技术领域technical field
本发明属于生物技术领域，具体涉及miR-197在作为肝癌检测标志物中的应用。The invention belongs to the field of biotechnology, and in particular relates to the application of miR-197 as a liver cancer detection marker.
背景技术Background technique
肝细胞性肝癌(HepatocellularCarcinoma,HCC，以下简称肝癌)是世界范围内最常见的恶性肿瘤之一，其恶性程度高、侵袭力强、预后差，死亡率位居世界肿瘤死亡第二位。中国是肝癌高发区，据统计每年发患者数占全球约55％。肝癌发病比较隐匿，早期缺乏特异的临床表现，发现时往往已经属于中晚期。提高肝癌患者长期生存率的原则是早发现、早诊断、早治疗，其中的关键是早期诊断。Hepatocellular carcinoma (Hepatocellular Carcinoma, HCC, hereinafter referred to as liver cancer) is one of the most common malignant tumors in the world. China is a high-incidence area of liver cancer, and according to statistics, the number of patients with liver cancer accounts for about 55% of the world every year. The onset of liver cancer is relatively hidden, lacking specific clinical manifestations in the early stage, and it is often already in the middle and late stages when it is discovered. The principle of improving the long-term survival rate of patients with liver cancer is early detection, early diagnosis, and early treatment, and the key is early diagnosis.
目前，最常用的肝癌常规诊断方法有影像学检查、肝组织活检或细胞学检查以及肿瘤血清标志物检查。但是，影像学检查分辨率较差且存在难以检测的盲区，而肝穿刺活检取样过程复杂，易造成较大创伤且易产生假阴性结果，因此，目前最理想的肝癌诊断方法是非创性的肿瘤血清标志物检查。熟知的肝癌血清标志物比较多，如甲胎蛋白(AFP)、磷脂酰肌醇蛋白聚糖3(GPC-3)、α-L岩藻糖苷酶(AFU)等。但这些标志物在应用过程之中普遍存在敏感度或特异度不高、在高危人群筛查和复发监测中缺乏有效的预警作用等局限性。就当前临床现状来看，AFP仍是应用非常广泛的肝癌检测标志物，其阳性率最高也仅为66.7％。Currently, the most commonly used methods for routine diagnosis of liver cancer include imaging examination, liver biopsy or cytology examination, and tumor serum marker examination. However, the resolution of imaging examination is poor and there are blind spots that are difficult to detect, and the sampling process of liver biopsy is complicated, which is easy to cause large trauma and false negative results. Therefore, the most ideal method for the diagnosis of liver cancer is non-invasive tumor Serum marker examination. There are many well-known serum markers for liver cancer, such as alpha-fetoprotein (AFP), glypican 3 (GPC-3), and α-L fucosidase (AFU). However, these markers generally have limitations in the application process, such as low sensitivity or specificity, lack of effective early warning in high-risk population screening and recurrence monitoring. As far as the current clinical situation is concerned, AFP is still a very widely used marker for liver cancer detection, and its highest positive rate is only 66.7%.
近年研究发现，血液中存在丰富而稳定的miRNA，其表达谱具有明显的组织特异性，并且在不同疾病、不同肿瘤患者血清中也具有特异的表达谱，因此外周血miRNA作为潜在标志物在肿瘤诊断中具有明显优势(PNAS,2008,105(30):10513-18)，目前已经报道血清中的循环miRNA在多种肿瘤的诊断中具有较高的特异性和灵敏度(MolecularCancer,2010,9(306):1-9)。早在2009年就出现了有关肝癌的miRNA血清标志物研究，至今已报道的具备肝癌诊断潜在价值的miRNA就有miR-21、miR-122、miR-210等多种(Biomarkers,2009,14(7):529-38；MolCarcinog,2011,50(2):136-42；EurJCancer,2013,49(16):3442-9)。但是，上述研究普遍采用样本血清中原本就存在的核酸作为内源性参照，例如RNU6B、RNU44、RNU48、miR-16等。由于miR-16本身在肿瘤发生过程发挥功能，其表达水平并不稳定；而RNU6B等短序列RNA在血清中含量极低不易检测，必然无法避免病患间的个体差异以及实验过程造成的误差(BMCClinicalPathology,2014,14:27)。因此，如何选取血清miRNA实验的内源性参照物至今还存在很大争议(J.Cancer,2012,3:432-448)。In recent years, studies have found that there are abundant and stable miRNAs in blood, and their expression profiles have obvious tissue specificity, and they also have specific expression profiles in the serum of patients with different diseases and different tumors. Therefore, peripheral blood miRNAs are potential markers in tumors. It has obvious advantages in diagnosis (PNAS, 2008, 105(30): 10513-18), and it has been reported that circulating miRNA in serum has high specificity and sensitivity in the diagnosis of various tumors (Molecular Cancer, 2010, 9( 306): 1-9). As early as 2009, research on miRNA serum markers for liver cancer appeared, and the miRNAs with potential diagnostic value for liver cancer that have been reported so far include miR-21, miR-122, miR-210, etc. (Biomarkers, 2009, 14( 7): 529-38; MolCarcinog, 2011, 50(2): 136-42; EurJ Cancer, 2013, 49(16): 3442-9). However, the above-mentioned studies generally use nucleic acids that already exist in the sample serum as endogenous references, such as RNU6B, RNU44, RNU48, miR-16, etc. Because miR-16 itself plays a role in the process of tumorigenesis, its expression level is not stable; while the content of short-sequence RNA such as RNU6B in serum is extremely low and difficult to detect, it is inevitable that individual differences between patients and errors caused by the experimental process cannot be avoided ( BMC Clinical Pathology, 2014, 14:27). Therefore, how to select an endogenous reference substance for serum miRNA experiments is still controversial (J. Cancer, 2012, 3: 432-448).
Cel-miR-39是最早被发现的一批线虫miRNA之一(Science,2001,294:858-62)。由于线虫与哺乳动物在遗传上差距太大，cel-miR-39序列在哺乳动物等高等动物基因组内不存在，其对哺乳动物生命过程也不产生影响。因而在哺乳动物miRNA检测过程中，外加人工合成的cel-miR-39作为外源参照是目前控制样本间实验误差的一种可靠做法。Cel-miR-39 is one of the first nematode miRNAs discovered (Science, 2001, 294:858-62). Due to the large genetic gap between nematodes and mammals, the cel-miR-39 sequence does not exist in the genomes of mammals and other higher animals, and it does not affect the life process of mammals. Therefore, in the process of mammalian miRNA detection, adding artificially synthesized cel-miR-39 as an exogenous reference is a reliable way to control the experimental error between samples.
实时荧光定量PCR检测是一种通用的基因定量检测方法，它在PCR指数扩增期间通过连续监测荧光信号强弱的变化来即时测定特异性产物的量，并据此推断目的基因的初始量，而不需要取出PCR产物进行分离。实时定量PCR灵敏度高，通用性好，可重复性强，特别适用于大通量检测，已被广泛地应用于分子生物学研究的各个领域。Real-time fluorescent quantitative PCR detection is a general gene quantitative detection method, which can measure the amount of specific products in real time by continuously monitoring the changes in the intensity of fluorescent signals during the PCR exponential amplification period, and infer the initial amount of the target gene accordingly. There is no need to take out the PCR products for isolation. Real-time quantitative PCR has high sensitivity, good versatility, and strong repeatability. It is especially suitable for large-throughput detection and has been widely used in various fields of molecular biology research.
发明内容Contents of the invention
本发明的一个目的是提供检测miR-197表达量的物质的新用途。One object of the present invention is to provide a new application of the substance for detecting the expression level of miR-197.
本发明提供了检测miR-197表达量的物质在制备诊断或辅助诊断待测患者是否为肝癌患者的产品中的应用。The invention provides an application of a substance for detecting the expression level of miR-197 in the preparation of a product for diagnosing or assisting in diagnosing whether a patient to be tested is a liver cancer patient.
本发明还提供了检测miR-197表达量的物质在制备检测或辅助检测肝癌的产品中的应用。The present invention also provides the application of the substance for detecting the expression level of miR-197 in the preparation of products for detecting or assisting in the detection of liver cancer.
上述应用中，所述miR-197的核苷酸序列如序列表中序列1所示。In the above application, the nucleotide sequence of miR-197 is shown as sequence 1 in the sequence listing.
上述应用中，所述检测miR-197表达量的物质为如下a)或b)或c)：In the above application, the substance for detecting the expression level of miR-197 is as follows a) or b) or c):
a)扩增所述miR-197的引物；a) primers for amplifying said miR-197;
b)含有所述a)的PCR试剂组；b) the PCR reagent group containing said a);
c)含有所述a)或所述b)的试剂盒。c) A kit containing said a) or said b).
上述应用中，所述引物由序列表中序列2所示的单链DNA分子和序列表中序列3所示的单链DNA分子组成。In the above application, the primer consists of a single-stranded DNA molecule shown in sequence 2 in the sequence listing and a single-stranded DNA molecule shown in sequence 3 in the sequence listing.
本发明的另一个目的是提供一种检测miR-197表达量的物质。Another object of the present invention is to provide a substance for detecting the expression level of miR-197.
本发明提供的检测miR-197表达量的物质为如下1)或2)：The substances for detecting the expression level of miR-197 provided by the present invention are as follows 1) or 2):
1)诊断或辅助诊断待测患者是否为肝癌患者的产品；1) Products for diagnosing or assisting in diagnosing whether the patient under test is a liver cancer patient;
2)检测或辅助检测肝癌的产品；2) Products that detect or assist in the detection of liver cancer;
所述miR-197的核苷酸序列如序列表中序列1所示。The nucleotide sequence of miR-197 is shown as sequence 1 in the sequence listing.
上述物质中，所述检测miR-197表达量的物质为如下a)或b)或c)：Among the above substances, the substance for detecting the expression level of miR-197 is as follows a) or b) or c):
a)扩增所述miR-197的引物；a) primers for amplifying said miR-197;
b)含有所述a)的PCR试剂组；b) the PCR reagent group containing said a);
c)含有所述a)或所述b)的试剂盒；c) a kit containing said a) or said b);
所述引物由序列表中序列2所示的单链DNA分子和序列表中序列3所示的单链DNA分子组成。The primer consists of a single-stranded DNA molecule shown in sequence 2 in the sequence listing and a single-stranded DNA molecule shown in sequence 3 in the sequence listing.
本发明还有一个目的是提供一种检测待测患者是否为肝癌患者的试剂盒。Another object of the present invention is to provide a kit for detecting whether a patient to be tested is a liver cancer patient.
本发明提供的检测或辅助检测待测患者是否为肝癌患者的试剂盒包括上述检测miR-197表达量的物质。The kit for detecting or assisting in detecting whether a patient to be tested is a liver cancer patient provided by the present invention includes the above-mentioned substances for detecting the expression level of miR-197.
上述试剂盒中，所述试剂盒还包括记载有如下内容的诊断卡：In the above kit, the kit also includes a diagnostic card with the following contents:
1)若待测患者的miR-197的表达量高于健康人，且二者有显著差异，则待测患者为或候选为肝癌患者；2)若待测患者不满足上述步骤1)的条件，则待测患者不为或候选不为肝癌患者。1) If the expression level of miR-197 in the patient to be tested is higher than that of healthy people, and there is a significant difference between the two, the patient to be tested is or is a candidate for liver cancer; 2) If the patient to be tested does not meet the conditions of the above step 1) , the patient to be tested is not or the candidate is not a liver cancer patient.
上述试剂盒中，所述待测患者的miR-197的表达量高于健康人，且二者有显著差异为待测患者血清中miR-197的表达量比健康人血清中miR-197的表达量高18.8倍。In the above kit, the expression level of miR-197 in the patient to be tested is higher than that in the healthy person, and the significant difference between the two is that the expression level of miR-197 in the serum of the patient to be tested is higher than that in the serum of the healthy person. The amount is 18.8 times higher.
本发明的最后一个目的是提供上述试剂盒的新用途。A final object of the present invention is to provide a new use of the above kit.
本发明提供了上述试剂盒在制备诊断或辅助诊断待测患者是否为肝癌患者的产品中的应用。The invention provides the application of the above kit in the preparation of products for diagnosing or assisting in diagnosing whether a patient to be tested is a liver cancer patient.
本发明还提供了上述试剂盒在制备检测或辅助检测肝癌的产品中的应用。The present invention also provides the application of the above kit in the preparation of products for detection or auxiliary detection of liver cancer.
上述miR-197表达量为血清中miR-197的表达量。The above expression level of miR-197 is the expression level of miR-197 in serum.
本发明以实时荧光定量PCR技术为基础，以肝癌患者的血清miR-197为检测对象，采用线虫小RNAcel-miR-39为外源参照，通过比对正常人、肝癌患者及其他几种国内常见癌症(肝癌、食道癌、胃癌、结肠癌、肺癌)病例患者血清中miR-197的水平，发现miR-197在肝癌患者血清中的特异性高表达，因此，将血清中稳定存在的miR-197作为肝癌的血清标志物和分子靶标应用于肝癌临床诊断，用于制备以血清检测为基础的肝癌诊断产品，该产品将对肿瘤的治疗具有重要意义，有广阔的应用前景。The present invention is based on real-time fluorescent quantitative PCR technology, takes the serum miR-197 of liver cancer patients as the detection object, and uses nematode small RNA cel-miR-39 as the exogenous reference, and compares normal people, liver cancer patients and other domestic common The level of miR-197 in the serum of patients with cancer (liver cancer, esophageal cancer, gastric cancer, colon cancer, lung cancer) was found to be highly specifically expressed in the serum of patients with liver cancer. Therefore, miR-197 stably present in the serum As a serum marker and molecular target of liver cancer, it is used in the clinical diagnosis of liver cancer, and is used to prepare a liver cancer diagnostic product based on serum detection. This product will be of great significance to the treatment of tumors and has broad application prospects.
附图说明Description of drawings
图1为实时荧光定量PCR检测血清miRNA。图1A为PCR扩增曲线；图1B为扩增产物溶解曲线；图1C为扩增产物溶解峰。Figure 1 is real-time fluorescent quantitative PCR detection of serum miRNA. Figure 1A is the PCR amplification curve; Figure 1B is the dissolution curve of the amplification product; Figure 1C is the dissolution peak of the amplification product.
图2为各样本miR-197表达水平比较。Figure 2 is a comparison of the expression levels of miR-197 in each sample.
图3为肝癌患者与正常人之间miR-197表达差异的AUC分析。Figure 3 is the AUC analysis of the difference in miR-197 expression between liver cancer patients and normal people.
具体实施方式detailed description
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
实施例1、miR-197在作为检测肝癌标志物中的应用Example 1, the application of miR-197 as a marker for detecting liver cancer
一、待测血清样本的获得及信息1. Obtaining and information of serum samples to be tested
本发明所检测的血清样本均由甘肃省医学科学研究中心甘肃肿瘤血清资源库提供，每例病患血清均有对应的诊断记录及确切的病例信息。血清的取样经由事先通知并获得患者允许，符合社会伦理学相关标准。本发明共检测健康志愿者的血清样本66例，肝癌患者血清样本70例，食道癌患者血清样本20例，胃癌患者血清样本22例，结肠癌患者血清样本20例，肺癌患者血清样本21例。各癌症患者信息统计详见表1。The serum samples detected in the present invention are all provided by Gansu Tumor Serum Resource Bank of Gansu Medical Science Research Center, and each patient serum has a corresponding diagnosis record and exact case information. The sampling of serum was notified in advance and the patient's permission was obtained, which complied with the relevant standards of social ethics. The present invention detects 66 serum samples of healthy volunteers, 70 serum samples of liver cancer patients, 20 serum samples of esophageal cancer patients, 22 serum samples of gastric cancer patients, 20 serum samples of colon cancer patients, and 21 serum samples of lung cancer patients. The statistical information of each cancer patient is shown in Table 1.
表1、癌症患者信息汇总表Table 1. Summary table of cancer patient information
二、实时荧光定量PCR测定miR-197的表达水平2. Real-time fluorescent quantitative PCR to measure the expression level of miR-197
本发明以人工合成线虫39miRNA(cel-miR-39)为外参基因，对步骤一中的待测血清样本血清中miR-197的表达水平进行检测。miR-197的成熟核苷酸序列为CGGGUAGAGAGGGCAGUGGGAGG(序列1)。In the present invention, artificially synthesized nematode 39miRNA (cel-miR-39) is used as an external reference gene to detect the expression level of miR-197 in the serum of the serum sample to be tested in step 1. The mature nucleotide sequence of miR-197 is CGGGUAGAGAGGGCAGUGGGAGG (SEQ ID NO: 1).
1、血清RNA的提取1. Extraction of serum RNA
参照文献“张晓娟等，南京医科大学学报(自然科学版)，2011，31(4):529-31”中的实验方案，采用TRIzol法对血清样本的全RNA进行提取。具体步骤如下：Referring to the experimental protocol in the document "Zhang Xiaojuan et al., Journal of Nanjing Medical University (Natural Science Edition), 2011, 31(4):529-31", the total RNA of the serum sample was extracted by the TRIzol method. Specific steps are as follows:
(1)将步骤一的各个待测血清样本在冰上融化，待完全溶解后使用1000g的离心力在4℃下离心10分钟，去除血清中血细胞残片等杂质，收集上清。(1) Thaw each serum sample to be tested in step 1 on ice, and centrifuge at 4°C for 10 minutes with a centrifugal force of 1000g after complete dissolution to remove impurities such as blood cell fragments in the serum, and collect the supernatant.
(2)每例血清样本取400μL步骤(1)获得的上清，加入到1mL的TRIzol(ThermoFisherScientific,Waltham,MA,USA)中，同时加入10uL的浓度为1mM的人工合成的线虫miRNAcel-39类似物(广州复能基因有限公司，广州，广东，中国)，充分混匀之后静置5分钟。(2) For each serum sample, take 400 μL of the supernatant obtained in step (1), add it to 1 mL of TRIzol (ThermoFisher Scientific, Waltham, MA, USA), and add 10 uL of artificially synthesized nematode miRNAcel-39 with a concentration of 1 mM. (Guangzhou Funeng Gene Co., Ltd., Guangzhou, Guangdong, China), mix thoroughly and let stand for 5 minutes.
(3)将步骤(2)获得的产物通过氯仿萃取和异丙醇沉淀后，得到RNA，将其溶解到20μL去除RNA酶的灭菌水中，得到待测血清的RNA。(3) The product obtained in step (2) was extracted with chloroform and precipitated with isopropanol to obtain RNA, which was dissolved in 20 μL of RNase-removed sterilized water to obtain the RNA of the serum to be tested.
2、RNA反转录2. RNA reverse transcription
采用RNA的3’末端加A(腺嘌呤)法开展RNA的反转录。将步骤1获得的RNA样本、POLYA聚合酶、M-MLV反转录酶及5×的聚合反应Buffer混匀，配置成反转录体系，在37℃反应60分钟，得到反转录产物。反应体系中各组分含量及具体实施过程参照miRNAqRT-PCRDetectionKit(广州复能基因有限公司，广州，广东，中国)说明书。RNA reverse transcription was carried out by adding A (adenine) to the 3' end of RNA. Mix the RNA sample obtained in step 1, POLYA polymerase, M-MLV reverse transcriptase, and 5× polymerization reaction buffer, configure a reverse transcription system, and react at 37°C for 60 minutes to obtain a reverse transcription product. The content of each component in the reaction system and the specific implementation process refer to the instructions of miRNAqRT-PCRDetectionKit (Guangzhou Funeng Gene Co., Ltd., Guangzhou, Guangdong, China).
3、实时荧光定量PCR3. Real-time fluorescent quantitative PCR
以上述步骤2获得的反转录产物为模板，分别采用Hsa-miR-197特异性扩增引物和cel-miR-39特异性扩增引物进行实时荧光定量PCR，分别得到扩增产物，每个反应体系使用的模板的量保持一致。Hsa-miR-197特异性扩增引物：Using the reverse transcription product obtained in the above step 2 as a template, the Hsa-miR-197 specific amplification primer and the cel-miR-39 specific amplification primer were used to perform real-time fluorescent quantitative PCR, respectively, to obtain the amplification product, each The amount of template used in the reaction system was kept consistent. Hsa-miR-197 specific amplification primers:
Sence：5’-CGGGUAGAGAGGGC-3’(序列2)；Sence: 5'-CGGGUAGAGAGGGC-3' (sequence 2);
antisense：5’-TTTTTTTTTTTTTTTT-3’(序列3)；antisense: 5'-TTTTTTTTTTTTTTTT-3' (SEQ ID NO: 3);
cel-miR-39特异性扩增引物：cel-miR-39 specific amplification primers:
Sence：5’-TCACCGGGTGTAAATC-3’；Sence: 5'-TCACCGGGTGTAAATC-3';
antisense：5’-TTTTTTTTTTTTTTTT-3’。Antisense: 5'-TTTTTTTTTTTTTTTT-3'.
各个待测样本的实时荧光定量PCR的扩增曲线如图1A所示，曲线与基线相交处为PCR反应的循环数(Ct值)，此值可反映所检测miRNA的最初含量。各个待测样本的PCR产物的溶解曲线和溶解峰分别如图1B和图1C所示，从图中可以看出，溶解峰为单峰，说明荧光定量PCR反应为单一产物，PCR测定可信度高。也说明常规试验方法(TRIzol法提取RNA、实时荧光定量PCR进行miRNA检测)可实现待测血清样本的miRNA的提取和定量检测。The amplification curve of real-time fluorescent quantitative PCR for each sample to be tested is shown in Figure 1A, where the intersection of the curve and the baseline is the cycle number (Ct value) of the PCR reaction, which can reflect the initial content of the detected miRNA. The dissolution curve and dissolution peak of the PCR products of each sample to be tested are shown in Figure 1B and Figure 1C respectively. It can be seen from the figure that the dissolution peak is a single peak, indicating that the fluorescence quantitative PCR reaction is a single product, and the reliability of PCR measurement high. It also shows that the conventional test method (TRIzol method for RNA extraction, real-time fluorescent quantitative PCR for miRNA detection) can realize the extraction and quantitative detection of miRNA in the serum sample to be tested.
4、统计学分析判定miRNA相对表达量4. Statistical analysis to determine the relative expression of miRNA
参照文献“WangGK、ZhouJ,EuropeanHeartJournal,2010,31:659-66；JOURNALOFCLINICALONCOLOGY,2011,29(36):4781-8”中的统计策略，以人工合成的cel-miR-39为外源参照，使用cel-miR-39的Ct值以去除样品间误差，以正常人miRNA表达变化倍数的均值为基准点，采用ΔΔCt法计算各类癌症患者血清miRNA的相对表达变化倍数。计算公式如下：miRNA相对表达变化倍数＝2-ΔΔCt，其中ΔΔCt＝样本ΔCt–正常人ΔCt的均值；样本ΔCt＝样本miRNACt值–样本cel-miR-39Ct值；正常人ΔCt的均值＝所有正常人样本的(miRNACt值–cel-miR-39Ct值)的算术平均值。舍弃Ct值高于40的结果，按上述公式计算各个待测样本血清中的miR-197的相对表达变化倍数，采用Z检验判定数据间统计学差异，当p<0.05判定为差异具有显著性。并使用SPSS19.0进行受试者工作特征曲线(ROC,ReceiverOperatingCharacteristicCurve)分析，当AUC值>90％，则判定为准确性高。Referring to the statistical strategy in the literature "WangGK, ZhouJ, European HeartJournal, 2010, 31: 659-66; JOURNALOF CLINICALONCOLOGY, 2011, 29 (36): 4781-8", using artificially synthesized cel-miR-39 as an exogenous reference, using The Ct value of cel-miR-39 was used to remove the inter-sample error, and the average fold change of miRNA expression in normal people was used as the reference point, and the relative expression fold change of serum miRNA in various cancer patients was calculated by the ΔΔCt method. The calculation formula is as follows: miRNA relative expression change fold = 2 -ΔΔCt , where ΔΔCt = sample ΔCt - average value of normal ΔCt; sample ΔCt = sample miRNACt value - sample cel-miR-39Ct value; normal ΔCt average = all normal people Arithmetic mean of (miRNACt value - cel-miR-39Ct value) for samples. The results with a Ct value higher than 40 were discarded, and the relative expression changes of miR-197 in the serum of each sample to be tested were calculated according to the above formula, and the Z test was used to determine the statistical difference between the data. When p<0.05, the difference was considered significant. Receiver Operating Characteristic Curve (ROC, Receiver Operating Characteristic Curve) analysis was performed using SPSS 19.0. When the AUC value > 90%, it was judged as high accuracy.
各个待测样本的miR-197的表达量的数据统计结果如表2和图2所示，从表2中的中位数来看，肝癌患者血清样本中miR-197表达量相对于正常人增高18.8倍，而食道癌、胃癌、结肠癌、肺癌等癌症患者样本血清中的miR-197的表达量同正常人相比没有显著性差异。从图2中的相对表达变化倍数来看，肝癌患者血清样本中miR-197的相对表达变化倍数显著高于正常人、食道癌、胃癌、结肠癌、肺癌等癌症患者。The statistical results of the expression of miR-197 in each sample to be tested are shown in Table 2 and Figure 2. From the median in Table 2, the expression of miR-197 in serum samples of liver cancer patients is higher than that of normal people 18.8 times, while the expression of miR-197 in the serum of patients with esophageal cancer, gastric cancer, colon cancer, lung cancer and other cancer samples has no significant difference compared with normal people. From the relative expression changes in Figure 2, the relative expression changes of miR-197 in serum samples of patients with liver cancer were significantly higher than those in normal people, esophageal cancer, gastric cancer, colon cancer, lung cancer and other cancer patients.
表2、各样本miR-197表达水平的数据统计结果Table 2. Statistical results of miR-197 expression level in each sample
*：Z检验1的数据为各样本与正常人之间比对结果；#：Z检验2的数据为各样本与肝癌之间比对结果。*: The data of Z test 1 is the comparison result between each sample and normal people; #: The data of Z test 2 is the comparison result between each sample and liver cancer.
受试者工作特征曲线分析结果如图3所示，miR-197的特征曲线表现出较高的特异性和敏感度，其AUC达到0.946，大于90％，说明本发明的检测结果准确性高。The receiver operating characteristic curve analysis results are shown in Figure 3. The characteristic curve of miR-197 shows high specificity and sensitivity, and its AUC reaches 0.946, which is greater than 90%, indicating that the detection results of the present invention are highly accurate.
综上所述，本发明可确认肝癌患者血清miR-197较正常人有特异性高表达，可作为肝癌临床诊断标志物。因此，可通过检测待测患者的miR-197的表达量的方法来判断待测患者是否为肝癌患者，具体方法如下：In summary, the present invention can confirm that the serum miR-197 of patients with liver cancer is more specifically expressed than normal people, and can be used as a clinical diagnostic marker for liver cancer. Therefore, it is possible to determine whether the patient to be tested is a liver cancer patient by detecting the expression level of miR-197 in the patient to be tested, and the specific method is as follows:
1)若待测患者的miR-197的表达量高于健康人，且二者有显著差异，则待测患者为或候选为肝癌患者；1) If the expression level of miR-197 in the patient to be tested is higher than that of healthy people, and there is a significant difference between the two, the patient to be tested is or is a candidate for liver cancer;
2)若待测患者不满足上述步骤1)的条件，则待测患者不为或候选不为肝癌患者。2) If the patient to be tested does not meet the conditions of step 1) above, the patient to be tested is not or is not a candidate for liver cancer patients.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510724797.4ACN105238863A (en) | 2015-10-29 | 2015-10-29 | Application of miR-197 as liver cancer detection marker |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510724797.4ACN105238863A (en) | 2015-10-29 | 2015-10-29 | Application of miR-197 as liver cancer detection marker |
| Publication Number | Publication Date |
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| CN105238863Atrue CN105238863A (en) | 2016-01-13 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510724797.4APendingCN105238863A (en) | 2015-10-29 | 2015-10-29 | Application of miR-197 as liver cancer detection marker |
| Country | Link |
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| CN (1) | CN105238863A (en) |
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| WO2012089630A1 (en)* | 2010-12-30 | 2012-07-05 | Fondazione Istituto Firc Di Oncologia Molecolare (Ifom) | A METHOD TO IDENTIFY ASYMPTOMATIC HIGH-RISK INDIVIDUALS WITH EARLY STAGE LUNG CANCER BY MEANS OF DETECTING miRNAs IN BIOLOGIC FLUIDS |
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