CN111518883A - 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. Despite a continuous effort of forty years, despite a reduced incidence of coronary artery disease, more than 1/3 died of the disease in individuals who died after age 35. 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. The number of coronary heart disease intervention cases in 2018 is 915256 cases per year, and from the number of coronary heart disease intervention cases in 2018, the coronary heart disease intervention treatment in continental areas of China is continuously promoted in stable development in both quantity and quality. The death rate of coronary heart disease of mature residents in China is 11.67/10 ten thousand, the death rate of coronary heart disease of residents in rural areas is 110.91/10 ten thousand, and the number of deaths of coronary heart disease in China is listed at the top in the world.
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 the method belongs to a radioactive or traumatic detection means, is expensive and cannot be used as a screening and detecting item for routine early diagnosis of coronary heart disease. Meanwhile, besides coronary artery CT and coronary artery angiography, there is no good method for evaluating the postoperative efficacy of patients after coronary heart disease interventional therapy.
The prior patent (application number is 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 of 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 for 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 further aims to provide a primer for detecting the miRNA marker.
The invention further 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 diagnosis 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 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.
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: 11-12; 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: 14-15; 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: 17-18; 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: 20-21; 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: 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 plasma miRNA of the coronary heart disease patient, 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 idea 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, the graphs (a), (b), (c), (d), (e) and (f) are results 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 respectively.
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, the graphs (a), (b), (c), (d), (e) and (f) are graphs of the 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 patients with coronary heart disease 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 combinations 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: mu.L of crude RNA, 1. mu.L of 0.05. mu.M reverse transcription primer, 1U of PolyA Polymerase (polyadenylic acid Polymerase), 100U of MMLV (murine leukemia reverse transcriptase)) 1.5. mu.L of reaction buffer, RNase-free Water to 10. mu.L; keeping the temperature at 37 deg.C for 15min, at 42 deg.C for 15min, at 75 deg.C for 5min to inactivate enzyme, rapidly 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 MgCl24mM ATP, 2mM dNTP, pH 8.0; 10. mu.L of cDNA was obtained.
RT-qPCR reaction: taking 0.5 μ L of 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 XTOX (fluorescent reference reagent), and nucleic acid-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 MgCl2，5％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 ℃, 5min, denaturation 95 ℃, 10s, annealing 60 ℃, 40s, 40 cycles, time of 50 min, two duplicate wells per RT-qPCR reaction.
Using external reference cel-miR-54 as reference and using 2-ΔCtCalculating the relative expression quantity of the miRNA in the blood plasma, wherein the calculation formula of the delta Ct is as follows: Δ Ct ═ CtmiRNA-Ctcel-miR-54The 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, and then 35 mu L of crude RNA can synthesize 87.5 mu L of cDNA within about 35 minutes; each RT-qPCR reaction actually consumes 0.25 mu L of cDNA, so that 175 different miRNAs can be detected by 87.5uLcDNA (each miRNA has two multiple holes, and one miRNA actually needs to consume 0.5 mu LcDNA) within 50 minutes, therefore, 175 miRNAs can be detected within 75 minutes by every 20 mu L 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 miRNAs which are differentially expressed in each sample are obtained, 28 candidate miRNA markers (let-7i-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 and miR-149-5 p) are respectively utilized, the Ct value of miR-155-5p, miR-15b-3p, miR-186-5p, miR-187-3p, miR-208a-3p, miR-26a-5p, miR-27a-3p, miR-29c-3p, miR-320e, miR-499a-5p, miR-92a-3p and miR-92b-5p) is compared with the Ct value of cel-miR-54 to be drawn, and meanwhile, a negative control group (a cDNA template is not added in an RT-qPCR reaction system, and ddH is used for setting2O substitution), 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 plasma samples of 95 coronary heart disease patients and 60 high-risk normal control subjects, performing plasma miRNA direct amplification RT-qPCR (RT-qPCR detection method referring 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 products, obtaining Ct values of plasma miRNAs with differential expression in each sample and cel-miR-54, and using 2 Ct values-ΔCtCalculating the relative expression quantity of the miRNA in the blood plasma, wherein the calculation formula of the delta Ct is as follows: Δ Ct ═ CtmiRNA-Ctcel-miR-54Setting negative control group (not adding cDNA template in RT-qPCR reaction system, using ddH2O 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 amounts of the 6 miRNA markers are good in aggregation, and the relative expression amounts of the 6 miRNA markers in the plasma of the coronary heart disease patients are significantly 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-15b-5p, hsa-miR-29c-3p, hsa-miR-378b, hsa-miR-320e, hsa-miR-361-5p and hsa-miR-199a-3p), any combination of 2 to 5 of the 6 miRNA markers (2 marker combinations for hsa-miR-378b and hsa-miR-320e, 2 marker combinations for hsa-miR-378b and hsa-miR-15b-5p, 2 marker combinations for hsa-miR-320e and hsa-miR-15b-5p, 3 marker combinations for 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 ROC curve analysis result graph of the separate 6 miRNA markers in the coronary heart disease patients and the high-risk normal control subjects is 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 ROC curve analysis result graph shows that the AUC value of hsa-miR-15b-5p is 0.663, the AUC value of hsa-miR-29c-3p is 0.615, the AUC value of hsa-miR-378b is 0.784, and the ROC 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-199a-3p is 0.581; 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 ROC curve analysis result graph 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 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 any 2-5 of the 6 miRNA markers in the diagnosis of the coronary heart disease are 75.6-90.3% and 81.6-90.3%.
The above results show that compared with the combination of any 2-5 of the 6 miRNA markers alone and the 6 miRNA markers used for diagnosing coronary heart disease, the combination of the 6 miRNA markers hsa-miR-15b-5p, hsa-miR-29c-3p, hsa-miR-378b, hsa-miR-320e, hsa-miR-361-5p and hsa-miR-199a-3p has higher accuracy, stronger specificity and higher sensitivity in diagnosing whether a subject suffers from coronary heart disease, and has extremely excellent reference value for diagnosis.
Sensitivity and specificity results of any 2-5 of miRNA markers in table 36 in coronary heart disease patients 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 changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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<211>50
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
gtgcagggtc cgaggtcaga gccacctggg caattttttt ttttttctgc 50
<210>11
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
ctgggactgg acttggag 18
<210>12
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
cagtgcaggg tccgaggt 18
<210>13
<211>18
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
aaagcugggu ugagaagg 18
<210>14
<211>50
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
gtgcagggtc cgaggtcaga gccacctggg caattttttt ttttccttct 50
<210>15
<211>17
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
ctgggaaagc tgggttg 17
<210>16
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
cagtgcaggg tccgaggt 18
<210>17
<211>22
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
uuaucagaau cuccaggggu ac 22
<210>18
<211>50
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
gtgcagggtc cgaggtcaga gccacctggg caattttttt ttttgtaccc 50
<210>19
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
ctgggttatc agaatctcca g 21
<210>20
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
cagtgcaggg tccgaggt 18
<210>21
<211>22
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
acaguagucu gcacauuggu ua 22
<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 (9)
1. A plasma miRNA marker for coronary heart disease diagnosis is characterized in that the miRNA marker is any one or the 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 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.
2. The use of the miRNA marker of claim 1 or the detection reagent thereof in the preparation of a coronary heart disease diagnostic kit and/or a preparation.
3. A primer for detecting the miRNA marker of claim 1, wherein the reverse transcription primer of hsa-miR-15b-5p has a sequence 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: 11-12; 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: 14-15; 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: 17-18; 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: 20-21; 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.
4. The use of the primer of claim 3 in the preparation of a coronary heart disease diagnostic kit.
5. A coronary heart disease diagnostic kit comprising primers capable of detecting the miRNA marker of claim 1.
6. The kit according to claim 5, wherein the primer is the primer according to claim 3.
7. The kit of claim 5, further comprising a probe having a sequence as set forth in SEQ ID NO: shown at 25.
8. Use of the kit according to any one of claims 5 to 7 for the diagnosis of coronary heart disease.
9. The use of claim 8, wherein the coronary heart disease is coronary atherosclerosis.
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