Exosomal miRNA as a Molecular Marker for the Diagnosis of Dilated Cardiomyopathy Complicated with Heart Failure and Its Application

Abstract

The present invention belongs to the field of molecular diagnostic technology, and specifically relates to an exosomal miRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure and its application; the exosomal miRNA is any one or a combination of two or more molecular markers selected from miR-423-5p, miR-148a-3p, miR-185-5p, miR-320a-3p, miR-22-3p, miR-148b-3p, let-7i-5p, miR-103a-3p, miR-21-5p, and let-7d-5p. The specific nucleic acid sequences of the exosomal miRNA are shown in SEQ NO.1-10. Preferably, the miRNA is miR-423-5p, miR-148a-3p, or miR-148b-3p. The technical solution provided by the present invention effectively improves the detection accuracy of dilated cardiomyopathy complicated with heart failure. The molecular marker |log(FC)| > 0.5, and there are significant differential expressions in patients with dilated cardiomyopathy complicated with heart failure compared with the healthy control group. In addition, the molecular combination of the three preferably selected miRNAs has better diagnostic efficacy than a single molecule and has certain clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular diagnosis and treatment, and specifically relates to an exosomal miRNA 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 cardiovascular diseases, which can cause cardiac insufficiency and cardiac dysfunction. Although gene mutations have been identified as one of the causes of DCM, the use of gene biomarkers such as RNA for the early diagnosis of DCM has still been neglected. In addition, the changes in RNA can reflect the progression of the disease and serve as an indicator of the patient's prognosis. Therefore, it is beneficial to develop gene-based DCM diagnostic tools. RNA is usually unstable in the circulatory system, resulting in its infeasibility for clinical applications. Recently discovered exosomal miRNAs have the stability required for current diagnostic purposes. Therefore, a full understanding of exosomal miRNAs in DCM patients is crucial for clinical translation. In this study, we used next-generation sequencing technology based on plasma exosomal miRNAs to comprehensively characterize the expression of miRNAs in plasma exosomes of patients with chronic heart failure (CHF) DCM.

[0003] Numerous prior arts have disclosed the use of miRNAs as molecular markers for heart failure detection. For example, the Chinese patent application with the publication number CN104988216A discloses a serum miRNA related to chronic heart failure and its application. The present invention provides the use of a class of peripheral blood miRNAs in the risk assessment / diagnosis and prognosis of chronic heart failure as biomarkers. Among them, hsa-miR-4491, hsa-miR-1285-3p, hsa-miR-665, hsa-miR-660-3p, hsa-miR-206, hsa-miR-1268b, hsa-miR-130a-3p, and hsa-miR--330-3p are differentially expressed in the peripheral blood of patients with chronic heart failure; it is demonstrated that peripheral blood hsa-miR665, etc. can specifically diagnose chronic heart failure, and their expression levels are correlated with the ejection fraction, having the effect of evaluating the prognosis. Thus, by detecting the expression levels of the above miRNAs, it can be used to predict and assist in diagnosing existing chronic heart failure or evaluating the prognosis of chronic heart failure.

[0004] For another example, a Chinese invention patent application with the publication number CN108165625A discloses an application of miRNA in the preparation of reagents and kits for detecting heart diseases, as well as detection reagents and detection kits. The invention develops novel miRNAs, including miR-155-5p, miR-216a-5p, or miR-217F-5p, and finds that their expression is significantly increased in cardiomyocytes of patients with heart diseases. Therefore, miR-155-5p, miR-216a-5p, or miR-217F-5p provided in this invention can be used for risk prediction and detection of heart-related diseases. Additionally, the invention also provides detection reagents and detection kits for miR-155-5p, miR-216a-5p, or miR-217F-5p, which use real-time quantitative PCR technology to count the changes in microRNAs, and have the advantages of requiring a small amount of sample for analysis, high automation, and avoiding contamination. Moreover, according to paragraphs

[0002] and

[0111] -

[0112] of the patent specification, the technical solution provided by this invention is used for the diagnosis of heart failure.

[0005] Although the technical solution disclosed in the aforementioned invention patent can be used for the diagnosis of heart failure, it is not applicable to the diagnosis of dilated cardiomyopathy complicated with heart failure. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides an application of exosomal miRNA molecular markers for the diagnosis of dilated cardiomyopathy complicated with heart failure and its kit, which solves the problems raised in the above background art.

[0007] The technical solution of the present invention is as follows:

[0008] An exosomal miRNA as a molecular marker for the diagnosis of dilated cardiomyopathy complicated with heart failure, wherein the exosomal miRNA is any one or a combination of two or more molecular markers selected from miR-423-5p, miR-148a-3p, miR-185-5p, miR-320a-3p, miR-22-3p, miR-148b-3p, let-7i-5p, miR-103a-3p, miR-21-5p, and let-7d-5p.

[0009] Further, the exosome is a plasma exosome.

[0010] An application of an exosomal miRNA as a molecular marker for the diagnosis of dilated cardiomyopathy complicated with heart failure, wherein the exosomal miRNA is used as a marker for preparing a kit, microarray, or biochip for the diagnosis of dilated cardiomyopathy complicated with heart failure.

[0011] Further, the substance for detecting the expression level of the exosomal miRNA is a primer or a probe.

[0012] Furthermore, the primer sets for quantitatively detecting the expression level of the exosomal miRNA are as follows:

[0013] (1) Primers for miR-148b-3p: 5’-ATGTGCGTCAGTGCATCACAGA-3’

[0014] (2) Primers for miR-185-5p: 5’-AAGCGGATGGAGAGAAAGGCAG-3’

[0015] (3) Primers for let-7d-5p: 5’-CCACGGTCAGAGGTAGTAGGTTG-3’

[0016] (4) Primers for let-7i-5p: 5’-CGCGCGTGAGGTAGTAGTTTGT-3’

[0017] (5) Primers for miR-103a-3p: 5’-CAAGCCGAGCAGCATTGTACAG-3’

[0018] (6) Primers for miR-21-5p: 5’-GCGCGTAGCTTATCAGACTGA-3’

[0019] (7) Primers for miR-22-3p: 5’-ACTCCGGAAAGCTGCCAGTTG-3’

[0020] (8) Primers for miR-423-5p: 5’-AAGAATTGAGGGGCAGAGAGCG-3’

[0021] (9) Primers for miR-148a-3p: 5’-AACACGTGTCAGTGCACTACAGA-3’

[0022] (10) Primers for miR-320a-3p: 5’-ACGGCACAAAAGCTGGGTTGA-3’.

[0023] A kit for diagnosing dilated cardiomyopathy complicated with heart failure, the kit is used to detect the expression level of plasma exosomal miRNA, and the exosomal miRNA is any one or a combination of two or more molecular markers among miR-423-5p, miR-148a-3p, miR-185-5p, miR-320a-3p, miR-22-3p, miR-148b-3p, let-7i-5p, miR-103a-3p, miR-21-5p and let-7d-5p.

[0024] Beneficial effects

[0025] 1. Compared with the prior art, the application provided by the present invention has the following beneficial effects:

[0026] The present invention selects any one or a combination of two or more molecular markers from miR-423-5p, miR-148a-3p, miR-185-5p, miR-320a-3p, miR-22-3p, miR-148b-3p, let-7i-5p, miR-103a-3p, miR-21-5p and let-7d-5p as molecular markers for the expression level of exosomal miRNAs, effectively improving the detection accuracy of dilated cardiomyopathy complicated with heart failure. For the above molecules, |log(FC)| > 0.5, and the difference is statistically significant, showing obvious differential expression compared with the healthy control group.

[0027] The present invention discloses molecular markers for diagnosing dilated cardiomyopathy complicated with heart failure based on plasma exosomal miRNAs and their applications. RT-qPCR results show that ten human plasma exosomal miRNAs, such as miR-423-5p, miR-148a-3p, miR-185-5p, miR-320a-3p, miR-22-3p, miR-148b-3p, let-7i-5p, miR-103a-3p, miR-21-5p and let-7d-5p, are differentially expressed between patients with dilated cardiomyopathy complicated with heart failure and healthy control groups. The diagnostic efficacy of the ten plasma exosomal miRNAs was analyzed using the ROC curve. The results show that the AUC values of the above exosomal miRNAs are 0.926, 0.938, 0.827, 0.815, 0.728, 0.864, 0.827, 0.790, 0.753, and 0.691, respectively.

[0028] The present invention proves that the above ten plasma exosomal miRNAs can be used as molecular diagnostic markers for dilated cardiomyopathy complicated with heart failure. By preparing them into diagnostic kits, microarrays or biochips, and detecting the expression level of exosomal miRNAs in the plasma of subjects, rapid diagnosis and determination of dilated cardiomyopathy complicated with heart failure can be carried out: when the expression level of at least one miRNA among exosomal miR-423-5p, miR-148a-3p, miR-185-5p, miR-320a-3p, miR-22-3p, miR-148b-3p, let-7i-5p, miR-103a-3p, miR-21-5p and let-7d-5p is up-regulated, or the expression levels of miR-423-5p, miR-148a-3p and miR-148b-3p are all up-regulated, it indicates that the subject has dilated cardiomyopathy complicated with heart failure, which has important clinical application value.

[0029] 2. Compared with the prior art, the kit provided by the present invention has the following beneficial effects:

[0030] The kit provided by the present invention diagnoses dilated cardiomyopathy complicated with heart failure by detecting the expression level of plasma exosomal miRNA. The exosomal miRNA is any one or a combination of two or more molecular markers among miR-423-5p, miR-148a-3p, miR-185-5p, miR-320a-3p, miR-22-3p, miR-148b-3p, let-7i-5p, miR-103a-3p, miR-21-5p and let-7d-5p, which 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, |log(FC)| > 0.5, and there is a significant differential expression compared with the healthy control group. Description of the Drawings

[0031] Figure 1 It is the TEM image of plasma exosomes of the healthy control group and patients with dilated cardiomyopathy complicated with heart failure;

[0032] Figure 2 It is the result of particle size analysis of plasma exosomes of the healthy control group and patients with dilated cardiomyopathy complicated with heart failure;

[0033] Figure 3 It is the Western blot image of exosome-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;

[0034] Figure 4 It is the RT-qPCR verification of the differential expression of miRNA in patients with dilated cardiomyopathy complicated with heart failure and the healthy control group;

[0035] Figure 5 It is the diagnostic efficacy of miRNA for dilated cardiomyopathy complicated with heart failure;

[0036] Figure 6 It is the ROC curve analysis of the diagnostic efficacy of the combination of miR-423-5p, miR-148a-3p and miR-148b-3p molecular markers for dilated cardiomyopathy complicated with heart failure. Detailed Embodiments

[0037] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the 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 of the embodiments.

[0038] An exosomal miRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure, wherein the exosomal miRNA is any one or a combination of two or more molecular markers selected from miR-423-5p, miR-148a-3p, miR-185-5p, miR-320a-3p, miR-22-3p, miR-148b-3p, let-7i-5p, miR-103a-3p, miR-21-5p, and let-7d-5p.

[0039] In the present invention, the exosomes are plasma exosomes.

[0040] An application of an exosomal miRNA as a molecular marker for diagnosing dilated cardiomyopathy complicated with heart failure, wherein the exosomal miRNA is used as a marker for preparing a kit, microarray, or biochip for diagnosing dilated cardiomyopathy complicated with heart failure.

[0041] In the present invention, the substance for detecting the expression level of the exosomal miRNA is a primer or a probe.

[0042] In the present invention, the forward primer sequence combination for detecting the expression level of the exosomal miRNA by fluorescence quantitative method is as follows:

[0043] (1) Primer for miR-148b-3p: 5’-ATGTGCGTCAGTGCATCACAGA-3’

[0044] (2) Primer for miR-185-5p: 5’-AAGCGGATGGAGAGAAAGGCAG-3’

[0045] (3) Primer for let-7d-5p: 5’-CCACGGTCAGAGGTAGTAGGTTG-3’

[0046] (4) Primer for let-7i-5p: 5’-CGCGCGTGAGGTAGTAGTTTGT-3’

[0047] (5) Primer for miR-103a-3p: 5’-CAAGCCGAGCAGCATTGTACAG-3’

[0048] (6) Primer for miR-21-5p: 5’-GCGCGTAGCTTATCAGACTGA-3’

[0049] (7) Primer for miR-22-3p: 5’-ACTCCGGAAAGCTGCCAGTTG-3’

[0050] (8) Primer for miR-423-5p: 5’-AAGAATTGAGGGGCAGAGAGCG-3’

[0051] (9) Primer of miR-148a-3p: 5’-AACACGTGTCAGTGCACTACAGA-3’

[0052] (10) Primer of miR-320a-3p: 5’-ACGGCACAAAAGCTGGGTTGA-3’.

[0053] Reverse primer is as follows: 5’-ATCCAGTGCAGGGTCCGAGG-3’.

[0054] Preferably, the miRNA is miR-423-5p, miR-148a-3p, miR-148b-3p. A kit for diagnosing dilated cardiomyopathy complicated with heart failure, the kit is used to detect the expression level of plasma exosomal miRNA, and the exosomal miRNA is any one or a combination of two or more molecular markers among miR-423-5p, miR-148a-3p, miR-185-5p, miR-320a-3p, miR-22-3p, miR-148b-3p, let-7i-5p, miR-103a-3p, miR-21-5p and let-7d-5p.

[0055] Experimental verification

[0056] The present invention will be further described in detail below in conjunction with specific embodiments.

[0057] 1) Test reagents. The plasma samples of the healthy control group and the patients with dilated cardiomyopathy complicated with heart failure 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.

[0058] 2) Test method: Collect human peripheral blood using an EDTA anticoagulant blood collection tube, centrifuge at 2500g for 15 min, take the upper plasma and transfer it 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 exosomes. Extract exosomes using the Exoquick kit (System Biosciences). Add 5 μl of the obtained exosome suspension to a Formvar-carbon sample-loading copper grid, wash the copper grid with PBS solution, then place it on a 50 μl droplet of 1% glutaraldehyde solution for 5 min, and then place it in 100 μl of ddH2O for washing for 2 minutes. Stain with uranyl oxalate solution and methyl cellulose solution, blot off the excess liquid on filter paper, and dry it in air for 5 min. Use a JEOL-1230 transmission electron microscope to identify the morphology of the obtained exosomes. And analyze and identify the particle size and specific markers of the obtained exosomes using a ZetaView PMX110 particle tracker and Western blot.

[0059] 3) Analysis method: Use Graphpad Prism 8 software for statistical analysis and graphing. Continuous variables that conform to a normal distribution are expressed as mean ± standard deviation (mean ± SD). The comparison of data between two groups uses an independent samples t-test. Use SPSS22.0 software to calculate the area under the curve and graph it to evaluate the diagnostic efficacy of exosomal miRNAs. P < 0.05 indicates that the difference is statistically significant.

[0060] Example

[0061] Collect plasma from 14 healthy controls and 18 patients with dilated cardiomyopathy complicated with heart failure and store it in a -80°C refrigerator in a timely manner.

[0062] Extract exosomes from the above plasma samples using the Exoquick kit (System Biosciences), fix the obtained exosomes on a sample-loading copper grid, stain and dry them, and then use a JEOL-1230 transmission electron microscope to identify the morphology of the obtained exosomes; and analyze and identify the particle size and specific markers of the obtained exosomes using a ZetaView PMX 110 particle tracker and Western blot. Among them, the TEM images are shown in Figure 1; The results of nanoparticle tracking analysis (NTA) are shown in Figure 2 ; The results of Western blot are shown in Figure 3 (Alix, CD 63 and Hsp 70). Figures 1 to 3 The results showed that the extracted vesicles met the characteristics of exosomes in terms of morphology, particle size distribution, and specific marker labeling.

[0063] Previous studies have suggested that 10 exosomal miRNAs are associated with dilated cardiomyopathy complicated with heart failure, namely: miR-423-5p, miR-148a-3p, miR-185-5p, miR-320a-3p, miR-22-3p, miR-148b-3p, let-7i-5p, miR-103a-3p, miR-21-5p, and let-7d-5p; qRT-PCR was used to verify them, and the specific process is as follows:

[0064] 1. Extract total RNA from plasma exosomes: Take an appropriate amount of exosomes in a 1.5 ml EP tube, add 800 ul of Trizol reagent (Invitrogen), mix well, lyse for 10 min, centrifuge at 12000 rpm for 10 min, and take the supernatant. After shaking and mixing, place it at 4 °C for 10 min, then add 200 ul 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, centrifuge at 12000 rpm at 4 °C for 10 min again, and discard the supernatant; add 1 ml of 70% ethanol, centrifuge at 12000 rpm at 4 °C again; 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.

[0065] 2. Reverse transcription of RNA into cDNA: Use the Evo M-MLV reverse transcription kit (containing reagents for removing gDNA, for qPCR) II (Aikerui Biotech) kit to reverse transcribe RNA into cDNA. First, prepare the reaction mixture on ice according to Table 1 to eliminate the genomic DNA in the RNA. To ensure the accuracy of the reaction mixture preparation, when performing each reaction, the MasterMix should be prepared in an amount +2 of the number of reactions, and then aliquoted into each reaction tube, and finally add the RNA sample. Incubate at 42 °C for 2 minutes and store at 4 °C.

[0066] Table 1 Elimination of genomic DNA in RNA

[0067]

[0068] The reverse transcription reaction system and reaction conditions are shown in Tables 2 and 3:

[0069] Table 2 Reverse transcription reaction system

[0070]

[0071]

[0072] Table 3 Reverse transcription reaction conditions

[0073]

[0074] 3.qRT-PCR detection

[0075] Exosomal miRNA was detected using a Stepone plus fluorescent quantitative PCR instrument (Applied Biosystems).

[0076] The analysis was repeated three times for each sample. Table 4 shows 10 miRNAs and their forward primer sequences, and the universal reverse primer sequence is 5'-ATCCAGTGCAGGGTCCGAGG-3'. The primer pairs were designed and synthesized by Sangon Biotech Co., Ltd., and the sensitivity and specificity of the primers were explored by NCBI blast comparison analysis. Tables 6 and 7 are the qRT-PCR reaction system and reaction conditions, respectively, where the SYBR Green Pro Taq HS premixed qPCR kit (including Rox) was purchased from Aikerui Biotech Co., Ltd. Using U6 as an external reference, Log2(2-ΔΔCT) represents the relative expression level of the target gene.

[0077] Table 4 Exosomal miRNA and its forward primer sequences

[0078]

[0079]

[0080] Table 5 qRT-PCR detection reaction system

[0081]

[0082] Table 6 qRT-PCR detection reaction conditions

[0083]

[0084] Results Analysis

[0085] The results of RT-qPCR showed that miR-423-5p, miR-148a-3p, miR-185-5p, miR-320a-3p, miR-22-3p, miR-148b-3p, let-7i-5p, miR-103a-3p, miR-21-5p, and let-7d-5p were differentially expressed in patients with dilated cardiomyopathy complicated with heart failure: compared with the healthy group, the ten miRNAs in the diseased group were generally up-regulated, and the difference was statistically significant. The results are shown in Figure 4 .

[0086] Statistical analysis and graphing were performed using R language and SPSS 22.0, and the area under the curve was calculated to evaluate the diagnostic efficacy of exosomal miRNAs. The ROC curve analysis of the expression levels of ten plasma exosomal miRNAs is shown in Figure 5 . The results showed that the AUC values of the above exosomal miR-423-5p, miR-148a-3p, miR-185-5p, miR-320a-3p, miR-22-3p, miR-148b-3p, let-7i-5p, miR-103a-3p, miR-21-5p, and let-7d-5p were 0.926, 0.938, 0.827, 0.815, 0.728, 0.864, 0.827, 0.790, 0.753, and 0.691, respectively. The results are shown in Figure 5 .

[0087] The exosomal miR-423-5p, miR-148a-3p, and miR-148b-3p with higher AUC values were used as a combination of molecular markers for investigation. The results showed that the AUC value of the molecular marker combination reached 0.975, which was higher than that of individual indicators (P<0.001), and the sensitivity and specificity were 88.9% and 100%, respectively. It was proved that the combination of exosomal miRNAs had good diagnostic efficacy for dilated cardiomyopathy complicated with heart failure. The results are shown in Figure 6 .

[0088] As mentioned above, it is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and all should be covered by the protection scope of the present invention.

Claims

1. Use of a reagent for detecting an exosomal miRNA molecular marker combination in the preparation of a kit, microarray or biochip for diagnosing dilated cardiomyopathy combined with heart failure, wherein the exosomal miRNA molecular marker combination is miR-423-5p, miR-148a-3p and miR-148b-3p.

2. The use according to claim 1, characterized in that: The exosomes are plasma exosomes.

3. The use according to claim 2, characterized in that: The substance for detecting the expression amount of the exosomal miRNA is a primer or a probe.

4. The use according to claim 1, characterized in that: The primer combination for fluorescence quantitative detection of the exosomal miRNA expression is as follows: Primer for miR-148b-3p: 5′-ATGTGCGTCAGTGCATCACAGA-3′; Primer for miR-423-5p: 5′-AAGAATTGAGGGGCAGAGAGCG-3′; Primer for miR-148a-3p: 5′-AACACGTGTCAGTGCACTACAGA-3′.

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