In this study, the common 978 DEGs(952 up-regulated and 26 down-regulated) in keloid samples were markedly differentiated from normal skin and normal scar samples. According to the analysis of the PPI network, the core genes were as followed: GNG3, GNG13, POMC, ADCY8, NMUR1, SST, CCR3, GCG, CHRM2, NPY, OPRM1, PDYN and PYY. Meanwhile, the miR-335-5p and Sp1 were identified as the main upstream factors, of which Sp1 was found to be associated with NPY, OPRM1 and miR-335-5p modulated three central genes that were NPY, POMC, CHRM2. Except for PYY gene, these core genes were commonly enriched in G protein-coupled receptor signaling pathway” in GO_BP terms. It is noticed that NPY, POMC, CHRM2, OPRM1 all belong to the mocode1, which underlines the similarities of their functions.
In the keloid microenvironment, the mechano-physiological conditions are important, they are involved in mechanical tension and inflammatory tension and a vicious circle was formed in between. The mechanical information needs to be converted into a biological signal through the cell membrane mechanoreceptors. Besides integrins, there are non-integrins for accomplishing the transformation process such as G-protein coupled receptors [16]. In the present study, both OPRM1 and CHRM2 belong to a superfamily of the G-protein-coupled receptors, inferring their latent correlation with the mechanical tension. Moreover, these receptors were also involved in growth and progression of tumor [17, 18]. In the view of the abnormal proliferation of keloid fibroblasts is the major reason for keloid formation, the similar role of receptor genes is speculated in keloid.
Altered balance between proangiogenic and antiangiogenic is required for tumor growth beyond a certain size. Vascular endothelial growth factor (VEGF), periostin, and endostatin have been shown to be related to new vessel formation in keloid lesions [19, 20]. We found that NPY, which encodes another direct angiogenic stimulator, was up-regulated in the keloid samples in our study. NPY-stimulated VEGF production and secretion was found to contribute highly to angiogenesis activity in human breast cancer [21] and NPY also was identified as a promoter of prostate and breast cancer, affecting the proliferation and migration of cells[22, 23]. Furthermore, NPY was shown to participate in fibrogenesis of hepatic stellated cells, thereby contributing to hepatic cancer [24].
The deregulation of extracellular matrix deposition is one of the pathological processes in keloids. POMC mRNA was detected in keloid-derived fibroblast, which is in agreement with our results [25]. As the precursor of various active peptides, POMC-derived products were found to have biological roles under cytokine stimulation in the regulation of extracellular matrix deposition and inflammation[26],suggesting their potential functions during the development of keloids.
Among the 2049 predicted miRNAs, miR-335 had the highest degrees and targeted DEGs in the regulation network. MiR-335 has been verified as both a tumor suppressor and tumor promoter in various cancers[27]. One of the inhibition mechanisms of miR-335 is the activation of tumor suppressor p53 by alerting Rb1 to repress cell proliferation [28], and the antagonistic effect of miR-335 on miR-21 was shown to be mainly a pro-tumorigenic mechanism [29]. miR-21 involvement in keloids has also been reported[30], suggesting that miR-335 is involved in the underlying mechanism and neuropeptides, including NPY, POMC, and CHRM2, are involved downstream. This axis mechanism requires further validation. SP1 is a well-known TF that is involved in keloid pathogenesis mainly by regulating the extracellular matrix process of keloids[31]. However, a more detailed understanding of the underlying mechanism is required. We found that Sp1 formed an interaction network with NPY and OPRM1 in the TF–DEG network.