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) classifies gliomas into grades I-IV, of which grade I and Ⅱ 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, radio-therapy 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 difficult 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 infiltration 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 inflammation, and its activity is lost in type Ⅰ 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 [18–21]. In addition, mutations in members of the tricarboxylate transporter family in fruit fly, 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[24–26]. 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 first time. Under the background of clinical and RNA sequence data, 529 patients with LGG were retrospectively analyzed and histologically confirmed. 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 Ⅱ was higher than that in LGG tissues of WHO Ⅲ. 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.
The prognosis of LGG is affected by age, location, degree of nerve injury, resection extent, gene phenotype and so on. The prognosis and progression of LGG in children and adults are different, and adult LGG is more likely to develop into malignant LGG [28]. Because of the heterogeneity of LGG, the tumor usually shows different molecular characteristics, so the prognosis is different [28]. In recent years, the research on prognostic biomarkers of LGG has been gradually deepened and enriched. According to the status of IDH mutation and 1p/19q deletion, WHO (2016) can be divided into three categories: IDH wild type, IDH mutation and 1p/19q 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 Ⅱ gliomas had IDH1 mutations, with a mutation rate of 55% in WHO Ⅲ 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 confirmed the significance 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 significantly 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 infiltrating 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 diversified, but quite predictable and consistent tumor immune infiltration, and B cells support anti-tumor immune response[40, 41].
LGG has significant heterogeneity in genetics and immune level, and finding a suitable target is still a key factor affecting LGG immunotherapy [42]. In this study, we analyzed the correlation between SLC25A1 gene expression and LGG immune cell infiltration and the effect of immune cell infiltration on the prognosis of patients. Our results showed that different immune markers and immune infiltration levels were related to the expression of SLC25A1 in LGG. Among them, SLC25A1 was positively correlated with tumor immune T cells and pDC infiltration, and negatively correlated with Tcm, Thelper, TFH, Th1, Th2, aDC, B cells, Cytotoxic, Eosinophils, iDC, Macrophages, Mast, Neutrophils, CD56bright cells, CD56dim cells, NK cells, T cells. In addition, the infiltration levels of B cells, Marcophage and Neutrophil all affect the overall prognosis of 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 significant 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 infiltration 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 flexible [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 significantly higher than that of the control group. Second, the lack of specific 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 verified 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.