SLC39A1 Expression in Gliomas
The TCGA database was used to analyze the expression of SLC39A1 in gliomas. The results showed that the expression of SLC39A1 was significantly higher in gliomas than in paracancerous tissues (Fig. 1A). In order to verify this finding, we downloaded three data sets from the GEO database, GSE16011, GSE44971, and GSE11260, and analyzed SLC39A1 expression among them. The results showed that in the three data sets, SLC39A1 expression in gliomas was significantly higher than in the paracancerous tissues (Fig. 1B, C, D), consistent with the analysis results from the TCGA data set, suggest that SLC39A1 was highly expressed in gliomas.
High expression of SLC39A1 is associated with poor glioma prognosis
We employed the TCGA and CGGA databases to study the role of SLC39A1 in the prognosis of glioma patients. Kaplan-Meier analysis results showed that in the CGGA dataset, patients with high SLC39A1 expression had a shorter overall survival time (Figure 2A). Due to the significant difference between HGG and LGG, we further analyzed the prognostic value of SLC39A1 in LGG and HGG patients. The patients were divided into two groups based on SLC39A1expression level. We found that in the high expression group, the prognosis of both LGG or HGG patients was poor (Figure 2B, C). Receiver operating characteristic (ROC) curve analysis showed that SLC39A1 is a predictor of 1-year, 3-year, and 5-year survival (Figure. 2D, E, F). Similar analysis results were also obtained in the TCGA database (Supplementary Figure 1). The above results suggest that SLC39A1 is a factor of poor prognosis for patients with glioma.
SLC39A1 is an independent prognostic factor for patients with glioma
Since the prognosis of glioma patients is affected by a variety of clinicopathological factors, in order to further evaluate the prognostic value of SLC39A1 in glioma patients in the CGGA database, we used cox regression analysis. Univariate cox analysis showed that SLC39A1, PRS_type, Histology and Grade were high-risk factors, while IDH_mutation and 1p19q_codeletion were low-risk factors (Figure 1C). Multivariate cox analysis showed that SLC39A1 was independently related to the overall survival rate. These results suggest that SLC39A1 may be a new prognostic factor for gliomas.
The Relationship Between SLC39A1 Expression and Clinicopathological Parameters
In order to study the relationship between SLC39A1 expression and clinicopathological parameters, we downloaded clinical data for 1,018 gliomas from CGGA. Using the median level, we divided the samples into high and low SLC39A1 expression groups. Correlation analysis showed that SLC39A1 expression was significantly correlated with PRS type, histology, grade, age, chemo status, IDH mutation status, and 1p19q codeletion status (Figure 4). In addition, the 700 glioma clinical samples downloaded from the TCGA data had similar analysis results (Supplementary Figure 2).
Gene Enrichment Analysis
The CGGA database samples were divided into high and low expression groups using the median of SLC39A1 expression according to the results of glioma mRNA sequencing. Gene enrichment analysis was used to determine the enrichment functions and pathways of the differentially expressed genes between the two groups. GO analysis shows that the main functions in which SLC39A1 is enriched are extracellular matrix organization, extracellular structure organization, neutrophil activation, and leukocyte migration; KEGG analysis shows that the main pathways in which SLC39A1 is enriched are ECM-receptor interaction, Antigen processing and presentation, Leukocyte transendothelial migration, and the TNF signaling pathway. In addition, analysis on the TCGA dataset also obtains similar results (Supplementary Figure 3). These results suggest that SLC39A1 may be related to the proliferation, migration and immune response of gliomas.
Role of SLC39A1 in extracellular matrix tissue of gliomas
Based on the gene function enrichment analysis, SLC39A1 is mainly enriched in extracellular matrix and extracellular structure tissue. This suggests that SLC39A1 may be related to the proliferation, metastasis and invasion ability of gliomas. To further analyze this result, we used the CGGA and TCGA datasets to analyze the association between SLC39A1 expression and various invasion-related proteins. The results showed that SLC39A1 was significantly positively correlated with the expression of MMP2 and MMP9 (P<0.0001, Figure 6).
SLC39A1 promotes glioma progression in vitro
In order to study the role of SLC39A1 in gliomas, we stably silenced or overexpressed SLC39A1 by transfecting SLC39A1 siRNA or SLC39A1 overexpression plasmid. As shown in Figure 8A, the expression of SLC39A1 in the siRNA group (SLC39A1 knockout group) was significantly reduced (P<0.05), while its expression in the overexpression group (SLC39A1 overexpression group) was significantly increased, indicating a successful transfection.
We used CCK-8 to measure the effect of SLC39A1 on the proliferation of U87 cells. The results (Figure 7A) showed that compared with Con, cell proliferation in the overexpression group was significantly increased (P<0.05), while that in the siRNA group was significantly decreased (P<0.05). It is suggested that the abnormal expression of SLC39A1 promotes the proliferation of U87 cells.
We employed a flow cytometer to measure cell apoptosis. The results showed (Figure 7B) that cell apoptosis in the overexpression group (P<0.05) was significantly reduced compared with Con, while that in the siRNA group (P<0.05) was significantly increased, suggesting that the abnormal expression of SLC39A1 inhibits apoptosis of U87 MG cells.
SLC39A1 significantly increased the expression of MMP2 and MMP9
In order to further study the relationship between SLC39A1 and metalloproteinase MMP2/MMP9, we used RT-qPCR and western blot to measure expression of SLC39A1 and MMP2/MMP9 mRNA and protein in the different groups. As shown in Figure 8, cell proliferation in the overexpression group was significantly increased compared with the Con group (P<0.05), while that in the siRNA group was significantly decreased (P<0.05). Expression of SLC39A1 and MMP2/MMP9 mRNA and protein in the overexpression group was significantly increased compared with the Con group (P<0.05), while in the siRNA group it was significantly decreased (P<0.05), consistent with the co-expression analysis, indicating that SLC39A1 promotes MMP2/MMP9 expression.
SLC39A1 is associated with immune infiltration of gliomas
The infiltration of immune cells in tumors is closely related to clinical results. Therefore, we used the ESTIMATE algorithm to estimate tumor stroma cell and immune cell infiltration scores, and calculated the correlation between them and SLC39A1 expression.
The results showed that the SLC39A1 expression was significantly and positively correlated with ImmuneScore and StromalScore, and significantly and negatively correlated with TumorPurity (Figure 9A). We also used the CIBERSORT algorithm to analyze the expression levels of 22 immune cell subgroups, and evaluated their correlation with SLC39A1 expression. The results showed that T cells regulatory (T regs), T cells gamma delta, Macrophages M0, Macrophages M1, Macrophages M2 and Eosinophils were significantly positively correlated with SLC39A1 expression (P<0.05), among which Macrophages M2 had the strongest correlation; T cells CD4+ naïve NK cells activate, Monocytes, and Mast cells activated were significantly and negatively correlated with SLC39A1 expression, among which NK cells activate had the strongest correlation (Figure 9B, Table 1). In addition, we also evaluated the possible correlation between 22 immune cells, and the heat map showed that the ratios of the different tumor infiltration immune cell subgroups were weakly to moderately correlated (Figure 9C).