The mRNA Expression Levels of NLRP3 in Different Types of Human Cancers
Initially, we evaluated NLRP3 expression levels in different human tumors and matched normal tissues by analyzing TCGA RNA-seq data (Fig. 2A). As indicated in figure. 2A, NLRP3 was highly expressed in the tissues of bile duct cancer (CHOL), esophagus carcinoma (ESCA) and head and neck squamous cancer (HNSC) and significantly lower in bladder cancer (BLCA), thymoma (THYM) and lung squamous cell carcinoma (LUSC) tissues than in their respective normal tissues. These results demonstrated that NLRP3 was abnormally expressed in multiple tumors. In addition, we focused on the NLRP3 expression in stomach adenocarcinoma (STAD). We found that NLRP3 mRNA expression level was significantly higher in gastric adenocarcinoma tissue compared to normal counterparts (Fig. 2B).
Prognostic Significance of NLRP3 in STAD
Next, we investigated the prognostic value of NLRP3 expression in gastric adenocarcinoma using the Kaplan-Meier Plotter database. The results showed that overexpression of NLRP3 corresponded with unfavorable overall survival(OS) and progression free survival (PFS) in two different data sets of gastric cancer patients (Figure 3A-3B, OS HR 1.54, 95% CI 1.29–1.84, p=1.1e-6;HR 1.56, 95% CI 1.3–1.87, p=1.2e-6; Figure 3C-3D, PFS HR 1.59, 95% CI 1.28–1.98, p=2.7e-5;HR 1.63, 95% CI 1.27–2.09, p=0.00011). As we all know, recent advances in the field of gastric cancer have appeared a new classification based on molecular features (4), and this molecular is human epidermal growth factor receptor 2 (HER2). It has been demonstrated that HER2 is an important mediator of tumor cell proliferation and differentiation[13].Based on the status of HER2,one subtype is characterized by overexpression or amplification of HER2 and another is characterized by HER2 negative. As indicated in Figure.4, the high expression of NLRP3 predicted a worse prognosis no matter in HER2 negative or positive patients(Figure 4A-4B, Her-2 negative HR 1.47, 95% CI 1.17–1.85, p=0.00076;HR 1.62, 95% CI 1.28–2.05, p=4.7e-5; Figure 4C-4D, Her-2 positive HR 1.57, 95% CI 1.18–2.08, p=0.0016;HR 1.37, 95% CI 1.06–1.78, p=0.016).Next, cox regression analysis was done to determine the factors that could influence the survival outcome of gastric patients. Univariate analysis indicated that T stage (HR = 1.719, 95% CI 1.131-2.612,P-value =0.011), N stage (HR = 1.925, 95% CI 1.264-2.931,P-value =0.002), M stage (HR = 2.254, 95% CI 1.295-3.924 P-value =0.004), pathological stage (HR = 2.180, 95% CI 1.388-3.423,P-value < 0.001) ,primary therapy outcome (HR = 0.241, 95% CI 0.165-0.352,P-value<0.001), residual tumor (HR = 3.445, 95% CI 2.160-5.494,P-value<0.001) and the expression level of NLRP3 (HR = 1.620, 95% CI 1.154-2.276,P-value =0.005) were significantly associated with overall survival. Multivariate analysis showed that upregulated NLRP3 expression, stage IV, and progression of disease were independent prognostic factors of unfavorable prognosis(Table 1). For disease specific survival(DSS), univariate analysis revealed that T stage (HR = 2.089, 95% CI 1.192-3.660,P-value =0.011), N stage (HR = 1.807, 95% CI 1.075-3.0361.264-2.931,P-value =0.025), M stage (HR = 2.438, 95% CI 1.221-4.870, P-value =0.012), pathological stage (HR = 2.320, 95% CI 1.324-4.066,P-value =0.003) ,primary therapy outcome (HR = 0.136, 95% CI 0.088-0.212,P-value<0.001) and residual tumor (HR = 5.142, 95% CI 3.014-8.771,P-value<0.001) were significant factors associated with DSS(Table2).Multivariate analysis showed that primary therapy outcome and residue tumor were independent prognostic factors of DSS. In conclusion, we confirmed the prognostic value of NLRP3, its higher expression was associated with worse prognosis in gastric cancer patients.
Association between NLRP3 expression and clinicopathologic variables
Next, we evaluated the association between NLRP3 mRNA expression and several common clinicopathological parameters. Factors such as age, sex, T stage, lymph node metastasis, distant metastasis, pathologic stage, histologic grade and vital status were included for investigation. Based on the median value of NLRP3 expression level, patients were classified into high or low expression group. As summarized in Table 3, high expression of NLRP3 was closely associated with high T stage(p=0.036), high histologic grade (p< 0.001)and worse survival outcome(p=0.036) in patients with gastric carcinoma. No significant correlation between NLRP3 expression and other clinicopathological factors, such as age, gender, lymph node metastasis and distant metastasis was observed.The difference of NLRP3 expression level in different T stages, histologic grades and different survival outcomes was clearly shown in Figure.5, In total, our results verified the significant relationship between higher NLRP3 expression and several unfavorable clinicopathologic characteristics.
NLRP3-related signaling pathways in gastric carcinoma identified by GSEA
In order to investigate the potential molecular function of NLRP3 in the carcinogenesis of gastric carcinoma, gene set enrichment analysis (GSEA) was conducted between samples with low and high NLRP3 expression to predict NLRP3-related signaling pathways. We focused on the top 6 significantly upregulated pathways (Figure. 6A-6F).
In STAD patients, the significantly upregulated pathways enriched in the high NLRP3 group included “Initial triggering of complement“pathway, “FCGR activation"pathway,“CD22 mediated BCR regulation”pathway,“Role of phosholipids in phagocytosis”pathway,”Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell”pathway, and“Antigen activates B Cell Receptor(BCR) leading to generation of second messengers” pathway.Interestingly , all these 6 pathways have close relationship with immune system, “Initial triggering of complement“pathway, “FCGR activation” pathway, “Role of phosholipids in phagocytosis” pathway belong to pathways of the innate immune system, and the following 3 pathways are related to adaptive immune system pathways. “Initial triggering of complement“pathway took part in the activation of complement, while “FCGR-mediated signaling in phagocytosis”pathway and “Role of phosholipids in phagocytosis” pathway acted in the process of phagocytosis. Three pathways closely connected to adaptive immune functions are crucial in modifying the response of lymphoid cells. For example,“CD22 mediated BCR regulation”pathway could regulate B lymphocyte function through both ligand-dependent and ligand-independent mechanisms[14].
Analysis of genes co-expressed with NLRP3 in gastric carcinoma
To further explore the possible effect of NLRP3 in gastric carcinoma, we tried to identify genes that positively co-expressed with NLRP3 with data downloaded from TCGA. The top 50 genes positively correlated with NLRP3 were selected and displayed as co-expression heatmap(Fig. 7). Additionally, we focused on the top 6 genes and verified their relationships with NLRP3 by scatter plots. As shown in Figure 8A-8F, expression of CASS4, PIK3R5, LCP2, LILRA1, MNDA and TM6SF1 were all positively correlated with the NLRP3 expression. investigated their relationship with overall survival outcome in LUAD or SKCM patients. Next,we tried to explore whether these co-expressed genes had an impact on overall survival(OS). Kaplan-Meier survival analysis showed that the expression of some co-expressed genes such as LILRA1, ITGAX, PIK3R5, RASGRP4, MYOIF and CSF1R was negatively correlated the overall survival (Figure. 9A-9F).Therefore, we believe that NLRP3 and its co-expressed genes collectively contribute to the worse survival in gastric carcinoma patients.
Relationship between NLRP3 and tumor-infiltrating immune cells
Previous analyses suggested tumor-infiltrating lymphocytes as independent predictors for survival in cancer patients[15]. Hence, we assessed the correlation between NLRP3 mRNA expression and immune infiltration status in gastric carcinoma.NK cells, macrophages, dendritic cells (DCs), B cells and T cells were included in the analysis. As indicated in Figure.10, NLRP3 expression level had significant positive correlations with infiltrating level of NK cells(P<0.001), macrophages(P<0.001), B cells(P<0.001), T cells(P<0.001), T helper cells(P<0.001), total T helper cells(P<0.001), T regulatory cells(Treg)(P<0.001), T follicular helper cells(Tfh) (P<0.001),dendritic cells(DCs)(P<0.001) . However, the infiltrating level of T helper 1 cells (Th1) ,T helper 2 cells(Th2) and T helper 17 cells (Th17) had no relationship with the expression level of NLRP3. Further, we explored the correlations between NLRP3 and immune markers of a series of immune cells via TIMER databases. CD8+ T cells, T cells (general), B cells, neutrophils, dendritic cells, macrophages, monocyte and natural killer cells were included and different functional T cells, such as T helper 1(Th1) cells, T helper 2(Th2) cells, Tfh cells, T helper 17(Th17) cells, and Tregs, as well as exhausted T cells were analyzed. After the correlation adjustment by purity, the results revealed the NLRP3 expression level was significantly correlated with most immune marker sets of various immune cells and different T cells in gastric carcinoma. Markers of T cells, B cells, dendritic cells, macrophages as well as NK cells all showed significant correlations with NLRP3 expression (Table 4). We also found significant correlations between NLRP3 and marker genes of Treg and T cell exhaustion, such as PD-1, CTLA4, LAG3, and TIM3. In conclusion, these results demonstrated NLRP3 level was strongly correlated with immune infiltration, suggesting that NLRP3 may play a critical role in the gastric tumor microenvironment. Further studies need to be done to elucidate the exact role of NLRP3 in the carcinogenesis and tumor progression of gastric carcinoma.
Verification of the Expression of NLRP3 in gastric cancer samples
Based on the above bioinformatics analysis, we verified the expression of NLRP3 in two gastric cancer samples.The results of western blot showed that the relative expression level of NLRP3 was significantly higher in gastric cancer samples, compared with adjacent normal tissues (Figure 11A). Then the NLRP3 expression level was detected by qRT-PCR, and we found the relative mRNA level was higher in tumor tissues than in paired normal tissues (Figures 11B).The result demonstrated that NLRP3 might be identified as a biomarker for gastric cancer.
NLRP3 Promotes Cell Proliferation in gastric cancer ells
To characterize NLRP3 function involved in tumor progression, colony formation examination was performed.We used a common human gastric adenocarcinoma cell line SGC-7901 for in vitro study. Expression level of NLRP3 was upregulated by transfection of NLRP3-expressing lentivirus. As shown in Figure. 12, in NLRP3 overexpressed SGC-7901 cells, the number of successfully formed clones was significantly higher than vector-transfected cells, indicating that increased NLRP3 could significantly promote colony formation.The results suggested that NLRP3 promote cell proliferation in human gastric carcinoma cells.