Identification of hepatocellular-carcinoma-associated antigens and autoantibodies by serological proteome analysis combined with protein microarray
Identification of hepatocellular-carcinoma-associated antigens and autoantibodies by serological proteome analysis combined with protein microarray
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DOI：
10.1021/pr070525r
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发表时间：
2008-02-01
影响因子：
4.4
通讯作者：
Wang, Sheng-qi
中科院分区：
文献类型：
--
作者：
Li, Lan;Chen, Sul-hong;Wang, Sheng-qi
为了全面研究肝细胞癌（HCC）患者的自身抗体，我们使用了基于血清学和蛋白质组学技术的方法。从HepG 2细胞和HepG2.2.15细胞中提取的总蛋白通过二维凝胶电泳（2DE）分离，然后转移到聚偏二氟乙烯（PVDF）膜上，随后与来自HCC患者或来自正常对照的血清孵育。结果，鉴定出13种HCC相关抗原。通过Western blotting（WB）和蛋白质微阵列分析进一步验证了8个蛋白的抗原性。抗原微阵列分析结果显示，在慢性肝炎对照组中，角蛋白8和核纤层蛋白A/C的信号较强，因此，角蛋白8和核纤层蛋白A/C的自身抗体可能不是HCC特异性的。这两种抗原从随后的分析中去除。DEAD（Asp-Glu-Ala-Asp）box多肽3、真核细胞翻译延伸因子2（eEF 2）、凋亡诱导因子（AIF）、异质核核糖核蛋白A2（hnRNP A2）、前列腺结合蛋白和磷酸丙糖异构酶（TIM）在肝癌中的阳性率显著高于慢性肝炎和正常人。对DEAD盒多肽3、eEF 2、AIF和前列腺结合蛋白的阳性反应在HCC中比在任何其他癌症中显著更频繁。任何单个抗原在I期HCC中的敏感性范围为50 - 85%。当分析六种抗原的组合时，灵敏度增加到90%。因此，检测抗六种抗原的自身抗体对肝癌的早期诊断有一定的价值。
To comprehensively study autoantibodies in patients with hepatocellular carcinoma (HCC), we used an approach-based serology and proteomics technologies. Total proteins extracted from HepG2 cells and HepG2.2.15 cells were separated by two-dimensional gel electrophoresis (2DE) and then transferred onto polyvinylidene difluoride (PVDF) membranes, which were subsequently incubated with sera from HCC patients or from normal controls. As a result, 13 HCC-associated antigens were identified. Antigenicity of eight proteins was further confirmed using recombinant proteins by Western blotting (WB) and protein microarray. The results of antigen microarray analysis showed strong signals of keratin 8 and lamin A/C in chronic hepatitis controls; therefore, the autoantibodies to keratin 8 and lamin A/C may not be HCC-specific. These two antigens were removed from subsequent analyses. The frequencies of positive reactions to DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, eukaryotic translation elongation factor 2 (eEF2), apoptosis-inducing factor (AIF), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2), prostatic binding protein, and triosephosphate isomerase (TIM) were significantly higher, in HCC than in chronic hepatitis and normal individuals. Positive reactions to DEAD box polypeptide 3, eEF2, AIF, and prostatic binding protein were significantly more frequent in HCC than in any other cancer. The sensitivity of any individual antigen in HCC at stage I ranged from 50 to 85%. When the combinations of six antigens were analyzed, the sensitivity increased to 90%. We conclude that the detection of autoantibodies against the six antigens may have value on early diagnosis of HCC.