经功能和质谱定量确认的肝癌血清标志物的高危人群预警研究

Hepatoma carcinoma (HCC) is the first fatal malignant tumor in Guangxi province. Searching effective biomarkers of tumor has a great of importance role in carcinogenesis mechanism, early diagnosis and screening high risk population. On the basis of the two previous Natural Science Foundation of China（NSFC），this project employs multidimensional LC-MS/MS labeled with isobaric tags for relative and absolute quantitation (iTRAQ) to screen and identify 7 candidate proteins which may affect carcinogenesis in HCC in comparison to controls. The relationship between candidate proteins and carcinogenesis of HCC is verified both in vivo and vitro function tests. Through the establishment of the MRM initiated detection and sequencing (MIDAS) workflow by LC-4000 QTRAP mass spectrometry，candidate protein peptides as serum biomarkers are validated qualitatively through MRM technology. Target polypeptides are synthesized by multi-channel microfluid solid phase polypeptide synthesis chip in vitro，the expression level of serum biomarkers are further absolutely quantitatively detected through MIDAS workflow. Serum biomarkers associated with HCC verified by function tests and validated quantitatively through MRM technology, are used to establish a new method which is characteristic of integrated liquid chip with high throughput. New method is evaluated synthetically their clinical application feasibility by large sample multi-centre trials. The effects of serum markers combined with AFP applied to screen high-risk subject of HCC are evaluated by utilizing established sample library from high-risk subject in Fusui County with HCC high incidence of Guangxi. According to census work annually in high incidence area of HCC，serum biomarkers are applied to screen more than 2000 cases of high-risk personnel，providing reliable theoretical bases for early prevention and control of focus group in HCC high incidence areas.

肝癌是广西第一位癌症杀手。寻找有效的肿瘤标志物用于癌变机理研究、疾病的早期诊断及预后判断具有重要意义。本项目在前两个国家自然科学基金的基础上，基于iTRAQ标记结合LC-MALDI-TOF/TOF串联质谱技术，筛选和鉴定7个可能影响肝癌肿瘤发生发展的候选蛋白；对候选蛋白通过体内外功能实验，验证其与肝癌发生发展的关系；建立MIDAS技术方案,MRM技术对候选蛋白多肽作为血清标志物定性确认;微流控芯片体外合成目标多肽，对血清标志物表达水平的进行绝对定量检测；经功能验证和MRM定量确认的肝癌血清标志物，建立高通量集成式液态芯片的检测新方法，大样本多中心联合评价其临床应用的可行性；利用已建立广西扶绥肝癌高发区高危人群样本库，评价肝癌血清标志物联合AFP筛选肝癌高危人员的效果；结合每年高发区普查，将血清标志物应用于高发区2000多例高危人群筛查，为高发区重点人群的早期防治提供可靠的理论依据。

本项目基于定量血清蛋白组技术，筛选和鉴定9个可能影响肝癌肿瘤发生发展的候选蛋白。MRM技术对9个候选蛋白定性确认和相对定量检测，发现6个蛋白与血清蛋白组的结果一致，选择差异明显RBP4、GSN进入功能验证。转录组结合血清蛋白组筛选NEK2和HSP90α基因作为验证的候选标志物。对候选标志物RBP4、GSN、NEK2和 HSP90α进行体内外功能实验，发现GSN和 HSP90α主要与肝癌的转移有关。在2457例大样本中进行多中心验证候选标志物RBP4和NEK2，其中在407例正常对照，433例慢性乙肝和461例HCC中ELISA验证RBP4表达水平；在312例HCC，442例慢性乙肝和402例正常对照中ELISA验证NEK2表达水平，结果发现RBP4和NEK2在肝癌表达水平明显下降（p=0.000）。RBP4、NEK2、AFP联合诊断HCC，ROC曲线下面积达0.86，高于AFP单独诊断的0.67, P<0.05；联合RBP4、NEK2诊断AFP阴性的HCC，AUC达到0.918；在3万人高危人群队列中，2年随访发现19例HCC，其RBP4、NEK2表达水平明显低于对照。因此，RBP4、NEK2、AFP联合可明显提高HCC诊断效率，但在高危人群中的预警作用还有待进一步观察。