1. BTN3A2 expression levels in different human cancers and LUAD.
The study design is shown as a flowchart for the analysis of the BTN3A2 gene (Fig. 1A). To detect BTN3A2 expression in cancer and non-malignant tissues, the BTN3A2 mRNA contents in different cancer type tissues were analyzed using the sangerbox and TIMER data resources. The results illustrated that the BTN3A2 expression was lower in LUAD, Lung squamous cell carcinoma, as well as Kidney renal clear cell carcinoma in contrast with the non-malignant tissues (Fig. 1B). Moreover, data from TCGA demonstrated that the BTN3A2 content was lower in the LUAD group in contrast with the healthy group (Fig. 1C-1D). Interestingly, protein expression of BTN3A2 in LUAD was still much lower in contrast with that in non-malignant tissues from CPTAC samples (UALCAN) (Fig. 1E). To verify the histological level of BTN3A2, we used the Human Protein Atlas database. The results showed that BTN3A2 is upregulated in normal tissue and downregulated in LUAD tissues (Fig. 7F).
2. BTN3A2 transcription in subgroups of patients with LUAD, stratified based on gender, race, nodal status metastasis and other criteria (UALCAN).
To further analysis multiple clinic features of 515 LUAD samples in the TCGA exhibited low BTN3A2 mRNA content. BTN3A2 mRNA content was lower in LUAD patients in contrast with healthy individuals in subgroup analyses on the basis of gender, race, nodal metastasis, smoking, stages, as well as tumor grade (Fig. 2). Therefore, BTN3A2 levels may have promising LUAD diagnostic value.
3. Prognostic Potential of BTN3A2 in LUAD
To explore whether BTN3A2 expression was related to prognosis of LUAD patients, we evaluated the influence of BTN3A2 contents on OS and FP through the Kaplan-Meier plotter data resource. The data demonstrated the high BTN3A2 contents were linked to good prognosis in LUAD from 204820_s_at probe (OS HR = 0.71, P = 8.5e-08; FP HR = 0.71, P = 5.5e-04) (Fig. 3A-3B). The results were consistent with these in 209846_s_at probe and 212613_s_at probe from Kaplan-Meier plotter (Fig. 3C-3F). To further verify the Prognostic value of BTN3A2 in LUAD, PrognoScan database was used and the results showed that the low BTN3A2 expression group was linked to good lung cancer prognosis (OS GSE31210 HR = 1.55, P = 0.034; RFS GSE31210 HR = 1.26, P = 0.0093) (Fig. 3G-3H). Thus, our data suggest that low BTN3A2 content is a protective factor and results in good prognosis in LUAD.
4. GSEA showed the most significant pathways and genes related with BTN3A2 based on TCGA
To identify the different signal pathways activated in LUAD, we used GSEA between high and low BTN3A2 expression data sets. 6 hallmark gene-sets (including KEGG_T_CELL_RECEPTOR_SIGNALING_CASCADE, KEGG_B_CELL_RECEPTOR_SIGNALING_CASCADE, KEGG_NATURAL_KILLER_CELL_MEDIATED_CYTOTOXICITY, GO_IMMUNE_RECEPTOR_ACTIVITY, GO_IMMUNOLOGICAL_SYNAPSE, GO_T_CELL_ACTIVATION) were chosen for analysis (Fig. 4A-4F). The results showed that T cell receptor signaling cascade, B cell receptor signaling cascade, natural killer cell mediated cytotoxicity, immune receptor activity, immunological synapse, T cell activation are differentially enriched in BTN3A2 high expression phenotype of LUAD.
5.Relationship between expression of BTN3A2 and immune invasion in LUAD
To assess the potential relationship of immune invasion with BTN3A2 expression in LUAD, we used TIMER to conduct the following analysis. Firstly, the "Gene" module analysis revealed that BTN3A2 expression is remarkable positively correlation with invading levels of tumor purity, B cells, dendritic cells, CD8 + T cells, macrophages, neutrophil and CD4 + T cells in LUAD (Figure. 5A). Then, the "SCNA" module analysis revealed immune cell infiltration may relevant to altered BTN3A2 gene copy numbers, including B cells, dendritic cells, CD4 + T cells, macrophages, CD8 + T cells, and neutrophils in LUAD (Figure. 5B). Besides, the "SURVIVAL" module analysis demonstrated that high B cell and dendritic cell levels were linked to good prognosis of LUAD (Figure. 5C, P < 0.05, respectively).
To study the relationship between immune cell markers and BTN3A2 expression, we analyzed the markers of B cells, macrophages, CD8 + T cells, dendritic cells and neutrophils by TIMER data resource. Interestingly, we discovered that the expression levels of CD19 along with CD79A in B cells, CD8B and CD8A in CD8 + T cell, CEACAM8, ITGAM and CCR7 in neutrophils, T helper cells (Th1 and Th2), Macrophage, Dendritic cell have markedly positive related to BTN3A2 expression in LUAD (table).
The data imply that high BTN3A2 expression in LUAD may influence prognosis because of immune invasion.
6. hsa-miR-17-5p may be miRNA targets of BTN3A2
To further elucidate the miRNA targets of BTN3A2 in LUAD, we used miRarbase, starbase and miRDB dadabase to predicted miRNA-target genes of BTN3A2 showing in the venn plot (Fig. 6A). The result showed that it had 6 miRNA-target (hsa-miR-17-5p; hsa-miR-20a-5p; hsa-miR-93-5p; hsa-miR-106b-5p; hsa-miR-20b-5p; hsa-miR-519d-3p) of BTN3A2 and which was visualized in Fig. 6B. Then, we analyzed prognostic potential of 6 miRNA-target in lung cancer by Kaplan-Meier Plotter database. Interestingly, we found that only hsa-miR-17 had differential expression (P = 1.62E-12) and prognostic value (OS, HR = 1.4, P = 0.035) (Fig. 6C-6D). Moreover, the target site in the BTN3A2 3'UTR were predicted to pair with hsa-miR-17-5p by miRDB and RNA22Sites (Fig. 6E).
We have confirmed that BTN3A2 expression was relation to the immune infiltration in LUAD, and the height expression of BTN3A2 was also correlated with the better prognosis of LUAD. Therefore, we hypothesized that hsa-miR-17 expression that may be the miRNA targets of BTN3A2. Prognosis analysis on the basis of the expression of hsa-miR-17of LUAD in linked immune cells subtype was performed using Kaplan Meier plotter, and data illustrated that the high hsa-miR-17 content of LUAD in abundant B cells (HR = 0.03), abundant CD4 + memory T cells (HR = 0.042), abundant macrophages (HR = 0.04), abundant Regulatory T cells (HR = 0.031) and Type 2 T helper cells (HR = 0.0077) had poor prognosis respectively (Fig. 7A-7E). Thus, the results may provide a potential mechanism that hsa-miR-17-5p may be miRNA targets of BTN3A2.