抑癌基因DKK3过表达抑制人皮肤黑素瘤细胞迁移和侵袭的机制探讨  

Mechanism underlying the inhibitory effect of overexpression of tumor suppressor gene DKK3on migration and invasion of human cutaneous malignant melanoma cells

作　　者：李晶[1] 余音[1] 陈静 张欣 刘冬阳[1] 黎智[1] 刁庆春[1] 刘素桃[1] 金晶 王灿 Li Jing;Yu Yin;Chen Jing;Zhang Xin;Liu Dongyang;Li Zhi;Diao Qingchun;Liu Sutao;Jin Jing;Wang Can(Department of Dermatology,Chongqing Traditional Chinese Medicine Hospital,Chongqing 400011,China;Department of Radiotherapy,Cancer Hospital Affiliated to Chongqing University,Chongqing Cancer Institute,Chongqing Cancer Hospital,Chongqing 400030,China;Operating Room,Jing Men No.2People's Hospital,Jingmen 448000,HuBei,China)

机构地区：[1]重庆市中医院皮肤科,400011 [2]重庆大学附属肿瘤医院、重庆市肿瘤研究所、重庆市肿瘤医院放疗科 [3]湖北省荆门市第二人民医院手术室

出　　处：《中华皮肤科杂志》2018年第12期874-878,共5页Chinese Journal of Dermatology

摘　　要：目的 探讨DKK3在人皮肤恶性黑素瘤细胞及组织中的表达及转染DKK3对人黑素瘤细胞A375迁移与侵袭的影响.方法 通过Western印迹法分析DKK3在人黑素瘤细胞系(HM、A375、WM451、SK-MEL-1、Hs-695T、MDA-MB-435s、WM35)及色素痣细胞中的表达,实时荧光定量PCR检测2014年8月至2017年6月在重庆市中医院皮肤科收集的58例恶性黑素瘤(包括原发性黑素瘤与转移性黑素瘤)和30例色素痣组织中DKK3 mRNA的相对表达水平.分别转染pcDNA3.1(+)-Flag-Vector质粒(对照组)和pcDNA3.1 (+)-Flag-DKK3质粒(转染组)至恶性黑素瘤A375细胞中,通过实时荧光定量PCR和Western印迹法验证DKK3的过表达,及DKK3过表达对黑素瘤细胞迁移和侵袭相关的分子表达水平的影响;通过细胞平板划痕实验、Transwell迁移和侵袭实验分析DKK3对A375细胞迁移及侵袭的影响.采用SPSS 13.0软件进行统计学分析,两独立样本间的比较采用t检验;多组数据的比较采用单因素方差分析,组间两两比较采用LSD-t检验.结果 DKK3蛋白在人黑素瘤细胞系中表达缺失或低表达,在色素痣组织中高表达.原发性黑素瘤、转移性黑素瘤与色素痣中DKK3 mRNA表达水平(2-ΔΔCt)分别为(0.325±0.150)×10^-3、(0.142±0.210)×10^-3、(0.634±0.120)×10^-3,差异有统计学意义(F=46.57,P<0.05),转移性黑素瘤表达水平低于原发性黑素瘤和色素痣(LSD-t分别为2.48、3.12,均P<0.05).A375细胞转染DKK3后,细胞划痕迁移率(22.11%±5.1 1%)低于对照组(54.36%±23.22%)(t=2.36,P<0.001);迁移及侵袭实验中,转染组穿出小室细胞数(265±33、76±18)低于对照组(429±41、135±21),差异有统计学意义(t值分别为1.24、1.35,均P< 0.001).转染组A375细胞过表达DKK3后,上皮钙黏着蛋白和mRNA表达上升,神经钙黏着蛋白、波形蛋白、基质金属蛋白酶2(MMP2)、MMP7、MMP11蛋白和mRNA的表达下降.结论 DKK3在黑素瘤细胞系和组织中表达下调,DKK3过表达后A375细胞的迁移和侵袭受�Objective To investigate the expression of DKK3 in human cutaneous malignant melanoma cells and tissues,and to evaluate the effect of transfection with DKK3 gene on migration and invasion of a malignant melanoma cell line A375.Methods Western blot analysis was performed to measure the relative expression of DKK3 in human cutaneous melanoma cell lines HM,A375,WM451,SK-MEL-1,Hs-695T,MDA-MB-435s and WM35,as well as pigmented nevus tissues.Real-time fluorescence-based quantitative PCR (RT-PCR) was conducted to determine the mRNA expression of DKK3 in 58 melanoma tissues (including primary melanoma and metastatic melanoma) and 30 pigmented nevus tissues from Chongqing Traditional Chinese Medicine Hospital between August 2014 and June 2017.The pcDNA3.1 (+)-Flag-Vector (control group) and pcDNA3.1 (+)-Flag-DKK3 (transfection group) were transfected into A375 melanoma cells separately.RT-PCR and Western blot analysis were performed to verify the overexpression of DKK3,and to evaluate the effect of DKK3 overexpression on the expression of molecules related to the migration and invasion of melanoma cells.Cell scratch assay,Transwell migration and invasion assay were conducted to assess the effect of DKK3 on the migration and invasion of A375 cells.Statistical analysis was done by a two-sample t-test for comparisons between two groups,one-way analysis of variance (ANOVA) for intergroup comparison,and least significant difference (LSD)-t test for multiple comparisons with the SPSS 13.0 software.Results DKK3 protein was absent or lowly expressed in the human melanoma cell lines,but highly expressed in the pigmented nevus tissues.There were significant differences in the mRNA expression of DKK3 among the primary melanoma tissues (2-ΔΔCt:[0.325 ± 0.150] × 10-3),metastatic melanoma tissues ([0.142 ± 0.210] × 103) and pigmented nevus tissues ([0.634 ±:0.120] × 10-3,F =46.57,P < 0.05).In addition,the mRNA expression of DKK3 was significantly lower in the metastatic melanoma tissues than in the primary melanoma tissues and

关 键 词：黑色素瘤 基因 肿瘤抑制 细胞运动 细胞侵袭 DKK3 A375细胞 

分 类 号：R739.5[医药卫生—肿瘤]