3.1 Identification of DEMGs
We identified 1415 DEGs between normal and HCC samples, including 1015 up-regulated and 400 down-regulated genes (Fig. 1A, B). Then, only SNRPC was identified as a DEMG in HCC from the intersection of DEGs and m6A methylation genes (Fig. 1C). In both TCGA and the ICGC datasets, the expression levels of SNRPC in tumors were always higher than normal samples (Fig. 1D, E).
3.2 SNRPC is related to survival of HCC
A Kaplan-Meier survival curve was drawn, and the results showed that both in TCGA and ICGC databases, the low-SNRPC expression group had a high survival probability relative to the high-SNRPC expression group (p < 0.05) (Fig. 2A, B). This suggested that SNRPC impacted outcome in HCC patients.
3.3 Construction of a nomogram for HCC prognosis
Univariate and multivariate Cox regression analyses results showed that T (pathological stage) and SNRPC were significantly correlated with the prognosis of HCC (Fig. 2C, D). Therefore, a nomogram including T clinical factors and SNRPC expression values was constructed for predicting 1-, 3- and 5-year survival probabilities of HCC using samples from TCGA database (Fig. 2E). A calibration curve demonstrated that our nomogram could accurately estimate the mortality of HCC (Fig. 2F).
3.4 Functional annotation
To gain insight into the potential mechanisms related to SNRPC, we first selected 64 DEGs with differences between the high- and low- SNRPC expression groups (Fig. 3A). Then, we found that GO terms including catalytic_activity_acting_on_DNA, catalytic_step_2_spliceosome and catalytic_ activity_acting_on_RNA were activated, while terms such as acute_inflammatory_response, alcohol_metabolic_process, alpha_amino_acid_catabolic_process, alpha_amino_acid_metabolic_process, blood_microparticle, cellular_amino_acid_catabolic_process and cellular_lipid_catabolic_process, were suppressed in the high-SNRPC expression group (Fig. 3B). In addition, KEGG pathways such as ribosome, DNA_replication, spliceosome and cell_cycle was up-regulated, while PPAR_ signaling_pathway, complement_and_coagulation_cascades, drug_metabolism_cytochrome_p450, fatty_acid_metabolism, retinol_metabolism and valine_leucine_and_isoleucine_degradation, were down-regulated in the high-SNRPC expression group (Fig. 3C).
In addition, compared to the low-expression group, there were 19 immune-related pathways dysregulated in the high-SNRPC expression group. Among them, ANTIGEN_PROCESSING_AND_PRESENTATION_OF_PEPTIDE_ANTIGEN, ANTIGEN_RECEPTOR_MEDIATED_SIGNALING_PATHWAY, ANTIGEN_PROCESSING_ AND_PRESENTATION, INNATE_IMMUNE_RESPONSE_ACTIVATING_CELL_ SURFACE_RECEPTOR_SIGNALING_PATHWAY, ANTIGEN_PROCESSING_ AND_PRESENTATION_OF_PEPTIDE_ANTIGEN_VIA_MHC_CLASS_I, ANTIGEN_PROCESSING_AND_PRESENTATION_OF_EXOGENOUS_PEPTIDE_ ANTIGEN_VIA_MHC_CLASS_I and SOMATIC_DIVERSIFICATION_OF_ IMMUNE_ RECEPTORS_VIA_GERMLINE_RECOMBINATION_WITHIN_A_SINGLE_LOCUS were inhibited, while DENDRITIC_CELL_ANTIGEN_PROCESSING_AND_PRESENTATION, IMMUNOGLOBULIN_BINDING, REGULATION_OF_HUMORAL_IMMUNE_RESPONSE, HUMORAL_IMMUNE_RESPONSE_MEDIATED_BY_CIRCULATING_IMMUNOGLOBULIN, B_CELL_MEDIATED_IMMUNITY, HUMORAL_IMMUNE_RESPONSE, LYMPHOCYTE_MEDIATED_IMMUNITY, ADAPTIVE_IMMUNE_RESPONSE, ADAPTIVE_IMMUNE_RESPONSE_BASED_ON_SOMATIC_RECOMBINATION_OF_
IMMUNE_RECEPTORS_BUILT_FROM_IMMUNOGLOBULIN_SUPERFAMILY_DOMAINS, REGULATION_OF_IMMUNE_EFFECTOR_PROCESS, IMMUNE_RECEPTOR_ ACTIVITY and NEGATIVE_REGULATION_OF_IMMUNE_SYSTEM_PROCESS were enhanced in the high-SNRPC expression group (Fig. 3D, E). These results implied that SNRPC was correlated with the immune system.
3.5 ESTIMATE analysis
The cells included in the tumor microenvironment impact tumor progress. In our study, we found that except for immune scores, the stromal scores, ESTIMATE scores and tumor purity were significantly different between the high- and low-SNRPC expression groups (Fig. 4A–D). We also found that a lower stromal score and a higher tumor purity were associated with poor survival in HCC patients.
3.6 Analysis of tumor microenvironment (TME)
MCP-counter and ssGSEA analyses were conducted to clear the abundance of various cells in TME. Immune cells such as T cells, CD8 T cells and neutrophils and stromal cells, such as endothelial cells and fibroblasts, were significantly different between the high- and low-SNRPC expression groups. Among these cell types, neutrophils, endothelial cells and fibroblasts were increased in the low-expression group, while others were decreased (Fig. 5A, B). Through ssGSEA analysis, additional sub-types of immune cells were counted. In the low-SNRPC expression group, only activated CD4 T cells were significantly decreased, while CD56dim natural killer cells, central memory CD8 T cells, effector memory CD8 T cells, eosinophils, immature dendritic cells, macrophages, memory B cells, monocytes, natural killer T cells, natural killer T cells, plasmacytoid dendritic cells, regulatory T cells, type 1 T helper cells and type 17 T helper cells were all increased (Fig. 5C, D). These results implied that both stromal cells and immune cells were abundant and were beneficial for HCC prognosis.
3.7 TIDE analysis
TIDE score is used for predicting the efficacy of anti-PD1 and anti-CTLA4 treatments. The high-SNRPC expression group acquired a higher TIDE score using samples from both TCGA and ICGC databases, which suggested that they had poor efficacy in immune checkpoint inhibitor therapies (Fig. 6A, B). Consistently, the high-SNRPC expression group also showed non-response to PD1 therapy by subclass mapping analysis (Fig. 6B, 6C).