Application of linc00987 as a breast cancer diagnostic marker

Abstract

The application discloses application of LINC00987 as a breast cancer diagnosis marker, relates to the technical field of molecular biology and medicine, and discloses that LINC00987 is a long intergenic non-coding RNA, is located in the 31th sub-band in the short arm p13 region of the 12th chromosome of human beings, and is 22828 bp long; the LINC00987 can be used as a marker in preparation of a breast cancer diagnosis product or a prognosis evaluation product; and the LINC00987 can be used as a target point in preparation of a drug for preventing and / or treating breast cancer; in breast cancer, with the progress of the disease, the expression of LINC00987 gradually decreases; through overexpression of LINC00987 in breast cancer, the application can effectively inhibit the proliferation and metastasis of breast cancer cells, thereby effectively inhibiting the growth of breast cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biomedical technology, and particularly relates to application of LINC00987 as a breast cancer diagnosis marker. BACKGROUND

[0002] Breast cancer is one of the most common malignant tumors in women, which has high heterogeneity. It not only shows diversity in histological characteristics, but also has complex gene expression and signal pathway differences at the molecular level. Luminal A type breast cancer is ER and / or PR positive, HER-2 negative, and Ki-67 low expression, which is the most common molecular subtype of breast cancer, accounting for 44.5%-69.0% of the total breast cancer. The malignancy of this subtype of breast cancer is low, the disease progresses slowly, the risk of recurrence is relatively low, and the prognosis is good. Luminal B type breast cancer is more common in older patients, and compared with Luminal A type, it has high expression of Ki-67. The cancer cells grow faster, and the prognosis is relatively poor. Among all types of breast cancer, HER-2 overexpression type usually accounts for less than 10%, which is ER negative, PR negative, HER-2 positive, and Ki-67 usually has a high expression level. This subtype of breast cancer has strong invasiveness and metastasis, poor prognosis, and high risk of recurrence. Triple-negative breast cancer refers to breast cancer with negative results of ER, PR and HER-2 detection. About 15% of breast cancer patients are triple-negative breast cancer. It has a very high malignancy, is not sensitive to endocrine therapy and targeted therapy, and is one of the most dangerous types of breast cancer, and often recurs and metastasizes in the early stage of cancer. 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, which is also the main cause of cancer death. Cancer cells first proliferate and invade in the primary tumor tissue, gradually penetrate the tissue barrier around the primary tumor, and escape from the primary tumor site. Then, the cancer cells enter the circulatory system and are transported over long distances through blood circulation or lymph circulation. Finally, the cancer cells escape from the blood vessels, colonize in distant tissues or organs, and continue to proliferate, forming new tumor lesions. The abnormal proliferation, invasion and metastasis of breast cancer cells seriously affect the treatment effect of breast cancer patients and are 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 the prognosis of patients.

[0004] Molecular markers play an important role in the early diagnosis, classification and prognosis evaluation of breast cancer. For example, molecular markers such as ER, PR and HER-2 have been widely used in the classification and targeted therapy of breast cancer. Studies have shown that high expression of HER-2 gene is closely related to the invasiveness and poor prognosis of breast cancer, and through anti-HER-2 targeted therapy, such as trastuzumab therapy, the survival rate of patients can be significantly improved. ER and PR as hormone receptors, their positive expression indicates that breast cancer is hormone-dependent, and such patients are more sensitive to endocrine therapy, and the positive status of ER and PR is positively correlated with the overall survival and disease-free survival of patients. However, the existing markers still have certain limitations in sensitivity and specificity, which cannot fully meet the needs of precise diagnosis and treatment of breast cancer. In some HER-2 positive breast cancer patients, due to the limitations of detection methods or the heterogeneity of tumor cells, the false positive rate of HER-2 detection can reach 20%. Therefore, it is urgent to explore new molecular markers to improve the diagnostic accuracy and treatment effect of breast cancer. SUMMARY

[0005] Therefore, the main purpose of the present application is to provide an application of LINC00987 as a breast cancer diagnostic marker. The present application is achieved by: 1. Using breast cancer transcriptome data from TCGA and GTEx databases, analyzing the expression difference of LINC00987 in breast cancer and normal breast tissue and its clinical significance; 2. Through single factor and multi-factor Cox regression analysis, survival curve is drawn, and the potential of LINC00987 as a breast cancer prognostic marker is analyzed; 3. Through fluorescence in situ hybridization (FISH) technology, the expression level of LINC00987 in breast cancer tissue and normal tissue adjacent to cancer is detected, and the relationship between its expression level and clinical pathological characteristics (such as tumor size, lymph node metastasis, pathological grade, etc.) is explored; 4. Through nuclear-cytoplasmic separation experiment and FISH technology, combined with multiple databases, the localization and function prediction of LINC00987 in breast cancer cells are determined; 5. Through small interfering RNA (siRNA) and overexpression vector, LINC00987 knockdown or overexpression breast cancer cell model is constructed, and MDA-MB-231 cells stably overexpressing LINC00987 are used to construct breast cancer subcutaneous xenotransplant tumor model; 6. Through CCK-8, clonogenic, flow cytometry (PI staining) and other cell experiments, as well as nude mouse subcutaneous tumor animal model, the relationship between the expression level of LINC00987 and the proliferation of breast cancer cells is explored; 7. Through transwell experiment, the effect of LINC00987 on the migration and invasion of breast cancer cells is further explored.

[0006] The present application identifies the expression difference of LINC00987 in breast cancer tissues and normal breast tissues, explores the correlation between the expression level and the clinical pathological characteristics of breast cancer patients and determines the role of LINC00987 in the biological function of breast cancer.

[0007] To achieve the above-mentioned purpose, the present application improves the following technical scheme:

[0008] In a first aspect of the present application, LINC00987 is provided as a marker for preparing a product for breast cancer diagnosis or prognosis evaluation. Wherein, LINC00987 is located in the 31st sub-band of the short arm p13 region of human chromosome 12, and based on the GRCh38 / hg38 reference genome, the start position is 9240073, the end position is 9262900, and the length is 22828bp.

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

[0010] Further, the expression level of LINC00987 is positively correlated with the prognosis of breast cancer; and LINC00987 is down-regulated in breast cancer patients.

[0011] In a second aspect of the present application, a product for predicting the prognosis of breast cancer is provided, and the product comprises a reagent or kit for detecting the marker of claim 1.

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

[0013] In a fourth aspect of the present application, the application of the above-mentioned LINC00987 as a target in preparing drugs for preventing and / or treating breast cancer is provided.

[0014] Further, the application is to improve the expression amount of LINC00987.

[0015] Further, the expression amount of LINC00987 is improved by RNA technology or construction of an overexpression vector of LINC00987.

[0016] Specifically, the overexpression vector of LINC00987 is constructed, including the following steps:

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

[0018] 2) Design the primer shown in sequence hLINC00987-XhoI-F: 5 '-CCG CTC GAG TCTAGA AGC TGG AAAAGG CAAGGA AAGA-3' and hLINC00987-BamHI-R: 5 '-CGC GGATCCATG GCT TTATTATATTGC TTT ATGTATAT-3', and take LINC00987-pcDNA3.1(+) as a template to carry out PCR amplification; obtain a target gene fragment;

[0019] 3) Perform XhoI and BamHII double enzyme digestion on the pLVX-P2A-Luc-T2A-Puro plasmid, recover a target fragment, and obtain a plasmid large fragment;

[0020] 4) Connect the recovered plasmid large fragment with the target gene fragment; after transformation and culture of the connection product, pick up a single colony, extract a plasmid, and perform double enzyme digestion verification; perform second-generation sequencing verification on the positive clone verified by enzyme digestion; and introduce the verified pLVX-hLINC00987-Luc-Puro plasmid into 293T cells to obtain a slow virus rLV-hLINC00987-Luc-Puro containing the target gene.

[0021] Optionally, the expression amount of LINC00987 can also be indirectly increased by inhibiting a negative regulatory factor through antisense RNA.

[0022] The fifth aspect of the present application also provides application of an expression agonist or an enhancement agent or an overexpression agent of LINC00987 in preparation of a drug for treating breast cancer.

[0023] The sixth aspect of the present application also provides a drug for treating breast cancer, wherein the drug comprises an agent for increasing the expression of LINC00987.

[0024] The present application has at least the following beneficial effects:

[0025] The present application discusses distribution and expression characteristics of LINC00987 in breast cancer and correlation between the same and clinical pathological characteristics of breast cancer patients; further studies the effect of LINC00987 on proliferation and migration and invasion ability of breast cancer cells and the relationship with prognosis;

[0026] 1) The results of nucleolus separation experiment and cell FISH show that LINC00987 is mainly located in the nucleus of breast cancer cells;

[0027] 2) Through bioinformatics analysis and tissue FISH experiment, it is found that the expression of LINC00987 in breast cancer tissues is significantly lower than that in normal tissues; the expression is significantly correlated with tumor size, metastasis and tumor stage;

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

[0029] 4) Low expression of LINC00987 promotes the proliferation, migration and invasion of breast cancer cells, while overexpression of LINC00987 inhibits the proliferation, migration and invasion of breast cancer cells and induces cell cycle arrest.

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

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

[0032] Figure 2 Figure is the schematic diagram of predicting the protein coding ability of LINC00987 by CPAT and CPC2 online databases.

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

[0034] Figure 4 Figure is the localization map of LINC000987 in breast cancer cells detected by nucleocytoplasmic separation experiment.

[0035] Figure 5 Figure is the localization map of LINC000987 in breast cancer cells detected by FISH (magnification: 200x).

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

[0037] Figure 7 Figure is the expression of LINC00987 in breast cancer tissues and paracancerous tissues of breast cancer patients detected by FISH (magnification: 200x).

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

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

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

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

[0042] Figure 12 Effect of LINC00987 overexpression on the proliferation ability of breast cancer cells, A—CCK-8 experiment; B—clonogenic assay. *P<0.05; ***P<0.001.

[0043] Figure 13 Figure for flow cytometry detection of LINC00987 overexpression breast cancer cell cycle changes.

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

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

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

[0047] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0048] In addition, the technical solutions of various embodiments of the present application can be combined with each other, but it must be based on the fact that a person of ordinary skill in the art can realize it. When the combination of technical solutions appears to be contradictory or unachievable, it should be considered that the combination of technical solutions does not exist and is not within the scope of protection required by the present application.

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

[0050] 1. Experimental materials

[0051] (1) Tissue specimens

[0052] The tissue chip was purchased from Shanghai Zhi Li Biological Technology Co., Ltd. and included cancer tissues and adjacent normal breast tissues. The inclusion criteria and exclusion criteria of the research subjects are as follows:

[0053] 1) Inclusion criteria

[0054] ① Patients aged ≥18 years, confirmed by pathology as breast cancer; ② First received breast cancer resection, without any adjuvant therapy before operation; ③ Paracancerous tissue is normal breast tissue with a distance of >2 cm from the cancer lesion; ④ The patient's basic information and postoperative pathological examination data are complete.

[0055] 2) Exclusion criteria

[0056] ① Exclude patients with other breast diseases other than breast cancer; ② Exclude patients with a history of other malignant tumors; ③ Exclude patients who have undergone neoadjuvant therapy before operation; ④ Exclude patients with other infectious diseases.

[0057] A total of 180 breast cancer patients were included in this study as research objects. The epidemiological data of all research objects included patient age, gender, case number, tumor location, tumor diameter, tumor stage, metastasis, ER, PR, and HER-2 status, and other information. All research objects gave informed consent, and this study was approved by the Ethics Committee of Huabei Polytechnic University and the Ethics Committee of Shanghai Zhuoli Biological Technology Co., Ltd. (Approval number: 2022025, ZLL-15-01).

[0058] (2) Cell lines

[0059] The MCF-10A normal breast epithelial cells used in this application and the MCF-7, SKBR3, and MDA-MB-231 breast cancer cells were purchased from Wuhan Punsun Life Science and Technology Co., Ltd. Among them, MCF-10A cells were cultured in a special medium provided by the company, and the other three breast cancer cells were cultured in DMEM high-sugar medium supplemented with 10% fetal bovine serum (Fetal Bovine Serum, FBS) and 1% penicillin-streptomycin (Penicillin-Streptomycin, PS). All cells were cultured in a constant temperature incubator containing 5% CO2 and set to 37°C.

[0060] MCF-7 and MDA-MB-231 cells were transfected with siNC and siLINC00987 small interfering RNA, and negative control (siNC) group and LINC00987 low expression (siLINC00987) group were set up, respectively. In addition, by screening MCF-7 and MDA-MB-231 cells infected with lentivirus, LINC00987 overexpression cells and control cells were constructed for subsequent experiments.

[0061] (3) Main reagents and sources

[0062] Table 1 Main experimental reagents

[0063] Experimental reagents Company DMEM high-sugar medium Gibco, USA FBS Gibco, USA PS Gibco, USA MCF-10A special medium Ponyer, China PBS Solabio, China 0.25% EDTA-trypsin Solabio, China 0.4% trypan blue Solabio, China Trizol Thermo, USA Anhydrous ethanol Yongda, China Isopropanol Huayue, China DEPC water Sigma, USA RevertAid First Strand cDNA Synthesis Kit Thermo, USA Realtime PCR Supermix SYBR green, with anti-Taq Beijing Hypermol, China Nuclear extraction kit Thermo, USA Fluorescent probes (target gene, U6-nuclear control, 18S- quality control) Ribo, China Fluorescent in situ hybridization kit Ribo, China 4% paraformaldehyde Biosharp, China Trition X-100 Solabio, China Tween-20 TGI, China 20x SSC (pH = 7.0) ZOMAN BIO, China 3% H2O2 Zhongshanjinqiao, China 3% citric acid + pepsin Bristol-Myers Squibb, China Xylene Yongda, China DAPI Solabio, China

[0064] (4) Main experimental instruments

[0065] Table 2 Main experimental instruments

[0066] Experimental instruments Company -80℃ ultra-low temperature refrigerator Thermo, China -20℃ refrigerator Haier, China CO2 constant temperature incubator Likang, China Constant temperature water bath Guohua Electrical Appliance, China High-speed low-temperature table centrifuge Eppendorf, Germany IX71 inverted fluorescence microscope Olympus, Japan Constant temperature metal bath Jide, China 7900HT fast real-time PCR instrument Applied Biosystems, USA Universal shaker / LSM900 confocal microscope Zeiss, Germany Electric heating incubator Test, China

[0067] 2. Experimental method

[0068] (1) Chromosomal localization, secondary structure and protein coding ability prediction of LINC00987

[0069] With the help of online databases, the related information of LINC00987 was analyzed in detail, including sequence information, chromosomal location, secondary structure and potential protein coding ability of LINC00987.

[0070] 1) LINC00987 localization in chromosome and secondary structure prediction

[0071] The sequence information and chromosomal location information of LINC00987 were obtained through two websites, UCSC Genome Browser (http: / / genome.ucsc.edu) and Vienna RNA Web Service (http: / / rna.tbi.univie.ac.at / ), and the secondary structure and minimum free energy of LINC00987 were predicted.

[0072] 2) LINC00987 protein coding ability prediction

[0073] In this study, the protein coding ability of LINC00987 was predicted by CPAT (http: / / lilab.research.bcm.edu / ) and CPC2 (http: / / cpc2.cbi.pku.edu.cn) online databases, and the classic coding genes GAPDH, ACTB and non-coding genes HOTAIR, XIST were used as controls.

[0074] (2) Expression difference of LINC00987 in breast cancer tissues

[0075] The transcriptome sequencing data of TCGA database and GTEx breast cancer and normal breast tissues were downloaded from UCSC XENA (https: / / xenabrowser.net / datapages / ) website. The above raw data was processed by TOIL process, the batch effect was corrected, the corresponding TCGA data of breast cancer and the corresponding normal tissue data in GTEx project were extracted and allowed to cross data sets and data. The expression difference of LINC00987 was analyzed by Wilcoxon signed rank test, and the data was visualized by R language (version 3.6.3) ggplot2 package.

[0076] The breast cancer lncRNA was used as the key word to screen the data set related to the expression of lncRNA in breast cancer tissue in the Gene Expression Omnibus (GEO) database. Among them, two data sets GSE119233 and GSE156229 were microarray detection of lncRNA expression profile in breast cancer and normal breast tissue. The matrix file containing gene expression data of the above two data sets was downloaded, and the lncRNA differential analysis was performed by R language limma package to verify the expression level of LINC00987 in breast cancer tissue.

[0077] (3) Survival analysis

[0078] Based on the breast cancer RNA-seq data and patient clinical data in TCGA, the patients were divided into high expression group and low expression group according to the median of LINC00987 expression level, and the Kaplan-Meier (KM) survival analysis was performed by using R language survival and survivalMiner package. Further, Cox regression analysis was used to explore the relationship between LINC00987 expression and overall survival (OS). That is, through single factor Cox regression analysis of patient age, tumor stage, ER, PR, HER-2 expression state and other pathological characteristics and LINC00987 expression level and the prognosis of patients, the possible independent prognostic factors were evaluated by multivariate Cox model. The risk ratio (Hazard Ratio, HR) of each index was evaluated by statistical method, and the numerical range of 95% confidence interval (Confidence Interval, CI) was determined.

[0079] (4) Cell culture

[0080] 1) Cell recovery

[0081] The cells to be recovered were removed from the liquid nitrogen storage container and quickly placed in a 37°C constant temperature water bath for thawing. After the frozen solution was fully melted, the cell suspension was transferred to a 15 mL centrifuge tube, 1 mL of complete medium was injected for gentle mixing, then centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, 3 mL of fresh complete medium was supplemented and mixed thoroughly, the cell suspension was transferred to the culture bottle and placed in a 5% CO2, 37°C constant temperature incubator for culture.

[0082] 2) Cell replacement and subculture

[0083] Before cell replacement, take out the cells and observe their morphology, pour the culture medium, wash with PBS twice, add DMEM complete medium, and continue to culture in a 5% CO2, 37°C constant temperature incubator.

[0084] When the cell density reaches or exceeds 80%, the cells are passaged. The following procedures are followed:

[0085] ① Use PBS to wash the cells twice. ② Add 1 mL of 0.25% EDTA-trypsin, digest for 1-2 minutes according to different cell types, observe the cell morphology under a microscope, and when the cell morphology is found to be round and ground glass-like, add 1 mL of complete medium to terminate digestion. ③ Blow the cells to mix, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes. ④ Discard the supernatant, add 3 mL of complete medium, and blow to mix to a single cell suspension. ⑤ Continue to culture the cells in the culture plate or culture bottle.

[0086] 3) Cell counting

[0087] 75% ethanol rinse the counting plate, dry, cover with a cover glass, and place in a clean bench. Mix the trypan blue dye and cell resuspension according to a ratio of 1:9, take 10 μL of the mixed cell suspension and drop it onto the counting plate, and count after 1 minute. Calculate the number of cells in the four large squares in the counting plate according to the formula: cell number / mL = four large square cell number / 4 x 10 4 It should be noted that if there are cells on the line of the counting plate, generally the upper line is not counted and the left line is not counted. Only complete cells are counted, and if cells are clustered together, they are counted as one cell. If the cells have been diluted before counting, the dilution factor needs to be multiplied.

[0088] 4) Cell freezing

[0089] According to the cell passage method, digest and resuspend the cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add the freshly prepared cell freezing solution, and gently blow to mix. Transfer the cell suspension to a freezing tube. Seal the freezing tube with a sealing film, place it in a programmed cooling box, and store it in a -80°C refrigerator overnight, then transfer it to a liquid nitrogen tank for long-term storage.

[0090] (5) Nucleocytoplasmic separation experiment

[0091] 1) Cell nucleus and cytoplasm RNA extraction

[0092] ① 0.25% EDTA-trypsin digestion and collection of cells, counting of cells, and adjustment of cell concentration to 1 x 10 7① Wash cells with pre-chilled PBS and centrifuge at 1000 rpm for 5 minutes. ② Discard the PBS, add 300 μL of pre-chilled cell separation buffer, gently tap the tube wall to 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 fraction from the supernatant into a new RNase-free centrifuge tube and place it on ice. This step achieves nucleoplasmic RNA separation. Extract the cytoplasmic fraction from the nucleus and proceed to step ⑦. ⑥ Add 300 μL of pre-chilled cell disruption buffer to the remaining precipitate of nuclei, vortex to dissolve the nuclei until the lysis buffer is homogeneous. Note that this step must be performed on ice. ⑦ RNA isolation: At room temperature, mix the lysis buffer from steps ⑤ and ⑥ with an equal volume of 2X lysis / binding buffer, repeatedly inverting the centrifuge tube to ensure thorough mixing; add 300 μL of anhydrous ethanol to the mixture and gently mix; filter the mixture through an adsorption column, centrifuge at 12000 g for 1 minute; wash twice with 700 μL of washing buffer 1, centrifuge at 12000 g for 1 minute; wash once with 500 μL of washing buffer 2 / 3, centrifuge at 12000 g for 1 minute; discard the washing buffer and continue centrifuging for 30 seconds; finally, elute the RNA with 40 μL of elution buffer preheated to 95 °C, centrifuge at 12000 g for 30 seconds to recover the RNA. Measure the concentration and purity of the extracted RNA and store at -80 °C for later use.

[0093] 2) RNA is reverse transcribed into cDNA

[0094] Follow the steps provided with the reverse transcription kit:

[0095] ① Based on the measured RNA concentration, add 2 μg RNA and 1 μL oligo(dT)18 primer to a sterile, RNase-free centrifuge tube placed on ice, and add RNase-free ultrapure water to a final volume of 12 μL. ② After mixing, briefly centrifuge, incubate at 65°C for 5 minutes, cool on ice, briefly centrifuge again, and cool. ③ Prepare the reverse transcription reagent (Table 3). ④ Mix the above reverse transcription reagent and add it to the centrifuge tube to bring the final reaction volume to 20 μL. ⑤ After mixing, briefly centrifuge, incubate at 42°C for 60 minutes, heat at 70°C for 5 minutes to terminate the reaction, measure the cDNA concentration and purity, and store at -80°C for later use.

[0096] Table 3 Reverse Transcription Reagent System

[0097] Reverse transcription reagent System (1 sample, μL) 5x Reaction Buffer 4 RibolockTMRNAse inhibitor (20U / μL) 1 10mMdNTPMix 2 RevertAidTMM-Mulv reverse transcriptase (200U / μL) 1

[0098] 3) Real-time quantitative PCR (RT-qPCR)

[0099] ① RT-qPCR primer design and synthesis.

[0100] The nucleic acid sequence information of human LINC00987, GAPDH and U6 was obtained by NCBI database retrieval, and then the primer design work was completed by using Primer Premier5 program. The designed primers were chemically synthesized by Beijing Nosy Biotechnology Co., Ltd. According to the instruction, different amounts of ddH2O were added to ensure that the primer concentration reached 100 μM, and it was stored at -20℃ for a long time. When performing RT-qPCR experiment, the final concentration of primers was diluted to 10 μM. The detailed primer sequences are as follows (Table 4):

[0101] Table 4 RT-qPCR primer information

[0102] Gene name Primer sequence GAPDHPF 5'-CTGGGCTACACTGAGGACC-3' GAPDHPR 5'-AAGTGGTCGTTGAGGGCAATG-3' U6PF 5'-TGCGGGTGCTCGCTTCGGCAGC-3' U6PR 5'-CCAGTGCAGGGTCCGAGGT-3' LINC00987PF 5'-ACGACGCACAATGCAAAGAC-3' LINC00987PR 5'-TGTTTTCTGCACTGACCCCA-3'

[0103] ②The RT-qPCR reaction system is as follows (Table 5):

[0104] Table 5 RT-qPCR reaction system

[0105]

[0106] ③According to the above reaction system, the reaction mixture was configured, 3 replicate wells were set for each sample, and the reaction mixture was sequentially added to the 384-well plate. ④Paste the sealing film, centrifuge at 3000 rpm for 3 minutes. ⑤Put the centrifuged 384-well plate into the real-time fluorescent quantitative PCR instrument, and the reaction program is as follows (Table 6):

[0107] Table 6 RT-qPCR reaction program

[0108]

[0109]

[0110] ⑥Take GAPDH as the internal reference to calculate the target gene value, and compare the relative expression amount of LINC00987 to the internal reference gene in different cells.

[0111] (6) Cell fluorescence in situ hybridization

[0112] 1) Put the cell climbing sheet into a 24-well culture plate, add 5×10 31) Cell culture: Cells were cultured in 24-well plates at a density of 5 x 105 cells per well for 24 hours. 2) Cell fixation: 600 μL of PBS was added to each well and the cells were washed for 5 minutes on a shaker. The PBS was discarded and 600 μL of 4% paraformaldehyde solution was added to each well and the cells were fixed at room temperature for 10 minutes. 3) Cell permeation: The cells were washed 3 times with 1 x PBS for 5 minutes each time. 1 mL of pre-cooled permeation solution was added to each well and the cells were permeated at 4°C for 5 minutes. The permeation solution was discarded and the cells were washed 3 times with 1 x PBS for 5 minutes each time. 4) Probe pre-hybridization: The pre-hybridization solution was pre-heated in a 37°C water bath. 200 μL of the pre-hybridization solution was added to each well and the cells were pre-hybridized at 37°C for 30 minutes. 5) Probe hybridization: 2.5 μL of probe (20 μM) was added to 100 μL of pre-heated hybridization solution at 37°C to obtain the probe hybridization solution. The pre-hybridization solution in each well was discarded and the probe hybridization solution was added to each well, taking care to avoid light. The cells were hybridized in a 37°C incubator overnight. 6) Post-hybridization treatment: The hybridization wash solutions I, II and III were pre-heated to 42°C. The cells were washed on a shaker in the following order to reduce background signals, taking care to avoid light: 1) The cells were washed 3 times with hybridization wash solution I for 5 minutes each time; 2) The cells were washed once with hybridization wash solution II for 5 minutes; 3) The cells were washed once with hybridization wash solution III for 5 minutes; and 4) The cells were washed once with 1 x PBS for 5 minutes. 7) Nucleus staining: 100 μL of 1 x DAPI staining solution was added to the cells, taking care to avoid light, and the cells were stained for 10 minutes. The cells were washed 3 times with 1 x PBS for 5 minutes each time. 8) Mounting and photographing: The cells were removed from the wells and mounted on glass slides. An anti-fluorescence quencher was added and a cover glass was mounted. The cells were photographed using a laser confocal microscope.

[0113] (7) Tissue fluorescence in situ hybridization

[0114] 1) Baking: Put the tissue chip into the oven and bake at 60°C for 1 hour. 2) Walking: First, put the tissue chip into xylene and soak for 10 minutes for deparaffinization; then put it into another xylene and soak for 10 minutes; then put it into 100% ethanol to remove xylene and soak for 10 minutes; then use 100% ethanol for 10 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 70% ethanol for 5 minutes, respectively; finally, rinse with tap water for 5 minutes. 3) Inactivation of endogenous enzymes: Add 3% H2O2 on the tissue chip and incubate at room temperature for 10 minutes to inactivate endogenous peroxidase, and then wash with ultrapure water for 5 minutes. 4) Exposure of RNA fragments: Add freshly diluted 3% citric acid pepsin on the tissue chip and digest in a 37°C incubator for 15 minutes. Wash with 1×PBS for 3 times, 5 minutes each time; wash with ultrapure water for 1 time, 5 minutes. 5) Post-fixation: Post-fixation solution (4% paraformaldehyde containing 1 / 1000 DEPC) is fixed at room temperature for 5 minutes, and washed with ultrapure water for 3 times, 5 minutes each time. 6) Probe pre-hybridization: Prepare a hybridization box before pre-hybridization, add 20% glycerol to the bottom of the box for moisturizing. Preheat the pre-hybridization solution in a 37°C water bath; add 200 μL of pre-hybridization solution to each tissue chip, and pre-hybridize at 37°C for 30 minutes. 7) Probe hybridization: Under light-proof conditions, add 2.5 μL of probe (20 μM) to 100 μL of preheated hybridization solution at 37°C, mix well by blowing, which is the probe hybridization solution. Discard the pre-hybridization solution and add the probe hybridization solution to the hybridization box, hybridize at 37°C overnight, pay attention to light-proof and moisturizing. 8) Post-hybridization treatment: Preheat hybridization washing solution I, II, III at 42°C; under light-proof conditions, wash the tissue chip with hybridization washing solution I for 3 times, 5 minutes each time, with hybridization washing solution II for 1 time, 5 minutes, with hybridization washing solution III for 1 time, 5 minutes, and with 1×PBS for 1 time, 5 minutes. 9) Nucleus staining: Add 100 μL of 1×DAPI staining solution and stain for 10 minutes; wash with 1×PBS for 3 times, 5 minutes each time, pay attention to light-proof operation. 10) Mounting and photographing: Add anti-fluorescence quencher on the tissue chip and cover with a coverslip. Take pictures under laser confocal microscope.

[0115] (8) siRNA transfection

[0116] 1) siRNA was synthesized by Shanghai Jimake Gene Biotechnology Co., Ltd., and the target sequence is as follows (Table 7):

[0117] Table 7 si-NC and si-LINC00987 target sequence

[0118]

[0119] 2) Cell plating: In the LINC00987 knockdown experiment, siNC as the control group, siLINC00987 as the knockdown group. After the MCF-7 and MDA-MB-231 cells with a fusion rate of 80% were digested, resuspended and counted according to the operation steps of cell subculture, the cells were inoculated into 6-well plates at a density of 2x10 5 3) Preparation of lipofectamine 2000 mixture: Take a sterile RNase-free 1.5 mL centrifuge tube, prepare the lipofectamine 2000 transfection mixture according to the ratio of 150 μL DMEM medium per well plus 3 μL lipofectamine 2000 transfection reagent, mix thoroughly, and then stand at room temperature for 5 minutes. 4) Dissolve siRNA dry powder / primer: Take the siRNA dry powder from the -20°C refrigerator, centrifuge at 2000 rpm for 3 minutes to ensure that the siRNA dry powder is precipitated at the bottom of the tube. According to the label on the tube, add the corresponding amount of DEPC water for dissolution, so that the final concentration is 20 μM / L. 5) Preparation of siRNA mixture: First, add 150 μL DMEM medium to a sterile RNase-free centrifuge tube, then add 5 μL of control (siNC) or experimental (siLINC00987) siRNA to each group of centrifuge tubes, so that the final concentration of transfection is 50 nM, and mix gently. 6) Take 150 μL of lipofectamine 2000 mixture prepared in step (3) and add it to each group of siRNA mixtures, mix gently, and then stand for 20 minutes. 7) During the standing process, take out the 6-well plate plated the day before, discard the culture medium, wash the cells with 1xPBS twice, add 700 μL of DMEM high glucose medium, and add the working solution after standing to the 6-well plate. 8) Incubate at 37°C in a 5% CO2 incubator for 5 hours, wash the cells with 1xPBS twice, then replace with 10% FBS-containing DMEM medium without PS. 9) Continue to culture for 24 hours, collect the cells, extract RNA, confirm the LINC00987 knockdown efficiency, and follow-up experiments are detected according to the transfection conditions.

[0120] (9) LINC00987 overexpression plasmid construction and lentivirus packaging

[0121] 1) LINC00987 overexpression plasmid construction

[0122] The full-length sequence of LINC00987 cDNA (NR_137429.2) was downloaded from the NCBI website, and pcDNA3.1(+)-LINC00987 overexpression plasmid and control pcDNA3.1(+) plasmid were synthesized by Shanghai Shengong Bioengineering Co., Ltd. Double enzyme digestion was performed using restriction endonucleases Xho I and Kpn I, and agarose gel electrophoresis was used for verification. At the same time, the enzyme digestion products were subjected to second-generation gene sequencing, and after comparison, it was confirmed that the sequence of the LINC00987 target fragment was consistent with that of the synthesized overexpression plasmid fragment.

[0123] 2) LINC00987 overexpression lentivirus packaging

[0124] The lentivirus packaging was completed by Beijing Xibei Hongcheng Biotechnology Co., Ltd., and the brief process was as follows: DNA primers hLINC00987-Xho I-F: 5'-CCG CTC GAG TCT AGA AGC TGG AAA AGG CAA GGA AAGA-3' and hLINC00987-BamH I-R: 5'-CGC GGA TCC ATG GCT TAT TAT ATT GCT TTAT G TAT AT-3' were designed and synthesized; PCR amplification was performed using the plasmid LINC00987-pcDNA3.1(+) as the template. The pLVX-P2A-Luc-T2A-Puro plasmid was treated with Xho I and BamH I double enzyme digestion, and the target fragment was recovered. The recovered plasmid large fragment was ligated with the target gene fragment. After the ligation product was transformed and cultured, single colonies were picked, the plasmid was extracted, and double enzyme digestion verification was performed. The positive clones that passed the enzyme digestion verification were subjected to second-generation sequencing verification. The verified pLVX-hLINC00987-Luc-Puro plasmid was introduced into 293T cells, and high-titer lentivirus rLV-hLINC00987-Luc-Puro containing the target gene was packaged according to the relevant method.

[0125] (10) Lentivirus infection and stable cell line screening

[0126] 1) Cell plating: when the logarithmically growing breast cancer cells (MCF-7 and MDA-MB-231) were grown to 80%-90% cell density, they were continuously passaged for 2-3 times. 2) When the cells were passaged to the 4th generation, the cells were digested and resuspended to prepare a single cell suspension, the cells were counted, and 2x10 5The density of cells was inoculated into 6-well plates and cultured overnight at 37°C in a 5% CO2 incubator. 3) According to the MOI = 50 virus titer, packaged lentivirus was added to the 6-well plate, and the transfection reagent polybrene was added. Serum-free DMEM high-glucose medium was used, and the cells were incubated at 37°C in a 5% CO2 incubator. 4) After 24 hours of virus infection, DMEM complete medium containing 10% FBS was used for continuous culture. 5) After 72 hours of lentivirus infection, the fluorescence intensity and fluorescence cell proportion of the cells were observed under a fluorescence microscope. According to the final concentration of 4 μg / mL, puromycin was added for resistance screening, and the medium was changed every 2-3 days (complete medium containing 4 μg / mL puromycin). 6) After continuous use of puromycin for one week, the proportion of fluorescent cells was observed, and when the fluorescent cells grew to 90% of the cell density, the cells were collected and the overexpression efficiency of LINC00987 was detected.

[0127] (11) Cell total RNA extraction, reverse transcription and RT-qPCR

[0128] 1) Cell total RNA extraction (Trizol method)

[0129] ① Lysis of cell samples: discard the culture medium, wash the cells twice with 1×PBS, add 1 mL Trizol to completely cover the cell surface, and repeatedly blow to completely lyse the cells. The supernatant was transferred to a new RNAase-free centrifuge tube. ② In a fume hood, let stand at room temperature for 5 minutes to allow the cells to fully lyse. ③ Add 200 μL chloroform, shake well for 15 seconds, and let stand at room temperature for 5 minutes. ④ 4°C, 13000g centrifugation, 15 minutes. ⑤ Carefully transfer the supernatant to a new RNAase-free centrifuge tube, add 500 μL isopropanol, mix well, and let stand at -20°C for 15 minutes. ⑥ 4°C, 12000g centrifugation for 12 minutes, discard the supernatant. ⑦ Add 1 mL 75% ethanol (DEPC water preparation, fresh preparation) to the centrifuge tube containing the precipitate, wash the precipitate, 4°C, 7500g centrifugation, 5 minutes. ⑧ Repeat step ⑦. ⑨ Let it dry at room temperature (5-10 minutes) to evaporate the ethanol, while avoiding complete drying of the RNA. ⑩ Add 20 μL DEPC water to each centrifuge tube, 55°C metal bath for 10 minutes to completely dissolve the RNA, measure the concentration and purity of the RNA, and store the sample in a -80°C refrigerator.

[0130] 2) RNA reverse transcription to cDNA

[0131] The experimental method is the same as above.

[0132] 3) RT-qPCR

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

[0134] (12) Cell proliferation ability detection

[0135] 1) CCK-8 experiment

[0136] ① Take the logarithmic growth period of each group of breast cancer cells, according to the cell passage procedure, after 0.25% EDTA-trypsin digestion and centrifugation, resuspend with complete culture medium, and mix into single cell suspension. ② Count the cells, adjust the cell concentration, and inoculate 5000 cells per 100 μL into 96-well plates. ③ CCK-8 and DMEM medium were prepared according to the ratio of 1:9 to prepare CCK-8 working solution. At 0, 24, 48, and 72 hours, discard the original culture medium in the 96-well plate, then add 100 μL CCK-8 working solution to each well, and incubate at 37°C in the dark for 2 hours. ④ After incubation, the optical density (OD) value was detected by multifunctional enzyme label instrument at 450 nm. ⑤ Record the OD value at different time points, and draw the cell proliferation fold line graph.

[0137] 2) Cloning experiment

[0138] ① Take the logarithmic growth period of each group of breast cancer cells for trypsin digestion, then resuspend the cells by centrifugation, count the cells, and dilute the cell suspension to a density of 200 cells / mL. ② Take 5 mL of diluted cell suspension and inoculate into 60 mm culture dish, and continue to culture in 37°C, 5% CO2 incubator. ③ Observe the cell state every day, and change the liquid in the culture dish every 5 days. ④ When most of the single cells form obvious clones (the number of single clone cells 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 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, wash the residual staining solution with 1×PBS, and take pictures after air drying. Count the number of clones in each group by Image J software and perform statistical analysis.

[0139] (13) Cell cycle detection

[0140] 1) Collect each group of cells in logarithmic growth phase, and collect the cell supernatant in a centrifuge tube for later use. Wash the cells with 1x PBS twice. 2) Digest the cells with 0.25% EDTA-trypsin, and when the cells are observed to be round and glassy under a microscope, stop the digestion with complete medium, mix the cell suspension gently with a pipette, and transfer the cell suspension to the centrifuge tube mentioned above, and centrifuge at 800 rpm for 5 minutes. Note that the blowing action should be gentle to avoid mechanical damage to the cells. 3) Discard the supernatant, add 1 mL of pre-cooled 1x PBS to wash the cells, and centrifuge at 800 rpm for 5 minutes. 4) Repeat the previous step. 5) Discard the supernatant, add 1 mL of 70% ethanol pre-cooled in an ice bath, and mix gently. Fix overnight (about 12 hours) at 4°C. 6) Centrifuge at 800 rpm for 5 minutes to precipitate the cells. 7) Add 1 mL of pre-cooled 1x PBS to resuspend the cells. 8) After centrifuging the cells again, slowly remove the upper liquid, and then tap the bottom of the tube gently to distribute the cells evenly, preventing them from clumping. 9) Prepare the propidium iodide (PI) staining solution according to the following table (Table 8): 10) Add 0.5 mL of PI staining solution to each sample tube, resuspend the cell pellet slowly and thoroughly, and incubate at 37°C for 30 minutes in the dark. Then place the cell suspension on ice, taking care to avoid light. 11) Detect red fluorescence signals at an excitation wavelength of 488 nm using flow cytometry, and record the light scattering data simultaneously.

[0141] Table 8 Propidium iodide staining solution configuration system

[0142] Reagent name 1 sample Staining buffer 0.5mL PI staining solution (20x) 25μL RNaseA (50x) 10μL Total volume 0.535mL

[0143] (14) Cell migration and invasion experiments

[0144] In the experimental setup, chambers without Matrigel were used to detect the migration ability of breast cancer cells, and chambers with Matrigel were used to detect the invasion ability. Through these two different experimental setups, the behavior characteristics of breast cancer cells in different environments can be more comprehensively understood.

[0145] 1) Transwell cell migration experiment

[0146] ① Count the cells, and adjust the cell density to 5x10 5 / mL. ③ Add 600 μL DMEM high glucose medium containing 20% FBS to the 24-well plate under the transwell chamber, and slowly place the transwell chamber into the 24-well plate, being careful not to generate bubbles. ④ Take 200 μL of the diluted cell suspension and slowly add it to the upper chamber of the transwell chamber, avoiding the generation of bubbles. Place the 24-well plate in a 37°C, 5% CO2 incubator for continued culture. ⑤ After 24 hours of continued culture, remove the chamber and gently wipe off the remaining cells in the upper chamber of the transwell chamber with a cotton swab wetted with PBS, and wash twice with 1x PBS. ⑥ Add 600 μL 4% paraformaldehyde to the 24-well plate, and place the chamber in it for 30 minutes of room temperature fixation, and wash twice with 1x PBS. ⑦ Add 600 μL 0.1% crystal violet staining solution to the 24-well plate, and stain for 15 minutes, and then rinse off the excess color with 1x PBS. ⑧ Observe the cell morphology under an IX71 inverted optical microscope and take photographs. Count the number of migrated cells in each group using Image J software, and perform statistical analysis.

[0147] 2) Transwell cell invasion experiment

[0148] ① The day before the experiment, take the Matrigel out of the -20°C refrigerator and slowly thaw it at 4°C. ② After pre-cooling the gun head and centrifuge tube on ice, mix the Matrigel with serum-free DMEM medium at a ratio of 1:8 on ice, and add 100 μL per well to the upper chamber of the transwell chamber. Incubate in a 37°C, 5% CO2 incubator for 5 hours, and slowly aspirate the residual liquid in the chamber. ③ The remaining experimental steps are consistent with the cell migration experiment, and the subsequent experimental operations are performed according to the experimental steps of the cell migration experiment.

[0149] (15) Construction of a subcutaneous tumor xenograft model in nude mice

[0150] 1) When the control (vector) group and LINC00987 overexpression (LINC00987) group of MDA-MB-231 cells in the logarithmic growth phase reached 80%-90% confluence, the cells were treated with 0.25% EDTA-trypsin, then centrifuged, and washed twice with 1x PBS to completely remove the residual serum components. 2) Resuspend the cells with pre-cooled 1x PBS, count the cells, and adjust the cell concentration so that the final concentration of the cell suspension is 3x10 74) The left thumb and index finger gently pinch the skin on the back of the neck of the nude mouse, while the ring finger and little finger firmly hold the tail of the nude mouse, so that the nude mouse is securely fixed in the palm. 5) 75% ethanol cotton ball disinfects the skin of the right upper limb of the axillary fossa of the nude mouse, uses a 1 mL syringe to suck 100 μL of cell suspension, and follows the "Z" shape to needle, slowly inoculates the cell suspension into the subcutaneous tissue of the axillary fossa of the nude mouse. 6) Normal feeding and watering, daily observation of the mental state, food intake, activity, etc. of the nude mice, measurement of the body weight of the nude mice every 2 days, and measurement of the long diameter and short diameter of the tumor using a vernier caliper, calculation of the tumor volume according to the formula volume = long diameter x short diameter2x 0.52, and good record keeping. 7) When the long diameter of the tumor of the nude mouse is about 15 mm, the nude mouse is sacrificed, the subcutaneous tumor tissue of the nude mouse is dissected and peeled off, weighed, and photographed. Part of the tissue is frozen in a-80°C refrigerator for later use, and the remaining tumor tissue is fixed with 4% paraformaldehyde for subsequent experiments.

[0151] (16) Pathological detection

[0152] 1) Fixation and embedding of tumor tissue

[0153] ① 4% paraformaldehyde was used to fix the tumor tissue, and after 24 hours of fixation, the tumor tissue was washed with ultrapure water. During the operation, gentle and delicate movements were required to avoid damaging the tumor tissue. ② Dehydration with 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol I, 95% ethanol II, and 100% ethanol for 1 hour each. ③ Xylene transparency for 1 hour. ④ Embedding of tumor tissue in heated and melted paraffin. ⑤ After the paraffin completely solidified, the embedded tissue was processed for sectioning, cut into thin sections with a thickness of 4 μM, and placed in a 40°C water bath for expansion and retrieval, followed by drying.

[0154] 2) Hematoxylin-eosin (HE) staining

[0155] ① Put the tissue section into a 60℃ oven, bake for 30 minutes, soak in xylene I for 20 minutes, and then continue to soak in xylene II for 20 minutes for dewaxing. ② Put the dewaxing slide into 100%, 100%, 95%, 85%, and 75% ethanol respectively for 5 minutes, and then rinse with water for 5 minutes. ③ Stain with hematoxylin for 5 minutes, and then rinse with water for 10 minutes. ④ Soak in 75% ethanol for 2 minutes, and then rinse with water for 30 seconds. ⑤ Re-blue with 0.6% ammonia water, and then rinse with water for 30 seconds. ⑥ Soak in 85% and 95% ethanol respectively for 5 minutes for dehydration. ⑦ Stain with eosin for 5 minutes, and then rinse with water for 30 seconds. ⑧ Dehydrate in 100% anhydrous ethanol for 3 times, and then clear in xylene for 2 times, each for 5 minutes. ⑨ Seal with neutral balsam.

[0156] 3) Immunohistochemistry (IHC) staining

[0157] ① Put the paraffin section into a 60℃ oven, bake for 30 minutes, and then soak in xylene I for 20 minutes, and then continue to soak in xylene II for 20 minutes for dewaxing. ② Put the dewaxing slide into 100%, 100%, 95%, 85%, and 75% ethanol respectively for 5 minutes, and then rinse with water for 5 minutes. ③ Add 3% H2O2 on the tissue section, incubate for 10 minutes, and then wash with 1×PBS for 2 times, each for 5 minutes. ④ Put the section into 0.01M citric acid buffer, heat with a microwave oven for 8 minutes, and then wash with 1×PBS for 2 times, each for 5 minutes when the temperature drops to room temperature. ⑤ Seal with 10% goat serum at room temperature for 60 minutes, remove the sealing liquid, add the diluted primary antibody Ki67 (dilution ratio is 1:200), incubate in a wet box, and then incubate at 4℃ overnight. ⑥ After removing the primary antibody, wash with 1×PBS for 2 times, each for 10 minutes. ⑦ Add the secondary antibody, incubate at room temperature for 30 minutes, wash with 1×PBS for 2 times, each for 10 minutes. ⑧ Incubate with DAB, observe the staining degree under a light microscope, and terminate the color development in time. ⑨ Stain with hematoxylin for 5 minutes, and then differentiate in 1% hydrochloric acid alcohol for a few seconds. ⑩ Put the tissue section into 50%, 75%, 85%, 95%, and 100% ethanol respectively for 5 minutes. Clear with xylene for 2 times, and then seal with neutral balsam. Observe and take photos under an optical microscope. Analyze the IHC average optical density (IOD) and positive area (Area) by using Image J software, and calculate the AOD by the formula AOD = IOD / Area.

[0158] 3、Statistical processing

[0159] SPSS23.0 and GraphPad Prism 8 software were used for statistical analysis and graph drawing. Measurement data were expressed as mean ± standard deviation, and the differences between two independent samples were analyzed by t-test. The differences among multiple samples were analyzed by one-way ANOVA. The differences between non-parameters were analyzed by chi-square test or Fisher's exact probability method. P<0.05 was considered to have a statistically significant difference.

[0160] The scheme proposed by the present application is specifically described below through specific examples:

[0161] Example 1 Basic information of LINC00987

[0162] 1. Localization of LINC00987 in chromosome

[0163] The sequence and location information of LINC00987 on the chromosome were obtained through the UCSC website. The results showed that LINC00987, as a long intergenic non-coding RNA, is located in the 31st sub-band of the short arm p13 region of human chromosome 12, with a length of 22828 bp. For specific location information, see Figure 1 .

[0164] 2. Prediction of LINC00987 protein coding ability

[0165] In this study, the protein coding ability of LINC00987 was predicted by two different algorithms of CPAT and CPC2 databases. The lower the score, the lower the possibility of encoding protein. The classic coding gene 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. The results are shown in Figure 2 , through CPAT and CPC2 database prediction, the protein coding ability value of LINC00987 was 0.0392 and 0.1619, respectively, which was close to the protein coding ability value of the known classic non-coding genes HOTAIR and XIST, and was much lower than the protein coding ability value predicted by the classic protein coding genes GAPDH and ACTB.

[0166] 3. Prediction of LINC00987 secondary structure

[0167] The complex secondary structure of lncRNAs is crucial for their diverse biological functions. By forming specific secondary structures, such as stem-loop structures and pseudoknots, various biological functions can be achieved. Therefore, studying the secondary structure of LINC00987 is of great significance for in-depth exploration and elucidation of its function in the development and progression of breast cancer. This study used the Vienna RNA Network service to predict the secondary structure of LINC00987. The results showed that LINC00987 exhibits complex and unique secondary structural features, including multiple stem-loop structures and hairpin motifs. These structural units are interwoven to form a stable spatial conformation. The minimum free energy of this lncRNA secondary structure was calculated to be -885.40 kcal / mol, indicating high thermodynamic stability. This stability may play an important supporting role in the biological functions of LINC00987, such as its interactions with other molecules and its intracellular localization. Figure 3 ).

[0168] 4. Subcellular localization of LINC00987

[0169] Subcellular localization of lncRNAs plays a crucial role in their function. Therefore, this study selected GAPDH, which is mainly expressed in the cytoplasm, and U6, a small nuclear RNA mainly expressed in the nucleus, as internal controls. The distribution of LINC00987 within cells was detected using nucleocytoplasmic separation experiments. The results showed that in MCF-7 and MDA-MB-231 cells, LINC00987 was mainly expressed in the nucleus, while its expression level in the cytoplasm was relatively low. Figure 4 )

[0170] Furthermore, this invention was validated using fluorescence in situ hybridization (FISH). The LINC000987 nucleic acid probe was labeled with Cy3 fluorescent dye, and the lncRNA sequence was detected and localized by detecting the fluorescence signal (Cy3, red fluorescence). 18S and U6 fluorescent probes were used as references for cytoplasmic and nuclear localization, respectively. FISH results showed that in MCF-7 cells, the fluorescence signal of the LINC00987 probe was mainly concentrated in the nuclear region, suggesting that LINC00987 is primarily localized in the nucleus. This is consistent with the conclusions obtained from the nucleocytoplasmic separation experiment, further confirming that LINC00987 is mainly distributed in the nucleus, while its distribution in the cytoplasm is relatively low. Figure 5 ).

[0171] Example 2: Expression of LINC00987 in breast cancer tissue samples

[0172] To investigate the expression levels of LINC00987 in breast cancer tissues, this invention first analyzed the differences in LINC00987 expression between breast cancer and normal breast tissues using the TCGA and GTEx databases. The unpaired sample differential analysis for breast cancer included 1099 breast cancer tissues and 292 normal breast tissues. Wilcoxon rank-sum test analysis revealed 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 paired sample differential analysis of breast cancer, 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).

[0173] Furthermore, this invention used FISH technology to detect the expression level of LINC00987 in 180 pairs of breast cancer tissues and adjacent normal tissues. The results showed that the expression level of LINC00987 in adjacent normal tissues was significantly higher than that in breast cancer tissues, with positive staining mainly concentrated in the cell nuclei. Figure 7 ).

[0174] Example 3: Relationship between LINC00987 expression level and clinicopathological features of breast cancer patients

[0175] Using the median relative expression level of LINC00987 as the cutoff value, breast cancer cases were divided into a low-expression group and a high-expression group. The differences in clinicopathological parameters between the two groups were analyzed using the chi-square test, and the results are shown in Table 9. Tumor size was used as the cutoff at 4 cm. In the ≤4cm group, 37 patients (41.11%) had low LINC00987 expression, and 55 patients (61.11%) had high expression; in the >4cm group, 53 patients (58.89%) had low expression, and 35 patients (38.89%) had high expression. Statistical analysis showed that the differences were statistically significant (χ²). 2 =7.204, P=0.007). This result indicates a correlation between tumor size and LINC00987 expression level; larger tumors showed a relatively higher proportion of low LINC00987 expression. Furthermore, patients were divided into two groups based on whether they had metastasized. In the non-metastatic group, 38 patients (42.22%) showed low LINC00987 expression, and 69 patients (76.67%) showed high expression. In the metastatic group, 52 patients (57.78%) showed low LINC00987 expression, and 21 patients (23.33%) showed high expression. The difference was statistically significant (χ² test). 2= 22.146, P < 0.001). It showed that the proportion of patients with low expression of LINC00987 was higher in patients with metastasis. According to the tumor stage, patients were divided into stage I, stage II, stage III+IV, 6 cases (6.67%) of stage I patients had low expression of LINC00987, and 46 cases (51.11%) had high expression; 54 cases (60.00%) of stage II patients had low expression of LINC00987, and 42 cases (46.67%) had high expression; 30 cases (33.33%) of stage III+IV patients had low expression of LINC00987, and 2 cases (2.22%) had high expression. Statistical analysis showed χ 2 = 7.204, P < 0.001, the difference was statistically significant. It showed that the tumor stage was significantly related to the expression level of LINC00987, and the later the tumor stage, the higher the proportion of patients with low expression of LINC00987. However, the pathological factors such as age, ER, PR and HER-2 status of patients had no significant correlation with the expression level of LINC00987, and the difference was not statistically significant (P > 0.05).

[0176] Table 9 Relationship between expression level of LINC00987 and clinical pathological characteristics of breast cancer patients

[0177]

[0178] Example 4 Analysis of LINC00987 expression level and prognosis of breast cancer patients

[0179] The above study showed that LINC00987 was lowly expressed in breast cancer and was significantly related to the progression of breast cancer. Therefore, the present application further studied the effect of the lncRNA on the prognosis of breast cancer. According to the median of LINC00987 expression, breast cancer patients were divided into low expression group and high expression group, and the overall survival (OS), disease specific survival (DFS) and progress free interval (PFI) of patients were analyzed to explore the correlation between the expression level of LINC00987 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. With the passage of time (in months), the survival probability of the high expression group and the low expression group of LINC00987 showed a downward trend, and the survival analysis results showed that the hazard ratio (HR) of OS was 0.68, and the 95% confidence interval (95% CI) was 0.49-0.94, which indicated that low expression of LINC00987 was related to poor 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).

[0180] The clinical pathological characteristics of breast cancer patients, such as age, tumor stage, ER, PR, HER-2 status, and the expression level of LINC00987 and other variables that may affect survival were included in the single factor Cox model for analysis (Table 10). The breast cancer patients were divided into two groups according to the age of 60 years old, and the single factor COX analysis was performed. Taking the low age (≤60 years old) group as the reference group, the HR of the high age (>60 years old) group was 2.024, and the 95% CI was 1.47-2.79, P<0.001, indicating that the breast cancer patients in the high age group faced a higher risk of death compared with the patients in the low age group. When the single factor COX regression analysis of tumor staging was performed, the patients in stage III & IV faced a higher risk of death compared with the patients in stage I & II (HR=2.37, 95% CI=1.69-3.32, P<0.001). According to the median of the expression level of LINC00987, the breast cancer patients were divided into LINC00987 low expression group and high expression group. Taking the low expression group as the reference, the single factor COX analysis results showed that the risk ratio of the LINC00987 high expression group was 0.68 (95% CI=0.49-0.939, P=0.019), indicating that the expression level of LINC00987 might be an important factor affecting the risk of breast cancer, and high expression of LINC00987 was a protective factor in the prognosis of breast cancer. In addition, the results of single factor COX regression analysis showed that the risk difference of ER, PR and HER-2 status was not statistically significant (P>0.05).

[0181] Table 10 Single factor Cox analysis of breast cancer patients

[0182]

[0183] On the basis of single factor COX regression analysis, the present application further takes the single factor COX regression variables such as age, tumor stage and LINC00987 expression of breast cancer patients into the multivariate Cox model for analysis (Table 11). With the low age (≤60 years) group as a reference, the HR of the high age (>60 years) group is 2.77 (95% CI=1.66-4.62, P<0.001), which indicates that after considering other factors, the breast cancer patients in the high age group face a higher risk of death relative to the patients in the low age group, and age growth is an independent risk factor for the death risk of breast cancer. In the multivariate COX regression analysis of tumor staging, patients in stage III and IV face a higher risk of death (HR=2.15, 95% CI=1.02-4.50, P<0.001) compared with patients in stage I and II, which shows that late stage disease is an independent risk factor for the death risk of breast cancer. With the low expression group of LINC00987 patients as a control group, the multivariate COX regression analysis shows that the HR of the high expression group is 0.56 (95% CI=0.34-0.94, P=0.028), which indicates that the expression level of LINC00987 can be an independent factor affecting the prognosis of breast cancer, and its high expression can be a key factor to reduce the death risk of breast cancer patients.

[0184] Table 11 Multivariate Cox analysis of breast cancer patients

[0185]

[0186] Example 5 LINC00987 interference and overexpression efficiency

[0187] To further study the role of LINC00987 in breast cancer cells, the present application transfects specific siRNA against LINC00987 into MCF-7 and MDA-MB-231 breast cancer cells, respectively, and sets the cells transfected with siNC as a control group. RT-qPCR experiment is used to detect the relative expression of LINC00987. Compared with the control group, the expression level of LINC00987 in the LINC00987 knockdown group is significantly reduced, and the knockdown efficiency reaches 65% (MCF-7: t=7.11, P=0.002; MDA-MB-231: t=10.24, P<0.001; Figure 9 ). The above results show that transfection of siLINC00987 in MCF-7 and MDA-MB-231 cells can significantly inhibit the expression of the lncRNA.

[0188] Subsequently, MCF-7 and MDA-MB-231 cells were infected with a lentiviral plasmid overexpressing LINC00987, and stable breast cancer cells overexpressing LINC00987 were obtained by puromycin screening. RT-qPCR was used to detect the expression of LINC00987. Compared with the control (Vector) group, the expression level of LINC00987 in the LINC00987 overexpression (LINC00987) group of MCF-7 and MDA-MB-231 cells was significantly up-regulated, 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 show that infection of LINC00987 overexpression plasmid in MCF-7 and MDA-MB-231 cells can significantly increase the expression of the lncRNA.

[0189] Example 6 Effect of LINC00987 on the in vitro proliferation ability of breast cancer cells

[0190] 1) LINC00987 knockdown increases the proliferation ability of breast cancer cells

[0191] The CCK-8 experiment was used to detect the activity of MCF-7 and MDA-MB-231 cells. The results showed that compared with the siNC group, the cell proliferation ability of 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 colony formation ability of MCF-7 and MDA-MB-231 cells after LINC00987 knockdown was detected by colony formation experiment, and the change of cell proliferation ability was further detected. The experimental results were consistent with the CCK-8 experimental results. The number of MCF-7 and MDA-MB-231 cell colonies in the siLINC00987 group was significantly higher than that in the siNC group, and the difference was statistically significant (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 ability of MCF-7 and MDA-MB-231 cells.

[0192] 2) LINC00987 overexpression breast cancer cell proliferation ability decreased

[0193] First, CCK-8 experiment was used to detect the OD value of breast cancer cells overexpressing LINC00987 at a specific time point. The results showed that the proliferation ability of breast cancer cells in the control (vector) group and the LINC00987 overexpression (LINC00987) group showed a gradually increasing trend with the passage of time. However, compared with the control group, the 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 difference was 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 clonogenic ability of breast cancer cells was detected by clonogenic assay. Compared with the control group, the number of colonies of MCF-7 and MDA-MB-231 cells overexpressing LINC00987 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 clonogenic assay experiments confirmed each other, and strongly proved the in vitro inhibitory effect of overexpression of LINC00987 on breast cancer cells.

[0194] 3) LINC00987 overexpression promotes breast cancer cell cycle arrest

[0195] To further explore the reason why LINC00987 inhibits the proliferation of breast cancer cells, flow cytometry (PI staining method) was used to detect the effect of overexpression of LINC00987 on the cell cycle of MCF-7 cells. Compared with the control group, a decrease in the proportion of G1 phase cells and an increase in the proportion of G2 phase cells were observed in MCF-7 cells overexpressing LINC00987, and the transition from G1 phase to G2 phase was blocked. The cell cycle arrest induced by overexpression of LINC00987 was significant, and the difference was statistically significant (P<0.05). Figure 13 From this result, it can be inferred that LINC00987 may inhibit the proliferation of MCF-7 cells by inducing cell cycle arrest.

[0196] Example 7 Effect of LINC00987 on the in vitro migration and invasion ability of breast cancer cells

[0197] The present application uses transwell migration and invasion experiments to evaluate the effect of knockdown or overexpression of LINC00987 on the metastasis of breast cancer cells. In the MCF-7 and MDA-MB-231 cell migration experiments, 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, and statistical analysis further confirmed that the 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 experiment, compared with the control group, the number of cells in the LINC00987 knockdown group of MCF-7 and MDA-MB-231 cells that passed through the transwell chamber aperture coated with Matrigel to the outer bottom wall of the chamber was significantly increased, and the difference was statistically significant (MCF-7: t = 6.51, P = 0.003; MDA-MB-231: t = 8.80, P<0.001; Figure 14B). The above results show that low expression of LINC00987 enhances the migration and invasion abilities of MCF-7 and MDA-MB-231 cells.

[0198] Example 8 Effect of LINC00987 on the in vivo proliferation ability of breast cancer cells

[0199] There is currently no clear evidence on whether LINC00987 also has the ability to inhibit tumor growth in a complex in vivo environment. Therefore, in this part of the experiment, MDA-MB-231 cells in the control group and the LINC00987 overexpression group were injected subcutaneously into the armpit of BALB / c-nu nude mice in the same number, and a nude mouse subcutaneous xenograft tumor model was successfully constructed (n = 6). The subcutaneous tumor formation was observed, and the tumor volume was measured every 3 days. The results are shown in Figures 8A and 8B. Figure 15 A and B, the tumor volume of the LINC00987 overexpression group was significantly smaller than that of the control group. In terms of body weight of nude mice, by collecting and analyzing the body weight data of the two groups of nude mice, it was found that the body weight of the control group and the LINC00987 overexpression group of nude mice had a certain upward trend with the progress of time, and the body weight data trend of the two groups of nude mice was close, and there was no significant difference in the change of body weight (P > 0.05; Figure 15 C). However, during tumor growth, with the passage of time, the tumor volume of the control group continued to increase, while the tumor volume growth of the LINC00987 overexpression group was relatively slow. When the tumor grew to the 10th day, the tumor volume of the LINC00987 overexpression group was significantly smaller than that of the control group, and the 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 show that LINC00987 also has the ability to inhibit the proliferation of breast cancer cells in vivo.

[0200] By observing the tumor cell morphology by HE staining, it can be seen that both the control group and the LINC00987 overexpression group exhibit typical tumor cell morphological characteristics. In contrast, the nuclei in the control group show more mitotic figures, and the number of mitotic figures observed in the nuclei of the LINC00987 overexpression group is relatively small Figure 16 A). The expression level of Ki-67 was detected by IHC technology to evaluate the effect of LINC00987 on the proliferation ability of breast cancer cells in vivo. The results show that in the LINC00987 overexpression group of nude mice tumor tissues, the expression level of Ki-67 is significantly reduced, which indicates that the proliferation activity of tumor cells is weakened, and the difference compared with the control group is statistically significant Figure 16B; 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 the growth rate of tumors.

[0201] The present application discloses the important biological function of LINC00987 in breast cancer, especially its change in cell substructure localization and expression level. First, through the verification of nuclear-cytoplasmic separation experiment and fluorescence in situ hybridization technology, it is confirmed that LINC00987 is to be located 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. Compared with normal breast tissue, the expression of MALAT1 in breast cancer tissue is significantly increased, its high expression is related to the poor prognosis of breast cancer patients, and it is more prone to recurrence 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 promotes the formation of p50 / p65 complex by inhibiting the transcription of NFKBIA and the expression of IκBα, activates the NF-κB signaling pathway, and thus enhances the proliferation and migration ability of breast cancer cells. In addition, HOTAIR is a known nuclear lncRNA, and overexpression of lncRNA HOTAIR induces histone H3 lysine 27 methylation and increases the invasiveness and metastatic ability of cancer in a PRC2-dependent manner. These research results show that the nuclear localization of lncRNA is closely related to its biological function, and may play an anticancer or pro-cancer role by regulating nuclear processes such as transcription regulation, chromatin remodeling, etc. This localization feature suggests that the function of LINC00987 in cells may be mainly through direct regulation of nuclear molecular mechanisms, thereby participating in the occurrence and progression of breast cancer.

[0202] Secondly, the expression level of LINC00987 in breast cancer tissue is significantly lower than that in normal breast tissue, and its low expression is closely related to the progression of breast cancer. Similar to the study of breast cancer, LINC00987 shows abnormal expression in various malignant tumors, including lung cancer, acute myeloid leukemia, and osteosarcoma, etc., and is closely related to the proliferation, migration and invasion ability of these malignant tumors.

[0203] In addition, low expression of LINC00987 is also closely related to the prognosis of breast cancer patients and is considered an independent prognostic risk factor for breast cancer. This result is consistent with the results of studies in other cancer types. In lung adenocarcinoma and glioblastoma, the expression of LINC00987 in cancer tissues is lower than that in normal tissues adjacent to cancer, and its low expression level is closely related to the poor prognosis of patients. In patients with acute myeloid leukemia and osteosarcoma, a higher expression level of LINC00987 is observed, and this high expression is associated with a poor OS of patients with acute myeloid leukemia and osteosarcoma. The importance of LINC00987 in the evaluation of cancer prognosis indicates that it may play a key role in the progression of breast cancer.

[0204] Abnormally expressed lncRNAs can affect the biological functions of tumor cells by regulating complex cell signaling pathways. For example, the overexpressed lncRNA HAGLROS promotes breast cancer cell proliferation, migration by inducing TAM polarization to M2, leading to poor prognosis of breast cancer patients

[60] . The underexpressed lncRNA BC069792 negatively regulates cyclin expression and shows a significant inhibitory effect on breast cancer cells in in vitro and in vivo experiments. At present, it has been confirmed that LINC00987 exhibits a unique expression pattern in several malignant tumors, thereby playing different roles in the biological functions of different malignant tumors. Overexpression of LINC00987 promotes the proliferation and invasion ability of acute myeloid leukemia cells, thereby accelerating the progression of tumors; in osteosarcoma, the silencing of LINC00987 regulates FNBP1 expression by absorbing miR-376a-5p, thereby inhibiting the proliferation, migration and invasion ability of osteosarcoma cells; in lung cancer, LINC00987 inhibits the proliferation, migration and invasion of lung cancer cells by targeting miR-375. In this study, with the help of LINC00987 knockdown and overexpression cell models, combined with a nude mouse xenotransplant tumor model, it is proved that low expression of LINC00987 enhances the proliferation, migration and invasion ability of breast cancer cells; overexpression of LINC00987 significantly slows down the proliferation rate and migration and invasion process of breast cancer cells; overexpression of LINC00987 in breast cancer cells can induce cell cycle arrest, which may be one of the reasons for its inhibition of cell proliferation. The expression of LINC00987 in breast cancer and other malignant tumors is different, and LINC00987 can regulate the proliferation and migration and invasion of malignant tumors through different mechanisms.

[0205] In summary, the present application confirms the important role of LINC00987 in breast cancer, especially in the subcellular localization, expression change, relationship with clinical pathological characteristics and influence on the proliferation and migration and invasion ability of breast cancer cells. LINC00987 may be a key molecular marker for the progression and prognosis of breast cancer.

[0206] The present application explores the distribution, expression characteristics of LINC00987 in breast cancer and its correlation with the clinical pathological characteristics of breast cancer patients; further studies the effect of LINC00987 on the proliferation and migration and invasion ability of breast cancer cells and its relationship with prognosis.

[0207] 1) The results of nucleo-cytoplasmic fractionation experiment and cell FISH show that LINC00987 is mainly located in the nucleus of breast cancer cells;

[0208] 2) Through bioinformatics analysis and tissue FISH experiment, it is found that the expression of LINC00987 in breast cancer tissue is significantly lower than that in normal tissue; its expression is significantly related to tumor size, metastasis and tumor stage;

[0209] 3) LINC00987 is an important independent risk factor for the prognosis of breast cancer patients, and its low expression is significantly related to poor prognosis;

[0210] 4) Low expression of LINC00987 promotes the proliferation, migration and invasion ability of breast cancer cells; while overexpression of LINC00987 inhibits the proliferation, migration and invasion of breast cancer cells and induces cell cycle arrest.

[0211] It should be noted that in this paper, the term "including", "containing" or any other variant thereof is intended to cover non-exclusive inclusion, so that the process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or includes elements inherent to such process, method, article or device. Without more limitation, the element defined by the statement "including a" does not exclude the presence of another identical element in the process, method, article or device including the element.

[0212] The above-mentioned application example serial numbers are only for description, not representing the advantages and disadvantages of the embodiments.

[0213] The above embodiments are only used to illustrate the technical solutions of the present application and not limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the purpose and scope of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. Use of LINC00987 in the preparation of a drug for treating breast cancer.

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