Application of LINC00987 as breast cancer diagnosis marker

Abstract

The invention discloses application of LINC00987 as a breast cancer diagnosis marker, and relates to the technical field of molecular biomedicine. The LINC00987 is used as a long inter-gene non-coding RNA (Ribonucleic Acid), is positioned in the 31st subband in a p13 region of a short arm of a 12th chromosome of human, and has the length of 22828bp; the gene can be used as a marker in preparation of breast cancer diagnosis products or prognosis evaluation products. The invention also discloses application of the compound as a target spot in preparation of drugs for preventing and / or treating breast cancer. In the breast cancer, the expression of the LINC00987 is gradually reduced along with the disease progress, and the proliferation and metastasis of breast cancer cells can be effectively inhibited by overexpressing the LINC00987 in the breast cancer, so that the growth of the breast cancer is effectively inhibited.

Description

Technical Field

[0001] The present invention relates to the field of molecular biomedical technology, and in particular to the use of LINC00987 as a diagnostic marker for breast cancer. Background Art

[0002] Breast cancer, one of the most common malignancies in women, is highly heterogeneous, exhibiting not only diverse histological features but also complex differences in gene expression and signaling pathways at the molecular level. Luminal A breast cancer is ER and / or PR positive, HER2 negative, and has low Ki-67 expression. It is the most common molecular subtype of breast cancer, accounting for between 44.5% and 69.0% of all breast cancers. This subtype is characterized by a lower degree of malignancy, a slower progression, and a relatively low risk of recurrence, resulting in a favorable prognosis. Luminal B breast cancer is more common in older patients and, compared with Luminal A, has higher Ki-67 expression. Its cancer cells grow more rapidly and have a relatively poorer prognosis. Among all breast cancer types, HER2-overexpressing subtypes typically account for less than 10%. These subtypes are ER-negative, PR-negative, HER2-positive, and typically have elevated Ki-67 expression. This subtype is more aggressive and metastatic, with a poorer prognosis and a higher risk of recurrence. Triple-negative breast cancer refers to breast cancer that is negative for ER, PR, and HER-2. Approximately 15% of breast cancer patients have triple-negative breast cancer. It is highly malignant and insensitive to both endocrine and targeted therapies, making it one of the most aggressive types of breast cancer and often recurs and metastasizes in the early stages of the disease. This heterogeneity leads to significant differences in disease progression, treatment response, and prognosis among breast cancer patients.

[0003] The unlimited proliferation and metastasis ability of cancer cells is the main difference between malignant tumors and benign tumors, and it is also the main cause of death from cancer. Cancer cells first proliferate and invade indefinitely in the primary tumor tissue, gradually penetrating the tissue barrier around the primary tumor and leaving the location of the primary tumor; then, cancer cells enter the circulatory system and are transported long distances through the blood circulation or lymphatic circulation; eventually, cancer cells escape from the blood vessels, colonize in distant tissues or organs, and continue to proliferate, forming new tumor lesions. The biological characteristics of breast cancer cells, such as abnormal proliferation, invasion, and metastasis, seriously affect the treatment effect of breast cancer patients and are the key factors leading to poor prognosis of patients. Therefore, in-depth exploration of the biological behavior of breast cancer cells and the search for efficient molecular markers have become the key to improving the treatment effect of breast cancer and improving patient prognosis.

[0004] Molecular markers play a crucial role in the early diagnosis, classification, and prognostic assessment of breast cancer. For example, molecular markers such as ER, PR, and HER-2 have been widely used in breast cancer classification and targeted therapy. Studies have shown that high HER-2 gene expression is closely associated with breast cancer aggressiveness and poor prognosis. Targeted anti-HER-2 therapies, such as trastuzumab, can significantly improve patient survival. ER and PR are hormone receptors, and their positive expression indicates hormone-dependent breast cancer, making such patients more sensitive to endocrine therapy. Furthermore, ER and PR positivity is positively correlated with overall survival and disease-free survival. However, existing markers still have limitations in sensitivity and specificity, and cannot fully meet the needs of precision diagnosis and treatment of breast cancer. In some patients with HER-2-positive breast cancer, the false-positive rate of HER-2 testing can reach 20% due to limitations in detection methods or tumor cell heterogeneity. Therefore, there is an urgent need to explore new molecular markers to improve the diagnostic accuracy and treatment efficacy of breast cancer. Summary of the Invention

[0005] In view of this, the main purpose of the present invention is to provide an application of LINC00987 as a diagnostic marker for breast cancer. The present invention comprises the following steps: 1. Using breast cancer transcriptome data from the TCGA and GTEx databases, the expression differences of LINC00987 in breast cancer and normal breast tissues and their clinical significance are analyzed; 2. By using univariate and multivariate Cox regression analysis and plotting survival curves, the potential of LINC00987 as a prognostic marker for breast cancer is analyzed; 3. By using fluorescence in situ hybridization (FISH) technology, the expression levels of LINC00987 in breast cancer tissues and adjacent normal tissues are detected, and the relationship between its expression levels and clinical pathological characteristics (such as tumor size, lymph node metastasis, pathological grade, etc.) is explored; 4. By using nuclear cytoplasm fractionation experiments and FISH technology in combination with multiple databases, the localization and functional prediction of LINC00987 in breast cancer cells are clarified; 5. By using small interfering RNA (siRNA) 6. The LINC00987 knockdown or overexpression breast cancer cell models were constructed using siRNA (interfering RNA, siRNA) and overexpression vectors, and a subcutaneous breast cancer xenograft model was constructed using MDA-MB-231 cells stably overexpressing LINC00987. 6. The relationship between LINC00987 expression level and breast cancer cell proliferation was investigated using cell experiments such as CCK-8, colony formation, flow cytometry (PI staining), and a nude mouse subcutaneous xenograft tumor model. 7. The effect of LINC00987 on breast cancer cell migration and invasion was further investigated using transwell assays.

[0006] This study clarified the differential expression of LINC00987 in breast cancer tissue and normal breast tissue, explored the correlation between its expression level and the clinicopathological characteristics of breast cancer patients, and determined its role in the biological function of breast cancer.

[0007] To achieve the above objectives, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a method for using LINC00987 as a marker in the preparation of a breast cancer diagnostic or prognostic assessment product. LINC00987 is located in subband 31 of the p13 region of the short arm of human chromosome 12, specifically based on the GRCh38 / hg38 reference genome, with a start position of 9240073 and an end position of 9262900, and a length of 22,828 base pairs.

[0009] Furthermore, the product is a reagent or kit for detecting the expression level of LINC00987.

[0010] Furthermore, the expression level of LINC00987 is positively correlated with the prognosis of breast cancer; and the expression of LINC00987 is downregulated in breast cancer patients.

[0011] In a second aspect, the present invention provides a product for predicting the prognosis of breast cancer, wherein the product comprises a reagent or a kit for detecting the marker according to claim 1.

[0012] In a third aspect, the present invention provides the use of the above-mentioned LINC00987 as a target in screening candidate drugs for preventing or treating breast cancer.

[0013] In a fourth aspect, the present invention provides the use of the above-mentioned LINC00987 as a target in the preparation of a drug for preventing and / or treating breast cancer.

[0014] Furthermore, the application is to increase the expression level of LINC00987.

[0015] Furthermore, the expression level of LINC00987 was increased by RNA technology or by constructing an overexpression vector of LINC00987.

[0016] Specifically, constructing an overexpression vector of LINC00987 includes the following steps:

[0017] 1) Synthesize pcDNA3.1(+)-LINC00987 overexpression plasmid;

[0018] 2) Design primers with the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, and perform PCR amplification using LINC00987-pcDNA3.1(+) as a template to obtain the target gene fragment;

[0019] 3) The pLVX-P2A-Luc-T2A-Puro plasmid was double-digested with XhoⅠ and BamHI to recover the target fragment and obtain the large plasmid fragment;

[0020] 4) Ligating the recovered large plasmid fragment with the target gene fragment; after transformation and culture of the ligation product, single colonies were picked, and the plasmid was extracted and double-enzyme digestion verification was performed. Positive clones with correct enzyme digestion verification were verified by next-generation sequencing; the verified pLVX-hLINC00987-Luc-Puro plasmid was introduced into 293T cells to obtain the lentivirus rLV-hLINC00987-Luc-Puro containing the target gene.

[0021] Alternatively, negative regulatory factors can be inhibited by antisense RNA, thereby indirectly increasing the expression of LINC00987.

[0022] In a fifth aspect, the present invention further provides the use of an expression agonist, an enhancing agent, or an overexpression agent of LINC00987 in the preparation of a drug for treating breast cancer.

[0023] In a sixth aspect, the present invention further provides a drug for treating breast cancer, wherein the drug comprises an agent that increases the expression of LINC00987.

[0024] The beneficial effects of the present invention include at least:

[0025] This study explored the distribution and expression characteristics of LINC00987 in breast cancer and its association with the clinicopathological features of breast cancer patients. It also investigated its effects on the proliferation, migration, and invasion of breast cancer cells and their relationship with prognosis.

[0026] 1) Nuclear-cytoplasmic fractionation experiments and cell FISH results showed that LINC00987 was mainly localized in the nuclei of breast cancer cells;

[0027] 2) Bioinformatics analysis and tissue FISH experiments revealed that LINC00987 expression in breast cancer tissues was significantly lower than that in normal tissues; its expression was significantly correlated with tumor size, metastasis, and tumor stage;

[0028] 3) LINC00987 is an important independent risk factor for prognosis in breast cancer patients, and its low expression is significantly associated with poor prognosis;

[0029] 4) Low expression of LINC00987 promoted the proliferation, migration and invasion of breast cancer cells; whereas overexpression of LINC00987 inhibited the proliferation, migration and invasion of breast cancer cells and induced cell cycle arrest.

[0030] Specific detection of LINC00987 can achieve the purpose of diagnosing and / or prognostic evaluation of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The localization map of LINC00987 on the chromosome.

[0032] Figure 2 Schematic diagram of the protein coding capacity of LINC00987 predicted by the CPAT and CPC2 online databases.

[0033] Figure 3 The secondary structure diagram of LINC00987: the free energy is -885.40 kcal / mol.

[0034] Figure 4 The localization map of LINC000987 in breast cancer cells was detected by nuclear-cytoplasmic fractionation experiments.

[0035] Figure 5 FISH detection of LINC000987 localization in breast cancer cells (magnification: 200×).

[0036] Figure 6 Schematic diagram of the expression levels of LINC00987 in breast cancer tissues and normal breast tissues.

[0037] Figure 7 FISH detection of LINC00987 expression in cancerous tissues and adjacent adjacent tissues of breast cancer patients (magnification: 200×).

[0038] Figure 8 Survival analysis of LINC00987 in breast cancer patients, A—overall survival; B—disease-free survival; C—progression-free interval.

[0039] Figure 9 Figure 2 is the knockdown efficiency of LINC00987.

[0040] Figure 10 Figure 2 is the overexpression efficiency diagram of LINC00987.

[0041] Figure 11 Effect of LINC00987 knockdown on the proliferation ability of breast cancer cells.

[0042] Figure 12Effect of LINC00987 overexpression on breast cancer cell proliferation. A—CCK-8 assay; B—Clonogenic assay. *P < 0.05; ***P < 0.001.

[0043] Figure 13 Flow cytometry analysis of LINC00987-overexpressing breast cancer cell cycle changes.

[0044] Figure 14 Effects of LINC00987 knockdown on the migration and invasion abilities of breast cancer cells.

[0045] Figure 15 Effect of LINC00987 overexpression on the proliferation ability of breast cancer in vivo.

[0046] Figure 16 HE staining and Ki67 immunohistochemical staining (magnification: 200×), A is HE staining and Ki-67 immunohistochemical staining; B is quantitative analysis of Ki67 expression. ***P<0.001. DETAILED DESCRIPTION

[0047] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0048] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that ordinary technicians in this field can implement it. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0049] The materials and methods used in the experiment are as follows:

[0050] 1. Experimental Materials

[0051] (1) Tissue specimens

[0052] Tissue microarrays were purchased from Shanghai Zhuoli Biotechnology Co., Ltd. and included cancer tissue and adjacent normal breast tissue. The inclusion and exclusion criteria for the study subjects were as follows:

[0053] 1) Inclusion criteria

[0054] ① Patients aged ≥18 years with pathologically confirmed breast cancer; ② Patients undergoing first-time breast cancer resection without any preoperative adjuvant treatment; ③ Paracancerous tissue is normal breast tissue >2 cm away from the cancer lesion; ④ Patient basic information and postoperative pathological examination data are complete.

[0055] 2) Exclusion criteria

[0056] ① Patients with other breast diseases other than breast cancer were excluded; ② Patients with a history of other malignant tumors were excluded; ③ Patients who had received neoadjuvant therapy before surgery were excluded; ④ Patients with other infectious diseases were excluded.

[0057] This study enrolled 180 patients with breast cancer. Epidemiological data for all subjects included age, sex, case number, tumor location, tumor diameter, stage, metastasis, ER, PR, and HER-2 status. All subjects provided informed consent. This study was approved by the Ethics Committee of North China University of Technology and the Ethics Committee of Shanghai Zhuoli Biotechnology Co., Ltd. (Approval No.: 2022025, ZLL-15-01).

[0058] (2) Cell lines

[0059] The MCF-10A normal mammary epithelial cells and the three breast cancer cells, MCF-7, SKBR3, and MDA-MB-231, used in the present invention were purchased from Wuhan Punosai Life Science Co., Ltd. MCF-10A cells were cultured in a specialized culture medium provided by the company, while the other three breast cancer cells were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). All cells were cultured in a constant-temperature incubator set at 37°C with 5% CO2.

[0060] MCF-7 and MDA-MB-231 cells were transfected with siNC and siLINC00987 small interfering RNAs to establish a negative control (siNC) group and a LINC00987 low-expression (siLINC00987) group, respectively. Furthermore, LINC00987-overexpressing cells and control cells were constructed by screening lentiviral-infected MCF-7 and MDA-MB-231 cells for subsequent experiments.

[0061] (3) Main reagents and sources

[0062] Table 1 Main experimental reagents

[0063]

[0064]

[0065] (4) Main experimental instruments

[0066] Table 2 Main experimental instruments

[0067] Experimental instruments company -80℃ ultra-low temperature refrigerator Thermo Fisher Scientific, China -20℃ Refrigerator Haier, China CO2 constant temperature incubator Likang, China Constant temperature water bath Guohua Electric, China High-speed low-temperature desktop centrifuge Eppendorf, Germany IX71 inverted fluorescence microscope Olympus, Japan Constant temperature metal bath Jidi, China 7900HT Fast Real-Time PCR Instrument Applied Biosystems, USA Universal shaker / LSM900 confocal microscope Zeiss, Germany Electric incubator Tester, China

[0068] 2. Experimental methods

[0069] (1) Chromosomal location, secondary structure, and protein coding capacity prediction of LINC00987

[0070] With the help of online databases, relevant information of LINC00987 was analyzed in detail, including the sequence information, chromosomal location, secondary structure and potential protein coding ability of LINC00987.

[0071] 1) Chromosome localization and secondary structure prediction of LINC00987

[0072] The sequence information and chromosomal location information of LINC00987 were obtained through the UCSC genome browser (http: / / genome.ucsc.edu) and the Vienna RNA Web Service (http: / / rna.tbi.univie.ac.at / ), and the secondary structure and minimum free energy of LINC00987 were predicted.

[0073] 2) Prediction of protein coding capacity of LINC00987

[0074] In this study, the protein coding ability of LINC00987 was predicted using the CPAT (http: / / lilab.research.bcm.edu / ) and CPC2 (http: / / cpc2.cbi.pku.edu.cn) online databases, with the classic coding genes GAPDH and ACTB and the non-coding genes HOTAIR and XIST as controls.

[0075] (2) Differential expression of LINC00987 in breast cancer tissues

[0076] Transcriptome sequencing data for breast and normal breast tissues from the TCGA database and the GTEx database were downloaded from the UCSC XENA website (https: / / xenabrowser.net / datapages / ). The raw data were processed using the TOIL pipeline to correct for batch effects. The corresponding TCGA data for breast cancer and the corresponding normal tissue data from the GTEx project were extracted, allowing for cross-dataset merging. The differential expression of LINC00987 was analyzed using the Wilcoxon signed rank test, and the data were visualized using the ggplot2 package in the R language (version 3.6.3).

[0077] This study used breast cancer lncRNA as a keyword to identify datasets related to lncRNA expression in breast cancer tissue from the Gene Expression Omnibus (GEO) database. The GSE119233 and GSE156229 datasets, which contain microarray-based lncRNA expression profiles from breast and normal breast tissue, were downloaded. LncRNA differential analysis was performed using the R language limma package to verify the expression level of LINC00987 in breast cancer tissue.

[0078] (3) Survival analysis

[0079] Based on breast cancer RNA-seq data from TCGA and patient clinical data, patients were divided into high-expression and low-expression groups according to the median LINC00987 expression level. Kaplan-Meier (KM) survival analysis was performed using the survival and survMiner packages in R. Cox regression analysis was further used to explore the relationship between LINC00987 expression and overall survival (OS). Univariate Cox regression was used to analyze the relationship between patient age, tumor stage, ER, PR, and HER-2 expression status, and LINC00987 expression level and patient prognosis. A multivariate Cox model was used to identify potential independent prognostic factors. Statistical methods were used to estimate the hazard ratio (HR) of each indicator and determine the numerical range of its 95% confidence interval (CI).

[0080] (4) Cell culture

[0081] 1) Cell recovery

[0082] Remove the cells to be revived from the liquid nitrogen storage container and quickly thaw in a 37°C water bath. Once the cryopreservation solution has fully thawed, transfer the cell suspension to a 15mL centrifuge tube. Add 1mL of complete culture medium and gently mix. Centrifuge at 1000 rpm for 5 minutes, remove the supernatant, add 3mL of fresh complete culture medium, and mix thoroughly. Transfer the cell suspension to a culture flask and culture in a 5% CO2, 37°C incubator.

[0083] 2) Cell culture medium replacement and subculturing

[0084] Before changing the cell culture medium, remove the cells, observe their morphology, discard the culture medium, wash twice with PBS, add DMEM complete culture medium, and continue culturing in a 5% CO2, 37°C constant temperature incubator.

[0085] When the cell density reaches or exceeds 80%, perform cell passage. Follow the steps below:

[0086] ① Wash the cells twice with PBS. ② Add 1 mL of 0.25% EDTA-trypsin and digest for 1–2 minutes, depending on the cell type. Observe cell morphology under a microscope. When cells become rounded and exhibit a ground-glass appearance, add 1 mL of complete culture medium to terminate digestion. ③ Mix the cells by pipetting, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes. ④ Discard the supernatant, add 3 mL of complete culture medium, and mix thoroughly by pipetting to create a single-cell suspension. ⑤ Plate the cells in a culture plate or flask and continue culturing.

[0087] 3) Cell counting

[0088] Rinse the counting slide with 75% ethanol, dry it, cover it with a coverslip, and place it in a clean bench. Mix the trypan blue dye and cell resuspension solution in a ratio of 1:9. Pipette 10 μL of the mixed cell suspension onto the counting slide and let it stand for 1 minute before counting. Calculate the number of cells in the four large squares in the counting slide according to the formula: number of cells / mL = number of cells in the four large squares / 4×10 4 Note that if cells are located above the counting line, the upper line should be counted, not the lower line, and the left line should be counted, not the right line. Only intact cells should be counted; if cells are clustered, they should be counted as a single cell. If cells have been diluted before counting, multiply by the dilution factor.

[0089] 4) Cell cryopreservation

[0090] Following cell passaging procedures, digest and resuspend the cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add freshly prepared cell freezing solution, and gently pipette to mix. Transfer the cell suspension into cryovials. Seal the tubes with parafilm, place them in a programmed cooling box, freeze at -80°C overnight, and then transfer to a liquid nitrogen tank for long-term storage.

[0091] (5) Nuclear-cytoplasmic separation experiment

[0092] 1) Nuclear and cytoplasmic RNA extraction

[0093] ① 0.25% EDTA-trypsin digestion and collection of cells, cell count, and adjust the cell concentration to 1 × 10 7 / mL, wash the cells with pre-cooled PBS, and centrifuge at 1000rpm for 5 minutes. ② Discard the PBS, add 300μL of ice-bath pre-cooled cell separation buffer, flick the tube wall, and resuspend the cells. ③ Incubate the cell suspension on ice for 10 minutes to fully lyse the cells. The cell suspension will quickly become clear. ④ Centrifuge at 4℃, 500g for 5 minutes to separate the nucleus and cytoplasm. ⑤ Gently aspirate the cytoplasmic component in the supernatant into a new RNase-free centrifuge tube and place it on ice for later use. This step realizes the separation of nuclear and cytoplasmic RNA, extracts the cytoplasm from the nucleus, and proceeds to step ⑦. ⑥ Add 300μL of ice-bath pre-cooled cell disruption buffer to the remaining precipitated nuclei, vortex to dissolve the nuclei until the lysis solution is uniform. Note that this step needs to be performed on ice. ⑦ RNA Isolation: At room temperature, combine the lysates from steps ⑤ and ⑥ with an equal volume of 2X Lysis / Binding Buffer. Invert the tube repeatedly to ensure thorough mixing. Add 300 μL of anhydrous ethanol to the mixture and mix gently. Filter the mixture through an adsorption column and centrifuge at 12,000 g for 1 minute. Wash twice with 700 μL of Wash Buffer 1 and centrifuge at 12,000 g for 1 minute. Wash once with 500 μL of Wash Buffer 2 / 3 and centrifuge at 12,000 g for 1 minute. Discard the wash buffer and continue centrifugation for 30 seconds. Finally, elute the RNA with 40 μL of Elution Buffer preheated to 95°C and centrifuge at 12,000 g for 30 seconds to recover the RNA. Measure the concentration and purity of the extracted RNA and store at -80°C until further use.

[0094] 2) RNA reverse transcription into cDNA

[0095] According to the instructions provided by the reverse transcription kit, follow the steps below:

[0096] ① Based on the measured RNA concentration, add 2μg RNA and 1μL oligo(dT)18 primer to a sterile, RNase-free centrifuge tube placed on ice, and add RNase-free ultrapure water to make up to 12μL. ② After mixing, centrifuge briefly, incubate at 65℃ for 5 minutes, cool on ice, centrifuge briefly again, and cool. ③ Prepare reverse transcription reagents (Table 3). ④ After mixing, add the above reverse transcription reagents to a centrifuge tube so that the final reaction volume is 20μL. ⑤ After mixing, centrifuge briefly, incubate at 42℃ for 60 minutes, heat at 70℃ for 5 minutes, terminate the reaction, measure the cDNA concentration and purity, and store at -80℃ for later use.

[0097] Table 3 Reverse transcription reagent system

[0098]

[0099]

[0100] 3) Real-time quantitative PCR (RT-qPCR)

[0101] ① RT-qPCR primer design and synthesis.

[0102] The nucleic acid sequence information of human LINC00987, GAPDH, and U6 was retrieved from the NCBI database, and primer design was completed using the Primer Premier5 program. The designed primers were chemically synthesized by Beijing Nosai Biotechnology Co., Ltd. Different amounts of ddH2O were added according to the instructions to ensure that the primer concentration reached 100 μM and the primers were stored at -20°C for a long time. For RT-qPCR experiments, the primers were diluted to a final concentration of 10 μM. The detailed primer sequences are shown in the following table (Table 4):

[0103] Table 4 RT-qPCR primer information

[0104] Gene name Primer sequences serial number GAPDHPF 5'-CTGGGCTACACTGAGGACC-3' SEQ ID NO: 1 GAPDHPR 5'-AAGTGGTCGTTGAGGGCAATG-3' SEQ ID NO:2 U6PF 5'-TGCGGGTGCTCGCTTCGGCAGC-3' SEQ ID NO:3 U6PR 5'-CCAGTGCAGGGTCCGAGGT-3' SEQ ID NO:4 LINC00987PF 5'-ACGACGCACAATGCAAAGAC-3' SEQ ID NO:5 LINC00987PR 5'-TGTTTTCTGCACTGACCCCA-3' SEQ ID NO:6

[0105] ②The RT-qPCR reaction system is as follows (Table 5):

[0106] Table 5 RT-qPCR reaction system

[0107]

[0108] ③ Prepare the reaction mixture according to the above reaction system, set up 3 replicate wells for each sample, and add the reaction mixture to the 384-well plate in sequence. ④ Apply sealing film and centrifuge at 3000 rpm for 3 minutes. ⑤ Place the centrifuged 384-well plate into the real-time fluorescence quantitative PCR instrument. The reaction program is as shown in Table 6:

[0109] Table 6 RT-qPCR reaction procedure

[0110]

[0111]

[0112] ⑥ Using GAPDH as the internal reference, calculate the target gene 2 -△△Ct The relative expression levels of LINC00987 and reference genes were compared in different cells.

[0113] (6) Fluorescence in situ hybridization

[0114] 1) Place the cell slides into a 24-well culture plate and add 5×10 3 cells and culture for 24 hours. 2) Cell fixation: Add 600 μL PBS to each well, place on a shaker, and wash for 5 minutes; discard the PBS, add 600 μL 4% paraformaldehyde solution to each well, and fix at room temperature for 10 minutes. 3) Cell permeabilization: Wash three times with 1× PBS, 5 minutes each time; add 1 mL of pre-cooled permeabilization solution to each well and permeabilize at 4°C for 5 minutes; discard the permeabilization solution, then wash three times with 1× PBS, 5 minutes each time. 4) Probe prehybridization: Preheat the prehybridization solution in a 37°C water bath; add 200 μL of prehybridization solution to each well and prehybridize at 37°C for 30 minutes. 5) Probe hybridization: Under light-proof conditions, add 2.5 μL of probe (20 μM) to 100 μL of hybridization solution preheated at 37°C to obtain probe hybridization solution; discard the prehybridization solution in each well, add probe hybridization solution to each well, pay attention to the light-proof operation, and hybridize overnight in a 37°C incubator. 6) Post-hybridization treatment: Before hybridization, preheat Hybridization Wash Solutions I, II, and III to 42°C. Wash cells on a shaker in the following order to reduce background signal, protecting the slide from light: ① Wash the slide three times with Hybridization Wash Solution I for 5 minutes each; ② Wash the slide once with Hybridization Wash Solution II for 5 minutes; ③ Wash the slide once with Hybridization Wash Solution III for 5 minutes; ④ Wash the slide once with 1× PBS for 5 minutes. 7) Nuclear staining: Add 100 μL of 1× DAPI staining solution to the slide and stain for 10 minutes in the dark. Wash the slide three times with 1× PBS for 5 minutes each. 8) Mounting and photography: Remove the slide and place it on a glass slide. Apply anti-fluorescence quencher, cover the slide with a coverslip, and photograph using a laser confocal microscope.

[0115] (7) Tissue fluorescence in situ hybridization

[0116] 1) Baking: Bake the tissue microarray in an oven at 60°C for 1 hour. 2) Dewaxing: First, dewax the tissue microarray by soaking it in xylene for 10 minutes. Then, soak it in another xylene bath for 10 minutes. Then, soak it in 100% ethanol, remove the xylene, and soak it for 10 minutes. Then, soak it in 100% ethanol for 10 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 70% ethanol for 5 minutes. Finally, rinse it with tap water for 5 minutes. 3) Inactivation of endogenous enzymes: Add 3% H2O2 to the tissue microarray and incubate it at room temperature for 10 minutes to inactivate endogenous peroxidase. Then, wash it with ultrapure water for 5 minutes. 4) Expose RNA fragments: Add pepsin freshly diluted in 3% citric acid to the tissue microarray and digest it in a 37°C incubator for 15 minutes. Wash it three times with 1× PBS for 5 minutes each, and once with ultrapure water for 5 minutes. 5) Post-fixation: Fix with post-fixative solution (4% paraformaldehyde containing 1 / 1000 DEPC) at room temperature for 5 minutes, then wash three times with ultrapure water for 5 minutes each. 6) Probe pre-hybridization: Before pre-hybridization, prepare a hybridization box and add 20% glycerol to the bottom of the box for moisturizing. Pre-hybridization solution should be preheated in a 37°C water bath in advance; add 200μL of pre-hybridization solution to each tissue chip and pre-hybridize at 37°C for 30 minutes. 7) Probe hybridization: In the dark, add 2.5μL of probe (20μM) to 100μL of pre-heated hybridization solution at 37°C and mix thoroughly by pipetting. This is the probe hybridization solution. Discard the pre-hybridization solution, add the probe hybridization solution, place it in the hybridization box, and hybridize overnight at 37°C, keeping it away from light and moisturizing. 8) Post-Hybridization Treatment: Preheat Hybridization Wash Solutions I, II, and III at 42°C in advance. Wash the tissue microarray three times with Hybridization Wash Solution I for 5 minutes each, once with Hybridization Wash Solution II for 5 minutes, once with Hybridization Wash Solution III for 5 minutes, and once with 1× PBS for 5 minutes, all in the dark. 9) Nuclear Staining: Add 100 μL of 1× DAPI staining solution for 10 minutes. Wash three times with 1× PBS for 5 minutes each, protecting the microarray from light. 10) Mounting and Photography: Apply anti-fading agent to the tissue microarray and mount with a coverslip. Photograph using a laser confocal microscope.

[0117] (8) siRNA transfection

[0118] 1) siRNA was synthesized by Shanghai Gene Biotechnology Co., Ltd. The target sequences are as follows (Table 7):

[0119] Table 7 si-NC and si-LINC00987 target sequences

[0120]

[0121] 2) Cell plating: In the LINC00987 knockdown experiment, siNC was used as the control group and siLINC00987 was used as the knockdown group. MCF-7 and MDA-MB-231 cells with a confluency of 80% were digested and resuspended according to the cell passaging procedure. After counting, 2×10 cells were plated per well. 5 Seed cells into a 6-well plate at a density of 100 μg / cell and incubate the plate in a 37°C, 5% CO2 incubator overnight. 3) Prepare the lipofectamine 2000 mixture: In a sterile, RNase-free 1.5mL centrifuge tube, add 150 μL of DMEM medium to 3 μL of lipofectamine 2000 transfection reagent per well to prepare the lipofectamine 2000 transfection mixture. Mix thoroughly and let stand at room temperature for 5 minutes. 4) Dissolve the siRNA powder / primers: Remove the siRNA powder from the -20°C freezer and centrifuge at 2000 rpm for 3 minutes, ensuring that the siRNA powder settles at the bottom of the tube. Dissolve the siRNA powder according to the instructions on the tube label, adding the appropriate amount of DEPC water to a final concentration of 20 μM / L. 5) Prepare the siRNA mixture: first add 150 μL of DMEM medium to a sterile RNase-free centrifuge tube, then add 5 μL of control (siNC) or experimental (siLINC00987) siRNA to each centrifuge tube to make the final transfection concentration 50 nM, and gently pipette to mix. 6) Take 150 μL of the lipofectamine 2000 mixture prepared in step (3) and add it to each group of siRNA mixtures, mix gently, and let it stand for 20 minutes. 7) During the waiting period, remove the 6-well plate laid the day before, discard the culture medium, wash the cells twice with 1×PBS, add 700 μL of DMEM high-glucose culture medium, and add the working solution after the standing period to the 6-well plate. 8) Incubate in a 37°C, 5% CO2 incubator for 5 hours, wash the cells twice with 1×PBS, and then replace with DMEM culture medium containing 10% FBS and no PS. 9) Continue culturing for 24 hours, collect cells, extract RNA, confirm the LINC00987 knockdown efficiency, and perform subsequent experimental tests according to the transfection conditions.

[0122] (9) Construction of LINC00987 overexpression plasmid and lentiviral packaging

[0123] 1) Construction of LINC00987 overexpression plasmid

[0124] The full-length LINC00987 cDNA (NR_137429.2) sequence was downloaded from the NCBI website. The pcDNA3.1(+)-LINC00987 overexpression plasmid and the control pcDNA3.1(+) plasmid were synthesized by Shanghai Sangon Biotechnology Co., Ltd. Double digestion with restriction endonucleases Xho I and Kpn I was performed and verified by agarose gel electrophoresis. Simultaneously, next-generation sequencing was performed on the digestion products, and alignment confirmed that the LINC00987 target fragment was identical to the synthesized overexpression plasmid fragment.

[0125] 2) LINC00987 overexpression lentiviral packaging

[0126] The lentiviral packaging was completed by Beijing Xibei Hongcheng Biotechnology Co., Ltd. The brief process was as follows: DNA primers hLINC00987-XhoI-F: 5'-CCG CTC GAG TCT AGAAGC TGG AAA AGG CAA GGAAAGA-3' (SEQ ID NO: 11) and hLINC00987-BamHI-R: 5'-CGC GGA TCCATG GCTTTATTATATTGCTTT ATG TATAT-3' (SEQ ID NO: 12) were designed and synthesized. PCR amplification was performed using the plasmid LINC00987-pcDNA3.1(+) as a template. The pLVX-P2A-Luc-T2A-Puro plasmid was double-digested with XhoI and BamHI, and the target fragment was recovered. The recovered large plasmid fragment was then ligated with the target gene fragment. After transformation and culture of the ligation product, a single colony was picked, and the plasmid was extracted and double-enzyme digestion was performed. Positive clones that showed correct enzyme digestion were verified by next-generation sequencing. The verified pLVX-hLINC00987-Luc-Puro plasmid was introduced into 293T cells and packaged according to relevant methods to produce high-titer lentivirus rLV-hLINC00987-Luc-Puro containing the target gene.

[0127] (10) Lentivirus infection and stable cell line screening

[0128] 1) Cell plating: Breast cancer cells (MCF-7 and MDA-MB-231) in the logarithmic growth phase were grown to 80%-90% cell density and passaged 2-3 times. 2) When the cells reached the fourth passage, digested and resuspended the cells to prepare a single cell suspension, counted the cells, and plated at 2×10 cells per well. 5Cells were seeded into 6-well plates at a density of 100 cells / well and cultured overnight in a 37°C, 5% CO2 incubator. 3) At an MOI of 50, the packaged lentivirus was added to the 6-well plate, along with the transfer reagent polybrene. Serum-free DMEM high-glucose medium was used and the cells were cultured in a 37°C, 5% CO2 incubator. 4) 24 hours after viral infection, the culture medium was switched to DMEM complete medium containing 10% FBS and continued. 5) 72 hours after lentiviral infection, the fluorescence intensity and proportion of fluorescent cells were observed under a fluorescence microscope. Puromycin was added to a final concentration of 4 μg / mL for resistance selection, and the medium was changed every 2-3 days (complete medium containing 4 μg / mL puromycin). 6) After one week of continuous puromycin selection, the proportion of fluorescent cells was observed. When the fluorescent cells grew to 90% cell density, the cells were harvested and the LINC00987 overexpression efficiency was tested.

[0129] (11) Total cell RNA extraction, reverse transcription and RT-qPCR

[0130] 1) Total RNA extraction from cells (Trizol method)

[0131] ① Lyse the cell sample: Discard the culture medium, wash the cells twice with 1× PBS, add 1 mL of Trizol to completely cover the cell surface, pipette repeatedly to completely lyse, and transfer the supernatant to a new RNase-free centrifuge tube. ② In a fume hood, let the tube stand at room temperature for 5 minutes to fully lyse the cells. ③ Add 200 μL of chloroform, shake thoroughly for 15 seconds, and let it stand at room temperature for 5 minutes. ④ Centrifuge at 13,000 g at 4°C for 15 minutes. ⑤ Carefully aspirate the supernatant into a new RNase-free centrifuge tube, add 500 μL of isopropanol, mix thoroughly, and let it stand at -20°C for 15 minutes. ⑥ Centrifuge at 12,000 g at 4°C for 12 minutes, and discard the supernatant. ⑦ Add 1 mL of 75% ethanol (prepared with DEPC water, freshly prepared) to the centrifuge tube containing the pellet, wash the pellet, and centrifuge at 7,500 g at 4°C for 5 minutes. ⑧ Repeat step ⑦. ⑨ Air dry at room temperature (5-10 minutes) to evaporate the ethanol while preventing the RNA from drying out completely. ⑩ Add 20 μL of DEPC water to each centrifuge tube and place in a 55°C metal bath for 10 minutes to completely dissolve the RNA. Measure the RNA concentration and purity, and store the sample in a -80°C freezer.

[0132] 2) RNA reverse transcription into cDNA

[0133] The experimental method is the same as above.

[0134] 3) RT-qPCR

[0135] The experimental method is the same as above.

[0136] (12) Cell proliferation ability detection

[0137] 1) CCK-8 assay

[0138] ① Take breast cancer cells from each group in the logarithmic growth phase, digest them with 0.25% EDTA-trypsin, centrifuge them, resuspend them in complete medium, and mix them evenly to form a single-cell suspension according to the cell passaging procedure. ② Count the cells, adjust the cell concentration, and inoculate them into a 96-well plate at a ratio of 5000 cells per 100 μL. ③ CCK-8 working solution was prepared by mixing CCK-8 and DMEM culture medium in a ratio of 1:9. At 0, 24, 48, and 72 hours, the original culture medium in the 96-well plate was discarded, and 100 μL of CCK-8 working solution was added to each well. Incubate at 37°C in the dark for 2 hours. ④ After incubation, the absorbance (Optical Density, OD) value was measured at 450 nm using a multifunctional microplate reader. ⑤ Record the OD values ​​at different time points and draw a cell proliferation line graph.

[0139] 2) Clone formation assay

[0140] ① Breast cancer cells from each group in the logarithmic growth phase were trypsinized, resuspended by centrifugation, counted, and diluted to a density of 200 cells / mL. ② 5 mL of the diluted cell suspension was inoculated into a 60 mm culture dish and cultured in a 37°C, 5% CO2 incubator. ③ Observe the cells daily and change the medium in the culture dish every 5 days. ④ When most of the individual cells have formed distinct colonies (the number of cells in a single clone exceeds 50), discard the culture medium, wash the cells twice with 1× PBS, and add 1 mL of 4% paraformaldehyde solution to each culture dish and fix for 30 minutes. ⑤ Discard the 4% paraformaldehyde solution, then wash the cells twice with 1× PBS. Add 1 mL of 0.1% crystal violet to each culture dish and stain for 15 minutes. ⑥ Discard the crystal violet, rinse the remaining stain with 1× PBS, and then dry the cells and photograph them. The number of colonies in each group was counted using Image J software, and statistical analysis was performed.

[0141] (13) Cell cycle detection

[0142] 1) Take cells from each group in the logarithmic growth phase and collect the cell supernatant into a centrifuge tube for later use. Wash the cells twice with 1×PBS. 2) Digest the cells with 0.25% EDTA-trypsin. When the cell morphology becomes round and ground glass-like under the microscope, stop the digestion with complete medium. Use a pipette to gently pipette to mix, transfer the cell suspension to the above centrifuge tube, and centrifuge at 800rpm for 5 minutes. Be careful to be gentle in pipetting to avoid mechanical damage to the cells. 3) Discard the supernatant, add 1mL of pre-cooled 1×PBS to wash the cells, and centrifuge at 800rpm for 5 minutes. 4) Repeat the previous step. 5) Discard the supernatant, add 1mL of ice-bath pre-cooled 70% ethanol, and gently pipette to mix. Fix at 4℃ overnight (about 12 hours). 6) Centrifuge at 800rpm for 5 minutes to precipitate the cells. 7) Add 1mL of pre-cooled 1×PBS and resuspend the cells. 8) After the cells are centrifuged a second time, slowly remove the upper layer of liquid, then tap the bottom of the tube to evenly distribute the cells to prevent them from aggregating. 9) Prepare propidium iodide (PI) staining solution (Table 8) according to the following table: 10) Add 0.5 mL of PI staining solution to each tube of sample, slowly and thoroughly resuspend the cell pellet, and incubate at 37°C in the dark for 30 minutes. Then, place the cell suspension on ice, protecting from light. 11) Detect red fluorescence signal by flow cytometry at an excitation wavelength of 488 nm, while simultaneously recording light scattering data.

[0143] Table 8 Propidium iodide staining solution preparation system

[0144] Reagent name 1 sample Staining buffer 0.5mL PI staining solution (20×) 25 μL RNase A (50×) 10 μL Total volume 0.535mL

[0145] (14) Cell migration and invasion assay

[0146] In the experimental setup, the chamber without Matrigel was used to detect the migration ability of breast cancer cells, and the chamber with Matrigel was used to detect the invasion ability. Through these two different experimental settings, we can have a more comprehensive understanding of the behavioral characteristics of breast cancer cells in different environments.

[0147] 1) Transwell cell migration assay

[0148] ① Breast cancer cells in each group in the logarithmic growth phase were digested with 0.25% EDTA-trypsin, centrifuged, and the supernatant discarded. The cells were then resuspended in serum-free DMEM medium. ② Cell counts were performed and the cell density was adjusted to 5×10 5 / mL. ③ Add 600 μL of DMEM high-glucose medium containing 20% ​​FBS to the bottom chamber of the 24-well plate. Slowly lower the transwell chamber into the 24-well plate, being gentle and avoiding bubbles. ④ Slowly add 200 μL of the diluted cell suspension to the upper chamber of the transwell chamber, avoiding bubbles. Place the 24-well plate in a 37°C, 5% CO2 incubator for continued incubation. ⑤ After 24 hours of incubation, remove the chamber and gently wipe any remaining cells from the upper chamber with a PBS-moistened cotton swab. Wash twice with 1× PBS. ⑥ Add 600 μL of 4% paraformaldehyde to the 24-well plate, place the chamber in it, and fix at room temperature for 30 minutes. Wash twice with 1× PBS. ⑦ Add 600 μL of 0.1% crystal violet stain to the 24-well plate and stain for 15 minutes. Rinse with 1× PBS to remove any excess stain. ⑧ Observe cell morphology and photograph under an inverted light microscope. The number of migrated cells in each group was counted using Image J software and statistical analysis was performed.

[0149] 2) Transwell cell invasion assay

[0150] ① Remove the Matrigel from the -20°C freezer one day in advance and slowly thaw at 4°C. ② After pre-chilling the pipette tip and centrifuge tube on ice, mix the Matrigel with serum-free DMEM medium at a ratio of 1:8 on ice. Add 100 μL / well dropwise to the upper chamber of the transwell. Incubate in a 37°C, 5% CO2 incubator for 5 hours. Slowly aspirate any remaining liquid in the chamber. ③ The remaining experimental steps are consistent with those for the cell migration assay. Perform subsequent experimental procedures according to the experimental procedures for the cell migration assay.

[0151] (15) Construction of nude mouse subcutaneous transplant tumor model

[0152] 1) MDA-MB-231 cells in the control (vector) and LINC00987 overexpression (LINC00987) groups in the logarithmic growth phase were grown to 80%-90% confluence and then digested with 0.25% EDTA-trypsin. The cells were then centrifuged and washed twice with 1× PBS to completely remove residual serum components. 2) The cells were resuspended in pre-chilled 1× PBS, counted, and the cell concentration was adjusted to a final concentration of 3×10 73) Twelve 4-week-old SPF-grade female BALB / c-nu nude mice were randomly divided into a control (vector) group and a LINC00987 overexpression (LINC00987) group, with 6 BALB / c-nu nude mice in each group. Each nude mouse was ear-tagged using ear tag forceps for subsequent recording, and each nude mouse was weighed. 4) Gently pinch the skin on the back of the nude mouse's neck with the thumb and index finger of the left hand, while the ring finger and pinky finger firmly grasp the nude mouse's tail to securely fix the nude mouse in the palm of the hand. 5) Disinfect the skin of the nude mouse's right upper arm axilla with a 75% ethanol cotton ball. Use a 1mL syringe to aspirate 100μL of the cell suspension and slowly inoculate the cell suspension subcutaneously into the nude mouse's axilla in a "Z" pattern. 6) Feed and water the mice normally, observe their mental state, food intake, activity level, etc. daily, measure their weight every 2 days, and use a vernier caliper to measure the long and short diameters of the tumor. According to the formula, volume = long diameter × short diameter 2 Calculate the tumor volume using the value of 0.52 × 0.52 and keep a record. 7) When the long diameter of the nude mouse tumor reaches approximately 15 mm, sacrifice the nude mouse, dissect and remove the subcutaneous tumor tissue, weigh it, and photograph it. A portion of the tissue should be frozen at -80°C until further use. The remaining tumor tissue should be fixed with 4% paraformaldehyde for subsequent experiments.

[0153] (16) Pathological examination

[0154] 1) Fixation and embedding of tumor tissue

[0155] ① Fix the tumor tissue with 4% paraformaldehyde. After 24 hours of fixation, rinse the tissue with ultrapure water. Be gentle and meticulous during the procedure to avoid damaging the tumor tissue. ② Dehydrate the tissue using a gradient of 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol I, 95% ethanol II, and 100% ethanol for 1 hour each. ③ Transparent the tissue in xylene for 1 hour. ④ Embed the tumor tissue in melted paraffin. ⑤ After the paraffin has completely solidified, section the embedded tissue into 4 μM slices. Spread and remove the slices in a 40°C water bath, then bake them dry.

[0156] 2) Hematoxylin-eosin (HE) staining

[0157] ① Place the tissue sections in a 60°C oven for 30 minutes. Dewax by soaking in xylene I for 20 minutes and then in xylene II for another 20 minutes. ② Soak the slides to be dewaxed in 100%, 100%, 95%, 85%, and 75% ethanol, sequentially for 5 minutes each, then rinse in running water for 5 minutes. ③ Stain with hematoxylin solution for 5 minutes and rinse with water for 10 minutes. ④ Soak in 75% ethanol for 2 minutes and rinse with water for 30 seconds. ⑤ Bluing with 0.6% ammonia solution and rinse with water for 30 seconds. ⑥ Dehydrate by soaking in 85% and then 95% ethanol for 5 minutes each. ⑦ Stain with eosin solution for 5 minutes and rinse with water for 30 seconds. ⑧ Dehydrate in 100% anhydrous ethanol three times and clear in xylene twice for 5 minutes each. ⑨ Mount the slides with neutral gum.

[0158] 3) Immunohistochemistry (IHC) staining

[0159] ① Dewax the paraffin sections by baking them in a 60°C oven for 30 minutes, then soaking them in xylene I for 20 minutes and then in xylene II for another 20 minutes. ② Soak the slides to be dewaxed in 100%, 100%, 95%, 85%, and 75% ethanol for 5 minutes each, followed by a 5-minute rinse in water. ③ Add 3% H₂O₂ to the tissue sections and incubate for 10 minutes. Wash twice with 1× PBS for 5 minutes each. ④ Heat the sections in 0.01M citrate buffer in a microwave for 8 minutes. After cooling to room temperature, wash twice with 1× PBS for 5 minutes each. ⑤ Block with 10% goat serum at room temperature for 60 minutes. Remove the blocking solution and add diluted primary antibody Ki67 (1:200 dilution) and incubate in a humidified chamber at 4°C overnight. ⑥ After removing the primary antibody, wash twice with 1× PBS for 10 minutes each. ⑦ Add secondary antibody and incubate at room temperature for 30 minutes. Wash twice with 1× PBS for 10 minutes each. ⑧ Incubate with DAB and observe the extent of staining under a light microscope, stopping color development promptly. ⑨ Counterstain with hematoxylin for 5 minutes and differentiate in 1% hydrochloric acid alcohol for a few seconds. ⑩ Soak tissue sections in 50%, 75%, 85%, 95%, and 100% ethanol for 5 minutes, respectively. The slides were permeabilized with xylene twice and sealed with neutral gum. The cells were observed and photographed under an optical microscope. Image J software was used to analyze the IHC average optical density (IOD) and positive area (Area), and the AOD was calculated using the formula: average optical density (AOD) = IOD / Area.

[0160] 3. Statistical processing

[0161] SPSS 23.0 and GraphPad Prism 8 software were used for statistical analysis and graphics. Continuous data were expressed as mean ± standard deviation. Differences between two independent samples were analyzed using the t-test. Differences between multiple groups were analyzed using one-way analysis of variance. Nonparametric differences were analyzed using the chi-square test or Fisher's exact test. A P value < 0.05 was considered statistically significant.

[0162] The solution proposed by the present invention is described in detail below through specific embodiments:

[0163] Example 1 Basic Information of LINC00987

[0164] 1. Localization of LINC00987 in chromosomes

[0165] The sequence and chromosome location information of LINC00987 were obtained from the UCSC website. The results showed that LINC00987, as a long intergenic non-coding RNA, is located in subband 31 of the p13 region of the short arm of human chromosome 12, with a length of 22,828 bp. For specific location information, see Figure 1 shown.

[0166] 2. Prediction of protein coding capacity of LINC00987

[0167] This study used databases with two different algorithms, CPAT and CPC2, to predict the protein coding ability of LINC00987. The lower the score, the lower the possibility of protein coding. The classic coding genes GAPDH and ACTB were used as positive controls for coding genes, and the non-coding genes HOTAIR and XIST were used as positive controls for non-coding genes. Figure 2 The protein coding capacity values ​​of LINC00987 predicted by the CPAT and CPC2 databases were 0.0392 and 0.1619, respectively, which are close to the protein coding capacity values ​​of the known classical non-coding genes HOTAIR and XIST, and much lower than the protein coding capacity values ​​predicted by the classical protein-coding genes GAPDH and ACTB.

[0168] 3. LINC00987 secondary structure prediction

[0169] The complex secondary structure of lncRNA is crucial for it to perform various biological functions. By forming specific secondary structures, such as stem-loop structures and pseudoknots, it can achieve a variety of biological functions. Therefore, studying the secondary structure of LINC00987 is of great significance for in-depth exploration and explanation of its function in the occurrence and development of breast cancer. This study used the Vienna RNA network service to predict the secondary structure of LNC00987. The results showed that LINC00987 presented complex and unique secondary structural characteristics, including motifs such as multiple stem-loop structures and hairpin structures. These structural units are intertwined with each other to form a stable spatial conformation. It was calculated that the minimum free energy of the lncRNA secondary structure is -885.40 kcal / mol, indicating that its structure has high thermodynamic stability. This stability may play an important supporting role in the biological functions of LINC00987, such as interactions with other molecules and localization in cells. ( Figure 3 ).

[0170] 4. LINC00987 subcellular localization

[0171] LncRNA subcellular localization plays a crucial role in its function. Therefore, this study selected GAPDH, which is mainly expressed in the cytoplasm, and small nuclear RNA U6, which is mainly expressed in the nucleus, as internal controls. The nuclear-cytoplasmic fractionation experiment was used to detect the distribution of LINC00987 in cells. The results showed that in MCF-7 and MDA-MB-231 cells, LINC00987 was mainly expressed in the nucleus, with a low expression level in the cytoplasm ( Figure 4 )

[0172] In addition, the present invention is verified by fluorescence in situ hybridization experiments. The LINC000987 nucleic acid probe is labeled with Cy3 fluorescent dye, and the lncRNA sequence is detected and located by detecting the fluorescent signal (Cy3, red fluorescence). The 18S fluorescent probe and the U6 fluorescent probe are used as references for cytoplasmic and nuclear localization, respectively. The FISH results showed that in MCF-7 cells, the fluorescent signal of the LINC00987 probe was mainly concentrated in the nuclear region, suggesting that LINC00987 is mainly located in the nucleus, which is consistent with the conclusion drawn from the nuclear-cytoplasmic separation experiment, and further confirmed that LINC00987 is mainly distributed in the nucleus, and the distribution in the cytoplasm is relatively small ( Figure 5 ).

[0173] Example 2 Expression of LINC00987 in breast cancer tissue samples

[0174] To explore the expression level changes of LINC00987 in breast cancer tissues, the present invention first used the TCGA and GTEx databases to analyze the expression differences of LINC00987 in breast cancer tissues and normal breast tissues. In the unpaired sample difference analysis of breast cancer, a total of 1099 breast cancer tissues and 292 normal breast tissues were included. The Wilcoxon rank sum test analysis found that the expression level of LINC00987 in breast cancer tissues was significantly lower than that in normal breast tissues, and the difference was statistically significant ( Figure 6 A, P<0.001). In the breast cancer paired sample difference analysis, a total of 113 pairs of breast cancer and adjacent normal tissues were included. The results showed that the expression level of LINC00987 in breast cancer tissues was significantly lower than that in adjacent normal tissues, and the difference was statistically significant ( Figure 6 B, P < 0.001).

[0175] Furthermore, the present invention used FISH technology to detect the expression level of LINC00987 in 180 pairs of breast cancer tissues and adjacent normal tissues, and found that the expression level of LINC00987 in adjacent normal tissues was significantly higher than that in breast cancer tissues, and the positive staining was mainly concentrated in the cell nucleus ( Figure 7 ).

[0176] Example 3 Relationship between LINC00987 expression level and clinicopathological characteristics of breast cancer patients

[0177] Using the median of LINC00987 relative expression as the cutoff value, breast cancer cases were divided into a low LINC00987 expression group and a high LINC00987 expression group. The chi-square test was used to analyze the differences in various clinical pathological parameters between the two groups of patients, and the results are shown in Table 9. The tumor size was divided into two groups based on 4 cm. In the ≤4 cm group, there were 37 patients (41.11%) with low LINC00987 expression and 55 patients (61.11%) with high LINC00987 expression; in the >4 cm group, there were 53 patients (58.89%) with low LINC00987 expression and 35 patients (38.89%) with high LINC00987 expression. After statistical analysis, the difference was statistically significant (χ 2 =7.204, P=0.007). This result indicates that tumor size is correlated with LINC00987 expression levels, and patients with larger tumors have a relatively higher proportion of low LINC00987 expression. In addition, patients were divided into two groups according to whether the tumor had metastasis. In the non-metastasis group, LINC00987 was low expressed in 38 cases (42.22%) and high expressed in 69 cases (76.67%); in the metastasis group, LINC00987 was low expressed in 52 cases (57.78%) and high expressed in 21 cases (23.33%). The difference was statistically significant by chi-square test (χ 2=22.146, P<0.001). This indicates that the proportion of patients with low LINC00987 expression is higher in patients with metastasis. According to tumor stage, patients were divided into stage I, stage II, and stage III+IV. In stage I patients, there were 6 cases of low LINC00987 expression (6.67%) and 46 cases of high LINC00987 expression (51.11%); in stage II patients, there were 54 cases of low LINC00987 expression (60.00%) and 42 cases of high LINC00987 expression (46.67%); in stage III+IV patients, there were 30 cases of low LINC00987 expression (33.33%) and 2 cases of high LINC00987 expression (accounting for 2.22%). Statistical analysis results showed that χ 2 =7.204, P < 0.001, indicating a statistically significant difference. This suggests that tumor stage is significantly correlated with LINC00987 expression, with the proportion of patients with low LINC00987 expression increasing with advanced tumor stage. However, pathological factors such as patient age, ER, PR, and HER-2 status were not significantly correlated with LINC00987 expression levels, with no statistically significant differences (P > 0.05).

[0178] Table 9 Relationship between LINC00987 expression level and clinicopathological characteristics of breast cancer patients

[0179]

[0180] Example 4 Analysis of LINC00987 expression level and prognosis of breast cancer patients

[0181] The above studies have shown that LINC00987 is lowly expressed in breast cancer and is significantly associated with breast cancer progression. Therefore, the present invention further studies the effect of this lncRNA on breast cancer prognosis. Breast cancer patients were divided into a low-expression group and a high-expression group based on the median LINC00987 expression. The patients' overall survival (OS), disease-free survival (DFS), and progression-free survival (PFI) were analyzed to explore the association between LINC00987 expression levels and the prognosis of breast cancer patients. In the KM survival curve, the blue curve represents the low-expression group and the orange curve represents the high-expression group. Over time (in months), the survival probability of both the LINC00987 high expression group and the low expression group showed a downward trend. The results of survival analysis showed that the hazard ratio (HR) for OS was 0.68, and the 95% confidence interval (95% CI) was 0.49-0.94, indicating that low LINC00987 expression was associated with a worse prognosis, and the survival difference between the two groups was statistically significant ( Figure 8A, P = 0.019). However, no significant correlation was found between the expression level of LINC00987 and DFS and PFI ( Figure 8 B, C, P>0.05).

[0182] Variables potentially influencing survival, such as age, tumor stage, ER, PR, and HER-2 status, and LINC00987 expression, were included in a univariate Cox model analysis (Table 10). Breast cancer patients were divided into two groups based on age (≤60 years) for univariate Cox regression analysis. The HR for the older age group (>60 years) was 2.024, with a 95% CI of 1.47-2.79 (P < 0.001), using the younger age group (≤60 years) as the reference group. This suggests that older age groups face a higher risk of death compared to younger age groups. Univariate Cox regression analysis of tumor stage revealed that patients with stage III & IV breast cancer faced a higher risk of death compared to those with stage I & II (HR = 2.37, 95% CI = 1.69-3.32, P < 0.001). Based on the median LINC00987 expression level, breast cancer patients were divided into a low-expression group and a high-expression group. Using the low-expression group as the baseline, univariate COX regression analysis showed that the hazard ratio for the high-expression group was 0.68 (95% CI = 0.49-0.939, P = 0.019), suggesting that LINC00987 expression may be an important factor affecting the risk of breast cancer and that high LINC00987 expression is a protective factor for breast cancer prognosis. Furthermore, univariate COX regression analysis showed no statistically significant differences in risk among ER, PR, and HER-2 statuses (P > 0.05).

[0183] Table 10 Univariate Cox analysis of breast cancer patients

[0184]

[0185] Based on the univariate COX regression analysis, the present invention further incorporated statistically significant variables such as age, tumor stage, and LINC00987 expression level of breast cancer patients into the multivariate Cox model for analysis (Table 11). Taking the younger age group (≤60 years old) as the reference, the HR for the older age group (>60 years old) was 2.77 (95% CI = 1.66-4.62, P < 0.001). This suggests that after considering other factors, breast cancer patients in the older age group face a higher risk of death than those in the younger age group, and increasing age is an independent risk factor for breast cancer mortality. In the multivariate COX regression analysis of tumor stage, patients in stages III and IV faced a higher risk of death than those in stages I and II (HR = 2.15, 95% CI = 1.02-4.50, P < 0.001), indicating that advanced disease is an independent risk factor for breast cancer mortality. Using the LINC00987 low expression group as the control group, multivariate COX regression analysis showed that the HR of the high expression group was 0.56 (95% CI = 0.34-0.94, P = 0.028), indicating that the expression level of LINC00987 may be an independent factor affecting the prognosis of breast cancer, and its high expression may be a key factor in reducing the risk of death in breast cancer patients.

[0186] Table 11 Multivariate Cox analysis of breast cancer patients

[0187]

[0188] Example 5 LINC00987 interference and overexpression efficiency

[0189] To further investigate the role of LINC00987 in breast cancer cells, the present invention transfected specific siRNA targeting LINC00987 into MCF-7 and MDA-MB-231 breast cancer cells, respectively, and set up cells transfected with siNC as a control group. RT-qPCR experiments were used to detect the relative expression of LINC00987. Compared with the control group, the expression level of LINC00987 in the LINC00987 knockdown group was significantly reduced, with a knockdown efficiency of 65% (MCF-7: t = 7.11, P = 0.002; MDA-MB-231: t = 10.24, P < 0.001; Figure 9 The above results showed that transfection of siLINC00987 in MCF-7 and MDA-MB-231 cells could significantly inhibit the expression of this lncRNA.

[0190] Subsequently, LINC00987-overexpressing lentiviral plasmids were used to infect MCF-7 and MDA-MB-231 cells, and puromycin selection was performed to obtain breast cancer cells stably overexpressing LINC00987. RT-qPCR was used to detect LINC00987 expression. Compared with the control (Vector) group, the expression level of LINC00987 in MCF-7 and MDA-MB-231 cells in the LINC00987-overexpressing group (LINC00987) was significantly upregulated, and the difference was statistically significant (MCF-7: t = 44.76, P < 0.001; MDA-MB-231: t = 6.82, P = 0.002; Figure 10 The above results showed that infection of MCF-7 and MDA-MB-231 cells with LINC00987 overexpression plasmid significantly increased the expression of this lncRNA.

[0191] Example 6 Effect of LINC00987 on the proliferation ability of breast cancer cells in vitro

[0192] 1) LINC00987 knockdown increases the proliferation ability of breast cancer cells

[0193] The present invention uses CCK-8 assay to detect the activity of MCF-7 and MDA-MB-231 cells. The results show that compared with the siNC group, the cell proliferation ability of breast cancer cells in the siLINC00987 knockdown group was significantly enhanced at 24, 48 and 72 hours (MCF-7: t 0h =0.23, P 0h =0.830; t 24h =39.69, P 24h <0.001; t 48h =11.55, P 48h <0.001; t 72h =28.48, P 72h <0.001; MDA-MB-231: t 0h =2.53, P 0h =0.065; t 24h =53.61, P 24h <0.001; t 48h =20.72, P 48h <0.001; t 72h =12.47, P 72h <0.001; Figure 11A). The clone formation ability of MCF-7 and MDA-MB-231 cells after LINC00987 knockdown was tested using a clone formation assay, and the changes in cell proliferation were further examined. The experimental results were consistent with the results of the CCK-8 assay. The number of MCF-7 and MDA-MB-231 cell clones formed in the siLINC00987 group was significantly higher than that in the siNC group (MCF-7: t = 3.70, P = 0.021; MDA-MB-231: t = 3.69, P = 0.021; Figure 11 B) The results of these two experiments confirmed that low expression of LINC00987 significantly enhanced the proliferation capacity of MCF-7 and MDA-MB-231 cells.

[0194] 2) LINC00987 overexpression reduces the proliferation ability of breast cancer cells

[0195] First, the CCK-8 assay was used to detect the OD values ​​of breast cancer cells overexpressing LINC00987 at specific time points. The results showed that the proliferation ability of breast cancer cells in the control (vector) group and the LINC00987 overexpression (LINC00987) group showed a trend of gradual increase over time. However, compared with the control group, the cell proliferation ability of MCF-7 and MDA-MB-231 cells in the LINC00987 overexpression group was significantly decreased at 24, 48, and 72 hours, and the differences were statistically significant (MCF-7: t 0h =1.48, P 0h =0.213; t 24h =4.06, P 24h =0.015; t 48h =3.39, P 48h =0.027; t 72h =2.98, P 72h =0.041; MDA-MB-231:t 0h =2.36, P 0h =0.078; t 24h =12.51, P 24h <0.001; t 48h =14.73, P 48h <0.001; t 72h =28.86, P 72h <0.001; Figure 12A). Next, the in vitro clonal formation ability of breast cancer cells was tested by clonogenic assay. Compared with the control group, the number of clones formed by LINC00987-overexpressing MCF-7 and MDA-MB-231 cells was significantly reduced (MCF-7: t = 3.44, P = 0.026; MDA-MB-231: t = 8.62, P = 0.001). Figure 12 B) CCK-8 and colony formation assays corroborated each other, strongly demonstrating the inhibitory effect of LINC00987 overexpression on breast cancer cells in vitro.

[0196] 3) LINC00987 overexpression promotes breast cancer cell cycle arrest

[0197] To further explore the reasons why LINC00987 inhibits breast cancer cell proliferation, flow cytometry (PI staining) was used to detect the effect of LINC00987 overexpression on the MCF-7 cell cycle. Compared with the control group, the proportion of cells in the G1 phase decreased and the proportion of cells in the G2 phase increased in MCF-7 cells overexpressing LINC00987, and the cell cycle was arrested at the transition stage from G1 to G2. LINC00987 overexpression induced a significant cell cycle arrest, and the difference was statistically significant ( Figure 13 , P<0.05). These results suggest that LINC00987 may inhibit the proliferation of MCF-7 cells by inducing cell cycle arrest.

[0198] Example 7 Effect of LINC00987 on the migration and invasion ability of breast cancer cells in vitro

[0199] The present invention used transwell migration and invasion assays to evaluate the effects of knockdown or overexpression of LINC00987 on the metastatic ability of breast cancer cells. In the MCF-7 and MDA-MB-231 cell migration assays, compared with the siNC group, the number of cells in the siLINC00987 group that passed through the specific membrane structure of the transwell chamber to the outer bottom wall was significantly increased. Statistical analysis further confirmed that this difference was significant (MCF-7: t = 4.20, P = 0.014; MDA-MB-231: t = 6.35, P = 0.003; Figure 14 A). In the invasion assay, compared with the control group, the number of cells in the LINC00987 knockdown groups of MCF-7 and MDA-MB-231 cells that passed through the pores of the Matrigel-lined transwell chamber and reached the bottom wall of the chamber was significantly increased (MCF-7: t = 6.51, P = 0.003; MDA-MB-231: t = 8.80, P < 0.001; Figure 14B) The above results indicate that low expression of LINC00987 enhances the migration and invasion abilities of MCF-7 and MDA-MB-231 cells.

[0200] Example 8 Effect of LINC00987 on the Proliferation Ability of Breast Cancer Cells in Vivo

[0201] There is still a lack of clear evidence as to whether LINC00987 has the ability to inhibit tumor growth in complex environments in vivo. Therefore, in this part of the experiment, MDA-MB-231 cells in the control group and the LINC00987 overexpression group were injected into the subcutaneous tissue of BALB / c-nu nude mice at the same number, and a subcutaneous xenograft tumor model was successfully established in nude mice (n=6). The subcutaneous tumor formation was observed. Figure 15 As shown in A and B, the subcutaneous tumor volume of nude mice in the LINC00987 overexpression group was significantly smaller than that in the control group. In terms of nude mouse weight, by collecting and analyzing the weight data of the two groups of nude mice, it was found that the weight of nude mice in the control group and the LINC00987 overexpression group showed a certain upward trend over time. The weight data of the two groups of nude mice showed similar trends, and there was no significant difference in their weight changes ( Figure 15 C, P>0.05). However, during tumor growth, the tumor volume of the control group continued to increase over time, while the tumor volume of the LINC00987 overexpression group grew relatively slowly. When the tumor grew to day 10, the tumor volume of the LINC00987 overexpression group was significantly smaller than that of the control group, and this difference was statistically significant (P<0.05; Figure 15 D). In addition, the tumor weight of the LINC00987 overexpression group was significantly lower than that of the control group (P<0.001; Figure 15 E) These results indicate that LINC00987 also has the ability to inhibit breast cancer cell proliferation in vivo.

[0202] By HE staining, tumor cell morphology was observed, and it was found that both the control group and the LINC00987 overexpression group exhibited typical tumor cell morphological characteristics. In contrast, the cell nuclei in the control group showed more mitotic figures, while the number of mitotic figures observed in the cell nuclei of the LINC00987 overexpression group was relatively small ( Figure 16 A). IHC technology was used to detect Ki-67 expression levels and evaluate the effect of LINC00987 on breast cancer cell proliferation in vivo. The results showed that the Ki-67 expression level in nude mouse tumor tissues in the LINC00987 overexpression group was significantly reduced, indicating that the proliferation activity of tumor cells was weakened. This finding was statistically significant compared with the control group ( Figure 16B, C; P < 0.001). These results indicate that overexpression of LINC00987 can effectively inhibit the division and proliferation activity of tumor cells in vivo, thereby slowing down tumor growth.

[0203] The present invention reveals the important biological functions of LINC00987 in breast cancer, especially its changes in cellular substructure localization and expression levels. First, through nuclear cytoplasm fractionation experiments and fluorescence in situ hybridization technology verification, it was confirmed that LINC00987 is localized in the nucleus in MCF-7 and MDA-MB-231 cells. Similar studies have shown that other lncRNAs such as MALAT1, SPRY4-IT1, HOTAIR, etc. also play an important role in the nucleus. For example, MALAT1, a classic lncRNA, was first discovered in non-small cell lung cancer and was confirmed to be closely related to tumor metastasis. Subsequent studies have found that it also has important biological functions in various tumors including breast cancer

[50] . Compared with normal breast tissue, the expression of MALAT1 in breast cancer tissue is significantly increased, and its high expression is associated with poor prognosis of breast cancer patients, and is more likely to relapse and metastasis. This may be because MALAT1 promotes the progression of breast cancer by activating different signaling pathways such as PI3K / AKT / mTOR or POSTN / Hippo / YAP. LncRNA SPRY4-IT1 is also a nuclear lncRNA that is highly expressed in breast cancer. It inhibits NFKBIA transcription and IκBα expression, promoting the formation of the p50 / p65 complex and activating the NF-κB signaling pathway, thereby enhancing the proliferation and migration of breast cancer cells. Furthermore, HOTAIR is a known nuclear lncRNA. Overexpression of HOTAIR induces histone H3 lysine 27 methylation and increases cancer invasiveness and metastasis in a PRC2-dependent manner. These findings suggest that the nuclear localization of lncRNAs is closely related to their biological functions, potentially exerting tumor suppressor or promoter effects by regulating nuclear processes such as transcriptional regulation and chromatin remodeling. This localization suggests that the intracellular function of LINC00987 may primarily participate in the development and progression of breast cancer by directly regulating nuclear molecular mechanisms.

[0204] Secondly, LINC00987 expression levels in breast cancer tissue are significantly lower than in normal breast tissue, and its low expression is closely associated with breast cancer progression. Similar to breast cancer studies, LINC00987 is abnormally expressed in a variety of malignant tumors, including lung cancer, acute myeloid leukemia, and osteosarcoma, and is closely associated with the proliferation, migration, and invasion of these malignancies.

[0205] In addition, low expression of LINC00987 is closely associated with the prognosis of breast cancer patients and is considered an independent prognostic risk factor for breast cancer. This result is consistent with research results in other cancer types. In lung adenocarcinoma and glioblastoma, the expression of LINC00987 in cancer tissue is lower than that in adjacent normal tissue, and its low expression level is closely associated with the patient's poor prognosis. In patients with acute myeloid leukemia and osteosarcoma, higher expression levels of LINC00987 were observed, and this high expression was associated with poor OS in patients with acute myeloid leukemia and osteosarcoma. The importance of LINC00987 in cancer prognostic assessment suggests that it may play a key role in the progression of breast cancer.

[0206] Abnormally expressed lncRNAs may affect the biological functions of tumor cells by regulating complex cell signaling pathways. For example, the highly expressed lncRNA HAGLROS promotes the proliferation and migration of breast cancer cells by inducing TAM polarization to M2, leading to poor prognosis in breast cancer patients

[60] . On the other hand, the low-expressed lncRNA BC069792 negatively regulates the expression of cell cycle proteins and exhibits significant inhibitory effects on breast cancer cells in both in vitro and in vivo experiments. Currently, it has been confirmed that LINC00987 exhibits unique expression patterns in several malignant tumors, thereby playing different roles in the biological functions of different malignant tumors. Highly expressed LINC00987 promotes the proliferation and invasion of acute myeloid leukemia cells, thereby accelerating the progression of the tumor; in osteosarcoma, silencing of LINC00987 regulates FNBP1 expression by adsorbing miR-376a-5p, inhibiting the proliferation, migration and invasion of osteosarcoma cells; in lung cancer, LINC00987 inhibits the proliferation, migration and invasion of lung cancer cells by targeting miR-375. This study, using LINC00987 knockdown and overexpression cell models, combined with a nude mouse xenograft tumor model, demonstrated that low-expression of LINC00987 enhanced the proliferation, migration, and invasion of breast cancer cells. Overexpression of LINC00987 significantly slowed the proliferation, migration, and invasion of breast cancer cells. LINC00987 overexpression induced cell cycle arrest in breast cancer cells, potentially contributing to its inhibitory effect. LINC00987 expression varies across various malignancies, including breast cancer, and LINC00987 regulates tumor proliferation, migration, and invasion through distinct mechanisms.

[0207] In summary, this study confirms the important role of LINC00987 in breast cancer, especially its subcellular localization in breast cancer cells, expression changes, relationship with clinical pathological characteristics, and effect on the proliferation, migration and invasion ability of breast cancer cells. LINC00987 may be a key molecular marker for breast cancer progression and prognosis.

[0208] This study explored the distribution and expression characteristics of LINC00987 in breast cancer and its association with the clinicopathological features of breast cancer patients; further investigated its effects on the proliferation, migration and invasion of breast cancer cells and its relationship with prognosis.

[0209] 1) Nuclear-cytoplasmic fractionation experiments and cell FISH results showed that LINC00987 was mainly localized in the nuclei of breast cancer cells;

[0210] 2) Bioinformatics analysis and tissue FISH experiments revealed that LINC00987 expression in breast cancer tissues was significantly lower than that in normal tissues; its expression was significantly correlated with tumor size, metastasis, and tumor stage;

[0211] 3) LINC00987 is an important independent risk factor for prognosis in breast cancer patients, and its low expression is significantly associated with poor prognosis;

[0212] 4) Low expression of LINC00987 promoted the proliferation, migration and invasion of breast cancer cells; whereas overexpression of LINC00987 inhibited the proliferation, migration and invasion of breast cancer cells and induced cell cycle arrest.

[0213] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0214] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0215] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, and all of these should be included in the scope of the claims of the present invention.

Claims

1. Use of LINC00987 as a marker in the preparation of products for breast cancer diagnosis or prognosis assessment.

2. The use according to claim 1, characterized in that The product is a reagent or kit for detecting the expression level of LINC00987.

3. The use according to claim 2, characterized in that The expression level of LINC00987 is positively correlated with the prognosis of breast cancer.

4. A product for predicting the prognosis of breast cancer, characterized in that: The product comprises a reagent or a kit for detecting the marker according to claim 1.

5. Application of LINC00987 as a target in screening candidate drugs for the prevention or treatment of breast cancer.

6. Use of LINC00987 as a target in the preparation of drugs for the prevention and / or treatment of breast cancer.

7. The use according to claim 6, characterized in that The application is to increase the expression level of LINC00987.

8. The use according to claim 7, characterized in that Increase the expression level of LINC00987 by RNA technology or constructing an overexpression vector of LINC00987.

9. Use of an expression agonist, enhancer, or overexpression agent of LINC00987 in the preparation of a drug for treating breast cancer.

10. A drug for treating breast cancer, characterized in that: The drug includes an agent that increases the expression of LINC00987.

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