Application of a new molecular marker in the diagnosis or prognosis of breast cancer

Abstract

The present invention discloses the application of a novel molecular marker in the diagnosis or prognosis assessment of breast cancer. Specifically disclosed are the application of the molecular marker lncRNA LIVAR and / or a substance for detecting the molecular marker in the diagnosis, auxiliary diagnosis, screening, prognosis assessment, and risk assessment of breast cancer, as well as its application as a target in the prevention or treatment of breast cancer. The expression level of LIVAR in breast cancer patients is significantly upregulated, and the high expression of LIVAR is significantly correlated with a poor prognosis of breast cancer (including triple-negative breast cancer). Experiments have shown that inhibiting the expression of LIVAR can significantly inhibit the migration and / or invasion ability of breast cancer cells, and can significantly inhibit the proliferation of breast cancer cells. It shows that it can be used as a target for the treatment of breast cancer, the substance that inhibits the expression of LIVAR can be used to prepare a drug for treating breast cancer, and the substance that detects LIVAR can be used for the diagnosis or prognosis of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and medical diagnosis, and relates to the application of a novel molecular marker in the diagnosis or prognosis evaluation of breast cancer, and specifically to a screening method for long non-coding RNA related to breast cancer and its application. Background Art

[0002] Genome-wide transcriptome studies have shown that protein-coding regions comprise only 2% of the human genome, with over 90% of the sequence being transcribed into non-coding RNAs (ncRNAs) that do not control protein synthesis. Long non-coding RNAs (lncRNAs) are a class of transcripts exceeding 200 nucleotides in length. They are characterized by diverse types, multiple modes of action, and high numbers. They exhibit spatial and temporal specificity and can regulate gene expression at multiple levels, participating in diverse biological processes such as proliferation, apoptosis, metastasis, metabolism, and drug resistance. Accumulating evidence indicates that abnormal lncRNA expression and genetic polymorphisms are closely associated with tumor development and progression. Therefore, lncRNAs hold promise as novel tumor diagnostic markers and therapeutic targets for diagnosis, treatment, and recurrence monitoring.

[0003] According to 2020 data released by the Global Cancer Observatory, breast cancer has become the most common type of cancer. Breast cancer patients can be divided into four subtypes based on the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and KI67: luminal A (Luminal A), luminal B (Luminal B), HER2-positive, and triple-negative breast cancer (TNBC). Triple-negative breast cancer (ER, PR, and HER2-negative) lacks hormone receptors and accounts for approximately 15% of breast cancer cases. It is insensitive to targeted and endocrine therapies. Over 70% of patients with triple-negative breast cancer relapse within three years of surgery, resulting in a poor prognosis. Therefore, the search and identification of new, sensitive, and specific breast cancer molecular markers and their detection methods for the diagnosis or auxiliary diagnosis of breast cancer are of great significance for guiding clinical treatment and assessing prognosis, and hold broad application prospects. Summary of the Invention

[0004] The present invention aims to provide a molecular marker useful for breast cancer prognosis, diagnosis, auxiliary diagnosis, screening, risk assessment, and / or postoperative recurrence or metastasis risk assessment, and / or to provide a therapeutic target for breast cancer. The technical problems to be solved are not limited to the technical subject matter described herein, and those skilled in the art will clearly understand other technical subjects not mentioned herein through the following description.

[0005] To achieve the above objectives, the present invention first provides any of the following applications of a molecular marker and / or a substance for detecting the molecular marker:

[0006] A1) Use in the diagnosis of breast cancer or in the preparation of products for the diagnosis of breast cancer;

[0007] A2) Use in the auxiliary diagnosis of breast cancer or in the preparation of products for auxiliary diagnosis of breast cancer;

[0008] A3) Use in breast cancer screening or in the preparation of products for breast cancer screening;

[0009] A4) Use in the assessment of breast cancer prognosis or in the preparation of products for the assessment of breast cancer prognosis;

[0010] A5) Use in assessing breast cancer risk or in preparing products for assessing breast cancer risk;

[0011] A6) Use in assessing the risk of recurrence or metastasis of breast cancer after surgery or in preparing products for assessing the risk of recurrence or metastasis of breast cancer after surgery;

[0012] A7) Use in the prevention or treatment of breast cancer or in the preparation of a medicament for the prevention or treatment of breast cancer;

[0013] A8) Use in preventing or treating triple-negative breast cancer or in preparing a medicament for preventing or treating triple-negative breast cancer;

[0014] A9) Use in inhibiting breast cancer cell proliferation or in preparing an agent or drug for inhibiting breast cancer cell proliferation;

[0015] A10) Use in inhibiting the migration, metastasis, spread and / or invasion of breast cancer cells or in preparing an agent or drug for inhibiting the migration, metastasis, spread and / or invasion of breast cancer cells;

[0016] The molecular marker may be a lncRNA (long non-coding RNA), which may be named LIVAR (liver cell viability associated lncRNA). The lncRNA LIVAR may be any of the following:

[0017] B1) RNA whose nucleotide sequence is SEQ ID No. 1;

[0018] B2) a polynucleotide derived from a human and having more than 80% identity with B1);

[0019] Preferably, the breast cancer may be triple-negative breast cancer.

[0020] Furthermore, the expression level of the lncRNA LIVAR is significantly upregulated in breast cancer patients, and high LIVAR expression is significantly correlated with a poor breast cancer prognosis. The lncRNA LIVAR can be used as a molecular marker in molecular diagnostic fields such as diagnosis, auxiliary diagnosis, screening, prognosis assessment, risk assessment, or postoperative recurrence or metastasis risk assessment for breast cancer.

[0021] Furthermore, the gene encoding lncRNA LIVAR is the LIVAR gene, and the nucleotide sequence of the LIVAR gene is shown in SEQ ID No.4.

[0022] Furthermore, the lncRNA LIVAR may be of human origin.

[0023] As used herein, identity refers to nucleotide sequence identity. Nucleotide sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, nucleotide sequence identity can be calculated using Advanced BLAST 2.1 by using blastp as the program, setting the Expect value to 10, all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. The identity value (%) can then be obtained.

[0024] Herein, the greater than 80% identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0025] In the above application, the substance for detecting the molecular marker may include reagents and / or instruments for detecting the molecular marker by reverse transcription-polymerase chain reaction, real-time fluorescence quantitative PCR technology, transcriptome sequencing technology, Northern blot, in situ hybridization technology, gene chip technology, Nanopore sequencing technology or PacBio sequencing technology.

[0026] In the above application, the substance for detecting the molecular marker may be a reagent and / or instrument for detecting the expression level of the lncRNA LIVAR.

[0027] Furthermore, the reagent for detecting the expression level of the lncRNA LIVAR includes a probe that specifically recognizes lncRNA LIVAR and / or a primer that specifically amplifies lncRNA LIVAR.

[0028] Furthermore, the reagent for detecting the expression level of the lncRNA LIVAR includes a probe that specifically recognizes the LIVAR gene and / or a primer that specifically amplifies the LIVAR gene.

[0029] The probe may be a DNA probe, an RNA probe, a cDNA probe, a cRNA probe or an oligonucleotide probe.

[0030] The present invention also provides a kit for breast cancer diagnosis, auxiliary diagnosis, screening, prognosis assessment, risk assessment, or postoperative recurrence or metastasis risk assessment, which may include any of the substances described herein for detecting the molecular markers.

[0031] Furthermore, the test sample of the kit may be a blood sample (such as whole blood, plasma, serum), a tissue sample, a saliva sample, a sputum sample, a body fluid sample, etc., but is not limited thereto.

[0032] The various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0033] The present invention also provides any of the following uses of a substance for inhibiting the expression of the molecular marker:

[0034] C1) Use in the prevention or treatment of breast cancer or in the preparation of a medicament for the prevention or treatment of breast cancer;

[0035] C2) Use in preventing or treating triple-negative breast cancer or in preparing a drug for preventing or treating triple-negative breast cancer;

[0036] C3) Use in inhibiting breast cancer cell proliferation or preparing an agent or drug for inhibiting breast cancer cell proliferation;

[0037] C4) Use of the method in inhibiting the migration, metastasis, spread and / or invasion of breast cancer cells or in preparing an agent or drug for inhibiting the migration, metastasis, spread and / or invasion of breast cancer cells.

[0038] Furthermore, the inhibition of the expression of the molecular marker (lncRNA LIVAR) can be achieved by techniques well known to those skilled in the art, such as knocking down lncRNA by antisense oligonucleotides ASO, knocking down lncRNA by shRNA or siRNA delivered by plasmids or viruses, and inhibiting the expression of lncRNA using CRISPRi (gene suppression / silencing) technology.

[0039] Furthermore, the substance that inhibits the expression of the molecular marker includes but is not limited to one or more of nucleic acid molecules, carbohydrates, small molecule compounds, gene editing vectors, lentiviruses or adeno-associated viruses.

[0040] Furthermore, the nucleic acid molecule includes but is not limited to antisense oligonucleotides (such as antisense RNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), microRNA (microRNA) or short hairpin RNA (shRNA).

[0041] Furthermore, the antisense oligonucleotide (such as antisense RNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), microRNA (microRNA) or short hairpin RNA (shRNA) is used to inhibit the expression of the molecular marker (lncRNA LIVAR).

[0042] The antisense oligonucleotide refers to a nucleic acid fragment that is complementary to the target gene (lncRNA LIVAR or LIVAR gene), and can bind to the target gene through the principle of base complementarity, thereby inhibiting, blocking, or reducing the expression of lncRNA LIVAR. The antisense oligonucleotide can be antisense DNA or antisense RNA, and can be artificially synthesized or expressed in vivo by methods well known to those skilled in the art; the double-stranded RNA (dsRNA), small interfering RNA (siRNA), microRNA (miRNA) or short hairpin RNA (shRNA) refers to RNA that inhibits or reduces the expression of lncRNA LIVAR through RNA interference (RNAi), and can be prepared by methods well known to those skilled in the art.

[0043] In the above application, the substance that inhibits the expression of the molecular marker includes a substance that silences or knocks out the LIVAR gene, and the LIVAR gene may be a gene encoding the molecular marker lncRNA LIVAR.

[0044] Furthermore, the nucleotide sequence of the LIVAR gene may be shown as SEQ ID No.4.

[0045] In the above applications, the substance for silencing or knocking out the LIVAR gene may include but is not limited to siRNA, sgRNA, microRNA, shRNA and / or antisense oligonucleotides.

[0046] In the above application, the siRNA may be si-LIVAR, the nucleotide sequence of the sense strand of the si-LIVAR may be SEQ ID No. 2, and the nucleotide sequence of the antisense strand may be SEQ ID No. 3.

[0047] The present invention also provides a pharmaceutical composition, which may include any of the substances described herein for inhibiting the expression of the molecular markers, and the pharmaceutical composition may have at least one of the following uses:

[0048] D1) Prevention or treatment of breast cancer;

[0049] D2) Prevent or treat triple-negative breast cancer;

[0050] D3) inhibits breast cancer cell proliferation;

[0051] D4) Inhibit breast cancer cell migration, metastasis, spread and / or invasion.

[0052] Furthermore, the pharmaceutical composition may further include one or more pharmaceutically acceptable carriers.

[0053] The pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant.

[0054] The present invention also provides a device for breast cancer diagnosis, auxiliary diagnosis, screening, prognosis assessment, risk assessment, or postoperative recurrence or metastasis risk assessment. The device may include any substance for detecting the molecular marker described herein and a computer-readable storage medium storing a computer program, wherein the computer program causes the computer to execute the following steps: diagnose, auxiliary diagnosis, screening, prognosis assessment, risk assessment, or postoperative recurrence or metastasis risk assessment of breast cancer based on the expression level of the molecular marker in the sample.

[0055] The breast cancer described herein may be triple-negative breast cancer.

[0056] Herein, the products include but are not limited to reagents, kits, chips, test strips, detection cards, high-throughput sequencing platforms or biosensors.

[0057] The product described herein may include any of the substances described herein for detecting the molecular markers.

[0058] The present invention also provides a method for diagnosing, assisting in the diagnosis, or screening of breast cancer, which may include: obtaining a test sample from a subject, then detecting the expression level of the molecular marker (lncRNA LIVAR) in the test sample using any of the substances for detecting the molecular marker described herein, and performing diagnosis, assisting in the diagnosis, or screening of breast cancer based on the expression level of the molecular marker.

[0059] The present invention also provides a method for evaluating the prognosis, risk, or postoperative recurrence or metastasis risk of breast cancer, which may include: obtaining a test sample from a subject, then detecting the expression level of the molecular marker (lncRNA LIVAR) in the test sample using any of the substances for detecting the molecular marker described herein, and performing a prognosis, risk, or postoperative recurrence or metastasis risk assessment for breast cancer based on the expression level of the molecular marker.

[0060] The present invention also provides a method for preventing or treating breast cancer, which may include administering to a subject any of the substances for inhibiting the expression of the molecular marker (lncRNA LIVAR) or the pharmaceutical composition described herein.

[0061] In the above method, the subject may include a patient suffering from breast cancer.

[0062] In the above method, the breast cancer may include triple-negative breast cancer, luminal A breast cancer, luminal B breast cancer or HER2-positive breast cancer.

[0063] The molecular marker (lncRNA LIVAR) is also within the protection scope of the present invention.

[0064] Any of the substances for detecting the molecular markers described herein is also within the scope of protection of the present invention.

[0065] Any substance described herein that inhibits the expression of the molecular markers is also within the scope of protection of the present invention.

[0066] The present invention also provides any substance described herein for inhibiting the expression of the molecular marker, for use in preventing or treating breast cancer.

[0067] The purpose of the above-mentioned applications and methods may be disease diagnosis, disease prognosis and / or disease treatment, or their purpose may be non-disease diagnosis, non-disease prognosis and non-disease treatment; their direct purpose may be to obtain information on intermediate results of disease diagnosis, disease prognosis and / or disease treatment, or their direct purpose may be non-disease diagnosis, non-disease prognosis and / or non-disease treatment.

[0068] The present invention aims to address the shortcomings of existing technologies by providing a novel molecular marker associated with breast cancer, as well as a kit and device for use in such diagnostic methods. The molecular marker developed by the present invention is LIVAR (liver cell viability associated lncRNA), a molecular target associated with breast cancer diagnosis and prognosis. It is a reliable molecular marker that can be used in the preparation of kits for detecting and assessing breast cancer prognosis, diagnosing and monitoring breast cancer patients, and identifying further treatment options to improve efficacy.

[0069] After extensive and in-depth research, the inventors mined data from public databases and discovered that the expression level of the long noncoding RNA LIVAR is significantly upregulated in breast cancer patients. High LIVAR expression is significantly associated with a poor prognosis in breast cancer (including triple-negative breast cancer). Based on this finding, they developed a molecular marker that can be used in molecular diagnostics for breast cancer, including diagnosis, auxiliary diagnosis, screening, prognosis assessment, risk assessment, and postoperative recurrence or metastasis risk assessment. Kaplan-Meier survival analysis results indicate that high LIVAR expression is associated with a poor prognosis in breast cancer. Phenotypic experiments, such as cell migration and proliferation, further demonstrated that LIVAR is associated with the proliferation and migration of breast cancer cells. In summary, the present invention has identified LIVAR, a breast cancer diagnostic and prognostic predictor that can be used to assess a patient's likelihood of developing breast cancer or subsequently developing the disease.

[0070] The present invention also designed siRNA targeting the LIVAR gene as a substance to inhibit the expression of the molecular marker of the present invention (lncRNALIVAR), and used it to knock down LIVAR gene expression. The results further verified that knocking down the LIVAR gene and inhibiting LIVAR expression can significantly inhibit the migration and / or invasion ability of breast cancer cells, and significantly inhibit breast cancer cell proliferation. This indicates that LIVAR can affect breast cancer cell proliferation and can serve as a drug target for breast cancer treatment. Substances that inhibit LIVAR expression can be used to prepare drugs for breast cancer treatment. It also further verified that substances used to detect LIVAR can be used in the diagnosis, auxiliary diagnosis, or prognosis of breast cancer.

[0071] In summary, the development of the breast cancer molecular marker lncRNA LIVAR and substances that inhibit the expression of the molecular marker for diagnosis, auxiliary diagnosis or prognosis evaluation of the present invention has important clinical application value for the diagnosis, prognosis and risk prediction of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 This is a graph showing LIVAR expression and survival analysis in breast cancer tissues in Example 1 of the present invention. Figure 1Middle A is the difference analysis of LIVAR expression between breast cancer tissue and normal breast tissue. Figure 1 Figure B is the Kaplan-Meier survival analysis result of breast cancer patients. Figure 1 Figure C shows the Kaplan-Meier survival analysis results of patients with triple-negative breast cancer.

[0073] Figure 2 The experimental results of Example 2 of the present invention show the effect of LIVAR on cell migration ability. The cells used in the experiment are the triple-negative breast cancer cell line MDA-MB-231. Among them, si-LIVAR is the experimental group in which LIVAR is knocked down in cells, and si-NC is the corresponding negative control group.

[0074] Figure 3 The results of the experiment on the effect of LIVAR on cell proliferation activity in Example 3 of the present invention show the effect of knocking down the LIVAR gene on cell proliferation activity in MDA-MB-231 cells. si-LIVAR is the knockdown experimental group, and si-NC is the negative control group.

[0075] Figure 4 This is the receiver operating curve for LIVAR as a molecular marker for the diagnosis of breast cancer. DETAILED DESCRIPTION

[0076] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0077] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0078] The following examples were processed using GraphPad Prism 9 statistical software. The experimental results are expressed as mean ± standard deviation. An unpaired t-test was used. P < 0.05 (*) indicates a statistically significant difference, P < 0.01 (**) indicates a statistically significant difference, and P < 0.001 (***) indicates an extremely significant difference.

[0079] The MDA-MB-231 cells in the following examples were derived from the American Type Culture Collection (ATCC) Cat# CRM-HTB-26.

[0080] Some of the sequences involved in the following examples are shown in Table 1.

[0081] Table 1. lncRNA LIVAR related sequences

[0082]

[0083] Example 1. LIVAR expression and survival analysis in breast cancer tissue

[0084] After extensive research and analysis, the present inventors identified a novel prognostic-associated long noncoding RNA (lncRNA) between tumor and non-cancerous samples in breast cancer data. This lncRNA, named LIVAR (liver cell viability-associated lncRNA), is highly expressed in breast cancer samples and is associated with poor patient survival. In the triple-negative breast cancer subtype, upregulation of LIVAR is also associated with a poorer prognosis. As a novel molecular marker, LIVAR's application in assessing cancer survival prognosis has not been reported. Therefore, we propose that LIVAR could be an effective therapeutic target in breast cancer, representing a promising new strategy to improve patient treatment outcomes.

[0085] The nucleotide sequence of LIVAR is shown in SEQ ID No. 1:

[0086] The gene encoding lncRNA LIVAR is the LIVAR gene, and the nucleotide sequence of the LIVAR gene is shown in SEQ ID No. 4:

[0087] The inventors downloaded breast cancer RNA-Seq and clinical data from The Cancer Genome Atlas (TCGA) and analyzed LIVAR expression in 1,109 tumor tissues and 113 normal breast tissues. Breast cancer samples were then retained. Based on LIVAR expression and survival, a Youden index of 3.5 was calculated as the optimal cutoff value. Using this cutoff value as the divergence point, the samples were divided into high-expression and low-expression groups for Kaplan-Meier survival analysis. Tumor subtypes were then categorized based on clinical data. The Youden index for triple-negative samples was also calculated, resulting in an optimal cutoff value of 4.5, which was also used to distinguish between high- and low-expression groups for survival analysis.

[0088] The analysis results are as follows Figure 1 As shown, Figure 1 Figure A shows that the expression level of LIVAR in breast cancer tumor tissue is significantly higher than that in normal tissue. Figure 1 The Kaplan-Meier curves in B and C show that the overall survival of the LIVAR high expression group in breast cancer samples is poor ( Figure 1 B), and in the triple-negative classification there is also a poor prognosis ( Figure 1 The results indicate that LIVAR can be used as a molecular marker for the diagnosis and prognosis of breast cancer and as an effective therapeutic target for breast cancer.

[0089] Example 2: Effect of LIVAR on cell migration ability

[0090] After knocking down the LIVAR gene in breast cancer cells, a cell migration experiment was performed. The steps are as follows:

[0091] (1) MDA-MB-231 cells were transfected with si-LIVAR to knock down LIVAR gene expression, and si-NC served as the corresponding negative control group;

[0092] The sequences of the siRNA used to knock down LIVAR gene expression (i.e., si-LIVAR) are as follows:

[0093] si-LIVAR positive chain: 5'-GGUUGGGACAGAUCUAUUUTT-3' (SEQ ID No. 2),

[0094] si-LIVAR antisense strand: 5′-AAAUAGAUCUGUCCCAACCTT-3′ (SEQ ID No. 3).

[0095] The nucleotide sequence of the negative control si-NC was: 5'-UUCUCCGAACGUGUCACGUTT-3'.

[0096] Cells were seeded in six-well plates. After the cells adhered, a transfection complex consisting of siRNA and transfection reagent was added. Serum-free medium was used during transfection. After approximately 6 hours in an incubator, the medium was replaced with complete medium and cultured for an additional 48 hours before proceeding.

[0097] (2) Prepare cell suspension, count the number of cells using a cell counter, dilute to an appropriate concentration with DMEM medium, and add to a 24-well plate with an upper chamber. Each upper chamber is added with 1 × 10 4 -5 × 10 4 400 μL cell suspension of the appropriate cell number was added, and then 600 μL complete culture medium containing DMEM + FBS (10×) + pen / strep (100×) was added to the lower chamber. 3-4 replicate wells were plated in each group and cultured at a constant temperature of 37°C and 5% CO2 for 24-48 hours.

[0098] (3) Fix the cells in the lower chamber with methanol and stain with 0.1% or 0.5% crystal violet. Observe the cells under a microscope and take photos. Count the number of migrated cells (cells / chamber).

[0099] The experimental results are as follows Figure 2 As shown, knocking down the LIVAR gene significantly reduced the number of migrating breast cancer cells and significantly inhibited their migration and / or invasion abilities. This suggests that LIVAR can regulate cell migration and could serve as a drug target for breast cancer treatment. Substances that inhibit LIVAR expression could be used to prepare drugs for breast cancer treatment. Substances used to detect LIVAR could be used in the diagnosis, auxiliary diagnosis, or prognosis of breast cancer.

[0100] Example 3: Effect of LIVAR on cell proliferation activity

[0101] Proliferation experiments were performed using cells with LIVAR knockdown. The steps are as follows:

[0102] (1) Cell suspensions were prepared from MDA-MB-231 cells transfected with si-LIVAR and si-NC control, and inoculated into 96-well plates. Five replicate wells were inoculated in each group, and 200 μL per well contained 2 × 10 3 Cells were plated in 5 plates and cultured in a 37°C, 5% CO2 incubator.

[0103] The sequences of the siRNA used to knock down LIVAR gene expression (i.e., si-LIVAR) are as follows:

[0104] si-LIVAR positive chain: 5'-GGUUGGGACAGAUCUAUUUTT-3' (SEQ ID No. 2),

[0105] si-LIVAR antisense strand: 5′-AAAUAGAUCUGUCCCAACCTT-3′ (SEQ ID No. 3).

[0106] The nucleotide sequence of the negative control si-NC was: 5'-UUCUCCGAACGUGUCACGUTT-3'.

[0107] (2) After the cells have fully adhered, take one of the 96-well plates from step (1) and prepare the reagent by adding 10 μL of CCK-8 per 100 μL of culture medium. Then, add 200 μL of the reagent to the wells containing the inoculated cells using a liquid exchange method. Protect the plate from light during the operation and return it to the incubator. After 1 hour, measure the absorbance at 450 nm using a microplate reader and record the value.

[0108] (3) After 24, 48, 72, and 96 hours, take a 96-well plate from step (1) and replace the culture medium in each replicate well with 200 μL of culture medium + CCK-8 reagent. Incubate in an incubator for 1 hour and then measure the absorbance. Calculate the absorbance values ​​measured for 5 consecutive days and plot a curve with time as the horizontal axis and absorbance as the vertical axis.

[0109] The experimental results are as follows Figure 3 As shown, knocking down the LIVAR gene weakened the proliferation activity of breast cancer cells, indicating that LIVAR can regulate cell proliferation. This indicates that reducing LIVAR expression can significantly inhibit the growth of breast cancer cells, that is, inhibit their proliferation. This further demonstrates that LIVAR can affect breast cancer cell proliferation and could serve as a drug target for breast cancer treatment. Substances that inhibit LIVAR expression could be used to prepare drugs for breast cancer treatment. Substances used to detect LIVAR can be used in the diagnosis, auxiliary diagnosis, or prognosis of breast cancer.

[0110] Example 4: Performance of LIVAR as a molecular marker for breast cancer diagnosis

[0111] Based on information on estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) in breast cancer samples and clinical data downloaded from TCGA, samples negative for all three were designated as triple-negative. Survival status, age, and TNM stage of patients in this subtype were then analyzed using receiver operating characteristic (ROC) curves for multi-indicator prediction.

[0112] The results are as follows Figure 4 As shown in the data, the area under the ROC curve of LIVAR as a molecular marker for the diagnosis of triple-negative breast cancer is 0.829, indicating that the use of LIVAR for the diagnosis of breast cancer, especially triple-negative breast cancer, has good specificity and high sensitivity. As a new molecular marker for breast cancer, it has great potential value and can provide a powerful molecular biological tool for the screening, diagnosis or auxiliary diagnosis of breast cancer.

[0113] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Use of a substance for detecting the expression level of a molecular marker in the preparation of a product for diagnosing triple-negative breast cancer, wherein the molecular marker is lncRNA, and the lncRNA is an RNA with a nucleotide sequence of SEQ ID No.

1.

2. The use according to claim 1, characterized in that The substances for detecting the molecular markers include reagents for detecting the molecular markers by reverse transcription-polymerase chain reaction, real-time fluorescence quantitative PCR technology, transcriptome sequencing technology, Northern blot, in situ hybridization technology, gene chip technology, Nanopore sequencing technology or PacBio sequencing technology.

3. The use according to claim 1, characterized in that The substance for detecting the molecular marker is a reagent for detecting the expression level of the lncRNA in claim 1.

4. Use of a substance that inhibits the expression of the molecular marker of claim 1 in the preparation of a drug for treating triple-negative breast cancer; the substance is si-LIVAR, the nucleotide sequence of the sense strand of the si-LIVAR is SEQ ID No. 2, and the nucleotide sequence of the antisense strand is SEQ ID No. 3.

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