Identification marker for early chronic obstructive pulmonary disease, and use thereof in diagnostic kit

Abstract

The present invention relates to the technical field of genetic engineering. Particularly provided are an identification marker for early chronic obstructive pulmonary disease and the use thereof in a diagnostic kit. The identification marker for the early chronic obstructive pulmonary disease is screened, which is miRNA1255a in serum-derived exosomes; in addition, biological function analysis after sequencing proves that among a plurality of target genes of miRNA1255a, the tissue inhibitor of metalloprotease (TIMP-2) and CD36, which are genes related to the protease / antiprotease balance, are involved in the Ca ion signaling pathway, and moreover, the expression of CD36 and TIMP-2 proteins proves that said two proteins are negatively correlated with miRNA1255a. Moreover, it is found in detection results that the expression of TIMP-2 and the expression of CD36 are all downregulated when the expression of miRNA1255a is upregulated. The biomarker for the early chronic obstructive pulmonary disease is provided, which can easily screen potential patients with early onset of chronic obstructive pulmonary disease.

Description

Early chronic obstructive pulmonary disease identification markers and their application in diagnostic kits

[0001] The present application claims priority to the Chinese patent application No. CN202410735686.2, filed on June 07, 2024, and entitled "Early chronic obstructive pulmonary disease identification markers and their application in diagnostic kits", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the technical field of genetic engineering, and specifically relates to early chronic obstructive pulmonary disease identification markers and their application in diagnostic kits. BACKGROUND

[0003] Chronic obstructive pulmonary disease (COPD) is a common preventable and treatable lung disease with persistent respiratory symptoms and airflow limitation. COPD is a complex and systemic heterogeneous disease that progresses, and is generally believed to be related to abnormal reactions of the lungs to harmful gases or particles.

[0004] In some cases, inter-alpha-trypsin inhibitor heavy chain H3, lymphatic endothelial hyaluronan receptor 1, paraoxonase 1, alanine-glyoxylate aminotransferase, immunoglobulin kappa variable region 3D-11, fodrin 2, apolipoprotein F, dopamine beta-hydroxylase, muscle creatine kinase, FAM20C Golgi-associated secretory pathway kinase, or fat storage-inducing transmembrane protein 1 can be used as a marker related to the phenotype of frequent acute exacerbation of COPD (CN117110617A).

[0005] Clinically, lung function tests are often used as the gold standard for early diagnosis of COPD, but a series of methods including imaging examination, echocardiogram examination, electrocardiogram examination, blood routine examination, blood gas analysis examination, and sputum culture are required for diagnosis. At present, there is a lack of simple and effective early methods for the diagnosis of COPD in clinical practice.

[0006] SUMMARY

[0007] In view of the deficiencies in the prior art, the present application provides early chronic obstructive pulmonary disease identification markers and their application in diagnostic kits.

[0008] The present application provides early chronic obstructive pulmonary disease identification markers, which are any one of the following four:

[0009] 1) miRNA1255a;

[0010] 2) miRNA1255a and protein CD36;

[0011] 3) miRNA1255a and protein TIMP-2;

[0012] 4) miRNA1255a, protein CD36 and protein TIMP-2;

[0013] The nucleotide sequence of the miRNA1255a is shown as SEQ ID NO. 1, the amino acid sequence of the protein CD36 is shown as SEQ ID NO. 2, and the amino acid sequence of the protein TIMP-2 is shown as SEQ ID NO. 3; the miRNA1255a, the protein CD36 and the protein TIMP-2 are derived from serum exosomes.

[0014] The application provides application of the early COPD identification marker or the reagent for detecting expression level of the early COPD identification marker in preparation of an early COPD detection kit.

[0015] The application further provides a kit comprising the reagent for detecting expression level of the early COPD identification marker.

[0016] The application further provides a method for detecting early COPD based on the early COPD identification marker, comprising the following steps: detecting expression level of miRNA1255a, protein CD36 or protein TIMP-2 in a sample to be tested, and judging whether the sample to be tested is a COPD sample according to expression levels of the miRNA1255a, the protein CD36 and the protein TIMP-2.

[0017] When the expression level of the miRNA1255a is higher than the average expression level of miRNA1255a of normal people, and the expression of the protein CD36 and / or the protein TIMP-2 is lower than the average expression level of the protein CD36 and / or the protein TIMP-2 of normal people, the sample to be tested is a COPD sample.

[0018] In addition to the above, the sample to be tested is a non-COPD sample.

[0019] Compared with the prior art, the application has the following beneficial technical effects:

[0020] The application finds a biomarker of early COPD, can very simply screen possible patients with early COPD, does not need to perform a lung function test, and is beneficial to general screening of diseases. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed in the embodiments will be briefly introduced as follows.

[0022] Fig. 1 is a result of analyzing the expression difference of miRNA in serum exosomes between healthy people and patients with chronic obstructive pulmonary disease by miRNA expression profiling in the present application;

[0023] Fig. 2 is a result of target gene function enrichment analysis of miRNA1255a in the present application;

[0024] Fig. 3 is a result of analyzing the expression level difference of genes between samples and performing pathway enrichment analysis on differentially expressed genes by enriching the target genes of differential miRNA in the present application;

[0025] Fig. 4 is a result of analyzing the up-regulation of miRNA1255a in people with small airway lesions but normal lung function by miRNA expression profiling in the present application;

[0026] Fig. 5 is a result of analyzing the expression of CD36 and TIMP2 protein levels in serum exosomes of non-smoking and smoking volunteers in people with small airway lesions but normal lung function by single exosome sequencing technology in the present application;

[0027] Fig. 6 is a result of analyzing the expression of CD36 or TIMP2 in serum samples of 16 randomly selected people in the present application;

[0028] Fig. 7 is a result of simultaneously detecting the expression of CD36 and TIMP2 in 24 people in the present application. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present application will be described clearly and completely in the embodiments of the present application in combination with the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0030] Extracellular vesicles (EVs) are emerging as mediators of intercellular communication and are highly valued, especially exosomes, which are considered to be a very promising nanoscale application drug for the diagnosis and treatment of various diseases. Exosomes are double-membrane vesicles secreted by cells to the extracellular space, with a size range of 30-100 nm. It can selectively encapsulate intracellular proteins, RNA and lipids, and specifically bind to target cells through adhesion molecules carried on its surface, thereby participating in intercellular communication.

[0031] MicroRNA(miRNA) is a kind of endogenous, about 20-24 nucleotides small RNA, several miRNAs can also regulate the same gene. The expression of a certain gene can be finely regulated by the combination of several miRNAs. It is estimated that miRNA regulates one-third of human genes. The level of miRNA in different tissues and different development stages has significant difference, and this miRNA expression pattern has differentiation phase and time sequence, which suggests that miRNA is likely to be a molecule involved in the regulation of gene expression, and thus has important significance.

[0032] The present application uses high-throughput sequencing technology, combined with biological function analysis method, collects a large number of human serum of patients with chronic obstructive pulmonary disease, extracts exosome RNA, and establishes RNA difference database of different levels of chronic obstructive pulmonary disease through second-generation sequencing. It is found that miRNA1255a in the exosome of early patients is abnormal, and the early chronic obstructive pulmonary disease identification marker is screened out, which is miRNA1255a in serum-derived exosome. And through the biological function analysis after sequencing, among the numerous target genes of miRNA1255a, the genes related to the balance of protease and antiprotease, metalloproteinase tissue inhibitor TIMP-2 and CD36, are involved in Ca ion signal pathway, and the expression of the two proteins is negatively correlated with miRNA1255a.

[0033] And it is found in the detection result that when the expression of miRNA1255a increases, the expressions of TIMP-2 and CD36 decrease at the same time. Therefore, another two early chronic obstructive pulmonary disease identification markers-protein CD36 and protein TIMP-2 are screened out. At the same time, it is proved that the combined analysis of the simultaneous expression changes of protein CD36 and protein TIMP-2 has higher accuracy than the analysis of a single protein.

[0034] The RNA sequence of miRNA1255a is shown as SEQ ID NO. 1, the amino acid sequence of protein CD36 is shown as SEQ ID NO. 2, and the amino acid sequence of protein TIMP-2 is shown as SEQ ID NO. 3.

[0035] The sequences shown in SEQ ID NO. 1-SEQ ID NO. 3 of the present application are as follows:

[0036] In the early stage, the present application collects 64 cases of normal people and 103 cases of patients with different levels of chronic obstructive pulmonary disease, extracts serum exosome RNA through a kit, and establishes RNA difference expression database of normal people and different levels of chronic obstructive pulmonary disease by using second-generation sequencing technology. It is found that miRNA1255a in the exosome of early patients is abnormal.

[0037] Specifically, first, an sRNA library is constructed, and miRNA sequence information can be obtained by sRNA sequencing library and high-throughput sequencing. The construction process of the sRNA library includes: sample RNA quality inspection, 5' end and 3' end adapter, reverse transcription reaction, PCR enrichment, fragment selection and sRNA library quality inspection. Then the library is detected for concentration by Qubit dsDNA HS Assay, and the fragment distribution range of the library is detected by Agilent 2100 Bioanalyzer High Sensitivity DNA Assay, and the main peak of the library is about 150 bp. Through sRNA classification annotation, and expression analysis, differential expression analysis, correlation analysis of gene expression levels between samples, clustering analysis of the expression pattern of differential genes, it is found that the expression of miRNA1255a is increased in patients with COPD.

[0038] After high-throughput sequencing, the target genes of miRNA1255a are predicted, and the target genes obtained by prediction are subjected to KEGG functional enrichment analysis, and it is found that they indeed participate in mechanisms related to the pathogenesis of COPD, such as Ca ion signaling pathway. As shown in FIG. 1 and FIG. 2, the results of miRNA expression profile analysis of the expression difference of miRNA in serum exosomes between healthy people and patients with COPD. Among them, 27 miRNAs are up-regulated, and 19 are down-regulated. Among them, miRNA1255a is up-regulated. Through functional enrichment analysis of the target genes of miRNA1255a, the KEGG results obtain mechanisms related to the pathogenesis of COPD, such as Ca ion signaling pathway. It is indicated that miRNA1255a is closely related to the pathogenesis of COPD.

[0039] As shown in FIG. 3, the target genes of miRNA1255a are enriched, and then the results of gene expression level difference between samples and pathway enrichment analysis of differentially expressed genes are analyzed. Moreover, through biological function analysis after sequencing, among the numerous target genes of miRNA1255a, the genes metalloproteinase tissue inhibitor TIMP-2 and CD36 related to the balance between proteases / anti-proteases are involved in the Ca ion signaling pathway. And the expression of these two proteins is found to be negatively correlated with miRNA1255a. Specifically, as shown in FIG. 4 and FIG. 5, by extracting exosomes in serum and detecting the expression of CD36 and TIMP-2 protein levels in serum exosomes through single exosome sequencing technology, it is finally found that in the population of smoking, miRNA-1255a in serum exosomes is up-regulated, while CD36 / TIMP-2 protein co-expression is decreased. FIG. 4 and FIG. 5 together show that in the population with small airway lesions but normal lung function, miRNA1255a is up-regulated, while the levels of CD36 and TIMP-2 proteins are decreased. It is indicated that the three have the possibility of being early diagnostic markers.

[0040] Figure 6 is a random single detection of CD36 or TIMP-2 protein expression in serum samples of 16 people in the present application by enzyme-linked immunosorbent assay. The present application is divided into A and B groups of 8 people according to the expression, and it is found that when the single CD36 expression is different, the expression of group A is significantly higher than that of group B, but only looking at this one indicator, the A group and the B group indeed have no distinction of whether they suffer from COPD. The same is true when the single TIMP-2 expression is different.

[0041] Figure 7 is the expression of CD36 and TIMP-2 protein in 24 people in the present application by enzyme-linked immunosorbent assay. In the case of 4 known normal people, 4 people with simultaneous reduction of CD36 and TIMP-2 protein expression were found, and they were indeed early patients with COPD.

[0042] In summary, Figures 6 and 7 show that simply taking CD36 or TIMP-2 as a single marker for judging COPD is not accurate. The combination of miRNA1255a expression up-regulation and CD36 and TIMP2 protein level down-regulation can greatly improve the accuracy of marker judgment.

[0043] It should be noted that in this paper, terms such as "including", "containing" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or includes elements inherent to such process, method, article or equipment.

[0044] Although the above embodiment makes a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which are within the protection scope of the present application.

Claims

1. An early COPD identification marker characterized in that, The early COPD identification marker is any one of the following four: 1) miRNA1255a; 2) miRNA1255a and protein CD36; 3) miRNA1255a and protein TIMP-2; 4) miRNA1255a, protein CD36 and protein TIMP-2; The nucleotide sequence of the miRNA1255a is shown in SEQ ID NO. 1, the amino acid sequence of the protein CD36 is shown in SEQ ID NO. 2, and the amino acid sequence of the protein TIMP-2 is shown in SEQ ID NO. 3; the miRNA1255a, protein CD36 and protein TIMP-2 are derived from serum exosomes.

2. Use of the early COPD identification marker or reagent for detecting the expression level of the early COPD identification marker in claim 1 in the preparation of an early COPD detection kit.

3. Use according to claim 2, wherein the compound is ###0002### When the expression of the miRNA1255a increases, the expression of the protein TIMP-2 and / or the protein CD36 decreases.

4. A kit characterized in that, The kit comprises a reagent for detecting the expression level of the early COPD identification marker in claim 1.

5. A method of detecting early COPD based on the early COPD identification marker of claim 1, characterized by, The method comprises the following steps: detecting the expression level of miRNA1255a, protein CD36 or protein TIMP-2 in a sample to be tested, and determining whether the sample to be tested is a COPD sample according to the expression level of the miRNA1255a, protein CD36 and protein TIMP-2: When the expression level of the miRNA1255a increases relative to the average expression level of miRNA1255a in normal people, and the expression of the protein CD36 and / or protein TIMP-2 decreases relative to the average expression level of the protein CD36 and / or protein TIMP-2 in normal people, the sample to be tested is a COPD sample; Except for the above, the sample to be tested is a non-COPD sample.

6. The method of claim 5, wherein, The sample to be tested comprises a serum exosome sample.

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