应用蛋白质组学分析结直肠癌促泌素和糖调节蛋白78蛋白变化及其意义

目的分析结直肠癌、配对正常黏膜组织差异表达蛋白质,寻找与结直肠癌发生、发展相关的分子标记物。方法固相pH梯度双向凝胶电泳分离结直肠癌患者配对癌及正常黏膜组织的总蛋白,液相色谱串联质谱鉴定差异表达蛋白质点。半定量逆转录聚合酶链反应（RT—PCR）、Western blot及免疫组织化学（EnVision法）方法检测促泌素（SCGN）及糖调节蛋白78（GRP78）两个差异表达蛋白质及组织定位,并检测54例神经内分泌肿瘤SCGN的表达。结果比较结直肠癌、配对正常黏膜组织蛋白质表达图谱,共获得5倍以上差异蛋白质点35个,癌组织中表达上调15个、下调20个。质谱分析鉴定14个蛋白质。RT-PCR及Western blot证实结直肠癌组织SCGN低表达,免疫组织化学染色发现正常结直肠黏膜神经内分泌细胞及98％（53／54）的神经内分泌肿瘤SCGN阳性。GRP78蛋白在结直肠癌组织中的表达明显高于正常黏膜组织,但在mRNA水平上癌组织和正常组织之间差异无统计学意义。结论双向凝胶电泳结合质谱分析是筛选肿瘤异常表达蛋白质的有效手段,所获得的35个差异表达候选蛋白质可能与结直肠癌的发生、发展有关。SCGN是一个候选神经内分泌标记物,GRP78蛋白表达增高可能涉及翻译后修饰。

Differential expression of secretagogin and glucose-related protein 78 in colorectal carcinoma: a proteome study

OBJECTIVE: To identify the differentially expressed proteins or peptides and potential biomarkers of tumorigenesis for colorectal cancers. METHODS: Immobilized pH gradient two-dimensional gel electrophoresis (2-DE) was used to separate and obtain the differentially expressed protein spots between colorectal cancers and matched normal mucosa. Liquid chromatography/mass spectrometry (LC-MS/MS) was used to characterize these proteins. Selected candidate proteins were further studied by Western blot, semi-quantitative RT-PCR and immunohistochemical staining. RESULTS: Thirty-five protein spots showed marked expression changes (more than 5-fold) in colorectal carcinoma compared to normal mucosa. Fifteen proteins were up regulated and 20 were down regulated. Fourteen of these proteins were identified by tandem mass spectrometry, among which secretagogin (SCGN) was down-regulated and glucose-related protein (GRP) 78 was up-regulated in the tumors. The SCGN down-regulation was further supported by Western blot and RT-PCR analyses. Immunohistochemistry revealed that SCGN was strongly expressed in neuroendocrine cells of the colonic crypts and 53 of 54 (98%) neuroendocrine tumors. At protein level, although GRP78 was up regulated in colorectal carcinoma, there was no difference in the mRNA expression level between the tumor and paired normal mucosa. CONCLUSIONS: The 2-DE combined with MS is a powerful tool for screening potential tumor biomarkers. The differentially expressed candidate proteins identified by 2-DE may be of significance in understanding the tumorigenesis of the colon cancer. SCGN is a potential biomarker for neuroendocrinal differentiation. GRP78 up-regulation in colorectal carcinomas may be related to its post-translational modification.