CN111518883B - Plasma miRNA marker for coronary heart disease diagnosis and application thereof - Google Patents
Plasma miRNA marker for coronary heart disease diagnosis and application thereof Download PDFInfo
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Abstract
The invention discloses a plasma miRNA marker for coronary heart disease diagnosis and application thereof. The invention provides a plasma miRNA marker for coronary heart disease diagnosis, wherein the miRNA marker is any one or combination of more of hsa-miR-15b-5p, hsa-miR-29c-3p, hsa-miR-378b, hsa-miR-320e, hsa-miR-361-5p or hsa-miR-199a-3 p; the sequence is shown as SEQ ID NO:1 to 6. The invention can diagnose whether the testee has coronary heart disease by carrying out differential analysis on the relative expression quantity of the miRNA markers in the plasma, and the AUC value of the combination of 6 miRNA markers is 0.971, the sensitivity is up to 92.8%, and the specificity is up to 89.5%; therefore, the miRNA marker has wide application prospect in preparation of coronary heart disease diagnosis kits and/or preparations.
Description
Technical Field
The invention belongs to the technical field of medical molecular biology. More particularly, relates to a plasma miRNA marker for coronary heart disease diagnosis and application thereof.
Background
Coronary Artery Disease (CAD) continues to be the most prevalent disease with morbidity and mortality worldwide. With a continuous effort of forty years, despite the reduced incidence of coronary artery disease, more than 1/3 of the individuals who die after age 35 still die of the disease. According to the 'Chinese cardiovascular disease report', the prevalence trend of cardiovascular disease risk factors is obvious, so that the number of cardiovascular disease patients is continuously increased, the number of cardiovascular disease patients is still rapidly increased in the next decade, and the death of cardiovascular diseases accounts for the first cause of the total death of urban and rural residents. The number of patients with cardiovascular diseases is calculated to be 2.9 million, wherein the number of patients with cerebral apoplexy is 1300 million, the number of patients with coronary heart disease is 1100 million, the number of patients with heart failure is 450 million, the number of patients with pulmonary heart disease is 500 million, the number of patients with rheumatic heart disease is 250 million, and the number of patients with congenital heart disease is 200 million.
Coronary heart disease is the short term of coronary atherosclerotic heart disease, the most common heart disease, and is also called ischemic cardiomyopathy, which refers to myocardial dysfunction and/or organic lesions caused by coronary artery stenosis and insufficient blood supply.
With the intensive research on the pathogenesis of coronary heart disease, it is now recognized that various molecular mechanisms are involved in the development of coronary heart disease, mainly including abnormal lipid metabolism of vascular cells, migration of vascular smooth muscle cells, inflammatory reaction caused by necrosis of vascular endothelial cells, stress reaction caused by hypoxia of cardiac muscle cells, and the like. At present, the common diagnostic methods of coronary heart disease are electrocardiogram, coronary artery CT or coronary artery angiography; the electrocardiogram diagnosis method is only suitable for patients with acute ischemia, and dynamic observation can be carried out to determine whether the patients are coronary heart diseases, while the patients with stable coronary heart diseases cannot be diagnosed clearly and have large use limitation; coronary artery CT or coronary artery angiography can clearly diagnose coronary heart disease, but such methods belong to radioactive or traumatic examination means, are expensive, and cannot be used as screening examination items for routine early diagnosis of coronary heart disease. Meanwhile, besides coronary artery CT and coronary artery angiography, there is also no good method for postoperative efficacy assessment of patients after coronary heart disease interventional therapy.
The prior patent (application number 201910678056.5) discloses a miRNA probe composition, a primer composition and a coronary heart disease diagnosis kit for coronary heart disease diagnosis, wherein the miRNA probe composition miRNA-29a-3p, miRNA-574-3p or miRNA-574-5p can be used as a diagnosis marker for early coronary heart disease, but the accuracy, sensitivity and specificity for diagnosing coronary heart disease are low. Therefore, the development of a coronary heart disease diagnosis method with higher diagnosis accuracy, higher sensitivity and stronger specificity has important significance in effectively controlling the incidence of the coronary heart disease and further researching the pathogenesis of the coronary heart disease.
Disclosure of Invention
The invention aims to overcome the defects of the existing coronary heart disease diagnosis method and provide a plasma miRNA marker for coronary heart disease diagnosis and application thereof.
The invention aims to provide a plasma miRNA marker for coronary heart disease diagnosis.
The invention also aims to provide application of the miRNA marker or the detection reagent thereof in preparation of a coronary heart disease diagnosis kit and/or preparation.
The invention also aims to provide a primer for detecting the miRNA marker.
The invention also aims to provide application of the primer in preparation of a coronary heart disease diagnosis kit.
The invention further aims to provide a coronary heart disease diagnostic kit.
The invention further aims to provide application of the kit in coronary heart disease diagnosis.
The above purpose of the invention is realized by the following technical scheme:
the invention firstly provides a plasma miRNA marker for coronary heart disease diagnosis, wherein the miRNA marker is any one or a combination of more than one of hsa-miR-15b-5p, hsa-miR-29c-3p, hsa-miR-378b, hsa-miR-320e, hsa-miR-361-5p or hsa-miR-199a-3 p; the sequence of the hsa-miR-15b-5p is shown in SEQ ID NO:1, the sequence of the hsa-miR-29c-3p is shown as SEQ ID NO:2, the sequence of the hsa-miR-378b is shown as SEQ ID NO:3, the sequence of hsa-miR-320e is shown in SEQ ID NO:4, the sequence of the hsa-miR-361-5p is shown as SEQ ID NO:5, the sequence of the hsa-miR-199a-3p is shown in SEQ ID NO: and 6.
Compared with a high-risk normal control subject, the miRNA marker has the advantages that the relative expression level of the miRNA marker in the plasma of a coronary heart disease patient is obviously increased, the corresponding AUC value is 0.581-0.971, and the accuracy and the sensitivity in diagnosing whether the subject suffers from the coronary heart disease are high; therefore, the following applications should also be within the scope of the present invention:
the miRNA marker or the detection reagent thereof is applied to the preparation of a coronary heart disease diagnosis kit and/or a preparation.
The invention also provides a primer for detecting the miRNA marker, and the sequence of the reverse transcription primer of hsa-miR-15b-5p is shown in SEQ ID NO:7, the sequences of the forward primer and the reverse primer are respectively shown as SEQ ID NO: 8-9; the sequence of the reverse transcription primer of the hsa-miR-29c-3p is shown in SEQ ID NO:10, the sequences of the forward primer and the reverse primer are respectively shown as SEQ ID NO:11 to 12; the sequence of the reverse transcription primer of hsa-miR-378b is shown in SEQ ID NO:13, the sequences of the forward primer and the reverse primer are respectively shown as SEQ ID NO:14 to 15; the sequence of the reverse transcription primer of hsa-miR-320e is shown in SEQ ID NO:16, the sequences of the forward primer and the reverse primer are respectively shown as SEQ ID NO:17 to 18; the sequence of the reverse transcription primer of the hsa-miR-361-5p is shown as SEQ ID NO:19, the sequences of the forward primer and the reverse primer are respectively shown as SEQ ID NO:20 to 21; the sequence of the reverse transcription primer of the hsa-miR-199a-3p is shown as SEQ ID NO:22, the sequences of the forward primer and the reverse primer are respectively shown as SEQ ID NO:23 to 24.
The application of the primer in the preparation of the coronary heart disease diagnostic kit also belongs to the protection scope of the invention.
The invention also provides a coronary heart disease diagnosis kit, which comprises a primer capable of detecting the miRNA marker.
Preferably, the primer is SEQ ID NO: primers shown in 7 to 24.
Preferably, the kit is a real-time fluorescent quantitative PCR detection kit.
Preferably, the kit further comprises a probe, and the sequence of the probe is shown in SEQ ID NO: shown at 25.
In addition, the application of the kit in coronary heart disease diagnosis also falls into the protection scope of the invention.
Preferably, the coronary heart disease is coronary atherosclerosis.
The invention has the following beneficial effects:
the invention provides a plasma miRNA marker for coronary heart disease diagnosis and application thereof. The invention can diagnose whether the subject has coronary heart disease by carrying out differential analysis on the relative expression of miRNA markers in the plasma; compared with the coronary artery CT or coronary artery radiography, the method belongs to the non-invasive non-radiation examination, greatly facilitates the clinical use and relieves the pain of patients.
The 6 miRNA markers screened by the invention have certain accuracy in diagnosing whether a subject suffers from coronary heart disease, and can be used as coronary heart disease diagnosis markers, and the AUC value of the combination of the 6 miRNA markers is 0.971, so that the sensitivity and specificity of coronary heart disease diagnosis are obviously improved, the sensitivity is up to 92.8%, the specificity is up to 89.5%, and the diagnosis reference value is extremely excellent; the real-time fluorescence quantitative PCR technology is used for directly detecting the miRNA in the blood plasma of the patient with coronary heart disease, and compared with the traditional method, the detection efficiency is obviously improved, the quantification is more accurate, and the method is economical and convenient; in addition, miRNA in the blood of the patient with coronary heart disease is released into the blood before the intracellular protein marker, and the detected plasma level can be used as a marker for assisting early diagnosis of coronary heart disease; therefore, the miRNA marker provided by the invention provides an effective basis for clinical accurate prevention, early diagnosis and individualized intervention treatment of coronary heart disease, provides a theoretical basis for research of miRNA in blood plasma of coronary heart disease, provides a new thought for clinical molecular diagnosis of coronary heart disease, and has certain theoretical significance and potential practical value.
Drawings
Fig. 1 is a classification criterion for coronary angiography of coronary patients versus high-risk normal control subjects, wherein the "arrows" indicate the location of the vascular stenosis.
Figure 2 is a plasma miRNA specific expression profile of a subject in comparison to a high risk normal control; wherein, the CDH-2 and the CDH-3 represent patients with coronary heart disease; both "Nor-2" and "Nor-3" represent high risk normal control subjects.
Figure 3 is a volcano plot of plasma primary screening for differentially expressed mirnas in patients with coronary heart disease and high risk normal control subjects.
Figure 4 is a heatmap of plasma rescreening of patients with coronary heart disease and high risk normal control subjects for differentially expressed mirnas.
FIG. 5 is a graph of the results of the differences in expression of 6 miRNA markers in coronary heart disease patients versus high risk normal control subjects; wherein, (a), (b), (c), (d), (e) and (f) are respectively the result graphs of the expression difference of hsa-miR-15b-5p, hsa-miR-29c-3p, hsa-miR-378b, hsa-miR-320e, hsa-miR-361-5p and hsa-miR-199a-3p in patients with coronary heart disease and high-risk normal control subjects.
FIG. 6 is a graph showing the results of ROC curve analysis of 6 miRNA markers alone in patients with coronary heart disease and high-risk normal control subjects; wherein, (a), (b), (c), (d), (e) and (f) are graphs of results of ROC curve analysis of hsa-miR-15b-5p, hsa-miR-29c-3p, hsa-miR-378b, hsa-miR-320e, hsa-miR-361-5p and hsa-miR-199a-3p in coronary heart disease patients and high-risk normal control subjects respectively.
FIG. 7 is a graph showing the results of ROC curve analysis of any 2-5 combinations of 6 miRNA markers in patients with coronary heart disease and high-risk normal control subjects.
FIG. 8 is a graph showing the results of ROC curve analysis of the combination of 6 miRNA markers in patients with coronary heart disease and high-risk normal control subjects.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 mapping of plasma miRNA-specific expression profiles of patients with coronary heart disease
1. Clinical sample collection and clinical data organization
Blood samples of all study subjects were from the second hospital of peony, heilongjiang province, and the plasma collection procedure was: for patients with coronary artery angiography results showing coronary artery occlusion and non-occlusion, collecting peripheral blood in a blood collection tube containing EDTA anticoagulant, centrifuging at 4 deg.C under 3000g centrifugal force for 10 min, and collecting supernatant as plasma; subpackaging the plasma sample into a 20 mu L system, and storing at-80 ℃; meanwhile, the system collects pathological data of the patient, including but not limited to coronary artery imaging results, blood routine detection results and the like.
2. Screening of patients with coronary heart disease and high-risk Normal controls
Selecting patients with Coronary Heart Disease (CHD) from the collected clinical plasma, with average age of 56 + -9 years; high risk normal control subjects (CK) were selected, with a mean age of 51 ± 11 years.
The classification criteria of coronary artery angiography of patients with coronary heart disease and high-risk normal control subjects are shown in fig. 1, and it can be seen that the coronary artery angiography results of patients with coronary heart disease show significant stenosis of the blood vessel, while the coronary artery angiography results of high-risk normal control subjects show no stenosis of the blood vessel.
3. Calculating relative expression quantity of plasma miRNA and drawing expression map
(1) Experimental methods
Randomly mixing 18 plasma samples of different groups into 3 mixed plasma samples, and carrying out real-time fluorescent quantitative PCR (RT-qPCR) detection on plasma miRNA for drawing a plasma miRNA specific expression map.
Crude extraction of RNA: incubating 20. Mu.L of plasma and 20. Mu.L of lysate for 15 minutes at 50 ℃, denaturing for 5 minutes at 95 ℃ and centrifuging for 5 minutes at 4 ℃ under 13000g of centrifugal force; 35 μ L of crude RNA was obtained.
And (3) cDNA synthesis: taking 4 μ L of crude RNA,1 μ L of 0.05 μ M reverse transcription primer, 1U of PolyA Polymerase (poly A Polymerase), 100U of MMLV (murine leukemia reverse transcriptase), 1.5 μ L of reaction buffer, and RNase-free Water to make up to 10 μ L; keeping the temperature at 37 ℃ for 15min, keeping the temperature at 42 ℃ for 15min, keeping the temperature at 75 ℃ for 5min to inactivate the enzyme, then quickly placing on ice, and standing for 2min to terminate inactivation; wherein the reaction buffer comprises the following components in final concentration: 200mM Tris-HCl,600mM NaCl,40mM MgCl 2 4mM ATP,2mM dNTP, pH 8.0; 10. Mu.L of cDNA was obtained.
RT-qPCR reaction: taking 0.5 μ L of the diluted one-time cDNA,2 μ L of 10 XTaq reaction buffer (Taq enzyme reaction buffer), 0.5 μ L of 2.5mM dNTP (deoxynucleotide mixture), 4 μ L of 1 μ M upstream amplification primer, 4 μ L of 1 μ M downstream amplification primer, 5 μ L of 1 μ M Taqman fluorescent probe primer, 0.5 μ L Taq DNA polymerase (Taq DNA polymerase), 0.2 μ L100 XROX (fluorescent reference reagent), and Nuclear-free Water to make up to 20 μ L; wherein, the Taq reaction buffer comprises the following components in final concentration: 20mM Tris-HCl,50mM KCl,2mM MgCl 2 ，5％Glycerol，pH 8.5。
RT-qPCR detection adopts a probe method, a PCR running instrument is an ABI Step-One-Plus thermal cycler, 0.5 mu L of cDNA diluted by One time is added into each 20 mu LRT-qPCR detection system, and RT-qPCR detection conditions are as follows: pre-denaturation 95 ℃,5 minutes, denaturation 95 ℃,10s, annealing 60 ℃,40s,40 cycles, time for 50 minutes, and two duplicate wells per RT-qPCR reaction.
Using external reference cel-miR-54 as reference and using 2 -ΔCt Calculating the relative expression quantity of the miRNA in the blood plasma, wherein the calculation formula of the delta Ct is as follows: Δ Ct = Ct miRNA -Ct cel-miR-54 The test method is two-tailed Student's test.
(2) Results of the experiment
The specific expression profiles of the plasma miRNAs of patients with coronary heart disease and high-risk normal control subjects are shown in figure 2, and it can be seen that the relative expression amounts of the plasma miRNAs of the patients with coronary heart disease and the high-risk normal control subjects are differentially expressed.
The volcanic map of the differential expression miRNA in the plasma of the coronary heart disease patient and the high-risk normal control subject is shown in figure 3, and it can be seen that the specific miRNA shows the trend of up-regulation or down-regulation of the expression in the plasma of the coronary heart disease patient.
In addition, from the above experiments, it can be seen that: every 4 mu L of crude RNA is reversely transcribed to generate 10 mu L of cDNA, so that 87.5 mu L of cDNA can be synthesized by 35 mu L of crude RNA, and the time is about 35 minutes; each RT-qPCR reaction actually consumes 0.25 mul of cDNA, then 175 different miRNAs can be detected by 87.5 mul of cDNA (each miRNA has two multiple holes, and actually detects one miRNA and consumes 0.5 mul of cDNA), the time is taken for 50 minutes, therefore, 175 miRNAs can be detected in 75 minutes per 20 mul of plasma; the invention shortens the time for diagnosing whether the coronary heart disease exists or not and has high detection efficiency.
Example 2 rescreening of plasma miRNA markers in coronary patients and high-risk normal control subjects
1. Experimental methods
Plasma samples of 18 coronary heart disease patients and 12 high-risk normal control subjects are selected to carry out plasma miRNA direct amplification RT-qPCR (RT-qPCR detection method refers to example 1), ct values of plasma miRNA in different expressions in each sample are obtained, respectively using 28 candidate miRNA markers (let-7 i-5p, miR-126-3p, miR-133b, miR-1-3p, miR-145-5p, miR-15b-5p, miR-16-2-3p, miR-16-5p, miR-199a-3p, miR-199a-5p, miR-199b-3p, miR-29b-3p, miR-378b, miR-361-5p, miR-409-3p, miR-149-5p, miR-155-5p, miR-15b-3p, miR-186-5p, miR-187-3p, miR-208a-3p, miR-26a-5p, mDrawing the Ct value ratio of iR-27a-3p, miR-29c-3p, miR-320e, miR-499a-5p, miR-92a-3p and miR-92b-5 p) to the Ct value of cel-miR-54, and setting a negative control group (cDNA template is not added in an RT-qPCR reaction system, ddH is used for 2 O instead), all RT-qPCR were performed in 2 replicates.
2. Results of the experiment
The heat map of the plasma rescreening of the differentially expressed mirnas of the coronary heart disease patient and the high-risk normal control subject is shown in fig. 4, and it can be seen that the expression amounts of the 28 candidate miRNA markers in the plasma of the coronary heart disease patient and the high-risk normal control subject are significantly different.
Example 3 analysis of expression differences and evaluation of clinical Performance of plasma miRNA markers in coronary atherosclerotic heart disease patients and high-risk Normal control subjects
1. Differential analysis of expression of plasma miRNA markers in coronary heart disease patients and high-risk normal control subjects
(1) Experimental methods
Selecting 95 coronary heart disease patients and 60 high-risk normal control subjects, performing plasma miRNA direct amplification RT-qPCR (RT-qPCR detection method refers to example 1) by using primers and probes (containing FAM luminescent group and BHQ1 quenching group and modified by ZEN naphthyl azo group, FAM/cagagcac/ZEN/ctgggcaattt/BHQ 1) of miRNA markers in table 1 and external reference standard substances, obtaining Ct values of plasma miRNA in different expressions in each sample compared with Ct values of cel-miR-54, and using 2 Ct values of plasma miRNA in each sample -ΔCt Calculating the relative expression quantity of the miRNA in the blood plasma, wherein the calculation formula of the delta Ct is as follows: Δ Ct = Ct miRNA -Ct cel-miR-54 Setting negative control group (RT-qPCR reaction system without cDNA template and ddH 2 O substitution), results are expressed as mean ± SD (mean ± SD), test method is two-tailed Student's test, all RT-qPCR are performed in 2 replicates.
TABLE 1 miRNA markers and sequences of primers, probes and external reference standards thereof
(2) Results of the experiment
The result graph of the expression difference of the 6 miRNA markers in the coronary heart disease patients and the high-risk normal control subjects is shown in fig. 5, wherein (a), (b), (c), (d), (e), (f) are the result graphs of the expression difference of hsa-miR-15b-5p, hsa-miR-29c-3p, hsa-miR-378b, hsa-miR-320e, hsa-miR-361-5p and hsa-miR-199a-3p in the coronary heart disease patients and the high-risk normal control subjects respectively, and it can be seen that the plasma relative expression amount aggregation of the 6 miRNA markers is good, and the relative expression amount of the 6 miRNA markers in the plasma of the coronary heart disease patients is remarkably increased (p < 0.01) compared with the high-risk normal control subjects.
2. Clinical efficacy evaluation of plasma miRNA markers
(1) Experimental methods
To evaluate the individual 6 miRNA markers (hsa-miR-15 b-5p, hsa-miR-29c-3p, hsa-miR-378b, hsa-miR-320e, hsa-miR-361-5p and hsa-miR-199a-3 p), any combination of 2 to 5 of the 6 miRNA markers (2 marker combinations of hsa-miR-378b and hsa-miR-320e, 2 marker combinations of hsa-miR-378b and hsa-miR-15b-5p, 2 marker combinations of hsa-miR-320e and hsa-miR-15b-5p, 3 marker combinations of hsa-miR-320e, hsa-miR-15b-5p and hsa-miR-378b, the combination of 4 markers of hsa-miR-320e, hsa-miR-15b-5p, hsa-miR-378b and hsa-miR-29c-3p, and the combination of 5 markers of hsa-miR-320e, hsa-miR-15b-5p, hsa-miR-378b, hsa-miR-29c-3p and hsa-miR-361-5 p) and the combination of 6 miRNA markers as coronary heart disease diagnosis markers, carries out risk assessment on the plasma miRNA expression results of the patients with coronary heart disease and high-risk normal control subjects, and evaluates the value of the miRNA markers in coronary heart disease diagnosis through an ROC curve.
Sensitivity (sensitivity) is taken as an ordinate to represent a true positive rate, 1-specificity (specificity) is taken as an abscissa to represent a false positive rate, ROC curve analysis is carried out, and the larger the area (AUC value) under the ROC curve is, the higher the diagnosis accuracy is; on the ROC curve, the point closest to the top left of the graph is the cut-off value for high sensitivity and specificity.
(2) Results of the experiment
The results of ROC curve analysis of the individual 6 miRNA markers in the coronary heart disease patients and the high-risk normal control subjects are shown in FIG. 6, wherein (a), (b), (c), (d), (e) and (f) are hsa-miR-15b-5p, hsa-miR-29c-3p, hsa-miR-378b, hsa-miR-320e, hsa-miR-361-5p and hsa-miR-199a-3p in the coronary heart disease patients and the high-risk normal control subjects respectively, and the results of ROC curve analysis of the hsa-miR-15b-5p, the AUC value of hsa-miR-29c-3p is 0.615, the AUC value of hsa-miR-b is 0.784, the AUC value of hsa-miR-320e is 0.811, the AUC value of hsa-miR-361-5p is 0.603, and the AUC value of hsa-miR-3 a-199 a-3p is 0.199; the results show that the 6 miRNA markers have certain accuracy in diagnosing whether the subject suffers from coronary heart disease, and can be used as coronary heart disease diagnosis markers.
The result of ROC curve analysis of any 2-5 of the 6 miRNA markers in the coronary heart disease patient and the high-risk normal control subject is shown in FIG. 7, and it can be seen that the AUC value of the combination of 2, 3, 4 or 5 of the 6 miRNA markers is 0.849-0.961; demonstrating improved accuracy of any 2-5 combinations of 6 miRNA markers compared to 6 miRNA markers alone.
The ROC curve analysis result graph of the combination of 6 miRNA markers in the coronary heart disease patients and the high-risk normal control subjects is shown in fig. 8, and it can be seen that the AUC value of the combination of 6 miRNA markers is 0.971; demonstrating that the accuracy of the combination of 6 miRNA markers in diagnosing whether a subject has coronary heart disease is very high.
In addition, the results of the sensitivity and specificity of the combination of the 6 miRNA markers and the 6 miRNA markers in the patients with coronary heart disease and the high-risk normal control subjects are shown in table 2, and it can be seen that the sensitivity and specificity of the 6 miRNA markers alone for the diagnosis of coronary heart disease are 55.7% to 76.5% and 56.3% to 76.6%, while the sensitivity and specificity of the combination of the 6 miRNA markers for the diagnosis of coronary heart disease are 92.8% and 89.5%.
TABLE 2 combination of 6 miRNA markers alone and 6 miRNA markers in coronary heart disease patients with high risk of normal control subjects sensitivity and specificity results
The sensitivity and specificity results of the combination of any 2-5 of the 6 miRNA markers in the coronary heart disease patients and the high-risk normal control subjects are shown in Table 3, and it can be seen that the sensitivity and specificity of the combination of any 2-5 of the 6 miRNA markers for the diagnosis of coronary heart disease are 75.6-90.3% and 81.6-90.3%.
The above results show that compared with the independent 6 miRNA markers and the combination of any 2-5 of the 6 miRNA markers for diagnosing the coronary heart disease, the combination of the hsa-miR-15b-5p, hsa-miR-29c-3p, hsa-miR-378b, hsa-miR-320e, hsa-miR-361-5p and hsa-miR-199a-3p 6 miRNA markers has higher accuracy, stronger specificity and higher sensitivity in diagnosing whether a subject suffers from the coronary heart disease, and has extremely excellent diagnosis reference value.
TABLE 3 sensitivity and specificity results of any 2-5 combinations of 6 miRNA markers in patients with coronary heart disease and high-risk normal control subjects
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.
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<213> Artificial Sequence (Artificial Sequence)
<400> 12
cagtgcaggg tccgaggt 18
<210> 13
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gtgcagggtc cgaggtcaga gccacctggg caattttttt ttttttctgc 50
<210> 14
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ctgggactgg acttggag 18
<210> 15
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cagtgcaggg tccgaggt 18
<210> 16
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gtgcagggtc cgaggtcaga gccacctggg caattttttt ttttccttct 50
<210> 17
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ctgggaaagc tgggttg 17
<210> 18
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
cagtgcaggg tccgaggt 18
<210> 19
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gtgcagggtc cgaggtcaga gccacctggg caattttttt ttttgtaccc 50
<210> 20
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ctgggttatc agaatctcca g 21
<210> 21
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
cagtgcaggg tccgaggt 18
<210> 22
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gtgcagggtc cgaggtcaga gccacctggg caattttttt tttttaacca 50
<210> 23
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
ctgggacagt agtctgcaca t 21
<210> 24
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
cagtgcaggg tccgaggt 18
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
cagagccacc tgggcaattt 20
<210> 26
<211> 22
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
aggauaugag acgacgagaa ca 22
<210> 27
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gtgcagggtc cgaggtcaga gccacctggg caattttttt tttttgttct 50
<210> 28
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ctgggaggat atgagacgac g 21
<210> 29
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
cagtgcaggg tccgaggt 18
Claims (2)
1. The application of primers and probes for detecting plasma miRNA markers in preparation of kits or reagents is characterized in that the plasma miRNA markers consist of hsa-miR-15b-5p, hsa-miR-29c-3p, hsa-miR-378b, hsa-miR-320e, hsa-miR-361-5p and hsa-miR-199a-3 p; the sequence of hsa-miR-15b-5p is shown in SEQ ID NO:1, the sequence of the hsa-miR-29c-3p is shown as SEQ ID NO:2, the sequence of hsa-miR-378b is shown in SEQ ID NO:3, the sequence of hsa-miR-320e is shown in SEQ ID NO:4, the sequence of the hsa-miR-361-5p is shown as SEQ ID NO:5, the sequence of the hsa-miR-199a-3p is shown in SEQ ID NO: and 6.
2. The use of claim 1, wherein the reverse transcription primer of hsa-miR-15b-5p has the sequence shown in SEQ ID NO:7, the sequences of the forward primer and the reverse primer of the hsa-miR-15b-5p are respectively shown in SEQ ID NO: 8-9; the sequence of the reverse transcription primer of the hsa-miR-29c-3p is shown in SEQ ID NO:10, the sequences of the forward primer and the reverse primer of the hsa-miR-29c-3p are respectively shown in SEQ ID NO:11 to 12; the sequence of the reverse transcription primer of hsa-miR-378b is shown in SEQ ID NO:13, the sequences of the forward primer and the reverse primer of the hsa-miR-378b are respectively shown in SEQ ID NO:14 to 15; the sequence of the reverse transcription primer of hsa-miR-320e is shown in SEQ ID NO:16, the sequences of the forward primer and the reverse primer of hsa-miR-320e are respectively shown in SEQ ID NO:17 to 18; the sequence of the reverse transcription primer of the hsa-miR-361-5p is shown as SEQ ID NO:19, the sequences of the forward primer and the reverse primer of the hsa-miR-361-5p are respectively shown in SEQ ID NO:20 to 21; the sequence of the reverse transcription primer of the hsa-miR-199a-3p is shown as SEQ ID NO:22, the sequences of the forward primer and the reverse primer of the hsa-miR-199a-3p are respectively shown in SEQ ID NO:23 to 24; the sequence of the probe in the kit or the reagent is shown as SEQ ID NO: shown at 25.
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| CN112575079B (en) * | 2020-12-31 | 2023-02-24 | 郑州大学第一附属医院 | Exosome miRNA (micro ribonucleic acid) serving as molecular marker for diagnosing type 2 diabetes combined coronary heart disease and application of exosome miRNA |
| CN113549688B (en) * | 2021-08-27 | 2023-08-04 | 河北医科大学第二医院 | A set of molecular markers for diagnosing coronary artery disease |
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