RCC is the sixth most commonly diagnosed cancer in men and the tenth in women worldwide, accounting for 5% and 3% of all cancer diagnoses, respectively[53]. KIRC, being the major pathological subtype, has a higher risk of recurrence, metastasis, and poorer prognosis[54].Numerous studies indicate that m6A RNA methylation regulators are linked to the development and progress of human cancers [55], and prognostic models based on m6A RNA modification-related lncRNA have improved understanding of KIRC[21, 56]. However, few studies have examined m7G-related lncRNA in predicting KIRC patient prognosis. This study seeks to establish a prognostic model for KIRC patients' lncRNA to assess its clinical usefulness and explore its correlation with immune cell infiltration and immune checkpoint sites in a systematic manner.
We obtained transcriptome and clinical data of KIRC samples and their corresponding normal controls from TCGA and ICGC databases. Using weighted gene co-expression network analysis, differential analysis, and machine learning, we identified eight differentially expressed m7G-related lncRNAs as biomarkers.
Differential expression analysis revealed that the eight biomarkers were significantly down-regulated in KIRC. LncRNA and m7G correlation analysis showed that all the final genes we obtained were lncRNAs associated with NUDT4. Studies have suggested that NUDT4 is significantly downregulated in various cancers, including KIRC[57-59],, which matches our results. Research in other cancer types has also indicated that NUDT4 downregulation affects tumor cell proliferation by impacting the m7G gene[60].Research indicates that the absence of certain m7G-related regulatory factors is linked to disease. For example, the lack of the METTL1/WDR4 complex impacts tRNA function and translation of multiple mRNAs, leading to abnormal cell cycle progression and proliferation[61].Thus, combining our results with previously published findings, we propose that downregulation of lncRNA in diseases leads to m7G downregulation, resulting in decreased RNA stability and impaired translation and regulation, ultimately causing the development of various diseases.
Only a few studies have indicated that LINC00645 is downregulated in KIRC, but its role remains unclear[33, 62].The remaining seven biomarkers have been scarcely reported in KIRC. Our study reports for the first time on the prognostic value of RP4-655J12.4, RP11-321G12.1, RP11-195B3.1, CTD-2626G11.2, AP000696.2, PTCSC3, and RP11-528A4.2 in KIRC. Additionally, previous research has suggested that AP000696.2, LINC00645, and PTCSC3 have potential as biomarkers for different cancers.Additionally, previous research has suggested that AP000696.2, LINC00645, and PTCSC3 have potential as biomarkers for different cancers.AP000696.2 exhibits superior predictive performance compared to traditional tumor markers in esophageal squamous cell carcinoma and may affect patient prognosis and treatment by regulating angiogenesis[63, 64].LINC00645 is a potential biomarker for acute rejection and graft loss in kidney allografts[65] and was also enriched in the same pathway in our GSEA analysis. Additionally, it can serve as an independent prognostic factor for lung cancer patients[66]. Experimental data suggest that PTCSC3 has antitumor properties, as its overexpression inhibits thyroid papillary carcinoma cell proliferation and development in vitro and in vivo by suppressing glycolysis and promoting PGK1 ubiquitin-mediated degradation[67].
Furthermore, K-M analysis revealed that the expression levels of PTCSC3 and RP11-321G12.1 were significantly associated with the prognosis of KIRC patients. We then used univariate and multivariate Cox regression analyses to examine the association between AP000696.2, PTCSC3, clinical features of KIRC patients, and their prognosis. Based on these prognostic factors, a column chart was constructed to accurately predict the prognosis of KIRC patients.
Pearson correlation analysis was conducted to explore the correlation between the biomarkers and clinical features, as well as their expression levels in different clinical feature subgroups. The results showed that AP000696.2 was positively correlated with patient staging, T-staging, and M-staging in the TCGA-KIRC dataset. Additionally, ROC curves of the biomarkers had an area under the curve greater than 0.95 in both the TCGA-KIRC dataset and the validation set, indicating their potential as diagnostic biomarkers for KIRC by accurately distinguishing KIRC samples from healthy controls.
To identify signaling pathways related to the occurrence and development of KIRC, GSEA was performed on the eight biomarkers. The results showed that these biomarkers were mainly involved in signaling pathways including allograft rejection, cytokine-cytokine receptor interactions, oxidative phosphorylation, chemokine signaling pathway, and other pathways.
Studies have found that certain lncRNAs can serve as potential biomarkers for detecting acute rejection after kidney transplantation[65, 68], providing valuable prognostic information. This suggests that the eight biomarkers identified in our study may have significant prognostic value in KIRC patients.
As early as 2013, the Cancer Genome Atlas research on KIRC found that metabolic changes play a critical role in disease progression, including alterations in the pentose phosphate pathway, fatty acid synthesis pathway, and tricarboxylic acid cycle, which are associated with poor prognosis[69, 70]. Given that oxidative phosphorylation participates in these pathways to provide energy conversion, we speculate that RP4-655J12.4 and CTD-2626G11.2 may affect metabolic pathways through their involvement in oxidative phosphorylation, ultimately leading to poor prognosis for KIRC patients.
Interestingly, the cytokine-cytokine receptor interaction pathway plays a crucial role in adaptive inflammatory host defenses, cell growth, differentiation, cell death, angiogenesis, and developmental and repair processes aimed at restoring homeostasis[71, 72]. This pathway is commonly enriched in the development of liver cancer[73] and colorectal cancer[74]. Studies have shown that chemokines and cytokines, such as TNF-α, IL-2, and chemokine CCL2, play a role in the formation of the cancer microenvironment and are responsible for the migration of inflammatory cells and cancer cells[75].
In addition, we found that LINC00645 plays an oncogenic role in endometrial cancer and glioma with high specificity[76, 77]. It has been reported that LINC00645 can induce the activation of EMT and enhance the migratory and invasive abilities of tumor cells by regulating the expression of miRNA-205-3p and its target gene ZEB1 through the induction of the reverse transforming growth factor TGF-β[78].These findings provide promising directions for elucidating the potential molecular mechanisms underlying the lncRNA characteristics of KIRC.
The multifunctionality of lncRNAs depends on their subcellular localization. If lncRNAs are located in the cytoplasm, they can act as ceRNAs and regulate mRNA stability or translation[79]. Our research results indicate that most biomarkers are mainly located in the cytoplasm, suggesting that they may participate in post-transcriptional regulatory pathways.
In our research results, the infiltration levels of 28 immune cells showed significant differences between KIRC samples and normal samples. Immune cells and inflammatory cytokines in the tumor microenvironment can affect tumor development and occurrence. Tumor cells inhibit T cell activation through immune checkpoint, thereby avoiding anti-tumor immune attacks and accelerating tumor deterioration, which is also the main mechanism of cancer immune escape[80]. Therefore, targeting immune checkpoint inhibitors is a significant method for tumor immunotherapy[81].Our findings indicate changes in the immune microenvironment of KIRC, with most primary immune cells showing significantly higher infiltration levels than normal samples. Additionally, eight biomarkers were found to be significantly negatively correlated with the infiltration levels of immune cells, suggesting their potential involvement in regulating the immune microenvironment of KIRC.
Immunotherapy is mainly represented by immune checkpoint blockade (ICB) and chimeric antigen receptor T cell therapy (CAR-T)[82, 83]. Currently, it is also a first-line treatment for metastatic KIRC[84], with immune checkpoint inhibitors such as PD-1 and CTLA4 being widely used in clinical practice, such as nivolumab and Ipilimumab. ICB has shown significant efficacy in solid tumors, including melanoma, non-small cell lung cancer, and renal cell carcinoma[82].The upregulation of immune checkpoint molecules, including CTLA-4, PD-1, and PD-L1, has been shown to contribute to tumor immune escape[85]. In addition, some lncRNAs have been found to participate in regulating the expression of PD-1 by affecting the function of specific miRNAs. For example, the interaction between lncRNA SNHG14 and miR-5590-3p upregulates Zinc finger E-box-binding homeobox 1 (ZEB1) to activate the PD-1/PD-L1 immune checkpoint, leading to the inactivation of CD8+ T cells and promoting immune escape of tumor cells in diffuse large B-cell lymphoma[86].
Immune checkpoints are essential predictive indicators for assessing responses to immunotherapy. Our study found that eight lncRNAs exhibited significant negative correlations with some common immune checkpoint sites (PDCD1, PDCD1LG2, CTLA4, HAVCR2, and LAG3) in KIRC. Specifically, RP4-655J12.4, PTCSC3, and AP000696.2 were negatively correlated with CD274. These findings suggest that the eight lncRNA model may play a role in evaluating patients' response to immune checkpoint blockade therapy. However, there is currently no relevant report explaining the mechanism of action of these eight lncRNAs in immune dysregulation in KIRC.
Our study primarily focused on m7G methylation-related lncRNAs and identified eight DE-m7G-lncRNAs that could serve as biomarkers for KIRC. This may provide a new research direction for the study and treatment of KIRC.