WO2021159562A1 - 一种与泛肿瘤辅助诊断相关的循环miRNA和癌胚miRNA标志物及其应用 - Google Patents

一种与泛肿瘤辅助诊断相关的循环miRNA和癌胚miRNA标志物及其应用 Download PDF

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WO2021159562A1
WO2021159562A1 PCT/CN2020/077198 CN2020077198W WO2021159562A1 WO 2021159562 A1 WO2021159562 A1 WO 2021159562A1 CN 2020077198 W CN2020077198 W CN 2020077198W WO 2021159562 A1 WO2021159562 A1 WO 2021159562A1
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mirna
pan
tumor
markers
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朱伟
周鑫
刘平
陈彦
丁强
夏添松
耿向南
高峰
邹璇
单霞
朱丹霞
徐德文
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朱伟
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

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  • the invention belongs to the fields of genetic engineering and oncology, and relates to a circulating miRNA and carcinoembryonic miRNA markers related to pan-tumor auxiliary diagnosis and applications thereof.
  • Cancer is one of the leading causes of death worldwide. In recent years, although people have gained a deeper understanding of the molecular mechanisms and clinical features of cancer and developed more treatment methods and methods, the prognosis of cancer patients is still very poor. Due to the lack of obvious symptoms and sufficiently sensitive biomarkers, many patients are already at an advanced stage when they are diagnosed, and have lost the opportunity for surgery. Early detection and intervention are key strategies to improve the long-term outcome of cancer patients. Today, pathological examination of tissue samples is still the gold standard for cancer diagnosis. However, these methods rely too much on the tester's experience, and their invasive detection methods limit their feasibility for screening diseases in large-scale populations. The existing non-invasive tumor markers, such as CEA, CA19-9, etc., have low sensitivity or specificity in cancer diagnosis. Therefore, there is an urgent need to find new tumor markers, promote early intervention and treatment, and prolong the survival period of patients.
  • MicroRNAs are a class of small non-coding RNA molecules with a length of about 22 nucleotides. They are widely involved in various life activities through post-transcriptional regulation, including tumor occurrence, invasion and metastasis. Studies have found that the expression of miRNA is up-regulated and down-regulated in tumors to varying degrees, laying the foundation for its ability as an emerging tumor marker. In 2008, Mitchell detected free miRNA in peripheral blood and found that it can stably exist in peripheral blood and can be used as a non-invasive marker for tumor diagnosis. Studies have confirmed the potential diagnostic value of circulating miRNAs in thyroid cancer, gastric cancer, lung cancer, breast cancer, colorectal cancer and other tumors.
  • this study uses Exiqon miRNA qPCR panel chip and qRT-PCR-based relative quantification method to study the serum and plasma of large samples of pan-tumor patients, aiming to find circulating miRNAs with potential diagnostic value for pan-tumor.
  • this study uses Exiqon miRNA qPCR panel chip and qRT-PCR-based relative quantification method to study the serum and plasma of large samples of pan-tumor patients, aiming to find circulating miRNAs with potential diagnostic value for pan-tumor.
  • carcinoembryonic miRNA markers are obtained, which has better diagnostic value for pan-tumor.
  • a diagnostic kit for pan-tumor is designed based on this type of miRNA, it will promote the level of diagnosis and treatment of tumors in my country, and also provide ideas for further research on pan-tumors in the future.
  • the purpose of the present invention is to provide a circulating carcinoembryonic miRNA marker related to pan-tumor auxiliary diagnosis.
  • Another object of the present invention is to provide a circulating miRNA marker related to the auxiliary diagnosis of pan-tumor.
  • Another object of the present invention is to provide the application of the above-mentioned circulating miRNA markers and their primers in the preparation of pan-tumor auxiliary diagnostic kits and in the preparation of drugs for treating pan-tumors.
  • Another object of the present invention is to provide a pan-tumor auxiliary diagnosis kit or auxiliary treatment drug.
  • the circulating carcinoembryonic miRNA marker includes serum carcinoembryonic miRNA markers miR-19b-3p, miR-20a-5p, miR223-3p, miR- 93 ⁇ 5p, miR ⁇ 25 ⁇ 3p, miR ⁇ 425 ⁇ 5p, miR ⁇ 19a ⁇ 3p, miR ⁇ 92a ⁇ 3p and miR ⁇ 20b ⁇ 5p; and plasma carcinoembryonic miRNA markers miR ⁇ 106a ⁇ 5p, miR ⁇ 146a A combination of two or more miRNAs among ⁇ 5p, miR ⁇ 18a ⁇ 5p, miR ⁇ 20b ⁇ 5p, miR ⁇ 210 ⁇ 3p, miR ⁇ 21 ⁇ 5p, miR ⁇ 409 ⁇ 3p and miR ⁇ 584 ⁇ 5p; preferred , Including serum carcinoembryonic miRNA markers miR-19b-3p, miR-20a-5p, miR223-3p, miR93-5p, miR
  • the circulating miRNA marker includes serum markers let-7b-5p (ugagguaguagguugugugguu), miR106a-5p (aaaagugcuuacagugcagguag), miR-10b-5p (uacccuguagaaccgaauuugug), miR ⁇ 122 ⁇ 5p (uggagugugacaaugguguuug), miR ⁇ 133a ⁇ 3p (uuugguccccuucaaccagcug), miR ⁇ 140 ⁇ 3p (uaccacaggguagaaccacgg), miR ⁇ 143 ⁇ 3p (ugagaugaagcacuguagcuc), miR ⁇ 146a ⁇ 5p (ugagaacuga3auuc) (cuagacugaagcuccuugagg), miR-16-5p(uagcagcacguaaauauuggcg), miR-192-5p(cugaccuaugaauugacagcc), miR-192-5p(cugaccuaugaauugacag
  • a primer for circulating carcinoembryonic miRNA markers related to pan-tumor assisted diagnosis contains serum carcinoembryonic miRNA markers miR-19b-3p, miR-20a-5p, miR223-3p, miR93-5p , miR-25-3p, miR-425-5p, miR-19a-3p, miR-92a-3p and miR-20b-5p; and plasma carcinoembryonic miRNA markers miR-106a-5p, miR-146a-5p, The combination of two or more miRNA primers among miR-18a-5p, miR-20b-5p, miR2103p, miR21-5p, miR409-3p and miR584-5p; preferably, Contains serum carcinoembryonic miRNA markers miR-19b-3p, miR-20a-5p, miR223-3p, miR93-5p, miR25-3p, miR425-5p, miR19a-3p, miR ⁇ 92a ⁇ 3p and miR ⁇ 20b ⁇
  • a primer for circulating miRNA markers related to pan-tumor assisted diagnosis contains serum markers let-7b-5p, miR-106a-5p, miR-10b-5p, miR-122-5p, miR-133a- 3p, miR-140-3p, miR-143-3p, miR-146a-5p, miR-151a-3p, miR-16-5p, miR-192-5p, miR-195-5p, miR-19a-3p, miR-19b-3p, miR204-5p, miR-20a-5p, miR-20b-5p, miR21-5p, miR221-3p, miR223-3p, miR223-5p, miR 24 ⁇ 3p, miR ⁇ 25 ⁇ 3p, miR ⁇ 296 ⁇ 5p, miR ⁇ 29c ⁇ 3p, miR ⁇ 423 ⁇ 3p, miR ⁇ 425 ⁇ 5p, miR ⁇ 451a, miR ⁇ 484, miR ⁇ 485 ⁇ 3p, miR ⁇ 590 ⁇ 5p, miR ⁇ 92a
  • the above kit may also include reagents commonly used in PCR technology, such as reverse transcriptase, buffer, dNTPs, MgCl 2 , DEPC water and Taq enzyme, etc.; it may also contain standard substances and/or reference substances.
  • reagents commonly used in PCR technology such as reverse transcriptase, buffer, dNTPs, MgCl 2 , DEPC water and Taq enzyme, etc.; it may also contain standard substances and/or reference substances.
  • the amplification primers of each miRNA marker can be purchased from the market.
  • the primers of the circulating miRNA markers used in the embodiments of the present invention are the specific miRNA stem-loop RT-PCR primers synthesized and produced by Guangzhou Ruibo Company, but Not limited to this.
  • the technical solution of the present invention to solve the problem includes: (1) Establishing a uniform standard specimen library and database: collecting blood samples that meet the standards using standard operating procedures (SOP), and systematically collecting complete demographic and clinical data. (2) Serum and plasma miRNA differential expression profile analysis: analyze the differentially expressed circulating miRNAs in 15 common tumor patients and age-sex matched normal controls, and conduct further large-scale multi-stage verification of differentially expressed miRNAs. (3) Through multi-stage verification, it is clear that these miRNAs can diagnose 15 common tumors.
  • SOP standard operating procedures
  • the inventors used standard operating procedures (SOP) to collect blood samples that met the standards, systematically collected complete demographic and clinical data, and used Exiqon miRNA qPCR panel chips and qRT-PCR methods.
  • SOP standard operating procedures
  • Study sample selection 15 types of pan-tumor patients who are newly treated, have not undergone surgery, radiotherapy and chemotherapy intervention and are pathologically confirmed as common clinically common tumors (including: nasopharyngeal carcinoma, thyroid carcinoma, esophageal carcinoma, lung squamous cell carcinoma, lung adenocarcinoma) , Cardia cancer, gastric adenocarcinoma, pancreatic cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, endometrial cancer, cervical cancer and ovarian cancer) and neonatal cord blood.
  • the normal control is a group of normal adults who undergo physical examination in the hospital.
  • Exiqon miRNA qPCR panel chip preliminary screening Use TRIZOL-LS reagents to extract RNA from serum and plasma samples, and perform qRT-PCR operations to obtain preliminary screening results.
  • RNA extraction is performed on each serum and plasma sample using AM1556 kit (ABI), cDNA sample is obtained by reverse transcription reaction, PCR primer and SYBR Green fluorescent dye are added for PCR reaction . By comparing the Ct value of spike-in control, the relative quantification method is used to obtain the relative content of miRNA in the sample.
  • pan-tumor patients including: nasopharyngeal cancer, thyroid cancer, esophageal cancer, lung squamous cell carcinoma, lung adenocarcinoma, cardia cancer, gastric adenocarcinoma, pancreatic cancer, colon cancer, rectal cancer, etc.
  • circulating miRNA as a new type of biomarker, has the characteristics of good stability, minimally invasive and easy access, high sensitivity and specificity. The development and utilization of such molecular markers will provide new directions for the diagnosis and further treatment of various diseases including tumors.
  • Figure 1 Experimental flow chart (except for serum samples from cardia cancer, gastric cancer, and ovarian cancer, and plasma samples from ovarian cancer, which used three stages: preliminary screening, validation set, and test set. All other studies used preliminary screening, validation set, and test set. And additional verification set four stages)
  • FIG. 1 18 and 16 miRNAs highly expressed in umbilical cord blood serum and plasma, respectively
  • A Serum sample
  • B Plasma sample
  • Figure 3 Overview of miRNAs that can be used to diagnose corresponding tumors obtained from 15 common clinical pan-tumors.
  • A miRNA obtained from 15 tumor types corresponding to the tumor; B: circulating miRNA (left image: serum; right image: plasma) and carcinoembryonic miRNA (red marker) for diagnosing pan-tumor Position on chromosome
  • Figure 4 The ability of serum carcinoembryonic miRNA (A-H) and plasma carcinoembryonic miRNA (I-N) to diagnose pan-tumor.
  • Figure 5 The expression of carcinoembryonic miRNA in pan-tumor in the TCGA tissue bank.
  • pan-tumor patients from the First affiliated Hospital of Nanjing Medical University from 2010 to 2018 (including: nasopharyngeal cancer, thyroid cancer, esophageal cancer, lung squamous cell carcinoma, lung adenocarcinoma, cardia cancer, and gastric adenocarcinoma.
  • Pancreatic cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, endometrial cancer, cervical cancer and ovarian cancer as well as neonatal umbilical cord blood and serum and plasma samples of the normal physical examination population.
  • tumor specimens (2050 serum specimens and 1781 plasma specimens), 248 cord blood specimens (120 serum specimens and 128 plasma specimens), and a total of 4,148 normal adult controls (2119 serum specimens) were selected. And 2029 plasma samples) as the experimental samples for the initial screening of Exiqon miRNA qPCR panel chip and subsequent series of qRT-PCR verification.
  • the selected patients' serum and plasma samples were from patients who were newly treated, who had not undergone surgery, radiotherapy and chemotherapy, and were pathologically confirmed as tumors.
  • Umbilical cord blood is the baby's cord blood collected immediately after the baby is delivered. And systematically collected demographic and clinical data of these samples. The total number of specific tumor types, cord blood and normal control samples and the number of cases by stages are shown in Table 1 in the attached table.
  • pan-tumor or umbilical cord blood samples and 10 normal controls were randomly selected from pan-tumor, umbilical cord blood, normal control serum and plasma samples, and 10 normal controls were mixed into corresponding ones.
  • 2/3/4 of the tumor or umbilical cord blood serum and plasma mixed samples and 1 normal mixed sample (a mixed sample is composed of 10 200ul serum or plasma samples combined to form a 2ml sample).
  • RNA Transfer the aqueous phase to a new centrifuge tube.
  • the aqueous phase is mixed with isopropanol to precipitate the RNA in it.
  • the amount of isopropanol added is: add 1ml TRIZOL-LS reagent while homogenizing each sample while adding 0.5ml isopropanol and 5ul glycogen. Let stand for half an hour at 4°C to allow RNA to precipitate out as much as possible. Centrifuge at 13,000g for 15 minutes at 4°C.
  • RNA/50ml serum/plasma Usually ⁇ 5 ⁇ g RNA/50ml serum/plasma can be obtained.
  • Dilute template RNA Use DEPC water to dilute 20-25ng template RNA to 14ul.
  • reaction solution Dissolve 5 ⁇ Reaction Buffer and DEPC water on ice, and shake and mix. Enzyme mix is placed in an ice box at -20°C, flick and mix before use, and then place on ice. All reagents are used after centrifugation.
  • Reverse transcription reaction and thermal inactivation After incubating the reaction solution at 42°C for 60 minutes, incubate at 95°C for 5 minutes to inactivate the reverse transcriptase.
  • Dilute the cDNA template Dilute the cDNA template obtained by the RT reaction by 110 times with nuclease free water (for example, add 2180ul nuclease free water to 20 ⁇ l of the reaction solution).
  • Real-time PCR amplification Perform Real-time PCR amplification and melting curve analysis according to the reaction conditions in the table below.
  • GenEx qPCR analysis software www.exiqon.com/mirna-pcr-analysis
  • ⁇ Ct(group 1) average Ct–average of HK genes’Ct for group 1array
  • ⁇ Ct(group 2) average Ct–average of HK genes’Ct for group 2array
  • the corresponding tumor and cord blood serum and plasma differentially expressed miRNAs were obtained (the serum/plasma mixed samples of each tumor and cord blood have more than 1.5 times the difference compared with the normal sample). Selected as a candidate miRNA).
  • the training set, test set and additional validation set serum samples for cardia cancer, gastric cancer, ovarian cancer and plasma samples for ovarian cancer are used Preliminary screening, validation set and test set three stages), using qRT-PCR-based relative quantification method for verification, the specific steps are:
  • Serum/plasma RNA extraction Use ABI's serum RNA extraction kit (AM1556), refer to the kit instructions, draw 200ul of each sample to extract RNA, and finally dissolve it with 100ul DEPC water.
  • reaction system is evenly mixed, centrifuged briefly, placed in a real-time quantitative PCR machine, and reacted with the following procedures:
  • Data analysis Use SPSS 16.0 software for statistical analysis, and identify miRNAs that are uniformly differentially expressed in the training set, test set, and additional verification set as diagnostic markers for the tumor. See Table 2 in the attached table for the uniformly differentially expressed miRNAs in the serum and plasma of pan-tumor patients at each stage.
  • the differentially expressed miRNA in cord blood serum and the differentially expressed miRNA in plasma were obtained ( Figure 2).
  • the differentially expressed miRNA in cord blood serum is shown in Table 3 of the attached table.
  • the differentially expressed miRNAs in cord blood plasma are shown in Table 4 of the attached table.
  • the differential miRNAs in all 15 tumors and cord blood were analyzed ( Figure 3), and 9 serum carcinoembryonic miRNAs (miR-19b-3p, miR-20a-5p, miR-223-3p, miR-93) were obtained.
  • ⁇ 5p, miR ⁇ 25 ⁇ 3p, miR ⁇ 425 ⁇ 5p, miR ⁇ 19a ⁇ 3p, miR ⁇ 92a ⁇ 3p and miR ⁇ 20b ⁇ 5p) and 8 plasma carcinoembryonic miRNAs (respectively miR ⁇ 106a ⁇ 5p, miR ⁇ 146a ⁇ 5p, miR ⁇ 18a ⁇ 5p, miR ⁇ 20b ⁇ 5p, miR ⁇ 210 ⁇ 3p, miR ⁇ 21 ⁇ 5p, miR ⁇ 409 ⁇ 3p and miR ⁇ 584-5p).
  • serum carcinoembryonic miRNA A-H respectively: nasopharyngeal carcinoma, thyroid cancer, colon cancer, rectal cancer, pancreatic cancer, lung squamous cell carcinoma, breast cancer and endometrial cancer
  • plasma carcinoembryonic miRNA I -N are: nasopharyngeal cancer, esophageal cancer, gastric cancer, lung squamous cell carcinoma, lung adenocarcinoma and endometrial cancer
  • the research team further analyzed the expression of these carcinoembryonic miRNAs in pan-tumor tissues in the TCGA database. As shown in Figure 5, most of these miRNAs are highly expressed in pan-tumors. The mechanism of tumors and these miRNAs provide new ideas for the treatment of pan-tumor.
  • the kit includes a batch of serum and plasma miRNA qRT-PCR primers, as well as common reagents required for corresponding PCR technology, such as: reverse transcriptase, buffer, dNTPs, MgCl2, DEPC water, fluorescent probes, RNase inhibitors , Taq enzymes, etc., can be selected according to the specific experimental methods used. These commonly used reagents are well known to those skilled in the art, and there can also be standards and controls (such as quantitatively standardized normal human samples, etc.). The value of this kit is that only serum/plasma is needed and no other tissue samples are needed.
  • the most streamlined fluorescence method is used to detect the expression of miRNA in serum/plasma samples to assist in diagnosing the possibility of pan-tumor in the patient from which the sample is derived.
  • Serum and plasma miRNA detection is convenient and accurate, which greatly improves the sensitivity and specificity of disease diagnosis. Therefore, putting this kit into practice can help guide diagnosis and further individualized treatment.

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Abstract

一种与泛肿瘤辅助诊断相关的循环miRNA标志物和癌胚miRNA标志物及其应用，循环miRNA和癌胚miRNA作为新型的生物标志物，具有稳定性好、微创易获取、灵敏性和特异性高的特点。这类分子标志物的开发利用将为包括肿瘤在内的各种疾病的诊断以及进一步治疗提供新的方向。将更有针对性的得出具有临床诊断潜能的泛肿瘤循环miRNA标记物和癌胚miRNA标志物。研究证实了这组miRNA作为诊断泛肿瘤的无创标记物的可靠性以及可重复性。

Description

一种与泛肿瘤辅助诊断相关的循环miRNA和癌胚miRNA标志物及其应用 技术领域
本发明属于基因工程及肿瘤学领域，涉及一种与泛肿瘤辅助诊断相关的循环miRNA和癌胚miRNA标志物及其应用。
背景技术
癌症是全球主要的死亡原因之一。近年来，虽然人们对癌症的分子机制和临床特征有了更深入的了解并开发了更多的治疗手段及方法，但是癌症患者的预后仍然很差。由于缺乏明显的症状和以及足够敏感的生物标志物，许多患者被诊断时已处于晚期，失去了手术机会。早期检测和干预是改善肿瘤患者长期结局的关键策略。如今，对组织标本的病理检查仍是诊断癌症的金标准。但是，这些方法过于依赖检测者经验，且其侵入性的检测方法限制了其在大规模人群中筛查疾病的可行性。现有的无创性肿瘤标志物，如CEA、CA19‐9等，在癌症诊断中的敏感性或特异性不高。因此，迫切需要寻找新型的肿瘤标志物，促进早期干预和治疗，延长病人的生存期。
微小RNA(miRNAs)是一类长度在22个左右的核苷酸的小非编码RNA分子，其通过转录后调控广泛参与了各种生命活动过程，包括肿瘤的发生、侵袭及转移等。研究发现，miRNA的表达在肿瘤中有不同程度的上调和下调，为其能够作为一种新兴的肿瘤标志物奠定了基础。2008年，Mitchell在外周血中检测到游离的miRNA，发现其能够稳定存在于外周血中，并且可以作为诊断肿瘤的无创标志物。现已有研究证实了循环miRNA在甲状腺癌、胃癌、肺癌、乳腺癌、结直肠癌等肿瘤中的潜在诊断价值。但由于研究方法及纳入人群的差异，导致研究结果不完全一致。因此，本研究利用Exiqon miRNA qPCR panel芯片以及基于qRT‐PCR的相对定量法，通过对大样本的泛肿瘤患者血清及血浆的研究，旨在寻找对泛肿瘤具有潜在诊断价值的循环miRNA。同时通过纳入脐带血进行研究，与泛肿瘤循环miRNA进行比较分析，得到癌胚miRNA标志物，对泛肿瘤具有更好的诊断价值。若根据这类miRNA设计针对于泛肿瘤的诊断试剂盒，将会推动我国肿瘤的诊治水平，也为将来对泛肿瘤的进一步研究提供思路。
发明内容
本发明的目的在于提供一种与泛肿瘤辅助诊断相关的循环癌胚miRNA标志物。
本发明的另一目的在于提供一种与泛肿瘤辅助诊断相关的循环miRNA标志物。
本发明的另一目的在于提供上述循环miRNA标志物及其引物在制备泛肿瘤辅助诊断试剂盒以及在制备治疗泛肿瘤的药物中的应用。
本发明的又一目的在于提供一种泛肿瘤辅助诊断试剂盒或辅助治疗药物。
本发明的目的可以通过以下技术方案实现：
一种与泛肿瘤辅助诊断相关的循环癌胚miRNA标志物，该循环癌胚miRNA标志物包含血清癌胚miRNA标志物miR‐19b‐3p,miR‐20a‐5p,miR‐223‐3p,miR‐93‐5p,miR‐25‐3p,miR‐425‐5p,miR‐19a‐3p,miR‐92a‐3p和miR‐20b‐5p；以及血浆癌胚miRNA标志物miR‐106a‐5p,miR‐146a‐5p,miR‐18a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐21‐5p,miR‐409‐3p和miR‐584‐5p中两种或两种以上miRNA的组合；优选的，包含血清癌胚miRNA标志物miR‐19b‐3p,miR‐20a‐5p,miR‐223‐3p,miR‐93‐5p,miR‐25‐3p,miR‐425‐5p,miR‐19a‐3p,miR‐92a‐3p和miR‐20b‐5p以及血浆癌胚miRNA标志物miR‐106a‐5p,miR‐146a‐5p,miR‐18a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐21‐5p,miR‐409‐3p和miR‐584‐5p十七种miRNA标志物。
一种与泛肿瘤辅助诊断相关的循环miRNA标志物，该循环miRNA标志物包含血清标志物let‐7b‐5p(ugagguaguagguugugugguu)，miR‐106a‐5p(aaaagugcuuacagugcagguag)，miR‐10b‐5p(uacccuguagaaccgaauuugug)，miR‐122‐5p(uggagugugacaaugguguuug)，miR‐133a‐3p(uuugguccccuucaaccagcug)，miR‐140‐3p(uaccacaggguagaaccacgg)，miR‐143‐3p(ugagaugaagcacuguagcuc)，miR‐146a‐5p(ugagaacugaauuccauggguu)，miR‐151a‐3p(cuagacugaagcuccuugagg)，miR‐16‐5p(uagcagcacguaaauauuggcg)，miR‐192‐5p(cugaccuaugaauugacagcc)，miR‐195‐5p(uagcagcacagaaauauuggc)，miR‐19a‐3p(ugugcaaaucuaugcaaaacuga)，miR‐19b‐3p(ugugcaaauccaugcaaaacuga)，miR‐204‐5p(uucccuuugucauccuaugccu)，miR‐20a‐5p(uaaagugcuuauagugcagguag)，miR‐20b‐5p(caaagugcucauagugcagguag)，miR‐21‐5p(uagcuuaucagacugauguuga)，miR‐221‐3p(agcuacauugucugcuggguuuc)，miR‐223‐3p(ugucaguuugucaaauacccca)，miR‐223‐5p(cguguauuugacaagcugaguu)，miR‐24‐3p(uggcucaguucagcaggaacag)，miR‐25‐3p(cauugcacuugucucggucuga)，miR‐296‐5p(agggcccccccucaauccugu)，miR‐29c‐3p(uagcaccauuugaaaucgguua)，miR‐423‐3p(agcucggucugaggccccucagu)，miR‐425‐5p(aaugacacgaucacucccguuga)，miR‐451a(aaaccguuaccauuacugaguu)，miR‐484(ucaggcucaguccccucccgau)，miR‐485‐3p(gucauacacggcucuccucucu)，miR‐590‐5p(gagcuuauucauaaaagugcag)，miR‐92a‐3p(uauugcacuugucccggccugu)，miR‐92b‐3p(uauugcacucgucccggccucc)和miR‐93‐5p(caaagugcuguucgugcagguag)以及血浆标志物let‐ 7b‐5p(ugagguaguagguugugugguu)，miR‐106a‐5p(aaaagugcuuacagugcagguag)，miR‐122‐5p(uggagugugacaaugguguuug)，miR‐125b‐5p(ucccugagacccuaacuuguga)，miR‐140‐3p(uaccacaggguagaaccacgg)，miR‐142‐3p(uguaguguuuccuacuuuaugga)，miR‐144‐3p(uacaguauagaugauguacu)，miR‐146a‐5p(ugagaacugaauuccauggguu)，miR‐151a‐5p(ucgaggagcucacagucuagu)，miR‐17‐5p(caaagugcuuacagugcagguag)，miR‐181a‐5p(aacauucaacgcugucggugagu)，miR‐185‐5p(uggagagaaaggcaguuccuga)，miR‐18a‐5p(uaaggugcaucuagugcagauag)，miR‐192‐5p(cugaccuaugaauugacagcc)，miR‐193b‐3p(aacuggcccucaaagucccgcu)，miR‐19b‐3p(ugugcaaauccaugcaaaacuga)，miR‐205‐5p(uccuucauuccaccggagucug)，miR‐20a‐5p(uaaagugcuuauagugcagguag)，miR‐20b‐5p(caaagugcucauagugcagguag)，miR‐210‐3p(cugugcgugugacagcggcuga)，miR‐2110(uuggggaaacggccgcugagug)，miR‐21‐5p(uagcuuaucagacugauguuga)，miR‐221‐3p(agcuacauugucugcuggguuuc)，miR‐223‐3p(ugucaguuugucaaauacccca)，miR‐25‐3p(cauugcacuugucucggucuga)，miR‐409‐3p(gaauguugcucggugaaccccu)，miR‐425‐5p(aaugacacgaucacucccguuga)，miR‐451a(aaaccguuaccauuacugaguu)，miR‐486‐5p(uccuguacugagcugccccgag)，miR‐584‐5p(uuaugguuugccugggacugag)，miR‐92b‐3p(uauugcacucgucccggccucc)和miR‐93‐5p(caaagugcuguucgugcagguag)中的一种或多种；优选为包含上述所有血清标志物和血浆标志物。
上述的循环癌胚miRNA标志物或/和循环miRNA标志物在辅助诊断泛肿瘤中的应用。
上述的循环癌胚miRNA标志物或/和循环miRNA标志物在制备泛肿瘤辅助诊断试剂盒或制备治疗泛肿瘤药物中的应用。
一种与泛肿瘤辅助诊断相关的循环癌胚miRNA标志物的引物，该引物包含血清癌胚miRNA标志物miR‐19b‐3p,miR‐20a‐5p,miR‐223‐3p,miR‐93‐5p,miR‐25‐3p,miR‐425‐5p,miR‐19a‐3p,miR‐92a‐3p和miR‐20b‐5p；以及血浆癌胚miRNA标志物miR‐106a‐5p,miR‐146a‐5p,miR‐18a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐21‐5p,miR‐409‐3p和miR‐584‐5p中两种或两种以上miRNA引物的组合；优选的，包含血清癌胚miRNA标志物miR‐19b‐3p,miR‐20a‐5p,miR‐223‐3p,miR‐93‐5p,miR‐25‐3p,miR‐425‐5p,miR‐19a‐3p,miR‐92a‐3p和miR‐20b‐5p以及血浆癌胚miRNA标志物miR‐106a‐5p,miR‐146a‐5p,miR‐18a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐21‐5p,miR‐409‐3p和miR‐584‐5p十七种miRNA标志物的引物。
一种与泛肿瘤辅助诊断相关的循环miRNA标志物的引物，该引物包含血清标志物let‐7b‐5p,miR‐106a‐5p,miR‐10b‐5p,miR‐122‐5p,miR‐133a‐3p,miR‐140‐3p,miR‐143‐3p,miR‐146a‐ 5p,miR‐151a‐3p,miR‐16‐5p,miR‐192‐5p,miR‐195‐5p,miR‐19a‐3p,miR‐19b‐3p,miR‐204‐5p,miR‐20a‐5p,miR‐20b‐5p,miR‐21‐5p,miR‐221‐3p,miR‐223‐3p,miR‐223‐5p,miR‐24‐3p,miR‐25‐3p,miR‐296‐5p,miR‐29c‐3p,miR‐423‐3p,miR‐425‐5p,miR‐451a,miR‐484,miR‐485‐3p,miR‐590‐5p,miR‐92a‐3p,miR‐92b‐3p和miR‐93‐5p；以及血浆标志物let‐7b‐5p,miR‐106a‐5p,miR‐122‐5p,miR‐125b‐5p,miR‐140‐3p,miR‐142‐3p,miR‐144‐3p,miR‐146a‐5p,miR‐151a‐5p,miR‐17‐5p,miR‐181a‐5p,miR‐185‐5p,miR‐18a‐5p,miR‐192‐5p,miR‐193b‐3p,miR‐19b‐3p,miR‐205‐5p,miR‐20a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐2110,miR‐21‐5p,miR‐221‐3p,miR‐223‐3p,miR‐25‐3p,miR‐409‐3p,miR‐425‐5p,miR‐451a,miR‐486‐5p,miR‐584‐5p,miR‐92b‐3p和miR‐93‐5p中的一种或多种miRNA的引物；优选为包含上述所有血清标志物和血浆标志物的引物。
上述的引物在辅助诊断泛肿瘤或制备泛肿瘤辅助诊断试剂盒中的应用。
一种泛肿瘤辅助诊断试剂盒，该试剂盒中含有血清标志物let‐7b‐5p,miR‐106a‐5p,miR‐10b‐5p,miR‐122‐5p,miR‐133a‐3p,miR‐140‐3p,miR‐143‐3p,miR‐146a‐5p,miR‐151a‐3p,miR‐16‐5p,miR‐192‐5p,miR‐195‐5p,miR‐19a‐3p,miR‐19b‐3p,miR‐204‐5p,miR‐20a‐5p,miR‐20b‐5p,miR‐21‐5p,miR‐221‐3p,miR‐223‐3p,miR‐223‐5p,miR‐24‐3p,miR‐25‐3p,miR‐296‐5p,miR‐29c‐3p,miR‐423‐3p,miR‐425‐5p,miR‐451a,miR‐484,miR‐485‐3p,miR‐590‐5p,miR‐92a‐3p,miR‐92b‐3p和miR‐93‐5p；以及血浆标志物let‐7b‐5p,miR‐106a‐5p,miR‐122‐5p,miR‐125b‐5p,miR‐140‐3p,miR‐142‐3p,miR‐144‐3p,miR‐146a‐5p,miR‐151a‐5p,miR‐17‐5p,miR‐181a‐5p,miR‐185‐5p,miR‐18a‐5p,miR‐192‐5p,miR‐193b‐3p,miR‐19b‐3p,miR‐205‐5p,miR‐20a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐2110,miR‐21‐5p,miR‐221‐3p,miR‐223‐3p,miR‐25‐3p,miR‐409‐3p,miR‐425‐5p,miR‐451a,miR‐486‐5p,miR‐584‐5p,miR‐92b‐3p和miR‐93‐5p中的一种或多种miRNA的引物；优选为包含上述所有血清标志物和血浆标志物的引物。
一种泛肿瘤辅助诊断试剂盒，该试剂盒中含有血清癌胚miRNA标志物miR‐19b‐3p,miR‐20a‐5p,miR‐223‐3p,miR‐93‐5p,miR‐25‐3p,miR‐425‐5p,miR‐19a‐3p,miR‐92a‐3p和miR‐20b‐5p以及血浆癌胚miRNA标志物miR‐106a‐5p,miR‐146a‐5p,miR‐18a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐21‐5p,miR‐409‐3p和miR‐584‐5p中两种或两种以上miRNA的引物；进一步优选的，该试剂盒包含癌胚血清miR‐19b‐3p,miR‐20a‐5p,miR‐223‐3p,miR‐93‐5p,miR‐25‐3p,miR‐425‐5p,miR‐19a‐3p,miR‐92a‐3p和miR‐20b‐5p以及癌胚血浆miR‐106a‐5p,miR‐146a‐5p,miR‐18a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐21‐5p,miR‐409‐3p和miR‐584‐5p十七种miRNA的引物。
上述的试剂盒中还可以包括PCR技术常用的试剂，如逆转录酶，缓冲液，dNTPs，MgCl 2，DEPC水和Taq酶等；还可以含有标准品和/或对照品。
本发明所涉及的与泛肿瘤诊断相关的循环miRNA标志物let‐7b‐5p，miR‐106a‐5p，miR‐ 10b‐5p，miR‐122‐5p，miR‐125b‐5p，miR‐133a‐3p，miR‐140‐3p，miR‐142‐3p，miR‐143‐3p，miR‐144‐3p，miR‐146a‐5p，miR‐151a‐3p，miR‐151a‐5p，miR‐16‐5p，miR‐17‐5p，miR‐181a‐5p，miR‐185‐5p，miR‐18a‐5p，miR‐192‐5p，miR‐193b‐3p，miR‐195‐5p，miR‐19a‐3p，miR‐19b‐3p，miR‐204‐5p，miR‐205‐5p，miR‐20a‐5p，miR‐20b‐5p，miR‐210‐3p，miR‐2110，miR‐21‐5p，miR‐221‐3p，miR‐223‐3p，miR‐223‐5p，miR‐24‐3p，miR‐25‐3p，miR‐296‐5p，miR‐29c‐3p，miR‐409‐3p，miR‐423‐3p，miR‐425‐5p，miR‐451a，miR‐484，miR‐485‐3p，miR‐486‐5p，miR‐584‐5p，miR‐590‐5p，miR‐92a‐3p，miR‐92b‐3p和miR‐93‐5p中的每种miRNA的序列均已公开，但是将各miRNA标志物进行组合作为泛肿瘤辅助诊断标志物需要本领域技术人员付出创造性劳动。各miRNA标志物的扩增引物均可通过市场购买获得，本发明实施例中使用的循环miRNA标志物的引物为购自广州锐博公司所合成生产的特异性miRNA茎环RT‐PCR引物，但不限于此。
具体地说，本发明解决问题的技术方案包括：(1)建立统一标准的标本库和数据库：以标准操作程序(SOP)采集符合标准的血液样本，系统收集完整的人口学资料和临床资料。(2)血清及血浆miRNA差异表达谱分析：分析包括常见的15类常见的肿瘤患者和年龄性别匹配的正常对照人群中差异表达的循环miRNA，并对差异表达miRNAs进行进一步大样本多阶段验证。(3)通过多阶段的验证，明确这些miRNA诊断15类常见的肿瘤的能力。(4)通过纳入并分析脐带血与正常成年人群中差异表达的循环miRNA，与泛肿瘤的miRNA差异表达谱进行比较分析获得癌胚miRNA，明确癌胚miRNA诊断泛肿瘤的能力。(5)循环miRNA诊断试剂盒的研制：根据泛肿瘤患者与正常人群血清及血浆中的差异表达miRNA以及癌胚miRNA开发miRNAs诊断试剂盒，实现对临床常见的15类泛肿瘤患者的无创辅助诊断。(6)分析这些miRNA在泛肿瘤组织中的表达情况，揭示这些miRNA与临床常见的15类泛肿瘤的关系，为将来研制可能与这些miRNA相关的治疗泛肿瘤的药物提供依据。
本发明人以标准操作程序(SOP)采集符合标准的血液样本，系统收集完整的人口学资料、临床资料，并采用了Exiqon miRNA qPCR panel芯片以及qRT‐PCR方法等。
具体来说研究的实验方法主要包括以下几个部分：
1.研究样本选择：初治、未行手术以及放化疗干预且经病理确认为临床常见的15类泛肿瘤患者(具体包括：鼻咽癌、甲状腺癌、食管癌、肺鳞癌、肺腺癌、贲门癌、胃腺癌、胰腺癌、结肠癌、直肠癌、前列腺癌、乳腺癌、子宫内膜癌、宫颈癌和卵巢癌)以及新生儿脐带血。正常对照为在医院进行体检的正常成年人群。
2.Exiqon miRNA qPCR panel芯片初筛：利用TRIZOL‐LS试剂对血清、血浆样本进行RNA 提取，并进行qRT‐PCR操作获得初筛结果。
3.训练集、测试集以及额外验证集：利用AM1556试剂盒(ABI公司)对每个血清、血浆样本进行RNA提取，通过逆转录反应得到cDNA样品，加入PCR引物和SYBR Green荧光染料进行PCR反应。通过比对spike‐in control的Ct值，利用相对定量法获得样本中的miRNA的相对含量。
4.统计分析：运用χ 2检验、配对t检验以及非参数秩和检验比较miRNA表达水平在不同研究组中的差异。分析泛肿瘤患者中的差异miRNA与脐带血中的差异miRNA具有一致的表达趋势，则认定为癌胚miRNA。通过计算ROC曲线分析证实血清及血浆miRNA的诊断价值。
本发明研究组目前通过对临床常见的15类泛肿瘤患者(具体包括：鼻咽癌、甲状腺癌、食管癌、肺鳞癌、肺腺癌、贲门癌、胃腺癌、胰腺癌、结肠癌、直肠癌、前列腺癌、乳腺癌、子宫内膜癌、宫颈癌和卵巢癌)以及新生儿脐带血的血清及血浆中的miRNA进行系统的表达分析，现已发现了一组具有临床诊断潜能的泛肿瘤循环miRNA标志物(血清标志物let‐7b‐5p,miR‐106a‐5p,miR‐10b‐5p,miR‐122‐5p,miR‐133a‐3p,miR‐140‐3p,miR‐143‐3p,miR‐146a‐5p,miR‐151a‐3p,miR‐16‐5p,miR‐192‐5p,miR‐195‐5p,miR‐19a‐3p,miR‐19b‐3p,miR‐204‐5p,miR‐20a‐5p,miR‐20b‐5p,miR‐21‐5p,miR‐221‐3p,miR‐223‐3p,miR‐223‐5p,miR‐24‐3p,miR‐25‐3p,miR‐296‐5p,miR‐29c‐3p,miR‐423‐3p,miR‐425‐5p,miR‐451a,miR‐484,miR‐485‐3p,miR‐590‐5p,miR‐92a‐3p,miR‐92b‐3p和miR‐93‐5p以及血浆标志物let‐7b‐5p,miR‐106a‐5p,miR‐122‐5p,miR‐125b‐5p,miR‐140‐3p,miR‐142‐3p,miR‐144‐3p,miR‐146a‐5p,miR‐151a‐5p,miR‐17‐5p,miR‐181a‐5p,miR‐185‐5p,miR‐18a‐5p,miR‐192‐5p,miR‐193b‐3p,miR‐19b‐3p,miR‐205‐5p,miR‐20a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐2110,miR‐21‐5p,miR‐221‐3p,miR‐223‐3p,miR‐25‐3p,miR‐409‐3p,miR‐425‐5p,miR‐451a,miR‐486‐5p,miR‐584‐5p,miR‐92b‐3p和miR‐93‐5p。以及癌胚miRNA标志物：其中血清癌胚miRNA标志物miR‐19b‐3p,miR‐20a‐5p,miR‐223‐3p,miR‐93‐5p,miR‐25‐3p,miR‐425‐5p,miR‐19a‐3p,miR‐92a‐3p和miR‐20b‐5p；血浆癌胚miRNA标志物miR‐106a‐5p,miR‐146a‐5p,miR‐18a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐21‐5p,miR‐409‐3p和miR‐584‐5p)。
本发明的有益效果：
1.相较于传统的肿瘤标志物，循环miRNA作为新型的生物标志物，具有稳定性好、微创易获取、灵敏性和特异性高的特点。这类分子标志物的开发利用将为包括肿瘤在内的各种疾病的诊断以及进一步治疗提供新的方向。
2.研究者通过Exiqon miRNA qPCR panel芯片以及基于qRT‐PCR的相对定量法，对泛肿瘤以及脐带血和正常对照人群血清及血浆中的差异表达miRNA进行严密、多阶段的验证和评 价。证实了循环miRNA以及癌胚miRNA作为诊断泛肿瘤的无创标记物的可靠性以及可重复性。
3.研究者发现利用TCGA数据库分析发现，血清癌胚miRNA标志物miR‐19b‐3p,miR‐20a‐5p,miR‐223‐3p,miR‐93‐5p,miR‐25‐3p,miR‐425‐5p,miR‐19a‐3p,miR‐92a‐3p和miR‐20b‐5p以及血浆癌胚miRNA标志物miR‐106a‐5p,miR‐146a‐5p,miR‐18a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐21‐5p,miR‐409‐3p和miR‐584‐5p在泛肿瘤组织标本中大部分都是高表达的状态，与癌胚miRNA在相应肿瘤中的表达一致。这些结果将给未来研究miRNA对于泛肿瘤的机制以及针对这些miRNA对于泛肿瘤的治疗提供新的思路。
附图说明
图1：实验流程图(除了贲门癌、胃癌、卵巢癌的血清样本以及卵巢癌的血浆样本采用了初筛、验证集和测试集三阶段，其余所有研究均采用初筛、验证集、测试集和额外验证集四阶段)
图2：在脐带血中的血清及血浆中分别高表达的18和16个miRNA
A：血清样本；B：血浆样本
图3：在15种临床常见的泛肿瘤中获得的可用于诊断相对应肿瘤的miRNA总览。
A：根据15种肿瘤相对应的瘤种获得的用于诊断该肿瘤的miRNA；B：用于诊断泛肿瘤的循环miRNA(左图：血清；右图：血浆)及癌胚miRNA(红色标记)在染色体上的位置
图4：血清癌胚miRNA(A‐H)和血浆癌胚miRNA(I‐N)诊断泛肿瘤的能力。
图5：癌胚miRNA在TCGA组织库中泛肿瘤中的表达情况。
具体实施方式
发明人于2010年至2018年从南京医科大学第一附属医院收集了大量的泛肿瘤患者(具体包括：鼻咽癌、甲状腺癌、食管癌、肺鳞癌、肺腺癌、贲门癌、胃腺癌、胰腺癌、结肠癌、直肠癌、前列腺癌、乳腺癌、子宫内膜癌、宫颈癌和卵巢癌)以及新生儿脐带血和正常体检人群的血清及血浆样品，通过对样品资料的整理，从中选择了3831例肿瘤标本(2050例血清标本和1781例血浆标本)、248例脐带血标本(120例血清标本和128例血浆标本)以及相匹配的共4148例正常成年人对照(2119例血清标本和2029例血浆标本)的样本作为Exiqon miRNA qPCR panel芯片初筛和后续一系列qRT‐PCR验证的实验样品。所选择的患者血清及血浆样本均来自于初治、未行手术以及放化疗干预且经病理确认为肿瘤的病人。脐带血为婴儿分娩之后即立即采集的婴儿脐带血。并系统采集了这些样本的人口学资料、临床 资料。具体各瘤种、脐带血及正常对照样本的总例数及分阶段的例数见附表中的表1。
参照流程图(图1)，从泛肿瘤、脐带血以及正常对照血清、血浆样本中各随机选择了20/30/40例泛肿瘤或者脐带血样本以及10例正常对照，分别混合成了相对应的肿瘤或者脐带血血清、血浆混合样本各2/3/4例和正常混合样本1个(一个混合样本由10例200ul血清或血浆样本汇合形成2ml的样本)。对这些混合样本进行Exiqon miRNA qPCR panel芯片初筛和分析，具体步骤参照Exiqon miRNA qPCR panel芯片的说明书：
1.血清/血浆抽提
取出血清/血浆样品，样品化冻后3000g离心5min去除一些碎片及一些不溶成分。转移上清至新的1.5ml管中，加入750ul TRIZOL‐LS后，剧烈震荡5s。
2.两相分离
匀浆后样品于15到30℃孵育5分钟。每1ml的TRIZOL‐LS试剂匀浆的样品中加入0.2ml的氯仿，盖紧管盖。手动剧烈振荡管体15秒后，15到30℃孵育2到3分钟。4℃下13,000g离心15分钟。
3.RNA沉淀
将水相转移到新离心管中。水相与异丙醇混合以沉淀其中的RNA，加入异丙醇的量为：每个样品匀浆时加入1ml TRIZOL‐LS试剂的同时加0.5ml的异丙醇和5ul的糖元。4℃静置半小时，让RNA尽可能的析出。于4℃13,000g离心15分钟。
4.RNA清洗
移去上清液，每1ml TRIZOL‐LS试剂匀浆的样品中加入至少1ml的75％(v/v)乙醇，清洗RNA沉淀。静置10分钟，然后4℃下10000g离心5分钟。
5.重新溶解RNA沉淀
去除乙醇溶液，空气中干燥RNA沉淀5‐10分钟，加入无RNA酶的水用枪反复吹打几次，然后55到60℃孵育10分钟。
6.测量浓度：
通常能得到～5μg RNA/50ml血清/血浆。
7.cDNA合成
(1)稀释模板RNA：使用DEPC水将20－25ng模板RNA稀释至14ul。
(2)准备反应液：将5×Reaction Buffer以及DEPC水置于冰上溶解，并震荡混匀。Enzyme mix置于‐20℃冰盒，使用前轻弹混匀后置于冰上。所有试剂均离心后使用。
(3)配置反应液：配置下表中的反应液
Figure PCTCN2020077198-appb-000001
(4)混合并离心试剂：震荡或者抽吸混匀反应液后离心，以保证所有溶液彻底混合均匀。
(5)逆转录反应及热失活：将反应液于42℃温育60分钟后，于95℃温育5分钟以失活逆转录酶。
8.Real‐Time PCR
试剂：
Nuclease free water(Exiqon)
SYBR TM Green master mix(Exiqon)
cDNA模板
ROX(Invitrogen)
miRNA PCR ARRAY(Exiqon)
仪器：
ABI PRISM7900system(Applied Biosystems)
(1)准备Real‐time PCR试剂：将制备的cDNA模板，DEPC水和SYBR TM Green master mix置于冰上溶解15‐20分钟。
(2)稀释cDNA模板：将RT反应获得的cDNA模板用nuclease free water稀释110倍(例如，向20μl反应液中加入2180ul nuclease free water)。
(3)混合所有反应试剂：
A.将PCR板简单离心后，移去封膜。
B.将110倍稀释的cDNA模板与2×SYBR Green master mix按照1：1混合。
C.倒置混匀反应液并离心。
D.将混合反应液加入板中的每个孔。
E.重新封好PCR板。
(4)将PCR板简单低温离心
(5)Real‐time PCR扩增：根据下表中的反应条件进行Real‐time PCR扩增和溶解曲线分析。
Real‐time PCR循环条件如下表：
Figure PCTCN2020077198-appb-000002
数据分析：采用ΔΔCt方法
使用PCR仪器附带的软件进行初步数据分析，获得原始的Cq值(Cp或Ct，依仪器不同名称可能不同)。
我们建议使用GenEx qPCR分析软件(www.exiqon.com/mirna‐pcr‐analysis)对数据进行标准和深入的数据分析。
a.计算每个处理组中的每个通路相关基因的ΔCt。
ΔCt(group 1)＝average Ct–average of HK genes’Ct for group 1array
ΔCt(group 2)＝average Ct–average of HK genes’Ct for group 2array
b.计算2个PCR Array(或两组)中每个基因的ΔΔCt。
ΔΔCt＝ΔCt(组2)‐ΔCt(组1)
备注：通常组1是对照，组2是实验组。
c.通过2‐ΔΔCt计算组2与组1对应基因的表达差异。
在芯片初筛后，分别得到了相对应的肿瘤及脐血相对应的血清和血浆差异表达miRNA(每个肿瘤、脐血的血清/血浆混合样本中相对于正常样本都超过了1.5倍差异可被选为备选miRNA)。
对于初筛得到的各瘤种及脐血中差异表达的血清miRNA和血浆miRNA，通过训练集、测试集及额外验证集(贲门癌、胃癌、卵巢癌的血清样本以及卵巢癌的血浆样本采用了初筛、验证集和测试集三阶段)，采用基于qRT‐PCR的相对定量法进行验证，具体步骤为：
1.血清/血浆RNA提取：选用ABI公司血清RNA提取试剂盒(AM1556)，参照试剂盒说明，每个样本吸取200ul进行提取RNA，并最后用100ul DEPC水进行溶解。
2.cDNA的制备：
1)采用50μL反应体系进行逆转录实验
Figure PCTCN2020077198-appb-000003
Figure PCTCN2020077198-appb-000004
以上反应体系混匀，瞬时离心后，以下列程序进行反应：
Figure PCTCN2020077198-appb-000005
2)上述反应之后再反应体系中再加入如下反应物
Figure PCTCN2020077198-appb-000006
3.qPCR
1)采用5μL反应体系，按以下比例进行试验
Figure PCTCN2020077198-appb-000007
反应体系混匀，瞬时离心后，放置于实时定量PCR仪中，以下列程序进行反应：
Figure PCTCN2020077198-appb-000008
Figure PCTCN2020077198-appb-000009
反应结束后添加溶解曲线。
数据分析：利用SPSS 16.0软件进行统计分析，对在训练集、测试集以及额外验证集中均一致差异表达的miRNA认定为该肿瘤的诊断标志物。在泛肿瘤患者血清及血浆中各阶段一致差异表达的miRNA见附表中的表2。
同时，通过脐带血与正常成人样本的分析，获得脐血血清差异表达miRNA以及血浆差异表达miRNA(图2)，脐血血清中差异表达的miRNA见附表的表3。脐血血浆中差异表达的miRNA见附表的表4。
对所有15种肿瘤及脐血中的差异miRNA进行分析(图3)，得到9个血清癌胚miRNA(分别为miR‐19b‐3p,miR‐20a‐5p,miR‐223‐3p,miR‐93‐5p,miR‐25‐3p,miR‐425‐5p,miR‐19a‐3p,miR‐92a‐3p和miR‐20b‐5p)及8个血浆癌胚miRNA(分别为miR‐106a‐5p,miR‐146a‐5p,miR‐18a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐21‐5p,miR‐409‐3p和miR‐584‐5p)。进一步分析这些癌胚miRNA对相应的肿瘤的诊断能力。如图4，血清癌胚miRNA(A‐H分别为：鼻咽癌、甲状腺癌、结肠癌、直肠癌、胰腺癌、肺鳞癌、乳腺癌和子宫内膜癌)及血浆癌胚miRNA(I‐N分别为：鼻咽癌、食管癌、胃癌、肺鳞癌、肺腺癌和子宫内膜癌)能很好的诊断出相应的肿瘤患者。
研究组之后进一步对这些癌胚miRNA在TCGA数据库中泛肿瘤组织中的表达进行了分析，如图5所示，这些miRNA大多数在泛肿瘤中呈高表达，这些结果将给未来研究miRNA对于泛肿瘤的机制以及针对这些miRNA对于泛肿瘤的治疗提供新的思路。
试剂盒包括一批血清及血浆miRNA qRT‐PCR引物，还可以有相应PCR技术所需的常用试剂，如：逆转录酶，缓冲液，dNTPs，MgCl2，DEPC水，荧光探针，RNA酶抑制剂，Taq酶等，可根据具体采用的实验方法选用，这些常用试剂都是本领域技术人员熟知的，另外还可以有标准品和对照(如定量标化的正常人样本等)。此试剂盒的价值在于只需要血清/血浆而不需要其它组织样品，通过最精简的荧光法检测血清/血浆样本中miRNA的表达含量，来辅助诊断该样本来源患者的患泛肿瘤的可能性。血清及血浆miRNA检测方便，且定量精确，大大提高疾病诊断的敏感性和特异性，因此将此试剂盒投入实践，可以帮助指导诊断以及进一步的个体化治疗。
Figure PCTCN2020077198-appb-000010
Figure PCTCN2020077198-appb-000011
Figure PCTCN2020077198-appb-000012
Figure PCTCN2020077198-appb-000013
Figure PCTCN2020077198-appb-000014

Claims (11)

  1. 一种与泛肿瘤辅助诊断相关的循环癌胚miRNA标志物，其特征在于该循环癌胚miRNA标志物包含血清癌胚miRNA标志物miR‐19b‐3p,miR‐20a‐5p,miR‐223‐3p,miR‐93‐5p,miR‐25‐3p,miR‐425‐5p,miR‐19a‐3p,miR‐92a‐3p和miR‐20b‐5p；以及血浆癌胚miRNA标志物miR‐106a‐5p,miR‐146a‐5p,miR‐18a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐21‐5p,miR‐409‐3p和miR‐584‐5p中两种或两种以上miRNA的组合；优选的，包含血清癌胚miRNA标志物miR-19b-3p,miR-20a-5p,miR-223-3p,miR-93-5p,miR-25-3p,miR-425-5p,miR-19a-3p,miR-92a-3p和miR-20b-5p以及血浆癌胚miRNA标志物miR-106a-5p,miR-146a-5p,miR-18a-5p,miR-20b-5p,miR-210-3p,miR-21-5p,miR-409-3p和miR-584-5p十七种miRNA标志物。
  2. 权利要求1所述的循环癌胚miRNA标志物在辅助诊断泛肿瘤、制备泛肿瘤辅助诊断试剂盒或制备治疗泛肿瘤药物中的应用。
  3. 一种与泛肿瘤辅助诊断相关的循环癌胚miRNA标志物的引物，其特征在于该引物包含血清癌胚miRNA标志物miR‐19b‐3p,miR‐20a‐5p,miR‐223‐3p,miR‐93‐5p,miR‐25‐3p,miR‐425‐5p,miR‐19a‐3p,miR‐92a‐3p和miR‐20b‐5p；以及血浆癌胚miRNA标志物miR‐106a‐5p,miR‐146a‐5p,miR‐18a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐21‐5p,miR‐409‐3p和miR‐584‐5p中两种或两种以上miRNA引物的组合；优选的，包含血清癌胚miRNA标志物miR‐19b‐3p,miR‐20a‐5p,miR‐223‐3p,miR‐93‐5p,miR‐25‐3p,miR‐425‐5p,miR‐19a‐3p,miR‐92a‐3p和miR‐20b‐5p；以及血浆癌胚miRNA标志物miR‐106a‐5p,miR‐146a‐5p,miR‐18a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐21‐5p,miR‐409‐3p和miR‐584‐5p十七种miRNA的引物。
  4. 权利要求3所述的引物在辅助诊断泛肿瘤或制备泛肿瘤辅助诊断试剂盒中的应用。
  5. 一种泛肿瘤辅助诊断试剂盒，其特征在于该试剂盒中含有血清癌胚miRNA标志物miR‐19b‐3p,miR‐20a‐5p,miR‐223‐3p,miR‐93‐5p,miR‐25‐3p,miR‐425‐5p,miR‐19a‐3p,miR‐92a‐3p和miR‐20b‐5p；以及血浆癌胚miRNA标志物miR‐106a‐5p,miR‐146a‐5p,miR‐18a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐21‐5p,miR‐409‐3p和miR‐584‐5p中两种或两种以上miRNA引物的组合；优选的，含有血清癌胚miRNA标志物miR‐19b‐3p,miR‐20a‐5p,miR‐223‐3p,miR‐93‐5p,miR‐25‐3p,miR‐425‐5p,miR‐19a‐3p,miR‐92a‐3p和miR‐20b‐5p；以及血浆癌胚miRNA标志物miR‐106a‐5p,miR‐146a‐5p,miR‐18a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐21‐5p,miR‐409‐3p和miR‐584‐5p十七种miRNA的引物。
  6. 一种与泛肿瘤辅助诊断相关的循环miRNA标志物，其特征在于该循环miRNA标志物包含血清标志物let‐7b‐5p,miR‐106a‐5p,miR‐10b‐5p,miR‐122‐5p,miR‐133a‐3p,miR‐140‐3p,miR‐143‐3p,miR‐146a‐5p,miR‐151a‐3p,miR‐16‐5p,miR‐192‐5p,miR‐195‐5p,miR‐19a‐3p,miR‐19b‐3p, miR‐204‐5p,miR‐20a‐5p,miR‐20b‐5p,miR‐21‐5p,miR‐221‐3p,miR‐223‐3p,miR‐223‐5p,miR‐24‐3p,miR‐25‐3p,miR‐296‐5p,miR‐29c‐3p,miR‐423‐3p,miR‐425‐5p,miR‐451a,miR‐484,miR‐485‐3p,miR‐590‐5p,miR‐92a‐3p,miR‐92b‐3p和miR‐93‐5p；以及血浆标志物let‐7b‐5p,miR‐106a‐5p,miR‐122‐5p,miR‐125b‐5p,miR‐140‐3p,miR‐142‐3p,miR‐144‐3p,miR‐146a‐5p,miR‐151a‐5p,miR‐17‐5p,miR‐181a‐5p,miR‐185‐5p,miR‐18a‐5p,miR‐192‐5p,miR‐193b‐3p,miR‐19b‐3p,miR‐205‐5p,miR‐20a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐2110,miR‐21‐5p,miR‐221‐3p,miR‐223‐3p,miR‐25‐3p,miR‐409‐3p,miR‐425‐5p,miR‐451a,miR‐486‐5p,miR‐584‐5p,miR‐92b‐3p和miR‐93‐5p中的一种或多种。
  7. 权利要求6所述的循环miRNA标志物在辅助诊断泛肿瘤、制备泛肿瘤辅助诊断试剂盒或制备治疗泛肿瘤药物中的应用。
  8. 一种与泛肿瘤辅助诊断相关的循环miRNA标志物的引物，其特征在于该引物包含血清标志物let‐7b‐5p,miR‐106a‐5p,miR‐10b‐5p,miR‐122‐5p,miR‐133a‐3p,miR‐140‐3p,miR‐143‐3p,miR‐146a‐5p,miR‐151a‐3p,miR‐16‐5p,miR‐192‐5p,miR‐195‐5p,miR‐19a‐3p,miR‐19b‐3p,miR‐204‐5p,miR‐20a‐5p,miR‐20b‐5p,miR‐21‐5p,miR‐221‐3p,miR‐223‐3p,miR‐223‐5p,miR‐24‐3p,miR‐25‐3p,miR‐296‐5p,miR‐29c‐3p,miR‐423‐3p,miR‐425‐5p,miR‐451a,miR‐484,miR‐485‐3p,miR‐590‐5p,miR‐92a‐3p,miR‐92b‐3p和miR‐93‐5p；以及血浆标志物let‐7b‐5p,miR‐106a‐5p,miR‐122‐5p,miR‐125b‐5p,miR‐140‐3p,miR‐142‐3p,miR‐144‐3p,miR‐146a‐5p,miR‐151a‐5p,miR‐17‐5p,miR‐181a‐5p,miR‐185‐5p,miR‐18a‐5p,miR‐192‐5p,miR‐193b‐3p,miR‐19b‐3p,miR‐205‐5p,miR‐20a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐2110,miR‐21‐5p,miR‐221‐3p,miR‐223‐3p,miR‐25‐3p,miR‐409‐3p,miR‐425‐5p,miR‐451a,miR‐486‐5p,miR‐584‐5p,miR‐92b‐3p和miR‐93‐5p中一种或多种miRNA的引物。
  9. 权利要求8所述的引物在辅助诊断泛肿瘤或制备泛肿瘤辅助诊断试剂盒中的应用。
  10. 一种泛肿瘤辅助诊断试剂盒，其特征在于该试剂盒中含有循环miRNA中血清let‐7b‐5p,miR‐106a‐5p,miR‐10b‐5p,miR‐122‐5p,miR‐133a‐3p,miR‐140‐3p,miR‐143‐3p,miR‐146a‐5p,miR‐151a‐3p,miR‐16‐5p,miR‐192‐5p,miR‐195‐5p,miR‐19a‐3p,miR‐19b‐3p,miR‐204‐5p,miR‐20a‐5p,miR‐20b‐5p,miR‐21‐5p,miR‐221‐3p,miR‐223‐3p,miR‐223‐5p,miR‐24‐3p,miR‐25‐3p,miR‐296‐5p,miR‐29c‐3p,miR‐423‐3p,miR‐425‐5p,miR‐451a,miR‐484,miR‐485‐3p,miR‐590‐5p,miR‐92a‐3p,miR‐92b‐3p和miR‐93‐5p以及血浆let‐7b‐5p,miR‐106a‐5p,miR‐122‐5p,miR‐125b‐5p,miR‐140‐3p,miR‐142‐3p,miR‐144‐3p,miR‐146a‐5p,miR‐151a‐5p,miR‐17‐5p,miR‐181a‐5p,miR‐185‐5p,miR‐18a‐5p,miR‐192‐5p,miR‐193b‐3p,miR‐19b‐3p,miR‐205‐5p,miR‐20a‐5p,miR‐20b‐5p,miR‐210‐3p,miR‐2110,miR‐21‐5p,miR‐221‐3p,miR‐223‐3p,miR‐25‐3p,miR‐409‐3p,miR‐425‐5p,miR‐ 451a,miR‐486‐5p,miR‐584‐5p,miR‐92b‐3p和miR‐93‐5p中一种或多种miRNA的引物。
  11. 根据权利要求5或10所述的泛肿瘤辅助诊断试剂盒，其特征在于该试剂盒中还包括PCR技术常用的试剂。
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