CN108774641A - Coronary heart disease lncRNA expression spectrum discrimination identification biomarkers and related application - Google Patents

Abstract

The present invention provides coronary heart disease lncRNA expression spectrum discrimination identification biomarkers and related applications；Specifically, the application the present invention provides the reagent material and/or instrument and equipment of following lncRNA levels in sample of the detection from test individual in preparing the detecting system for diagnosis of coronary heart disease risk：ENST00000444488.1 and/or uc010yfd.1.The expression of ENST00000444488.1 increases, the expression of uc010yfd.1 declines, and test individual coronary heart disease risk increases.

Description

Coronary heart disease lncRNA expression profile identification and identification of biomarkers and related applications

technical field

The present invention relates to the recognition and identification of biomarkers of coronary heart disease lncRNA expression profiles and related applications. Specifically, the present invention relates to reagent materials and materials for detecting the levels of 7 differentially expressed lncRNAs such as ENST00000444488.1 in samples from individuals to be tested. The invention also relates to a detection system for assessing the risk of coronary heart disease.

Background technique

Coronary artery disease (CAD) is the leading cause of death in the world, including acute myocardial infarction (AMI), which is known for its severity and complexity, and is the main cause of morbidity and death in the global population. The "China Cardiovascular Disease Report 2017" report shows that the total prevalence of CAD in China in 2013 was 10.2‰, and the mortality rate of coronary heart disease among Chinese urban and rural residents in 2015 continued to rise since 2012. Although great progress has been made in the prevention, drug and intervention policies of coronary heart disease worldwide in recent years, the individual, medical and social economic burdens of cardiovascular disease are still gradually increasing, and the situation is not optimistic. Therefore, the diagnosis of coronary heart disease is particularly important. At the level of molecular biology, screening biomarkers will help to better diagnose coronary heart disease, and can also provide insights into the pathogenesis of coronary heart disease and the study of new targeted treatments. There is active help.

Atherosclerosis is the main pathogenesis of coronary heart disease, but also a chronic inflammatory disease. Cardiovascular disease risk factors, including hypertension, dyslipidemia, smoking, excessive drinking and other chronic risk factors stimulate the vascular endothelium for a long time, increase oxidative stress, induce inflammatory response, lead to endothelial dysfunction, and promote the occurrence of atherosclerosis; subsequently, foam cells Formation, smooth muscle cells proliferate and migrate to the subendothelium to promote the formation of fibrous plaques; the inflammatory response intensifies, leading to plaque rupture and thrombus formation, leading to acute coronary syndrome (acute coronary syndrome, ACS). Therefore, the process of atherosclerosis is actually a complex process involving the interaction of different types of cells, which is regulated by different coding genes and non-coding genes (Wierda, RJ, Geutskens, SB, Jukema, JW, et al., Epigenetics in atherosclerosis and inflammation, J Cell Mol Med, 2010; 14:1225-1240).

Long non-coding RNA (long non-coding RNA, lncRNA) is a kind of RNA molecule whose transcript length is greater than 200nt and does not encode protein. It has a low degree of conservation and is species-specific. A number of studies have proved that lncRNAs are involved in biological processes such as cell transduction, chromosome modification, transcription and translation regulation, and the occurrence of various diseases. Foam cell formation and vascular inflammation play important regulatory roles (Zhou, T, Ding, JW, Wang, XA, et al., Long noncoding RNAs and atherosclerosis, Atherosclerosis, 2016; 248:51-61). Myocardial infarction associated transcript (MIAT) is the earliest lncRNA to identify AMI risk factors, and its single single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) can change the expression of MIAT, leading to the risk of AMI Susceptibility (Ishii, N, Ozaki, K, Sato, H, et al., Identification of a novel non-coding RNA, MIAT, that confers risk of myocardial infarction, Journal of human genetics, 2006; 51:1087-1099). Genome-wide association studies (GWAS) of coronary heart disease have shown that the region located on chromosome 9p21 is the strongest susceptibility region for coronary heart disease (Helgadottir, A, Thorleifsson, G, Manolescu, A, et al., A common variant on chromosome 9p21 affects the risk of myocardial infarction, Science (New York, N.Y.), 2007; 316:1491-1493; McPherson, R, Pertsemlidis, A, Kavaslar, N, et al., A common allele on chromosome9 associated with coronary heart disease, Science ( New York, N.Y.), 2007; 316:1488-1491; Samani, NJ, Erdmann, J, Hall, AS, et al., Genomewide association analysis of coronary artery disease, The New England journal of medicine, 2007; 357:443- 453), this region contains a functional RNA INK4 locus antisense non-coding RNA ANRIL (antisense non-coingRNA in the INK4locus, ANRIL), which has a direct regulatory effect on cardiovascular disease (Holdt, LM, Hoffmann, S, Sass, K , et al., Alu elements in ANRIL non-coding RNA at chromosome9p21modulate atherogenic cell functions through trans-regulation of gene networks, PLoS genetics, 2013; 9:e1003588). A number of studies have shown that ANRIL is expressed in vascular endothelial cells, smooth muscle cells, macrophages, inflammatory cells, and common carotid arteries in the process of atherosclerosis, regulates target genes in cis, promotes cell proliferation, inhibits cell apoptosis, and phenotypes Consistent with the performance of atherosclerosis.

In order to explain how lncRNAs participate in the regulation of the occurrence and development of CAD, differentially expressed lncRNAs were systematically identified in patients with coronary heart disease, and potential functional lncRNAs were screened as biomarkers to further explain their role in the mechanism of atherosclerosis will be very necessary. The current diagnosis of coronary heart disease is based on typical symptoms, electrocardiogram and cardiac troponin (cTnI and cTnT), but these diagnostic methods have their own limitations and deficiencies. Therefore, it is of great significance to find new biomarkers with better sensitivity and specificity to diagnose coronary heart disease.

Contents of the invention

One purpose of the present invention is to find new biomarkers with better sensitivity and specificity for diagnosing coronary heart disease.

In a study, the inventors of this case obtained the genome-wide lncRNA/mRNA expression profile of coronary heart disease through lncRNA/mRNA microarray analysis, including 1210 differentially expressed lncRNAs and 890 differentially expressed mRNAs. The present invention selected 7 differentially expressed lncRNAs: ENST00000444488.1, uc010yfd.1, ASO3973, ENST00000602558.1, ENST00000561165.1, RNA147299_p0403_imsncRNA819 and ENST00000415255.1, and repeated verification in 7 large samples of independent coronary heart disease cases and controls The differentially expressed lncRNAs were consistent with the results of the expression profile microarray, among which ENST00000444488.1 was significantly increased to 2.6 times in CAD group PBMCs (P<0.01), uc010yfd.1, ASO3973, ENST00000602558.1, ENST00000561165.1, RNA147299_p0403_0ENST50ncRNA819 and .1 Significantly decreased to 37%, 81%, 79%, 87%, 83% and 65% in PBMCs of CAD group respectively (P<0.01). Through the ROC curve analysis, the AUC of 6 lncRNAs (ENST00000444488.1, uc010yfd.1, ASO3973, ENST00000602558.1, ENST00000561165.1 and RNA147299_p0403_imsncRNA819) was 0.599-0.799, which has a certain diagnostic efficacy for coronary heart disease. The AUCs of ENST00000444488.1 and uc010yfd.1 were 0.799 (0.762-0.837) and 0.779 (0.744-0.815), respectively. The AUC of the combination of ENST00000444488.1 and uc010yfd.1 rose to 0.851 (0.819-0.884), the sensitivity was 0.707, and the specificity was 0.844, which has higher diagnostic value. Therefore, ENST00000444488.1 and uc010yfd.1 can be used as new lncRNA biomarkers; further combining the four common risk factors of age, BMI, blood sugar and HDL model, the ROC curve area AUC is as high as 0.902 (0.876-0.928), and the sensitivity is 0.736, and the specificity is 0.922, so the model has higher diagnostic efficiency. Among the 412 patients with coronary heart disease in an independent large sample, there were 290 AMI patients and 122 non-AMI patients. Compared with non-AMI patients, the expression of ENST00000444488.1 was significantly increased to 1.5 times in AMI patients. ROC curve analysis showed that its The AUC was 0.758 (0.716-0.800), the sensitivity was 0.632, and the specificity was 0.773, which indicated that ENST00000444488.1 had the efficacy of diagnosing AMI.

Thereby, on the one hand, the present invention provides the application of following lncRNA as the biomarker of diagnosis coronary heart disease:

ENST00000444488.1 and/or uc010yfd.1.

On the other hand, the present invention also provides the application of reagent materials and/or instruments and equipment for detecting the following lncRNA levels in the sample from the individual to be tested in the preparation of a detection system for diagnosing the risk of coronary heart disease:

ENST00000444488.1 and/or uc010yfd.1.

According to specific embodiments of the present invention, ASO3973, ENST00000602558.1, ENST00000561165.1, RNA147299_p0403_imsncRNA819 and/or ENST00000415255.1 can be used to assist ENST00000444488.1 and/or uc010yfd.1 in further diagnosing coronary heart disease.

According to a specific embodiment of the present invention, the lncRNA level includes the expression level in peripheral blood mononuclear cells.

According to a specific embodiment of the present invention, in the relevant application of the present invention for diagnosing coronary heart disease, the lncRNA includes ENST00000444488.1 and/or uc010yfd.1; preferably further includes ASO3973, ENST00000602558.1, ENST00000561165.1, RNA147299_p0403_imsncRNA819 and ENST000004152 one or more of .

According to a specific embodiment of the present invention, in the related application of the present invention for diagnosing coronary heart disease, the lncRNA includes ENST00000444488.1. Preferably, one or more of uc010yfd.1, ASO3973, ENST00000602558.1, ENST00000561165.1, RNA147299_p0403_imsncRNA819 and ENST00000415255.1 are further included. More preferably include uc010yfd.1; further preferably include one or more of ASO3973, ENST00000602558.1, ENST00000561165.1, RNA147299_p0403_imsncRNA819 and ENST00000415255.1.

According to a specific embodiment of the present invention, the expression level of ENST00000444488.1 increases, and the risk of coronary heart disease of the individual to be tested increases.

According to a specific embodiment of the present invention, the expression level of uc010yfd.1 is reduced, and the risk of coronary heart disease of the individual to be tested increases.

According to a specific embodiment of the present invention, further, the expression level of ASO3973, ENST00000602558.1, ENST00000561165.1, RNA147299_p0403_imsncRNA819 and/or ENST00000415255.1 is reduced, and the risk of coronary heart disease of the individual to be tested increases.

In a more specific embodiment of the present invention, the detection system includes detecting the expression levels of ENST00000444488.1 and uc010yfd.1, age, BMI, blood sugar and HDL of the individual to be tested.

On the other hand, the present invention also provides the application of reagent materials and/or instruments for detecting the level of ENST00000444488.1 in samples from individuals to be tested in the preparation of a detection system for diagnosing the risk of acute myocardial infarction;

Preferably, when the expression level of ENST00000444488.1 increases, the risk of acute myocardial infarction in the individual to be tested increases.

On the other hand, the present invention also provides a detection system for assessing the risk of coronary heart disease, which includes: reagent materials and/or instruments for detecting the following lncRNA levels in samples from individuals to be tested:

ENST00000444488.1 and/or uc010yfd.1. Preferably, the lncRNA includes ENST00000444488.1 and uc010yfd.1; preferably further includes one or more of ASO3973, ENST00000602558.1, ENST00000561165.1, RNA147299_p0403_imsncRNA819 and ENST00000415255.1.

According to a specific embodiment of the present invention, the expression level of ENST00000444488.1 increases, and the risk of coronary heart disease of the individual to be tested increases.

According to a specific embodiment of the present invention, in the present invention, the individuals to be tested are East Asian populations, including Chinese, Filipinos, Chinese-Americans, Chinese-Singaporeans, and Malaysians of Singaporean origin. For specific testing, the sample may come from blood, urine, saliva, gastric juice, hair or biopsy of the individual to be tested, preferably blood.

The expression level of lncRNA can be detected by any available technique in the art.

According to a specific embodiment of the present invention, in the present invention, the reagent materials and/or instruments and equipment for detecting the expression level of the lncRNA can be the reagent materials and equipment used in any feasible technology for detecting the expression level of the lncRNA /or equipment, etc.

According to a specific embodiment of the present invention, the detection system for assessing the risk of coronary heart disease of the present invention includes a detection unit and an evaluation unit, wherein:

The detection unit includes reagent materials and/or equipment for detecting the expression level of the lncRNA in the sample from the individual to be tested, for obtaining the detection result of the expression level of the lncRNA of the individual to be tested;

The evaluation unit includes a processing unit for performing evaluation processing according to the detection result of the detection unit. Among them, the expression level of ENST00000444488.1 increases, the expression level of uc010yfd.1 decreases, and the risk of coronary heart disease of the individual to be tested increases.

The detection system for evaluating the risk of coronary heart disease in the present invention can be a virtual device, as long as it can realize the functions of the detection unit and the evaluation unit. The detection unit may include various detection reagent materials and/or detection instruments and equipment, etc.; the data analysis unit may be any that can analyze and process the detection results of the detection unit to obtain a risk assessment of coronary heart disease The calculation instrument, module or virtual device of the status, for example, can pre-create a corresponding data chart for various possible test results and corresponding disease risk conditions, and compare the test results of the detection unit with the data chart to obtain the crown. Heart disease risk assessment results.

Applying the technology of the invention can provide positive help to the pathogenesis of coronary heart disease and the research of new targeted treatment means.

Description of drawings

Figure 1A-Figure 1D are cluster analysis and volcano plots of differentially expressed lncRNA/mRNA in biological expression profiles. Among them, Figure 1A: differentially expressed lncRNAs; Figure 1B: differentially expressed mRNAs; Y-axis: hierarchical clustering of CAD case and control groups; X-axis: hierarchical clustering of expression levels of lncRNAs and mRNAs. Green and red indicate significantly different downregulated and upregulated expression changes, respectively. Figure 1C: volcano plot of differentially expressed lncRNAs; Figure 1D: volcano plot of differentially expressed mRNAs; X-axis: log2 (Fold Change); Y-axis: log10 (corrected P value). Red dots and green dots indicate that lncRNAs or mRNAs are significantly up-regulated or down-regulated 2-fold.

Figure 2A-Figure 2H show the analysis results of qRT-PCR repeated verification of 7 differentially expressed lncRNAs. Among them, Fig. 2A-Fig. 2G: the expression changes of lncRNA ENST00000444488.1, uc010yfd.1, ASO3973, ENST00000602558.1, ENST00000561165.1, ENST00000415255.1 and RNA147299_p0403_ims in 412 cases of coronary heart disease and 295 healthy controls; Fig. 2H : Comparison of expression fold changes of 7 screened lncRNAs in microarray and qRT-PCR. The Y-axis indicates the log2 differential expression ratio between cases and controls, Healthy is the control group, CAD is the coronary heart disease case group, ** indicates that compared with the control group, P<0.001.

Figure 3A-Figure 3D show the ROC curve analysis results of ROC curve area analysis of lncRNAENST00000444488.1 and uc010yfd.1 in 412 cases of CAD and 295 cases of healthy controls. Among them, Figure 3A: AUC of ENST00000444488.1; Figure 3B: AUC of uc010yfd.1; Figure 3C: AUC of combining ENST00000444488.1 and uc010yfd.1; Figure 3D: combining age, BMI, blood glucose and HDL and ENST00000444488.1 and AUC of the uc010yfd.1 model.

Figure 4A-Figure 4D shows the analysis results of the area under the ROC curve of the four lncRNAs in CAD. Among them, Figure 4A: AUC result of lncRNAENST00000602558.1; Figure 4B: AUC result of ASO3973; Figure 4C: AUC result of RNA147299_p0403_imsncRNA819; Figure 4D: AUC result of ENST00000561165.1.

Figure 5A and Figure 5B show the analysis results that lncRNAENST00000444488.1 has diagnostic value for acute myocardial infarction. Among them, Figure 5A: Analysis of the expression level of lncRNA ENST00000444488.1 in AMI patients; Figure 5B: ROC curve analysis of lncRNA ENST00000444488.1 in AMI.

Detailed ways

In order to understand the present invention more clearly, the present invention will now be further described with reference to the following examples. The examples are for illustration only and do not limit the invention in any way.

The experimental methods without specific conditions indicated in the examples are conventional methods and conventional conditions well known in the art, or according to the conditions suggested by the manufacturer.

Embodiment one

The research protocol of this embodiment includes lncRNA chip screening stage and repeated verification stage.

1. lncRNA chip screening stage:

A total of 141 cases of coronary heart disease cases and healthy controls were collected and divided into healthy group and coronary heart disease case group. AgilentlncRNA/mRNA Human Gene Expression Microarray V4.0 (4×180K) was applied, based on the multiple of difference >2, P value <0.05, prediction Screening criteria such as target genes and positional relationship with CAD or dyslipidemia susceptibility loci. In the present invention, seven lncRNAs differentially expressed in coronary heart disease were selected as candidate lncRNAs for further verification.

2. Repeat the verification phase:

A total of 707 CHD case-control samples were collected (412 CHD cases and 295 healthy controls), and qRT-PCR was used to detect the expression levels of 7 candidate lncRNAs in peripheral blood mononuclear cells (PBMCs) of the case-control samples. Six lncRNAs were screened for receiver operating characteristic curve (ROC) analysis.

3. Subject selection criteria for coronary heart disease case-control studies

This study protocol has been approved by the Ethics Committee of Fuwai Hospital, Chinese Academy of Medical Sciences, and all subjects participating in the study signed the informed consent.

3.1 lncRNA/mRNA expression profile chip samples

A total of 141 coronary heart disease case-control lncRNA/mRNA chip research samples were male, including 93 cases in the case group, who were diagnosed with coronary heart disease at Fuwai Hospital of the Chinese Academy of Medical Sciences from January 2011 to May 2014. The 48 samples of the healthy control group came from healthy individuals recruited in the Shijingshan community from September 2013 to June 2014.

3.2 qRT-PCR repeated verification of coronary heart disease case-control population

3.2.1 Coronary Heart Disease Case Population

The case group consisted of 412 CAD patients recruited from Fuwai Hospital of the Chinese Academy of Medical Sciences between January 2011 and May 2014. The inclusion criteria for the case group were as follows:

1) Inclusion criteria:

a) Han nationality, male, aged 30-70 years;

b) Left main coronary artery ≥ 50% stenosis or major stenosis at least There are 70% narrow coronary heart disease patients. Acute myocardial infarction: typical symptoms of chest pain, lasting for more than 30 minutes, electrocardiogram with ST-segment elevation in two consecutive leads (limb lead ≥ 0.1m, chest lead ≥ 0.2mV) and dynamic changes; myocardial injury markers increased And higher than 2 times the normal value, there is a dynamic evolution within 24 hours. Unstable angina includes initial exertional, exacerbated, and spontaneous angina. Stable angina pectoris: short-term chest pain episodes induced by exercise or other increases in myocardial oxygen demand, accompanied by changes in the ST-T segment of the electrocardiogram, sublingual nitroglycerin may relieve the pain.

2) Exclusion criteria:

If the patient has any of the following conditions, exclude it: 1) congenital heart disease; 2) rheumatic valvular disease; 3) cardiomyopathy; 4) stroke; 5) diabetes; 6) malignant tumor; 7) acute or chronic Infectious disease; 8) Severe liver or kidney dysfunction; 9) Secondary hypertension; 10) Thyroid disease; 11) Familial hypercholesterolemia; 12) Cognitive dysfunction, dementia, severe mental illness; 13 ) did not sign the informed consent.

3.2.2 Healthy control group

The 295 samples of the healthy control group came from healthy individuals recruited from September 2013 to June 2014 in Shijingshan Community and Henan Province. Inclusion criteria: aged 30-60 years, excluding coronary heart disease, diabetes, heart valve disease, congenital heart disease, cardiomyopathy, heart failure, secondary hypertension, stroke, hypertension grade 2 or above, familial high blood pressure Cholesterolemia, severe kidney and liver disease, and thyroid disease.

4. Receiver operating characteristic curve analysis

Receiver operating characteristic (ROC) curve analysis was performed on the lncRNA expression detection data of patients with coronary heart disease and healthy controls to study whether differentially expressed lncRNAs in coronary heart disease could be used as potential diagnostic biomarkers for coronary heart disease.

5. Experimental method

The main experimental methods used in the embodiment include:

(1) Isolation of peripheral blood mononuclear cells (PBMCs)

1) Using gradient centrifugation, dilute and mix the anticoagulated whole blood and Hank’s solution at a ratio of 1:1;

2) Slowly spread the diluted whole blood on the lymphocyte separation medium along the wall of the centrifuge tube at a ratio of 2:1, and centrifuge at 2800g for 20min at room temperature;

3) Take out the centrifuge tube and divide the tube into four layers (the upper layer is plasma and Hank's solution, the lower layer is red blood cells and granulocytes, the middle layer is lymphocyte separation liquid, and the white cloud is visible to the naked eye between the plasma layer and the separation liquid. Thin layer, rich in mononuclear cells and platelets);

4) After absorbing the uppermost layer of plasma, collect the mononuclear cells at the interface between the plasma layer and the lymphocyte separation medium, absorb all the PBMCs as much as possible, and transfer them to a new centrifuge tube;

5) Add 12ml of Hank’s solution, evenly suspend the cells, centrifuge at 2250g for 10min at low temperature, discard the supernatant, and repeat again;

6) Centrifuge at 2250g for 3min, and discard the remaining Hank's solution in the upper layer;

7) Add 1ml TRIzol to the isolated PBMCs, pipette the cells slowly until uniform, then transfer to a new 1.5ml RNase-free Eppendorf tube, and store in a -80°C ultra-low temperature refrigerator.

(2) Extraction of total cellular RNA

After the cells were washed, they were lysed with TRIzol reagent for 5 minutes, and then the cells were collected. Add 100 μl of isoamyl alcohol:chloroform (1:49) to each sample, shake vigorously, wait for the layers to stand, centrifuge to absorb the supernatant, and add an equal volume of isoamyl alcohol to the supernatant. propanol, the product was ethanol-precipitated at -80°C overnight. The next day, it was centrifuged and washed to obtain RNA precipitate, which was dissolved in DEPC water, and the RNA concentration was determined by Nano Drop 2000 (Thermo Scientific, USA).

(3) Elimination of genomic DNA in RNA

The TURBO DNase Treatment and Removal Reagents kit from Life Technologies was used to eliminate genomic DNA contamination. The specific operation steps and system are as follows:

1) According to the RNA concentration determined by NanoDrop 2000, calculate the volume required for 2 μg RNA, and configure according to the system described in Table 1 below:

Table 1

Reagent Dosage RNA 2μg TURBO Dnase Buffer 1.5μl TURBO DNAse 0.34μl Nuclease-free water Make up 13.2μl

2) Incubate at 37°C for 30 minutes;

3) Add 1.5 μl DNase Inactivation Reagent, let stand at room temperature for 5 minutes, flick and mix every 1.5 minutes;

4) Centrifuge at 10000 g for 90 s, absorb the supernatant, and measure the RNA concentration using a NanoDrop2000 spectrophotometer.

(4) Total RNA reverse transcription

Reverse transcription was performed with the Transcriptor First Strand cDNA (Roche, Switzerland) kit (the method was operated according to the standard procedure recommended by the kit)

1) The reaction system is recorded in the following table 2:

Table 2

Reagent volume RNA 1μg Trancripot RT Reaction Buffer 4μl Deoxynucleotide Mix 2μl Random Hexamer Primer 2μl Protecor RNase Inhibitor 0.5μl Transcriptor Reverse Transcriptase 0.5μl RNAase-free water Make up 20μl

2) The reaction conditions are as follows in Table 3:

table 3

temperature time 25°C 10min 50℃ 1h 85°C 5min 4°C hold

Take 10 μl of cDNA for 4-fold dilution, store it at -20°C, and store the rest at -80°C for long-term storage.

(5) Real-time quantitative PCR (qRT-PCR)

ABI 7900T fluorescent quantitative PCR instrument (Applied Biosystems, USA) was used to detect the mRNA levels of lncRNAs and related genes. Take 10 μl of 4-fold diluted cDNA as a template, set 3 parallel wells for each sample, and use GAPDH as an internal reference gene, and use the 2- ^ΔΔCt method to calculate the relative expression level of mRNA.

Calculated as follows:

Relative expression level=2 ^{-[(target gene Ct-internal reference gene Ct)-(control target gene Ct mean-control group internal reference gene Ct mean)]} , that is, 2- ^△△Ct .

1) The reaction system is recorded in the following table 4:

Table 4

Reagent volume 2×Master Mix 5μl H ₂ O 3.6μl Primer F (10μM) 0.2μl Primer R (10μM) 0.2μl cDNA 1μl

2) The reaction conditions are recorded in the following table 5:

table 5

(6) qRT-PCR primers

The primers used in the qRT-PCR reaction are listed in Table 6 below:

Table 6

(7) Statistical analysis

Continuous variables with normal distribution were compared using independent sample t-test to compare the means between ^two groups, categorical variables were tested with χ2 test, variables with non-normal distribution were tested with Mann-Whitney U test, and data with normal distribution were tested with Shapiro-Wilk test. Fisher's exact test was used for significant GO enrichment and pathway analysis; the expression of lncRNA was analyzed and explained by the statistical difference between coronary heart disease patients and healthy controls; ROC curve was constructed and calculated using the "pROC" package in R statistical software (Version 3.3.3) Calculate the area to evaluate the diagnostic value of lncRNA among CAD patients; Logistic regression is used to analyze the risk association between lncRNA, population characteristics and CAD; the experimental data results are expressed as mean ± standard deviation (mean ± SD), with P<0.05 Indicates that the difference is statistically significant.

6. Experimental research results

(1) Basic characteristics of the research population

The basic characteristics of biological expression profiling chip CAD case-control samples are shown in Table 7.

Table 7 Basic characteristics of biological expression profiling chip CAD case-control samples

variable healthy control group CAD case group P-Value* quantity 48 93 age 53±1.7 50±6.4 0.002 BMI, kg/ ^m2 26.0±3.2 26.5±3.4 0.402 blood sugar, mmol/L 5.2±0.5 5.1±0.7 0.643 TG, mg/dl 151.8 (93.3,229.6) 151.3 (121.2,192) 0.490 LDL, mg/ml 95.8±29.5 90.9±28.8 0.351 TC, mg/dl 179.6±32.5 152.6±33.3 <0.0001 HDL, mg/ml 51.5±14.3 37.3±8.7 <0.0001 smoking, % 89.6 100 0.004 Drinking, % 75 100 <0.0001

Note: Continuous variables are mean ± standard deviation, and categorical variables are expressed as percentages;

BMI, body mass index;

TC, total cholesterol;

TG, triglycerides;

LDL, low-density lipoprotein;

HDL, high-density lipoprotein;

*Indicates the statistical test results of each index in the CAD case group and the healthy control group.

See Table 8 for the basic characteristics of CAD case-control samples repeatedly verified by qRT-PCR.

Table 8 Basic characteristics of qRT-PCR repeated validation of CAD case-control samples

variable healthy control group CAD case group P-Value* quantity 295 412 age 50.7±12.3 53.4±7.8 0.692 BMI, kg/ ^m2 25.5±3.4 26.0±3.1 0.088 HDL, mmol/L 46.9±10.1 38.7±8.8 <0.0001 LDL, mmol/L 101.8±25.0 98.5±32.1 0.040 blood sugar, mmol/L 5.18±0.6 5.00±0.6 <0.0001 TC, mmol/L 180.7±27.9 161.2±37.3 <0.0001 TG, mmol/L 119 (84.7-172.8) 129.0 (101.0-145.6) 0.014 creA, mmol/L 85.3±14.6 77.7±13.8 <0.0001 hypertension,% 38 32 0.144 Type (AMI/UA/SA) 290/82/40

Note: Continuous variables are mean ± standard deviation, and categorical variables are expressed as percentages;

BMI, body mass index;

TC, total cholesterol;

TG, triglycerides;

LDL, low-density lipoprotein;

HDL, high-density lipoprotein;

creA, creatinine;

AMI, acute myocardial infarction;

UA, unstable angina;

SA, stable angina;

*Indicates the statistical test results of each index in the CAD case group and the healthy control group.

(2) Expression spectrum chip analysis

Differentially expressed genes were screened from the expression profiling results with the fold change >2 and P<0.05 as the differential expression screening criteria. Compared with the control group, there were 1210 differentially expressed lncRNAs and 890 differentially expressed mRNAs among the differentially expressed genes in the CHD case group; 833 lncRNAs and 427 mRNAs were significantly up-regulated, and 377 lncRNAs and 463 mRNAs were significantly down-regulated. Cluster analysis and volcano plots of differentially expressed genes are shown in Figure 1A-Figure 1D. Among them, Figure 1A: differentially expressed lncRNAs; Figure 1B: differentially expressed mRNAs; Y-axis: hierarchical clustering of CAD case and control groups; X-axis: hierarchical clustering of expression levels of lncRNAs and mRNAs. Green and red indicate significantly different downregulated and upregulated expression changes, respectively. Figure 1C: volcano plot of differentially expressed lncRNAs; Figure 1D: volcano plot of differentially expressed mRNAs; X-axis: log2 (Fold Change); Y-axis: log10 (corrected P value). Red dots and green dots indicate that lncRNAs or mRNAs are significantly up-regulated or down-regulated 2-fold.

(3) qRT-PCR verification of differentially expressed lncRNAs

Based on screening principles such as multiple of difference >2, P<0.05, predicted target genes, and positional relationship between lncRNAs and CAD or dyslipidemia susceptibility sites, 7 lncRNAs were selected from the expression profile chip analysis results for repeated verification in the present invention. Among them, the SNP susceptibility loci of ENST00000602558.1, ENST00000561165.1 and ENST00000415255.1 near coronary heart disease and dyslipidemia are shown in Table 9.

Table 9 ENST00000602558.1, ENST00000561165.1 and ENST00000415255.1 susceptibility loci near coronary heart disease and dyslipidemia

Using the qRT-PCR method, 7 lncRNAs were verified in PBMCs of 412 patients with coronary heart disease and 295 healthy controls, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference. See Figures 2A-2H for junctions. Among them, Figure 2A-Figure 2G: qRT-PCR repeatedly verified the expressions of lncRNA ENST00000444488.1, uc010yfd.1, ASO3973, ENST00000602558.1, ENST00000561165.1, ENST00000415255.1 and RNA144239_p0 in 412 cases of coronary heart disease and 295 healthy controls Change level; Figure 2H: Comparison of the expression fold changes of the 7 screened lncRNAs of the present invention in chip and qRT-PCR. The Y-axis indicates the log2 differential expression ratio between cases and controls, Healthy is the control group, CAD is the coronary heart disease case group, ** indicates that compared with the control group, P<0.001. The results showed that the expression trend of 7 lncRNAs in the repeated verification results of large samples was consistent with the expression trend in the microarray data (Figure 2H), among which ENST00000444488.1 was significantly increased to 2.6 times in the PBMCs of the CAD group (P<0.01 ), uc010yfd.1, ASO3973, ENST00000602558.1, ENST00000561165.1, RNA147299_p0403_imsncRNA819 and ENST00000415255.1 were significantly decreased to 37%, 81%, 79%, 87%, 83% and 65% in CAD group PBMCs, respectively (Fig. - Figure 2G, P<0.01), due to the Ct value of ENST00000415255.1 >30, it will not be included in the follow-up study in the follow-up study.

(4) Identification of new lncRNA biomarkers for coronary heart disease

A) ROC curve analysis to evaluate the diagnostic performance of 6 lncRNAs

ROC curve was used to evaluate the diagnostic efficacy of ENST00000444488.1, uc010yfd.1, ASO3973, ENST00000602558.1, ENST00000561165.1 and RNA147299_p0403_imsncRNA819 in the diagnosis of coronary heart disease. The area under the ROC curve (Area under the ROC curve, AUC) of the six lncRNAs is shown in Figure 3A-Figure 3D and Figure 4A-Figure 4D. Among them, Figure 3A: AUC of ENST00000444488.1; Figure 3B: AUC of uc010yfd.1; Figure 3C: AUC of combining ENST00000444488.1 and uc010yfd.1; Figure 3D: combining age, BMI, blood glucose and HDL and ENST00000444488.1 and AUC of the uc010yfd.1 model. Figure 4A: the area under the ROC curve of lncRNAENST00000602558.1 in CAD; Figure 4B: the AUC result of ASO3973; Figure 4C: RNA147299_p0403_imsncRNA819; Figure 4D: the AUC result of ENST00000561165.1.

ENST00000602558.1、ASO3973、RNA147299_p0403_imsncRNA819和ENST00000561165.1的AUC分别为0.625(0.581-0.669)、0.639(0.595-0.682)、0.625(0.582-0.668)和0.599(0.555-0.644)(图4A-图4B)， The diagnostic efficiency is low.

The AUCs of ENST00000444488.1 and uc010yfd.1 were 0.799 (0.762-0.837) and 0.779 (0.744-0.815), respectively (Fig. 3A-Fig. 3B). The AUC of the combination of ENST00000444488.1 and uc010yfd.1 was 0.851 (0.819-0.884), the sensitivity was 0.707, and the specificity was 0.844 (Fig. 3C), which was higher than the AUC of any single lncRNA, so the combination of 2 lncRNAs had a higher crown The diagnostic value of heart disease can be used as a new lncRNA biomarker for coronary heart disease. Further combining the two lncRNAs markers with four common risk factors of age, BMI, blood sugar and HDL, the AUC reached 0.902 (0.876-0.928), the sensitivity was 0.736, and the specificity was 0.922 (Figure 3D), so the model has Higher diagnostic efficiency.

B) lncRNAENST00000444488.1 can be used as a biomarker for identifying AMI

Figure 5A and Figure 5B show the analysis data of lncRNAENST00000444488.1 with diagnostic value for acute myocardial infarction.

Compared with 112 non-AMI patients, the expression of ENST00000444488.1 was significantly increased to 1.5 times in 290 AMI patients (P<0.001) (Fig. 5A).

The present invention further uses ROC curve analysis to evaluate the effectiveness of ENST00000444488.1 in diagnosing AMI. The results showed that its AUC was 0.758 (0.716-0.800), its sensitivity was 0.632, and its specificity was 0.773 ( FIG. 5B ), indicating that ENST00000444488.1 has the efficacy of diagnosing AMI.

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<110> Fuwai Hospital, Chinese Academy of Medical Sciences

<120> Coronary heart disease lncRNA expression profile identification and identification of biomarkers and related applications

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Claims (10)

1. Applications of the lncRNA below 1. as the biomarker of diagnosis of coronary heart disease：

   ENST00000444488.1 and/or uc010yfd.1.
2. 2. the reagent material and/or instrument and equipment that detect lncRNA levels in the sample from test individual are being prepared for diagnosing Application in the detecting system of coronary heart disease risk, the lncRNA include：

   ENST00000444488.1 and/or uc010yfd.1.
3. 3. application according to claim 2, wherein lncRNA levels include expression in peripheral blood mononuclear cells.
4. 4. application according to claim 2, wherein lncRNA further comprise ASO3973, ENST00000602558.1, One kind or more in ENST00000561165.1, RNA147299_p0403_imsncRNA819 and ENST00000415255.1 Kind.
5. 5. application according to claim 2, wherein lncRNA includes ENST00000444488.1；Include preferably further uc010yfd.1；

   Still more preferably include ASO3973, ENST00000602558.1, ENST00000561165.1, RNA147299_ It is one or more in p0403_imsncRNA819 and ENST00000415255.1.
6. 6. application according to claim 2, wherein the expression of ENST00000444488.1 increases, test individual hat Worry risk increases.
7. 7. application according to claim 2, wherein the expression of uc010yfd.1 reduces, test individual coronary disease sufferer Sick risk increases.
8. 8. application according to claim 2, wherein the detecting system includes detection test individual The expression of ENST00000444488.1 and uc010yfd.1 and age, BMI, blood glucose and HDL situations.
9. 9. the reagent material and/or instrument and equipment of ENST00000444488.1 levels exist in sample of the detection from test individual Prepare the application in the detecting system for diagnosing acute myocardial infarction risk；

   Preferably, the expression of ENST00000444488.1 increases, and test individual acute myocardial infarction risk increases.
10. 10. a kind of detecting system of assessment incidence of coronary heart disease risk comprising：It detects following in the sample from test individual The reagent material and/or instrument and equipment of lncRNA levels：

    ENST00000444488.1 and/or uc010yfd.1；

    The expression of ENST00000444488.1 increases, and the expression of uc010yfd.1 reduces, test individual coronary disease sufferer Sick risk increases.