基于LC-MS/MS筛选宫颈病变和宫颈癌发生发展过程中的新型蛋白标志物

LC-MS/MS-based screening of new protein biomarkers for cervical precancerous lesions and cervical cancer

目的筛选可用来指示宫颈癌从癌前病变到癌变的血浆蛋白标志物,并分析其潜在机制与功能。方法收集健康人(Control组),低度鳞状上皮内病变(LSIL)、高度鳞状上皮内病变(HSIL)、宫颈癌(CC)和治疗后患者血浆样本富集低丰度蛋白。LC-MS/MS检测样本的全多肽类型与序列,检测结果用Proteome Discoverer 2.2进行数据库检索,筛选差异蛋白类型与种类(要求Coverage≥20%,且Unique Peptides≥2蛋白质),LSIL、HSIL、CC组分别与Control组进行T检验比较,P≤0.05视为表达有显著差异的蛋白。通过观察差异蛋白在各组中的色谱图筛选出能提示宫颈病变及宫颈癌的潜在标志物及预后效能评估标记物,并通过靶向蛋白组学技术,在上述实验的基础上样本量对潜在蛋白标记物在各组的含量进行验证实验。进一步通过蛋白功能、功能富集及共表达分析明确差异蛋白的功能探讨其作为标志物的意义与病理机制。结果基于LC-MS/MS和蛋白组学分析发现LSIL、HSIL组相较于Control组分别存在9个异常表达蛋白,CC组相较于Control组存在5个差异蛋白。其中ORM2、HPR在LSIL组相对于Control组的表达存在一定差异,可能是指示宫颈癌癌前病变的潜在标志物;F9在LSIL、HSIL、CC组中相对于Control组,随着疾病的发展呈上升趋势,是一种潜在的指示宫颈病变程度的蛋白标记物;CFI、AFM蛋白在预后组和CC组差异明显,是一类潜在的评价疗效和预后的蛋白指标。各组中的差异蛋白在功能富集分析中发现与补体和凝血级联反应通路有关。结论 F9、CFI、AFM、HPR、ORM2五种新型蛋白可能是指示宫颈病变程度、疗效和预后的潜在标志物,联合分子标志物来评估宫颈病变的发生发展可以增强诊断的灵敏度和特异性,具有一定的临床应用前景。

Objective To screen potential plasma protein biomarkers for the progression of cervical precancerous lesions into cervical carcinoma and analyze their functions. Methods Plasma samples obtained from healthy control subjects, patients with low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), cervical cancer (CC), and patients with CC after treatment were enriched for low-abundance proteins for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The MS data of the samples were analyzed using Discoverer 2.2 software, and the differential proteins (peptide coverage ≥20%, unique peptides≥2) were screened by comparison of LSIL, HSIL and CC groups against the control group followed by verification using target proteomics technology. Protein function enrichment and coexpression analyses were carried out to explore the role of the differentially expressed proteins as potential biomarkers and their pathological mechanisms. Results Compared with the control group, both LSIL group and HSIL group showed 9 differential proteins; 5 differentially expressed proteins were identified in CC group. The proteins ORM2 and HPR showed obvious differential expressions in LSIL and HSIL groups compared with the control group, and could serve as potential biomarkers for the progression of cervical carcinoma. The expression of F9 increased consistently with the lesion progression from LSIL to HSIL and CC, suggesting its value as a potential biomarker for the progression of cervical cancer. CFI and AFM protein levels were obviously decreased in treated patients with CC compared with the patients before treatment, indicating their predictive value for the therapeutic efficacy. Protein function enrichment analysis showed that all these differentially expressed proteins were associated with the complement system and the coagulation cascades pathway. Conclusion We identified 5 new protein biomarkers (F9, CFI, AFM, HPR, and ORM2) for cervical precancerous lesions and for prognostic evaluation of CC, and combined detection of these biomarkers may help in the evaluation of the development and progression of CC and also in improving the diagnostic sensitivity and specificity of cervical lesions.