Correlation between mRNAsi and clinical characteristics in GAC
First, we explored the association between mRNAsi and clinical features in GC. As shown, when compared with normal samples, mRNAsi of tumor samples was significantly higher (Fig. 1a). Next, the results of survival analysis indicated that overall survival of patients with lower scores was worse than those with higher mRNAsi index (Fig. 1b). Further, in terms of clinical features, patients with gastric adenocarcinoma were classified into groups by age, gender, TNM stage, tumor stage, histopathological types, respectively; for which, mRNAsi was not associated with age (P = 0.133), gender (P = 0.780), T (P = 0.140), N (P = 0.579), M (P = 0.598) stage, but had a decline trend with the improved tumor stage (Fig. 1c-h). However, in terms of histological subtype, mRNAsi in intestinal-type was significantly higher than that in diffuse-type (Fig. 1i).
Screening of DEGs and stemness-related modules and key genes in IGC and DGC
The above results indicated that there may exist specific DEGs which control the stemness of IGC and DGC, respectively. Therefore, we further filtered the modules and key genes with stemness properties. First, we compared the DEGs between IGC or DGC and normal samples, respectively. In IGC, 7494 DEGs were discerned, among which 5946 were over-expressed and 1548 were down-regulated (Fig. 2a and Additional file 1:Table S1); in DGC, 5424 DEGs were discerned, among which 4647 were up-regulated and 777 were down-regulated (Fig. 3a and Additional file 1: Table S2).
Furthermore, WGCNA was used to construct DEG coexpression network with β = 5 (scale-free R2 = 0.90) and β = 8 (scale-free R2 = 0.90) as soft thresholds, and 14 and 10 gene modules were obtained for subsequent analysis (Fig. 2b,c; Fig. 3b,c). And then, the gene expression status of relevant modules were explored to elucidate the relationship between the corresponding modules and mRNAsi in IGC and DGC samples(Fig. 2d-f; Fig. 3d-f). Here, we noted that the brown module of intestinal-type (R2=0.73, P= 3E-18) and the brown module of diffuse-type (R2=0.9, P=9e-20) both showed a positive correlation between stemness properties and gene expression. Then, we applied all the brown module to screen the key genes related with mRNAsi, with the selection criteria of cor. GS> 0.5, cor. MM> 0.8 and cor. GS> 0.8, cor. MM> 0.8 respectively. Finally, seven stemness-related genes including BUB1, RAD54L, EXO1, NCAPH, DDIAS, XRCC2, RACGAP1 were obtained for intestinal-type; 43 key genes for the diffuse-type, including DLGAP5, DTL, NCAPG2, KIF11, NCAPH, EZH2, ORC6, MTHFD2, BUB1, RAD54L, XRCC2, BUB1B, HMMR, KIF18A, KIF2C, NCAPG，EME1，PLK4，GINS1，PARPBP，SPAG5，ZWILCH, RAD51AP1, RACGAP1, CCDC138, TPX2, SPC25, MAD2L1, CCNB2，DKC1，KNSTRN，ZWINT，RAD51, NUSAP1, CHAF1A, SGO1, FEN1, CCNB1, RRM2, CDCA8, MND1, CCT6A, DBF4. Then we plotted their expression tendency in normal and tumor samples and discovered that the candidate genes were overexpressed in IGC and DGC, respectively (Fig. 4a, 4b). However, taking the intersection of key genes involved in IGC and DGC, BUB1, NCAPH, XRCC2 and 2QA were left, which were more highly expressed in IGC.
Validation of stemness-related key genes in Oncomine database
Next, the mRNAsi-related key genes were verified in Oncomine database. As shown in Fig. 5a-g, 7 key genes in IGC were highly expressed. Except for CHAFIA, 42 key genes showed higher expression in DGC. Fig. 5h-n showed 7 representative genes in DGC compared with normal tissues, and the remaining were shown in Supplementary Figure S1.
The cellular functions and pathway analysis of stemness-related key genes
In IGC, the GO analysis results indicated that key genes participated in biological process of meiotic cell cycle. The main cellular component manifested enrichment mainly at condensed nuclear density. In addition, the main molecular function enriched these genes in DNA-dependent ATPase activity, and the KEGG analysis demonstrated that the major pathway was homologous recombination (HR) (Fig. 6a, 6c). In DGC, GO analysis results indicated that key gene participated in the biological process of nuclear division. The major cellular component suggested enrichment mainly at the chromosomal region. And the main molecular function was catalytic activity. KEGG analysis showed that the main pathway was the cell cycle (Fig. 6b,6d).
Correlation between stemness-related key genes at transcription and protein levels
We explored the interactive relation of the above key genes using Pearson correlation and found that the candidate genes in the brown module of IGC or DGC had strong correlation, with the minimum correlation coefficient of 0.57 and 0.58, respectively (Figure 7a, 8). Next, we built the protein-protein interaction network using the STRING online tool. As shown in Fig. 7b and Fig. 9a, the key genes of the two types of GC formed a close interaction relationship respectively. In IGC, the most crucial key proteins were EXO1 (5 edges) and RAD54L (5 edges) (Figure 7c). The most important key proteins in DGC were CCNB1 (37 edges) and RAD51AP1 (37 edges) (Figure 9b). And BUB1 had high connectivity in both.