The Role of M6A-related CBLL1 in the Prognosis and Immune Infiltration of Pan-cancer


 Background

The modification of N6-methyladenosine (m6A) plays an important role in physiology and disease progression. The relationship between the role of m6a-related gene CBLL1 in pan-carcinoma and tumor immune infiltrates has remained unknown.
Methods

To explore the expression level of CBLL1 methylation in pan-cancer in SMART database, and download mRNA expression, mutation and clinical data in UCSC database, to analyze the expression level of CBLL1, and the relationship between CBLL1 expression and clinicopathological features, prognosis, mutation and immune microenvironment in pan-cancer. CIBERSORT was used to analyze the relationship between the expression of CBLL1 and the infiltration of pan-carcinoma immune cells. The mRNA expression data of UCSC database were used to analyze the correlation between CBLL1 expression and pan-cancer immunomodulations, checkpoints and receptor molecules. Gene Set enrichment analysis (GSEA) revealed the possible mechanism of CBLL1 in the regulation of pan-cancer progression.
Results

The levels of CBLL1 methylation and mRNA expression in pan-cancer tissues were abnormal. The level of CBLL1 is related to the age, race, clinical stage and treatment effect of patients with pan-carcinoma and associated with the prognosis of patients with KIRC, LUSC, THCA, THYM, MESO, PRAD, STAD, and UVM. Univariate COX regression analysis showed that expression of CBLL1 was a risk factor for poor prognosis in patients with KICH, KIRC, LAML, THYM, KIRC, PCPG, OV, PRAD, STAD, GBM and UVM.The expression level of CBLL1 was correlated with BLCA, BRCA, COAD, LAML, LGG, LUAD, LUSC, SARC, STAD, THCA, THYM and UVM tumor mutational burden (TMB), and with ACC, BRCA, CESC, COAD, DLBC, HNSC, PRAD, READ, SARC, STAD, TGCT, THCA and UCEC microsatellite instability (MSI). The expression level of CBLL1 was correlated with pan-cancer stromal cells and immune cells. The expression of CBLL1 is related to pan-cancer immunomodulators, checkpoints and receptor molecules. GSEA found that CBLL1 may participate in the progression of pan-cancer through B cell receptor singaling pathway, mRNA binding, immunoglobulin receptor binding, Positive Regulation of cell cycle phase transition and other mechanisms.
Conclusions

CBLL1 is abnormally expressed in patients with pan-carcinoma, which is expected to be a biomarker for prognosis, mutation and immune infiltration in patients with pan-carcinoma.

YTH domain containing 3 (YTHDF3), etc [7,8]. Under pathological conditions, the abnormal modi cation of m6A is closely related to diseased progression. For example, Guo et al. reported lower ALKBH5 m6A related gene expression and patients with pancreatic cancer (PC) associated with poor prognosis. Increasing ALKBH5 expression reduced the proliferation, migration and invasion of PC cells in vitro and inhibited tumor growth in vivo. Interfering with the expression of ALKBH5 promotes the progress of PC. ALKBH5 can activate PER1, in a m6A-YTHDF2 -dependent manner and increase the expression of PER1, which can lead to the activation of ATM-CHK2-P53/CDC25C signal pathway, which in turn inhibits the progress of PC [9]. Meng et al. found that the expression of METTL3 in nasopharyngeal carcinoma (NPC) tissues was signi cantly higher than that in the adjacent normal tissues. METTL3 expression was associated with T staging and lymph node metastasis in NPC patients, and the Overall survival (OS) of NPC patients with elevated METTL3 expression was poorer. Knocking down the expression of METTL3 in SUNE-1 cells can inhibit cell viability and migration ability, and reduce the expression of EZH2 protein. M6A modi cation plays a role in the process of METTL3 binding to EZH2 [10]. Zhang et al. found that there was a negative correlation between the expression of YTHDF2 and the prognosis of patients with hepatocellular carcinoma (HCC). Interfering with the expression of YTHDF2 can damage the stemness of HCC cells. On the contrary, increasing the expression of YTHDF2 increased the phenotype of cancer stem cell (CSC). The increase or decrease of YTHDF2 expression leads to the change of m6A level and OCT4 protein expression in OCT4 mRNA 5'-untranslated region (UTR), and affects OCT4 translation. Interfering with the expression of YTHDF2 reduced the ability of transplanted tumor in vivo in nude mice [11]. Studies have reported that the m6a-related gene cbl proto-oncogene like 1 (CBLL1) is highly expressed in the tissues of non-small cell lung cancer (NSCLC).
The increased expression of CBLL1 (Hakai) is related to the size of tumor in patients with NSCLC. The overexpression of CBLL1 promotes the cell cycle transition of G1-S, which leads to cell proliferation and invasion. Interfering with the expression of CBLL1 gene inhibits cell invasion by increasing the expression of adhesion proteins, and reduces the expression of MMP2 and MMP9 in cells [12,13]. Compared with healthy human colon tissue, Hakai is highly expressed in adenoma and colon adenocarcinoma, and is related to patient stage. The overexpression of Hakai can enhance cell transformation and invasion, inhibit the expression of E-cadherin and promote the expression of N-cadherin. Hakai can signi cantly induce tumor growth and local invasion in nude mice [14].
Overexpression of Hakai inhibits the proliferation and migration of breast cancer cells [15]. However, the role of CBLL1 in prognosis, mutation and immune in ltration in pan-cancer is still unknown. In this study, we aimed to use SMART, UCSC and CIBERSORT to explore the level of CBLL1 methylation and mRNA expression in pan-cancer, and to analyze the relationship between CBLL1 mRNA expression and clinicopathological features, prognosis, mutation, immune microenvironment, immune cell in ltration in pan-cancer patients. The mRNA expression data of UCSC database were used to analyze the correlation between CBLL1 expression level and pan-cancer immunomodulators, checkpoints and receptor molecules, as well as the possible mechanism of regulating pan-cancer progression.

Materials And Methods
SMART database SMART (http://www.bioinfo-zs.com/smartapp/) database is an online database website for analysis based on the Cancer Genome Atlas (TCGA) data [16]. The methylation expression levels of CBLL1 in 33 cancers were analyzed by using SMART database to explore whether the methylation expression levels of CBLL1 in pan-carcinoma showed differential expression changes.

UCSC database
The pan-cancer mRNA expression data, clinical data and mutation data were downloaded from UCSC database [17]. The expression data of CBLL1 mRNA in 33 kinds of cancer tissues were extracted and analyzed.The clinicopathological features of 33 cancer patients were extracted and sorted out to analyze the correlation between CBLL1 expression level and age, race, clinical stage, treatment effect and prognosis (OS, Disease-speci c survival (DSS), Disease-free interval (DFI) and Progression-free interval (PFI)) of patients with pan-cancer. Univariate COX analyzed the correlation between the expression of CBLL1 and OS, DSS, DFI and PFI in patients with pan-cancer. The tumor mutational burden (TMB) in each tumor sample was calculated, and the MSI score of tumor microsatellite instability (MSI) was downloaded [18]. The correlation between CBLL1 expression level and TMB and MSI was analyzed. The contents of stromal cells and immune cells in 33 kinds of cancers were scored, and the relationship between the expression of CBLL1 mRNA and the microenvironment of pan-cancerous tumors was analyzed. The mRNA expression data of UCSC database was used to analyze the correlation between CBLL1 expression level and pan-cancer immunomodulators, checkpoints and receptor molecules. The median value of CBLL1 expression was used to analyze the gene expression data, and GSEA was used to analyze the possible mechanism of CBLL1 regulating the progression of pan-cancer [19]. CIBERSORT CIBERSORT (https://cibersort.stanford.edu/) is a kind of bioinformatics algorithms which can calculate according to the gene expression pro les of immune cells. We used CIBERSORT to evaluate the relative proportion of 22 immune cell types in 33 cancers. The scores of immune cells were downloaded from The Cancer Imaging Archive (TCIA) (https://tcia.at/home) database, and the correlation between the expression level of CBLL1 and tumor immune cells was analyzed. Screening criteria: P < 0.05 [20].

Results
Abnormal CBLL1 methylation and mRNA expression in pan-cancerous tissues In SMART database, we found 17 CBLL1 gene probes and abnormal CBLL1 methylation levels in pan-cancerous tissues ( Fig. 1 and The expression level of CBLL1 is associated with TMB and MSI in patients with pan-cancer The mutation load of pan-cancer tumor was calculated based on the expression data of pan-cancer mRNA in UCSC database, and the correlation between the expression level of CBLL1 mRNA and TMB of pan-cancer patients was analyzed. In other words, the expression level of CBLL1 mRNA was correlated with TMB in patients with BLCA, BRCA, COAD, LAML, LGG, LUAD, LUSC, SARC, STAD, THCA, THYM and UVM ( Fig. S19A and Table 1); The expression level of CBLL1 mRNA was correlated with MSI in patients with pancancer. In detail, the expression level of CBLL1 mRNA was associated with ACC, BRCA, CESC, COAD, DLBC, HNSC, PRAD, READ, SARC, STAD, TGCT, THCA and MSI in patients with UCEC ( Fig. S19B and Table 2). Table 1 The expression level of CCBL1 is related to TMB in patients with pan-cancer.  The expression level of CBLL1 mRNA was correlated with tumor microenvironment (Fig. 5, Fig. S20 and Table 3). Figure 5 shows the correlation between CBLL1 mRNA expression level and tumor stromal cells in the top 9 P values. In detail, the expression level of CBLL1 was correlated with tumor stromal cells such as TGCT, LGG, SARC, GBM, LUSC, BLCA, BRCA, THCA, PCPG and so on ( Fig. 5 and Table 3). Figure S20 shows the correlation between CBLL1 mRNA expression level and tumor immune cells in the top 9 P values. In detail, the expression level of CBLL1 was correlated with tumor immune cells such as BRCA, LGG, THCA, GBM, LUSC, SARC, UCEC, PCPG, LIHC and so on ( Fig. S20 and Table 3).

The expression level of CBLL1 is related to immunomodulators, checkpoints and receptor molecules in pan-cancerous tumors
To further explore the relationship between CBLL1 mRNA expression and tumor immune markers, in order to understand the role of CBLL1 in pan-cancer immune escape. immunomodulators include immunostimulators, immunoinhibitors and MHC molecules. We found that the expression level of CBLL1 was associated with pan-cancer immunomodulators (Fig. 7A-C).  Fig. 7C). In addition, the expression level of CBLL1 was associated with pancancerous immune checkpoint molecules (Fig. 7D). For example, the expression level of CBLL1 was correlated with BLCA checkpoint molecules CCL26, CCL14, CCL23, CCL21, CCL28; BRCA checkpoint molecules CCL26, CCL3, CCL17, CCL23, CX3CL1, CXCL2; CESC checkpoint molecules CXCL2, CXCL3, CCL3, CXCL16, CCL13 (P < 0.05, Fig. 7D). In addition, the expression level of CBLL1 was associated with receptor molecules (Fig. S22 and Table 4). For example, the expression level of CBLL1 is related to BLCA receptor molecules CCR9, CCR8, CCR4, CCR7 and CXCR5; BRCA receptor molecules CCR10, CCR8, CCR9, CCR4, CXCR3, etc; CESC molecules CCR8, XCR1, CCR2, CCR6, CCR4, etc (Table 4). The possible mechanism of the involvement of CBLL1 in the progression of pan-cancer The expression of CBLL1 mRNA is related to the prognosis and immunity of patients with pan-cancer. Therefore, we use GSEA to analyze the possible mechanism of CBLL1 involved in tumor progression. The results showed that CBLL1 was involved in the development of pan-cancer through a variety of signal pathways (Fig. 8, Fig. S23  M6A inhibitor therapy inhibited this migration and invasion [21]. WTAP is highly expressed in (GC) tissues of gastric cancer, and the patients with increased expression of WTAP suggest a poor prognosis, and WTAP is an independent risk factor for the prognosis of patients with GC [22]. However, there are few reports on the value of CBLL1 in cancer. In this study, we found that the level of CBLL1 methylation was abnormally expressed in a variety of cancers in the SMART database. Abnormal methylation rate often leads to changes in the level of its gene, which in turn affects the prognosis of tumor patients [23]. Therefore, we analyzed the transcriptome data, clinical and mutation data of TCGA database and found that CBLL1 was highly expressed in CHOL, COAD, ESCA and other cancer tissues, while low expression in THCA and UCEC tissues. The expression level of CBLL1 is related to the age, race, clinical stage and therapeutic effect of patients with pan-cancer. In addition, the expression of CBLL1 was correlated with the prognosis of patients with KIRC, LUSC, THCA, THYM, MESO, PRAD, STAD and UVM. Univariate COX regression analysis found that the expression of CBLL1 mRNA was a prognostic risk factor in patients with KICH, KIRC, LAML, THYM, PCPG, OV, PRAD, STAD, GBM and UVM. This suggests that CBLL1 is related to the occurrence and progression of tumor and is expected to become a target molecule for cancer therapy.
Programmed death inhibitor-1 (PD-1) protein or its has achieved signi cant clinical e cacy in the treatment of a variety of tumors.
TMB and MSI are used as biomarkers to evaluate the therapeutic effect of PD-1 antibody and microsatellite instability is also one of the tumor progression [24,25]. We found that the expression level of CBLL1 was correlated with TMB in patients with BLCA Cancer is considered to be a disease of tumor microenvironment [26].  [28][29][30]. For example, cyclic actin promotes gastric cancer progression through sponge miRNA-331-3p and regulation of TGFBR1 mRNA expression [28]. The expression of TNFSF9 was down-regulated in liver cancer tissues and cells. Overexpression of TNFSF9 can inhibit the proliferation, migration and invasion of Huh7 and SMMC-7721 cells in vitro, and inhibit the growth and metastasis of HCC in vivo [29]. We found that the expression level of CBLL1 was associated with pan-cancer immunomodulators, checkpoints and receptor molecules. At present, CBLL1 is mainly involved in cancer progression by regulating EMT-related E-cadherin and N-cadherin pathways [13,14,31,32]. For example, interfering with CBLL1 gene expression can inhibit cell migration and invasion by increasing E-cadherin protein expression and reducing MMP2 and MMP9, and regulate cell sensitivity to cisplatin through AKT pathway. Overexpression of Hakai can enhance cell transformation and invasion, inhibit the expression of E-cadherin and promote the expression of N-cadherin [14]. Through GSEA, we found that CBLL1 can participate in tumor progression through mRNA binding, B cell receptor singaling

Conclusions
This study found that CBLL1 methylation and mRNA expression were abnormally expressed in pan-cancer, which is expected to be a marker of prognosis, mutation and tumor immune in ltration in cancer patients.

Declarations
Ethics approval and consent to participate Not Applicable.

Consent for publication
All authors agree to publish the manuscript Availability of data and material The datasets generated for this study are available on request to the corresponding author.