The Role of CBLL1 in the Prognosis and Immune Inltration of Pan-Cancer

This study aims to explore the role of CBLL1 in pan-carcinoma and tumor immune inltrates. Methods Download mRNA expression, mutation and clinical data in UCSC database, to analyze the relationship between CBLL1 expression and clinicopathological vlaue, and immune microenvironment in pan-cancer. CIBERSORT was used to analyze the relationship between CBLL1 expression and the inltration 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. Results The levels of CBLL1 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, and with ACC, BRCA, CESC, COAD, DLBC, HNSC, PRAD, READ, SARC, STAD, TGCT, THCA and UCEC microsatellite instability. The expression level of CBLL1 was correlated with cancer stromal cells and immune cells. The expression of CBLL1 is related to pan-cancer immunomodulators, checkpoints and receptor molecules. Overexpression of Hakai inhibits the proliferation and migration of breast cancer cells [5]. However, the role of CBLL1 in prognosis, mutation and immune inltration in pan-cancer is still unknown. In this study, we aimed to use SMART, UCSC and CIBERSORT to explore the level of CBLL1 mRNA expression in pan-cancer, and to analyze the relationship between CBLL1 mRNA expression and clinicopathological features, prognosis, mutation, immune microenvironment, immune cell inltration 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.


Data download and visual analysis
The pan-cancer mRNA expression data, clinical data and mutation data were downloaded from UCSC database [6]. 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 [7]. 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.
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 [8].

Results
Abnormal CBLL1 mRNA expression in pan-cancerous tissues In the UCSC database, we found abnormal expression of CBLL1 in a variety of tumors ( Fig. 1 and S1). In detail, CBLL1 is highly The expression of CBLL1 is related to the age, race, clinical stage and therapeutic effect of patients with pan-cancer After collating and analyzing the clinicopathological features of 33 cancer patients, it was found that the expression level of CBLL1 was correlated with the age of Breast invasive carcinoma (BRCA), CHOL, Kidney renal papillary cell carcinoma (KIRP), LIHC and Pancreatic adenocarcinoma (PAAD) patients ( The expression level of CBLL1 is associated with TMB and MSI in patients with pan-cancer 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. 5A 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. 5B and Table 2).
The expression level of CBLL1 mRNA is related to the microenvironment of pan-cancer The expression level of CBLL1 mRNA was correlated with tumor microenvironment ( Fig. 6 and Table 3). Figure 6 shows the correlation between CBLL1 mRNA expression level and tumor stromal cells via 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. 6 and Table 3). Figure S5 shows the correlation between CBLL1 mRNA expression level and tumor immune cells via 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, etc ( Fig. S5 and Table 3).
The expression level of CBLL1 is related to the immune cells of pan-cancerous tumors  Table S1). In addition, the expression level of CBLL1 was associated with various immune in ltrating cells in pan-cancer. For example, the expression level of CBLL1 was signi cantly correlated with immune cells such as BRCA B cells naive, B cells memory, T cells CD8, T cells CD4 memory activated, Tregs; HNSC B cells naive, B cells memory, T cells CD4 memory resting, NK cells activated; PRAD B cells naive, B cells memory, Plasma cells, T cells CD8, T cells CD4 memory resting (P < 0.05, Additional e 1: Supplementary table S1).

Discussion
A variety of molecular abnormal expressions occur during the development of cancer. For example, METTL14 is signi cantly upregulated in breast cancer (BC) tissues compared with normal tissues. The overexpression of METTL14 enhanced the migration and increased expression of WTAP suggest a poor prognosis, and WTAP is an independent risk factor for the prognosis of patients with GC [10]. However, there are few reports on the value of CBLL1 in cancer. In this study, we 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 [11,12]. We found that the expression level of CBLL1 was correlated with TMB in patients with BLCA, BRCA, COAD, LAML, LGG, LUAD, LUSC, SARC, STAD, THCA, THYM and UVM, and with MSI in patients with ACC, BRCA, CESC, COAD, DLBC, HNSC, PRAD, READ, SARC, STAD, TGCT, THCA and UCEC, which indicates that CBLL1 may be a biomarker of treatment and prognosis in patients with pan-cancer.
Cancer is considered to be a disease of tumor microenvironment [13]. Tumor microenvironment is a complex and dynamic cell population, which is composed of tumor epithelial cells, tumor immune cells, broblasts, immunosuppressive cells, adipocytes, endothelial cells and so on. The interaction between tumor microenvironment and tumor cells is a key factor in immune escape, physiological tolerance and local and systemic invasiveness of malignant cells [14].  [15][16][17]. For example, cyclic actin promotes gastric cancer progression through sponge miRNA-331-3p and regulation of TGFBR1 mRNA expression [15]. 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 [16]. We found that the expression level of CBLL1 was associated with pan-cancer immunomodulators, checkpoints and receptor molecules. For example, CBLL1 expression level is signi cantly correlated with BRCA immunostimulators (LGALS9, IL10RB, CD160, KDR, TGFBR1, etc), immunoinhibitors (TNFRSF4, TNFRSF14, ULBP1, MICB,  TNFSF18,  In summary, this study found that CBLL1 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.     The expression level of CCBL1 is related to TMB and MSI in patients with pan-cancer (A)TMB; (B)MSI.