CN111748624B - Biomarker for predicting whether liver cancer is recurrent - Google Patents

Abstract

The invention relates to the field of medical molecular diagnosis, in particular to a protein molecule used as a biomarker of liver cancer and a prognosis method thereof; the biomarker for predicting liver cancer recurrence comprises one or more protein molecules of LAS1L protein, CLTB protein, JAGN1 protein, ALYREF protein and HNRNPA3 protein. The invention adopts LC-MS/MS mass spectrometry to carry out mass spectrometry on a large amount of clinical samples, and then 5 representative protein molecules are screened out in protein detection. Finally determining that 5 protein molecules have good detection benefit through the multiple difference (more than 2 or less than 0.5) of the corresponding molecular contents of the cancer tissues of the patients without liver cancer recurrence and the cancer tissues of the patients with liver cancer recurrence; the LAS1L protein, the CLTB protein, the JAGN1 protein, the ALYREF protein and the HNRNPA3 protein are used as biomarkers to carry out liver cancer prognosis diagnosis on a subject, and the method is simple and easy to implement, safe and effective in diagnosis process, easy to accept by patients, unified in diagnosis standard and less influenced by subjective factors.

Description

Translated fromChinese

用于预测肝癌是否复发的生物标志物Biomarkers for predicting recurrence of liver cancer

技术领域technical field

本发明涉及医学分子诊断领域，具体涉及一种用于预测肝癌是否复发的生物标志物。The invention relates to the field of medical molecular diagnosis, in particular to a biomarker for predicting whether liver cancer recurs.

背景技术Background technique

肝癌是消化系统常见的恶性肿瘤之一，发病率居世界第6位，死亡率居第三位，每年约有25万人死于肝癌。肝癌由于起病隐匿，恶性程度高，治愈率低，是严重威胁人类健康和生活质量的恶性肿瘤之一，且肝癌的发病率每年都在持续地上升。中国是全世界肝癌每年新增病例最多的国家,占全世界总发病率的一半,且欧美国家发病率虽不高但也在逐年增加。肝细胞性肝癌（HCC）是肝癌最主要的组织学类型，占原发性肝恶性肿瘤的70%～85%，在我国更是占到了约90%。迄今为止，HCC的发病机理仍然不明确，但病毒感染、黄曲霉素B1摄入和酗酒等被认为是HCC主要的致病因素。其中10%～40%慢性B型肝炎病毒携带者最终将发展为肝细胞癌，全世界每年估计超过1百万患者死于B型肝炎病毒（hepatitis B virus，HBV）和C型肝炎病毒相关的肝癌。在中国和日本，75%～80%HCC发病与肝脏慢性病毒感染关系密切，其中B型肝炎病毒感染率占50%～55%，而世界范围内80%～90%的HCC病例合并有肝硬化。因此，肝脏HBV感染及肝硬化是HCC最主要的高危因素和癌前病变过程。Liver cancer is one of the common malignant tumors of the digestive system. Its incidence rate ranks sixth in the world and its mortality rate ranks third. About 250,000 people die of liver cancer every year. Due to its insidious onset, high degree of malignancy and low cure rate, liver cancer is one of the malignant tumors that seriously threaten human health and quality of life, and the incidence of liver cancer continues to rise every year. China is the country with the largest number of new cases of liver cancer every year in the world, accounting for half of the total incidence in the world, and the incidence in European and American countries is not high, but it is also increasing year by year. Hepatocellular carcinoma (HCC) is the most important histological type of liver cancer, accounting for 70% to 85% of primary liver malignancies, and it accounts for about 90% in my country. So far, the pathogenesis of HCC is still unclear, but viral infection, aflatoxin B1 intake, and alcohol abuse are considered to be the main pathogenic factors of HCC. Among them, 10% to 40% of chronic hepatitis B virus carriers will eventually develop into hepatocellular carcinoma, and it is estimated that more than 1 million patients die each year from hepatitis B virus (hepatitis B virus, HBV) and hepatitis C virus-related diseases worldwide. liver cancer. In China and Japan, 75% to 80% of HCC cases are closely related to liver chronic viral infection, among which hepatitis B virus infection rate accounts for 50% to 55%, and 80% to 90% of HCC cases worldwide are combined with liver cirrhosis . Therefore, liver HBV infection and liver cirrhosis are the most important high-risk factors and precancerous lesion process of HCC.

肝癌的目前治疗手段主要有介入栓塞，手术切除，射频消融，生物靶向治疗等，但是即便如此，肝癌的预后仍旧较差。究其原因，肝癌术后的复发占有相当大的比例。因此，肝癌术后定期随访监测患者肿瘤相关指标，早期发现和诊断肝癌复发转移，是肝癌预后研究的重要课题。临床上常用的肿瘤标记物甲胎蛋白（ＡＦＰ）敏感性低，部分肝癌术后患者肿瘤复发未能及时检出。肝脏穿刺病理活检用于肝癌诊断及其复发诊断最为可靠，但对患者的损伤较大。影像学手段被广泛应用于术后随访中，根据与甲胎蛋白的结合能够更好地发现复发的肿瘤。上述方法虽都有一定的优势，但是都需要患者定期复查，这对于很多依从性较差的患者来说很难实现。The current treatment methods for liver cancer mainly include interventional embolization, surgical resection, radiofrequency ablation, and biological targeted therapy, but even so, the prognosis of liver cancer is still poor. The reason is that postoperative recurrence of liver cancer accounts for a considerable proportion. Therefore, regular follow-up and monitoring of tumor-related indicators in patients after liver cancer surgery, early detection and diagnosis of liver cancer recurrence and metastasis are important topics in the study of liver cancer prognosis. Alpha-fetoprotein (AFP), a commonly used clinical tumor marker, has low sensitivity, and tumor recurrence in some postoperative patients with liver cancer cannot be detected in time. Liver biopsy is the most reliable method for the diagnosis of liver cancer and its recurrence, but it is more harmful to patients. Imaging methods are widely used in postoperative follow-up, and recurrent tumors can be better detected based on the combination with alpha-fetoprotein. Although the above methods have certain advantages, they all require regular review by patients, which is difficult for many patients with poor compliance.

肝癌复发的来源一是多中心发生，即肝癌根治性切除后，由于肝癌生长的土壤（肝硬化）和其它促癌因素的继续存在，再发新的肿瘤；二是单中心发生，即原先切除病灶术前、术中癌细胞经门静脉途径播散，发生肝内复发和肝外转移。目前对影响肝癌术后预后因素的研究，主要集中于对肿瘤大小、数目、分化程度、脉管侵犯、甲胎蛋白水平等临床病理因素的分析，而这些对肝癌肝切除后的复发尚不能进行较准确的预测。因此，针对肝癌复发这一科学难题，如何对切除术后肿瘤复发进行有效的预测，如何预防肝癌术后的肿瘤复发均对提高肝癌的疗效有重大的临床意义，探索和建立简单、快速、敏感性高和特异性强的分子预测指标和新的治疗靶点已成为提高临床疗效的迫切需要。The source of liver cancer recurrence is multi-center occurrence, that is, after radical resection of liver cancer, new tumors recur due to the continued existence of the soil where liver cancer grows (cirrhosis) and other cancer-promoting factors; the second is single-center occurrence, that is, original resection Cancer cells disseminated through the portal vein before and during operation, and intrahepatic recurrence and extrahepatic metastasis occurred. At present, the research on the factors affecting the prognosis of liver cancer after hepatectomy mainly focuses on the analysis of clinicopathological factors such as tumor size, number, degree of differentiation, vascular invasion, and alpha-fetoprotein level, but these factors cannot be analyzed for the recurrence of liver cancer after hepatectomy. more accurate predictions. Therefore, in view of the scientific problem of liver cancer recurrence, how to effectively predict tumor recurrence after resection and how to prevent tumor recurrence after liver cancer surgery are of great clinical significance to improve the curative effect of liver cancer. Exploring and establishing a simple, fast and sensitive Highly specific and specific molecular predictors and new therapeutic targets have become an urgent need to improve clinical efficacy.

因此，如果在手术切除的标本中加以分析，并且能够准确地预测患者复发的风险，那么就可以让高危复发人群加以重视，提高肝癌生存期。Therefore, if it is analyzed in surgically resected specimens and the risk of recurrence can be accurately predicted, then high-risk recurrence groups can be paid attention to and the survival period of liver cancer can be improved.

发明内容Contents of the invention

（一）解决的技术问题(1) Solved technical problems

针对现有技术的不足，本发明提供了用于预测肝癌是否复发的生物标志物，采用质谱分析的方法检测生物标志物的含量来判断肝癌复发的风险；该方法简单，实用，且通过多种小分子的检测可以更好地提高检测方法的灵敏度与特异性。Aiming at the deficiencies of the prior art, the present invention provides biomarkers for predicting the recurrence of liver cancer, and uses mass spectrometry to detect the content of the biomarkers to determine the risk of liver cancer recurrence; the method is simple, practical, and through a variety of The detection of small molecules can better improve the sensitivity and specificity of the detection method.

（二）技术方案(2) Technical solutions

为实现以上目的，本发明通过以下技术方案予以实现：To achieve the above object, the present invention is achieved through the following technical solutions:

用于预测肝癌复发的生物标志物，包括LAS1L蛋白，CLTB蛋白，JAGN1蛋白，ALYREF蛋白和HNRNPA3蛋白中的一种或几种蛋白分子。The biomarkers for predicting liver cancer recurrence include one or several protein molecules in LAS1L protein, CLTB protein, JAGN1 protein, ALYREF protein and HNRNPA3 protein.

用于预测肝癌复发诊断的生物标志物的预后方法，包括以下步骤：采用LC-MS/MS质谱分析法检测待测样本中LAS1L蛋白，CLTB蛋白，JAGN1蛋白，ALYREF蛋白或HNRNPA3蛋白的蛋白质表达量强度值。The prognostic method for the biomarkers used to predict the diagnosis of liver cancer recurrence, comprising the following steps: detecting the protein expression of LAS1L protein, CLTB protein, JAGN1 protein, ALYREF protein or HNRNPA3 protein in the sample to be tested by LC-MS/MS mass spectrometry strength value.

优选地，样本中LAS1L蛋白的蛋白质表达量强度值大于52085687.5时，被判为复发患者，否则被判为术后未复发患者，且假阳性率为22.9%。Preferably, when the protein expression intensity value of LAS1L protein in the sample is greater than 52085687.5, it is judged as a relapse patient, otherwise it is judged as a non-relapse patient after surgery, and the false positive rate is 22.9%.

优选地，样本中CLTB蛋白的蛋白质表达量强度值大于941197795时，被判为复发患者，否则被判为术后未复发患者，且假阳性率为22.9%。Preferably, when the protein expression intensity value of CLTB protein in the sample is greater than 941197795, it is judged as a relapse patient, otherwise it is judged as a non-relapse patient after surgery, and the false positive rate is 22.9%.

优选地，样本中JAGN1蛋白的蛋白质表达量强度值大于467852297时，被判为复发患者，否则被判为术后未复发患者，且假阳性率为8.6%。Preferably, when the protein expression intensity value of the JAGN1 protein in the sample is greater than 467852297, it is judged as a relapse patient, otherwise it is judged as a non-relapse patient after surgery, and the false positive rate is 8.6%.

优选地，样本中ALYREF蛋白的蛋白质表达量强度值小于149218152时，被判为复发患者，否则被判为术后未复发患者，且假阳性率为22.9%。Preferably, when the protein expression intensity value of the ALYREF protein in the sample is less than 149218152, it is judged as a relapse patient, otherwise it is judged as a non-relapse patient after surgery, and the false positive rate is 22.9%.

优选地，样本中HNRNPA3蛋白的蛋白质表达量强度值小于4386125605时，被判为复发患者，否则被判为术后未复发患者，且假阳性率为20%。Preferably, when the protein expression intensity value of the HNRNPA3 protein in the sample is less than 4386125605, it is judged as a relapse patient, otherwise it is judged as a non-relapse patient after surgery, and the false positive rate is 20%.

一种诊断肝癌是否复发的试剂盒，包括特异性检测LAS1L蛋白的试剂、特异性检测CLTB蛋白的试剂、特异性检测JAGN1蛋白的试剂、特异性检测ALYREF蛋白和特异性检测HNRNPA3蛋白的试剂中的一种或几种。A reagent kit for diagnosing recurrence of liver cancer, comprising reagents for specifically detecting LAS1L protein, reagents for specifically detecting CLTB protein, reagents for specifically detecting JAGN1 protein, reagents for specifically detecting ALYREF protein, and reagents for specifically detecting HNRNPA3 protein one or several.

优选地，特异性检测LAS1L蛋白的试剂是特异性识别LAS1L蛋白核酸的引物或探针；特异性检测CLTB蛋白的试剂是特异性识别CLTB蛋白核酸的引物或探针；特异性检测JAGN1蛋白的试剂是特异性识别JAGN1蛋白核酸的引物或探针；特异性检测ALYREF蛋白的试剂是特异性识别ALYREF蛋白核酸的引物或探针；特异性检测HNRNPA3蛋白的试剂是特异性识别HNRNPA3蛋白核酸的引物或探针；试剂可用于检测组织样本。Preferably, the reagent for specific detection of LAS1L protein is a primer or probe for specific recognition of LAS1L protein nucleic acid; the reagent for specific detection of CLTB protein is a primer or probe for specific recognition of CLTB protein nucleic acid; the reagent for specific detection of JAGN1 protein It is a primer or probe that specifically recognizes the nucleic acid of the JAGN1 protein; the reagent for specifically detecting the ALYREF protein is a primer or probe for specifically recognizing the nucleic acid of the ALYREF protein; the reagent for specifically detecting the HNRNPA3 protein is a primer or probe for specifically recognizing the nucleic acid of the HNRNPA3 protein Probe; Reagent can be used to detect a tissue sample.

（三）有益效果(3) Beneficial effects

与现有技术相比，本发明具有如下有益效果：Compared with the prior art, the present invention has the following beneficial effects:

（1）本发明采用LC-MS/MS质谱分析法检测待测样本，将大量临床样本进行质谱分析后，通过将肝癌术后未复发患者癌组织与肝癌术后复发患者癌组织相应分子含量的差异倍数（大于2或小于0.5）确定5个蛋白分子具有良好的检验效益。该5个蛋白分子（即LAS1L蛋白，CLTB蛋白，JAGN1蛋白，ALYREF蛋白和HNRNPA3蛋白）可以作为诊断肝癌复发的生物标志物。(1) The present invention uses LC-MS/MS mass spectrometry to detect the samples to be tested. After a large number of clinical samples are analyzed by mass spectrometry, the corresponding molecular content of cancer tissues of patients with non-recurrence after liver cancer surgery and cancer tissues of patients with recurrence of liver cancer after surgery The multiple of difference (greater than 2 or less than 0.5) determines that 5 protein molecules have good inspection power. The five protein molecules (ie, LAS1L protein, CLTB protein, JAGN1 protein, ALYREF protein and HNRNPA3 protein) can be used as biomarkers for the diagnosis of liver cancer recurrence.

（2）本发明以LAS1L蛋白，CLTB蛋白，JAGN1蛋白，ALYREF蛋白和HNRNPA3蛋白为生物标志物对被试者进行肝癌是否复发诊断，简单易行、诊断过程安全有效、易为病人所接受、诊断标准统一且受主观因素影响较小。(2) The present invention uses LAS1L protein, CLTB protein, JAGN1 protein, ALYREF protein and HNRNPA3 protein as biomarkers to diagnose liver cancer recurrence in subjects. The standard is uniform and less affected by subjective factors.

（3）本发明通过质谱分析检测出的生物标志物，该方法可以为日后的抗肝癌药物的研发提供新的治疗靶点和思路。(3) The biomarkers detected by mass spectrometry in the present invention can provide new therapeutic targets and ideas for the development of anti-liver cancer drugs in the future.

附图说明Description of drawings

图1为LAS1L蛋白的蛋白质表达量强度值的ROC曲线图；Fig. 1 is the ROC curve diagram of the protein expression intensity value of LAS1L protein;

图2为肝癌复发组织和术后未复发组织中LAS1L蛋白的蛋白质表达量强度值图；Figure 2 is a graph showing the protein expression intensity of LAS1L protein in recurrent liver cancer tissues and postoperative non-recurrence tissues;

图3为CLTB蛋白的蛋白质表达量强度值的ROC曲线图；Fig. 3 is the ROC curve diagram of the protein expression intensity value of CLTB protein;

图4为肝癌复发组织和术后未复发组织中CLTB蛋白的蛋白质表达量强度值图；Figure 4 is a graph showing the protein expression intensity of CLTB protein in liver cancer recurrence tissue and postoperative non-recurrence tissue;

图5为JAGN1蛋白的蛋白质表达量强度值的ROC曲线图；Fig. 5 is the ROC curve diagram of the protein expression intensity value of JAGN1 protein;

图6为肝癌复发组织和术后未复发组织中JAGN1蛋白的蛋白质表达量强度值图；Figure 6 is a graph showing the protein expression intensity of JAGN1 protein in recurrent liver cancer tissues and postoperative non-recurrence tissues;

图7为ALYREF蛋白的蛋白质表达量强度值的ROC曲线图；Fig. 7 is the ROC curve diagram of the protein expression intensity value of ALYREF protein;

图8为肝癌复发组织和术后未复发组织中ALYREF蛋白的蛋白质表达量强度值图；Fig. 8 is a graph showing the protein expression intensity value of ALYREF protein in recurrent liver cancer tissue and postoperative non-recurrence tissue;

图9为HNRNPA3蛋白的蛋白质表达量强度值的ROC曲线图；Fig. 9 is the ROC curve diagram of the protein expression intensity value of HNRNPA3 protein;

图10为肝癌复发组织和术后未复发组织中HNRNPA3蛋白的蛋白质表达量强度值图；Figure 10 is a graph showing the protein expression intensity of HNRNPA3 protein in recurrent liver cancer tissues and postoperative non-recurrence tissues;

图11为ALYREF蛋白和HNRNPA3蛋白组合的蛋白质表达量强度值的ROC曲线图；Fig. 11 is the ROC curve diagram of the protein expression intensity value of the combination of ALYREF protein and HNRNPA3 protein;

图12为肝癌复发组织和术后未复发组织中ALYREF蛋白和HNRNPA3蛋白组合的蛋白质表达量强度值图。Fig. 12 is a graph showing the protein expression intensity value of the combination of ALYREF protein and HNRNPA3 protein in recurrent liver cancer tissues and postoperative non-recurrence tissues.

具体实施方式Detailed ways

为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the present invention Examples, not all examples. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

与肝癌诊断相关的生物标志物的筛选Screening of biomarkers associated with the diagnosis of liver cancer

1、实验步骤1. Experimental steps

（1）蛋白质样品信息(1) Protein sample information

样本：分别取自肝癌复发患者癌组织的样本5例和取自术后未复发患者的肝癌组织35例。Samples: 5 samples of cancer tissues from patients with recurrent liver cancer and 35 samples of liver cancer tissues from patients without recurrence after surgery.

（2）样本预处理(2) Sample pretreatment

样品采用SDT（4%(w/v)十二烷基磺酸钠，100mM Tris/HCl pH7.6，0.1M二硫苏糖醇）裂解法提取蛋白质，然后采用BCA法进行蛋白质定量；每个样品取适量蛋白质采用Filter aided proteome preparation（FAS）法进行胰蛋白酶酶解，采用C18 Cartridge对肽段进行脱盐，肽段冻干后加入40μL 0.1%甲酸溶液复溶，肽段定量（OD280）。Samples were extracted by SDT (4% (w/v) sodium dodecylsulfonate, 100mM Tris/HCl pH7.6, 0.1M dithiothreitol) lysis method, and then protein quantification was carried out by BCA method; each An appropriate amount of protein was taken from the sample for trypsin enzymolysis by Filter aided proteome preparation (FAS) method, peptides were desalted with C18 Cartridge, and 40 μL of 0.1% formic acid solution was added to redissolve the peptides after freeze-drying, and the peptides were quantified (OD280).

BCA法进行蛋白质定量，是根据吸光值可以推算出蛋白浓度，在碱性条件下，蛋白将Cu²⁺还原为Cu⁺，Cu⁺与BCA试剂形成紫颜色的络合物，两分子BCA螯合一个Cu⁺。将该水溶性复合物在562nm处的吸收值，与标准曲线对比，即可计算待测蛋白的浓度。The BCA method is used for protein quantification, and the protein concentration can be calculated according to the absorbance value. Under alkaline conditions, the protein reduces Cu²⁺ to Cu⁺ , and Cu⁺ forms a purple-colored complex with BCA reagent, and two molecules of BCA are chelated. a Cu⁺ . The concentration of the protein to be tested can be calculated by comparing the absorption value of the water-soluble complex at 562 nm with the standard curve.

（3）LC-MS/MS数据采集(3) LC-MS/MS data acquisition

每份样品采用纳升流速的HPLC液相系统Easy nLC进行分离。Each sample was separated using an HPLC liquid phase system Easy nLC with nanoliter flow rate.

其中：缓冲液A液为0.1%甲酸水溶液，B液为0.1%甲酸乙腈水溶液（乙腈为84%）。Among them: buffer solution A is 0.1% formic acid aqueous solution, and B solution is 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 84%).

色谱柱以95%的A液平衡，样品由自动进样器上样到上样柱（Thermo ScientificAcclaim PepMap100, 100μm*2cm, nanoViper C18），经过分析柱（Thermo scientificEASY column, 10cm, ID75μm, 3μm, C18-A2）分离，流速为300nL/min。The chromatographic column was equilibrated with 95% liquid A, and the sample was loaded from the autosampler to the loading column (Thermo ScientificAcclaim PepMap100, 100μm*2cm, nanoViper C18), and passed through the analytical column (Thermo scientificEASY column, 10cm, ID75μm, 3μm, C18 -A2) Separation with a flow rate of 300nL/min.

样品经色谱分离后用Q-Exactive质谱仪进行质谱分析。检测方式为正离子，母离子扫描范围300–1800m/z，一级质谱分辨率为70,000 at 200m/z，AGC（Automatic gaincontrol）target为1e6，Maximum IT为50ms，动态排除时间（Dynamic exclusion）为60.0s。多肽和多肽碎片的质量电荷比按照下列方法采集：每次全扫描（full scan）后采集20个碎片图谱（MS2 scan），MS2 Activation Type为HCD，Isolation window为2m/z，二级质谱分辨率17, 500 at 200m/z，Normalized Collision Energy为30eV，Underfill为0.1%。The samples were separated by chromatography and then analyzed by Q-Exactive mass spectrometer. The detection method is positive ions, the precursor ion scanning range is 300–1800m/z, the primary mass spectrometry resolution is 70,000 at 200m/z, the AGC (Automatic gaincontrol) target is 1e6, the Maximum IT is 50ms, and the dynamic exclusion time (Dynamic exclusion) is 60.0s. The mass-to-charge ratio of peptides and peptide fragments is collected according to the following method: 20 fragment spectra (MS2 scan) are collected after each full scan (full scan), MS2 Activation Type is HCD, Isolation window is 2m/z, and the resolution of MS2 17, 500 at 200m/z, Normalized Collision Energy is 30eV, Underfill is 0.1%.

（4）蛋白质鉴定和定量分析(4) Protein identification and quantitative analysis

质谱分析原始数据为RAW文件，用软件MaxQuant软件（版本号1.5.3.17）进行查库鉴定及定量分析。The raw data of mass spectrometry analysis is a RAW file, and the software MaxQuant software (version number 1.5.3.17) is used for library search identification and quantitative analysis.

iBAQ Intensity是基于iBAQ算法得到的样品X中的蛋白质表达量，近似等于该样品中的蛋白质绝对浓度。LFQ Intensity是基于LFQ算法得到的样品X的蛋白相对表达量，常应用于组间比较。一般Labelfree选择其中之一作为定量结果。iBAQ Intensity is the protein expression in sample X obtained based on the iBAQ algorithm, which is approximately equal to the absolute protein concentration in the sample. LFQ Intensity is the relative protein expression of sample X obtained based on the LFQ algorithm, which is often used for comparison between groups. Generally, Labelfree chooses one of them as the quantitative result.

iBAQ（Intensity-based absolute quantification）和LFQ属于Maxquant软件提供的两种不同的蛋白质定量算法。iBAQ (Intensity-based absolute quantification) and LFQ belong to two different protein quantification algorithms provided by Maxquant software.

iBAQ一般用于样本的蛋白质绝对定量，主要算法是基于该蛋白质鉴定到的肽段的强度之和与理论肽段个数的比值。iBAQ is generally used for the absolute quantification of proteins in samples, and the main algorithm is based on the ratio of the sum of the intensities of peptides identified by the protein to the number of theoretical peptides.

LFQ一般用于多组间的两两定量比较，主要算法是经过肽段和蛋白质多层的pair-wise矫正。本专利采用LFQ进行蛋白质定量。LFQ is generally used for pairwise quantitative comparisons between multiple groups, and the main algorithm is pair-wise correction of multiple layers of peptides and proteins. This patent uses LFQ for protein quantification.

（5）统计学分析(5) Statistical analysis

对符合同组三次重复数据中至少有两个非空值的数据进行比值计算和统计学分析，包含各比较组的LFQ或者iBAQ强度值比值和P-value；初步筛选出各组间的差异物。Perform ratio calculation and statistical analysis on the data with at least two non-null values in the three repeated data of the same group, including the LFQ or iBAQ intensity value ratio and P-value of each comparison group; preliminarily screen out the differences between the groups .

进一步根据P-value验证差异蛋白物是否具有显著性。选择同时具有多维统计分析Fold change>2或<0.5认为癌组织和癌旁组织之间该蛋白物的含量存在明显的倍数差异，并且筛选出单变量统计分析P value<0.05的蛋白物，作为具有显著性差异的蛋白质；从而得到差异蛋白分子。然后利用SPSS软件作出差异蛋白物ROC曲线，并计算其曲线下面积（AUC），进而判断其诊断价值。具体判断方法为AUC线下面积大于0.7，且P<0.05，利用约登指数最大时候的阈值标准（cut off值）作为判断肿瘤与否的阈值标准（倍数大于2者认为大于阈值为肿瘤检测阳性，倍数小于0.5者认定小于阈值者为肝癌检测阳性），从而得到较高的敏感度和特异度。Further verify whether the difference protein is significant according to P-value. Select the protein with multidimensional statistical analysis Fold change>2 or <0.5 to consider that there is a significant fold difference in the content of the protein between cancer tissue and paracancerous tissue, and screen out the protein with univariate statistical analysis P value<0.05 as the protein with Significantly different proteins; resulting in differential protein molecules. Then use SPSS software to draw ROC curve of differential protein, and calculate its area under the curve (AUC), and then judge its diagnostic value. The specific judgment method is that the area under the AUC line is greater than 0.7, and P<0.05, and the threshold standard (cut off value) when the Youden index is the largest is used as the threshold standard for judging whether the tumor is or not (multiples greater than 2 are considered positive for tumor detection) , and those whose multiple is less than 0.5 are considered to be positive for liver cancer if the multiple is less than the threshold), thus obtaining higher sensitivity and specificity.

（6）生物信息学分析(6) Bioinformatics analysis

① GO功能注释① GO function notes

利用Blast2GO对目标蛋白质集合进行GO注释，过程大致可以归纳为序列比对（Blast）、GO条目提取（Mapping）、GO注释（Annotation）和InterProScan补充注释（Annotation Augmentation）等四个步骤。Using Blast2GO to perform GO annotation on the target protein collection, the process can be roughly summarized into four steps: sequence alignment (Blast), GO entry extraction (Mapping), GO annotation (Annotation) and InterProScan supplementary annotation (Annotation Augmentation).

② KEGG通路注释② KEGG pathway annotation

利用KAAS（KEGG Automatic Annotation Server）软件，对目标蛋白质集合进行KEGG通路注释。Use KAAS (KEGG Automatic Annotation Server) software to perform KEGG pathway annotation on the target protein collection.

③ GO注释和KEGG注释的富集分析③ Enrichment analysis of GO annotation and KEGG annotation

采用Fisher精确检验（Fisher’s Exact Test），比较各个GO分类或KEGG通路在目标蛋白质集合和总体蛋白质集合中的分布情况，对目标蛋白质集合进行GO注释或KEGG通路注释的富集分析。Fisher's Exact Test was used to compare the distribution of each GO classification or KEGG pathway in the target protein set and the overall protein set, and enrichment analysis of GO annotation or KEGG pathway annotation was performed on the target protein set.

④ 蛋白质聚类分析④ Protein cluster analysis

首先对目标蛋白质集合的定量信息进行归一化处理（归一化到（-1,1）区间）。然后，使用Complexheatmap R包（R Version 3.4）同时对样品和蛋白质的表达量两个维度进行分类（距离算法：欧几里得，连接方式：Average linkage），并生成层次聚类热图。Firstly, the quantitative information of the target protein set is normalized (normalized to (-1,1) interval). Then, the Complexheatmap R package (R Version 3.4) was used to classify the two dimensions of sample and protein expression at the same time (distance algorithm: Euclidean, connection method: Average linkage), and a hierarchical clustering heat map was generated.

⑤ 蛋白质相互作用网络分析⑤ Protein interaction network analysis

基于STRING（http://string-db.org/）数据库中的信息查找目标蛋白质之间的相互作用关系，并使用CytoScape软件（版本号：3.2.1）生成相互作用网络并对网络进行分析。Based on the information in the STRING (http://string-db.org/) database, the interaction relationship between target proteins was found, and the interaction network was generated and analyzed using CytoScape software (version number: 3.2.1).

（7）差异表达蛋白质筛选(7) Differentially expressed protein screening

以倍数变化大于2.0倍（上调大于2倍或者下调小于0.5）且P value小于0.05的标准筛选差异表达蛋白质，各比较组的差异表达蛋白质数目。Differentially expressed proteins were screened by the criteria of fold change greater than 2.0 times (up-regulation greater than 2 times or down-regulation less than 0.5) and P value less than 0.05, and the number of differentially expressed proteins in each comparison group.

（8）实验基本原理(8) Basic principles of the experiment

非标记定量蛋白质组学（Label-free）技术近年来已成为重要的质谱定量方法。Label-free技术的定量原理主要有两种：首先，spectrum counts类的非标记定量方法发展较早，也形成了多种定量算法，但核心原理都是根据MS2的鉴定结果作为定量的基础，各种方法的差别在于后期算法对高通量数据的修正；第二种非标记定量方法的原理是以MS1为基础，计算每个肽段信号在LCMS色谱上的积分。本发明采用的Maxquant算法即基于第二种原理。Label-free quantitative proteomics (Label-free) technology has become an important mass spectrometry quantitative method in recent years. There are two main quantification principles of Label-free technology: First, the non-label quantification method of spectrum counts developed earlier, and a variety of quantification algorithms have also been formed, but the core principle is based on the identification results of MS2 as the basis of quantification, each The difference between the two methods lies in the correction of the high-throughput data by the later algorithm; the principle of the second non-labeled quantitative method is based on MS1, and the integral of each peptide signal on the LCMS chromatogram is calculated. The Maxquant algorithm adopted in the present invention is based on the second principle.

2、实验结果2. Experimental results

经质谱数据分析和将复发患者肝癌组织与未复发患者肝癌组织（术后未复发）的蛋白分子进行比较，从而得到5个蛋白分子，可以作为与肝癌复发的生物标志物。After analyzing the mass spectrometry data and comparing the protein molecules of liver cancer tissues of recurrent patients with those of non-recurring patients (no recurrence after surgery), 5 protein molecules were obtained, which can be used as biomarkers for liver cancer recurrence.

为了评估蛋白分子的蛋白质表达量强度值对肝癌复发的诊断效能，本发明采用了ROC曲线分析，AUC为ROC曲线下的面积，是最常用的评价ROC曲线特征的参数，也是重要的试验准确度指标。若AUC在0.7以下，则表示诊断的准确率较低；AUC在0.7以上，则可以满足临床诊断的要求。In order to evaluate the diagnostic efficiency of the protein expression intensity value of the protein molecule on the recurrence of liver cancer, the present invention adopts ROC curve analysis, and AUC is the area under the ROC curve, which is the most commonly used parameter for evaluating the characteristics of the ROC curve, and is also an important test accuracy. index. If the AUC is below 0.7, it means that the diagnostic accuracy is low; if the AUC is above 0.7, it can meet the requirements of clinical diagnosis.

具体结果与分析如下：The specific results and analysis are as follows:

（1）采用LC-MS/MS质谱分析法，检测到LAS1L蛋白在肝癌复发组织与术后未复发组织中存在差异。(1) Using LC-MS/MS mass spectrometry, it was detected that there were differences in LAS1L protein between recurrent liver cancer tissues and non-recurrence tissues after surgery.

研究发现，LAS1L蛋白在肝癌复发样本显著性上调了11.68倍，p值<0.05。The study found that LAS1L protein was significantly up-regulated by 11.68 times in recurrent liver cancer samples, with a p value of <0.05.

由图1可知，LAS1L蛋白的AUC为0.806>0.7，说明LAS1L蛋白具有较好的判断效果，可以作为肝癌是否复发的生物标志物。It can be seen from Figure 1 that the AUC of LAS1L protein is 0.806>0.7, indicating that LAS1L protein has a good judgment effect and can be used as a biomarker for liver cancer recurrence.

在LAS1L蛋白的cut off值为52085687.5时，灵敏度为80%，特异度为77.1%。当进行个体检测时，LAS1L蛋白的蛋白质表达量强度值大于52085687.5时，被判为复发患者，否则被判为未复发患者（假阳性率为22.9%）。When the cut off value of LAS1L protein was 52085687.5, the sensitivity was 80%, and the specificity was 77.1%. When performing individual detection, if the protein expression intensity value of LAS1L protein was greater than 52085687.5, it was judged as a relapsed patient, otherwise it was judged as a non-relapsed patient (false positive rate was 22.9%).

由图2可知，肝癌复发组织样本主要分布在检测阈值（图2中实线）以上，术后未复发组织主要分布在检测阈值以下，说明肝癌复发组织和术后未复发组织的蛋白质表达量强度值相差甚大，该检测阈值检测效果良好。It can be seen from Figure 2 that the recurrent liver cancer tissue samples are mainly distributed above the detection threshold (solid line in Figure 2), and the postoperative non-recurrence tissues are mainly distributed below the detection threshold, indicating the protein expression intensity of liver cancer recurrence tissues and postoperative non-recurrence tissues The values are very different, and the detection threshold has a good detection effect.

综上所述，LAS1L蛋白可以作肝癌复发的生物标志物。In summary, LAS1L protein can be used as a biomarker for liver cancer recurrence.

（2）采用LC-MS/MS质谱分析法，检测到CLTB蛋白在肝癌复发组织与术后未复发组织中存在差异。(2) Using LC-MS/MS mass spectrometry, it was detected that there were differences in CLTB protein between recurrent liver cancer tissues and non-recurrence tissues after surgery.

研究发现，CLTB蛋白在肝癌复发样本显著性上调了6.68倍，p值<0.05。The study found that CLTB protein was significantly up-regulated by 6.68 times in the recurrent liver cancer samples, with a p value of <0.05.

由图3可知，CLTB蛋白质的AUC为0.806>0.7，说明CLTB蛋白具有较好的判断效果，可以作为肝癌是否复发的生物标志物。It can be seen from Figure 3 that the AUC of CLTB protein is 0.806>0.7, indicating that CLTB protein has a good judgment effect and can be used as a biomarker for liver cancer recurrence.

在CLTB蛋白的蛋白质表达量强度值为941197795时，灵敏度为80%，特异度为77.1%。当进行个体检测时，CLTB蛋白的蛋白质表达量强度值大于941197795时，被判为复发患者，否则被判为未复发患者（假阳性率为22.9%）。When the protein expression intensity value of CLTB protein was 941197795, the sensitivity was 80%, and the specificity was 77.1%. When performing individual detection, if the protein expression intensity value of CLTB protein was greater than 941197795, it was judged as a relapse patient, otherwise it was judged as a non-relapse patient (false positive rate was 22.9%).

由图4可知，肝癌复发组织样本主要分布在检测阈值（图4中实线）以上，术后未复发组织主要分布在检测阈值以下，说明肝癌复发组织和术后未复发组织的蛋白质表达量强度值相差甚大，该检测阈值检测效果良好。It can be seen from Figure 4 that the recurrent liver cancer tissue samples are mainly distributed above the detection threshold (solid line in Figure 4), and the postoperative non-recurrence tissues are mainly distributed below the detection threshold, indicating the protein expression intensity of liver cancer recurrence tissues and postoperative non-recurrence tissues The values are very different, and the detection threshold has a good detection effect.

综上所述，CLTB蛋白可以作为肝癌复发的生物标志物。In summary, CLTB protein can be used as a biomarker for HCC recurrence.

（3）采用LC-MS/MS质谱分析法，检测到JAGN1蛋白在肝癌复发组织与术后未复发组织中存在差异。(3) Using LC-MS/MS mass spectrometry, it was detected that there were differences in the JAGN1 protein between recurrent liver cancer tissues and non-recurrence tissues after surgery.

研究发现，JAGN1蛋白在肝癌复发样本显著性上调了2.45倍，p值＜0.05。The study found that the JAGN1 protein was significantly up-regulated by 2.45 times in the recurrent liver cancer samples, with a p value < 0.05.

由图5可知，JAGN1蛋白的AUC为0.840>0.7，说明JAGN1蛋白具有较好的判断效果，可以作肝癌是否复发的生物标志物。It can be seen from Figure 5 that the AUC of JAGN1 protein is 0.840>0.7, indicating that JAGN1 protein has a good judgment effect and can be used as a biomarker for liver cancer recurrence.

在JAGN1蛋白的蛋白质表达量强度值为467852297时，灵敏度为80%，特异度为91.4%。JAGN1蛋白的当进行个体检测时，蛋白质表达量强度值大于467852297时，被判为复发患者，否则被判为未复发患者（假阳性率为8.6%）。When the protein expression intensity value of JAGN1 protein was 467852297, the sensitivity was 80% and the specificity was 91.4%. When the individual detection of JAGN1 protein is performed, if the protein expression intensity value is greater than 467852297, it is judged as a relapse patient, otherwise it is judged as a non-relapse patient (the false positive rate is 8.6%).

由图6可知，肝癌复发组织样本主要分布在检测阈值（图6中实线）以上，术后未复发组织主要分布在检测阈值以下，说明肝癌复发组织和术后未复发组织的蛋白质表达量强度值相差甚大，该检测阈值检测效果良好。It can be seen from Figure 6 that the recurrent liver cancer tissue samples are mainly distributed above the detection threshold (solid line in Figure 6), and the postoperative non-recurrence tissues are mainly distributed below the detection threshold, indicating the protein expression intensity of liver cancer recurrence tissues and postoperative non-recurrence tissues The values are very different, and the detection threshold has a good detection effect.

综上所述，JAGN1蛋白可以作为肝癌复发的生物标志物。In summary, JAGN1 protein can be used as a biomarker for HCC recurrence.

（4）采用LC-MS/MS质谱分析法，检测到ALYREF蛋白在复发组织与未复发组织中存在差异。(4) Using LC-MS/MS mass spectrometry, it was detected that there were differences in ALYREF protein between relapsed and non-relapsed tissues.

研究发现，ALYREF蛋白在肝癌复发样本显著性下调了0.19倍，p值<0.05。The study found that the ALYREF protein was significantly down-regulated by 0.19 times in the recurrent liver cancer samples, with a p value of <0.05.

由图7可知，ALYREF蛋白的AUC为0.937>0.7，说明ALYREF蛋白具有较好的判断效果，可以作为肝癌是否复发的生物标志物。It can be seen from Figure 7 that the AUC of the ALYREF protein is 0.937>0.7, indicating that the ALYREF protein has a good judgment effect and can be used as a biomarker for liver cancer recurrence.

在ALYREF蛋白的cut off值为149218152时，灵敏度为100%，特异度为77.1%。当进行个体检测时，ALYREF蛋白的蛋白质表达量强度值小于149218152时，被判为复发患者，否则被判为术后未复发患者（假阳性率为22.9%）。When the cut off value of ALYREF protein was 149218152, the sensitivity was 100%, and the specificity was 77.1%. When performing individual detection, if the protein expression intensity value of ALYREF protein is less than 149218152, it is judged as a relapsed patient, otherwise it is judged as a postoperative non-relapsed patient (false positive rate 22.9%).

由图8可知，肝癌复发组织样本主要分布在检测阈值（图8中实线）以下，未复发组织主要分布在检测阈值以上，说明肝癌复发组织和未复发组织的蛋白质表达量强度值相差甚大，该检测阈值检测效果良好。It can be seen from Figure 8 that the recurrent liver cancer tissue samples are mainly distributed below the detection threshold (solid line in Figure 8), and the non-recurrent tissues are mainly distributed above the detection threshold, indicating that the protein expression intensity values of liver cancer recurrent tissues and non-recurrent tissues are very different. This detection threshold detection works well.

综上所述，ALYREF蛋白可以作为肝癌复发的生物标志物。In summary, ALYREF protein can be used as a biomarker for HCC recurrence.

（5）采用LC-MS/MS质谱分析法，检测到HNRNPA3蛋白在复发组织与未复发组织中存在差异。(5) Using LC-MS/MS mass spectrometry, it was detected that there were differences in the HNRNPA3 protein between relapsed and non-relapsed tissues.

研究发现，HNRNPA3蛋白在肝癌复发样本显著性下调了0.49倍，p值<0.05。The study found that the HNRNPA3 protein was significantly down-regulated by 0.49 times in the recurrent liver cancer samples, with a p value of <0.05.

由图9可知，HNRNPA3蛋白的AUC为0.920>0.7，说明HNRNPA3蛋白具有较好的判断效果，可以作为肝癌复发是否的生物标志物。It can be seen from FIG. 9 that the AUC of HNRNPA3 protein is 0.920>0.7, indicating that HNRNPA3 protein has a good judgment effect and can be used as a biomarker for liver cancer recurrence.

在HNRNPA3蛋白的蛋白质表达量强度值为4386125605时，灵敏度为100%，特异度为80%。当进行个体检测时，HNRNPA3蛋白的蛋白质表达量强度值小于4386125605时，被判为复发患者，否则被判为术后未复发患者（假阳性率为20%）。When the protein expression intensity value of HNRNPA3 protein was 4386125605, the sensitivity was 100%, and the specificity was 80%. When performing individual detection, when the protein expression intensity value of HNRNPA3 protein is less than 4386125605, it is judged as a relapsed patient, otherwise it is judged as a postoperative non-relapsed patient (false positive rate is 20%).

由图10可知，肝癌复发组织样本主要分布在检测阈值（图10中实线）以下，未复发组织主要分布在检测阈值以上，说明肝癌复发组织和未复发组织的蛋白质表达量强度值相差甚大，该检测阈值检测效果良好。It can be seen from Figure 10 that the recurrent liver cancer tissue samples are mainly distributed below the detection threshold (solid line in Figure 10), and the non-recurrent tissues are mainly distributed above the detection threshold, indicating that the protein expression intensity values of liver cancer recurrent tissues and non-recurrent tissues are very different. This detection threshold detection works well.

综上所述，HNRNPA3蛋白可以作为肝癌复发的生物标志物。In summary, HNRNPA3 protein can be used as a biomarker for HCC recurrence.

（6）ALYREF蛋白和HNRNPA3蛋白组合在制备早期肝癌诊断试剂盒中的应用。(6) Application of the combination of ALYREF protein and HNRNPA3 protein in the preparation of a diagnostic kit for early liver cancer.

本发明还提供一种肝癌的诊断方法，步骤为：采用二元逻辑回归分析计算P（肝癌术后复发概率）的数值，SPSS软件二元逻辑回归后得到的公式为：The present invention also provides a method for diagnosing liver cancer, the steps of which are: using binary logistic regression analysis to calculate the value of P (recurrence probability after liver cancer surgery), and the formula obtained after binary logistic regression by SPSS software is:

其中a为ALYREF蛋白的蛋白质表达量强度值，b为HNRNPA3蛋白的蛋白质表达量强度值；若检测P（肝癌术后复发概率）大于0.0694114则被判为会复发患者，否则被判为未复发患者。Where a is the protein expression intensity value of ALYREF protein, and b is the protein expression intensity value of HNRNPA3 protein; if the detection P (recurrence probability after liver cancer surgery) is greater than 0.0694114, it is judged as a patient who will relapse, otherwise it is judged as a patient without recurrence .

如图11所示，组合蛋白的AUC为0.977>0.7，说明具有较好的判断效果，即组合蛋白可以作为肝癌是否复发的生物标志物。As shown in Figure 11, the AUC of the combined protein is 0.977>0.7, which shows that it has a good judgment effect, that is, the combined protein can be used as a biomarker for liver cancer recurrence.

在组合蛋白的蛋白质表达量强度值为0.0694114时，灵敏度为100%，特异度为88.6%。当进行个体检测时，组合蛋白的蛋白质表达量强度值大于0.0694114时，被判为复发患者，否则被判为术后未复发患者（假阳性率为11.4%）。When the protein expression intensity value of the combined protein was 0.0694114, the sensitivity was 100%, and the specificity was 88.6%. When performing individual detection, when the protein expression intensity value of the combination protein is greater than 0.0694114, it is judged as a relapsed patient, otherwise it is judged as a postoperative non-relapsed patient (false positive rate 11.4%).

由图12可知，肝癌复发组织样本主要分布在检测阈值（图12中实线）以上，未复发组织主要分布在检测阈值以上，说明肝癌复发组织和未复发组织的蛋白质表达量强度值相差甚大，该检测阈值检测效果良好。It can be seen from Figure 12 that the recurrent liver cancer tissue samples are mainly distributed above the detection threshold (solid line in Figure 12), and the non-recurrent tissues are mainly distributed above the detection threshold, indicating that the protein expression intensity values of liver cancer recurrent tissues and non-recurrent tissues are very different. This detection threshold detection works well.

鉴于上述结果，ALYREF蛋白和HNRNPA3蛋白组成的组合蛋白可以作为肝癌复发的生物标志物。In view of the above results, the combined protein composed of ALYREF protein and HNRNPA3 protein can be used as a biomarker for liver cancer recurrence.

实施例1Example 1

用于预测肝癌复发的生物标志物，包括LAS1L蛋白。Biomarkers for predicting liver cancer recurrence, including LAS1L protein.

用于预测肝癌复发肝癌诊断的生物标志物的预后方法，包括以下步骤：采用LC-MS/MS质谱分析法检测待测样本中LAS1L蛋白的蛋白质表达量强度值；样本中LAS1L蛋白的蛋白质表达量强度值大于52085687.5时，被判为复发患者，否则被判为未复发患者（假阳性率为22.9%）。The prognostic method for the biomarkers used to predict the diagnosis of liver cancer recurrence, comprising the following steps: using LC-MS/MS mass spectrometry to detect the intensity value of the protein expression level of the LAS1L protein in the sample to be tested; the protein expression level of the LAS1L protein in the sample When the intensity value is greater than 52085687.5, it is judged as a relapse patient, otherwise it is judged as a non-relapse patient (the false positive rate is 22.9%).

一种诊断肝癌是否复发的试剂盒，包括特异性检测LAS1L蛋白的试剂，特异性检测LAS1L蛋白的试剂是特异性识别LAS1L蛋白核酸的探针。A reagent kit for diagnosing recurrence of liver cancer, including a reagent for specifically detecting LAS1L protein, the reagent for specifically detecting LAS1L protein is a probe for specifically recognizing the nucleic acid of the LAS1L protein.

实施例2Example 2

用于预测肝癌复发的生物标志物，包括CLTB蛋白。Biomarkers for predicting liver cancer recurrence, including CLTB protein.

用于预测肝癌复发肝癌诊断的生物标志物的预后方法，包括以下步骤：采用LC-MS/MS质谱分析法检测待测样本中CLTB蛋白的蛋白质表达量强度值；样本中CLTB蛋白的蛋白质表达量强度值大于941197795时，被判为复发患者，否则被判为未复发患者（假阳性率为22.9%）。The prognostic method of the biomarker used for predicting the diagnosis of liver cancer recurrent liver cancer comprises the following steps: using LC-MS/MS mass spectrometry to detect the protein expression level value of CLTB protein in the sample to be tested; the protein expression level of CLTB protein in the sample When the intensity value is greater than 941197795, it is judged as a relapse patient, otherwise it is judged as a non-relapse patient (the false positive rate is 22.9%).

一种诊断肝癌是否复发的试剂盒，包括特异性检测CLTB蛋白的试剂，特异性检测CLTB蛋白的试剂是特异性识别CLTB蛋白核酸的探针。A reagent kit for diagnosing whether liver cancer recurs, including a reagent for specifically detecting CLTB protein, the reagent for specifically detecting CLTB protein is a probe for specifically recognizing nucleic acid of the CLTB protein.

实施例3Example 3

用于预测肝癌复发的生物标志物，包括JAGN1蛋白。Biomarkers for predicting liver cancer recurrence, including JAGN1 protein.

用于预测肝癌复发肝癌诊断的生物标志物的预后方法，包括以下步骤：采用LC-MS/MS质谱分析法检测待测样本中JAGN1蛋白的蛋白质表达量强度值；样本中JAGN1蛋白的蛋白质表达量强度值大于467852297时，被判为复发患者，否则被判为未复发患者（假阳性率为8.6%）。The prognostic method of the biomarker used for predicting the diagnosis of liver cancer recurrent liver cancer comprises the following steps: using LC-MS/MS mass spectrometry to detect the intensity value of the protein expression level of the JAGN1 protein in the sample to be tested; the protein expression level of the JAGN1 protein in the sample When the intensity value is greater than 467852297, it is judged as a relapse patient, otherwise it is judged as a non-relapse patient (the false positive rate is 8.6%).

一种诊断肝癌是否复发的试剂盒，包括特异性检测JAGN1蛋白的试剂，特异性检测JAGN1蛋白的试剂是特异性识别JAGN1蛋白核酸的探针。A kit for diagnosing whether liver cancer recurs, comprising a reagent for specifically detecting JAGN1 protein, and the reagent for specifically detecting JAGN1 protein is a probe for specifically recognizing JAGN1 protein nucleic acid.

实施例4Example 4

用于预测肝癌复发的生物标志物，包括ALYREF蛋白。Biomarkers for predicting liver cancer recurrence, including ALYREF protein.

用于预测肝癌复发肝癌诊断的生物标志物的预后方法，包括以下步骤：采用LC-MS/MS质谱分析法检测待测样本中ALYREF蛋白的蛋白质表达量强度值；样本中ALYREF蛋白的蛋白质表达量强度值小于149218152时，被判为复发患者，否则被判为术后未复发患者（假阳性率为22.9%）。The prognostic method of the biomarker used for predicting the diagnosis of liver cancer recurrent liver cancer comprises the following steps: using LC-MS/MS mass spectrometry to detect the protein expression level value of the ALYREF protein in the sample to be tested; the protein expression level of the ALYREF protein in the sample When the intensity value is less than 149218152, it is judged as a recurrence patient, otherwise it is judged as a postoperative non-recurrence patient (the false positive rate is 22.9%).

一种诊断肝癌是否复发的试剂盒，包括特异性检测ALYREF蛋白的试剂，特异性检测ALYREF蛋白的试剂是特异性识别ALYREF蛋白核酸的探针。A reagent kit for diagnosing recurrence of liver cancer comprises a reagent for specifically detecting the ALYREF protein, and the reagent for specifically detecting the ALYREF protein is a probe for specifically recognizing the nucleic acid of the ALYREF protein.

实施例5Example 5

用于预测肝癌复发的生物标志物，包括HNRNPA3蛋白。Biomarkers for predicting liver cancer recurrence, including HNRNPA3 protein.

用于预测肝癌复发肝癌诊断的生物标志物的预后方法，包括以下步骤：采用LC-MS/MS质谱分析法检测待测样本中HNRNPA3蛋白的蛋白质表达量强度值；样本中HNRNPA3蛋白的蛋白质表达量强度值小于4386125605时，被判为复发患者，否则被判为术后未复发患者（假阳性率为20%）。The prognostic method for the biomarkers used to predict the diagnosis of liver cancer recurrence, comprising the following steps: using LC-MS/MS mass spectrometry to detect the protein expression intensity value of the HNRNPA3 protein in the sample to be tested; the protein expression level of the HNRNPA3 protein in the sample When the intensity value is less than 4386125605, it is judged as a recurrence patient, otherwise it is judged as a postoperative non-relapse patient (false positive rate is 20%).

一种诊断肝癌是否复发的试剂盒，包括特异性检测HNRNPA3蛋白的试剂，特异性检测HNRNPA3蛋白的试剂是特异性识别HNRNPA3蛋白核酸的探针。A reagent kit for diagnosing whether liver cancer recurs, including a reagent for specifically detecting HNRNPA3 protein, and the reagent for specifically detecting HNRNPA3 protein is a probe for specifically recognizing HNRNPA3 protein nucleic acid.

实施例6Example 6

肝癌诊断的生物标志物，包括ALYREF蛋白和HNRNPA3蛋白。Biomarkers for liver cancer diagnosis, including ALYREF protein and HNRNPA3 protein.

肝癌诊断的生物标志物的预后方法，包括以下步骤：采用二元逻辑回归分析计算P（肝癌术后复发概率）的数值，SPSS软件二元逻辑回归后得到的公式为：The prognostic method of the biomarkers for the diagnosis of liver cancer comprises the following steps: using binary logistic regression analysis to calculate the value of P (probability of recurrence after liver cancer operation), the formula obtained after binary logistic regression by SPSS software is:

其中：a为ALYREF蛋白的蛋白质表达量强度值，b为HNRNPA3蛋白的蛋白质表达量强度值；若检测P（肝癌术后复发概率）大于0.0694114则判为会复发患者，否则判为未复发患者。Among them: a is the protein expression intensity value of ALYREF protein, b is the protein expression intensity value of HNRNPA3 protein; if the detection P (recurrence probability after liver cancer surgery) is greater than 0.0694114, it is judged as a patient who will relapse, otherwise it is judged as a patient without recurrence.

当进行个体检测时，组合蛋白的蛋白质表达量强度值大于0.0694114时，被判为复发患者，否则被判为术后未复发患者（假阳性率为11.4%）。When performing individual detection, when the protein expression intensity value of the combination protein is greater than 0.0694114, it is judged as a relapsed patient, otherwise it is judged as a postoperative non-relapsed patient (false positive rate 11.4%).

本发明以LAS1L蛋白，CLTB蛋白，JAGN1蛋白，ALYREF蛋白和HNRNPA3蛋白中至少一种蛋白作为生物标志物对被试者进行肝癌诊断，简单易行、诊断过程安全有效、易为病人所接受、诊断标准统一受个人主观因素影响较小。In the present invention, at least one protein among LAS1L protein, CLTB protein, JAGN1 protein, ALYREF protein and HNRNPA3 protein is used as a biomarker to diagnose liver cancer in subjects, which is simple and easy to implement, safe and effective in the diagnosis process, and easy to be accepted by patients. Standard unification is less affected by personal subjective factors.

以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: it can still be described in the foregoing embodiments Modifications are made to the recorded technical solutions, or equivalent replacements are made to some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (1)

1. The application of a reagent for detecting one or more of LAS1L protein, CLTB protein, JAGN1 protein and HNRNPA3 protein in preparing a kit for predicting liver cancer recurrence diagnosis.

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