葡萄膜黑色素瘤特异基因GST-pi和LASP1在其转移分子机制中的作用研究

Uveal melanoma is the most common intraocular malignancies in adults. Since about half of patients die of systemic metastasis, especially liver metastasis, it has become the limiting step in the therapy for uveal melanoma. In our previous studies, we identified two metastasis-related protein glutathione-S-transferase-pi(GST-pi) and LIM and SH3 protein 1(LASP1) by detecting the variation of protein patterns of uveal melanomas with mass spectrometry. In addition, we found that miR-21 was significantly up-regulated in uveal melanomas using miRNA microarray. Current studies have indicated that GST-pi and LASP1 may be regulated by anti-oncogene p53 which could be suppressed by miR-21. Accordingly, we speculate that in uveal melanoma the up-regulated miR-21 may inhibit the expression of wild type p53 gene, which inactivates its inhibition on GST-pi and LASP1, and the increased expression of GST-pi and LASP1 might promote the liver metastasis of uveal melanomas subsequently. Based on the hypothesis above, we are planning to detect the interaction among miR-21, p53, GST-pi, and LASP1 to study the related molecular mechanism furtherly, and then carry out clinical verification. We hope to propose a novel molecular theory for the liver metastasis of uveal melanoma preliminarily, and develop new ideas for clinical diagnosis and treatment for uveal melanoma.

葡萄膜黑色素瘤（UM）患者中约有一半死于肝转移，能否抑制肝转移是降低患者死亡率，提高诊疗效果的关键所在。我们在前期工作中发现：蛋白质-谷胱甘肽S转移酶pi(GST-pi)和LIM和SH3蛋白1(LASP1)与UM肝转移密切相关。通过miRNA表达谱检测，我们还发现UM中与肿瘤侵袭转移密切相关的miR-21表达显著上调。GST-pi和LASP1主要受到抑癌基因p53的调控，而p53的表达又受到miR-21抑制。据此我们形成假说：miR-21在UM中表达上调抑制了野生型p53的表达，从而使其对GST-pi和LASP1的表达抑制减弱，后两者表达上调介导了UM的肝转移。本研究拟探讨miR-21对p53、GST-pi、LASP1表达的影响以及对它们对UM细胞侵袭转移能力的影响，深入研究相关分子机制，并进行临床验证。UM肝转移分子机理的阐明将有助于临床阻断或抑制UM的肝转移，提高UM患者的诊治效果。

葡萄膜黑色素瘤（UM）患者中约有一半死于肝转移，能否抑制肝转移是降低患者死亡率，提高诊疗效果的关键所在。 本研究拟探讨 miRNA-21 对 p53、GST-pi、LASP1 表达的影响以及对它们对 UM 细胞侵袭转移能力的影响，深入研究相关分子机制。利用Real Time-PCR检测miRNA-21、p53、GST-pi 和 LASP1在UM细胞系和正常葡萄膜组织的本底表达水平；利用iTRAQ蛋白检测技术对3个梭型细胞型和3个上皮样细胞型的UM标本的蛋白质谱进行分析研究，重点关注促使肿瘤细胞迁徙的蛋白；利用ProteinPilot软件进行iTRAQ蛋白检测分析发现在p53生物过程的信号转导、信使RNA剪接、核糖核苷三磷酸分解过程及精氨酸分解和ATP代谢过程、脂肪酸氧化和蛋白质聚泛素化的过程中都表现显著高浓度（(FDR < 0.001)；建立miRNA-21过表达和抑制的UM系，进行体外和体内的细胞功能和蛋白和mRNA的研究，结果显示miRNA-21、p53及GST-Pi在葡萄膜黑色素瘤细胞系中的表达丰度为高，LASP1的表达丰度为中； 成功构建miRNA-21过表达和抑制的葡萄膜黑色素瘤细胞系OCM-1、MuM-2B和M619，发现microRNA-21对细胞的增殖、转移和侵袭能力都有相应的影响，且在MuM-2B细胞系的迁徙和转移明显（P<0.01）；Real Time-PCR检测p53和其下游的LASP-1及GST-pi显示野生型p53在microRNA-21过表达时下调，抑制时上调，下游的LASP-1则在过表达时上调，抑制时下调，GST-pi则只在MuM-2B细胞系中表现较明显。皮下裸鼠模型示OCM-1细胞系在miRNA-21过表达时增殖能力增强，抑制时则减弱。本研究初步阐明了miRNA-21对p53、GST-pi、LASP1表达的影响以及它们对细胞侵袭转移能力的影响，为葡萄膜黑色素瘤侵袭转移机制的研究提供了新的思路。