沉默ATAD3A对黑素瘤A375细胞增殖、侵袭和迁移的影响及机制研究  

Effect of ATAD3A gene silencing on the proliferation,invasion and migration of A375 human melanoma cells and its mechanisms

作　　者：罗茂 罗卓夫 毕建军 许飏 Luo Mao;Luo Zhuofu;Bi Jianjun;Xu Yang(Department of Dermatology,The Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan,China;Department of Dermatology,People′s Hospital of Chongqing Yubei District,Chongqing 401120,China)

机构地区：[1]西南医科大学附属医院皮肤科,四川泸州646000 [2]重庆市渝北区人民医院皮肤科,401120

出　　处：《中华皮肤科杂志》2020年第7期539-545,共7页Chinese Journal of Dermatology

摘　　要：目的探讨沉默三磷酸腺苷酶家族蛋白3A(ATAD3A)对黑素瘤A375细胞增殖、侵袭和迁移能力的影响。方法2019年8-12月在重庆市渝北区人民医院收集经病理确诊的3例黑素瘤患者的黑素瘤组织及癌旁组织,应用Western印迹检测ATAD3A在上述组织中的表达。分别采用沉默ATAD3A慢病毒和空载慢病毒感染黑素瘤A375细胞系,构建沉默ATAD3A(shATAD3A)实验组和空载对照(shCtrl)组,经实时荧光定量聚合酶链反应(qRT-PCR)和Western印迹验证干扰效率。采用CCK-8实验、克隆形成实验比较两组细胞的增殖能力和克隆形成能力,采用Transwell细胞侵袭实验、划痕实验比较两组细胞的侵袭、迁移能力,采用Western印迹检测两组细胞自我更新相关蛋白(NANOG、SOX2、OCT4)和侵袭、迁移相关蛋白[基质金属蛋白酶2(MMP2)、波形蛋白、SLUG]的表达水平。两组间各指标的比较采用独立样本t检验。结果Western印迹显示,相对于癌旁组织,ATAD3A在3例黑素瘤组织中高表达(t=10.825,P<0.001)。qRT-PCR和Western印迹显示,shATAD3A组A375细胞ATAD3A mRNA为0.230±0.073,shCtrl组为1.000±0.244,两组比较,t=9.461,P<0.001,蛋白表达水平分别为0.279±0.267和0.867±0.115,t=8.595,P=0.002,表明成功构建沉默ATAD3A的黑素瘤A375细胞系。克隆形成实验和CCK-8实验显示,shATAD3A组克隆形成率低于shCtrl组(22.667%±2.510%比43.667%±5.030%,t=6.464,P=0.003),且细胞增殖活性自第2天开始直至第4天均显著低于shCtrl组。划痕实验及Transwell细胞侵袭实验显示,与shCtrl组相比,shATAD3A组划痕愈合减慢,划痕愈合率自第12 h开始(32.920%±4.642%比49.302%±1.448%,t=5.835,P=0.004)至24 h明显降低,通过Transwell小室下室的侵袭细胞数减少(68.330±13.050比234.330±19.139,t=12.411,P<0.001)。Western印迹显示,shATAD3A组细胞NANOG、SOX2、OCT4、MMP2、波形蛋白、SLUG的表达水平均显著下降(P<0.05或0.001)。结论ATAD3A在黑素瘤组织中高表达,沉默ATAD3A能抑制黑Objective To evaluate the effect of ATPase family AAA-domain containing protein 3A(ATAD3A)gene silencing on the proliferation,invasion and migration of A375 human melanoma cells.Methods From August to December in 2019,melanoma and paracancerous tissues were collected from 3 patients with pathologically diagnosed melanoma in People′s Hospital of Chongqing Yubei District,and Western blot analysis was performed to measure the protein expression of ATAD3A in the above tissues.Cultured A375 human melanoma cells were divided into 2 groups to be infected with a lentiviral vector carrying shATAD3A(shATAD3A group)and an empty vector(shCtrl group)respectively,and real-time fluorescence-based quantitative PCR(qRT-PCR)and Western blot analysis were performed to verify the interference efficiency.Cell counting kit-8(CCK8)assay and colony formation assay were performed to compare cell proliferative ability and colony-formation ability respectively between the 2 groups,and Transwell invasion assay and wound healing assay to compare invasive and migratory abilities respectively between the above 2 groups.Western blot analysis was performed to determine the expression of cell self-renewal-related proteins(NANOG,SRY-related high-mobility-group box protein SOX2,octamer-binding protein 4[OCT4])and invasion-and migration-related proteins(matrix metalloproteinase 2[MMP2],vimentin,zinc-finger transcription factor SLUG)in the 2 groups.Two-independent-sample t test was used to compare the experimental indices between the 2 groups.Results Western blot analysis showed that ATAD3A was significantly highly expressed in the 3 melanoma tissues compared with the paracancerous tissues(t=10.825,P<0.001).qRT-PCR and Western blot analysis showed that the mRNA and protein expression of ATAD3A in A375 cells was significantly lower in the shATAD3A group(0.230±0.073,0.279±0.267,respectively)than in the shCtrl group(1.000±0.244,0.867±0.115,respectively;t=9.461,8.595,respectively;P<0.001 or=0.002),indicating that the ATAD3A gene-silenced A375 cell

关 键 词：痣和黑素瘤 腺苷三磷酸酶类 细胞增殖 细胞迁移分析 肿瘤侵润 三磷酸腺苷酶家族蛋白3A 

分 类 号：R739.5[医药卫生—肿瘤]