The mRNA expression levels of MTUS1 in different types of human tumors.
In order to interrogate the expression level of MTUS1 in different human tumors and healthy tissues, we primarily analyzed the mRNA level of MTUS1 in multiple tumors and normal tissues by using the Oncomine database. The results showed that the expression of MTUS1 in head and neck cancer, kidney cancer, lymphoma, myeloma and other cancers was higher than that in normal tissues, while in brain and CNS cancer, breast cancer, colorectal cancer, head and neck cancer, kidney cancer, ovarian cancer, sarcoma, and lung cancer, MTUS1 mRNA expression was lower compared to their corresponding adjacent normal controls (Figure 1A). In order to further examine which cancers have differences in MTUS1 mRNA expression level, we detected the MTUS1 expression using the TIMER database. We found that MTUS1 expression in CHOL, KICH, STAD and THCA was found to be significantly higher compared to normal tissues. In contrast, MTUS1 was expressed at lower levels in 9 types of cancer (BLCA, BRCA, COAD, KIRC, KIRP, LUSC, PRAD, READ, UCEC) than in the corresponding normal controls (Figure 1B). Immediately following, we analyzed MTUS1 RNA expression data of the CRC tissues and normal tissues from TCGA (September 2021) using the Wilcoxon rank-sum test. The CRC tissues exhibited significantly lower MTUS1 mRNA expression levels than the normal tissues (Figure 1C). Additionally, the MTUS1 mRNA expression levels in 50 paired CRC and adjacent non-tumorous tissues were compared using Wilcoxon matched-pairs signed-rank test. Similarly, the result showed that MTUS1 mRNA was down-regulated expression in the CRC tissues.
Relationship between MTUS1 expression and clinicopathological parameters of colorectal cancer.
Because expression of MTUS1 was markedly up-regulated in CRC tissues, we further explored analyze the mRNA expression profiles of MTUS1 based on clinicopathological parameters. The Clinical characteristic data of 643 patients with CRC were downloaded from TCGA database in September 2021 (Table 1). The expression of MTUS1 was significantly decreased in CRC compared with normal tissues according to the analysis results of the clinicopathological factors including gender, age, pathologic stage, T stage, N stage, M stage, CEA level, neoplasm type and survival status (Fig 2A-I). It is noteworthy that the expression of MTUS1 was apparently decreased in grade IV than in grade I and grade II in the tumor grade regard (Fig 2C). For the nodal metastasis status, MTUS1 expression was significantly downregulated in N2 compared to N0 (Fig 2E). Logistic regression was performed to further investigate the connection between MTUS1 expression and the clinicopathologic features of patients with cancer. All patients were divided into MTUS1 mRNA high and low expression groups using median expression (50%) as the cut-off. The logistic regression analysis demonstrated that decreased MTUS1 expression in CRC strongly associated with Pathologic stage (stage III/IV vs. stage I/II, OR = 0.626, p= 0.004), N stage (N1/N2 vs. N0, OR = 1.67, p= 0.008), and Neoplasm type (READ vs. COAD, OR = 0.699, p= 0.048) (Table 2). These results revealed that low expression of MTUS1 promoted tumor progression, the lymph node metastasis and distant metastasis of patients with CRC and MTUS1 may also have diagnostic and prognostic implications.
Association between MTUS1 expression and survival prognosis in patients with colorectal cancer.
To further identify the prognostic potential of MTUS1 in colorectal cancer, Kaplan-Meier survival analysis was performed using data from the TCGA. As shown in Fig 3, lower expression of MTUS1 is positively correlated with poor overall survival (HR=0.62, P = 0.009; Fig 3A) and disease specific survival (Fig 3B, HR=0.55, P = 0.012). As shown in Table 3, the univariate Cox regression analysis demonstrated that low MTUS1 expression was observably correlated with poor OS [P=0.009, HR= 0.625, 95% CI (0.439-0.890)]. Among other clinicopathologic factors, age, pathologic stage, T stage, N stage, M stage and CEA level is also strongly associated with OS (Table 3). In multivariate Cox regression analysis, the down-regulated MTUS1 expression, higher pathological stage are independent prognostic factors of worse outcome (Table 3). From this COX model, we additionally constructed nomogram to predict the survival probability at 1, 3, and 5 years (Fig 4A). Figure 4B demonstrates the calibration plots to verify the reliability of this prognostic model.
Diagnostic Value of MTUS1 in colorectal cancer.
The results and the analysis presented above suggest that a significant difference in MTUS1 expression between tumor and non-tumor tissue was observed and MTUS1 is an independent prognostic factor. Given that low MTUS1 expression correlates with poor outcomes, ROC curves were plotted and the areas under the ROC curves (AUC) were computed to further analyze the diagnostic value of MTUS1 for CRC and the larger AUC, the higher the diagnostic value. The results show that MTUS1 expression had a modest diagnostic value for in patients (AUC=0.880; Fig 5A) and for patients with stage I&II of cancer (AUC =0.857; Fig 5B), while MTUS1 has demonstrated a high diagnostic value for patients with stage III&IV of cancer (AUC =0.915; Fig 5C). Unfortunately, MTUS1 has low diagnostic value in the assessment of pathological stages (AUC =0.587; Fig 5D). These results fully demonstrate that MTUS1 exhibit the diagnostic ability to identify CRC from the general population and is expected to become a promising diagnostic markers.
Identification and enrichment analysis of key MTUS1-interacting genes and proteins.
To investigate mechanism of MTUS1 in CRC, we identified key genes related to MTUS1 and performed a series of pathway enrichment analyses on these molecule. As shown in Figure 6A, the protein-protein interaction (PPI) network containing 51 nodes and 280 edges for MTUS1 was constructed using the STRING database. The ten genes most significantly associated with MTUS1 were AGTR2, CEP170B, ANKRD28, PMFBP1, CWH43, ANKRD52, PPP6R2, UPP2, BDKRB2 and LGI3. Subsequently, we exhumed the top 100 genes that correlated with MTUS1 expression in the CRC cohort and the first 50 positively and negatively correlated genes are shown in the heat map (Figure 6B-C). Based on these two sets of data we performed cross analysis and obtained a common gene, namely, SEC24A (Figure 6D). We further assessed the relationship between MTUS1 and SEC24A and the Spearman correlation coefficients were calculated, the result is shown in Figure 6E. Gene ontology analyses and KEGG pathway analysis of 150 genes from these two data sets revealed that these gene are involved in different pathways and biological process associated with cancer, as shown in Figure 6F-G. KEGG pathways include the HIF-1 signaling pathway, the leukocyte transendothelial migration, the cGMP-PKG signaling pathway, the chemokine signaling pathway and the sphingolipid signaling pathway. GO_BP (biological process) was mainly associated with cell growth, TRAIL-activated apoptotic signaling pathway, positive regulation of epithelial cell migration, regulation of apoptotic signaling pathway and adenylate cyclase-modulating G protein-coupled receptor signaling pathway. GO_MF (molecular function) was mainly related to exonuclease activity, 3'-5' exonuclease activity, G protein-coupled receptor binding, nucleobase-containing compound kinase activity and protein phosphatase binding. GO_CC (cell component) terms were cytosolic large ribosomal subunit, ESCRT complex, cytosolic part, cytosolic ribosome and heterotrimeric G-protein complex. These results revealed that it is highly likely that MTUS1 and MTUS1-related genes were involved in biological processes associated with the onset and progression of tumors, such as immune cell infiltration,tumor cell proliferation, cell migration and apoptosis of cells.
MTUS1 expression is correlated with immune Infiltration Level in colorectal cancer.
Previous research has demonstrated that the density of TILs within a tumour were an independent predictor of favorable disease free and overall survival (18-20). Therefore, the relationship between MTUS1 expression and the degree of various immune cell infiltration were assessed by ssGSEA algorithm (Fig 7A). As shown in Fig7B, MTUS1 expression was significantly positively correlated with T cells, CD8 T cells, activated dendritic cells, macrophages, T helper cells, Th1 cells,Th2 cells, central memory T cell (Tcm), effector memory T cell (Tem) and T follicular helper cell (TFH) infiltration and can negatively regulates infiltration of natural killer (NK) cells and regulatory T cells (Tregs). Next, to better understand the role of MTUS1 in CRC, we investigate the relationship between MTUS1 expression and immune infiltration and whether the copy number variation of MTUS1 is related to the infiltration levels of immune cells using TIMER. We found that MTUS1 expression correlates significantly with tumor purity, infiltrating levels of CD8 + T cells and neutrophils both COAD and READ. MTUS1 also was related to immune infiltration of B cells, CD4 T cells, macrophages and dendritic cells in COAD. The results are shown in Figure8A. Moreover, FIG 8B-C showed that the copy number variation of MTUS1 had different degrees of correlation with the infiltration levels of 6 kinds of immune cells. Results shown above suggest that that MTUS1 be involved in the recruitment of immune cells. To further confirm the correlation between MTUS1 expression and immune infiltrating cells in colorectal cancer,we analyzed the immune markers of T cells, CD8+T cells, B cells, monocytes, TAMS,M1 and M2 macrophages, neutrophils, NK cells, dendritic cells,Th1 cells,Th2 cells, Th17 cells, TFH cells,Tregs cells, and T cell exhaustion by using the GEPIA web tool. The results showed that the expression level of MTUS1 in tumor tissues (particularly colon cancer tissues) and most of the immune marker sets of immune cells are closely related (Table 4).