CN111286536A

CN111286536A - Gene marker for diagnosing myocardial infarction and application thereof

Info

Publication number: CN111286536A
Application number: CN202010329892.5A
Authority: CN
Inventors: 步芳芳
Original assignee: Qilu Hospital of Shandong University Qingdao
Current assignee: Qilu Hospital of Shandong University Qingdao
Priority date: 2020-04-24
Filing date: 2020-04-24
Publication date: 2020-06-16
Anticipated expiration: 2040-04-24
Also published as: CN111286536B

Abstract

The invention relates to an acute myocardial infarction gene marker and application thereof. The expression level of the gene marker LINC01065 provided by the invention in peripheral blood mononuclear cells of patients with acute myocardial infarction is obviously higher than that of normal human peripheral blood mononuclear cells, and the expression level of LINC01065 is obviously reduced after the patients are treated, which indicates that the gene marker provided by the invention can be used for diagnosing patients with acute myocardial infarction.

Description

Gene marker for diagnosing myocardial infarction and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a gene marker for diagnosing myocardial infarction.

Background

Acute myocardial infarction is a cardiovascular disease with high morbidity and mortality, mainly due to acute necrosis of myocardial tissue caused by persistent severe ischemia. Early, accurate diagnosis can ensure that the patient receives timely reperfusion therapy, thereby significantly reducing mortality. Currently, the widely used biological marker for diagnosing myocardial infarction in clinical practice is troponin (cTnI), but the concentration of troponin in plasma may be falsely increased in certain cardiac and non-cardiac diseases such as severe heart failure, atrial fibrillation, chronic kidney disease, severe sepsis, myocarditis, etc. Therefore, it is necessary to search for biomarkers with higher specificity and sensitivity in the diagnosis of acute myocardial infarction.

The transcriptome in the human genome is more than 90%, but only around 2% of the genes can encode proteins, the remainder being non-coding RNA. Among non-coding RNAs, microRNAs of 21-23nt in length have been shown to play important roles in a variety of biological and pathological processes. Long non-coding RNA is a type of non-coding RNA with transcript length more than 200nt discovered in recent years. The long-chain non-coding RNA is widely involved in cell signal transduction and influences processes such as cell metabolism, cell growth, differentiation, apoptosis and death. Since long non-coding RNAs tend to form relatively stable secondary structures, they can be detected in blood. At present, the research of long-chain non-coding RNA in myocardial infarction is less, and compared with the traditional biochemical markers, the long-chain non-coding RNA has high stability, so that the search for the long-chain non-coding RNA with specificity and sensitivity to the myocardial infarction has important significance for early diagnosis of the myocardial infarction.

Disclosure of Invention

The present invention aims to provide a marker for diagnosing myocardial infarction.

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

a gene marker for diagnosing acute myocardial infarction is LINC01065, and the sequence of LINC01065 is shown in SEQ ID NO. 1.

In addition, the invention provides application of LINC01065 in preparation of a kit for diagnosing acute myocardial infarction.

Preferably, the kit is a fluorescent quantitative PCR kit.

Preferably, the kit comprises a primer pair for specifically detecting LINC01065 expression.

Preferably, the forward primer sequence of the primer pair is shown as SEQ ID NO. 2; the reverse primer sequence of the primer pair is shown as SEQ ID NO. 3.

Preferably, the kit further comprises a GAPDH primer pair, SYBR Green fluorochrome, ddH 2O.

In addition, the invention also provides a kit for diagnosing myocardial infarction, which is a fluorescent quantitative PCR kit and is used for detecting the expression of LINC01065 in peripheral blood mononuclear cells.

The invention has the advantages of

The invention discovers that the long-chain non-coding RNA LINC01065 is highly expressed in peripheral blood mononuclear cells of patients with acute myocardial infarction for the first time, and the expression level of LINC01065 is reduced after treatment, which indicates that LINC01065 can be used as a marker for diagnosing acute myocardial infarction.

Meanwhile, when the LINC01065 fluorescent quantitative PCR kit provided by the invention is used for detecting the expression of LINC01065, a patient with acute myocardial infarction can be effectively diagnosed, and acute severe viral myocarditis and acute myocardial infarction can be effectively distinguished.

Drawings

FIG. 1 expression differences of LINC01065 in the normal, acute myocardial infarction and treatment groups.

FIG. 2 ROC curves for the normal group and acute myocardial infarction group.

FIG. 3 ROC curves for acute myocardial infarction group and treatment group.

FIG. 4 shows the difference in the expression of LINC01065 in the normal group and in the acute severe viral myocarditis group.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. It should be understood that the present invention may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein.

The study mainly collected peripheral blood (42 cases) of acute myocardial infarction patients and peripheral blood (42 cases) of normal people in 12h of acute chest pain occurrence in Qilu hospital (Qingdao) of Shandong university and peripheral blood of acute myocardial infarction patients after treatment.

Diagnostic criteria for acute myocardial infarction: 1 the change in the cardiac biomarker troponin exceeds the upper reference value and there is at least one evidence of myocardial ischemia: (1) clinical symptoms of cardiac ischemia; (2) typical ECG changes indicating new myocardial ischemia, such as ST segment changes, new left bundle branch block; (3) pathological Q waves appear on leads at different parts of the electrocardiogram. (4) Imaging evidence indicates new viable myocardial loss or new regional myocardial wall motion abnormalities. The selected patients with acute myocardial infarction all perform coronary angiography to prompt that the coronary stenosis is more than 50 percent, and strictly follow the globally unified definition of acute myocardial infarction recommended to adopt myocardial infarction in China by the cardiovascular subcontract of the Chinese medical society and the like. The normal people are chest pain patients who have coronary heart disease eliminated by coronary angiography detection.

Exclusion criteria: the diagnosis of cardiomyopathy, atrial fibrillation, severe ventricular arrhythmia, malignant tumor, chronic hepatorenal disease, and patients who have recently undergone surgical operations.

All patients received routine treatment, informed and signed consent.

Example 1

1. Peripheral blood mononuclear cell isolation

(1) Taking 2ml of fresh anticoagulation blood, centrifuging at the rotating speed of 3500 rpm for 10 minutes, separating the blood into two layers, wherein the upper layer is faint yellow plasma, and the lower layer is blood cells;

(2) removing the upper light yellow plasma layer, adding normal saline according to the proportion of 1:1, and gently mixing with an aseptic sterilizing gun head;

(3) injecting 4ml of lymphocyte separation solution into a 15ml enzyme-free centrifuge tube, carefully pouring physiological saline of mixed blood cells, rotating at 1500 rpm, and centrifuging for 20 minutes;

(4) the liquid is divided into three layers, wherein the uppermost layer is faint yellow plasma, the middle layer is milk white, the lower layer is red blood cells, milk white lymphocyte layer cells are carefully collected and put into a 2ml centrifugal tube, and the centrifugal tube is centrifuged for 5 minutes at 10000 rpm;

(5) removing supernatant, retaining precipitate, washing for 2 times to obtain mononuclear cells.

RNA extraction

(1) Adding 1ml of Trizaol into the mononuclear cells, and blowing the cells for 5min on ice by using a pipette;

(2) adding 200 mu L of chloroform, reversing the mixture up and down, uniformly mixing the mixture for 20 times, standing the mixture for 5 minutes at room temperature, rotating the mixture at 12000 rpm, and centrifuging the mixture for 15 minutes;

(3) carefully transferring the upper aqueous phase into a new enzyme-free centrifuge tube, adding isopropanol with the same volume, uniformly mixing, and standing on ice for 10 min;

(4) 13000 r/min, centrifuging for 10min at 4 ℃, discarding the supernatant, and keeping the precipitate;

(5) adding 500 μ L of precooled 70% ethanol to dissolve the precipitate, 12000 r/min, centrifuging at 4 ℃ for 3min, discarding the supernatant, drying at room temperature for 5-10min, and adding 40 μ L of DEPC water to obtain the RNA.

3. Reverse transcription reaction

Reference Tiangen biological reverse transcription kit:

(1) gDNA removal reaction system

Composition of matter	Amount of the composition used
		5×g DNA Buffer	2 μL
TotaL RNA
		2 μg
RNase-Free ddH2O			Make up to 10. mu.L

Reaction conditions are as follows: the mixture was placed in a PCR apparatus at 42 ℃ for 3min on ice.

(2) Reverse transcription reaction system

Composition of matter	Amount of the composition used
		10×King RT Buffer	2 μL
FastKing RT Enzyme Mix	1 μL
		FQ-RT Primer Mix	2 μL
Reaction solution in step (1)	10 μL
		RNase-Free ddH2O	5μL

Reaction conditions are as follows: incubate at 42 ℃ for 15min, incubate at 95 ℃ for 3min, and then place on ice.

4. Fluorescent quantitative PCR

Reference is made to the Invitrogen 2xSYBR Green PCR Master Mix instructions:

reaction system:

reaction conditions are as follows: 10min at 95 ℃; 95 ℃ for 15s, 62 ℃ for 60s for 40 cycles.

The primer sequence is as follows:

LINC01065

Forward primer 5'-TTCTTCCAGGGTTGCCTGCA- 3'

Reverse primer 5'- TGCTGGCTCCTTCTCTTCATGA- 3'

GAPDH

Forward primer 5'- ACGTGTCAGTGGTGGACCTG- 3'

Reverse primer 5'- GTGTAGCCCAGGATGCCCTT- 3'

by using 2^-△△CtThe method processes real-time quantitative PCR data, calculates the expression change of LINC01065, and the experimental result is shown in FIG. 1.

Results of the experiment

The experimental results are shown in fig. 1, from which it can be seen that the relative expression amount (3.951 ± 0.269) of LINC01065 in peripheral blood mononuclear cells in the acute myocardial infarction group is significantly higher than that in the normal group, and the difference is statistically significant compared with that in the normal group.

The expression level of LINC01065 in the treated group was 1.261 ± 0.087, and the difference was statistically significant compared to the acute myocardial infarction group, but not significantly different compared to the normal group (P value of 0.525). Shows that LINC01065 can be used as marker for diagnosing acute myocardial infarction and as prognostic marker for acute myocardial infarction.

The ROC curves for the normal group and the acute myocardial infarction group are shown in fig. 2, and it can be seen from the figure that the AUC values of the ROC curves are 0.9450, std. Error 0.2177, 95% confidence interval 0.9023 to 0.9877, and P value < 0.0001. Shows that LINC01065 has excellent diagnostic value as a diagnostic marker of acute myocardial infarction.

The ROC curves for the acute myocardial infarction group and the treatment group are shown in fig. 3, and it can be seen from the figure that the AUC values of the ROC curves are 0.927, std. Error 0.02698, 95% confidence interval 0.8746 to 0.9803, and P value < 0.0001. Shows that LINC01065 has excellent diagnostic value as a marker for acute myocardial infarction prognosis.

Example 2

Fluorescent quantitative PCR kit for diagnosing acute myocardial infarction

1. Composition of matter

LINC01065 primer pair, GAPDH primer pair, SYBR Green fluorescent dye, and ddH 2O.

LINC01065

Forward primer 5'-TTCTTCCAGGGTTGCCTGCA- 3'

Reverse primer 5'- TGCTGGCTCCTTCTCTTCATGA- 3'

GAPDH

Forward primer 5'- ACGTGTCAGTGGTGGACCTG- 3'

Reverse primer 5'- GTGTAGCCCAGGATGCCCTT- 3'

2. Mode of addition

2. Reaction conditions

10min at 95 ℃; 95 ℃ for 15s, 62 ℃ for 60s for 40 cycles.

Example 3

20 patients with acute severe viral myocarditis were tested for the expression of LINC01065 in peripheral blood mononuclear cells.

1. Collecting peripheral blood of 20 patients with acute severe viral myocarditis;

2. the peripheral blood mononuclear cell separation, RNA extraction, reverse transcription reaction and fluorescence quantitative PCR reaction were performed as in example 1, comparing the expression difference of LINC01065 in peripheral blood mononuclear cells of patients with acute severe viral myocarditis and normal human peripheral blood mononuclear cells.

Results of the experiment

The experimental results are shown in FIG. 4, and it can be seen from the graph that the relative expression level of LINC01065 in peripheral blood mononuclear cells of patients with acute severe viral myocarditis is 1.106 + -0.089, and the difference between the relative expression level and the normal group is not statistically significant. Thus indicating that the detection of the expression level of LINC01065 can not diagnose the acute severe viral myocarditis.

Because the clinical symptoms of acute severe viral myocarditis and acute myocardial infarction are similar, misdiagnosis often occurs in clinic, but the LINC01065 gene marker provided by the invention has high expression in patients with acute myocardial infarction, but has no obvious increase in patients with acute severe viral myocarditis. Therefore, when the LINC01065 fluorescent quantitative PCR kit provided by the invention is used for detecting the expression of LINC01065, a patient with acute myocardial infarction can be effectively diagnosed, and acute severe viral myocarditis and acute myocardial infarction can be effectively distinguished.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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<120> gene marker for diagnosing myocardial infarction and application thereof

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tggacttctc tgacatctac tagaccacat taaatgaatc acagccttga aactgaactg 180

gaaggacttc cttcaagatt tttatctaat tcctgttgga cagtgtaaaa tgccacttct 240

tccagggttg cctgcatatc atcaaaaggg aaatagtact catcgaagat gtgcctttgc 300

taggaactag gacaagggtg gcctttataa attcatgaag agaaggagcc agcatatgaa 360

cttgctgcct atctttagca aggagatgca aatgacatcc acaaacctct tctgctgtat 420

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Claims

1. A gene marker for diagnosing acute myocardial infarction is characterized in that the marker is LINC01065, and the sequence of LINC01065 is shown in SEQ ID No. 1.

Application of LINC01065 in preparation of kit for diagnosing acute myocardial infarction.

3. The use according to claim 2, wherein the kit is a fluorescent quantitative PCR kit.

4. The use according to claim 2, wherein said kit comprises a primer pair for specifically detecting LINC01065 expression.

5. The use of claim 4, wherein the forward primer sequence of the primer pair is shown as SEQ ID No. 2; the reverse primer sequence of the primer pair is shown as SEQ ID NO. 3.

6. The use of claim 2, wherein the kit further comprises GAPDH primer pair, SYBRGreen fluorochrome, ddH 2O.

7. A kit for diagnosing acute myocardial infarction is characterized by being a fluorescent quantitative PCR kit and used for detecting the expression of LINC01065 in peripheral blood mononuclear cells.

8. The kit of claim 7, wherein the kit comprises a primer pair for specifically detecting LINC01065 expression.

9. The kit according to claim 8, wherein the forward primer sequence of the primer pair is shown as SEQ ID No. 2; the reverse primer sequence of the primer pair is shown as SEQ ID NO. 3.

10. The kit of claim 7, further comprising a GAPDH primer pair, SYBRGreen fluorochrome, ddH 2O.