小鼠黑色素瘤转移相关蛋白的差异蛋白质组学分析

Differential Proteomic Analysis of Metastasis-associated Proteins in Mice Melanoma

目的:分析B16-F10黑色素移植瘤及其肺转移瘤的差异表达蛋白,以筛选黑色素瘤转移相关的分子标志。方法:应用荧光差异凝胶电泳(two-dimensional differential gel electrophoresis,2D-DIGE)结合基质辅助激光解析电离飞行时间质谱技术(matrixassisted laser desorption ionisation time-of-flight mass spectrometry,MALDI-TOF-MS)分离鉴定B16-F10黑色素移植瘤及其肺转移瘤的差异表达蛋白,部分差异蛋白经Real-time PCR进行mRNA表达水平验证。结果:Decyder6.0软件分析结果显示2D-DIGE图谱分辨率高、重复性好,30个蛋白点在实验组和对照组间存在表达差异(Ratiol≥2,P<0.01),经质谱分析和数据库查询鉴定出9个蛋白在实验组表达上调,包括肌红蛋白(myoglobin,MB)、波形蛋白(vimentin,VIM)、磷酸甘油激酶1(phosphoglycerate kinase1,PGK1)、磷酸丙糖异构酶(Triosephosphate isomerase,TPI或TIM)、重链结合蛋白(heavy-chain bindingprotein,BiP)、α-烯醇化酶(α-enolase或enolase1)、β-肌动蛋白(β-actin)、γ-肌动蛋白(γ-actin)、层连蛋白结合蛋白(laminin-binding protein),这些蛋白主要参与了细胞骨架构成、糖酵解等生物学过程。Real-time PCR结果显示糖酵解酶PGK1及TPI mRNA表达水平在实验组显著高于对照组(P=0.001,0.003),变化趋势与蛋白质组学相一致。结论:小鼠黑色素瘤转移过程与多种蛋白的异常表达有关,糖酵解酶PGK1、TPI可能参与了黑色素瘤的转移过程。

Objective： To investigate differentially expressed protein profiles in B16-F10 grafted melanoma and its metastasis in the lung in order to identify molecular markers of melanoma metastasis. Methods： Differentially expressed proteins in B16-F10 grafted melanoma and its metastatic lesion in the lung were isolated and identified by fluorescence two-dimensional differential gel electrophoresis （2D-DIGE） coupled with matrix assisted laser desorption ionisation time-of-flight mass spectrometry （MALDI-TOF-MS）. Some of identified proteins were further confirmed by Real-time PCR analysis. Results： High resolutional images of differential gel electrophoresis were obtained and 9 of 30 differentially expressed proteins （IRatiol ≥2, P〈0.01） were identified by MALDI-TOF-MS. The expression of Myoglobin （MB）, vimentin （VIM）, phosphoglycerate kinase 1 （PGK1）, Triosephosphate isomerase （TPI or TIM）, heavy-chain binding protein （BiP）, α-enolase, β-actin, γ-actin, and laminin-binding protein were up-regulated in the experimental group compared with the control group. These proteins were involved in the cytoskeletal formation, glycolysis and so on. Real-time PCR analysis showed up-regulation of mRNA expression of PGK1 and TPI in the experimental group （P=0.001 and 0.003）, which was in consistent with the results of proteomic analysis. Conclusion： A variety of abnormally expressed proteins contribute to the metastasis of mice melanoma. Glycolytic enzymes PGK1 and TPI may be involved in this process.