3.1 Differential expression analysis and clinicopathological correlation analysis
The comparative analysis of MDK and TIMP1 expression in LUAD samples against normal samples indicated that both genes are substantially overexpressed in LUAD patients (Fig. 1A and 1B). qRT-PCR and WB were applied to validate the expression levels of MDK and TIMP1 in lung epithelial and LUAD cells. Results demonstrated that, compared to the control group, the expression levels of MDK and TIMP1 in LUAD cells are significantly elevated (Fig. 1C and 1D).
The correlation analysis of MDK and TIMP1 expression levels with clinical pathology revealed that TIMP1 expression is linked to the survival and death of LUAD patients, with higher levels observed in deceased patients (Fig. 1E) (P = 0.016). The expression levels of MDK and TIMP1 are also related to age, gender, TNM staging, and tumor stage, but these relationships do not hold statistical significance (Figure S1).
The Kaplan-Meier curves showed that LUAD patients with lower expression of MDK and TIMP1 experienced prolonged OS, DFI, DSS, and PFI (Fig. 2). ROC analysis highlighted that MDK and TIMP1 had AUCs of 0.943 and 0.875, respectively, indicating their strong predictive potential for patient prognosis (Fig. 3A, 3B). Univariate Cox regression analysis pointed to the prognostic significance of TNM stage, tumor stage, and TIMP1 (P < 0.05) (Fig. 3C). Multivariate Cox regression analysis substantiated the significant prognostic value of TNM stage and TIMP1 (P < 0.05) (Fig. 3D), establishing TIMP1 as an independent prognostic factor in LUAD.
3.2 Enrichment and interaction analyses
The KEGG pathway analysis for the 50 genes with expression patterns similar to MDK from the GEPIA database revealed significant enrichment of these genes in the pathways of Various types of N-glycan biosynthesis, Proteasome, and N-Glycan biosynthesis (Fig. 4A); The GO analysis for these genes showed that these genes were mainly involved in peptidase activator activity and endopeptidase activator activity (Fig. 4B). For the 50 genes with similar expression to TIMP1, the GO analysis highlighted a concentration in cellular components such as the collagen-containing extracellular matrix and in molecular functions such as serine-type endopeptidase activity, serine-type peptidase activity, serine hydrolase activity, and endopeptidase activity (Fig. 4C); The KEGG analysis showed that these genes were enriched in pathways like Cytoskeleton in muscle cells and Protein digestion and absorption (Fig. 4D).
To predict the functions of MDK and TIMP1, we employed the GeneMANIA website to identify genes with analogous functions and build a protein-protein interaction (PPI) network involving MDK, TIMP1, and these genes, while also projecting their possible functions. The findings showed that these genes were predominantly associated with functions like regulation of metallopeptidase activity, negative regulation of peptidase activity, response to UV-A, extracellular matrix organization, regulation of peptidase activity, negative regulation of proteolysis, and regulation of membrane protein ectodomain proteolysis (Fig. 5).
Using the edgeR package, we carried out differential expression analysis on the high and low expression groups for MDK and TIMP1, followed by KEGG enrichment analysis for the upregulated and downregulated genes with the clusterProfiler package. DEGs identified as upregulated by MDK were predominantly enriched in pathways such as Ribosome, DNA replication, Proteasome, Spliceosome, and Carbon metabolism (Fig. 6A); whereas the downregulated DEGs were mainly enriched in pathways like Neuroactive ligand-receptor interaction, Alcoholism, Calcium signaling pathway, Olfactory transduction, and Steroid hormone biosynthesis (Fig. 6B). Under the influence of TIMP1, the upregulated DEGs were primarily enriched in pathways including Ribosome, Viral protein interaction with cytokine and cytokine receptor, Intestinal immune network for IgA production, Complement and coagulation cascades, and Rheumatoid arthritis (Fig. 6C); the downregulated DEGs were predominantly found in pathways such as Staphylococcus aureus infection, Viral carcinogenesis, Cortisol synthesis and secretion, Olfactory transduction, and Alcoholism (Fig. 6D).
3.3 Mutation profiles of MDK and TIMP1
Predictive modeling of MDK and TIMP1 mutations disclosed that MDK was mainly affected by Amplifications, Deep Deletions, and a limited number of Missense Mutations (Fig. 7A, 7C). The mutation profile of TIMP1 was characterized mainly by Amplification and Deep Deletion (Fig. 7B, 7D). Additionally, we predicted the mutation patterns for MDK and TIMP1 in their respective high and low expression groups. The data revealed that in the MDK high expression group, the principal gene mutation type was Missense Mutation, predominantly manifesting as SNPs, with C > A being the leading single nucleotide mutation. The genes with the highest mutation rates were TTN, RYR2, and CSMD3 (Fig. 8A). In the MDK low expression group, the main mutation types were Missense Mutations and SNPs, with C > A as the most recurrent single nucleotide mutation, and the top three mutated genes by rate are TTN, MUC16, and CSMD3 (Fig. 8B). For TIMP1, the mutation profiles for both high and low expression groups were analogous, with Missense Mutations as the main type, SNPs as the principal mutation category, and C > A as the predominant single nucleotide mutation. The genes with the top mutation rates were consistently TTN, MUC16, and CSMD3 (Figs. 8C and 8D).
Building on our predictions, we examined the impact of MDK and TIMP1 mutations on patient survival compared to non-mutation groups, and observed that the mutation groups had some influence on DSS, OS, PFI, and DFI, but these influences were not statistically significant as per P-values (Figure S2). This finding suggests that mutations in MDK and TIMP1 are not the primary influences on LUAD progression.
3.4 Immune attributes of MDK and TIMP1
We assessed the correlation of MDK and TIMP1 with immune cells via the TIMER website. Results indicated that MDK expression was negatively linked to tumor purity, exhibiting a negative correlation with the infiltration levels of CD8+T cells, macrophages, and neutrophils, and a positive correlation with dendritic cells, CD4+T cells, and B cells (Fig. 9A). TIMP1 expression correlated negatively with tumor purity and positive correlated with immune cell infiltration levels (Fig. 9B).
We also conducted an analysis of the influence of MDK and TIMP1 expression levels on immune subtypes, finding that these genes have distinct impacts in the C1-C6 immune subtypes (Fig. 10A, 10B). The TIDE scores, which measure immune evasion, were higher in the MDK high expression group, but the difference was not statistically significant (Fig. 10C). The high expression group of TIMP1 had a higher TIDE score, suggesting a lower likelihood for benefiting from immune intervention (P = 0.0047) (Fig. 10D).
Moving on, we analyzed the IPS data for MDK and TIMP1 expression groups. Results indicated that the IPS_ctla4_neg_IPS_pd1_neg group in the MDK high expression group was statistically significant in contrast with the low expression group (P < 0.05) (Fig. 11A). For TIMP1, the IPS_ctla_neg_IPS_pd1_pos and IPS_ctla_pos_IPS_pd1_pos groups in the high expression group showed statistically significant differences from the low expression group (P < 0.01) (Fig. 11B). Additionally, we applied the “estimate” scoring system to assess the stromal, immune, and ESTIMATE scores for the high and low expression groups of MDK and TIMP1. The MDK low expression group had higher stromal, immune, and ESTIMATE scores (P < 0.001, P < 0.05, P < 0.01 respectively) (Fig. 11C). The TIMP1 high expression group displayed higher stromal, immune, and ESTIMATE scores (P < 0.001) (Fig. 11D).
Using ssGSEA, we determined the levels of immune cell and immune function infiltration for the high and low expression groups of MDK and TIMP1. The low expression group of MDK demonstrated higher infiltration levels of aCDs, B_cells, DCs, Macrophages, Mast_cells, Neutrophils, T_helper_cells, TIL, and Treg cells (Fig. 12A). The MDK low expression group also displayed elevated infiltration of immune functions CCR and Type_II_IFN_Response (Fig. 12B). The TIMP1 high expression group had greater infiltration of B_cells, CD8+_T_cells, DCs, iDCs, Macrophages, pDCs, T_helper_cells, Tfh, Th1_cells, Th2_cells, TIL, and Treg cells (Fig. 12C). Additionally, this group had higher levels of immune functions including APC_co_inhibition, APC_co_stimulation, CCR, Check-point, Cytolytic_activity, HLA, Inflammation-promoting, Parainflammation, T_cell_co-inhibition, and T_cell_co-stimulation (Fig. 12D). Heat maps of immune cells and immune function pathways revealed higher infiltration in the MDK low expression group (Fig. 13A) and in the TIMP1 high expression group (Fig. 13B).