CARDIOVASCULAR EVENTS AND/OR DEATH IN PATIENTS WITH

CORONARY HEART DISEASE BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for predicting major adverse cardiovascular events and/or death in patients suffering from coronary heart disease, and more particularly to the method for analyzing the microRNA expression of a coronary heart disease patient to predict whether the patient is at high risk for major adverse cardiovascular events and/or death.

2. Description of the Prior Arts

Coronary heart disease is the most common disease caused by atherosclerotic degeneration. Due to cholesterol or fat accumulates on the inner wall of arterial blood vessels to form a blood clot, and the blood clot would block the coronary artery and cause heart hypoxia. It causes common cardiovascular symptoms such as chest pain, shortness of breath, upper limb pain, night sweats, and vomiting. If the artery is completely blocked by the blood clot, and then oxygen cannot be transported the heart, which would lead to a heart attack or heart tissue damage.

Patients with coronary heart disease are at a much higher risk of major adverse cardiovascular events and deaths than the average person. It is estimated that patients with coronary heart disease have a risk of major adverse cardiovascular events (MACE) or death of more than 20% within five years. Major adverse cardiovascular events include recurrent angina pectoris, acute myocardial infarction, severe arrhythmia, heart failure, cardiac death, nonfatal myocardial infarction, non-fatal stroke, death from stroke, etc. The main factors are hypertension, diabetes, hyperlipidemia, atrial fibrillation, smoking, and poor living habits.

More than one-third of patients in developing countries die from coronary heart disease, and the average age of the disease is gradually reduced. In addition, the number of deaths from coronary heart disease has increased year by year. According to statistics from the World Health Organization, the number of deaths due to coronary heart disease increased by 40% in 2013 compared with 1990. Patients with coronary heart disease are like an untimely bomb due to patients and their families do not know when it will be diseased or dead and cause great stress.

At present, the methods for diagnosing coronary heart disease include: electrocardiogram, cardiac ultrasound, cardiac tomography and other imaging diagnostic methods and biomarkers. Because there is no biomarker for predicting MACE or death in patients with coronary heart disease currently, patients with coronary heart disease can only be tracked by regular cardiac imaging diagnosis, and a good life style to reduce the incidence of adverse cardiovascular events.

MicroRNA (miRNA) is a ribonucleic acid (RNA) molecule about 21 to 23 nucleotides long and regulates the expression of other genes in eukaryotes widely. miRNA is transcribed from DNA but does not translate into proteins (belonging to non-coding RNA). The miRNA binds to the target messenger ribonucleic acid (mRNA), thereby inhibiting the expression of the gene after transcription, and plays an important role in regulating gene expression, cell cycle, and development timing of the organism. Current research has found that miRNA has many functions, such as cancer markers, early cancer detection biomarkers, and exogenous miRNA that can be detected in the blood. SUMMARY OF THE INVENTION

The object of the present invention is to provide a method for predicting major adverse cardiovascular events and/or death in patients with coronary heart disease by detecting the microRNA expression.

To achieve the above objective, the technical method of the present invention is to analyze the expression amount of at least one target microRNA in the serum of coronary heart disease patients, and the target microRNA is selected from one or any combination of: has-miR-1468, has-miR-1307, has-miR-200c, has-miR-574, has-miR-432, has-miR-3615, has-miR-3605, and has-miR-181a-2. When the expression of target microRNA in the patient is lower than one predetermined value, it indicates that the patient belongs to a high-risk group that will suffer from MACE or death in the future. With the prognostic information provided by this method, clinicians will be able to custom-design a better treatment strategy to improve the clinical outcomes of patients with coronary heart disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a comparison diagram of target miRNA expression levels in example 1 of the present invention.

FIG. 2 is a Kaplan-Meier survival analysis diagram of target has-miR-1468 in example 2 of the present invention.

FIG.3 is a Kaplan-Meier survival analysis diagram of target has-miR-1307 superscripted in example 2 of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

Without intent to limit the scope of the invention, exemplary instruments, apparatus, methods and their related results according to the embodiments of the present invention are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the invention without regard for any particular theory or scheme of action.

Example 1

This example is used to analyzes the difference of microRNA expression in patients with coronary heart disease who have suffered from MACE or death and those who have no MACE, and then to find out detecting biomarker.

Plasma samples were selected from blood sample in the present example as follows.

No MACE: After 5 years of follow-up, the plasma samples from patients with coronary heart disease who have not suffered from major adverse cardiovascular events or death.

Recurrent MACE: After 5 years of follow-up, the plasma samples from patients with coronary heart disease who have suffered from major adverse cardiovascular events or death.

First, the samples of two above groups were analyzed by next-generation sequencing to analyze the type and the expression of miRNA. The results were shown in FIG. 1. The plasma levels of the eight miRNAs were lower in the patients with

MACE or death than those without it. As shown in Table 1, the AUC’s under the cutoff value of eight miRNAs all exceeded 0.64 through ROC curve (AUC), and survival analysis showed all of these were significant negative predictors of MACE or death in patients with coronary heart disease.

Table 1 _' Plasma miRNAs that are significant predictors as cardiovascular events

5 or death in CAD patients

Example 2 Survival test

In this example, the occurrence of MACE or death of the patients with coronary heart disease and the expression of has-miR-1468 and has-miR-1307 in vivo were observed by long-term follow-up and Kaplan-Meier survival analysis.

10 As shown in Fig’s 2 and 3, the coronary heart disease patients with expression of has-miR-1468 and has-miR-1307 lower than the RPKM cut-off value were prone to have MACE or death, and the survival rate of Kaplan-Meier survival analysis decreased significantly via long-term follow-up. Conversely, in the coronary heart disease patients with expression of has-miR-1468 and has-miR-1307 higher than the 15 RPKM cut-off value, the physiological conditions were stable. Therefore, the expressions of these target miRNAs compared with their RPKM cut-off value could be used as a basis for predicting whether a MACE or death would occur in patients with coronary heart disease.

In summary, the expressions of the target miRNAs found in the present embodiment in patients with coronary heart disease were related to the major adverse cardiovascular events and death. In the patients with coronary heart disease, those with expression of single or multiple miRNAs lower than RPKM cut-off values would have higher risk for future MACE or death. Thus, the single or combined target miRNAs of the present invention could be used to predict the risk of MACE or death in the future. That is to say, the method proposed by the present invention has important values for clinical decision making and patient care.

The experimental methods of miRNA expression detection were not limited to the following: reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative real-time PCR (qPCR), digital droplet PCR (ddPCR), microarray, serial analysis of gene expression (SAGE), next-generation RNA sequencing, massively parallel signature sequencing (MPSS), ELISA, in situ hybridization (ISH), mass spectrometry (MS), RNA pull-down, and single nucleotide polymorphisms (SNPs).