ZNF407通过TGF-β/Smad信号通路抑制皮肤黑色素瘤迁移与侵袭的影响  

Mechanisms of ZNF407 in regulating cutaneous melanoma by inhibiting TGF-β/Smad pathway

作　　者：李晶[1] 余音[1] 刘素桃[1] 黎智[1] 王雨晴 王灿 LI Jing;YU Yin;LIU Sutao;LI Zhi;WANG Yuqing;WANG Can(Department of Dermatology,Chongqing Hospital of Traditional Chinese Medicine,Chongqing 400011,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：《中国美容整形外科杂志》2024年第8期477-482,共6页Chinese Journal of Aesthetic and Plastic Surgery

基　　金：重庆市自然科学基金(cstc2020jcyj-msxm X0355);重庆市卫生健康委员会、重庆市科学技术局2024年科卫联合医学科研项目(2024MSXM055)

摘　　要：目的探讨锌指蛋白ZNF407在黑色素瘤(melanoma)中的表达和功能,及对黑色素瘤迁移与侵袭的影响和作用机制.方法自2015年1月至2021年1月,收集重庆市中医院皮肤科的黑色素瘤临床样本42例和20例正常色素痣组织,以黑色素瘤组织和细胞系为研究对象,通过免疫蛋白印迹实验和实时荧光PCR检测ZNF407在黑色素瘤组织中相对表达,实时荧光PCR检测ZNF407在黑色素瘤细胞系中相对表达;Pearson相关性检测分析ZNF407与TGF-β1在黑色素瘤中的表达相关性;以黑色素瘤细胞系A375细胞为研究对象进行ZNF407基因过表达,分别转染pcDNA3.1(+)-Flag-ZNF407(实验组)和pcDNA3.1(+)-Flag-Vector(对照组),未转染为空白对照组.Transwell实验验证实验组和对照组中A375细胞迁移和侵袭的影响;半定量PCR和实时荧光PCR检测ZNF407对细胞上皮-间质转化(epithelial-mesenchymal transition,EMT)相关mRNA表达水平的影响,免疫蛋白印迹实验检测ZNF407对EMT相关蛋白表达水平的影响;免疫蛋白印迹实验检测ZNF407对TGF-β信号通路的影响.结果免疫蛋白印迹实验和实时荧光PCR显示:与色素痣细胞组织相比,ZNF407蛋白和mRNA在黑色素瘤组织中表达下调(P<0.001);实时荧光PCR显示:与正常色素痣细胞相比,ZNF407 mRNA在黑色素瘤细胞中表达下调(P<0.001).Transwell迁移实验显示:与对照组相比,实验组穿出小室细胞数(116±17)低于对照组(265±27,P<0.01)和空白对照组细胞(259±28,P<0.01);侵袭实验显示:实验组穿出小室细胞数(82±14)低于对照组(215±31,P<0.01)和空白对照组细胞(209±32,P<0.01).免疫蛋白印迹实验显示:过表达ZNF407后,实验组Vimentin、N-cadherin、Twist和MMP7的蛋白表达下调;E-cadherin的蛋白表达上调(P<0.001);半定量PCR和实时荧光PCR显示:过表达ZNF407后,实验组中干细胞标记物ABCG2、Snail2和OCT4 mRNA表达下调(P<0.001).Pearson相关性分析显示:黑色素瘤组织中ZNF407 mRNA与TGF-β1的表达呈负相关(P=0Objective To study the relative expression and function of the nuclear transcription factor ZNF407 in melanoma and its influence and mechanism on the migration and invasion.Methods From January 2015 to January 2021,42 cases of cutaneous malig-nant melanoma and 20 cases of pigmented nevus in Department of Dermatology,Chongqing Traditional Chinese Medicine Hospital were collected.We examined the expression of ZNF407 in melanoma cell lines and tissues through semi-quantitative polymerase chain Reaction(qRT-PCR)and westernblot.Pearson correlation analysis detection was used to analysis mRNA expression of ZNF407 and TGF-β-1.Melanoma cell line A375 was set as target cells,pcDNA3.1(+)-Flag-ZNF407(experimental group)and pcDNA3.1(+)-Flag-Vector(negative control group)were transfected,respectively.The non transfected group was the set as blank control group.Transwell assay was used to detect the effects of celluar migration and invasion;the effects of ZNF407 on the expression levels of epithelial mesenchymal transition(EMT)related mRNA were detected by semi quantitative PCR and real-time quantitative PCR.Westernblots were used to ex-plore the mechanisms of ZNF407 in epithelial-mesenchymal transition and the mechanisms of ZNF407 on TGF/βsignaling pathway.Results ZNF407 protein and mRNA were downregulated in human malignant melanoma tissues compared with normal tissues and its mRNA was down-regulated in melanoma cell lines(P<0.001).Compared with the control group,transwell migration assay showed that the number of cells in the ZNF407 group(116±17)was lower than that in the control group(265±27,P<0.01)and blank control group(259±28,P<0.01).Transwell invasion assay showed:The number of invated cells in the experimental group(82±14)was lower than that in the control group(215±31,P<0.01)and blank control group(209±32,P<0.01).Westernblot showed that overexpression of ZNF407 resulted in downregulation of protein expression of Vimentin,N-cadherin,Twist,and MMP7;The protein expression of E-cadherin was upregulated(P<0.001);R

关 键 词：黑色素瘤 ZNF407 基因表达 迁移和侵袭 TGF-Β/SMAD 

分 类 号：R739.5[医药卫生—肿瘤]