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

Description

[0002] The present disclosure belongs to the technical field of genetic engineering, and specifically relates to an identification marker for early-stage chronic obstructive pulmonary disease and use thereof in a diagnostic kit. BACKGROUND

[0003] Chronic obstructive pulmonary disease (COPD) is a common, preventable and treatable lung disease characterized by persistent respiratory symptoms and airflow limitation. COPD is a progressively developing, complex, systemic, heterogeneous disease, typically related to the abnormal response of the lung to harmful gases or particles.

[0004] In some cases, biomarkers such as the inter-alpha-trypsin inhibitor heavy chain H3, lymphatic vessel endothelial hyaluronan receptor 1, paraoxonase 1, alanine glyoxylate aminotransferase, immunoglobulin kappa variable region 3D-11, cytokinin 2, apolipoprotein F, dopamine P-hydroxylase, muscle creatine kinase, FAM20C Golgi-associated secretory pathway kinase or fat storage-induced transmembrane protein 1 have been identified as associated with the frequent acute exacerbation phenotype of COPD (CN 117110617 A).

[0005] Clinically, pulmonary function tests are often used as the gold standard for the diagnosis of early-stage COPD, but a series of methods including imaging examinations, echocardiography, electrocardiography, complete blood count, blood gas analysis and sputum culture are required for diagnosis. At present, there is a lack of simple and effective diagnostic methods for COPD (especially in its early stage) in clinical practice. SUMMARY

[0006] In view of the deficiencies in the prior art, the present disclosure provides an identification marker for early-stage chronic obstructive pulmonary disease and use thereof in a diagnostic kit.

[0007] The present disclosure provides an identification marker for early-stage chronic obstructive pulmonary disease (COPD), where the identification marker for early-stage COPD is any one of the followings: l)miRNA1255a; 2) miRNAl255a and protein CD36; 3) miRNA1255a and protein TIMP-2; and 4) miRNA1255a, protein CD36 and protein TIMP-2; the nucleotide sequence of the miRNA1255a is set forth in SEQ ID NO.l, the amino acid sequence of the protein CD36 is set forth in SEQ ID NO.2, and the amino acid sequence of the protein TIMP-2 is set forth in SEQ ID NO.3; and the miRNA1255a, protein CD36 and protein TIMP-2 are derived from serum exosomes.

[0008] The present disclosure provides use of the identification marker for early-stage COPD described in the aforementioned technical solution or a reagent for detecting an expression level of the identification marker for early-stage COPD described in the aforementioned technical solution in the preparation of a detection kit for early-stage COPD.

[0009] The present disclosure also provides a kit including a reagent for detecting an expression level of the identification marker for early-stage COPD described in the aforementioned technical solution.

[0010] The present disclosure further provides a method for detecting early-stage COPD based on the identification marker for early-stage COPD described in the aforementioned technical solution, including the following steps: detecting an 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 based on the expression levels of the miRNA1255a, protein CD36 and protein TIMP-2: when the expression level of miRNA1255a increases relative to an average expression level of miRNA1255a in healthy subjects, and the expression level of the protein CD36 and / or protein TIMP-2 decreases relative to an average expression level of protein CD36 and / or protein TIMP-2 in healthy subjects, the sample to be tested is a COPD sample; except for the above cases, the sample to be tested is a non-COPD sample.

[0011] Compared with the conventional technology, embodiments of the present disclosure have the following beneficial effects.

[0012] In the present disclosure, a biomarker for early-stage COPD is identified, allowing for a easy screening of potential patients with early-stage COPD, without the need for lung function tests, thus facilitating widespread disease screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] To describe the embodiments of the present disclosure or the conventional technology more clearly, the accompanying drawings required for the examples will be briefly described below.

[0014] FIG. 1 shows analysis results of the difference in the expression of miRNA in serum exosomes between healthy individuals and COPD patients by miRNA expression profile according to the present disclosure;

[0015] FIG. 2 shows results of function enrichment analysis on target genes of miRNA1255a according to the present disclosure;

[0016] FIG. 3 shows results of analyzing inter-sample gene expression differences by enriching target genes of differentially expressed miRNAs, along with pathway enrichment of differentially expressed genes in the present disclosure;

[0017] FIG. 4 shows results of miRNA expression profiling in the present disclosure, where miRNA1255a is up-regulated in smokers with small airway lesions, despite having normal lung function;

[0018] FIG. 5 shows results of CD36 and TIMP2 protein expressions in serum exosomes from non-smoking volunteers and smoking volunteers in a smoker population with small airway lesions but normal lung function, as analyzed by single exosome sequencing technique in the present disclosure;

[0019] FIG. 6 shows results of randomly and individually detecting the expression of CD36 or TIMP2 in serum samples of 16 individuals in the present disclosure;

[0020] FIG. 7 is the result of simultaneously detecting CD36 and TIMP2 expression in 24 individuals in the present disclosure. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] The embodiments of the present disclosure will be clearly and completely described below with reference to the accompanying drawings in the examples of the present disclosure. Apparently, the examples described are merely some rather than all embodiments of the present disclosure. On the basis of the examples of the present disclosure, all other embodiments that would have been obtained by those of ordinary skill in the art without involving any inventive effort shall fall within the protection scope of the present disclosure.

[0022] Extracellular vesicles (EVs) have emerged as a mediator for intercellular communication and have garnered significant attention. In particular, exosomes are considered to be a very promising nanoscale applied drug for the diagnosis and treatment of various diseases. Exosomes are heterogeneous double-membrane vesicles actively secreted by cells, with a diameter ranging from 30-100 nm. They can selectively encapsulate proteins, RNA and lipids within cells, and specifically bind to target cells by adhesion molecules present on their surface and the like, thereby participating in intercellular communication.

[0023] MicroRNA (miRNA) is a class of endogenous small RNAs with a length of about 20-24 nucleotides. Several miRNAs can also regulate the same gene. The expression of a specific gene can be finely regulated by the combination of several miRNAs. It is estimated that miRNAs regulate one-third of human genes. The levels of miRNAs exhibit significant differences across various tissues and developmental stages. These expression patterns of miRNAs are differentiated in both spatial and temporal aspects, suggesting that miRNAs may serve as molecules involved in regulating gene expression and are therefore of great significance.

[0024] In the present disclosure, high-throughput sequencing technique in combination with biological function analysis methods were utilized. A large number of human serum from patients with COPD were collected to extract exosome RNA, and a database including the differentially expressed RNA from patients with different levels of COPD was established by next-generation sequencing. It was found that miRNA1255a in exosomes from early-stage patients was abnormally expressed, and thereby an identification marker for early-stage COPD is identified, namely miRNA1255a in serum-derived exosomes. Moreover, by post-sequencing biological function analysis, among many target genes of miRNA1255a, the genes related to the balance of protease / antiprotease, tissue inhibitors of metalloproteinase, TIMP-2 and CD36, were found to involve in the Calcium ion signalling pathway. The expressions of the two proteins are negatively correlated with miRNA1255a.

[0025] The detection results showed that when the expression of miRNA1255a increased, the expressions of TIMP-2 and CD36 decreased at the same time. Therefore, two other identification markers for early-stage COPD were identified, specifically protein CD36 and protein TIMP-2. In addition, the present disclosure proves that the combined analysis of the simultaneous expression changes of protein CD36 and protein TIMP-2 is more accurate than the analysis of a single protein.

[0026] The RNA sequence of the miRNA1255a is set forth in SEQ ID NO.l, the amino acid sequence of the protein CD36 is set forth in SEQ ID NO.2, and the amino acid sequence of the protein TIMP-2 is set forth in SEQ ID NO.3.

[0027] The sequences set forth in SEQ ID NO.l to SEQ ID NO.3 described in the present disclosure are as follows: SEQ ID NO.l: 5'-AGGAUGAGCAAAGAAAGUAGAUU-3' SEQ ID NO.2: MGCDRNCGLIAGAVIGAVLAVFGGILMPVGDLLIQKTIKKQVVLEEG TIAFKNWVKTGTEVYRQFWIFDVQNPQEVMMNSSNIQVKQRGPYTYRVRFLAKENVTQ DAEDNTVSFLQPNGAIFEPSLSVGTEADNFTVLNLAVAAASHIYQNQFVQMILNSLINKS KSSMFQVRTLRELLWGYRDPFLSLVPYPVTTTVGLFYPYNNTADGVYKVFNGKDNISKV AIIDTYKGKRNLS YWESHCDMINGTDAASFPPFVEKSQVLQFF S SDICRSIYAVFESDVNL KGIPVYRFVLPSKAFASPVENPDNYCFCTEKIISKNCTSYGVLDISKCKEGRPVYISLPHFL YASPDVSEPIDGLNPNEEEHRTYLDIEPITGFTLQFAKRLQVNLLVKPSEKIQVLKNLKRN YIVPILWLNETGTIGDEKANMFRSQVTGKINLLGLIEMILLSVGVVMFVAFMISYCACRS KTIK' SEQ ID NO.3: MGAAARTLRLALGLLLLATLLRPADACSCSPVHPQQAFCNADVVIRA KAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPEKDIEFIYTAPSSAVCGVSLDVGGKK EYLIAGKAEGDGKMHITLCDFIVPWDTLSTTQKKSLNHRYQMGCECKITRCPMIPCYISS PDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP.

[0028] In the early stage of the present disclosure, serum samples from 64 healthy subjects and 103 patients with different grades of COPD were collected, serum exosome RNA was extracted using a kit, and a database of differentially expressed RNA for healthy subjects and patients with different grades of COPD was established by using a next-generation sequencing technique. It was found that the expression of miRNA1255a was abnormal in exosomes from early-stage patients.

[0029] Specifically, first, a small RNA (sRNA) library was constructed, allowing for the attainment of miRNA sequence information and high-throughput sequencing. The construction process of sRNA library involved sample RNA quality inspection, 5' and 3' end adaptor addition, reverse transcription, enrichment by polymerase chain reaction (PCR), fragment selection and SRNA library quality inspection. Then, the Qubit dsDNA HS Assay was used to detect the concentration of the library, and the Agilent 2100 Bioanalyzer High Sensitivity DNA Assay was used to detect the fragment distribution of the library. The main peak of the library was about 150 bp. By sRNA classification and annotation, as well as expression level analysis, differential expression analysis, correlation analysis of gene expression levels between samples, and cluster analysis of differential gene expression patterns, it was found that miRNA1255a expression was increased in patients with COPD.

[0030] After high-throughput sequencing, the target genes of miRNA1255a were predicted, and KEGG functional enrichment analysis was performed on the predicted target genes. It was found that the target genes were indeed involved in mechanisms related to the pathogenesis of COPD, such as the Calcium ion signalling pathway. The results of analyzing the difference in expression of miRNA in serum exosomes between healthy subjects and COPD patients by miRNA expression profile are shown in FIGS. 1 and 2. In particular, 27 miRNAs were upregulated and 19 were downregulated. In particular, miRNA1255a was upregulated. Functional enrichment analysis was performed on the target genes of miRNA1255a, and the KEGG results revealed mechanisms related to the pathogenesis of COPD, such as the Calcium ion signalling pathway. This indicated that miRNA1255a was closely related to the pathways involved in the development of COPD.

[0031] The results of analysis of the differences in gene expression levels between samples by enriching target genes of miRNA1255a, and the pathway enrichment analysis on differentially expressed genes are shown in FIG. 3. Moreover, by post-sequencing biological function analysis, among many target genes of miRNA1255a, the genes related to the balance of protease / antiprotease, tissue inhibitors of metalloproteinase, TIMP-2 and CD36, were involved in the Calcium ion signalling pathway. Preliminary verification of the expression of the two proteins revealed that they were negatively correlated with miRNA1255a. Specifically, as shown in FIGS. 4 and 5, by extracting exosomes from serum and detecting the expression levels of CD36 and TIMP-2 proteins in serum exosomes using single exosome sequencing technique, it was finally found that in smokers, miRNA-1255a in serum exosomes was upregulated, while the expression of CD36 / TIMP-2 proteins was decreased simultaneously. FIGS. 4 and 5 together indicated that in patients with small airway lesions but normal lung function, miRNA1255a expression was upregulated, while CD36 and TIMP-2 protein levels were decreased. This suggests the possibility of these three as the diagnostic marker for early-stage COPD.

[0032] FIG. 6 shows the expression of CD36 or TIMP-2 protein in serum samples from 16 random individuals, as detected by enzyme linked immunosorbent assay in the present disclosure. In the present disclosure, the participants were divided into two groups, according to the expression level, A and B, with 8 individuals in each group. It was found that when the CD36 was differentially expressed separately, its expression in group A was significantly higher than that in group B. However, if only this indicator did not effectively differentiate between individuals with and without COPD within group A and group B. The same was true for TIMP-2 expression when analyzed separately.

[0033] FIG. 7 shows the expression of CD36 and TIMP-2 proteins as detected simultaneously in a cohort of 24 individuals by enzyme-linked immunosorbent assay in the present disclosure. Four assumed healthy subjects were found to have reduced expression of CD36 and TIMP-2 proteins, and they were finally identified to be patients with early-stage COPD.

[0034] In summary, FIGS. 6 and 7 indicate that it is inaccurate to use CD36 or TIMP-2 alone as a marker for COPD. The combination of upregulated miRNA1255a expression and decreased CD36 and TIMP2 protein levels will greatly improve the accuracy of marker judgment.

[0035] It should be noted that the term "comprise", "include", or any other variant thereof herein is intended to encompass a non-exclusive inclusion, such that a process, method, article, or device that includes a series of elements not only includes those elements, but also includes other elements not explicitly listed, or elements that are inherent to such a process, method, article, or system.

[0036] Although the examples described above have provided a detailed description of the present disclosure, they are only a part of, rather than all of the embodiments of the present disclosure. All other embodiments that can be obtained according to the examples of the present disclosure without creative efforts shall fall within the protection scope of the present disclosure.

Claims

1. An identification marker for early-stage chronic obstructive pulmonary disease (COPD), wherein the identification marker for early-stage COPD is any one of the followings:l)miRNA1255a;2) miRNAl255a and protein CD36;3) miRNA1255a and protein TIMP-2; and4) miRNA1255a, protein CD36 and protein TIMP-2;the nucleotide sequence of the miRNA1255a is set forth in SEQ ID NO.1, the amino acid sequence of the protein CD36 is set forth in SEQ ID NO.2, and the amino acid sequence of the protein TIMP-2 is set forth in SEQ ID NO.3; and the miRNA1255a, protein CD36 and protein TIMP-2 are derived from serum exosomes.

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

3. The use of claim 2, wherein when an expression of the miRNA1255a increases, an expression of the protein TIMP-2 and / or the protein CD36 decreases.

4. A kit, comprising a reagent for detecting an expression level of the identification marker for early-stage COPD of claim 1.

5. A method for detecting early-stage COPD based on the identification marker for early-stage COPD of claim 1, wherein the method comprises the following steps: detecting an 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 based on the expression levels of the miRNA1255a, protein CD36 and protein TIMP-2:when the expression level of miRNA1255a increases relative to an average expression level of miRNA1255a in healthy subjects, and the expression level of the protein CD36 and / or protein TIMP-2 decreases relative to an average expression level of protein CD36 and / or protein TIMP-2 in healthy subjects, the sample to be tested is a COPD sample;otherwise, 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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