抑癌基因ADCY2/4/5/8在皮肤黑色素瘤中的作用研究  

Role of antioncogenes ADCY2/4/5/8 in cutaneous melanoma

作　　者：李晶[1] 黎智[1] 余音[1] 刘素桃[1] 刁庆春[1] 王灿 Li Jing;Li Zhi;Yu Yin;Liu Sutao;Diao Qingchun;Wang Can(Department of Dermatology,Chongqing Traditional Chinese Medicine Hospital,Chongqing Hospital of Integrated Traditional Chinese and Western Medicine,Chongqing Institute of Traditional Chinese Medicine,Chongqing First People's Hospital,Chongqing 400011,China;Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment,Chongqing University Cancer Hospital,Chongqing 400030,China)

机构地区：[1]重庆市中医院,重庆市中西医结合医院,重庆市中医研究院重庆市第一人民医院皮肤科,重庆400011 [2]重庆大学附属肿瘤医院肿瘤转移与个体化诊治转化研究重庆市重点实验室,重庆400030

出　　处：《中华皮肤科杂志》2024年第8期698-708,共11页Chinese Journal of Dermatology

基　　金：重庆市自然科学基金(cstc2020jcyj-msxmX0355);重庆市科卫联合医学科研项目(2024MSXM055)。

摘　　要：目的探讨腺苷酸环化酶(ADCY)2/4/5/8在皮肤恶性黑色素瘤中的表达、作用及功能机制,验证ADCY2/4/5/8对黑色素瘤细胞A375增殖和迁移的影响。方法通过基因表达谱分析(GEPIA)和人类蛋白质图谱(HPA)数据库分析ADCY2/4/5/8 mRNA和蛋白的表达以及基因表达与预后的关联,UALCAN、DeMth和cBioPortal数据库分析ADCY2/4/5/8基因甲基化和突变状态,DAVID和STRING数据库对ADCY2/4/5/8基因进行基因富集分析和通路分析。qPCR检测黑色素瘤A375细胞及色素痣FF细胞中ADCY2/4/5/8 mRNA的相对表达。将A375细胞分为实验组和对照组分别转染ADCY2/4/5/8过表达质粒和对照空载体,采用CCK8和Transwell实验检测过表达ADCY2/4/5/8对A375细胞增殖和迁移的影响,qPCR检测过表达ADCY2/4/5/8基因对A375细胞增殖、迁移相关基因mRNA相对表达的影响,Western印迹法检测过表达ADCY2/4/5/8基因对A375细胞环磷酸腺苷(cAMP)信号通路的影响。组间比较采用单因素方差分析和重复测量方差分析,组间两两比较采用LSD-t检验。结果GEPIA数据库分析显示,558例正常组织中,ADCY2、ADCY4、ADCY5和ADCY8 mRNA在黑色素瘤组织(n=461)中表达下调(P<0.01)。对GEPIA数据库中黑色素瘤ADCY2/4/5/8 mRNA的表达分层分析显示,ADCY2 mRNA(P=0.015)和ADCY8 mRNA(P=0.038)的表达与肿瘤分期相关,表达越低,分期越晚。HPA数据库30例黑色素瘤临床样本和正常组织中,ADCY2/4/5/8蛋白在黑色素瘤组织较正常组织表达降低(P<0.001)。UALCAN和DeMth数据库分析显示,与正常组织相比,ADCY2/4/5/8在黑色素瘤组织和转移性黑色素瘤组织中甲基化水平升高(P<0.001)。DAVID、STRING分析示,ADCY2/4/5/8基因可能通过激活cAMP信号通路抑制细胞增殖和迁移。qPCR检测显示,A375细胞中ADCY2/4/5/8 mRNA的相对表达均低于FF细胞。CCK8实验显示,培养48、72 h时,过表达ADCY2/4/5/8实验组A375细胞存活率均低于对照组(均P<0.05)。Transwell实验显示,过表达ADCY2/4/5/8�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 tu

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 

分 类 号：R739.5[医药卫生—肿瘤]