CN112877424A - Biomarker related to myocardial infarction and application thereof - Google Patents

Abstract

The invention discloses a biomarker related to myocardial infarction and application thereof, wherein the biomarker comprises LINC01191 and/or MXD 1. Experiments prove that LINC01191 and MXD1 are up-regulated in patients with myocardial infarction, and the fact that LINC01191 and/or MXD1 can be used for predicting or diagnosing myocardial infarction is prompted. The invention also discloses a method for identifying and evaluating the effect of the medicament and/or the operation treatment and/or the physical treatment on the myocardial infarction.

Description

Biomarker related to myocardial infarction and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a biomarker related to myocardial infarction and application thereof.

Background

With the change of life style and dietary structure of modern society, the incidence of myocardial infarction increases year by year, and the myocardial infarction becomes an important disease seriously harming the health of people in China. Myocardial infarction belongs to a physical and mental disease, has the characteristics of sudden onset of disease, quick change of illness state, more complications, poorer prognosis and high mortality, not only increases the social and economic burden, but also has great influence on the aspects of physiology, psychology, life quality and the like of patients. Therefore, prevention and treatment of myocardial infarction is currently an important issue. The method has the advantages of timely and accurate diagnosis of myocardial infarction and important significance for actively taking effective treatment measures to ensure that the heart is timely reperfused to reduce the death rate and ensure good prognosis of patients. The current serum biomarkers for clinical diagnosis of myocardial infarction mainly include creatine kinase isoenzyme, myoglobin, troponin and the like, however, in view of the defects of the above markers in sensitivity and specificity, the search for high-sensitivity and high-specificity biomarkers is still the focus of current research.

With the development of high throughput transcriptome sequencing technology, researchers have found that more than 90% of transcriptomes in the human genome are non-coding RNAs, and micrornas (mirnas) among them have been thought to be involved in a variety of biological processes, including the development and progression of diseases. With the continued discovery of disease-associated mirnas, researchers have focused on another non-coding RNA, Long non-coding RNA (LncRNAs). LncRNAs are more than 200 nucleotide units in length and have no or only limited protein coding capacity. LncRNAs have the functions of regulating chromatin and regulating genes according to the positions in cells. LncRNAs can be stably expressed in cell lines and tissues, have relatively stable properties in various body fluids, and particularly LncRNAs in circulation can resist factors such as freezing, thawing or enzyme degradation, so that research on LncRNAs related to myocardial infarction provides scientific basis for searching noninvasive biomarkers with high specificity and sensitivity for myocardial infarction, and provides possibility for developing new more effective treatment.

Disclosure of Invention

The invention aims to research the biomarkers related to myocardial infarction, thereby providing a new means for diagnosing myocardial infarction.

In order to achieve the purpose, the invention adopts the following technical scheme:

the present invention provides, in a first aspect, a system for diagnosing myocardial infarction, the system comprising an input device for inputting an expression amount of a molecular marker, an output device for outputting a diagnosis result of myocardial infarction; wherein the molecular marker comprises LINC01191 and/or MXD 1.

The "LINC 01191" refers to Gene with Gene ID of 440900.

The "MXD 1" refers to a Gene whose Gene ID is 4084.

Further, the system includes a computing device, the computing device including a memory and a processor; the memory having stored therein a computer program, the processor being configured to execute the computer program stored in the memory; and the computing device is used for analyzing the possibility of the myocardial infarction risk result according to the expression quantity of the molecular marker.

Further, the system further comprises a device for detecting the expression level of the molecular marker.

Furthermore, the detection device comprises a real-time quantitative PCR instrument, a real-time quantitative PCR primer, a high-throughput sequencing platform, a detection chip and a chip signal reader.

Furthermore, the chip comprises a probe for detecting the expression level of the marker.

Furthermore, the chip also comprises an internal reference probe, wherein the internal reference probe is a probe for detecting the expression level of GAPDH or beta-Actin.

Further, the real-time quantitative PCR primers comprise real-time quantitative PCR primers for detecting the expression level of the molecular marker.

Further, the real-time quantitative PCR primer also comprises an internal reference primer, wherein the internal reference primer is a real-time quantitative PCR primer for detecting GAPDH or beta-Actin.

In a second aspect, the invention provides the use of an agent for detecting a molecular marker comprising LINC01191 and/or MXD1 in the manufacture of a product for use in diagnosing myocardial infarction.

Further, the reagent comprises a primer, a probe and an antibody.

The primer of the present invention can be prepared by chemical synthesis, appropriately designed by referring to known information using a method known to those skilled in the art, and prepared by chemical synthesis.

The probe of the present invention may be prepared by chemical synthesis, by appropriately designing with reference to known information using a method known to those skilled in the art, and by chemical synthesis, or may be prepared by preparing a gene containing a desired nucleic acid sequence from a biological material and amplifying it using a primer designed to amplify the desired nucleic acid sequence.

The probe that hybridizes to the nucleic acid sequence of a gene may be DNA, RNA, a DNA-RNA chimera, PNA, or other derivatives. The length of the probe is not limited, and any length may be used as long as specific hybridization and specific binding to the target nucleotide sequence are achieved. The length of the probe may be as short as 25, 20, 15, 13 or 10 bases in length. Also, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or more, even for the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and at most, usually not more than 30 base pairs, and the length complementary to the nucleotide sequence of interest is optimally 15 to 25 base pairs. The probe self-complementary sequence is preferably less than 4 base pairs so as not to affect hybridization efficiency.

Further, detecting the molecular marker is performed by:

1) obtaining a subject sample;

2) determining the expression level of said molecular marker in said sample.

In a third aspect, the invention provides a product for diagnosing myocardial infarction, which comprises a reagent for detecting the expression level of a molecular marker, wherein the molecular marker comprises LINC01191 and/or MXD 1.

Furthermore, the product comprises a chip, a kit, test paper or a high-throughput sequencing platform; the high-throughput sequencing platform is a special tool, and with the development of high-throughput sequencing technology, the construction of a gene expression profile of a person becomes very convenient work. By comparing the gene expression profiles of patients with diseases and normal people, the abnormality of which gene is related to the disease can be easily analyzed. Therefore, the knowledge that the abnormality of the LINC01191 and/or MXD1 gene is related to the occurrence of myocardial infarction in high-throughput sequencing also belongs to the novel application of the LINC01191 and/or MXD1 provided by the invention and is also within the protection scope of the invention.

Further, the kit comprises a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry kit, an immunohistochemical detection kit, an ELISA kit and an electrochemiluminescence detection kit.

In the present invention, the kit further comprises a container, instructions for use, a positive control, a negative control, a buffer, an auxiliary agent or a solvent, and instructions for use with the kit, wherein the instructions describe how to use the kit for detection, and how to use the detection results to determine the development of a disease and select a treatment regimen.

The kit of the present invention may contain a plurality of different reagents suitable for practical use (e.g., for different detection methods), and is not limited to the reagents listed so far, and is included in the scope of the present invention as long as the reagents are used for diagnosing myocardial infarction based on the detection of LINC01191 and/or MXD1 genes.

In a fourth aspect, the present invention provides a method for identifying and assessing the effect of a pharmaceutical agent and/or a surgical and/or physical therapy on myocardial infarction, said method comprising:

(1) collecting a subject having a myocardial infarction to provide a first sample;

(2) obtaining a gene expression profile from the first sample;

(3) administering to or on the subject one or more drug candidates and/or performing one or more physical or surgical treatments;

(4) providing a second sample from the subject in step (3);

(5) obtaining a gene expression profile from the second sample;

(6) comparing the gene expression profile obtained in steps (2) and (5) with a reference gene expression profile; and

(7) assessing whether the one or more drug candidates and/or treatments are effective against myocardial infarction based on the comparison in step (6);

further, the genes in the step (2) and the step (5) are LINC01191 and/or MXD 1.

In a fifth aspect, the invention provides a pharmaceutical composition comprising an agent that inhibits the expression of LINC01191 and/or MXD1 genes.

Further, the pharmaceutical compositions comprise the active ingredient in combination with one or more pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the dosage form envisaged. Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, sucrose, ethanol, glycerol and the like, various preservatives, lubricants, dispersants, flavoring agents. Moisturizers, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like may also be added as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration, and the formulations that may be used in such compositions may be in the form of their original compounds as such, or optionally in the form of their pharmaceutically acceptable salts. The compositions so formulated may be administered in any suitable manner known to those skilled in the art, as desired. In using the pharmaceutical composition, a safe and effective amount of the medicament of the present invention is administered to a human.

The appropriate dose of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the method of preparation, the mode of administration, the age, body weight, sex, disease state, diet, time of administration, route of administration, excretion rate and reaction sensitivity of the patient, and a skilled physician can easily determine the prescription and the dose of administration effective for the desired treatment or prevention.

The sixth aspect of the invention provides the use of the pharmaceutical composition provided by the fifth aspect of the invention in the preparation of a medicament for preventing or treating myocardial infarction.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, some terms are explained as follows:

the term "sample" as used herein refers to a composition obtained or derived from a subject (e.g., an individual of interest) that comprises cells and/or other molecular entities to be characterized and/or identified based on, for example, physical, biochemical, chemical, and/or physiological characteristics. For example, the phrase "disease sample" or variants thereof refers to any sample obtained from a subject of interest that is expected or known to contain the cells and/or molecular entities to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, and tissue culture fluids, tissue extracts such as homogenized tissue, cell extracts, and combinations thereof.

The term "hybridization" as used herein generally refers to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences that under appropriate conditions will form a thermodynamically stable double-stranded structure. The term "hybridization" as used herein may refer to hybridization under stringent or non-stringent conditions.

The term "primer" as used herein refers to 7 to 50 nucleic acid sequences capable of forming a base pair (base pair) complementary to a template strand and serving as a starting point for replication of the template strand. The primers are generally synthesized, but naturally occurring nucleic acids may also be used. The sequence of the primer does not necessarily need to be completely identical to the sequence of the template, and may be sufficiently complementary to hybridize with the template. Additional features that do not alter the basic properties of the primer may be incorporated. Examples of additional features that may be incorporated include, but are not limited to, methylation, capping, substitution of more than one nucleic acid with a homolog, and modification between nucleic acids.

The term "chip," also referred to as an "array," as used herein, refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as "microarrays," can generally be produced using either mechanosynthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid-phase synthesis methods. The array may comprise a flat surface, or may be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. The array may be packaged in a manner that allows for diagnostic or other manipulation of the fully functional device.

The term "treatment" as used herein generally relates to the treatment of a human or animal (e.g., as applied by a veterinarian) wherein some desired therapeutic effect may be achieved, for example, inhibiting the development of a condition (including reducing the rate of development, halting development), ameliorating the condition, and curing the condition. Treatment as a prophylactic measure (e.g., prophylaxis) is also included. The use of a patient who has not yet developed a condition but who is at risk of developing the condition is also encompassed by the term "treatment".

The term "molecular marker" as used herein refers to a molecular indicator having a specific biological property, biochemical characteristic or aspect, which can be used to determine the presence or absence of a particular disease or condition and/or the severity of a particular disease or condition. It can be used universally with "biomarker" and "gene marker".

The term "excipient", as used herein, refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

The invention has the advantages and beneficial effects that:

the invention discovers for the first time that whether a subject suffers from myocardial infarction can be predicted or diagnosed by detecting the expression level of LINC01191 and/or MXD1 genes.

The present invention discloses a method for identifying and assessing the effect of a pharmaceutical agent and/or a surgical and/or physical treatment on myocardial infarction.

Drawings

FIG. 1 is a box plot of LINC01191 differential expression;

FIG. 2 is a ROC graph of LINC01191 diagnosis of acute myocardial infarction;

fig. 3 is a ROC graph of LINC01191 and MXD1 combined diagnosis of acute myocardial infarction.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

Example 1 screening of differentially expressed genes in acute myocardial infarction

1. Data source

Downloading acute myocardial infarction (AIM) data set GSE66360 from GEO database, sample size control: AIM 50: 49.

2. Differential expression analysis

Differential expression analysis was performed using the "limma" package in R software, with screening criteria for differential genes as adj. p value < 0.05.

3. Results of the experiment

The analysis result shows that the biomarkers LINC01191 and MXD1 related to the invention are up-regulated in samples of patients suffering from acute myocardial infarction, as shown in Table 1 and figure 1.

TABLE 1 differential expression of LINC01191, MXD1

Gene	AveExpr	t	P.Value
				LINC01191	5.34	3.57	0.00
MXD1	9.01	5.28	0.00

Example 2 diagnostic Performance validation

1. Experimental methods

Receiver Operating Curves (ROCs) were plotted using the R package "pROC" (version 1.15.0), AUC values, sensitivity and specificity were analyzed, and the diagnostic efficacy of the markers alone or in combination was judged. When the diagnosis efficiency of the index combination is judged, logistic regression is carried out on the expression level of each gene, the probability of whether each individual suffers from cancer is calculated through a fitted regression curve, different probability division threshold values are determined, and the sensitivity, specificity, accuracy and the like of the combined detection scheme are calculated according to the determined probability division threshold values.

2. Results of the experiment

(1) ROC curve analysis of LINC01191

Referring to table 2 and fig. 2, the diagnosis efficacy of LINC01191 is shown, and the experimental results show that LINC01191 has a good diagnosis effect on acute myocardial infarction.

TABLE 2 diagnostic efficacy of LINC01191

Gene	AUC	Sensitivity of the composition	Specificity of
				LINC01191	0.76	0.64	0.80

(2) ROC curve analysis for LINC01191+ MXD1 combined diagnosis

The AUC values of the LINC01191 and MXD1 genes are shown in Table 3, and the ROC curve of the LINC01191+ MXD1 combined diagnosis is shown in FIG. 3.

TABLE 3 AUC values of genes

Gene	AUC
		LINC01191	0.76
MXD1	0.79
		LINC01191+MXD1	0.88

According to the experimental results, the diagnosis effect of the LINC01191+ MXD1 combination on acute myocardial infarction is better than that of a single marker, and the diagnosis effect is better.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims (10)

1. A system for diagnosing myocardial infarction, comprising an input means for inputting an expression amount of a molecular marker, an output means for outputting a result of diagnosis of myocardial infarction; wherein the molecular marker comprises LINC01191 and/or MXD 1;

preferably, the system further comprises a computing device comprising a memory and a processor; the memory having stored therein a computer program, the processor being configured to execute the computer program stored in the memory;

preferably, the system further comprises a means for detecting the expression level of the molecular marker; preferably, the detection device comprises a real-time quantitative PCR instrument, a real-time quantitative PCR primer, a high-throughput sequencing platform, a detection chip and a chip signal reader;

preferably, the chip comprises a probe for detecting the expression level of the marker; preferably, the chip also comprises an internal reference probe, wherein the internal reference probe is a probe for detecting the expression level of GAPDH or beta-Actin;

preferably, the real-time quantitative PCR primer comprises a real-time quantitative PCR primer for detecting the expression amount of the molecular marker; preferably, the real-time quantitative PCR primer further comprises an internal reference primer, wherein the internal reference primer is a real-time quantitative PCR primer for detecting GAPDH or beta-Actin.

2. Use of a reagent for detecting a molecular marker in the manufacture of a product for diagnosing myocardial infarction, wherein the molecular marker comprises LINC01191 and/or MXD 1.

3. The use of claim 2, wherein said reagents comprise primers, probes, antibodies.

4. Use according to claim 2, characterized in that the detection of the molecular marker is carried out by:

1) obtaining a subject sample;

2) determining the expression level of said molecular marker in said sample.

5. A product for diagnosing myocardial infarction, which comprises a reagent for detecting the expression level of a molecular marker, wherein the molecular marker comprises LINC01191 and/or MXD 1.

6. The product of claim 5, wherein the product comprises a chip, a kit, a strip, or a high throughput sequencing platform.

7. The product of claim 6, wherein the kit comprises a qPCR kit, an immunoblot detection kit, an immunochromatographic detection kit, a flow cytometric assay kit, an immunohistochemical detection kit, an ELISA kit, and an electrochemiluminescent detection kit; preferably, the kit further comprises instructions for assessing whether the subject is suffering from or susceptible to myocardial infarction.

8. A method for identifying and assessing the effect of a pharmaceutical agent and/or a surgical and/or physical therapy on myocardial infarction, the method comprising:

(1) collecting a subject having a myocardial infarction to provide a first sample;

(2) obtaining a gene expression profile from the first sample;

(3) administering to or on the subject one or more drug candidates and/or performing one or more physical or surgical treatments;

(4) providing a second sample from the subject in step (3);

(5) obtaining a gene expression profile from the second sample;

(6) comparing the gene expression profile obtained in steps (2) and (5) with a reference gene expression profile; and

(7) assessing whether the one or more drug candidates and/or treatments are effective against myocardial infarction based on the comparison in step (6);

preferably, the genes in step (2) and step (5) are LINC01191 and/or MXD 1.

9. A pharmaceutical composition comprising an agent that inhibits the expression of LINC01191 and/or MXD1 gene.

10. Use of the pharmaceutical composition of claim 9 for the preparation of a medicament for the prevention or treatment of myocardial infarction.

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