甲状腺癌细胞中分化相关蛋白的筛选分析

Screening and analysis of differentiation-related proteins in the thyroid cancer cells

目的检测正常甲状腺细胞系、中分化甲状腺癌细胞系和低分化甲状腺癌细胞系蛋白质表达谱,寻找与甲状腺癌分化相关的生物标志物。方法应用液相色谱(liquid chromatogram,LC)与质谱(Mass Spectrum,MS)联用分析方法,找出正常甲状腺细胞与中、低分化甲状腺癌细胞系间的差异蛋白。利用生物信息学方法对差异蛋白进行分析,并利用Western blot检测THJ-16T、FTC133、Nthy-ori3-1等3个细胞系中I型胶原α1(collagen type Iα1,COL1A1)、I型胶原α2(collagen type Iα2,COL1A2)、内凹陷蛋白(caveolin-1,Cav-1)、纤维束蛋白同源物1(fascin 1,FSCN1)、硫氧化还原蛋白(thioredoxin,TRX)、高迁移率族蛋白A1(high mobility group protein A1,HMGA1)、超氧化物歧化酶2(superoxide dismutase,SOD2)、Rho关联含卷曲螺旋结合蛋白激酶1(Rho-associated protein kinase 1,Rock-1)、角蛋白7(cytoskeletal 7,KRT7)、角蛋白8(cytoskeletal 8,KRT8)的表达,利用免疫细胞化学方法进一步验证,从而筛选出分化相关蛋白。结果通过分析获得中分化与低分化甲状腺癌细胞细胞系差异表达蛋白质178个。生物信息学研究表明,这些差异蛋白质主要参与细胞内吞信号、T-淋巴细胞介导的细胞凋亡、维生素C运输等通路,且通过Western blot检测发现COL1A1、Cav-1、TRX、Rock-1的表达与细胞的分化能力呈正相关,部分蛋白表达水平使用免疫细胞化学验证,与Western blot结果相符。结论COL1A1、Cav-1、TRX、Rock-1可能是分化相关的标记物,此为进行大样本的临床检测提供了线索。

Objective To identify biomarkers related to the differentiation of thyroid cancer by detecting the protein expression profiles of normal thyroid cell lines,moderately differentiated thyroid cancer cell lines and poorly differentiated thyroid cancer cell lines.Methods Liquid chromatography(LC)and mass spectrometry(MS)were used to identify the differential proteins between normal thyroid cells and moderately and poorly differentiated thyroid cancer cells.Using bioinformatics methods to analyze differential protein and Western blot to detect the expression of collagen type Iα1(COL1A1),collagen type Iα2(COL1A2),caveolin-1(Cav-1),fascin 1(FSCN1),thioredoxin(TRX),high mobility group protein A1(HMGA1),superoxide dismutase 2(SOD2),Rock-1(Rho-associated protein kinase 1),cytokeratin-7(KRT7)and cytokeratin-8(KRT8)in three cell lines THJ-16T,FTC133 and Nthy-ori3-1,and further validation was performed by immunocytochemistry as a screening approach for differentiation-related proteins.Results 178 differentially expressed proteins were obtained from the medium and poorly differentiated thyroid cancer cell lines.Bioinformatics studies have indicated that these differential proteins are mainly involved in endocytosis signaling,T-lymphocyte-mediated apoptosis,Vitamin C transport and other pathways.Western blot showed that the expression of COL1A1,Cav-1,TRX and Rock-1 were positively correlated with the ability of cells differentiation.The results of immunocytochemistry were consistent with those of Western blots.Conclusion COL1A1,Cav-1,TRX and Rock-1 are candidates of differentiation-related markers,which provided clues for clinical detection of large samples.