CN113186273A - circRNA detection kit for predicting antiplatelet curative effect of P2Y12 receptor antagonist and application thereof - Google Patents
circRNA detection kit for predicting antiplatelet curative effect of P2Y12 receptor antagonist and application thereof Download PDFInfo
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Abstract
The invention discloses a circRNA detection kit for predicting antiplatelet curative effect of a P2Y12 receptor antagonist and application thereof. The circRNA detection kit comprises a reagent for detecting the relative expression quantity of the platelet circFAM 13B; the reagent for detecting the relative expression quantity of the platelet circFAM13B comprises a circFAM13B amplification primer, a reverse transcription reaction reagent and a fluorescent quantitative PCR reaction reagent; the amplification primer of the circFAM13B consists of a single-stranded DNA molecule shown in a sequence 2 in a sequence table and a single-stranded DNA molecule shown in a sequence 3 in the sequence table. The kit provided by the invention is simple to prepare and convenient to use, can quickly and accurately predict the antiplatelet reactivity of the P2Y12 receptor antagonist of Chinese patient groups, and provides an epigenetics reference index for individualized antiplatelet treatment of the P2Y12 receptor antagonist.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a platelet circFAM13B as a marker in preparation of a product for predicting the antiplatelet curative effect of a P2Y12 receptor antagonist, in particular to application of a substance for detecting the expression level of the platelet circFAM13B in preparation of a product for predicting the antiplatelet curative effect of a P2Y12 receptor antagonist.
Background
Acute Coronary Syndrome (ACS) is one of the common cardiovascular diseases that seriously jeopardize human health, and although the mortality rate of ACS has been greatly reduced in the past decade and the annual mortality rate of coronary heart disease has decreased by 34.4%, the risk of mortality in patients experiencing recurring events remains high. P2Y12 receptor antagonists in combination with aspirin antiplatelet therapy are the currently accepted standard for ACS treatment and the gold standard for prevention of intra-stent thrombosis following Percutaneous Coronary Intervention (PCI). Research proves that the antiplatelet reactivity of the P2Y12 receptor antagonist has individual difference, and the individual difference factors causing the antiplatelet reactivity of the P2Y12 receptor antagonist comprise disease states, combined medication and drug dosage and pharmacogenomics factors (CYP2C19 gene polymorphism). However, these known factors are not sufficient to account for individual differences in platelet reactivity. Therefore, how to predict the difference of antiplatelet reactivity of the P2Y12 receptor antagonist in clinic and realize personalized antiplatelet drug treatment of ACS patients is always a hotspot of research in the field of cardiovascular clinical pharmacology and a difficult problem to be solved urgently. To this end, there is a need to explore new biomarkers to compensate for the lack of association between genotype, phenotype and disease in pharmacogenomic studies.
In recent years, epigenetic studies on platelets have found that platelets contain abundant non-coding RNAs, especially small RNAs (micrornas) and circular RNAs (circrnas), in addition to messenger RNAs (mrnas). Sanger HL in 1976 first discovered circRNA in viruses. Subsequently, research into circRNA has been carried out, and in recent years, research into circRNA has been attracting much attention. It was found that platelets are rich in circRNA and are produced and present predominantly in a cleaved form with exonic cyclization. As circRNA is more stable than linear RNA, and the closed-loop end structure of the circRNA can protect the circRNA from degradation by exonuclease, the circRNA has potential as a clinical biomarker. Research shows that circRNA has correlation with diseases such as atherosclerosis, myocardial infarction, heart failure and the like. However, the platelet circRNAs are not yet known to be reactive with platelets and the regulatory mechanisms in platelets.
Disclosure of Invention
The invention aims to provide application of platelet-derived circFAM13B as a marker in prediction of antiplatelet curative effect of a P2Y12 receptor antagonist.
In order to achieve the above object, the present invention firstly provides use of a substance for detecting an expression level of platelet circFAM13B in any one of the following (a1) - (a 4):
(a1) predicting the therapeutic efficacy of the P2Y12 receptor antagonist on anti-platelet therapy;
(a2) preparing a product for predicting the curative effect of the P2Y12 receptor antagonist on anti-platelet therapy;
(a3) predicting antiplatelet reactivity of a P2Y12 receptor antagonist;
(a4) preparing products for predicting antiplatelet reactivity of the P2Y12 receptor antagonist.
In the above application, the P2Y12 receptor antagonist is clopidogrel or ticagrelor.
In the application, the substance for detecting the expression quantity of the platelet circFAM13B comprises a reagent and/or an apparatus for detecting the relative expression quantity of the platelet circFAM 13B;
the reagent for detecting the relative expression quantity of the platelet circFAM13B specifically comprises a circFAM13B amplification primer, a reverse transcription reaction reagent and a fluorescent quantitative PCR reaction reagent.
Further, the amplification primer of the circFAM13B consists of a single-stranded DNA molecule shown in a sequence 2 in a sequence table and a single-stranded DNA molecule shown in a sequence 3 in the sequence table.
The reverse transcription reaction reagent can be a reagent in Thermo First cDNA Synthesis Kit.
The fluorescent quantitative PCR reaction reagent may specifically include 2 XSG Green qPCR Mix (with ROX).
Further, the relative expression amount is the relative expression amount of the reference internal reference gene of circFAM 13B.
The reference gene may specifically be a GAPDH gene. The reagents also include a primer pair for amplifying the GAPDH gene.
The calculation method of the relative expression amount specifically comprises the following steps: using cDNA of a person to be tested as a template, adopting the primer pair to carry out real-time fluorescence quantitative PCR, and then using 2-△△CtAnd (4) calculating. The cDNA of the testee is cDNA obtained from platelets extracted from peripheral blood of the testee.
In order to achieve the purpose, the invention also provides a circRNA detection kit related to P2Y12 receptor antagonist individualized antiplatelet therapy, which can detect the expression level of platelet circFAM13B closely related to antiplatelet sensitivity of a P2Y12 receptor antagonist, is used for predicting antiplatelet curative effect of a P2Y12 receptor antagonist and further provides an epigenetic reference index for P2Y12 receptor antagonist individualized antiplatelet therapy.
The kit provided by the invention comprises a substance for detecting the expression quantity of the platelet circFAM 13B; the kit has the function of predicting the curative effect of the P2Y12 receptor antagonist on antiplatelet treatment or predicting the antiplatelet reactivity of the P2Y12 receptor antagonist.
In the kit, the P2Y12 receptor antagonist is clopidogrel or ticagrelor.
In the kit, the substance for detecting the expression quantity of the platelet circFAM13B comprises a reagent and/or an apparatus for detecting the relative expression quantity of the platelet circFAM 13B;
the reagent for detecting the relative expression quantity of the platelet circFAM13B specifically comprises a circFAM13B amplification primer, a reverse transcription reaction reagent and a fluorescent quantitative PCR reaction reagent.
Further, the amplification primer of the circFAM13B consists of a single-stranded DNA molecule shown in a sequence 2 in a sequence table and a single-stranded DNA molecule shown in a sequence 3 in the sequence table.
The reverse transcription reaction reagent can be a reagent in Thermo First cDNAsynthesis Kit.
The fluorescent quantitative PCR reaction reagent may specifically include 2 XSG Green qPCR Mix (with ROX).
Further, the relative expression amount is the relative expression amount of the reference internal reference gene of circFAM 13B.
The reference gene may specifically be a GAPDH gene. The reagents also include a primer pair for amplifying the GAPDH gene.
The calculation method of the relative expression amount specifically comprises the following steps: using cDNA of a person to be tested as a template, adopting the primer pair to carry out real-time fluorescence quantitative PCR, and then using 2-△△CtAnd (4) calculating. The cDNA of the testee is cDNA obtained from platelets extracted from peripheral blood of the testee.
In the above kit, when said P2Y12 receptor antagonist is clopidogrel, said kit further comprises a data processing device a; the data processing device A consists of a data input module A, a data recording module A, a data comparison module A and a conclusion output module A;
the data input module A is used for inputting a relative expression quantity value of circFAM13B in the blood platelets of the testee;
the data recording module A is used for storing the relative expression quantity value of the circFAM13B in the blood platelets of the testee;
the data comparison module A is used for comparing the relative expression quantity value of the circFAM13B in the platelets of the testee with a threshold value;
the conclusion output module A is used for displaying a conclusion that if the relative expression amount of circFAM13B in the platelet of the subject is more than 1.179, the platelet of the subject has high reactivity; if the relative expression quantity of circFAM13B in the blood platelet of the subject is less than 0.596, the blood platelet of the subject has low reactivity; if the relative expression quantity of circFAM13B in the blood platelet of the subject is between 0.596 and 1.179 (including 0.596 and 1.179), the blood platelet of the subject has moderate reactivity;
when the P2Y12 receptor antagonist is ticagrelor, the kit further comprises a data processing device b; the data processing device B consists of a data input module B, a data recording module B, a data comparison module B and a conclusion output module B;
the data input module B is used for inputting a relative expression quantity value of the circFAM13B in the blood platelet of the person to be tested;
the data recording module B is used for storing the relative expression quantity value of the circFAM13B in the blood platelets of the testee;
the data comparison module B is used for comparing the relative expression quantity value of the circFAM13B in the blood platelets of the testee with a threshold value;
the conclusion output module B is used for displaying a conclusion that if the relative expression quantity of the circFAM13B in the platelet of the subject is more than 1.636, the platelet of the subject has high reactivity; if the relative expression quantity of circFAM13B in the blood platelet of the subject is less than 0.565, the blood platelet of the subject has low reactivity; if the relative expression level of circFAM13B in the platelet of the subject is between 0.565 and 1.636 (including 0.565 and 1.636), the platelet of the subject has moderate reactivity.
The antiplatelet reactivity is measured by aiming at a reaction characteristic of patient platelets treated by a P2Y12 receptor antagonist after being activated by an ADP (adenosine diphosphate) stimulator through a TEG (TEG test) detection means, reflects the influence of an antithrombotic drug on the platelet reactivity, and has a value of judging the antiplatelet curative effect of a P2Y12 receptor antagonist drug clinically.
In a particular embodiment of the present invention,
when the P2Y12 receptor antagonist is clopidogrel, the high platelet reactivity means that the TEG-ADP inhibition rate is less than 50 percent; the low platelet reactivity means that the TEG-ADP inhibition rate is more than 80 percent; the moderate platelet reactivity means that the TEG-ADP inhibition rate is between 50% and 80%.
When the P2Y12 receptor antagonist is ticagrelor, the high platelet reactivity means a TEG-ADP inhibition rate of < 60%; the low platelet reactivity means that the TEG-ADP inhibition rate is more than 90 percent; the moderate platelet reactivity means that the TEG-ADP inhibition rate is between 60% and 90%.
The TEG-ADP inhibition rate refers to the platelet inhibition rate induced by Adenosine Diphosphate (ADP) detected by applying a platelet function detection-Thromboelastography (TEG) after 3 days of stable treatment of clopidogrel or ticagrelor, and a specific calculation method is described in the literature of Othman M, Kaur H.Thromboelastogry (TEG) [ J ]. Methods Mol Biol,2017,1646 (533-43.).
In practical application, when a certain body uses clopidogrel, a P2Y12 receptor antagonist, to perform antiplatelet therapy, if the relative expression level of circFAM13B in platelets is greater than 1.179, the reactivity of the platelets of the individual is high, the antiplatelet reactivity of the drug is weak, and the risk of thrombosis is high, and the antiplatelet drug is recommended to be increased or replaced by other powerful antiplatelet drugs; if the relative expression quantity of circFAM13B in the platelets is less than 0.596, the individual has low platelet reactivity, strong antiplatelet reactivity of the medicine and good antiplatelet curative effect; if the relative expression level of circFAM13B in the platelets is between 0.596 and 1.179, the individual has moderate platelet reactivity, moderate drug antiplatelet reactivity and good antiplatelet curative effect, and the current treatment is recommended to continue.
When an individual uses the P2Y12 receptor antagonist ticagrelor to carry out antiplatelet therapy, if the relative expression level of circFAM13B in the platelets is more than 1.636, the individual has high platelet reactivity, weak drug antiplatelet reactivity and high thrombosis risk, and the increase of the antiplatelet drug or the replacement of the antiplatelet drug by other powerful antiplatelet drugs is recommended; if the relative expression amount of circFAM13B in the platelets is less than 0.565, the individual has low platelet reactivity, strong drug antiplatelet reactivity and good antiplatelet curative effect; if the relative expression level of circFAM13B in the platelets is between 0.565 and 1.636, the individual has moderate platelet reactivity, moderate drug antiplatelet reactivity and good antiplatelet efficacy, and the current treatment is recommended to continue.
The application of circFAM13B as a marker in any one of the following (a1) - (a4) also belongs to the protection scope of the present invention:
(a1) predicting the therapeutic efficacy of the P2Y12 receptor antagonist on anti-platelet therapy;
(a2) preparing a product for predicting the curative effect of the P2Y12 receptor antagonist on anti-platelet therapy;
(a3) predicting antiplatelet reactivity of a P2Y12 receptor antagonist;
(a4) preparing products for predicting antiplatelet reactivity of the P2Y12 receptor antagonist.
In any of the above applications or kits, the nucleotide sequence of the circFAM13B (hsa _ circ _0001535) is shown as sequence 1 in the sequence table.
According to the invention, an ACS patient treated by a P2Y12 receptor antagonist (clopidogrel or ticagrelor) is taken as a research object, and annular RNA which is abundantly expressed in platelets is screened, and the result shows that the expression level of platelet annular RNA-circFAM13B in individuals with poor antiplatelet curative effect and ideal curative effect is remarkably different, namely the expression level of circFAM13B in individuals with case group (poor drug reactivity) is remarkably higher than that of individuals with control group (normal drug reactivity). Subsequent validation in ACS patients treated with P2Y12 receptor antagonist (clopidogrel or ticagrelor) antiplatelets demonstrated that the expression level of circFAM13B in these platelets correlates closely with the antiplatelet reactivity of the P2Y12 receptor antagonist in ACS patients. Therefore, the invention establishes a circFAM13B detection kit and a detection method related to the P2Y12 receptor antagonist individualized antiplatelet therapy. The circFAM13B detection kit related to the P2Y12 receptor antagonist individualized antiplatelet therapy is simple to prepare and convenient to use; the kit can be used for quickly and accurately predicting the antiplatelet reactivity and curative effect of the P2Y12 receptor antagonist of Chinese patient groups.
Drawings
FIG. 1 is a graph of the correlation of circFAM13B with P2Y12 receptor antagonist (FIG. left: clopidogrel; FIG. right: ticagrelor) antiplatelet reactivity.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The nucleotide sequence of circFAM13B (hsa _ circ _0001535) in the examples below is shown as sequence 1 in the sequence listing.
Example 1 correlation of the expression level of circFAM13B in platelets with the antiplatelet reactivity of P2Y12 receptor antagonists in ACS patients
First, experimental materials and methods
1. Case recruitment
1) Continuous recruitment of ACS patients stably treated with P2Y12 receptor antagonists
And (3) inclusion standard: patients with ACS treated with P2Y12 receptor antagonist (clopidogrel or ticagrelor) who participated voluntarily and signed an informed consent for human genetic testing, aged over 18 years, were continuously recruited to provide peripheral blood and were used for platelet RNA extraction. In order to reduce the influence of population stratification, all patients are Chinese Han nationalities, and have no blood relationship and history of foreign marriage. The total of 296 cases, 159 patients were treated with clopidogrel and 137 patients were treated with ticagrelor (see table 1, table 2 for case baseline).
Exclusion criteria: 1. contraindications for antiplatelet drug therapy are known; 2. acute and chronic inflammatory diseases; 3. non-steroidal anti-inflammatory drugs and opioid drugs are used; 4. liver and kidney dysfunction, life expectancy is less than 1 year; 5. those with recent bleeding tendencies, potential bleeding risks, or hematological disorders; 6. deep puncture and major surgical history in nearly 4 weeks; 7. other critically ill patients.
2) Screening of cases with different effects of anti-platelet reactivity
Platelet function detection-Thromboelastography (TEG) was used to detect Adenosine Diphosphate (ADP) -induced platelet inhibition rate 3 days after stable treatment of clopidogrel or ticagrelor according to the method in the literature "Othman M, Kaur H.Thromboelastogrphy (TEG) [ J ]. Methods Mol Biol,2017,1646 (533-43)", to detect the antiplatelet reactivity of P2Y12 receptor antagonist (based on the measurement of one response characteristic of P2Y12 receptor antagonist-treated platelets after activation by ADP stimulus by TEG detection means, showing the effect of antithrombotic drug on platelet reactivity and having a value for clinically judging the efficacy of antithrombotic drug), and to perform screening of cases with different antiplatelet reactivity effects by strict clinical judgment criteria, including case group (curative effect group) and control group (ideal curative effect group), wherein, 10 cases of clopidogrel-treated patients (case group and 5 cases of different curative effects of ACS-treated patients (curative effect group) and 12 cases of ticagrelor-treated patients (ideal curative effect group) 6 cases in each of the example group and the control group) (the baseline of cases is shown in tables 1 and 2). The magnitude of platelet reactivity is measured according to TEG criteria, which are as follows: in the ACS patients treated by clopidogrel, the TEG-ADP inhibition rate is more than 80 percent of the control group (ideal curative effect group), and the TEG-ADP inhibition rate is less than 50 percent of the case group (poor curative effect group); in ticagrelor-treated ACS patients, the TEG-ADP inhibition rate is more than 90% of that of a control group (ideal curative effect group), and the TEG-ADP inhibition rate is less than 60% of that of a case group (poor curative effect group).
TABLE 1 basic clinical characteristics of clopidogrel enrolled patients
TABLE 2 basic clinical characteristics of Ticagrelor enrolled patients
2. Whole blood platelets and RNA extraction thereof
1) Platelet extraction
3ml of peripheral blood in the sodium citrate anticoagulation vacuum blood collection tube is taken and placed for about 4 hours. Centrifuging at 100g room temperature for 15min, collecting 700 μ l of supernatant plasma with RNase-free and bacteria-free pipette tip to obtain platelet rich plasma, and transferring into new 1.5ml EP tube. Then centrifuging for 15min at the rotating speed of 2000g and the room temperature, removing the supernatant, and obtaining the precipitate as the platelet.
2) Platelet RNA extraction
Platelet RNA was extracted using SG high-purity total RNA extraction kit (SinoGene, cat # R1002, China) according to the procedures described in the kit instructions. The specific method comprises the following steps: blowing and beating the platelet sample obtained in the step 1) to precipitate and mixing the platelet sample uniformly. 1ml of SG TRIzol lysate was added to the sample. Shaking vigorously, and standing at room temperature for 2-3 min. Add 200. mu.l chloroform, vortex vigorously for 30 seconds, and let stand at room temperature for 5 minutes. The samples were centrifuged at 12000 Xg for 15min at 4 ℃. The supernatant was carefully transferred to an RNase-free 1.5ml centrifuge tube and mixed with an equal volume of isopropanol. Add 500. mu.l RNA Wash Buffer I to the column and centrifuge at 12000 Xg for 30 sec at 4 ℃. The filtrate was discarded. 13000 Xg, 4 degrees centrifugal 10 minutes. The supernatant was discarded and 1ml of 70% ethanol was added to rinse the precipitate. 13000 Xg, 5 minutes at 4 degrees. 70% ethanol was discarded and RNA was air dried. RNA was dissolved by adding 30. mu.l of DEPC-treated water. The concentration was determined (5. mu.l) and the remaining reverse transcribed cDNA was stored at-80 ℃ until use.
3) DNase I treatment (purpose: removal of genomic DNA
Preparing a reaction system according to a formula shown in table 3, and reacting the prepared reaction system under the following conditions: water bath at 37 ℃ for 30 minutes; inactivating the DNase I in water bath at 65 ℃ for 10 minutes to obtain a DNase I treatment reaction solution.
TABLE 3 reaction System
| RNA | 8.3 μ l (about 1 μ g) |
| 10×Reaction Buffer with MgCl2 | 1.2μl |
| DNase I (Fermentas, cat # EN0521, USA) | 2μl |
| Ribolock | 0.5μl |
| Total volume | 12μl |
3. Detection of platelets by fluorescent quantitative PCR method, circFAM13B
1) Design and Synthesis of primers
The primers were designed and synthesized by Sinojinda, Inc. for fluorescent quantitative PCR reaction.
circFAM13B(hsa_circ_0001535)：
An upstream primer: 5'-CAATGAAGCTATGCAGCAAGA-3' (SEQ ID NO: 2);
a downstream primer: 5'-CAAAAAGGTGCTGTTCCACA-3' (SEQ ID NO: 3).
GAPDH (internal reference gene):
an upstream primer: 5'-GAAGGTGAAGGTCGGAGTC-3', respectively;
a downstream primer: 5'-GAAGATGGTGATGGGATTTC-3' are provided.
2) Reverse transcription
The extracted platelet RNA (RNA after DNase I treatment) was reverse-transcribed using Thermo First cDNA Synthesis Kit (SinoGene, cat # Q1010, China) to obtain cDNA.
The reverse transcription reaction system was prepared according to the formulation shown in table 4, mixed well and centrifuged. Then the reverse transcription reaction system is reacted under the following conditions: water bath at 42 ℃ for 60 minutes; water bath at 85 deg.c for 10 min to deactivate reverse transcriptase.
TABLE 4 reverse transcription reaction System
| DNase I treatment reaction solution | 12μl |
| Random | 1μl |
| 5×Reaction Buffer | 4μl |
| RT enzyme | 1μl |
| dNTP(10mM) | 2μl |
| Total volume | 20μl |
3) Fluorescent quantitative PCR
Using cDNA obtained in the step 2) as a template, adopting the primer designed in the step 1), respectively amplifying circFAM13B and internal reference GAPDH by using 2 xSG Green qPCR Mix (with ROX) (SinoGene, the product number is # Q1002, China), respectively obtaining Ct value (cycle threshold), and respectively adopting the formula 2-△△CtCalculating the relative expression of circFAM13B in each sample, wherein the Δ Ct is Δ Ct (test sample) - Δ Ct (reference sample); Δ Ct (test sample) — Ct (test sample, target gene) — Ct (test sample, reference gene); Δ Ct (reference sample) — Ct (reference sample, target gene) — Ct (reference sample, reference gene); delta Ct (baseline sample) -3.543 for clopidogrel-treated ACS patients; delta Ct (baseline) of ACS patients treated with ticagrelor is-2.482. The specific operation process is shown in the specification. Three replicates per sample were performed.
A reaction system (15. mu.l system) for fluorescent quantitative PCR was prepared according to the formulation shown in Table 5, mixed well, centrifuged, and divided into 8-tube or 96-well PCR plates, with 3 PCR reactions per gene per sample in parallel. The PCR reaction conditions are shown in Table 6.
TABLE 5 fluorescent quantitative PCR reaction System
| 2×SG Green qPCR Mix | 7.5μl |
| Forward primer (10. mu.M) | 0.25μl |
| Reverse primer (10. mu.M) | 0.25μl |
| cDNA | 1μl |
| Sterile enzyme-free water | 6μl |
| Total volume | 15μl |
TABLE 6 PCR reaction conditions
Detecting the PCR amplification product obtained in step 3) by Sanger sequencing technology. The sequencing result is shown as a sequence 4 in the sequence table, the sequence comprises a T vector used for sequencing and a target gene sequence to be detected connected to the T vector, and the comparison with the sequence 1 shows that the target gene sequence is completely consistent with a PCR product sequence theoretically obtained after amplification by a circFAM13B primer, so that the platelet-derived circFAM13B is confirmed to be circular RNA, and the circFAM13B primer can specifically recognize and amplify the circFAM 13B.
Second, correlation analysis of platelet circFAM13B expression level and antiplatelet reactivity of P2Y12 receptor antagonist
Analysis of the differential expression of platelet circFAM13B in P2Y12 receptor antagonist case/control case using either the two independent sample mean T test or the Mann-Whitney U test, P <0.05 was significant.
The results are shown in FIG. 1 and show that: the relative expression level of circFAM13B in platelets was significantly higher in case group than in control group (clopidogrel: case group: 1.179 ± 0.232; control group: 0.596 ± 0.331, P ═ 0.020. ticagrelor: case group: 1.636 ± 1.105; control group: 0.565 ± 0.263, P ═ 0.025). For ACS patients taking P2Y12 receptor antagonist antiplatelet therapy, the relative expression amount of circFAM13B in platelets is higher than 1.179 (clopidogrel) or 1.636 (ticagrelor), which indicates that the P2Y12 receptor antagonist platelet reactivity is high, the antiplatelet reactivity of drugs is weak, and the thrombosis risk is high, and at this time, under the condition of combining clinical factors related to ischemia risk of patients and factors such as drug metabolizing enzyme CYP2C19 gene variation, the antiplatelet drug increment or other powerful antiplatelet drugs are considered to be replaced. The relative expression of the circFAM13B is lower than 0.596 (clopidogrel) or 0.565 (ticagrelor), which indicates that the P2Y12 receptor antagonist has low platelet reactivity, the drug has strong antiplatelet reactivity and good antiplatelet curative effect. The relative expression amount of the circFAM13B is between 0.596 and 1.179 (clopidogrel) or 0.565 and 1.636 (ticagrelor), so that the individual has moderate platelet reactivity, moderate drug antiplatelet reactivity and good antiplatelet curative effect, and the current treatment is recommended to continue.
In practical application, when an individual uses clopidogrel, a P2Y12 receptor antagonist, to perform antiplatelet therapy, if the relative expression level of circFAM13B in platelets is greater than 1.179, the individual has high platelet reactivity, weak drug antiplatelet reactivity and high thrombosis risk, and the increase of antiplatelet drugs or the replacement of the antiplatelet drugs with other powerful antiplatelet drugs is recommended; if the relative expression quantity of the circFAM13B is less than 0.596, the individual has low platelet reactivity, strong antiplatelet reactivity of the medicine and good antiplatelet curative effect; if the relative expression level of circFAM13B in the platelets is between 0.596 and 1.179, the individual has moderate platelet reactivity, moderate drug antiplatelet reactivity and good antiplatelet curative effect, and the current treatment is recommended to continue.
When an individual uses the P2Y12 receptor antagonist ticagrelor to carry out antithrombotic platelet therapy, if the relative expression level of circFAM13B in platelets is more than 1.636, the individual has high platelet reactivity, weak drug antiplatelet reactivity and high thrombosis risk, and the increase of the antiplatelet drug or the replacement of the antiplatelet drug by other powerful antiplatelet drugs is recommended; if the relative expression quantity of the circFAM13B is less than 0.565, the individual has low platelet reactivity, strong antiplatelet reactivity of the medicine and good antiplatelet curative effect; if the relative expression level of circFAM13B in the platelets is between 0.565 and 1.636, the individual has moderate platelet reactivity, moderate drug antiplatelet reactivity and good antiplatelet efficacy, and the current treatment is recommended to continue.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> first medical center of general hospital of people liberation force of China
<120> circRNA detection kit for predicting antiplatelet curative effect of P2Y12 receptor antagonist and application thereof
<160> 4
<170> PatentIn version 3.5
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gaatttacaa cagcaaagtg tggtgtgtaa taatgaagca gaaagtattc attgtgatgg 180
ggaaggatct aataaccaga ttgatattgc tgatgatatt attaatgcca gtgaaagtaa 240
cagagactgt tcaaaacctg tggctagcac taatttagac aatgaagcta tgcagcaaga 300
ttgtgtattt gagaatgaag aaaataccca g 331
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caatgaagct atgcagcaag a 21
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caaaaaggtg ctgttccaca 20
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ggggtaggga ccctcgagtc gacggtatcg ataagcttga tatcgaattc ccaatactca 60
atgaagctat gcagcaagat tgtgtatttg agaatgaaga aaatacccag cacccatata 120
tctcccatca gcatcctacc agcctctaca gatattttag aaagaacaat tagagcagct 180
gtggaacagc acctttttga gtattgggaa ttcctgcagc ccgggggatc cactagttct 240
agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc 300
gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 360
tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 420
ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 480
ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 540
ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 600
agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 660
catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 720
tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 780
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 840
ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 900
cgtggcgctt tctcatagct cacgctgtag tatctcagtt cggtgtaggt cgttcgctcc 960
aagctgggct gtgtgcacga accccccgtt cagcccgacc gctg 1004
Claims (10)
1. The application of the substance for detecting the expression quantity of the platelet circFAM13B in any one of the following (a1) - (a 4):
(a1) predicting the therapeutic efficacy of the P2Y12 receptor antagonist on anti-platelet therapy;
(a2) preparing a product for predicting the curative effect of the P2Y12 receptor antagonist on anti-platelet therapy;
(a3) predicting antiplatelet reactivity of a P2Y12 receptor antagonist;
(a4) preparing products for predicting antiplatelet reactivity of the P2Y12 receptor antagonist.
2. Use according to claim 1, characterized in that: the P2Y12 receptor antagonist is clopidogrel or ticagrelor.
3. Use according to claim 1 or 2, characterized in that: the substance for detecting the expression quantity of the platelet circFAM13B comprises a reagent and/or an apparatus for detecting the relative expression quantity of the platelet circFAM 13B;
and/or the reagent for detecting the relative expression quantity of the platelet circFAM13B comprises a circFAM13B amplification primer, a reverse transcription reaction reagent and a fluorescent quantitative PCR reaction reagent.
4. Use according to any one of claims 1 to 3, characterized in that: the amplification primer of the circFAM13B consists of a single-stranded DNA molecule shown in a sequence 2 in a sequence table and a single-stranded DNA molecule shown in a sequence 3 in the sequence table.
5. A kit comprising a substance for detecting the expression level of platelet circFAM 13B; the kit has the function of predicting the curative effect of the P2Y12 receptor antagonist on antiplatelet treatment or predicting the antiplatelet reactivity of the P2Y12 receptor antagonist.
6. The kit of claim 5, wherein: the P2Y12 receptor antagonist is clopidogrel or ticagrelor.
7. The kit according to claim 5 or 6, characterized in that: the substance for detecting the expression quantity of the platelet circFAM13B comprises a reagent and/or an apparatus for detecting the relative expression quantity of the platelet circFAM 13B;
and/or the reagent for detecting the relative expression quantity of the platelet circFAM13B comprises a circFAM13B amplification primer, a reverse transcription reaction reagent and a fluorescent quantitative PCR reaction reagent.
8. The kit according to any one of claims 5 to 7, wherein: the amplification primer of the circFAM13B consists of a single-stranded DNA molecule shown in a sequence 2 in a sequence table and a single-stranded DNA molecule shown in a sequence 3 in the sequence table.
9. The kit according to any one of claims 5 to 8, wherein: when the P2Y12 receptor antagonist is clopidogrel, the kit further comprises a data processing device a; the data processing device A consists of a data input module A, a data recording module A, a data comparison module A and a conclusion output module A;
the data input module A is used for inputting a relative expression quantity value of circFAM13B in the blood platelets of the testee;
the data recording module A is used for storing the relative expression quantity value of the circFAM13B in the blood platelets of the testee;
the data comparison module A is used for comparing the relative expression quantity value of the circFAM13B in the platelets of the testee with a threshold value;
the conclusion output module A is used for displaying a conclusion that if the relative expression amount of circFAM13B in the platelet of the subject is more than 1.179, the platelet of the subject has high reactivity; if the relative expression quantity of circFAM13B in the blood platelet of the subject is less than 0.596, the blood platelet of the subject has low reactivity; if the relative expression quantity of circFAM13B in the blood platelet of the subject is between 0.596 and 1.179, the reactivity of the blood platelet of the subject is moderate;
or, when the P2Y12 receptor antagonist is ticagrelor, the kit further comprises a data processing device b; the data processing device B consists of a data input module B, a data recording module B, a data comparison module B and a conclusion output module B;
the data input module B is used for inputting a relative expression quantity value of the circFAM13B in the blood platelet of the person to be tested;
the data recording module B is used for storing the relative expression quantity value of the circFAM13B in the blood platelets of the testee;
the data comparison module B is used for comparing the relative expression quantity value of the circFAM13B in the blood platelets of the testee with a threshold value;
the conclusion output module B is used for displaying a conclusion that if the relative expression quantity of the circFAM13B in the platelet of the subject is more than 1.636, the platelet of the subject has high reactivity; if the relative expression quantity of circFAM13B in the blood platelet of the subject is less than 0.565, the blood platelet of the subject has low reactivity; if the relative expression quantity of the circFAM13B in the blood platelet of the subject is between 0.565 and 1.636, the blood platelet of the subject has moderate reactivity.
Use of circFAM13B as a marker in any one of (a1) - (a4) as follows:
(a1) predicting the therapeutic efficacy of the P2Y12 receptor antagonist on anti-platelet therapy;
(a2) preparing a product for predicting the curative effect of the P2Y12 receptor antagonist on anti-platelet therapy;
(a3) predicting antiplatelet reactivity of a P2Y12 receptor antagonist;
(a4) preparing products for predicting antiplatelet reactivity of the P2Y12 receptor antagonist.
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2021
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| WO2017055487A2 (en) * | 2015-09-29 | 2017-04-06 | Max-Delbrück-Centrum Für Molekulare Medizin In Der Helmholtz-Gemeinschaft | A METHOD FOR DIAGNOSING A DISEASE BY DETECTION OF circRNA IN BODILY FLUIDS |
| WO2019060742A1 (en) * | 2017-09-22 | 2019-03-28 | Kymera Therapeutics, Inc | Protein degraders and uses thereof |
| WO2020118274A1 (en) * | 2018-12-08 | 2020-06-11 | Pfs Genomics, Inc. | Transcriptomic profiling for prognosis of breast cancer |
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Effective date of registration: 20230626 Address after: 100039 West courtyard, No. 28, Wukesong Fuxing Road, Haidian District, Beijing Patentee after: The second medical center of General Hospital of PLA Address before: Institute of Geriatrics, General Hospital of the Chinese people's Liberation Army, 28 Fuxing Road, Haidian District, Beijing 100853 Patentee before: The first medical center of PLA General Hospital |
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