NSUN6 expression was significantly upregulated in pan-cancer
NSUN6's expression in various cancer databases was first evaluated in this work in order to determine the level of NSUN6 expression in pan-cancer. Data from GTEx (Fig. 1A), CCLE (Fig. 1B), and TCGA (Fig. 1C) showed NSUN6 expression levels to be similar across the three datasets. Figure 1A shows that NSUN6 expression was lower in muscle and blood than in other tissues, but higher in the testis and pituitary than in other tissues. NSUN6 expression was lower in salivary glands than in other tissues, as depicted in Fig. 1B. Comparatively, the TCGA database shows that the NSUN6 gene is significantly expressed in COAD (ESCA) and HNSC (KIRC), as well as in LUAD (LUSC), PRAD (RAD), READ (STAD), and KIRP (KIRP). The expression of NSUN6 in the TCGA and GTEx datasets was further integrated in light of the small amount of normal sample data in TCGA. The final analysis revealed that NSUN6 expression was downregulated in CHOL, GBM, KICH, LIHC, OV, SKCM, TGCT, THCA, and UCEC, but elevated in BRCA, COAD, ESCA, HNSC, KIRC, KIRP, LAML, LGG, LUSC, PAAD, PRAD, READ, STAD, and UCS after integration of 27 tumor modules (Fig. 1D).
Prognostic analysis of NSUN6 in pan-cancer
In the above study, the expression levels of NSUN6 in various tumor tissues were revealed. To understand the relationship between NSUN6 and tumor prognosis, one-way Cox regression analysis was used to analyze NSUN6 expression levels and patient prognosis. Based on the TCGA database data, NSUN6 expressions were grouped into high and low expression groups according to the median value of NSUN6 expression in each tumor. It was further observed that NSUN6 was only expressed in ACC (HR = 1.1, 95CI%: 1.02–1.17, P = 0.0083), BLCA (HR = 0.97, 95CI%: 0.95–0.99, P = 0.0025), COAD(HR = 1.08, 95CI%: 1.02–1.14, P = 0.011), KIRC(HR = 1.07, 95CI%: 1.02–1.13, P = 0.0033), LGG(HR = 0.94, 95CI%: 0.91–0.97, P = 0.00033), PAAD (HR = 0.91, 95CI%: 0.84–0.98, P = 0.012), SARC(HR = 1.07, 95CI%: 1.01–1.12, P = 0.012) and T H C A (HR = 1.12, 95CI%: 1.02–1.22, P = 0.014) with of prognostic significance in 33 tumors (Fig. 2). In addition, the relationship between NSUN6 and ACC, BLCA, COAD, KIRC, LGG, PAAD, SARC, and THCA was further observed by K–M plotting survival curves (Fig. 3). It suggested that there was an association between NSUN6 and tumor prognosis, especially with LGG. Considering the possible existence of non-tumor-related deaths during follow- up, we analyzed the relationship between NSUN6 expression levels and prognostic DSS (disease-specific survival) in 33 tumors of TCGA. The results of the analysis showed that NSUN6 was related to ACC (HR = 1.1, 95CI%: 1.03–1.18, P = 0.0065), BLCA (HR = 0.96, 95CI%: 0.64–0.99, P = 0.0042), LGG (HR = 0.95, 95CI%: 0.91–0.98, P = 0.00088), PAAD (HR = 0.89, 95CI%: 0.81–0.97, P = 0.0078), PCPG (HR = 1.24, 95CI%: 1-1.54, P = 0.048) ,SARC(HR = 1.07, 95CI%: 1.01–1.13, P = 0.018), SKCM(HR = 0.96, 95CI%: 0.92-1, P = 0.036), and THCA(HR = 1.21, 95CI%: 1.09–1.33, P = 0.00017) revealing that NSUN6 and DSS had prognostic significance (Fig. 4). NSUN6 was also shown to be a predictive predictor of tumor DSS by the K–M survival curve (Fig. 5).
Tumor metabolic and immune signaling pathways involved in NSUN6
A GSEA analysis was performed to examine the signaling enrichment of KEGG and markers in both groups according to NSUN6 gene expression, which was separated into high and low groups, in order to better understand the relationship between NSUN6 involvement in cancer metabolism and tumor immunity. In addition, we identified the six signaling pathways that were most prevalent in each of the databases. (Figs. 6A-D).
Correlation between NSUN6 expression levels and immune checkpoints and immune neoantigens in pan-cancer
Immune checkpoints are a series of molecules that are expressed in immune cells and can modulate immune activity . On the contrary, new tumor antigens are newborn antigens encoded by mutant genes in tumor cells. The immune activity of new tumor antigen is used to promote the synthesis of new antigen vaccine . NSUN6's role in regulating the immune system was explored this time by examining the association between NSUN6 and immunological checkpoints and neoantigens. More than 40 checkpoints, including KICH, UVM, and NSUN6, were found to connect with NSUN6 expression levels in diverse cancers (Fig. 7A). In some cancers, NSUN6 was found to have an important function in modifying immunological checkpoints and may be able to boost immunity. In addition, we counted the amount of neoantigens present in each type of tumor in the current study. The results showed that NSUN6 was only correlated in BRCA (R = -0.128, P = 0.00055), THCA (R=-0.013,P = 0.0464) and STAD (R = 0.174, P = 0.0069) (Fig. 7B).
Correlation between NSUN6 and tumor immune infiltration and the tumor microenvironment in pan-cancer
The difference in the degree of infiltration of different immune cells is closely related to the progression and prognosis of the tumor. By contrast, the tumor microenvironment consists of a variety of cells, ECM, and related factors that have an important impact on the diagnosis, survival, and clinical treatment sensitivity of the tumor. As part of this work, we looked examined NSUN6's immune infiltration and the tumor microenvironment in various malignancies. A total of 3 most relevant tumors (BLCA, KIRC, KIRP) were identified after analyzing 33 tumors in the TCGA database. Among these tumors, NSUN6 was positively correlated with B cells (R = 0.165, P < 0.001), but negatively associated with CD4 + T cells (R = -0.286, P < 0.001), CD8 + T cells (R= -0.376, P < 0.001), Dendritic (R = -0.506, P < 0.001), Macrophages (R = -0.228, P < 0.001), and Neutrophil cells (R = -0.453, P < 0.001) in BLCA. In addition, NSUN6 was positively correlated with CD4 + T cells (R = 0.323, P < 0.001), CD8 + T cells (R = 0.139, P < 0.001), Neutrophils (R = 0.289, P < 0.001), macrophages (R = 0.127, P < 0.05) and dendritic cells (R = 0.113, P < 0.001) in KIRC. Furthermore, NSUN6 was positively correlated with CD4 + T cells (R = 0.144, P < 0.05), Neutrophil cells(R = 0.205, P < 0.001) ,but negatively related to B cells(R=-0.199,P < 0.001), CD8 + T cells(R=-0.307, P < 0.001), Macrophages(R=-0.219, P < 0.001) in KIRP (Fig. 8A).
Researchers in this study looked at the link between NSUN6 and tumor immune infiltration as well as the tumor microenvironment in pan-cancer. Each tumor sample was evaluated using the R package (ESTIMATE) and the association between NSUN6 expression levels and the immunological score, stromal score, and the immune score of ESTIMATE was analyzed individually. BLCA (R = − 0.44, p < 0.001), LGG (R = − 0.441, p < 0.001), PRAD (R = − 0.31, p < 0.001) stromal scores were negatively correlated with NSUN6 expression levels. We showed the top three tumors in which BLCA (R = − 0.44, p < 0.001), GBM (R = − 0.432, p < 0.001), THCA (R = − 0.23, p < 0.001) immune scores were negatively correlated with NSUN6 expression levels in 33 tumors. In addition, the immune scores of BLCA(R = − 0.44, P < 0.001), LGGG (R = − 0.441, P < 0.001), GBM (R = − 0.432, P < 0.001) ESTIMATE were found to be negatively correlated with NSUN6 expression levels (Fig. 8B). NSUN6 was found to be negatively related correlation with both tumor immune scores based on these findings.
TMB and MSI analyses of NSUN6 expression in pan-cancer
TMB can be used to assess the total number of substitutions and insertions/deletions per megabyte of exons in tumor samples and to predict the success and prognosis of immune-checkpoint therapy . MSI is caused by a mismatch repair (MMR) gene defect and is associated with tumorigenesis . We investigated the link between NSUN6 in TMB and MSI in the current study. A Spearman correlation analysis showed that NSUN6 expression levels were positively correlated with SKCM and STAD, and negatively with UCEC, UVM and BLCA, as well as TGCT, SARC, KIRC, KIRP, LGG and LIHC. This was in contrast to the correlations found between NSUN6 expression levels and SKCM, STAD, SARC, TGCT, DLBC and GBM (Fig. 9A). MSI cancers had NSUN6 expression levels identified using Spearman test analysis. According to Spearman test analysis, NSUN6 expression levels were positively connected with LUSC, LUAD, LGG, KIRP, HNSC, and GBM, ESCA, COAD, CHOL, UCEC, and THCA; negatively correlated with SKCM and TGCT; and unrelated to DLBC and LIHC (Fig. 9B).
In pan carcinoma, the expression of NSUN6 and MMR genes and methyl-transferases are linked
This study used the TCGA database to examine the association between the NSUN6 gene and the MMR gene family (MLH1, MSH2, MSH6, PMS2, EPCAM). All 33 cancers except LIHC and UCS had NSUN6 expression levels that were associated with MMR genes, according to the study (Fig. 10A). NSUN6 expression levels were also shown to be strongly and positively linked with the expression levels of both methyltransferases (Fig. 10B). According to these findings, NSUN6 is involved in cancer development and has epigenetic features that govern a variety of malignancies.