WGCNA is an informative method for detecting biologically relevant patterns using high dimensional data sets and had been widely applied in transcriptome analysis. It identifies gene modules by assign genes with strongly covarying patterns into groups and allows for the assessment of module relation to sample trait(19). The advantage of WGCNA lies in its focusing on the association between co-expression modules and clinical traits providing highly reliable and biologically significant results (20).
Lauren classification mainly distinguishes gastric cancer into two subtypes including diffuse and intestinal gastric cancer and the greatest advantage of this category system is it can perceive histological and biological trait of gastric cancer easily(21).
To our knowledge, the current study is the first one to utilize WGCNA method for identification of hub genes in Lauren gastric cancer subtype. We identified 12 gene modules in gastric cancer tissue samples and among these modules, blue and red were the most correlated with diffuse or intestinal subtype otherwise. Intramodular analysis showed MM correlated strongly with genes significance regarding to diffuse phenotype suggesting key genes in these two modules may make sense in diffuse subtype.
Involved in cytoskeleton remodeling, Actin is found to be critical for focal adhesion which is deficient in diffuse gastric cancer(7, 22, 23). In our study, result of gene functional enrichment analysis showed actin binding proteins were enriched in blue module that was positive with diffuse phenotype implied abnormal acting binding may occur in diffuse gastric cancer. In blue module, genes link to cation channel activity were also found to be enriched. Recent several publications has reported calcium channel protein such as TRPV4 and TRPM2 were involved in gastric cancer cell survival, invasion and disease progression (24–26). Taken our result into consideration, we propose this case should be confined in particular gastric cancer subtype.
As regarding to red module which is positive associated with intestinal gastric cancer, GO term including GO: 0140097 (catalytic activity, acting on DNA), GO: 0008017(microtubule binding), GO: 0003678(DNA helicase activity), GO: 0016538(cyclin-dependent protein serine/threonine kinase regulator activity) enriched among this module genes. Lei and colleagues identified 3 subtypes of gastric adenocarcinoma: proliferative, metabolic, and mesenchymal. Proliferative subtype is mainly consisted of intestinal gastric cancer(2, 27). As gene in these terms are involved in DNA replication and cell proliferation, this result provide a similar scenario for intestinal gastric cancer biology.
Hopefully, we identified 10 hub-genes in blue and red module. Nine of these genes including HSPB6, TNS1, PGM5, CPXM2, LIMS2, AOC3, CRYAB, ANGPTL1, and BOC were from blue module and associated with gastric cancer survival. As a member of tensin family, TNS1 is revealed to function as a scaffold for adhesion related signaling by binding to actin cytoskeleton and β1-integrin(28), and it had been studied in acute myeloid leukaemia and colorectal cancer. (29, 30). LIMS2,a member of PINCH proteins, that contain 5 LIM domain also plays important roles in cytoskeletal organization and cell–extracellular matrix adhesion, migration, proliferation, and survival(31).
HSPB6 and CRYAB are two members of small heat shock protein famlily (sHSPs) that confer cells the ability to survive under stress conditions (32, 33). Previous study discovered that mesenchymal stem cells overexpression HSPB6 rendered cell resistance to oxidative stress via increasing secretion of growth factors including VEGF, FGF-2, and IGF-1(34). On other side, CRYAB, also as a chaperone can interact with apoptosis-associated protein such as caspase-3, Bax, and Bcl-xS to inhibit cell apoptosis(35).Comprehensively, higher expression of these two gene means diffuse gastric cancer is more tolerant to stress.
In the present study, we also recognize CPXM2 to be a survival-related hub gene in diffuse gastric cancer. CPXM2 is a carboxypeptidases for C-terminal cleavage of peptide or protein have recently been validated to contribute the progression of liver cancer and osteosarcoma (36, 37). AOC3 that is an amine oxidase with copper as a cofactor is an inflammation-inducible endothelial cell molecule mediating leucocyte interactions with the blood vessels for its adhesive property (38). To date, AOC3 is has been showed to be a prognostic marker in breast cancer and astrocytomas(39, 40).