关键词: 黑色素瘤, 腺苷酸环化酶, 细胞增殖, 环AMP, 细胞迁移, 生物信息学, A375细胞

Abstract: 【Abstract】 Objective To determine the expression of adenylate cyclase genes ADCY2/4/5/8 in cutaneous melanoma, to explore their roles and mechanisms of action, and to verify their effect on the proliferation and migration of the melanoma cell line A375. Methods Data on the mRNA and protein expression of ADCY2/4/5/8, as well as the correlation between gene expression and prognosis, were analyzed through the Gene Expression Profiling Interactive Analysis （GEPIA） and Human Protein Atlas （HPA） databases; ADCY2/4/5/8 gene methylation and mutation status were analyzed through the UALCAN, DeMth, and cBioPortal databases; Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis were performed on the ADCY2/4/5/8 genes using the DAVID and STRING databases. qPCR was conducted to determine the relative mRNA expression of ADCY2/4/5/8 in the A375 melanoma cells and FF pigmented nevus cells. Cultured A375 cells were divided into experimental groups and control groups to be transfected with ADCY2/4/5/8 overexpression plasmids and empty vectors, respectively. Cell counting kit （CCK8） and Transwell assays were performed to evaluate the effect of ADCY2/4/5/8 overexpression on the proliferation and migration of A375 cells, qPCR was conducted to determine the relative mRNA expression of genes related to the proliferation and migration of A375 cells, and Western blot analysis to determine the expression of cyclic adenosine monophosphate （cAMP） signaling pathway-related proteins in A375 cells after the overexpression of ADCY2/4/5/8. One-way analysis of variance and repeated measures analysis of variance were used for the comparison among multiple groups, and least significant difference-t test was used for multiple comparisons. Results Bioinformatics analysis based on the GEPIA database showed that the mRNA expression of ADCY2, ADCY4, ADCY5, and ADCY8 was down-regulated in melanoma tissues （n = 461） compared with 30 normal tissues （n = 558, P < 0.01）; stratified analysis showed that the mRNA expression of ADCY2 （P = 0.015） and ADCY8 （P = 0.038） was correlated with tumor staging, and the lower the mRNA expression, the later the tumor stage. In the HPA database, the ADCY2/4/5/8 protein expression was reduced in 30 melanoma tissues compared with normal tissues （P < 0.001）. Analysis of the UALCAN and DeMth database data showed elevated methylation levels of ADCY2/4/5/8 in melanoma tissues and metastatic melanoma tissues compared with normal tissues （P < 0.001）. Analysis of the DAVID and STRING database data demonstrated that the ADCY2/4/5/8 gene may inhibit cell proliferation and migration by activating the cAMP signaling pathway. qPCR showed that the relative mRNA expression of ADCY2/4/5/8 was lower in A375 cells than in FF cells. CCK8 assay showed that the survival rates of A375 cells were significantly lower in the ADCY2/4/5/8 overexpression groups than in the corresponding control groups at 48 and 72 hours （all P < 0.05）. Transwell assay showed that the number of migratory A375 cells was significantly lower in the ADCY2/4/5/8 overexpression groups than in the corresponding control groups （all P < 0.01）. As qPCR revealed, the ADCY2/4/5/8 overexpression groups showed significantly upregulated relative mRNA expression of epithelial cadherin, but significantly downregulated relative mRNA expression of Ki67, vimentin, neural cadherin, and matrix metalloproteinase 2 compared with the corresponding control groups （all P < 0.001）. Western blot analysis showed that the protein expression of cAMP and phosphorylated protein kinase A （p-PKA） in A375 cells was significantly higher in the ADCY2/4/5/8 overexpression groups than in the corresponding control groups （all P < 0.001）, but there were no significant differences in the PKA protein expression levels between the ADCY2/4/5/8 overexpression groups and the corresponding control groups （all P > 0.05）. Conclusion The expression of ADCY2/4/5/8 was downregulated in melanoma tissues, and overexpressed ADCY2/4/5/8 could inhibit the proliferation and migration of melanoma cells, suggesting that ADCY2/4/5/8 may serve as potential tumor suppressor genes in the progression of melanoma.

Key words: Melanoma, Adenylate cyclase, Cell proliferation, Cyclic AMP, Cell migration, Bioinformatics, A375 cells