| 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 |
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| 引用本文： | 王应利,周玉梅,陶俊,靳扬扬,陈冉. 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[J]. 眼科新进展, 2018, 0(9): 937-941. DOI: 10.13389/j.cnki.rao.2018.0198 |
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| 作者姓名： | 王应利 周玉梅 陶俊 靳扬扬 陈冉 |
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| 作者单位： | 100028 北京市，煤炭总医院眼科 |
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| 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pGL3-Luc构建成融合表达载体pGL3-p286-Luc及pGL3-p2956-Luc，与pEV-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。pGL3-p2956-Luc及pGL3-p286-Luc重组质粒经ApaI和NheI双酶切可切出2956 bp和286 bp 2个条带，pEV-Brn-2重组质粒经EcoRI和BamHI双酶切可切出1329 bp条带。pEV-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。
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| 关 键 词： | Brn-2 MART-1基因启动子 脉络膜黑色素瘤细胞系 |
Expression of Brn-2 in human uveal melanoma cell lines and its effects on the activity of MART-1 gene promoter |
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| WANG Ying-Li,ZHOU Yu-Mei,TAO Jun,JIN Yang-Yang,CHEN Ran. Expression of Brn-2 in human uveal melanoma cell lines and its effects on the activity of MART-1 gene promoter[J]. Recent Advances in Ophthalmology, 2018, 0(9): 937-941. DOI: 10.13389/j.cnki.rao.2018.0198 |
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| Authors: | WANG Ying-Li ZHOU Yu-Mei TAO Jun JIN Yang-Yang CHEN Ran |
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| Affiliation: | Department of Ophthalmology，Meitan General Hospital，Beijing 100028，China |
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| Abstract: | 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 general pattern showed that Brn-2 strongly down-regulated MART-1 promoter activity both in p286 and p2956（P<0.05）.In the MART-1 negative cell lines Mel285,Brn-2 had no effect on the promoter activity（P>0.05）.Conclusion Brn-2 can be expressed in human uveal melanoma cells as well as down-regulate the activity of MART-1 gene promoter in MART-1 positive cell lines. |
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| Keywords: | Brn-2 MART-1 gene promoter uveal melanoma cell lines |
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