WO2020001473A1 - Utilisation d'un petit acide ribonucléique nucléolaire snord33 en tant que biomarqueur pour la préparation d'un kit de détection - Google Patents

Utilisation d'un petit acide ribonucléique nucléolaire snord33 en tant que biomarqueur pour la préparation d'un kit de détection 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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English (en)
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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Priority claimed from CN201811173295.7A external-priority patent/CN110656171B/zh
Application filed by 复旦大学 filed Critical 复旦大学
Publication of WO2020001473A1 publication Critical patent/WO2020001473A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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.

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Abstract

L'invention concerne l'utilisation d'un petit acide ribonucléique nucléolaire SNORD33 en tant que biomarqueur pour prédire l'efficacité du platine dans le cancer du sein triple négatif, et les kits et procédés de détection correspondants.
PCT/CN2019/092995 2018-06-28 2019-06-26 Utilisation d'un petit acide ribonucléique nucléolaire snord33 en tant que biomarqueur pour la préparation d'un kit de détection WO2020001473A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201810703342 2018-06-28
CN201810703342.8 2018-06-28
CN201811173295.7 2018-10-09
CN201811173295.7A CN110656171B (zh) 2018-06-28 2018-10-09 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

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016046640A2 (fr) * 2014-09-26 2016-03-31 Medical Prognosis Institute A/S Procédés de prédiction de la réactivité à un médicament
CN106729751A (zh) * 2015-11-20 2017-05-31 中国医学科学院肿瘤医院 一种microRNA分子用于检测和治疗乳腺癌的用途
CN107109488A (zh) * 2014-12-12 2017-08-29 麦迪韦逊前列腺治疗股份有限公司 预测对乳腺癌治疗剂的反应的方法和治疗乳腺癌的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016046640A2 (fr) * 2014-09-26 2016-03-31 Medical Prognosis Institute A/S Procédés de prédiction de la réactivité à un médicament
CN107109488A (zh) * 2014-12-12 2017-08-29 麦迪韦逊前列腺治疗股份有限公司 预测对乳腺癌治疗剂的反应的方法和治疗乳腺癌的方法
CN106729751A (zh) * 2015-11-20 2017-05-31 中国医学科学院肿瘤医院 一种microRNA分子用于检测和治疗乳腺癌的用途

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