阅读说明：本技术 一种用于冠心病诊断的血浆miRNA标志物及其应用 (Plasma miRNA marker for coronary heart disease diagnosis and application thereof ) 是由 苏明扬 苟德明 于 2020-04-02 设计创作，主要内容包括：本发明公开了一种用于冠心病诊断的血浆miRNA标志物及其应用。本发明提供了一种用于冠心病诊断的血浆miRNA标志物,所述miRNA标志物为hsa-miR-15b-5p、hsa-miR-29c-3p、hsa-miR-378b、hsa-miR-320e、hsa-miR-361-5p或hsa-miR-199a-3p中的任意一种或几种的组合；其序列依次如SEQ ID NO：1～6所示。本发明通过对血浆中的miRNA标志物的相对表达量进行差异分析,即可诊断受试者是否患有冠心病,且6个miRNA标志物的组合的AUC值为0.971,敏感性高达92.8％、特异性高达89.5％；因此,所述miRNA标志物在制备冠心病诊断试剂盒和/或制剂中具有广泛的应用前景。(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.)

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.

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.