WO2020001473A1 - Use of small nucleolar ribonucleic acid snord33 as biomarker for preparation of detection kit - Google Patents

Use of small nucleolar ribonucleic acid snord33 as biomarker for preparation of detection kit Download PDF

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WO2020001473A1
WO2020001473A1 PCT/CN2019/092995 CN2019092995W WO2020001473A1 WO 2020001473 A1 WO2020001473 A1 WO 2020001473A1 CN 2019092995 W CN2019092995 W CN 2019092995W WO 2020001473 A1 WO2020001473 A1 WO 2020001473A1
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snord33
plasma
platinum
rna
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PCT/CN2019/092995
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Chinese (zh)
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陈舌
王碧芸
张思
胡夕春
赵燕南
李懿
张力
徐莺莺
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复旦大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the invention belongs to the technical field of biological diagnostic detection, relates to the separation, qualitative and quantitative analysis of human plasma small nucleolar ribonucleic acid (SNORD33), and relates to the prediction of the efficacy of breast cancer patients. Specifically, it relates to the use of SNORD33 as a biomarker in the preparation of a detection kit, and more specifically, it relates to the use of the level of small nucleolar ribonucleic acid in the plasma to predict the efficacy of platinum-based drugs in triple-negative breast cancer patients. Effectiveness, duration of disease control, and prognosis of patients with negative breast cancer after receiving platinum drugs. The invention further relates to a prediction method for improving the effectiveness of platinum-based drugs.
  • TNBC triple negative breast cancer
  • ER estrogen receptor
  • PR progesterone receptor
  • HER2 human epidermal growth factor receptor 2
  • Existing predictive indicators including BRCA1 / 2 mutation, HRD-LOH, HRD-LST, HRD-TAI and other indicators, are still uncertain about the platinum curative effect of triple-negative breast cancer, and it is urgent to predict molecules with positive effects and convenient detection Markers predict the efficacy of platinum.
  • Small nucleolar ribonucleic acid is a kind of small molecule non-coding RNA, which is conservative in structure, stable in metabolism, multi-enriched in nucleoli, and widely present in nucleated cells; its main function is to participate in ribosomal RNA (RNA), small
  • RNA ribosomal RNA
  • the modification and maturation of various RNA precursors, including nuclear RNA (small RNA) affects the ribosome biosynthesis and the splicing function of spliceosome.
  • Small nucleolar RNAs often bind to specific RNA-binding proteins and play an important role in the development of diseases including genetics, hematopoietics, metabolism, and nervous system.
  • small nucleolar RNAs have been changed to varying degrees in various tumors such as B-cell lymphoma, prostate cancer, and breast cancer. After comparing 14 types of tumors including breast cancer, kidney cancer, and prostate cancer, 13 of them were found. There are specific small nucleoli RNA changes in tumors compared with normal tissues. These phenomena suggest that the occurrence and development of tumors are related to small nucleoli RNAs. At the same time, small nucleolar RNAs are abundant in plasma RNA and are related to intracellular Small nucleolar RNAs have a good correlation and can be used as a new biomarker for liquid biopsy to predict the prognosis and drug sensitivity of patients.
  • RNA SNORD33 As a biomarker for preparing a detection kit.
  • the purpose of the present invention is to provide the use of small nucleolar ribonucleic acid SNORD33 as a biomarker in the preparation of a detection kit based on the basis and status of the prior art.
  • the invention relates to a method for applying specific changes in plasma SNORD33 to predicting platinum treatment sensitivity in patients with triple-negative breast cancer, and the application of a primer for qualitatively or quantitatively detecting human plasma SNORD33 in a kit for predicting platinum curative effect.
  • Another object of the present invention is to provide a kit for in vitro prediction of triple negative breast cancer.
  • SNORD33 can be used as a biomarker to predict the efficacy of platinum-based drugs in TNBC patients; compared with the plasma samples of triple negative breast cancer subjects who received platinum-based treatment, SNORD33 Levels of plasma in triple negative breast cancer subjects who were not responding to platinum therapy were significantly reduced, and SNORD33 was present in plasma samples with high levels of triple negative breast cancer subjects who had progress-free survival -free survival (PFS) was significantly prolonged compared to subjects with low levels of SNORD33 in plasma samples; patients with TNBC with low levels of plasma small nucleolar RNA SNORD33 had low cisplatin treatment efficiency and short progression-free survival time Therefore, the present invention proposes a method of "predicting the efficacy of platinum-based treatment for TNBC patients by detecting plasma SNORD33 levels". It can predict the efficiency and duration of disease control after TNBC patients receive platinum, and choose whether to use platinum therapy according to the patient's plasma SNORD33 level before treatment, so as to achieve the purpose of personalized and precise treatment
  • the present invention solves problems such as the prediction of the efficacy of platinum drugs in TNBC patients by detecting SNORD33 in plasma; more specifically, it provides the use of small nucleolar RNA SNORD33 as a biomarker for preparing a detection kit.
  • a detection kit is prepared by using the small nucleolar RNA SNORD33 as a biomarker.
  • the primer for human plasma SNORD33 is qualitatively or quantitatively detected, and SNORD33 is detected in the plasma of the triple negative breast cancer patient subject by fluorescent quantitative PCR technology.
  • Levels in a sample comparing levels of SNORD33 in a plasma sample from a subject who is effective with the platinum treatment with SNORD33 in a plasma sample of a subject who is not effective with the platinum treatment; and determining that SNORD33 predicts platinum The best cutoff for class validity.
  • platinum-based drugs can be predicted by detecting the expression of SNORD33 in plasma samples from subjects with triple negative breast cancer; and the choice of platinum-based drugs can be guided according to the subject's plasma SNORD33 expression level; high levels of plasma SNORD33 For patients with platinum, the treatment with platinum drugs is better; for patients with low levels of plasma SNORD33, other anti-tumor drugs can be considered, so it can better guide the treatment of triple negative breast cancer patients.
  • the detection method for SNORD33 can be performed by any effective method, including one or more of the quantitative PCR method, RT-PCR method, Northern blotting method, RNase protection assay, Solexa sequencing technology, and biochip method. .
  • a fluorescent quantitative PCR method includes: (1) extracting total serum / plasma RNA from a subject, and obtaining a cDNA sample by an RNA reverse transcription reaction; (2) designing a primer with a small nucleolar RNA; (3) Add fluorescent probes for PCR reaction; (4) Detect and compare the change of the amount of small nucleolar RNA in the serum / plasma sample with respect to the set prediction threshold.
  • another RT-PCR method includes the steps of: (1) extracting the total serum / plasma RNA of the subject, and obtaining a cDNA sample by an RNA reverse transcription reaction; (2) using a primer designed by small nucleoside ribonucleic acid PCR reaction; (3) agarose gel electrophoresis of PCR products; (4) observation of results under UV light after EB staining.
  • the Northern blotting method includes the steps of: (1) extracting total serum / plasma RNA of the subject (for example, using Trizol reagent); (2) denaturing PAGE electrophoresis and membrane transfer experiments; (3) preparing isotopically labeled small Nucleolar ribonucleic acid probe; (4) membrane hybridization reaction; (5) isotope signal detection, such as phosphor screen scanning test results.
  • the RNase protection method includes the steps of: (1) synthesis of an antisense RNA probe, isotope labeling and purification; (2) extraction of serum / plasma total RNA from a subject (for example, extraction using Trizol reagent); (3) dissolve the extracted RNA in hybridization buffer and add antisense RNA probes for hybridization; (4) add RNase digestion solution for reaction; (5) perform electrophoresis and autoradiography; (6) analyze the results.
  • the Solexa sequencing technology method includes the steps of: (1) extracting total serum / plasma RNA of the subject (for example, using Trizol reagent); (2) performing PAGE electrophoresis to recover 80-90nt RNA molecules; (3) adaptor The primer is linked to the 3 'and 5' ends of the small RNA molecule; (4) RT-PCR is performed and sequenced; (5) data analysis and processing.
  • the biochip method includes the steps of: (1) latticeing all small nucleolar RNA pools in human plasma and preparing a biochip; (2) extracting total RNA from a subject's plasma (for example, using Trizol reagent) (3) Separation of small nucleolar RNA by column separation; (4) Fluorescent labeling of small nucleolar RNA using T4RNA ligase; (5) Hybridization reaction with biochip; (6) Data detection and analysis.
  • the serum / plasma used in the above method of the present invention is derived from a living body, tissue, organ, or / and cadaver of a subject.
  • the expression level of SNORD33 of the present invention can be used as a clear, effective and intuitive biomarker for predicting curative effect. It diagnoses and predicts the curative effect, efficiency, duration of disease control and prognosis of triple negative breast cancer patients after receiving platinum therapy, and guides the triple negative breast.
  • the medication for cancer patients achieves the purpose of individualized treatment;
  • the platinum drugs whose efficacy is predicted are all platinum chemotherapy drugs including cisplatin, carboplatin, oxaliplatin, loplatin, and nedaplatin; detectable specimens Including serum / plasma collected from the living body, tissue, organ and / or cadaver of the subject;
  • SNORD33 as a predictive biomarker of efficacy can be detected by any effective method, including fluorescent quantitative PCR method, RT-PCR method, Northern blotting method, One or more of RNase protection, Solexa sequencing technology, and biochip methods; detection of the efficacy of a platinum-based triple-negative breast cancer platinum-based efficacy prediction kit using primers containing human plasma SNORD33 by fluorescent quantitative PCR .
  • the present invention uses plasma SNORD33 for individualized and precise treatment: for subjects with high plasma SNORD33 levels, platinum drugs are selected to have better inhibition of triple negative breast cancer; for subjects with low plasma SNORD33 levels, Consider choosing other anti-tumor therapies. Plasma SNORD33 levels are used to guide the selection of platinum-based drugs in clinically triple-negative breast cancer patients.
  • the detection of SNORD33 in plasma has the following advantages: first, the plasma is easier to obtain than tissues and can be collected in daily routine diagnosis and treatment activities; secondly, SNORD33 reflects the overall physiological and pathological conditions of the body, and its detection has guiding significance Third, SNORD33 can reflect the current state of the body in real time, which is closer to the actual situation of the body than the original tissue, and it can also monitor its real-time changes through SNORD33.
  • the detection kit for SNORD33 in plasma using small nucleolar RNA SNORD33 as a biomarker is non-invasive, simple, and has a clear predictive effect. It predicts drugs from a new perspective of specific changes in small nucleolar RNA in plasma. The curative effect and guide the treatment, the present invention will help to establish a more sensitive and accurate technical solution for individualized treatment.
  • Figure 1 An experimental plot of the amplification curve and lysis curve of SNORD33 in plasma from triple negative breast cancer patients.
  • Figure 2 Changes in plasma SNORD33 in patients who failed to respond to platinum therapy for triple-negative breast cancer compared to plasma SNORD33 in effective patients.
  • Figure 3 Differences in progression-free survival time of patients with triple-negative breast cancer with low expression of plasma SNORD33 compared with patients with high expression of plasma SNORD33.
  • the purpose of this example is to explain the requirements and specific processing methods for the serum / plasma specimens collected by the subject.
  • Plasma Plasma should be collected and separated as fresh. Plasma must be separated and collected within 6 hours of blood collection. The collected whole blood is placed in EDTA anticoagulant tube, and the plasma is transferred to disposable RNase-free In a sterile microcentrifuge tube;
  • Serum samples can be stably stored at room temperature for 24 hours, 2-8 ° C for 7 days, and -18 ° C for 6 months.
  • the serum / plasma comes from the subjects who signed the informed consent, and the subjects were not treated with platinum drugs before taking the blood;
  • RNA extracted from serum / plasma samples can be stored stably for 12 hours at room temperature, 3 days at 2-8 ° C, and 3 months at -18 ° C;
  • RNA extraction from plasma samples Use Qiagen miRNeasy Mini Kit kit and follow the manufacturer's instructions to prepare positive and negative controls while preparing plasma samples.
  • the cDNA sample obtained after reverse transcription was diluted 10-fold with DEPC water.
  • the forward sequence of the SNORD33 primer is GGCCGGTGATGAGAA
  • the reverse sequence is CGAATGTGAGTGGGAGAA
  • the forward sequence of the internal reference U6 primer is CTCGCTTCGGCAGCACA
  • the reverse sequence is AACGCTTCACGAATTTGCGT;
  • Distribution reaction mixture Distribute 18ul / well of the mixture obtained in step (2) in the corresponding 96-well plate;
  • Reaction procedure Place the 96-well plate in the ABI7500 or ABI7300 real-time PCR instrument and follow TAKARA qPCR Premix kits perform qPCR reactions. Collect the fluorescence signal at the second step of each cycle, ie, 60 ° C.
  • the linear range of the SNORD33 standard in this embodiment is 10 6 -10 3 copies / ul;
  • Accuracy is the positive coincidence rate.
  • the 10-point positive reference P1-P10 (where the concentration of SNORD33 is> 20000 copy / ul) in the quality control sample is used to determine SNORD33.
  • the test results should be positive, that is, 10/10 ;
  • the specificity is the negative compliance rate.
  • the 10-point negative reference N1-N10 in the quality control sample (where the concentration of SNORD33 is less than 20000copy / ul) is used to measure SNORD33.
  • the test results should be negative, that is, 10/10 ;
  • the minimum detection threshold of SNORD33 in this patent should be ⁇ 10 3 copies / ul, and the standard of 10 3 copies / ul of SNORD33 should be detected 20 times. At least 17 times the detection results are higher than the minimum detection threshold.
  • the negative control after the extraction should be satisfied: 10 3 copies / ul ⁇ SNORD33 relative copy number ⁇ 104 copies / ul, the endogenous control Ct ⁇ 26.5;
  • the blank control should satisfy: the relative copy number of SNORD33 ⁇ 10 3 copies / ul, and the internal control Ct ⁇ 26.5;
  • the predicted cut-off value of SNORD33 expression level in plasma samples is 0.69. Below this cut-off value, it can be defined as that patients with triple negative breast cancer are not sensitive to platinum therapy.
  • the expression of SNORD33 in the test sample is greater than 0.69. Considering that the triple platinum-negative breast cancer patients are likely to be effective in subsequent platinum therapy;
  • kits for predicting the efficacy of triple-negative breast cancer Based on the quantitative PCR technology for the preparation of small nucleolar RNA kit for predicting the efficacy of triple-negative breast cancer, the special manufacturing process and process operation.
  • the experiments include commonly used SYBR, dNTP, and the preferred human plasma SNORD33 primers. Kit for detecting plasma SNORD33 levels before platinum treatment in triple-negative breast cancer patients, predicting the sensitivity and efficacy of patients receiving platinum therapy, using plasma SNORD33 levels to individualize and precision treatment, and guide clinical triple negative Selection of platinum drugs for breast cancer patients.

Abstract

Provided are the use of a small nucleolar ribonucleic acid SNORD33 as a biomarker for predicting the efficacy of platinum in triple negative breast cancer, and the corresponding kits and detection methods.

Description

小核仁核糖核酸SNORD33作为生物标记物用于制备检测试剂盒中的用途Use of small nucleolar RNA SNORD33 as a biomarker for preparing a detection kit 技术领域Technical field
本发明属于生物诊断检测技术领域，涉及人血浆小核仁核糖核酸(SNORD33)的分离、定性和定量分析，以及涉及乳腺癌患者的疗效预测。具体涉及SNORD33作为生物标记物用于制备检测试剂盒中的用途，更具体地，涉及一种利用血浆中的小核仁核糖核酸的水平预测三阴性乳腺癌患者铂类药物治疗的疗效，判断三阴性乳腺癌患者接受铂类药物后的有效率、疾病控制时长以及预后。本发明进一步涉及用于提高铂类药物有效性的预测方法。The invention belongs to the technical field of biological diagnostic detection, relates to the separation, qualitative and quantitative analysis of human plasma small nucleolar ribonucleic acid (SNORD33), and relates to the prediction of the efficacy of breast cancer patients. Specifically, it relates to the use of SNORD33 as a biomarker in the preparation of a detection kit, and more specifically, it relates to the use of the level of small nucleolar ribonucleic acid in the plasma to predict the efficacy of platinum-based drugs in triple-negative breast cancer patients. Effectiveness, duration of disease control, and prognosis of patients with negative breast cancer after receiving platinum drugs. The invention further relates to a prediction method for improving the effectiveness of platinum-based drugs.
技术背景technical background
资料记载了乳腺癌是全世界女性中最多罹患的侵袭性癌症，同时也是引起女性肿瘤相关死亡的最常见原因之一。三阴性乳腺癌(TNBC)是***受体(ER)阴性、孕激素受体(PR)阴性、人表皮生长因子受体2(HER2)阴性的一类分子亚型，占所有类型的乳腺癌中的12–17％。研究表明，TNBC是一类具有异质性的乳腺癌亚型，其恶性程度高，具有高侵袭及转移性，患者复发比例高、疾病进展时间短、总体生存率低，是亟待解决的难点问题。The data document that breast cancer is the most invasive cancer in women worldwide and one of the most common causes of tumor-related deaths in women. Triple negative breast cancer (TNBC) is a molecular subtype of estrogen receptor (ER) negative, progesterone receptor (PR) negative, and human epidermal growth factor receptor 2 (HER2) negative, accounting for all types of breast cancer 12-17% of the total. Studies have shown that TNBC is a heterogeneous breast cancer subtype with high malignancy, high invasion and metastasis, high recurrence rate, short disease progression time, and low overall survival rate, which are difficult problems to be solved urgently. .
精准医学时代的到来使得业内对TNBC的研究日益深入，使得越来越多的靶向药物应用到TNBC的治疗中，但靶向治疗在TNBC中的疗效仍不肯定，化疗仍具有不可替代的地位。近期有研究提示铂类药物在TNBC的治疗中有着重要作用；铂类化疗药物通过与双链DNA交联致其断裂、阻滞DNA复制、转录并最终导致细胞死亡；研究表明，部分三阴性乳腺癌有着与基底样乳腺癌相似的分子特征，分子表达的一致性高达70-90％，其中11.2％-14.3％的三阴性乳腺癌患者存在BRCA1/2突变，而BRCA1基因在DNA复制过程中，与DNA双链断裂时的同源重组修复密切相关；铂类化疗药物由于增加了DNA双链的断裂，导致BRCA1突变的乳腺癌细胞在DNA双链断裂后不能修复后出现停止生长，甚至死亡。铂类在临床的应用中也显示出良好的疗效，与其他化疗联合治疗时，其有效率可达64％，但仍有相当一部分患者对铂类药物不敏感或药物控制较短时间后即出现进展。现有的预测指标，包括BRCA1/2突变、HRD-LOH、HRD-LST、HRD-TAI等指标对三阴性乳腺癌铂类疗效的预测作用仍不肯定，亟需预测作用肯定且检测便利的分子标志物对 铂类的疗效进行预测。The advent of the era of precision medicine has made the industry's research on TNBC deeper and deeper, and more and more targeted drugs have been applied to the treatment of TNBC. However, the efficacy of targeted therapy in TNBC is still uncertain, and chemotherapy still has an irreplaceable status. . Recent studies suggest that platinum drugs play an important role in the treatment of TNBC; platinum chemotherapy drugs cross-link with double-stranded DNA, causing them to break, block DNA replication, transcription, and eventually cause cell death; studies have shown that some triple-negative breasts Cancer has similar molecular characteristics to basal-like breast cancer, with molecular expression consistency as high as 70-90%, of which 11.2% -14.3% of triple-negative breast cancer patients have BRCA1 / 2 mutations, and the BRCA1 gene is in the process of DNA replication, It is closely related to the repair of homologous recombination during DNA double-strand breaks; platinum chemotherapy drugs increase the DNA double-strand breaks, causing BRCA1 mutant breast cancer cells to stop growing and even die after DNA double-strand breaks cannot be repaired. Platinum has also shown good efficacy in clinical applications. When combined with other chemotherapy, its effective rate can reach 64%, but there are still a considerable number of patients who are not sensitive to platinum drugs or the drug control occurs after a short time. progress. Existing predictive indicators, including BRCA1 / 2 mutation, HRD-LOH, HRD-LST, HRD-TAI and other indicators, are still uncertain about the platinum curative effect of triple-negative breast cancer, and it is urgent to predict molecules with positive effects and convenient detection Markers predict the efficacy of platinum.
小核仁核糖核酸是一类小分子非编码RNA，结构保守，代谢稳定，多富集于核仁，广泛存在于有核细胞中；其主要功能是参与包括核糖体RNA(ribosomal RNA)、小核RNA(small nuclear RNA)在内的多种RNA前体的修饰和成熟过程，影响核糖体的生物合成以及剪接体的剪切组装功能。小核仁核糖核酸常通过与特定RNA结合蛋白质结合，在包括遗传、造血、代谢、神经***等疾病的发生发展中扮演重要作用。且小核仁核糖核酸在B细胞淋巴瘤、***癌、乳腺癌等多种肿瘤中不同程度的改变，且在通过对比包括乳腺癌、肾癌、***癌等14种肿瘤后发现，其中13种肿瘤较正常组织出现特定若干小核仁核糖核酸的改变，这些现象提示肿瘤的发生发展与小核仁核糖核酸的相关性；同时，小核仁核糖核酸在血浆RNA中的含量丰富，与细胞内小核仁核糖核酸相关性较好，可作为一种液体活检的新的生物标志物，预测患者的预后及药物敏感性。既往研究已在包括乳腺癌在内的多种肿瘤中，验证了血浆中的小核仁核糖核酸含量与患者肿瘤临床病理恶性程度以及预后的相关性，因此，研究人员猜想小核仁核糖核酸表达改变是肿瘤发生发展的重要因素，且小核仁核糖核酸完全可以作为一种新的疾病诊断以及疗效预测的标志物，其在疾病过程中发生的特异性变化，可以帮助了解疾病的发展阶段以及预测机体对药物的反应性，对小核仁核糖核酸的研究和分析将为肿瘤的诊断和治疗提供新途径。Small nucleolar ribonucleic acid is a kind of small molecule non-coding RNA, which is conservative in structure, stable in metabolism, multi-enriched in nucleoli, and widely present in nucleated cells; its main function is to participate in ribosomal RNA (RNA), small The modification and maturation of various RNA precursors, including nuclear RNA (small RNA), affects the ribosome biosynthesis and the splicing function of spliceosome. Small nucleolar RNAs often bind to specific RNA-binding proteins and play an important role in the development of diseases including genetics, hematopoietics, metabolism, and nervous system. And small nucleolar RNAs have been changed to varying degrees in various tumors such as B-cell lymphoma, prostate cancer, and breast cancer. After comparing 14 types of tumors including breast cancer, kidney cancer, and prostate cancer, 13 of them were found. There are specific small nucleoli RNA changes in tumors compared with normal tissues. These phenomena suggest that the occurrence and development of tumors are related to small nucleoli RNAs. At the same time, small nucleolar RNAs are abundant in plasma RNA and are related to intracellular Small nucleolar RNAs have a good correlation and can be used as a new biomarker for liquid biopsy to predict the prognosis and drug sensitivity of patients. Previous studies have verified the correlation between the content of plasma small nucleoside ribonucleic acid and the clinicopathological malignancy and prognosis of patients' tumors in various tumors including breast cancer. Therefore, the researchers speculated that small nucleoli RNA expression Change is an important factor in tumorigenesis and development, and small nucleoside RNA can be used as a new marker for disease diagnosis and efficacy prediction. The specific changes that occur during the disease process can help to understand the stage of disease development and Predicting the body's response to drugs, research and analysis of small nucleolar RNAs will provide a new way for the diagnosis and treatment of tumors.
基于现有技术的基础与现状，本申请的发明人拟提供小核仁核糖核酸SNORD33作为生物标记物用于制备检测试剂盒中的用途。Based on the basis and current status of the prior art, the inventor of the present application intends to provide the use of small nucleolar RNA SNORD33 as a biomarker for preparing a detection kit.
发明内容Summary of the invention
本发明的目的是基于现有技术的基础与现状，提供小核仁核糖核酸SNORD33作为生物标记物用于制备检测试剂盒中的用途。本发明涉及将血浆SNORD33的特异性变化应用于预测三阴性乳腺癌患者铂类治疗敏感性的方法，以及涉及定性或定量检测人血浆SNORD33的引物在预测铂类疗效的试剂盒中的应用。本发明的又一目的是提供一种体外辅助预测三阴性乳腺癌的试剂盒。The purpose of the present invention is to provide the use of small nucleolar ribonucleic acid SNORD33 as a biomarker in the preparation of a detection kit based on the basis and status of the prior art. The invention relates to a method for applying specific changes in plasma SNORD33 to predicting platinum treatment sensitivity in patients with triple-negative breast cancer, and the application of a primer for qualitatively or quantitatively detecting human plasma SNORD33 in a kit for predicting platinum curative effect. Another object of the present invention is to provide a kit for in vitro prediction of triple negative breast cancer.
基于本申请选取SNORD33作为研究对象的研究发现，治疗前血浆小核 仁核糖核酸SNORD33(small nucleolar RNA,C/D box 33，NC_000019.10)水平低的TNBC患者，其接受顺铂治疗的有效率低，无进展时间短；所以SNORD33可用作预测TNBC患者铂类药物疗效的生物标记物；与所述接受铂类药物治疗有效的三阴性乳腺癌受试者血浆样品相比，SNORD33在来自接受铂类药物治疗无效的三阴性乳腺癌受试者血浆样品中的水平显著减少，并且SNORD33在血浆样品中高水平的三阴性乳腺癌受试者，其接受铂类药物治疗的无进展生存时间(progression-free survival,PFS)较SNORD33在血浆样品中低水平所述受试者显著延长；血浆小核仁核糖核酸SNORD33水平低的TNBC患者，其接受顺铂治疗的有效率低，无进展生存时间短，因而，本发明提出“通过检测血浆SNORD33水平，以预测TNBC患者接受铂类治疗的疗效”的方法。可以预测TNBC患者接受铂类后有效率、疾病控制时长，并根据患者治疗前血浆SNORD33水平，选择是否进行铂类治疗，从而达到个性化、精准化的治疗的目的。Based on the research that SNORD33 was selected as the research object based on this application, it was found that patients with TNBC with low levels of small nucleolar RNA (C / Dbox 33, NC_000019.10) before treatment had a cisplatin-effective treatment. Low, short progression-free time; so SNORD33 can be used as a biomarker to predict the efficacy of platinum-based drugs in TNBC patients; compared with the plasma samples of triple negative breast cancer subjects who received platinum-based treatment, SNORD33 Levels of plasma in triple negative breast cancer subjects who were not responding to platinum therapy were significantly reduced, and SNORD33 was present in plasma samples with high levels of triple negative breast cancer subjects who had progress-free survival -free survival (PFS) was significantly prolonged compared to subjects with low levels of SNORD33 in plasma samples; patients with TNBC with low levels of plasma small nucleolar RNA SNORD33 had low cisplatin treatment efficiency and short progression-free survival time Therefore, the present invention proposes a method of "predicting the efficacy of platinum-based treatment for TNBC patients by detecting plasma SNORD33 levels". It can predict the efficiency and duration of disease control after TNBC patients receive platinum, and choose whether to use platinum therapy according to the patient's plasma SNORD33 level before treatment, so as to achieve the purpose of personalized and precise treatment.
本发明通过检测血浆中SNORD33解决TNBC患者铂类药物疗效预测等问题；更具体的，提供小核仁核糖核酸SNORD33作为生物标记物用于制备检测试剂盒中的用途。The present invention solves problems such as the prediction of the efficacy of platinum drugs in TNBC patients by detecting SNORD33 in plasma; more specifically, it provides the use of small nucleolar RNA SNORD33 as a biomarker for preparing a detection kit.
本发明的目的通过下述技术方案：The object of the present invention is through the following technical solutions:
采用所述的小核仁核糖核酸SNORD33作为生物标记物制备得检测试剂盒，通过定性或定量检测人血浆SNORD33的引物，用荧光定量PCR技术检测SNORD33在所述三阴性乳腺癌患者受试者血浆样本中水平；比较SNORD33在来自于所述铂类治疗有效的受试者的血浆样品中的水平与SNORD33在所述铂类治疗无效的受试者的血浆样品中的水平；以及确定SNORD33预测铂类有效性的最佳界值。A detection kit is prepared by using the small nucleolar RNA SNORD33 as a biomarker. The primer for human plasma SNORD33 is qualitatively or quantitatively detected, and SNORD33 is detected in the plasma of the triple negative breast cancer patient subject by fluorescent quantitative PCR technology. Levels in a sample; comparing levels of SNORD33 in a plasma sample from a subject who is effective with the platinum treatment with SNORD33 in a plasma sample of a subject who is not effective with the platinum treatment; and determining that SNORD33 predicts platinum The best cutoff for class validity.
通过受试者血浆SNORD33与界值的比较，预测三阴性乳腺癌患者铂类的疗效。By comparing the subject plasma SNORD33 with the cut-off value, the efficacy of platinum in patients with triple negative breast cancer can be predicted.
进一步，可以通过检测SNORD33在三阴性乳腺癌受试者的血浆样品中表达量预测铂类药物治疗的疗效；并且可根据受试者血浆SNORD33表达水平指导铂类药物的选用；对于血浆SNORD33高水平的受试者，选择铂类药物治疗疗效较好；对于血浆SNORD33低水平的受试者，可考虑选择其他抗肿瘤治疗药物，因此能够更好的指导三阴性乳腺癌患者的治疗。Further, the efficacy of platinum-based drugs can be predicted by detecting the expression of SNORD33 in plasma samples from subjects with triple negative breast cancer; and the choice of platinum-based drugs can be guided according to the subject's plasma SNORD33 expression level; high levels of plasma SNORD33 For patients with platinum, the treatment with platinum drugs is better; for patients with low levels of plasma SNORD33, other anti-tumor drugs can be considered, so it can better guide the treatment of triple negative breast cancer patients.
本发明中，对于SNORD33的检测方法可通过任何有效途径来进行，包括荧光定量PCR方法、RT-PCR方法、Northern blotting方法、RNase protection assay、Solexa sequencing technology以及生物芯片方法中的一种或几种。In the present invention, the detection method for SNORD33 can be performed by any effective method, including one or more of the quantitative PCR method, RT-PCR method, Northern blotting method, RNase protection assay, Solexa sequencing technology, and biochip method. .
本发明中，优选荧光定量PCR方法，其包括：(1)提取受试者血清/血浆总RNA，通过RNA逆转录反应得到cDNA样品；(2)用小核仁核糖核酸设计引物；(3)加入荧光探针进行PCR反应；(4)检测并比较血清/血浆样品相对于设定的预测界值中小核仁核糖核酸的量的变化。In the present invention, a fluorescent quantitative PCR method is preferred, which includes: (1) extracting total serum / plasma RNA from a subject, and obtaining a cDNA sample by an RNA reverse transcription reaction; (2) designing a primer with a small nucleolar RNA; (3) Add fluorescent probes for PCR reaction; (4) Detect and compare the change of the amount of small nucleolar RNA in the serum / plasma sample with respect to the set prediction threshold.
本发明中，另优选RT-PCR方法，其包括步骤：(1)提取受试者血清/血浆总RNA，通过RNA逆转录反应得到cDNA样品；(2)用小核仁核糖核酸设计的引物进行PCR反应；(3)进行PCR产物的琼脂糖凝胶电泳；(4)EB染色后紫外灯下观察结果。In the present invention, another RT-PCR method is preferred, which includes the steps of: (1) extracting the total serum / plasma RNA of the subject, and obtaining a cDNA sample by an RNA reverse transcription reaction; (2) using a primer designed by small nucleoside ribonucleic acid PCR reaction; (3) agarose gel electrophoresis of PCR products; (4) observation of results under UV light after EB staining.
本发明中，所述Northern blotting方法包括步骤：(1)提取受试者血清/血浆总RNA(例如使用Trizol试剂提取)；(2)变性PAGE电泳和膜转移实验；(3)制备同位素标记小核仁核糖核酸探针；(4)进行膜杂交反应；(5)同位素信号检测，如磷屏扫描检测结果。In the present invention, the Northern blotting method includes the steps of: (1) extracting total serum / plasma RNA of the subject (for example, using Trizol reagent); (2) denaturing PAGE electrophoresis and membrane transfer experiments; (3) preparing isotopically labeled small Nucleolar ribonucleic acid probe; (4) membrane hybridization reaction; (5) isotope signal detection, such as phosphor screen scanning test results.
本发明中，所述RNase protection assay方法包括步骤：(1)进行反义RNA探针的合成，同位素标记与纯化；(2)提取受试者血清/血浆总RNA(例如使用Trizol试剂提取)；(3)将提取后的RNA溶解在杂交缓冲液中并加入反义RNA探针进行杂交；(4)加入RNase消化液进行反应；(5)进行电泳与放射自显影；(6)分析结果。In the present invention, the RNase protection method includes the steps of: (1) synthesis of an antisense RNA probe, isotope labeling and purification; (2) extraction of serum / plasma total RNA from a subject (for example, extraction using Trizol reagent); (3) dissolve the extracted RNA in hybridization buffer and add antisense RNA probes for hybridization; (4) add RNase digestion solution for reaction; (5) perform electrophoresis and autoradiography; (6) analyze the results.
本发明中，所述Solexa sequencing technology方法包括步骤：(1)提取受试者血清/血浆总RNA(例如使用Trizol试剂提取)；(2)进行PAGE电泳回收80-90ntRNA分子；(3)将adaptor primer酶联在小RNA分子的3’与5’端；(4)进行RT-PCR反应后并进行测序；(5)数据分析与处理。In the present invention, the Solexa sequencing technology method includes the steps of: (1) extracting total serum / plasma RNA of the subject (for example, using Trizol reagent); (2) performing PAGE electrophoresis to recover 80-90nt RNA molecules; (3) adaptor The primer is linked to the 3 'and 5' ends of the small RNA molecule; (4) RT-PCR is performed and sequenced; (5) data analysis and processing.
本发明中，所述生物芯片方法包括步骤：(1)将人体血浆中全部小核仁核糖核酸库点阵并制备生物芯片；(2)提取受试者血浆总RNA(例如使用Trizol试剂提取)；(3)通过柱分离来分离小核仁核糖核酸；(4)利用T4RNA连接酶进行小核仁核糖核酸荧光标记；(5)与生物芯片进行杂交反应；(6)数据检测与分析。In the present invention, the biochip method includes the steps of: (1) latticeing all small nucleolar RNA pools in human plasma and preparing a biochip; (2) extracting total RNA from a subject's plasma (for example, using Trizol reagent) (3) Separation of small nucleolar RNA by column separation; (4) Fluorescent labeling of small nucleolar RNA using T4RNA ligase; (5) Hybridization reaction with biochip; (6) Data detection and analysis.
本发明所述上述方法中所使用的血清/血浆来源于受试者活体、组织、器 官或/和尸体。The serum / plasma used in the above method of the present invention is derived from a living body, tissue, organ, or / and cadaver of a subject.
前期实验结果显示，在三阴性乳腺癌患者血浆样品中，SNORD33水平在铂类治疗无效的受试者中显著低于治疗有效的受试者；并且SNORD33低水平的受试者，其铂类治疗的PFS时间显著短于SNORD33高水平的受试者。Previous experimental results showed that in plasma samples of triple-negative breast cancer patients, the level of SNORD33 was significantly lower in subjects who were ineffective in platinum therapy than in subjects who were ineffective in treatment; and in subjects with low levels of SNORD33, their platinum therapy was The PFS time was significantly shorter in subjects with high levels of SNORD33.
本发明SNORD33表达水平可以作为一种明确、有效、直观的疗效预测的生物标志物，诊断和预测三阴性乳腺癌患者接受铂类治疗后疗效、有效率、疾病控制时长以及预后，指导三阴性乳腺癌患者的用药，达到个体化治疗的目的；其预测疗效的铂类药物是包括顺铂、卡铂、奥沙利铂、洛铂、奈达铂在内的所有铂类化疗药物；可检测标本包括受试者活体、组织、器官和/或尸体中收集的血清/血浆；SNORD33作为疗效预测生物标志物可以通过任何有效途径来检测，包括荧光定量PCR方法、RT-PCR方法、Northern blotting方法、RNase protection assay、Solexa sequencing technology以及生物芯片方法中的一种或几种；通过荧光定量PCR方法，利用包含人血浆SNORD33的引物的三阴性乳腺癌铂类疗效预测试剂盒在受试者中进行检测。The expression level of SNORD33 of the present invention can be used as a clear, effective and intuitive biomarker for predicting curative effect. It diagnoses and predicts the curative effect, efficiency, duration of disease control and prognosis of triple negative breast cancer patients after receiving platinum therapy, and guides the triple negative breast. The medication for cancer patients achieves the purpose of individualized treatment; the platinum drugs whose efficacy is predicted are all platinum chemotherapy drugs including cisplatin, carboplatin, oxaliplatin, loplatin, and nedaplatin; detectable specimens Including serum / plasma collected from the living body, tissue, organ and / or cadaver of the subject; SNORD33 as a predictive biomarker of efficacy can be detected by any effective method, including fluorescent quantitative PCR method, RT-PCR method, Northern blotting method, One or more of RNase protection, Solexa sequencing technology, and biochip methods; detection of the efficacy of a platinum-based triple-negative breast cancer platinum-based efficacy prediction kit using primers containing human plasma SNORD33 by fluorescent quantitative PCR .
本发明利用血浆SNORD33进行个体化，精准化治疗：对于血浆SNORD33高水平的受试者，选择铂类药物对三阴性乳腺癌具有更好的抑制作用；对于血浆SNORD33低水平的受试者，可考虑选择其他抗肿瘤治疗药物。通过检测血浆SNORD33水平指导临床上三阴性乳腺癌患者的铂类药物的选用。The present invention uses plasma SNORD33 for individualized and precise treatment: for subjects with high plasma SNORD33 levels, platinum drugs are selected to have better inhibition of triple negative breast cancer; for subjects with low plasma SNORD33 levels, Consider choosing other anti-tumor therapies. Plasma SNORD33 levels are used to guide the selection of platinum-based drugs in clinically triple-negative breast cancer patients.
本发明所涉及的检测血浆中的SNORD33有以下优势：首先，血浆较组织容易获得，平时日常诊疗活动中都可以收集到；其次，SNORD33反映的是机体整体的生理病理情况，其检测具有指导意义；第三，SNORD33可以实时反映机体目前所处的状态，较原发灶组织更能贴近机体的实际情况，同时还可以通过SNORD33监控其实时变化。綜上，利用小核仁核糖核酸SNORD33作为生物标记物制备的检测试剂盒检测血浆中的SNORD33，无创简便且预测作用明确，从血浆的小核仁核糖核酸的特异性变化着一新角度预测药物的疗效，并指导治疗，本发明将有助于建立一种更敏感更精确的用于个体化治疗的技术方案。The detection of SNORD33 in plasma according to the present invention has the following advantages: first, the plasma is easier to obtain than tissues and can be collected in daily routine diagnosis and treatment activities; secondly, SNORD33 reflects the overall physiological and pathological conditions of the body, and its detection has guiding significance Third, SNORD33 can reflect the current state of the body in real time, which is closer to the actual situation of the body than the original tissue, and it can also monitor its real-time changes through SNORD33. In summary, the detection kit for SNORD33 in plasma using small nucleolar RNA SNORD33 as a biomarker is non-invasive, simple, and has a clear predictive effect. It predicts drugs from a new perspective of specific changes in small nucleolar RNA in plasma. The curative effect and guide the treatment, the present invention will help to establish a more sensitive and accurate technical solution for individualized treatment.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1：三阴性乳腺癌患者血浆中能检测到SNORD33的荧光定量PCR的扩增 曲线及溶解曲线的实验图。Figure 1: An experimental plot of the amplification curve and lysis curve of SNORD33 in plasma from triple negative breast cancer patients.
图2：三阴性乳腺癌铂类治疗无效的患者血浆SNORD33相对于有效患者血浆SNORD33的变化量。Figure 2: Changes in plasma SNORD33 in patients who failed to respond to platinum therapy for triple-negative breast cancer compared to plasma SNORD33 in effective patients.
图3：血浆SNORD33低表达的三阴性乳腺癌患者相对于血浆SNORD33高表达患者的顺铂治疗的无进展生存时间的差异。Figure 3: Differences in progression-free survival time of patients with triple-negative breast cancer with low expression of plasma SNORD33 compared with patients with high expression of plasma SNORD33.
具体实施方式detailed description
下面结合具体实施例，进一步阐述本发明。需要强调的是，这些实施例仅用于说明本发明而不用于限制本发明的范围。在不偏离与本发明范围的情况下，本发明的主要特征可以用于各种实施方式。除非另行定义，所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明中，这些等同物被认为处在本发明的范围之内，并且被权利要求所覆盖。文中所述的较佳实施方法与材料仅作示范之用。The present invention will be further described below with reference to specific embodiments. It should be emphasized that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The main features of the present invention can be applied to various embodiments without departing from the scope of the present invention. Unless otherwise defined, all professional and scientific terms have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be used in the present invention, and these equivalents are considered to be within the scope of the present invention and covered by the claims. The preferred implementation methods and materials described herein are for demonstration purposes only.
实施例1.样本要求及样本处理方法Example 1. Sample requirements and sample processing methods
本实施例的目的是说明对于受试者采集的血清/血浆标本的要求及具体处理方法。The purpose of this example is to explain the requirements and specific processing methods for the serum / plasma specimens collected by the subject.
样本要求：Sample requirements:
(1)样本类型：血浆，血浆以新鲜采集分离为好，采血6小时内必须分离、收集血浆，采集的全血放入EDTA素抗凝管，并将血浆转移至一次性使用的无RNA酶的无菌微量离心管中；(1) Sample type: Plasma, plasma should be collected and separated as fresh. Plasma must be separated and collected within 6 hours of blood collection. The collected whole blood is placed in EDTA anticoagulant tube, and the plasma is transferred to disposable RNase-free In a sterile microcentrifuge tube;
(2)血清样本置于室温可稳定保存24小时，2-8℃可稳定保存7天，-18℃一下可稳定保存6个月；(2) Serum samples can be stably stored at room temperature for 24 hours, 2-8 ° C for 7 days, and -18 ° C for 6 months.
(3)血清/血浆来自于签订了知情同意书的受试者，受试者在取血前未使用铂类药物的治疗；(3) The serum / plasma comes from the subjects who signed the informed consent, and the subjects were not treated with platinum drugs before taking the blood;
(4)血清/血浆样本提取出的RNA，室温可稳定保存12小时，2-8℃可稳定保存3天，-18℃一下可稳定保存3个月；(4) RNA extracted from serum / plasma samples can be stored stably for 12 hours at room temperature, 3 days at 2-8 ° C, and 3 months at -18 ° C;
处理方法：Approach:
(1)将全血样本处理成血浆：采集静脉血2ml，于5ml洁净离心管中，抗 凝。静置30分钟后，5000rpm离心5分钟，取其上清。如果样本不立即使用，需-18℃以下保存，本实施例所需血浆样本体积为200ul；(1) Process the whole blood sample into plasma: collect 2ml of venous blood and put it in a 5ml clean centrifuge tube to anticoagulate. After standing for 30 minutes, centrifuge at 5000 rpm for 5 minutes, and take the supernatant. If the sample is not used immediately, it needs to be stored below -18 ° C. The volume of plasma sample required in this example is 200ul;
(2)血浆样本的RNA提取：使用Qiagen miRNeasy Mini Kit试剂盒，遵循厂商说明进行操作，在制备血浆样品的同时，制备好阳性质控品及阴性质控品。(2) RNA extraction from plasma samples: Use Qiagen miRNeasy Mini Kit kit and follow the manufacturer's instructions to prepare positive and negative controls while preparing plasma samples.
实施例2.逆转录操作过程Example 2. Process of reverse transcription
逆转录体系如表1所示：The reverse transcription system is shown in Table 1:
表1Table 1
名称name 体积volume
2x miRNA逆转录反应缓冲液2x miRNA reverse transcription reaction buffer 5ul5ul
miRNA逆转录酶mixmiRNA reverse transcriptase mix 1ul1ul
RNARNA 1ul1ul
DEPC水DEPC water 3ul3ul
总体积total capacity 10ul10ul
实验操作：Experimental operation:
(1)计算反应体系：按照检测样本量计算逆转录反应体系所需体系量；(1) Calculate the reaction system: Calculate the system volume required for the reverse transcription reaction system according to the amount of sample detected;
(2)按照表1以及天根miRcute miRNA cDNA第一链合成试剂盒的配制反应体系，遵循厂商说明进行操作；(2) Prepare the reaction system according to Table 1 and the miRcute miRNA cute cDNA first strand synthesis kit, and follow the manufacturer's instructions;
(3)分布反应混合液：将步骤(2)所得混合液9ul/孔分布于相应96孔板中；(3) Distributing the reaction mixture: distribute 9ul / well of the mixture obtained in step (2) in the corresponding 96-well plate;
(4)加入样品：所有提取后的待测血浆样本RNA、提取后的阴性质控品及阳性质控品、标准品S1-S4、空白对照1ul置于(3)步骤中的96孔板中，贴膜；(4) Add samples: All extracted RNA from the plasma samples to be tested, extracted negative and positive controls, standards S1-S4, and blank 1ul are placed in the 96-well plate in step (3). , Film
(5)混匀：96孔PCR板置于涡旋震荡仪上涡旋约5s，然后置于板式离心机(1500rmp，30s)进行离心；(5) Mixing: Place the 96-well PCR plate on a vortex shaker for 5 seconds, and then place it in a plate centrifuge (1500rmp, 30s) for centrifugation;
反应程序：将96孔板置于PCR仪中，按照miRcute miRNA cDNA第一链合成试剂盒进；Reaction procedure: Place the 96-well plate in the PCR instrument and follow the miRcute miRNA and cDNA first strand synthesis kit;
将逆转录后所得的cDNA样品用DEPC水稀释10倍。The cDNA sample obtained after reverse transcription was diluted 10-fold with DEPC water.
实施例3.荧光定量PCR过程Example 3. Real-time PCR process
实验体系如表2所示：The experimental system is shown in Table 2:
表2Table 2
Figure PCTCN2019092995-appb-000001
Figure PCTCN2019092995-appb-000001
SNORD33引物正向序列为GGCCGGTGATGAGAA，反向序列为CGAATGTGAGTGGGAGAA，内参U6引物正向序列为CTCGCTTCGGCAGCACA，反向序列为AACGCTTCACGAATTTGCGT；The forward sequence of the SNORD33 primer is GGCCGGTGATGAGAA, the reverse sequence is CGAATGTGAGTGGGAGAA, the forward sequence of the internal reference U6 primer is CTCGCTTCGGCAGCACA, and the reverse sequence is AACGCTTCACGAATTTGCGT;
实验操作：Experimental operation:
(1)计算反应体系：按照检测样本量计算荧光定量PCR反应体系所需体系量；(1) Calculating the reaction system: Calculate the amount of system required for the fluorescent quantitative PCR reaction system according to the amount of detection samples;
(2)按照表2以及TAKARA
Figure PCTCN2019092995-appb-000002
qPCR Premix试剂盒的配制反应体系，遵循厂商说明进行操作； (2) According to Table 2 and TAKARA
Figure PCTCN2019092995-appb-000002
qPCR Premix kit preparation reaction system, follow the manufacturer's instructions to operate;
(3)分布反应混合液：将步骤(2)所得混合液18ul/孔分布于相应96孔板中；(3) Distribution reaction mixture: Distribute 18ul / well of the mixture obtained in step (2) in the corresponding 96-well plate;
(4)加入cDNA：所有逆转录所得的样品cDNA 2ul置于(3)步骤中的96孔板中，贴膜；(4) Adding cDNA: 2ul of all sample cDNA obtained by reverse transcription is placed in a 96-well plate in step (3), and the film is pasted;
(5)混匀：96孔PCR板置于涡旋震荡仪上涡旋约5s，然后置于板式离心机(1500rmp，30s)进行离心；(5) Mixing: Place the 96-well PCR plate on a vortex shaker for 5 seconds, and then place it in a plate centrifuge (1500rmp, 30s) for centrifugation;
3.反应程序：将96孔板置于ABI7500或ABI7300荧光定量PCR仪中，按照TAKARA
Figure PCTCN2019092995-appb-000003
qPCR Premix试剂盒进行qPCR反应。于每个循环的第二步，即：60℃收集荧光信号。 3. Reaction procedure: Place the 96-well plate in the ABI7500 or ABI7300 real-time PCR instrument and follow TAKARA
Figure PCTCN2019092995-appb-000003
qPCR Premix kits perform qPCR reactions. Collect the fluorescence signal at the second step of each cycle, ie, 60 ° C.
实施例4.产品的质量要求Example 4. Product quality requirements
本实施例中SNORD33标准品的线性范围为10 6-10 3拷贝/ul； The linear range of the SNORD33 standard in this embodiment is 10 6 -10 3 copies / ul;
准确度：准确度即阳性符合率，用质控标本中的10分阳性参考品P1-P10(其中SNORD33的浓度＞20000copy/ul)对SNORD33进行测定，检测结果均应为阳性，即10/10；对内对照U6进行测定，检测结果均应符合Ct＜26.5；Accuracy: Accuracy is the positive coincidence rate. The 10-point positive reference P1-P10 (where the concentration of SNORD33 is> 20000 copy / ul) in the quality control sample is used to determine SNORD33. The test results should be positive, that is, 10/10 ; For internal control U6, the test results should meet Ct <26.5;
特异度：特异度即阴性符合率，用质控标本中的10分阴性参考品N1-N10(其中SNORD33的浓度＜20000copy/ul)对SNORD33进行测定，检测结果均应为阴性，即10/10；对内对照U6进行测定，检测结果均应符合Ct＜26.5；Specificity: The specificity is the negative compliance rate. The 10-point negative reference N1-N10 in the quality control sample (where the concentration of SNORD33 is less than 20000copy / ul) is used to measure SNORD33. The test results should be negative, that is, 10/10 ; For internal control U6, the test results should meet Ct <26.5;
检测阈值：本专利SNORD33最低检测阈值应≤10 3拷贝/ul，检测SNORD33的10 3拷贝/ul的标准品20次，至少17次检测结果高于最低检测阈值的判读值； Detection threshold: The minimum detection threshold of SNORD33 in this patent should be ≤ 10 3 copies / ul, and the standard of 10 3 copies / ul of SNORD33 should be detected 20 times. At least 17 times the detection results are higher than the minimum detection threshold.
本申请对阳性质控品、阴性质控品和空白对照有效性的要求：Requirements for the validity of the positive control, negative control and blank control in this application:
经抽提后阳性质控品应满足：10 5拷贝/ul＜SNORD33相对拷贝数＜10 6拷贝/ul，内对照Ct＜26.5； After extraction positive control materials should satisfy: 105 copies / ul <SNORD33 relative copy number <106 copies / ul, the endogenous control Ct <26.5;
经抽提后阴性质控品应满足：10 3拷贝/ul＜SNORD33相对拷贝数＜10 4拷贝/ul，内对照Ct＜26.5； The negative control after the extraction should be satisfied: 10 3 copies / ul <SNORD33 relative copy number <104 copies / ul, the endogenous control Ct <26.5;
经抽提后空白对照应满足：SNORD33相对拷贝数＜10 3拷贝/ul，内对照Ct＜26.5； After extraction, the blank control should satisfy: the relative copy number of SNORD33 <10 3 copies / ul, and the internal control Ct <26.5;
精密度：批次内精密度的测定方法：阳性质控品、阴性质控品各平行检测10次，SNORD33检测应符合相对拷贝数的变异系数(CV,％)＜5％，内对照检测应符合Ct变异系数(CV,％)＜5％；Precision: The measurement method of the precision within the batch: Positive control and negative control are tested 10 times in parallel. The detection of SNORD33 should meet the coefficient of variation (CV,%) of the relative copy number <5%. The internal control test should Meet the Ct coefficient of variation (CV,%) <5%;
经过对临床常见的高浓度甘油三酯、胆红素、血红蛋白血浆样本的***研究，此类样本不影响本试剂盒的定性检测结果。After systematic research on high-concentration triglyceride, bilirubin, and hemoglobin plasma samples that are commonly used in clinical practice, such samples do not affect the qualitative test results of this kit.
实施例5.Example 5.
检测结果分析和判读Analysis and interpretation of test results
表达水平的计算：公式为SNORD33表达水平＝2 -ΔΔCt； Calculation of expression level: The formula is SNORD33 expression level = 2- ΔΔCt ;
受试者血浆样品中SNORD33表达水平结果如图2所示，铂类治疗有效的受试者(n＝13)血浆样品中SNORD33表达水平(2.7±0.8)显著高于铂类治疗无效的受试者(n＝17)血浆样品中SNORD33表达水平(0.5±0.1，P<0.001)；The results of the expression level of SNORD33 in the plasma samples of the subjects are shown in Figure 2. The expression level of SNORD33 (2.7 ± 0.8) in the plasma samples of subjects who were effective in platinum therapy (n = 13) was significantly higher than those ineffective in platinum therapy. (N = 17) SNORD33 expression level in plasma samples (0.5 ± 0.1, P <0.001);
血浆样品中SNORD33表达水平预测界值0.69，低于此界值可定义为三 阴性乳腺癌患者对铂类治疗不敏感；受试者血浆样品中SNORD33表达水平与无进展生存时间预测相关性结果如图3所示，血浆样品中SNORD33表达水平低的患者，其接受顺铂治疗后的无进展生存时间显著低于血浆样品中SNORD33表达水平高的患者(P＝0.005)；The predicted cut-off value of SNORD33 expression level in plasma samples is 0.69. Below this cut-off value, it can be defined as that patients with triple negative breast cancer are not sensitive to platinum therapy. The correlation between the expression level of SNORD33 in plasma samples and the prediction of progression-free survival time is as follows: As shown in Figure 3, patients with low SNORD33 expression levels in plasma samples had significantly less progression-free survival time after treatment with cisplatin than patients with high SNORD33 expression levels in plasma samples (P = 0.005);
本实施例结果的解释方法为：The interpretation method of the results of this embodiment is:
检测样品SNORD33表达量＞0.69，考虑此三阴性乳腺癌患者后续铂类药物治疗有效的可能性大；The expression of SNORD33 in the test sample is greater than 0.69. Considering that the triple platinum-negative breast cancer patients are likely to be effective in subsequent platinum therapy;
检测样本SNORD33表达量≤0.69，考虑此三阴性乳腺癌患者后续铂类药物治疗有效的可能性小。The expression of SNORD33 in the test sample was ≤0.69. Considering that the triple platinum-negative breast cancer patients were unlikely to be effective in subsequent platinum therapy.
实施例6Example 6
制备小核仁核糖核酸试剂盒Preparation of small nucleolar RNA kit
基于定量PCR技术用于预测三阴性乳腺癌铂类疗效的小核仁核糖核酸的试剂盒的专门制作工艺和流程操作，实验包括常用的SYBR、dNTP、以及优选的人血浆SNORD33的引物，通过制备的试剂盒，用于检测三阴性乳腺癌患者铂类治疗前血浆的SNORD33水平，预测患者接受铂类治疗的敏感性以及疗效，利用血浆SNORD33水平进行个体化，精准化治疗，指导临床上三阴性乳腺癌患者的铂类药物的选用。Based on the quantitative PCR technology for the preparation of small nucleolar RNA kit for predicting the efficacy of triple-negative breast cancer, the special manufacturing process and process operation. The experiments include commonly used SYBR, dNTP, and the preferred human plasma SNORD33 primers. Kit for detecting plasma SNORD33 levels before platinum treatment in triple-negative breast cancer patients, predicting the sensitivity and efficacy of patients receiving platinum therapy, using plasma SNORD33 levels to individualize and precision treatment, and guide clinical triple negative Selection of platinum drugs for breast cancer patients.

Claims (24)

  1. 小核仁核糖核酸SNORD33作为生物标记物用于制备检测试剂盒中的用途，所述的检测试剂盒基于定量PCR技术制备，检测的SNORD33水平用于预测三阴性乳腺癌铂类疗效。The use of small nucleolar RNA SNORD33 as a biomarker in the preparation of a detection kit, the detection kit is based on quantitative PCR technology, and the detected SNORD33 level is used to predict the efficacy of platinum in triple negative breast cancer.
  2. 根据权利要求1所述的用途，其特征在于，所述的检测试剂盒用于检测SNORD33水平，其包括：确定SNORD33在所述三阴性乳腺癌患者受试者血浆样本中水平；比较SNORD33在来自所述三阴性乳腺癌患者受试者的样品中的水平与设定的预测界值；其中与SNORD33在所述预测界值相比，SNORD33在来自所述三阴性乳腺癌患者受试者的样品中的水平降低是所述铂类药物治疗无效的指示，从而预测三阴性乳腺癌患者从铂类药物治疗中获益可能性较低。The use according to claim 1, wherein the detection kit is used to detect the level of SNORD33, comprising: determining the level of SNORD33 in the plasma sample of the subject of the triple negative breast cancer patient; comparing the SNORD33 in The level in the sample from the subject of the triple negative breast cancer patient and the set prediction threshold; wherein compared to the predicted threshold of SNORD33, SNORD33 is in the sample from the subject of the triple negative breast cancer patient A decrease in the level is an indicator of the ineffectiveness of the platinum drug treatment, thereby predicting that patients with triple negative breast cancer are less likely to benefit from platinum drug treatment.
  3. 根据权利要求2所述的用途，其特征在于，评价受试者的铂类药物治疗的敏感性，为测定受试者给予特定待测物后预测铂类药物的疗效。The use according to claim 2, characterized in that the sensitivity of the platinum drug treatment in the subject is evaluated, and the measurement is performed to predict the efficacy of the platinum drug after the subject is given a specific test substance.
  4. 根据权利要求3所述的用途，其特征在于，所述预测标记物用于评估待测药物的有效率以及疾病控制时长。The use according to claim 3, wherein the predictive marker is used to evaluate the effectiveness of the drug to be tested and the duration of disease control.
  5. 根据权利要求3所述的用途，其特征在于，所述预测标记物评价受试者三阴性乳腺癌的预后。The use according to claim 3, wherein the predictive marker evaluates the prognosis of triple negative breast cancer in a subject.
  6. 根据权利要求3所述的用途，其特征在于，所述评价受试者的所发生的疾病为乳腺癌，且组织学已确诊为三阴性乳腺癌，同时影像学或/和组织学确诊出现乳腺癌的复发转移。The use according to claim 3, wherein the disease occurring in the evaluation subject is breast cancer, and histology has been diagnosed as triple-negative breast cancer, and at the same time, imaging or / and histology confirmed the presence of breasts Cancer recurrence and metastasis.
  7. 根据权利要求3所述的用途，其特征在于，所述铂类药物包括顺铂、卡铂、奥沙利铂、洛铂、奈达铂在内的所有铂类化疗药物。The use according to claim 3, wherein the platinum drugs include all platinum chemotherapy drugs including cisplatin, carboplatin, oxaliplatin, loplatin, and nedaplatin.
  8. 一种评价受试者的铂类敏感性的方法，其特征在于，采用权利要求1的检测试剂盒测定受试者的血清/血浆中稳定存在且可检测的小核仁核糖核酸SNORD33水平。A method for evaluating a platinum sensitivity of a subject, characterized in that the detection kit of claim 1 is used to measure the level of the small nucleolar RNA SNORD33 that is stably present and detectable in the serum / plasma of the subject.
  9. 根据权利要求8所述的方法，其特征在于所述血清/血浆来源于受试者活体、组织、器官和/或尸体。The method according to claim 8, characterized in that the serum / plasma is derived from a living body, tissue, organ and / or cadaver of the subject.
  10. 根据权利要求8或9所述的方法，其特征在于SNORD33在血浆样本中水平可通过血浆样品中SNORD33的RNA含量检测。The method according to claim 8 or 9, characterized in that the level of SNORD33 in the plasma sample can be detected by the RNA content of SNORD33 in the plasma sample.
  11. 根据权利要求8或9所述的方法，其特征在于受试者的血浆样本为接受铂类药物治疗前收集的血浆样本。The method according to claim 8 or 9, characterized in that the subject's plasma sample is a plasma sample collected before receiving platinum drugs.
  12. 根据权利要求8或9所述的方法，其特征在于所述评价受试者的铂类药物治疗的敏感性，为测定受试者给予特定待测物后预测铂类药物的疗效。The method according to claim 8 or 9, characterized in that the evaluation sensitivity of the platinum drug treatment of the subject is to measure the subject to predict the curative effect of the platinum drug after the specific test substance is administered.
  13. 根据权利要求8或9所述的方法，其特征在于所述预测标记物用于评估待测药物的有效率以及疾病控制时长。The method according to claim 8 or 9, wherein the predictive marker is used to evaluate the effectiveness of the drug to be tested and the duration of disease control.
  14. 根据权利要求8或9所述的方法，其特征在于所述预测标记物评价受试者三阴性乳腺癌的预后。The method according to claim 8 or 9, characterized in that the predictive marker evaluates the prognosis of triple negative breast cancer in a subject.
  15. 根据权利要求8或9所述的方法，其特征在于所述测定受试者的血清/血浆中稳定存在且可检测的小核仁核糖核酸的方法，包括荧光定量PCR方法、RT-PCR方法、Northern blotting方法、RNase protection assay、Solexa sequencing technology以及生物芯片方法中的一种或几种。The method according to claim 8 or 9, characterized in that the method for measuring stable and detectable small nucleolar ribonucleic acid in serum / plasma of a subject comprises a fluorescent quantitative PCR method, an RT-PCR method, One or more of Northern blotting method, RNase protection assay, Solexa sequencing technology, and biochip method.
  16. 根据权利要求15所述的方法，其特征在于所述荧光定量PCR方法包括步骤：(1)提取受试者血清/血浆总RNA，通过RNA逆转录反应得到cDNA样品；(2)用小核仁核糖核酸设计引物；(3)加入荧光探针进行PCR反应；(4)检测并比较血清/血浆样品相对于设定的预测界值中小核仁核糖核酸的量的变化。The method according to claim 15, characterized in that the fluorescent quantitative PCR method comprises the steps of: (1) extracting the total serum / plasma RNA of the subject, and obtaining a cDNA sample by an RNA reverse transcription reaction; (2) using small nucleoli Design primers for RNA; (3) Add fluorescent probes for PCR reaction; (4) Detect and compare changes in the amount of small nucleolar RNA in the serum / plasma sample relative to the set prediction threshold.
  17. 根据权利要求15所述的方法，其特征在于所述RT-PCR方法包括步骤：(1)提取受试者血清/血浆总RNA，通过RNA逆转录反应得到cDNA样品；(2)用小核仁核糖核酸设计引物机型PCR反应；(3)进行PCR产物的琼脂糖凝胶电泳；(4)EB染色后紫外灯下观察结果。The method according to claim 15, characterized in that the RT-PCR method comprises the steps of: (1) extracting total serum / plasma RNA of the subject, and obtaining a cDNA sample by an RNA reverse transcription reaction; (2) using small nucleoli A primer-type PCR reaction was designed with RNA; (3) agarose gel electrophoresis of PCR products; (4) the results were observed under UV light after EB staining.
  18. 一种用于评价受试者的铂类敏感性的试剂盒，其特征在于所述试剂盒包括测定血清/血浆中稳定存在且可检测的小核仁核糖核酸的工具。A kit for evaluating a subject's platinum sensitivity, characterized in that the kit includes a tool for measuring stable and detectable small nucleolar RNA in serum / plasma.
  19. 根据权利要求18所述的试剂盒，其特征在于所述试剂盒包含人血清/血浆中的小核仁核糖核酸的引物。The kit according to claim 18, characterized in that the kit contains primers for small nucleolar RNAs in human serum / plasma.
  20. 根据权利要求18所述的试剂盒，其特征在于所述血清/血浆来源于受试者活体、组织、器官和/或尸体。The kit according to claim 18, wherein the serum / plasma is derived from a living body, tissue, organ, and / or cadaver of the subject.
  21. 根据权利要求18至20所述的试剂盒，其特征在于受试者的血清/血浆样本为接受铂类药物治疗前收集的血清/血浆样本。The kit according to claim 18 to 20, characterized in that the serum / plasma sample of the subject is a serum / plasma sample collected before receiving platinum drug treatment.
  22. 根据权利要求18至20所述的试剂盒，其特征在于所述评价受试者的铂类药物治疗的敏感性，为测定受试者给予特定待测物后预测铂类药物的疗效。The kit according to claim 18 to 20, characterized in that the evaluation sensitivity of the platinum drug treatment of the subject is to measure the subject to predict the curative effect of the platinum drug after the specific test substance is administered.
  23. 根据权利要求18至20所述的试剂盒，其特征在于所述预测标记物用于 评估待测药物的有效率以及疾病控制时长。The kit according to claims 18 to 20, characterized in that the predictive marker is used to evaluate the effectiveness of the test drug and the duration of disease control.
  24. 根据权利要求18至20所述的试剂盒，其特征在于所述预测标记物评价受试者三阴性乳腺癌的预后。The kit according to claims 18 to 20, wherein the predictive marker evaluates the prognosis of a triple negative breast cancer in a subject.
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Publication number Priority date Publication date Assignee Title
WO2016046640A2 (en) * 2014-09-26 2016-03-31 Medical Prognosis Institute A/S Methods for predicting drug responsiveness
CN106729751A (en) * 2015-11-20 2017-05-31 中国医学科学院肿瘤医院 A kind of microRNA molecule is used to detect and treat the purposes of breast cancer
CN107109488A (en) * 2014-12-12 2017-08-29 麦迪韦逊***治疗股份有限公司 The method predicted the method for the reaction to breast cancer treatment agent and treat breast cancer

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016046640A2 (en) * 2014-09-26 2016-03-31 Medical Prognosis Institute A/S Methods for predicting drug responsiveness
CN107109488A (en) * 2014-12-12 2017-08-29 麦迪韦逊***治疗股份有限公司 The method predicted the method for the reaction to breast cancer treatment agent and treat breast cancer
CN106729751A (en) * 2015-11-20 2017-05-31 中国医学科学院肿瘤医院 A kind of microRNA molecule is used to detect and treat the purposes of breast cancer

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