Correlation of Prognostic Biomarker SLC25A1 with Immune Inltrates in Low-Grade Gliomas

Background: Glioma is the most common primary malignant tumor of the central nervous system (CNS), and low-grade glioma (LGG) is an important pathological type of glioma. Immune inltration of tumor microenvironment is an independent predictor of survival and prognosis in LGG patients. SLC25A1 is a gene that has not been reported in LGG. We used the TCGA database to study the expression level of SLC25A1 in LGG tissues and its relationship with the prognosis of LGG patients and tumor immune cell inltration. Methods: Download gene expression prole data and clinical information of LGG patients from the TCGA database. The expression level of SLC25A1 gene in LGG tissues and normal tissues was compared. The expression level of SLC25A1 gene in LGG tissue samples with different WHO grades and its relationship with the prognosis of patients were analyzed. The correlation between clinical information and SLC25A1 expression was analyzed by Logistic regression. The effect of SLC25A1 on survival of LGG patients was evaluated by survival module. The correlation between SLC25A1 gene expression and tumor immune cell inltration in LGG and the effect of immune cell inltration on the prognosis of patients were analyzed by using the relevant modules of GEPIA. The gene groups related to SLC25A1 expression were analyzed by KEGG pathway enrichment analysis. In addition, TCGA dataset was used for gene set enrichment analysis (GSEA). Result: The expression level of SLC25A1 gene in LGG tissues was higher than that in normal tissues. The expression of SLC25A1 in WHO (cid:0) LGG tissues was higher than that in WHO (cid:0) tissues. Multivariate analysis showed that the up-regulated expression of SLC25A1 was an independent prognostic factor for good prognosis in patients with LGG. There was a correlation between the expression level of SLC25A1 and the level of immune cell inltration of LGG, and the latter affects the prognosis of patients with LGG. In addition, GSEA also found that Neuroactive ligand receptor interaction, cell adhesion molecules cams and so on were differentially enriched in the phenotypic pathway of high expression of SLC25A1. In the Kyoto Encyclopedia of Genome and Genome (KEGG), Neuroactive ligand-receptor interaction, Morphine addiction et al have been identied as differential enrichment pathways. Conclusion: SLC25A1 is the prognosis of LGG related to immune cell inltration, and may become a biomarker of LGG grade diagnosis, immunotherapy and prognosis.


Introduction
Glioma is a tumor that originates from neuroectoderm cells or precursor cells, including astrocytoma, oligodendroglioma and ependymoma, which account for 75% of primary malignant tumors in the adult brain [1]. The fatality rate and disability rate are both high [2]. According to the World Health Organization (WHO) central nervous system tumor classi cation standard, grades I-are low-grade gliomas (LGGs) and grades -IV are high-grade gliomas (HGGs).
LGG has a relatively good prognosis, but it may also progress to high-grade glioma with higher malignancy and aggressiveness. In recent years, advances in molecular genetics and biomarkers have brought new hope and direction for the precise diagnosis, individualized treatment and prognostic judgment of LGG [3,4]. Finding reliable and effective biomarkers is of great signi cance to the basic research and clinical treatment of LGG.
The mitochondrial citrate transporter SLC25A1, also known as CIC or CTP, belongs to a family of proteins embedded in the mitochondrial membrane. It promotes the out ow of tricarboxylic acid citric acid to the cytoplasm in exchange for dicarboxylic acid cytosolic malic acid [5][6][7]. Our previous work has shown that CIC is highly expressed in several tumor types, and its gene or chemical suppression has anti-tumor activity [8]. But there is no report about LGG and SLC25A1.
In this study, the LGG-related sequencing data and clinical information in the American Cancer Genome Atlas (TCGA) database were used for bioinformatics analysis, the R (v.3.6.3) software and Tumor Immune Estimation Resource (GEPIA) tools were used to analyze the expression differences of SLC25A1 in different tumor tissues and normal tissues, as well as the correlation between SLC25A1 and LGG of different WHO grades and its in uence on the prognosis of patients.
In order to understand tumor immune cell in ltration, this study further used the Tumor Immune Estimation Resource (TIMER) database to analyze the correlation between SLC25A1 and tumor in ltrating immune cells in LGG and the impact of tumor in ltrating cells on the prognosis of patients. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on the gene groups related to the expression of SLC25A1 in LGG to determine the potential pathway of SLC25A1 in the occurrence, development and immune cell in ltration of LGG.

General situation of tissue samples
A total of 529 LGG tissue samples downloaded from TCGA database were included in this study. All of them contained gene expression pro le sequencing data and clinical information, including age, sex, grade, overall survival, staging, distant metastasis, survival status and other information (Table 1). Among them, there were 224 cases of WHO grade LGG (48%) and 243cases of WHO grade LGG (52%). The median age of the patients was 40.5 years old (32,53).  Fig. 1).
In order to further evaluate the expression of SLC25A1 gene in LGG tissues, we analyzed the transcriptional level of SLC25A1 based on TCGA database. We found that the expression of SLC25A1 gene in LGG tissues was higher than that in normal tissues (p < 0.001, Fig. 2). In addition, SLC25A1 mRNA was also expressed differently in different age, sex, WHO grade, primary therapy outcome, IDH and 1p19q status groups. The results showed that the expression level of SLC25A1 in IDH Mut group was higher than that in WT group (P < 0.001), and the expression level of SLC25A1 in 1p19q non-codeletion group was lower than that in codeletion group (P = 0.031). In the primary therapy outcome group, SLC25A1 expression was higher in the PR group than in the PD group (P < 0.001), and higher in the CR group than in the PD group (P = 0.002). The average level in PR group was higher than that in SD group (P = 0.028). The expression of SLC25A1 in Male group was higher than that in Female group (P = 0.024). Most patients with high SLC25A1 expression were less than or equal to 40 years old (P = 0.003). The expression level of SLC25A1 gene in WHO grade LGG tissue was higher than that in WHO grade I LGG tissues, and the median difference between the two groups was 0.152(-0.265 --0.036), with statistical signi cance (P = 0.01, Fig. 3).

Correlation between clinical characteristics and SLC25A1 gene expression in patients with LGG
In this study, chi-square test was used to analyze the correlation between clinical characteristics and SLC25A1mRNA expression in patients with LGG (Table 2). According to the expression level of SLC25A1 mRNA, they were divided into high expression group and low expression group. The results showed that the high expression of SLC25A1 mRNA was related to IDH status (P < 0.001), Primary therapy outcome (< 0.001), Age (P = 0.019), Histological type (P = 0.015) and survival status (P < 0.001), respectively.

Diagnostic value of SLC25A1 mRNA expression in LGG
In this study, the diagnostic value of SLC25A1 mRNA expression in LGG was evaluated by ROC curve. The results showed that the area under the curve of SLC25A1 was 0.964, which had good diagnostic value (Fig. 4).

Relationship between tumor immune cell in ltration and SLC25A1 gene expression and its effect on prognosis of patients
Previous analysis showed that tumor in ltrating lymphocytes were independent predictors of sentinel lymph node status and survival in cancer patients [9]. Therefore, we try to nd out whether the expression of SLC25A1 is related to the immune in ltration of LGG. Spearman partial correlation analysis showed that the expression level of SLC25A1 gene was related to 24 kinds of immune cells (

Results of KEGG pathway enrichment analysis of gene groups positively related to SLC25A1 gene expression
As the high expression of SLC25A1 gene is a risk factor for the survival and prognosis of patients with LGG, we used UALCAN correlation analysis to obtain a total of 56494 genes related to SLC25A1 gene expression in LGG tissues, and used DAVID database for KEGG pathway enrichment analysis. The results showed that under the condition of p.adj < 0.05 and q value < 0.2, there were 1 BP, 13 CC, 37 MF and 1 KEGG (Table 4 and Fig. 11). GSEA is used to identify signaling pathways that are activated in LGG. The results showed that Neuroactive ligand receptor interaction, cell adhesion molecules cams, core matrisome, CD8 + T cell downstream pathway, acetylcholine neurotransmitter release cycle, barrestin pathway were differentially enriched in the negatively correlated with SLC25A1 mRNA expression phenotype (Fig. 12).

Discussion
Glioma is the most common primary malignant tumor of the central nervous system (CNS). It originates from glial cells in the brain and has a high degree of malignancy. The World Health Organization (WHO) (2016) classi es gliomas into grades I-IV, of which grade I and are low-grade gliomas (LGG) [10], accounting for 40%-50% of CNS tumors under 18 years of age.
LGG is characterized by slow growth and even growth stagnation [11]. Most of the patients with LGG are treated with comprehensive therapy, including surgery, radiotherapy and chemotherapy. However, the existing treatment methods can only improve the clinical symptoms of patients, cannot be cured, often occur drug resistance and tumor recurrence [12], and more than half of LGG can develop into high-level LGG that is di cult to treat [13].
In recent years, some new treatments, such as immunotherapy, have been adopted, but the prognosis of patients is still not ideal, which may be related to the lack of effective diagnosis and treatment targets. The SLC25A1 we studied in this article may provide a potential molecular target for the diagnosis, treatment and prognosis of LGG. The discovery of the relationship between SLC25A1 and tumor immune cell in ltration may contribute to the development of LGG immunotherapy.
The mitochondrial citrate transporter, SLC25A1 belongs to the family of ion transporters embedded in the mitochondrial membrane, and its defects have been indirectly related to a variety of human diseases [5,6,14].
The human SLC25A1 gene is located on the chromosome 22q11.21 [15,16]. The expression of SLC25A1 is induced by insulin and in ammation, and its activity is lost in type diabetes. SLC25A1 can also be expressed in a variety of cancer tissues, including ovarian cancer, colon cancer and so on [17]. Previous studies have also shown an increase in SLC25A1 activity in liver tumors [18][19][20][21]. In addition, mutations in members of the tricarboxylate transporter family in fruit y, INDY, which can prolong the lifespan, thus indicating that the citrate transporter pathway also controls longevity [22].
The potential prognostic effects of SLC25A1 in LGG have not been reported. Compared with normal cells, most tumor cells show metabolic changes, which is closely related to the development, progression and invasiveness of cancer [23]. These metabolic changes are largely due to the different utilization of citric acid from normal cells. In fact, it has been suggested that in cancer cells, citric acid is mainly exported from the mitochondria through CIC, and then cleaved in the cytoplasm by citrate lyase (CLY) to support lipid, acetyl-CoA and macromolecular biosynthesis [24][25][26]. This shift in citric acid metabolism from mitochondria to cytoplasm is thought to explain the acquisition of lipogenic phenotypes, as well as the high percentage of anaerobic glycolysis (Warburg effect), which represents the characteristics of many cancer cells [27]. Therefore, we studied the potential role of SLC25A1 in LGG and analyzed the expression of SLC25A1 in a large number of human glioma tumor specimens for the rst time. Under the background of clinical and RNA sequence data, 529 patients with LGG were retrospectively analyzed and histologically con rmed. Through the study, we observed that the expression of SLC25A1 in normal LGG tissues was different from that in tumor tissues, and the expression of SLC25A1 in LGG tissues was higher than that in normal tissues. In addition, the expression level of SLC25A1mRNA was related to tumor grade. The expression level in LGG tissues of WHO was higher than that in LGG tissues of WHO . The results of survival analysis showed that the expression level of SLC25A1 gene was related to the prognosis of LGG, and the prognosis of patients with high expression might be better. Multivariate Cox regression analysis showed that the level of SLC25A1 gene expression was an independent risk factor for the prognosis of patients with LGG. At the same time, the results of ROC curve suggest that SLC25A1 has a good diagnostic value. intact, IDH mutation and 1p/19q co-deletion. Among them, the prognosis of IDH wild type is the worst [29], while the expression of IDH mutation in LGG patients indicates a good prognosis [30]. In an article published by Professor Jiang et al. [31], 77% of WHO gliomas had IDH1 mutations, with a mutation rate of 55% in WHO and only 6% in WHO IV. And Professor Yan et al. [32] believe that IDH1 mutation is positively related to the prognosis of tumor treatment. Previous experiments have once again con rmed the signi cance of IDH1 mutation, that is, detection of IDH1 mutation can be used as an independent index to evaluate the prognosis of glioma patients. IDH1 mutation is more common in low-grade glioma patients, and the progression-free survival time of this type of patients is also relatively long [33]. An article published by Professor Brat et al. [34] pointed out that loss of heterozygosity of 1p/19q is common in oligodendrogliomas, and it is often accompanied by IDH mutation. The view of this study is that the molecular pathological typing based on IDH1 gene mutation combined with 1p/19q co-deletion can more accurately judge the recovery and survival of patients than traditional histopathological typing. The results of this study showed that the expression level of SLC25A1 in IDH mutation group and chromosome 1p19q co-deletion group was signi cantly higher than that in wild type group (P < 0.001) and non-deletion group (P < 0.001). The progress in the study of molecular markers will also promote the development of new technologies for brain tumors and further reduce the pain of patients while accurate diagnosis and treatment. The immunotherapy of LGG needs to be further studied.
Some studies have shown that tumor in ltrating immune cells are independent predictors of sentinel lymph node status and prognosis of tumor patients [9]. The genomic variation of tumor cells may produce tumor antigens, which are systematically recognized as non-self-components, thus triggering cellular immune response [35]. In recent years, it is considered that T cells are related to the positive prognosis of patients [36].
The role of B cells in solid tumors show that the expression of CD20 is related to the prolongation of overall survival in patients with breast and ovarian cancer [37]. Another previous study showed that there was a high correlation between T and B cell gene expression in all tumor types that had been evaluated [38,39]. The results of a previous study showed that there was a high correlation between T cell and B cell gene expression in all assessed tumor types. The expression of immune signals has a strong correlation between different types of immune cells, showing a diversi ed, but quite predictable and consistent tumor immune in ltration, and B cells support anti-tumor immune response [40,41].
LGG has signi cant heterogeneity in genetics and immune level, and nding a suitable target is still a key factor affecting LGG immunotherapy [42]. In this study, we analyzed the correlation between SLC25A1 gene LGG patients. Therefore, we think that SLC25A1 may have a potential effect on tumor immunity. In addition, it can also be used as a promising biomarker of cancer.
In this study, KEGG pathway enrichment analysis was carried out on the genes with signi cant correlation with SLC25A1 gene expression in LGG tissues, in order to obtain the pathways that mainly produce biological functions. The results showed that there were gene enrichment in Neuroactive ligand-receptor interaction, Morphine addiction and neuropeptide signaling pathway pathways, which suggested that most of the genes positively related to SLC25A1 gene expression might regulate the immune cell in ltration of LGG and affect the prognosis of patients through these pathways.
Finally, based on GSEA, we further studied the function of SLC25A1 and the possible mechanism of SLC25A1 affecting the progress and metastasis of LGG. GSEA has wide applicability and is one of the most used methods in path enrichment analysis. Compared with traditional pathway enrichment analysis such as Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG), GSEA can detect changes in the expression of gene sets rather than individual genes, and can detect subtle enrichment signals, which makes the results more reliable and exible [43]. With the development of GSEA, we found that Neuroactive ligand receptor interaction, cell adhesion molecules cams, core matrisome, CD8 tcr downstream pathway, acetylcholine neurotransmitter release cycle, barrestin pathway were differentially enriched in the negatively correlated with SLC25A1 mRNA expression phenotype Although our current study improves our understanding of the role of SLC25A1 in LGG, some limitations remain. First, the sample size of cancer patients in the TCGA database was signi cantly higher than that of the control group. Second, the lack of speci c details of patient medication and/or surgical treatment in public databases can also affect the evaluation of patient outcomes. Third, the protein level of SLC25A1 in KGG and its direct role in LGG progression and metastasis need to be further veri ed in vitro. Fourth, due to the limitations of GSEA, there are still few studies on SLC25A1, which may miss other important signaling pathways regulated by SLC25A1. Finally, this study is a retrospective study, and prospective studies should be conducted in the future to make up for the limitations of retrospective studies. Although this study has certain limitations, it does provide clues for studying the function of SLC25A1 in LGG, and provides targets and potential prognostic markers for the treatment of LGG.

Conclusion
SLC25A1 expression is a potential molecular marker for evaluating the prognosis of LGG patients. Signal pathways such as Neuroactive ligand-receptor interaction,neuropeptide signaling pathway regulated by SLC25A1 in LGG tissues may affect tumor immune cell in ltration, which in turn affects tumor occurrence, development and patient prognosis. As an independent prognostic factor of LGG patients, SLC25A1 has the potential to become a molecular target for the treatment of LGG.

Enrichment analysis of GESA
GSEA is a method that can be used for analysis and calculation to determine whether a priori de ned set of genes has consistent and statistically signi cant differences between the two biological states [43].       Association between clinicopathologic characteristics and survival outcome of LGG patient through multivariate Cox regression analysis. SLC25A1 can independently predict overall survival.

Figure 11
The Functions of SLC25A1 and Genes Signi cantly Associated with SLC25A1 Alterations. The functions of SLC25A1 and genes signi cantly associated with SLC25A1 alterations were predicted by the analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) by DAVID tools (https://david.ncifcrf.gov/summary.jsp).