阅读说明：本技术 用于诊断和治疗乳腺癌的生物标志物 (Biomarkers for diagnosis and treatment of breast cancer ) 是由 袁成良 巫奇 贾新建 魏伟 邹宁 蒋雪梅 于 2019-11-06 设计创作，主要内容包括：本发明公开了用于诊断和治疗乳腺癌的生物标志物,所述生物标志物为LINC02237。本发明公开了一种诊断乳腺癌的产品以及检测LINC02237的试剂在制备诊断乳腺癌的产品中的应用；本发明同时公开了LINC02237在构建预测乳腺癌的计算模型中的应用。(The invention discloses a biomarker for diagnosing and treating breast cancer, wherein the biomarker is LINC 02237. The invention discloses a product for diagnosing breast cancer and application of a reagent for detecting LINC02237 in preparation of a product for diagnosing breast cancer; the invention also discloses application of the LINC02237 in constructing a calculation model for predicting breast cancer.)

1. Application of a reagent for detecting LINC02237 in preparation of a product for diagnosing breast cancer.

2. The use of claim 1, wherein the breast cancer is Luminal type B breast cancer.

3. The use according to claim 2, wherein said product comprises reagents for detecting the expression level of LINC02237 by RT-PCR, real-time quantitative PCR, in situ hybridization, chip or high throughput sequencing platform.

4. The use according to claim 1, wherein the agent is selected from the group consisting of: a probe that specifically recognizes LINC 02237; or a primer for specifically amplifying the LINC02237, preferably, the primer sequence for specifically amplifying the LINC02237 is shown as SEQ ID NO. 3-4.

5. A product for diagnosing breast cancer, comprising an agent for detecting the expression level of LINC 02237.

6. The product according to claim 5, comprising a chip, a kit, a nucleic acid membrane strip, preferably wherein the chip comprises oligonucleotide probes specifically recognizing LINC 02237; the kit comprises a primer for specifically amplifying LINC02237 or an oligonucleotide probe for specifically recognizing LINC 02237; the nucleic acid membrane strip includes an oligonucleotide probe that specifically recognizes LINC 02237.

Use of LINC02237 in the construction of a computational model for predicting breast cancer.

Application of LINC02237 in preparing a pharmaceutical composition for treating breast cancer.

9. The use according to claim 8, wherein the pharmaceutical composition comprises an enhancer of LINC 02237.

10. A pharmaceutical composition, comprising an enhancer of LINC 02237.

Technical Field

The invention belongs to the field of biomedicine, and relates to a biomarker for diagnosing and treating breast cancer, wherein the biomarker is LINC 02237.

Background

The tumor is a new organism formed by abnormal gene level, abnormal function and local abnormal cell proliferation caused by the synergistic effect of internal and external tumorigenic factors. Common tumorigenic substances include chemical, viral and physical tumorigenics. The neoplastic tumor tissue differs from the normal tissue from which it originates in both cell morphology and tissue structure, and this difference is called the heterogeneity of the tumor tissue. Malignant tumors, known as cancers, are characterized by infiltration and metastasis, and do not have an intact envelope, and are not easily removed by surgery. In addition, it consumes a large amount of nutrients to propagate rapidly, and is extremely harmful to the body. For malignant tumors, our principle is early discovery, early diagnosis and early treatment. For patients who find later, the best chance of treatment is missed, and the curative effect of radiotherapy, chemotherapy, targeted drugs and other treatments on the patients with later period is very limited, so that the survival rate of the patients with later period is greatly reduced.

Breast cancer is a very common female disease, and its classification includes many clinical and pathological parameters, such as tumor solid size, whether lymph node metastasis occurs, histological grade, and patient age distribution; patients use a number of biomarkers in diagnostic and therapeutic procedures, mainly estrogen receptors (estrogen receptor), epidermal growth factor receptors (HER 2), Progestogen Receptors (PR), etc. With the continuous and intensive research on breast cancer, the latest typing results have classified breast cancer into 5 subtypes, HER2 overexpression type (ER)^-,PR^-And HER2⁺) Luminous type A (ER)⁺Or PR⁺And HER2^-) Luminous type B (ER)⁺Or PR⁺And HER2⁺) Basal-like (basal-like) type (ER)^-,PR^-And HER2^-) And normal-tissue (normal-like) type breast cancer (Mackay A, Weigel B, Gridiroadis A, et al. microarray-based class discovery for molecular classification of Breast cancer: analysis of Interactive Breast cancer [ J]J Natl Cancer Inst,2011,103(8): 662-. The new typing is more helpful for specific clinical treatment of breast cancer patients of different subtypes.

Long non-coding RNAs are RNAs with the length of more than 200 nucleotides and without protein translation function, and are widely expressed in transcriptional regulation. Currently, IncRNAs are mainly classified into five types, namely antisense IncRNA, intron IncRNA, lincRNA, promoter-related IncRNA and UTR-related IncRNA. LncRNA has spatiotemporal specificity, and expression patterns behave differently in different tissues. Compared with microRNA, lncRNA is longer in length and has similar mRNA structure. LncRNA can be combined with microRNA, mRNA and protein, and plays an important regulation and control role in cells. Currently, lncRNA is involved in biological activities such as gene transcription, epigenetic regulation, protein coding genes, chromatin organization, and the like. Meanwhile, lncRNAs play an important role in molecular mechanisms such as RNA splicing, X chromosome inactivation and the like. The lncRNA has the length similar to that of mRNA, has the structural characteristics of mRNA, and can be combined with transcription factors, microRNA and the like. Therefore, lncRNA has a dramatic regulatory role in the expression of protein-coding genes and other non-coding RNAs. In addition to the base sequence, lncrnas may bind to proteins, modulate protein activity, or perform other functions.

Although the biological function of most lncRNAs has not been determined, there have been many studies indicating that the expression of lncRNA is abnormal in the tissue cells of cancer patients. In studies on breast cancer, colorectal cancer, lung cancer, breast cancer and the like, a plurality of lncRNA are found to be related to cancer lesions. Based on the fact that the abnormal expression of some lncRNAs has been proved to be closely related to cancer, the use of lncRNAs as a marker for cancer diagnosis and prognosis is also a new research direction.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention aims to provide a biomarker related to the occurrence and development of breast cancer and application of the biomarker in diagnosis and treatment of breast cancer.

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

the invention provides application of a reagent for detecting LINC02237 in preparation of a product for diagnosing breast cancer.

Further, the breast cancer is Luminal B type breast cancer.

Further, the product comprises reagents for detecting the expression level of LINC02237 by RT-PCR, real-time quantitative PCR, in situ hybridization, chip, or high throughput sequencing platform.

Further, the agent is selected from: a probe that specifically recognizes LINC 02237; or a primer that specifically amplifies LINC 02237.

Further, the primer sequence of the specific amplification LINC02237 is shown in SEQ ID NO. 3-4.

The invention provides a product for diagnosing breast cancer, which comprises a reagent for detecting the expression level of LINC 02237.

Further, the product comprises a chip, a kit and a nucleic acid membrane strip.

Further, the chip comprises an oligonucleotide probe specifically recognizing LINC 02237; the kit comprises a primer for specifically amplifying LINC02237 or an oligonucleotide probe for specifically recognizing LINC 02237; the nucleic acid membrane strip includes an oligonucleotide probe that specifically recognizes LINC 02237.

Further, the primer sequence for specifically amplifying the LINC02237 is shown as SEQ ID No. 3-4.

The invention provides application of LINC02237 in constructing a calculation model for predicting breast cancer.

Further, the breast cancer is Luminal B type breast cancer.

The invention provides application of LINC02237 in preparing a pharmaceutical composition for treating breast cancer.

Further, the breast cancer is Luminal B type breast cancer.

Further, the pharmaceutical composition comprises an enhancer of LINC 02237.

Further, the promoter of LINC02237 is a substance that increases the level of LINC 02237.

The invention provides a pharmaceutical composition comprising an enhancer of LINC 02237.

Drawings

FIG. 1 is a graph showing the detection of the expression of LINC02237 gene in breast cancer tissue by QPCR.

FIG. 2 is a graph showing the detection of over-expressed LINC02237 by QPCR.

FIG. 3 is a graph showing the effect of LINC02237 on the proliferation of BT474 cells measured by the CCK-8 method.

FIG. 4 is a graph showing the effect of LINC02237 on the migratory invasion capacity of BT474 cells measured using a Transwell chamber.

Detailed Description

According to the invention, through extensive and intensive research, the expression of lncRNA in a breast cancer specimen in a tumor tissue and a normal tissue is detected by a high-throughput sequencing method, lncRNA with obvious expression difference is found, and the relation between the lncRNA and the occurrence of breast cancer is discussed, so that a better way and a better method are found for the diagnosis and the targeted therapy of the breast cancer. Through screening, the invention discovers that LINC02237 is remarkably up-regulated in breast cancer tissues for the first time, and suggests that LINC02237 can be used as a diagnostic marker and a therapeutic target of breast cancer.

The term "LINC 02237" is located on chromosome 8 with gene ID 105375706, and includes the LINC02237 gene and homologs, mutations, and isoforms thereof. The term encompasses full-length, unprocessed LINC02237, as well as any form of LINC02237 that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of LINC 02237. The term encompasses, for example, the LINC02237 gene, the gene sequence of human LINC02237 (NR _146282.1), and LINC02237 DNA from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats).

It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The expression level of the biomarker can be detected at the transcriptional level. The present invention may utilize any method known in the art for determining gene expression.

As used herein, the term "biomarker" refers to an indicator molecule or collection of molecules (e.g., predictive, diagnostic, and/or prognostic indicators) that can be detected in a sample and includes, for example, LINC 02237. The biomarker may be a predictive biomarker and serve as having a particular disease or condition. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA (e.g., mRNA)) with altered polynucleotide copy number (e.g., DNA copy number).

As used herein, an "amount" or "level" of a biomarker is a detectable level in a biological sample. These can be measured by methods known to those skilled in the art and disclosed herein.

The term "level of expression" or "expression level" generally refers to the amount of a biomarker in a biological sample. "expression" generally refers to the process by which information (e.g., gene coding and/or epigenetic information) is converted into structures present and operating in a cell. Thus, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., post-translational modifications of a polypeptide). In a particular embodiment of the invention, the "expression" refers to transcription into a polynucleotide.

"increased expression," "increased expression level," "increased level," "elevated expression level," or "elevated level" refers to increased expression or increased level of a biomarker in an individual relative to a control, such as a median expression level of the biomarker in an individual without a disease or disorder (e.g., cancer), an internal control (e.g., a housekeeping biomarker), or a sample from one patient group/population.

"reduced expression", "reduced expression level", "reduced expression level" or "reduced level" refers to reduced expression or reduced level of a biomarker in an individual relative to a control, such as a median expression level of the biomarker in an individual or an internal control (e.g., a housekeeping biomarker) that does not have a disease or disorder (e.g., cancer), or a sample from one patient group/population. In some embodiments, the reduced expression is little or no expression.

In the present invention, LINC02237 has reduced expression levels in breast cancer patients.

Chip, kit and nucleic acid membrane strip

The invention provides products, including but not limited to preparations, chips or kits, for detecting the expression level of the LINC02237 gene in a subject. Wherein the chip includes: a solid support; and oligonucleotide probes orderly fixed on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to part or all of the sequence shown in LINC 02237.

The solid phase carrier comprises an inorganic carrier and an organic carrier, wherein the inorganic carrier comprises but is not limited to a silicon carrier, a glass carrier, a ceramic carrier and the like; the organic vehicle includes a polypropylene film, a nylon film, and the like.

As used herein, "oligonucleotide" generally refers to a short, single-stranded polynucleotide that is less than about 250 nucleotides in length, although this is not required. The oligonucleotide may be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description for polynucleotides is equally and fully applicable to oligonucleotides.

The term "probe" refers to a molecule that binds to a specific 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.

These probes have 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 comprises a reagent for detecting the LINC02237 gene, and one or more substances selected from the following group: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent.

The kit of the invention can be also attached with an instruction manual of the kit, wherein the instruction manual describes how to adopt the kit for detection, how to judge the tumor development by using the detection result and how to select a treatment scheme.

The components of the kit may be packaged in aqueous medium or in lyophilized form. Suitable containers in the kit generally include at least one vial, test tube, flask, pet bottle, syringe, or other container in which a component may be placed and, preferably, suitably aliquoted. Where more than one component is present in the kit, the kit will also typically comprise a second, third or other additional container in which the additional components are separately disposed. However, different combinations of components may be contained in one vial. The kit of the invention will also typically include a container for holding the reactants, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials may be retained.

The nucleic acid membrane strip comprises a substrate and an oligonucleotide probe aiming at LINC02237 and fixed on the substrate; the substrate may be any substrate suitable for immobilizing oligonucleotide probes, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass plate, a silica gel wafer, a micro magnetic bead, or the like.

Calculation model

The invention provides application of LINC02237 in preparing a calculation model for predicting breast cancer. As the skilled artisan will appreciate, the measurement of two or more markers may be used to improve the diagnostic question in the survey. The biochemical markers may be determined individually, or in one embodiment of the invention, they may be determined simultaneously, for example using a chip or bead-based array technology. The concentration of the biomarkers is then interpreted independently, for example using individual retention of each marker, or a combination thereof.

In the present invention, the step of associating a marker level with a certain likelihood or risk may be carried out and carried out in different ways. Preferably, the measured concentrations of the gene and one or more other markers are mathematically combined and the combined value is correlated to the underlying diagnostic problem. The determination of marker values may be combined by any suitable prior art mathematical method.

Accelerator

The promoter for the LINC02237 refers to any substance which can improve the stability of the LINC02237 gene or an expression product, up-regulate the expression of the LINC02237, prolong the effective action time of the lncRNA LINC02237 or promote the transcription of the LINC02237 gene, can be used for the invention, and can be used as a substance which is useful for up-regulating the expression of the LINC02237 gene, thereby being used for preventing or treating breast cancer.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.