthermore, our study unraveled significant correlations between specific immune markers and the m6A-related genes. ADGRG2, as a G protein-coupled receptor, may be correlated with immune infiltrating cells, and its overexpression in tumors is generally associated with lower overall survival rates [49]. CTHRC1 is overexpressed in multiple tumor types to promote tumor initiation and progression. It regulates the activity of immune checkpoint genes through various signaling pathways and is associated with immune cell infiltration and the tumor microenvironment [41]. There is limited research on CPD and LRTM2 in the context of tumors. Still, it has been reported that CPD cleavage of C-terminal arginine generates nitric oxide, which plays a versatile regulatory role in various physiological processes and is closely associated with tumor invasion and tumor-induced angiogenesis [50]. In addition, we found that APC_co_stimulation, CCR, Macrophages, Parainflammation, Treg, and Type_II_IFN_Reponse were negatively correlated with LRTM2, Th1_cells were positively correlated with ADGRG2, CCR was negatively correlated with CPD, CCR was positively correlated with CTHRC1. These findings indicate that the four m6A-related genes could play an immunoregulation role in WT. In particular, LRTM2 may be involved in the immunosuppressive microenvironment of WT. Although the fact that there are few studies on LRTM2 in WT, it has potential research value in the future.

While this study provides valuable initial insights into m6A-associated prognostic biomarkers in Wilms tumor, there are some limitations regarding the study design. The analysis was performed on a single cohort of samples from the TARGET-WT database. We could not validate our findings on an independent GEO dataset due to the lack of publicly available cohorts with both Wilms tumor gene expression and survival outcome data. As more annotated datasets become available in resources like GEO, validation on external samples will be an important future direction to substantiate the prognostic utility of the identified biomarkers. Furthermore, the sample size of 121 WT patients, while sizable given the rarity of this pediatric cancer, may still limit the detection of more minor effects. Integrating multi-omic data beyond just transcriptomics could provide a more comprehensive understanding of the functional mechanisms of the identified biomarkers. Overall, this exploratory study offers a meaningful starting point for further research to build upon and more definitively characterize the clinical and biological significance of m6A-related genes in Wilms tumor.

Enhancing the immune activity of the WT tumor microenvironment may contribute to the treatment of WT. Still, it should be noted that different target cells have various therapeutic effects on WT. Immune checkpoint inhibitors (PD1 or PD-L1) have completely changed the treatment of many adult tumors because they can activate tumor-infiltrating lymphocytes to exert anti-tumor effects. People have placed similar hopes on treating recurrent or refractory solid tumors in children. However, current clinical trials have shown that this immunotherapy is little effective in treating WT, and childhood cancers are likely to follow a unique immune pathway [51]. This means that immune checkpoint inhibitors may not be suitable for treating WT. Other immunotherapy methods for WT are being explored, such as CAR-T cell therapy and cytotoxic T lymphocyte therapy. This immunotherapy for T cells shows a specific prospect and may be successfully applied to WT immunotherapy in the future [52]. Previous studies have primarily focused on applying immune checkpoint inhibitors in Wilms tumor (WT), while our research provides a novel perspective on WT immunotherapy. Our findings demonstrate a strong association between m6A-related genes and the immunosuppressive state of the WT tumor microenvironment, providing a theoretical foundation for developing immunotherapeutic strategies targeting these genes. Notably, the LRTM2 gene may become a promising target as it correlates negatively with multiple immune gene sets. Modulating LRTM2 could potentially enhance immune activity within the WT microenvironment.

Additionally, we predicted the potential therapeutic effects of these key genes, providing valuable leads for developing immunotherapeutic drugs for WT. In summary, this study comprehensively elucidates the WT immune microenvironment, uncovering the challenges faced by WT immunotherapy and offering essential data to support the development of novel treatment targets and drugs. It represents an innovative contribution to the field of WT immunotherapy.