The lesion area of GCMN becomes larger and the number of satellite nevi increases as the child grows. In addition,relevant complications may seriously affect the quality of life and even longevity15. These problems cannot be solved completely by surgical removal and new medical options based on the key disease-associated genes seem to be promising. Previously published studies mainly focused on the association between somatic mutations and clinical characteristics. In this study, we employed RNA-Seq to screen out 1163 DEGs, and found several novel biological processes and key genes that have not been investigated in GCMN.
GO classification and enrichment analysis revealed that several biological processes related to melanin synthesis were significantly enriched and the genes involved were up-regulated, which is consistent with the increased number of melanocytes and darker skin color of lesions16.In addition, primary biological processes of the DEGs were involved in the regulation of epidermis development, skin development, and keratinocyte differentiation. The thickened cuticle was also observed in diseased tissues with the HE staining results obtained, indicating that keratinocytes are strikingly different between pathological and normal tissues in terms of quantity and gene expression. A previous study suggested that melanocytes transfered melanin to keratinocytes through melanosomes, and the number of melanosomes in keratinocytes determined the degree of skin pigmentation17. We hypothesized that the two types of cells interact through direct contacting and cytokines secreting, and jointly participate in the progression of disease. Extracellular matrix (ECM) can provide the tissue with the mechanical support and mediate the cell-microenvironment interactions. Meanwhile, the extracellular structural changes may play facilitating roles in tumor progression18. Our results showed that GO analysis were linked to extracellular matrix synthesis, including collagen trimer and glycosaminoglycan biosynthetic process. ECM not only directly regulates cellular processes, such as adhesion, migration, proliferation and cell differentiation19, but also acts in binding, guiding and regulating signal molecules20. Tannous, et al. 15demonstrated that the structure of ECM in the lesion site was disordered and melanocytes were extensively infiltrated. This consistency implies that ECM is involved in the formation and development of GCMN by affecting the behavior of melanocytes.
Although no pathway was significantly enriched in KEGG analysis, pathway“Arachidonic acid metabolism”(padj=0.277) attracted our attention. Gledhill, et al. 21reported that arachidonic acid metabolism pathway exists in both keratinocytes and melanocytes, which can stimulate melanocyte proliferation and melanin production by secreting PGE2 and other regulatory factors. Furthermore, the arachidonic acid metabolic pathway has been proved to be activated or inhibited in some pigmented diseases, such as vitiligo22 and solar lentigo23. The role of over-expression of this pathway in GCMN lesions need to be further explored.
From the PPI network analysis, 10 genes were recognized as hub genes. Among them, PTGS2, EGF, SOX10 appeared most frequently in the top ten rankings and were involved in the mentioned biological processes. PTGS2, also known as COX-2, serves as an important role in prostaglandin production from arachidonic acid, and is highly expressed in inflammatory sites and melanoma24. Both animal experiments and cell experiments have confirmed that the growth, migration and invasion ability of melanoma cells were inhibited after the knockout of PTGS2 gene24,25. However ,it should be noted that compared with the normal group, PTGS2 gene was down-regulated in the diseased group in our study.
Epidermal growth factor (EGF) is commonly used to treat chronic ulcers of skin, such as diabetic foot ulcers26 and severe burn injuries27,as it can promote wound healing by inducing granulation tissue growth, angiogenesis and epidermal remodeling. In recent years, some studies have demonstrated that EGF has the inhibitory effects of skin pigmentation by blocking the activity of tyrosinase28. In addition, EGF is a typical anti-inflammatory and antioxidant cytokine, which could also reduce melanin production26,29.Kim, et al. 30proposed EGF-containing ointment had anti-pigmentation effects through performing a randomized controlled trial. In our study, it has been identified that the expression level of EGF was dramatically decreased in the lesions and may be related to local over-pigmentation.
As an essential transcription factor, Sox10 was the first gene in SOX family which was found to be involved in neural crest development31. By synergising with PAX3, SOX10 is capable of up-regulating the expression of the MITF(melanocyte master transcription factor)32 and further activating dopachrome tautomerase and tyrosinase33,the chain reaction can vastly promote melanocyte proliferation and synthesis of melanin. Numerous studies have been reported that SOX10 is highly expressed in tumors, especially melanoma, and plays an essential role in the proliferation, migration, and invasion of melanoma cells 34.Kaufman, et al. 35also found that overexpression of SOX10 in melanocytes significantly accelerated the formation of melanoma. Therefore, the overexpression of SOX10 in GCMN tissues is very likely to be linked to abnormal proliferation and high canceration of melanocytes and thus exploring the mechanisms of SOX10 in the occurrence and development of GCMN may be of great significance.
Several limitations exist in the present study because the sample size is not large enough. further experiments in vivo and in vitro are required to validate our findings and to explore the role of the hub genes and biological processes.