Brn-2在人脉络膜黑色素瘤细胞系中的表达及其对MART-1基因启动子活性的影响  

Expression of Brn-2 in human uveal melanoma cell lines and its effects on the activity of MART-1 gene promoter

作　　者：王应利 周玉梅 陶俊 靳扬扬 陈冉 WANG Ying-Li;ZHOU Yu-Mei;TAO Jun;JIN Yang-Yang;CHEN Ran

机构地区：[1]煤炭总医院眼科,北京市100028

出　　处：《眼科新进展》2018年第9期837-841,共5页Recent Advances in Ophthalmology

摘　　要：目的探讨黑色素抗原转录子Brn-2在人脉络膜黑色素瘤细胞系中的表达及其对T细胞可识别抗原-1(melanoma antigen recognized by T cells 1,MART-1)启动子活性的影响。方法采用RT-PCR和Western blot检测人脉络膜黑色素瘤细胞系92-1、92-2、Me1285和Ocm3细胞中Brn-2的mRNA和蛋白表达水平。将含不同长度MART-1基因启动子序列和荧光素酶报告基因的表达质粒p GL3-Luc构建成融合表达载体p GL3-p286-Luc及p GL3-p2956-Luc,与p EV-Brn-2融合表达质粒共转染脉络膜黑色素瘤细胞系细胞。分别测量四种细胞系中p286组、p2956组、p286+Brn-2组、p2956+Brn-2组细胞荧光素酶表达变化,并观察Brn-2对上述细胞MART-1基因启动子活性的影响。结果人脉络膜黑色素瘤细胞系92-1、92-2、Me1285、Ocm3细胞系均可检测到Brn-2 mRNA和蛋白的表达,Brn-2蛋白电泳带大致在相对分子质量47 000处。p GL3-p2956-Luc及p GL3-p286-Luc重组质粒经Apa I和Nhe I双酶切可切出2956 bp和286 bp 2个条带,p EV-Brn-2重组质粒经EcoRI和Bam HI双酶切可切出1329 bp条带。p EV-Brn-2重组质粒分别对人脉络膜黑色素瘤细胞系Ocm3、Mel285、92-2、92-1进行磷酸钙法转染,Western blot检测可见四种细胞系均表达Brn-2蛋白。缺乏Brn-2时,所有细胞的MART-1的p286均显示出活性,MART-1阳性细胞系(92-1、92-2、Ocm3)p286活性高于阴性细胞系(Mel285)。p2956活性不同细胞系表现不同,92-1、92-2中较高,Ocm3中其活性与细胞密度相关,MART-1阴性细胞系中p2956近乎无活性。共转染Brn-2后明显下调92-1、92-2、Ocm3中启动子p286及p2956活性(P<0.05);阴性细胞系Mel285中,Brn-2对启动子活性无影响(P>0.05)。结论 Brn-2表达于人脉络膜黑色素瘤细胞,并下调阳性细胞系MART-1启动子活性。Objective To investigate the expression of Brn-2 in human uveal melanoma cell lines and its effects on the activity of MART-1 gene promoter.Methods RT-PCR and Western blot were used to check the mRNA and protein level of Brn-2 in human uveal melanoma cell lines 92-1,92-2,Me1285 and Ocm3.Then the expression targeting vectors pGL3-p286-Luc and pGL3-p2956-Luc were constructed by the MART-1 core promoter （p286）/complete promoter （p2956） with luciferase reporter gene downstream of the promoter regions to investigate the effect of Brn-2 on the activity of MART-1 gene promoter.Transfection assays in uveal melanoma cells were performed using the constructed vectors described above in combination with pEV-Brn-2.The luciferase expression in p286,p2956,p286＋Brn-2 and p2956 ＋Brn-2 group was measured to evaluate Brn-2 influence on MART-1 gene promoter activity.Results The mRNA and protein of Brn-2 could be detected in 92-1,92-2,Me1285,Ocm3 cell lines.Brn-2 band was observed around 47 000.And 2956 bp band and 286 bp band were observed after reconstructed plasmid pGL3-p2956-Luc and pGL3-p286-Luc treated with ApaI and Nhel enzyme.Moreover,1329 bp band was observed following the treatment of pEV-Brn-2 reconstructed plasmid with EcoRI and BamHI enzyme,which was in line with our expectation.Reconstructed pEV-Brn-2 plasmid was transfected to Ocm3,Mel285,92-2,92-1 cell lines using calcium phosphate transfection.These cell lines were shown to express Brn-2 protein using Western blot.In the absence of co-transfected Brn-2,the core promoter presented activity in all cell lines.The activity in MART-1 positive cell lines （92-1,92-2,Ocm3） was higher than that in MART-1 negative cell lines （Mel285）.The activity of complete promoter varied in different cell lines.It showed a higher level in 92-1 and 92-2 but nearly no activity in Me1285;as to the Ocm3,its activity depended on the density of cells.The effect of co-transfected Brn-2 on MART-1 promoter activity also varied among cell lines.In 92-1,92-2 and Ocm3 lines,the gen

关 键 词：Brn-2 MART-1基因启动子 脉络膜黑色素瘤细胞系 

分 类 号：R773.4[医药卫生—眼科]