阅读说明：本技术 Linc00423在冠心病诊断中的应用 (Application of LINC00423 in coronary heart disease diagnosis ) 是由 杨宁 秦勤 宋衍秋 吴振华 董博 杨靖宇 高霞 于 2020-06-22 设计创作，主要内容包括：本发明公开了LINC00423在冠心病诊断中的应用。本发明发现了LINC00423在冠心病患者中表达上调,差异具有统计学意义。使用ROC曲线分析显示利用LINC00423诊断冠心病的AUC值高达90％以上,故LINC00423可作为诊断冠心病的生物标志物。(The invention discloses application of LINC00423 in coronary heart disease diagnosis. The invention discovers that the expression of LINC00423 is up-regulated in patients with coronary heart disease, and the difference has statistical significance. ROC curve analysis shows that the AUC value for diagnosing the coronary heart disease by using the LINC00423 is up to more than 90 percent, so the LINC00423 can be used as a biomarker for diagnosing the coronary heart disease.)

1. A biomarker for diagnosing coronary heart disease, wherein the biomarker is LINC 00423.

2. A reagent for detecting the biomarker of claim 1.

3. The reagent of claim 2, wherein the reagent comprises a reagent that detects the expression of LINC 00423.

4. The reagent of claim 3, wherein the reagent comprises a probe that specifically recognizes LINC00423, or a primer that specifically amplifies LINC 00423.

5. A product for diagnosing coronary heart disease, comprising an agent for detecting the expression level of LINC00423 in a sample.

6. The product of claim 5, wherein the product comprises a chip, a formulation or a kit.

7. The product of claim 6, wherein the chip comprises a solid support and a probe specific for LINC00423 immobilized on the solid support.

8. The product according to claim 6, wherein the kit comprises a probe specifically recognizing LINC00423, or a primer specifically amplifying LINC 00423.

9. The product of claim 5, wherein the sample comprises blood.

10. The application of a reagent for detecting LINC00423 in the preparation of a product for diagnosing coronary heart disease; preferably, the agent comprises an agent according to any one of claims 2 to 4.

Technical Field

The invention belongs to the field of biological medicine, and relates to application of LINC00423 in coronary heart disease diagnosis.

Background

Coronary heart disease is a common cardiovascular disease seriously harming human health, has complex and long pathogenesis, mainly consists of a plurality of pathogenic links such as cardiovascular endothelial cell damage, smooth muscle cell hyperplasia, platelet adhesion, lipid metabolism abnormality and the like, and relates to risk factors such as environment, heredity and the like. At present, the 'gold standard' for coronary heart disease diagnosis is coronary artery angiography, and the method is limited to be popularized due to objective reasons such as invasiveness and high cost of the detection method; the auxiliary examination for assisting the diagnosis of coronary heart disease includes circulating biomarkers such as inflammatory markers, proteolytic enzyme markers and plasma lipoproteins, and has limitations on the diagnosis specificity and sensitivity in combination with non-invasive examination methods such as electrocardiogram, exercise plate test, echocardiogram and the like. Therefore, the finding of specific molecular markers related to early diagnosis of coronary heart disease has profound significance for realizing early diagnosis and individualized treatment of coronary heart disease.

The rapid development and application of the high-throughput sequencing technology provide a more comprehensive and rapid analysis means for the research of the pathogenesis of the coronary heart disease and provide a new idea for the further treatment scheme of the coronary heart disease. In recent years, long-chain non-coding RNAs (lncRNAs) as key regulatory factors of gene expression attract extensive attention of scholars in the study of cardiovascular diseases such as coronary heart disease.

Long non-coding RNA (1 ncRNA) is a RNA molecule with the transcript length of more than 200nt and without protein coding capacity. Compared with protein coding sequences and microRNAs, the research on lncRNA is still in the initial stage, and the functions of lncRNA are to be further clarified. Recent studies have shown that in many pathological situations the expression levels of lncRNAs are altered and that lncRNAs may have multiple functions in physiological and pathological situations, such as modulating cell proliferation, apoptosis, cell cycle, cell migration, etc. The lncRNAs participate in a plurality of important signal transduction regulation processes such as genome imprinting, chromatin modification, transcription regulation, mRNA stability, protein translation regulation, microRNA function regulation, protein function regulation and the like. Therefore, the research on the function of lncRNA in coronary heart disease has important significance for disclosing the molecular mechanism of coronary heart disease pathogenesis and treating coronary heart disease.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a biomarker for diagnosing coronary heart disease and a product thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a biomarker for diagnosing coronary heart disease, wherein the biomarker is LINC 00423.

The invention also provides reagents for detecting the biomarkers described above.

Further, the reagent comprises a reagent for detecting the expression of LINC 00423.

Still further, the reagents include a probe that specifically recognizes LINC00423, or a primer that specifically amplifies LINC 00423.

The invention also provides application of the reagent for detecting LINC00423 in preparing a product for diagnosing coronary heart disease. The reagent comprises RT-PCR, real-time quantitative PCR, in-situ hybridization, northern blotting and reagents for detecting the expression level of LINC00423 by a chip or a high-throughput sequencing platform.

Further, the reagent comprises: a probe that specifically recognizes LINC 00423; or

And (3) primers for specifically amplifying LINC 00423.

The invention also provides a product for diagnosing coronary heart disease, which comprises a reagent for detecting the expression level of LINC00423 in a sample. The "sample" includes, but is not limited to, cells, tissues, organs, body fluids (blood, lymph, etc.), digestive fluids, expectoration, alveolar bronchial lavage, urine, stool, etc. Preferably, the sample is blood.

Further, the product comprises a chip, a preparation or a kit; the chip comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, wherein the oligonucleotide probes comprise oligonucleotide probes for detecting the transcription level of LINC00423 and aiming at LINC 00423; the kit comprises a primer or a probe for detecting the transcription level of LINC 00423.

The solid phase carrier can be made of various materials commonly used in the field of gene chip, such as but not limited to nylon membrane, glass or silicon slice modified by active group (such as aldehyde group, amino group, etc.), unmodified glass slice, plastic slice, etc.

"Probe" refers to a molecule that binds to a particular sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

The probe has a base sequence complementary to a specific base sequence of a target gene. Here, the term "complementary" may or may not be completely complementary as long as it is a hybrid. These polynucleotides usually have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% with respect to the specific nucleotide sequence. These probes may be DNA or RNA, and may be polynucleotides obtained by replacing nucleotides in a part or all of them with artificial Nucleic acids such as PNA (polypeptide Nucleic Acid), LNA (registered trademark, locked Nucleic Acid, bridge Nucleic Acid, crosslinked Nucleic Acid), ENA (registered trademark, 2 '-O, 4' -C-Ethylene-Bridged Nucleic acids), GNA (glyceronucleic Acid), and TNA (Threose Nucleic Acid).

The kit or the chip can be used for detecting the expression levels of a plurality of genes (for example, a plurality of genes related to coronary heart disease) including LINC00423, and can be used for simultaneously detecting a plurality of markers of the coronary heart disease, so that the accuracy of coronary heart disease diagnosis can be greatly improved.

One skilled in the art will recognize that the utility of the present invention is not limited to quantifying gene expression of any particular variant of the marker genes of the present invention. As a non-limiting example, the LINC00423 marker may have the cDNA sequence designated SEQ ID No. 1.

In the present invention, "expression" of LINC00423 means transcription and/or translation of a functional gene product. For non-protein encoding genes like LINC00423, "expression" can be deregulated at least two levels. First, at the DNA level, for example by deletion or disruption of the gene, or no transcription occurs (in both cases preventing synthesis of the relevant gene product). The loss of transcription can be caused, for example, by an epigenetic change (e.g., DNA methylation) or by a loss-of-function mutation. As used herein, a "loss of function" or "LOF" mutation is a mutation that prevents, reduces or eliminates the function of a gene product relative to a gain-of-function mutation that confers enhanced or new activity to a protein. Functional deletions can be caused by a wide variety of mutation types, including but not limited to deletions of entire genes or gene portions, splice site mutations, frameshift mutations caused by small insertions and deletions, nonsense mutations, missense mutations replacing essential amino acids, and mutations that prevent proper cellular localization of the product. This definition also includes mutations in the promoter or regulatory region of the LINC00423 gene if these mutations interfere with gene function. Null mutations are LOF mutations that completely disrupt the function of the gene product. Null mutations in one allele will typically reduce expression levels by 50%, but may have a severe impact on the function of the gene product. Notably, expression may also be deregulated due to gain-of-function mutations: by conferring new activities to the protein, the normal function of the protein is deregulated and the expressed functionally active protein is reduced. Vice versa, expression may be increased, for example, by gene replication or by lack of DNA methylation. Expression can also be deregulated due to gain-of-function mutations: by conferring new activities to the protein, the normal function of the protein is deregulated and the expressed functionally active protein is reduced. Vice versa, expression may be increased, for example, by gene replication or by lack of DNA methylation.

Second, at the RNA level, for example by lack of efficient translation-for example because of instability of the mRNA (e.g. by UTR variants), can lead to degradation of the mRNA prior to translation of the transcript. Or by lack of efficient transcription, e.g. because mutations induce new splice variants.

In the context of the present invention, "diagnosing coronary heart disease" includes determining whether a subject has suffered from coronary heart disease, determining whether a subject is at risk of suffering from coronary heart disease.

As used herein, the terms "LncRNA", "Long non-coding RNA", "Long non-coding RNA" and "Long non-coding RNA" are synonymous and used interchangeably and refer to a fragment of RNA transcribed by RNA polymerase II that does not encode a protein and is generally greater than 200bp in length.

LncRNA or a detection reagent thereof (e.g., an oligonucleotide capable of binding to LncRNA as a probe and further prepared as a chip) can be chemically synthesized according to the corresponding LncRNA sequence or a complementary sequence thereof in the database according to a conventional technique, or a cDNA sequence thereof can be used to prepare an expression vector and transcribe the expression vector into LncRNA.

Polynucleotide constructs

According to the human LncRNA sequence provided by the present invention, a polynucleotide construct that can be processed into LncRNA that can affect the expression of the corresponding mRNA after being introduced, i.e., the polynucleotide construct can up-regulate the amount of the corresponding LncRNA in vivo, can be designed. Accordingly, the present invention provides an isolated polynucleotide (construct) that can be transcribed into LncRNA by human cells.

Typically, the polynucleotide construct is located on an expression vector. Accordingly, the present invention also includes a vector comprising said LncRNA, or said polynucleotide construct. The expression vector usually further contains a promoter, an origin of replication, and/or a marker gene.

Methods well known to those skilled in the art can be used to construct the expression vectors required by the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as kanamycin, gentamicin, hygromycin, ampicillin resistance.

Chip and method for manufacturing the same

The LncRNA expression profiling chip usually contains up to several hundred probes, covers various RNAs, and utilizes the principle of DNA double-strand homologous complementation to detect the content of various RNAs in a sample at the whole genome level. Therefore, the transcription level of the RNA in the whole genome range in the sample to be tested can be detected at the same time.

By utilizing the LncRNA sequence, a corresponding LncRNA detection chip can be prepared, and the expression profile and the regulation mode of the LncRNA can be further researched.

Specifically, suitable probes can be designed based on the LncRNA of the present invention, and immobilized on a solid support to form an "oligonucleotide array". By "oligonucleotide array" is meant an array having addressable locations (i.e., locations characterized by distinct, accessible addresses), each addressable location containing a characteristic oligonucleotide attached thereto. The oligonucleotide array may be divided into a plurality of subarrays as desired.

The LncRNA chip can be prepared by a conventional method for manufacturing a biochip known in the art. For example, if a modified glass slide or silicon wafer is used as the solid support, and the 5' end of the probe contains a poly-dT string modified with an amino group, the oligonucleotide probe can be prepared into a solution, and then spotted on the modified glass slide or silicon wafer by using a spotting instrument, arranged into a predetermined sequence or array, and then fixed by standing overnight, so as to obtain the miRNA chip of the invention. If the nucleic acid does not contain amino modifications, the preparation can also be referred to: the "Gene diagnostic technique-non-Radioactive operation Manual" edited by Wangshen five; l.l.erisi, v.r.i.er, p.o.brown.expansion of the metabolic and genetic control of genetic compression a genetic scale, science, 1997; 278:680 and maliren, jiang china main edition biochip, beijing: chemical industry Press, 2000, 1-130.

Reagent kit

The kit of the present invention can be used to detect the expression of the LncRNA. Preferably, the kit further comprises a marker for marking the RNA sample and a substrate corresponding to the marker. Preferably, LncRNA contained in the kit of the present invention can also be used for performing a positive control. In addition, the kit may further include various reagents required for RNA extraction, PCR, hybridization, color development, and the like, including but not limited to: an extraction solution, an amplification solution, a hybridization solution, an enzyme, a control solution, a color development solution, a washing solution, an antibody, and the like. In addition, the kit can also comprise an instruction book and/or chip image analysis software.

Drawings

FIG. 1 shows a heat map of differentially expressed lncRNA;

FIG. 2 shows a statistical map of the differential expression of LINC 00423;

FIG. 3 shows a ROC plot.

Detailed Description

The present invention will be described in further detail with reference to examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.