Molecular marker for diagnosing dilated heart disease complicated with heart failure by using extracellular vesicle lncRNA and application of molecular marker

Abstract

The invention belongs to the technical field of molecular diagnosis, and particularly relates to an extracellular vesicle lncRNA as a molecular marker for diagnosing dilated heart disease complicated with heart failure. The molecular marker is NR045681, and the specific nucleic acid sequence of the extracellular vesicle lncRNA is as shown in SEQ NO. 5. According to the technical scheme provided by the invention, the detection accuracy of the dilated heart disease combined with heart failure is effectively improved, the P value of the combination of the molecular markers is less than 0.05, the log fold change (FC) is greater than 1, and the molecular markers have obvious differential expression compared with a healthy control group.

Description

Technical Field

[0001] The present invention relates to the field of molecular diagnosis technology, and more specifically, to an extracellular vesicle lncRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure and its application. Background Art

[0002] Dilated cardiomyopathy (DCM) is one of the most common hereditary cardiomyopathies, which can cause cardiac insufficiency and cardiac dysfunction. DCM has a high incidence rate, and the mortality rate during a 52-month follow-up is 42.24%, bringing a heavy burden to families and society (Journal of Clinical Cardiovascular Diseases, 2018, 34(05): 421-434.). Long non-coding RNA (lncRNA) refers to a transcript with a length greater than 200 nucleotides and no coding ability. The differential expression of lncRNA is related to various diseases. For example, the expression level of GAS5 is related to insulin sensitivity and glucose metabolism disorders (Identification of the long non-coding RNA GAS5 as a critical regulator of the M2 macrophage polarization in a mouse model of sepsis. Immunology. 2015.), the expression level of MALAT1 has significant changes in patients with myocardial infarction and heart failure (Overexpression of lncRNA MALATl correlates with poor prognosis and promotes invasion and metastasis in non-small cell lung cancer. J Cancer. 2017.), and the expression specificity of PCA3 in prostate cancer is even higher than that of traditional PSA, etc. (Hessels D et al., DD3(PCA3)-based molecular urine analysis for the diagnosis of prostate cancer. Eur Urol. 2003; 44: 8-15).

[0003] In the prior art, a patent application for invention with the publication number CN106550605A discloses a mitochondrial non-coding RNA for predicting disease progression in patients with heart failure and myocardial infarction. In this invention, UC004COS.4, UC022BQS.1, UC004COX.4, UC004COV.4, UC022BQW.1, UC004C0Z.1, UC011MFI.2 and UC022BQU.1 are differentially expressed in the peripheral blood of patients with heart failure and myocardial infarction; it is proved that the overexpression or underexpression of these 8 lncRNAs in the tested patients is more than 2 times compared with the control patients, which is related to the possibility of short-term or long-term survival after diagnosing chronic heart failure, and is related to cardiac remodeling after myocardial infarction. Thus, detecting the expression levels of the above lncRNAs can be used to predict the mortality of patients with chronic heart failure or the cardiac remodeling of patients with myocardial infarction.

[0004] Although the technical solution disclosed in the foregoing patent for invention can be used to predict the mortality of heart failure patients, its lncRNA is derived from mitochondria rather than plasma extracellular vesicles, and it is not applicable to the diagnosis of dilated cardiomyopathy complicated with heart failure. Therefore, it is necessary to develop a gene-based diagnostic tool for dilated cardiomyopathy complicated with heart failure. Extracellular vesicle lncRNAs have a certain stability and potential value for clinical application. In this study, we used next-generation sequencing technology based on plasma extracellular vesicle lncRNAs to comprehensively characterize the expression of lncRNAs in plasma extracellular vesicles of patients with chronic heart failure (CHF) DCM.

[0005] Therefore, it is very necessary to propose an extracellular vesicle lncRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure and its application to solve the above problems. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides an extracellular vesicle lncRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure and its application, and solves the problems raised in the above background art.

[0007] The present invention specifically adopts the following technical solutions to achieve the above object:

[0008] An extracellular vesicle lncRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure, and the molecular marker is NR_045681;

[0009] The nucleic acid sequence of the molecular marker NR_045681 is as shown in SEQ NO.5.

[0010] Furthermore, the extracellular vesicle is a plasma extracellular vesicle.

[0011] Application of extracellular vesicle lncRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure, including the above-mentioned molecular marker, and the extracellular vesicle lncRNA is used as a marker for preparing a kit, microarray or biochip for diagnosing dilated cardiomyopathy complicated with heart failure.

[0012] Furthermore, the substance for detecting the expression level of the extracellular vesicle lncRNA is a primer or a probe.

[0013] Furthermore, the primer combination for detecting the expression level of the extracellular vesicle lncRNA by fluorescence quantitative detection is as follows:

[0014] Primers for NR_045681:

[0015] Forward: GGCAGGTCCAAAGCCTTCA;

[0016] Reverse: CTACATGAATGCTGCAGATCCAA.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The combination of molecular markers selected in the present invention as molecular markers for the expression level of extracellular vesicle lncRNA effectively improves the detection accuracy of dilated cardiomyopathy complicated with heart failure. The P value of the combination of the molecular markers is <0.05, and the log fold change (FC) |>1, showing a significant differential expression compared with the healthy control group. Description of the Drawings

[0019] Figure 1 It is the TEM image of plasma extracellular vesicles of the healthy control group and patients with dilated cardiomyopathy complicated with heart failure;

[0020] Figure 2 It is the analysis result of the particle size of plasma extracellular vesicles of the healthy control group and patients with dilated cardiomyopathy complicated with heart failure;

[0021] Figure 3 It is the protein blot image of extracellular vesicle-specific markers. In the figure, Nor-Exo represents the healthy control; DCM-Exo represents the group of patients with dilated cardiomyopathy complicated with heart failure;

[0022] Figure 4 It is the RT-qPCR detection result of NR_045681;

[0023] Figure 5 It is the diagnostic efficacy of extracellular vesicle NR_045681. Detailed Embodiments

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0025] Please refer to Figures 1 to 5 , extracellular vesicle lncRNA is used as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure, and the molecular marker is NR_045681;

[0026] The nucleic acid sequence of the extracellular vesicle lncRNA is as shown in SEQ NO.5.

[0027] Specifically, the extracellular vesicle is a plasma extracellular vesicle.

[0028] The application of extracellular vesicle lncRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure includes the above-mentioned molecular marker, and the extracellular vesicle lncRNA is used as a marker for preparing a kit, microarray or biochip for diagnosing dilated cardiomyopathy complicated with heart failure.

[0029] Specifically, the kit is used for diagnosing dilated cardiomyopathy complicated with heart failure, the kit is used for detecting the expression level of plasma extracellular vesicle lncRNA, and the molecular marker is NR_045681.

[0030] Specifically, the substance for detecting the expression level of the extracellular vesicle lncRNA is a primer or a probe.

[0031] Experimental verification

[0032] The present invention will be further described in detail below with reference to specific embodiments.

[0033] 1) Test reagents. The plasma samples of the healthy control group and the coronary heart disease patients in the present invention are from the First Affiliated Hospital of Zhengzhou University. Other reagents or consumables used are commercially available or can be obtained by those skilled in the art through public channels.

[0034] 2) Test method: Collect human peripheral blood using an EDTA anticoagulant blood collection tube, centrifuge at 2500g for 15 min, transfer the upper plasma to a 2-ml sterile tube, and store it in a -80°C refrigerator. After taking out the plasma from the refrigerator, thaw it in a 25°C water bath, then transfer it to a centrifuge tube and centrifuge at 4°C at 3000g for 10 min to remove cell debris in the sample; after transferring the centrifuged supernatant without cell debris to a new centrifuge tube, centrifuge at 4°C at 10000g for 20 min to remove impurities in the sample. Take 1.6 ml of the supernatant, add 0.4 ml of Exoquick reagent (System Biosciences) and let it stand at 4°C for 30 minutes. After centrifugation (3000g × 30 minutes), discard the supernatant, and after centrifugation again (3000g × 5 minutes), discard the supernatant. The obtained precipitate is the extracellular vesicles. Extract extracellular vesicles using the Exoquick kit (System Biosciences). Add 5 μl of the obtained extracellular vesicle suspension to a Formvar-carbon-coated copper grid, wash the copper grid with PBS solution and then place it on a 50-μl droplet of 1% glutaraldehyde solution for 5 min, and then place it on 100 μl of ddH2O for washing for 2 minutes. Stain with uranyl oxalate solution and methyl cellulose solution, blot off the excess liquid on the filter paper, and air-dry for 5 min. Use a JEOL-1230 transmission electron microscope to identify the morphology of the obtained extracellular vesicles. Analyze and identify the particle size and specific markers of the obtained extracellular vesicles using a ZetaView PMX110 particle tracker and Western blotting.

[0035] 3) Analysis method: Use Graphpad Prism 8 software for statistical analysis and graphing. Continuous variables conforming to a normal distribution are expressed as mean ± standard deviation (mean ± SD), and an independent samples t-test is used for data comparison between two groups; use SPSS 22.0 software to calculate the area under the curve and graph to evaluate the diagnostic efficacy of extracellular vesicle lncRNA. P < 0.05 indicates that the difference is statistically significant.

[0036] Example

[0037] Collect plasma from 9 healthy controls and 20 patients with dilated cardiomyopathy complicated with heart failure, and store it in a -80°C refrigerator in a timely manner. The research was approved by the Ethics Committee; all patients signed a written informed consent form.

[0038] Extracellular vesicles were extracted from the above plasma samples using the Exoquick kit (System Biosciences). The obtained extracellular vesicles were fixed on a copper grid for loading samples. After staining and drying, the morphology of the obtained extracellular vesicles was identified using a JEOL-1230 transmission electron microscope. The particle size and specific markers of the obtained extracellular vesicles were analyzed and identified using a ZetaView PMX 110 particle tracker and Western blotting. Among them, the TEM images are shown in Figure 1 ; the results of nanoparticle tracking analysis (NTA) are shown in Figure 2 ; the Western blotting results are shown in Figure 3 (Alix, CD 63 and Hsp 70). Figures 1 to Figure 3 The results showed that the extracted vesicles met the characteristics of extracellular vesicles in terms of morphology, particle size distribution, and specific marker labeling.

[0039] Previous studies suggested that 9 extracellular vesicle lncRNAs were associated with dilated cardiomyopathy complicated with heart failure, namely: lnc-PGA3-1:1, ENST00000610279, lnc-PHLPP2-1:1, lnc-MTX1-1:6, NR_045681, lnc-EPHA7-2:1, NR_103488, ENST00000561588, and ENST00000431812. The specific nucleic acid sequences are shown in SEQ NO.1 to 9. qRT-PCR was used to verify them, and the specific process is as follows:

[0040] 1. Extract total RNA from plasma extracellular vesicles: Take an appropriate amount of extracellular vesicles in a 1.5 ml EP tube, add 800 μl of Trizol reagent (Invitrogen), mix well, lyse for 10 min, and centrifuge at 12000 rpm for 10 min to take the supernatant. After shaking and mixing, place it at 4°C for 10 min, then add 200 μl of chloroform, shake and mix well, place it at 4°C for 10 min, and then centrifuge at 12000 rpm (10 min); take the supernatant, add isopropanol equal to the volume of the supernatant, add a certain volume of glycogen, incubate overnight at -20°C, and centrifuge again at 12000 rpm at 4°C for 10 min to discard the supernatant; add 1 ml of 70% ethanol and centrifuge again at 12000 rpm at 4°C; when the RNA just becomes transparent, add an appropriate amount of Nuclease-free water and incubate in a 55°C water bath for 5 min to completely dissolve the RNA, and measure the concentration of the extracted RNA by ultraviolet analysis.

[0041] 2. RNA Reverse Transcription into cDNA: Use the Evo M-MLV Reverse Transcription Kit (containing reagent for removing gDNA, for qPCR) II (from Aikerui Biotech) to reverse transcribe RNA into cDNA. First, prepare the reaction mixture on ice according to Table 1 to eliminate genomic DNA in RNA. To ensure the accuracy of reaction mixture preparation, when conducting various reactions, MasterMix should be prepared in an amount of reaction number + 2, then aliquoted into each reaction tube, and finally add the RNA sample. Incubate at 42 °C for 2 minutes and store at 4 °C.

[0042] Table 1 Elimination of Genomic DNA in RNA

[0043]

[0044]

[0045] The reverse transcription reaction system and reaction conditions are shown in Tables 2 and 3:

[0046] Table 2 Reverse Transcription Reaction System

[0047]

[0048] Table 3 Reverse Transcription Reaction Conditions

[0049]

[0050] 3. qRT-PCR Detection

[0051] Analyze extracellular vesicle lncRNA using the Stepone plus real-time fluorescence quantitative PCR instrument (from Applied Biosystems), and each sample is analyzed in triplicate. Table 4 shows the nucleic acid sequences and primer sequences of 9 lncRNAs. The primer pairs were designed and synthesized by Sangon Biotech, and the sensitivity and specificity of the primers were explored using NCBI blast alignment analysis. Tables 5 and 6 show the qRT-PCR reaction system and reaction conditions respectively. The SYBR Green Pro TaqHS premixed qPCR kit (containing Rox) was purchased from Aikerui Biotech. Using U6 as an external reference, Log2(2-ΔΔCT) represents the relative expression level of the target gene.

[0052] Table 4 Nucleic Acid Sequences and Primer Sequences of Extracellular Vesicle lncRNA

[0053]

[0054]

[0055] Table 5 qRT-PCR Detection Reaction System

[0056]

[0057]

[0058] Table 6 Reaction conditions for qRT-PCR detection

[0059]

[0060] Result analysis

[0061] The results of RT-qPCR detection showed that there were differential expressions of NR_045681 in patients with dilated cardiomyopathy complicated with heart failure: compared with the healthy group, NR_045681 was up-regulated in the disease group, and the difference was statistically significant. The results are shown in Figure 4 .

[0062] Statistical analysis and graphing were performed using R language, and the area under the curve was calculated to evaluate the diagnostic efficacy of extracellular vesicle NR_045681. Its AUC was 0.889. The results are shown in Figure 5 .

[0063] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. The patent protection scope of the present invention shall be subject to the claims. All equivalent structural changes made by using the content of the specification and drawings of the present invention shall, by the same token, be included in the protection scope of the present invention.

Claims

1. The extracellular vesicle lncRNA is used as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure, and is characterized in that: The molecular marker is NR_045681; The nucleic acid sequence of the molecular marker NR_045681 is shown in SEQ NO.

5.

2. The extracellular vesicle lncRNA according to claim 1, as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure, is characterized in that: The extracellular vesicle is a plasma extracellular vesicle.

3. Use of extracellular vesicle lncRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure, including the molecular marker according to any one of claims 1-2, characterized in that: The extracellular vesicle lncRNA is used as a marker for preparing a kit, microarray or biochip for diagnosing dilated cardiomyopathy complicated with heart failure.

4. Use of the extracellular vesicle lncRNA according to claim 3 as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure, characterized in that: The substance for detecting the expression level of the extracellular vesicle lncRNA is a primer or a probe.

5. Use of the extracellular vesicle lncRNA according to claim 4 as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure, characterized in that: The primer combination for detecting the expression level of the extracellular vesicle lncRNA by fluorescence quantitative method is as follows: Primers for NR_045681: Forward: GGCAGGTCCAAAGCCTTCA; Reverse: CTACATGAATGCTGCAGATCCAA.

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