Extracellular vesicle lncRNA as a diagnostic biomarker for heart failure in patients with dilated cardiomyopathy and its application

Abstract

The application belongs to the technical field of molecular diagnosis, and particularly relates to extracellular vesicle lncRNA as a molecular marker for diagnosing heart failure combined with dilated cardiomyopathy. The molecular marker is NR_045681, and the specific nucleic acid sequence of the extracellular vesicle lncRNA is shown in SEQ ID NO. 5. The technical scheme provided by the application effectively improves the detection accuracy of heart failure combined with dilated cardiomyopathy. The P value of the combination of the molecular marker is less than 0.05, the |log fold change (FC)| is greater than 1, and the molecular marker has obvious differential expression relative to a healthy control group.

Description

Technical Field

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

[0002] Dilated cardiomyopathy (DCM) is one of the most common hereditary cardiomyopathies, which can cause heart failure and cardiac dysfunction. DCM has a high incidence rate, with a 52-month follow-up mortality rate of 42.24%, placing a heavy burden on families and society (Clinical Cardiovascular Disease Journal, 2018, 34(05): 421-434.). Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides that do not have coding capabilities. Differential expression of lncRNAs is associated with a variety of diseases. For example, the expression level of GAS5 is associated with 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 is significantly altered in patients with myocardial infarction and heart failure (Overexpression of lncRNA MALAT1 correlates with poor prognosis and promotes invasion and metastasis in non-small cell lung cancer. J Cancer. 2017.). The expression specificity of PCA3 in prostate cancer is even higher than that of traditional PSA (Hessels D et al., DD3(PCA3)-based molecular urine analysis for the diagnosis of prostate cancer. EurUrol. 2003; 44: 8-15).

[0003] A prior art patent application with 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, UC004COZ.1, UC011MFI.2, and UC022BQU.1 are differentially expressed in the peripheral blood of patients with heart failure and myocardial infarction. It demonstrates that these eight lncRNAs are overexpressed or lost more than twice in the tested patients compared to control patients, and are associated with the likelihood of short-term or long-term survival after diagnosis of chronic heart failure, as well as with cardiac remodeling after myocardial infarction. Therefore, detecting the expression levels of these lncRNAs can be used to predict mortality in patients with chronic heart failure or cardiac remodeling in patients with myocardial infarction.

[0004] While the aforementioned patented technologies can be used to predict mortality in heart failure patients, their lncRNAs originate from mitochondria rather than plasma extracellular vesicles, and are not suitable for diagnosing dilated cardiomyopathy complicated with heart failure. Therefore, developing gene-based diagnostic tools for dilated cardiomyopathy complicated with heart failure is necessary. Extracellular vesicle lncRNAs possess a certain degree of stability and have potential clinical application value. 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 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] To address the shortcomings of existing technologies, this invention provides extracellular vesicle lncRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure and its application, thus solving the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention specifically adopts the following technical solution:

[0008] Extracellular vesicle lncRNA is used as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure. The molecular marker is NR_045681.

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

[0010] Furthermore, the extracellular vesicles are plasma extracellular vesicles.

[0011] The application of extracellular vesicular lncRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure includes the aforementioned molecular markers, wherein the extracellular vesicular lncRNA is used to prepare markers for diagnosing dilated cardiomyopathy complicated with heart failure, such as kits, microarrays, or biochips.

[0012] Furthermore, the substances used to detect the expression level of the extracellular vesicle lncRNA are primers or probes.

[0013] Furthermore, the primer combination for quantitative real-time detection of extracellular vesicle lncRNA expression is as follows:

[0014] Primers for NR_045681:

[0015] Positive: 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 this invention serves as a molecular marker for the expression level of extracellular vesicle lncRNA, effectively improving the detection accuracy of dilated cardiomyopathy complicated with heart failure. The combination of molecular markers has a P value of <0.05 and |log fold change (FC)| >1, showing significant differential expression compared to the healthy control group. Attached Figure Description

[0019] Figure 1 TEM images of extracellular vesicles in the plasma of healthy controls and patients with dilated cardiomyopathy and heart failure;

[0020] Figure 2 The results of plasma extracellular vesicle diameter analysis were obtained from healthy controls and patients with dilated cardiomyopathy and heart failure.

[0021] Figure 3 These are protein imprinting images of extracellular vesicle-specific markers. In the image, Nor-Exo represents healthy controls, and DCM-Exo represents the dilated cardiomyopathy group with heart failure.

[0022] Figure 4 The RT-qPCR test result for NR_045681;

[0023] Figure 5 Diagnostic efficacy of extracellular vesicle NR_045681. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Please see 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 shown in SEQ ID NO.5.

[0027] Specifically, the extracellular vesicles are plasma extracellular vesicles.

[0028] The application of extracellular vesicular lncRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure includes the aforementioned molecular markers, wherein the extracellular vesicular lncRNA is used to prepare markers for diagnosing dilated cardiomyopathy complicated with heart failure, such as kits, microarrays, or biochips.

[0029] Specifically, the kit is used to diagnose dilated cardiomyopathy complicated with heart failure, and the kit is used to detect the expression level of extracellular vesicle lncRNA in plasma. The molecular marker is NR_045681.

[0030] Specifically, the substances used to detect the expression level of the extracellular vesicle lncRNA are primers or probes.

[0031] Experimental verification

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

[0033] 1) Test reagents: Plasma samples from the healthy control group and patients with coronary heart disease in this invention were obtained from the First Affiliated Hospital of Zhengzhou University. Other reagents or consumables used were commercially available or could be obtained by those skilled in the art through public channels.

[0034] 2) Experimental Method: Peripheral blood was collected using EDTA anticoagulant blood collection tubes. The tubes were centrifuged at 2500g for 15 min, and the supernatant plasma was transferred to a 2ml sterile tube and stored at -80℃. After removing the plasma from the freezer, it was thawed in a 25℃ water bath, then transferred to a centrifuge tube and centrifuged at 3000g for 10 min at 4℃ to remove cell debris. The supernatant after removing cell debris was transferred to a new centrifuge tube and centrifuged at 10000g for 20 min at 4℃ to remove impurities. 1.6ml of the supernatant was added to 0.4ml of Exoquick reagent (System Biosciences) and incubated at 4℃ for 30 minutes. After centrifugation (3000g × 30 min), the supernatant was discarded. The tubes were then centrifuged again (3000g × 5 min) and the supernatant was discarded. The resulting precipitate was the extracellular vesicle. The extracellular vesicles were extracted using the Exoquick kit (System Biosciences). The obtained 5 μl extracellular vesicle suspension was added to a Formvar-carbon copper grid. After washing the grid with PBS, it was placed on 50 μl of 1% glutaraldehyde solution for 5 min, followed by washing with 100 μl of dH2O for 2 min. The vesicles were stained with uranyl oxalate and methylcellulose solution, excess liquid was blotted on filter paper, and the vesicles were air-dried for 5 min. 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 PMX110 particle tracker and Western blotting.

[0035] 3) Analytical methods: Graphpad Prism 8 software was used for statistical analysis and graphing. Continuous variables conforming to a normal distribution were expressed as mean ± standard deviation (mean ± SD). Independent samples t-tests were used to compare data between two groups. SPSS 22.0 software was used to calculate the area under the curve and plot the results to evaluate the diagnostic efficacy of extracellular vesicle lncRNAs. P < 0.05 was considered statistically significant.

[0036] Example

[0037] Plasma was collected from 9 healthy controls and 20 patients with dilated cardiomyopathy and heart failure, and stored promptly at -80°C. The study was approved by the ethics committee; all patients signed written informed consent forms.

[0038] Extracellular vesicles were extracted from the plasma samples using the Exoquick kit (System Biosciences). The obtained extracellular vesicles were immobilized on a copper grid, stained, and dried. The morphology of the 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. TEM images are shown below. Figure 1 The results of nanoparticle tracking analysis (NTA) are shown below. Figure 2 The results of the Western blot are shown below. Figure 3 (Alix, CD 63 and Hsp 70). Figures 1 to 3 The results showed that the extracted vesicles conformed to the characteristics of extracellular vesicles in terms of morphology, particle size distribution, and specific marker labeling.

[0039] Previous studies have suggested an association between nine extracellular vesicle lncRNAs and dilated cardiomyopathy complicated with heart failure: lnc-PGA3-1:1, ENST00000610279, lnc-PHLPP2-1:1, lnc-MTX1-1:6, NR_045681, lnc-EPHA7-2:1, NR_103488, ENST00000561588, and ENST00000431812, with specific nucleic acid sequences shown in SEQ ID NO.1–9. These were validated using qRT-PCR, as detailed below:

[0040] 1. Extraction of total RNA from extracellular vesicles in plasma: Take an appropriate amount of extracellular vesicles into a 1.5 ml EP tube, add 800 μL Trizol reagent (Invitrogen), mix thoroughly, lyse for 10 min, centrifuge at 12000 rpm for 10 min, and collect the supernatant. After vortexing and mixing, incubate at 4°C for 10 min, then add 200 μL chloroform, vortex and mix again, incubate at 4°C for 10 min, and centrifuge at 12000 rpm for 10 min. Collect the supernatant, add an equal volume of isopropanol, add a certain volume of glycogen, incubate at -20°C overnight, and centrifuge again at 12000 rpm at 4°C for 10 min, discarding 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, incubate at 55°C for 5 min to completely dissolve the RNA, and determine the concentration of the extracted RNA by UV analysis.

[0041] 2. RNA Reverse Transcription to cDNA: RNA was reverse transcribed into cDNA using the Evo M-MLV Reverse Transcription Kit (including gDNA removal reagent for qPCR) II (Aikerui Biotechnology). First, prepare the reaction mixture on ice according to Table 1 to eliminate genomic DNA from the RNA. To ensure the accuracy of the reaction mixture preparation, prepare MasterMix by adding 2 to the reaction number before each reaction, then aliquot the mixture 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 the genome in RNA

[0043]

[0044] The reverse transcription reaction system and reaction conditions are shown in Tables 2 and 3:

[0045] Table 2 Reverse transcription reaction system

[0046]

[0047] Table 3 Reverse transcription reaction conditions

[0048]

[0049] 3. qRT-PCR detection

[0050] Extracellular vesicle lncRNAs were analyzed using the Stepone Plus real-time PCR instrument (Applied Biosystems), with each sample analyzed in triplicate. Table 4 shows the nucleic acid sequences of nine lncRNAs and their primer sequences. Primer pairs were designed and synthesized by Sangon Biotech, and NCBI BLAST alignment analysis was used to explore primer sensitivity and specificity. Tables 5 and 6 show the qRT-PCR reaction system and conditions, with the SYBR Green Pro TaqHS premixed qPCR kit (containing Rox) purchased from Aikerui Biotechnology. U6 was used as an external reference, and Log2(2-ΔΔCT) represents the relative expression level of the target gene.

[0051] Table 4. Nucleic acid sequences of extracellular vesicle lncRNAs and their primer sequences.

[0052]

[0053] Table 5 qRT-PCR detection reaction system

[0054]

[0055] Table 6 qRT-PCR detection reaction conditions

[0056]

[0057] Results Analysis

[0058] RT-qPCR results showed differential expression of NR_045681 in patients with dilated cardiomyopathy and heart failure: compared with the healthy group, NR_045681 was upregulated in the disease group, and the difference was statistically significant. (See attached figures). Figure 4 .

[0059] 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. The AUC was 0.889. (See attached table). Figure 5 .

[0060] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the description and drawings of the present invention shall also be included within the scope of protection of the present invention.

Claims

1. The application of a reagent for detecting the expression level of NR_045681 in extracellular vesicles of plasma in the preparation of a kit for diagnosing dilated cardiomyopathy complicated with heart failure, wherein the nucleotide sequence of NR_045681 is as shown in SEQ ID NO.

5.

2. The application according to claim 1, characterized in that: The reagents used to detect the expression level of NR_045681 in extracellular vesicles of plasma include primers or probes.

3. The application according to claim 2, characterized in that: The primers are real-time PCR primers, and their sequences are as follows: Forward primer: GGCAGGTCCAAAGCCTTCA; Reverse primer: CTACATGAATGCTGCAGATCCAA.

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