Application of RARRES2 gene in the diagnosis and treatment of brain metastasis of breast cancer

Abstract

The present invention discloses an application of RARRES2 gene in the diagnosis and treatment of brain metastasis of breast cancer. We first compared the gene expression differences between primary tumors of human breast cancer and brain metastases by mining public databases such as GEO and MET500, screened and compared the RARRES2 gene, and compared the expression of the RARRES2 gene in breast cancer bone metastasis, lung metastasis and liver metastasis. Secondly, single-cell transcriptome sequencing was performed on brain metastases of breast cancer patients. The results showed that the expression level of RARRES2 in brain metastasis of breast cancer was significantly lower than that in primary tumors, but the expression of RARRES2 in bone, lung and liver metastases of breast cancer was not significantly different from that in primary tumors, suggesting that it is specifically related to brain metastasis of breast cancer. By constructing RARRES2 overexpression and knockdown human breast cancer brain metastasis cell (MDA-MB-231) lines, it was found that RARRES2 overexpression significantly inhibited the proliferation and invasion of the cells. By constructing a luciferase-labeled RARRES2 overexpression breast cancer brain metastasis mouse model, it was found that RARRES2 overexpression can significantly inhibit the proliferation of breast cancer cells in the brain.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to the application of RARRES2 gene in the diagnosis and treatment of brain metastasis of breast cancer. Background Art

[0002] Breast cancer is the most common malignant tumor in women. According to the latest version of the 2016 China Malignant Tumor Epidemiology Analysis released by the National Cancer Center, the incidence of breast cancer ranks first among female malignant tumors and the mortality rate ranks fourth, which seriously endangers women's health. Among patients with metastatic breast cancer, the brain is one of the common metastatic sites, with an overall incidence of about 14%. Among them, the incidence of brain metastasis of human epidermal growth factor receptor 2 (HER2)-positive breast cancer can be as high as 35-50%, while the incidence of brain metastasis of triple-negative breast cancer can reach 46%. Compared with metastasis to other parts of the body, patients with breast cancer brain metastasis have a shorter survival period and a worse prognosis, with a mortality rate of up to 80% within 1 year. Because its mechanism is still unclear and there is a lack of effective targeted treatment methods, it is a difficult problem in the current field of breast cancer treatment. Therefore, it is urgent to clarify the mechanism of brain metastasis to provide new intervention strategies.

[0003] RARRES2 (retinoic acid receptor responder 2) Retinoic acid receptor responsive protein 2, also known as TIG2 or chemerin, is a multifunctional cytokine that regulates adipogenesis, metabolism, and inflammation by activating chemokine-like receptor 1 (CMKLR1). It can act as a ligand for CMKLR2 and can also bind to CC chemokine receptor-like 2 (CCRL2), but its affinity is lower than that of CMKLR1 or CMKLR2. RARRES2 can also act as an inflammatory adipokine, leading to increased secretion of pro-inflammatory and pro-diabetic adipokines. In recent years, the role of RARRES2 in tumor development has gradually attracted attention because its receptor CMKLR1 has been found to be expressed on the surface of a variety of tumor cells. RARRES2 is often knocked down in breast cancer, melanoma, and prostate cancer tissues compared with adjacent tissues. Previous studies have found that exogenous overexpression of RARRES2 in the tumor microenvironment of melanoma can significantly inhibit tumor growth, and similar phenomena have been observed in non-small cell lung cancer and hepatocellular carcinoma. At the same time, RARRES2 has also been found to have potential diagnostic and prognostic value in tumors such as acute lymphoblastic leukemia and adrenocortical carcinoma. Summary of the invention

[0004] In order to solve the existing clinical bottlenecks and technical problems, we first analyzed and compared the gene expression differences between human breast cancer primary tumors and breast cancer brain metastasis, breast cancer bone metastasis, breast cancer lung metastasis and breast cancer liver metastasis by using public databases such as Gene Expression Omnibus (GEO) MET500. Secondly, single-cell transcriptome sequencing was performed on brain metastasis tumor tissues of breast cancer patients. The analysis and comparison results showed that the expression level of RARRES2 in breast cancer brain metastasis was significantly lower than that in breast cancer primary tumors, while the expression level of RARRES2 in breast cancer bone metastasis, breast cancer lung metastasis and breast cancer liver metastasis was not significantly different from that in breast cancer primary tumors, suggesting that RARRES2 is specifically associated with breast cancer brain metastasis. By constructing a human breast cancer brain metastasis potential cell line (MDA-MB-231) with overexpression and knockdown of RARRES2, it was found that RARRES2 overexpression significantly inhibited the proliferation and invasion of MDA-MB-231 cells. By constructing a luciferase-labeled RARRES2 overexpression breast cancer brain metastasis mouse model, it was found that RARRES2 overexpression can significantly inhibit the proliferation of breast cancer brain metastasis in the brain.

[0005] The object of the present invention is to provide a product for the diagnosis or treatment of brain metastasis of breast cancer, the product comprising a reagent for detecting the expression level of a biomarker, wherein the biomarker is RARRES2.

[0006] The present invention adopts the following technical solutions:

[0007] In one aspect, the present invention provides the use of a reagent for detecting the RARRES2 gene in the preparation of a product for diagnosing tumors.

[0008] Furthermore, the reagents include oligonucleotide probes that specifically recognize the RARRES2 gene, primers that specifically amplify the RARRES2 gene, or binding agents that specifically bind to the protein encoded by the RARRES2 gene.

[0009] "Detecting expression" refers to determining the amount or presence of an RNA transcript or its expression product of an intrinsic gene. Methods for detecting intrinsic gene expression disclosed in the present invention, i.e., gene expression profiling, include methods based on polynucleotide hybridization analysis, methods based on polynucleotide sequencing, immunohistochemical methods, and proteomics-based methods. These methods generally detect expression products (e.g., mRNA) of the intrinsic genes described herein. In a preferred embodiment, PCR-based methods, such as reverse transcription PCR (RT-PCR) and array-based methods such as microarrays are used. "Microarray" refers to an ordered arrangement of hybridizable array elements, such as polynucleotide probes, on a substrate. The term "probe" refers to a molecule that can selectively bind to a particularly intended target biomolecule, such as a nucleotide transcript or protein encoded by or corresponding to an intrinsic gene. The probe can be synthesized by a person skilled in the art, or can be derived from a suitable biological preparation. The probe can be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0010] The oligonucleotide probe for the RARRES2 gene can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives. There is no limit to the length of the probe, as long as it completes specific hybridization and specifically binds to the target nucleotide sequence, any length is fine. The length of the probe can be as short as 25, 20, 15, 13 or 10 bases. Similarly, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or longer, or even the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and the longest is generally no more than 30 base pairs. The length complementary to the target nucleotide sequence is best at 15-25 base pairs. The probe self-complementary sequence is preferably less than 4 base pairs to avoid affecting the hybridization efficiency.

[0011] Furthermore, the tumors include breast cancer, melanoma, prostate cancer, acute lymphoblastic leukemia, and adrenocortical carcinoma.

[0012] Furthermore, the tumor is a brain metastasis of breast cancer.

[0013] Another aspect of the present invention provides a product for diagnosing tumors, characterized in that the product comprises a chip, a kit or a nucleic acid membrane strip capable of detecting the expression level of RARRES2.

[0014] Furthermore, the chip includes a gene chip and a protein chip, the gene chip includes an oligonucleotide probe for the RARRES2 gene for detecting the transcription level of the RARRES2 gene, and the protein chip includes a specific binder for the RARRES2 protein; the kit includes a gene detection kit and a protein detection kit, the gene detection kit includes a reagent or a chip for detecting the transcription level of the RARRES2 gene, and the protein detection kit includes a reagent or a chip for detecting the expression level of the RARRES2 protein.

[0015] The binding agent that specifically binds to the protein encoded by the RARRES2 gene is, for example, a receptor for protein RARRES2, a lectin that binds to protein RARRES2, an antibody to protein RARRES2, a peptide antibody (peptidebody) to protein RARRES2, a bispecific dual binding agent or a bispecific antibody format. Specific examples of specific binding agents are peptides, peptide mimetics, aptamers, spiegelmers, darpins, ankyrin repeat proteins, Kunitz-type domains, antibodies, single domain antibodies and monovalent antibody fragments.

[0016] Furthermore, the kit includes reagents for detecting the expression level of RARRES2 gene or protein by RT-PCR method, qRT-PCR method, biochip detection method, Southern blotting method, in situ hybridization method, and immunoblotting method.

[0017] Another aspect of the present invention provides the use of RARRES2 in constructing a computational model for predicting brain metastases of breast cancer.

[0018] The application of RARRES2 in constructing a computational model for predicting tumors, as known to the skilled artisan, can be implemented and realized in different ways, the step of associating the marker level with a certain probability or risk. In particular, mathematically combining the measured concentrations of the marker and one or more other markers, and associating the combined value with the underlying diagnostic question.

[0019] Preferably, the mathematical algorithm used in the marker combination is a logarithmic function. Preferably, the result of applying such mathematical algorithms or such logarithmic functions is a single value. According to the fundamental diagnostic problem, such values ​​can be easily associated with, for example, the risk of individual breast cancer brain metastasis or with other intended diagnostic purposes that help to assess patients with breast cancer brain metastasis. In a preferred manner, such logarithmic functions are obtained as follows: a) individuals are classified into groups, such as normal people, individuals with breast cancer brain metastasis risk, patients with breast cancer brain metastasis, etc., b) markers with significant differences between these groups are identified by univariate analysis, c) logarithmic regression analysis is used to assess the independent difference values ​​of these different groups of markers, d) logarithmic functions are constructed to combine independent difference values. In this type of analysis, markers are no longer independent, but represent a marker combination. The logarithmic function used to associate marker combinations with diseases preferably adopts an algorithm developed and obtained by applying statistical methods. For example, suitable statistical methods are discriminant analysis (DA) (i.e., linear, quadratic, regular DA), Kernel methods (i.e., SVM), non-parametric methods (i.e., k-nearest neighbor classifier), PLS (partial least squares), tree-based methods (i.e., logistic regression, CART, random forest methods, boosting / bagging methods), generalized linear models (i.e., logarithmic regression), principal component-based methods (i.e., SIMCA), generalized additive models, fuzzy logic-based methods, neural network- and genetic algorithm-based methods. The skilled person will have no problem selecting a suitable statistical method to evaluate the marker combination of the present invention and thereby obtain a suitable mathematical algorithm. In one embodiment, the statistical method used to obtain the mathematical algorithm used in the evaluation of breast cancer is selected from DA (i.e., linear, quadratic, regular discriminant analysis), Kernel methods (i.e., SVM), non-parametric methods (i.e., k-nearest neighbor classifier), PLS (partial least squares), tree-based methods (i.e., logistic regression, CART, random forest methods, boosting methods), or generalized linear models (i.e., logarithmic regression).

[0020] Another aspect of the present invention provides the use of RARRES2 in preparing a pharmaceutical composition for treating tumors.

[0021] Further, the pharmaceutical composition includes a promoter of RARRES2;

[0022] Further, the promoter specifically promotes the expression level of RARRES2;

[0023] Furthermore, the promoter is a RARRES2 overexpression vector or a RARRES2 protein;

[0024] Furthermore, the tumors include breast cancer, melanoma, prostate cancer, acute lymphoblastic leukemia, and adrenocortical carcinoma.

[0025] Furthermore, the pharmaceutical composition inhibits the growth and proliferation of tumor cells, and inhibits the migration of tumor cells.

[0026] The promoter refers to any substance that can increase the activity of RARRES2 protein, improve the stability of RARRES2 gene or protein, upregulate the expression of RARRES2 protein, increase the effective action time of RARRES2 protein, or promote the transcription and translation of RARRES2 gene. These substances can be used in the present invention as substances useful for upregulating RARRES2, and thus can be used to prevent or treat tumors. For example, the promoter includes nucleic acid promoter and protein promoter. The promoter includes but is not limited to a vector that increases the expression of RARRES2, a RARRES2 protein or its active peptide.

[0027] Many suitable vectors are known to those skilled in the art of molecular biology, and their selection depends on the desired function. Non-limiting examples of vectors include plasmids, cosmids, viruses, phages and other conventional vectors used in, for example, genetic engineering. Methods well known to those skilled in the art can be used to construct various plasmids and vectors.

[0028] As an optional mode of the present invention, the vector is a virus. The viral vector is used to introduce a non-endogenous nucleic acid sequence encoding a target-specific polypeptide. The viral vector can be a retroviral vector or a lentiviral vector. The viral vector can also include a nucleic acid sequence encoding a transduction marker.

[0029] Viral vectors suitable for use with the compositions of the invention include those that have been identified for human gene therapy applications. Suitable viral vectors include RNA virus-based vectors, such as retroviral-derived vectors, such as Moloney murine leukemia virus (MLV)-derived vectors, and include more complex retroviral-derived vectors, such as lentiviral-derived vectors. HIV-1-derived vectors belong to this category.

[0030] Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses (e.g., orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus type 1 and type 2 and Epstein-Barr virus and cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, but are not limited to, Norwalk virus, togavirus, flavivirus, reovirus, papillomavirus, hepatitis virus, and hepacivirus. Examples of retroviruses include avian leukosis sarcoma, mammalian C, B, D viruses, HTLV-BLV groups, lentiviruses, and foamy viruses.

[0031] As an optional mode of the present invention, the vector is an expression vector. The expression vector according to the present invention can direct the replication and expression of the nucleic acid molecule of the present invention in a host, and thus ensure the expression of the METTL7A of the present invention encoded thereby in a selected host.

[0032] Non-limiting examples of vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), λgt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (EdgeBiosystems), pTriEx-Hygro (Novagen), and pCINeo (Promega). Non-limiting examples of plasmid vectors suitable for Pichia pastoris include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all Invitrogen). Another vector suitable for expressing proteins in Xenopus embryos, zebrafish embryos, and a variety of mammalian and avian cells is the multi-purpose expression vector pCS2+.

[0033] Another aspect of the present invention provides a pharmaceutical composition for treating tumors, wherein the pharmaceutical composition comprises a promoter of RARRES2.

[0034] Further, the promoter specifically promotes the expression level of RARRES2;

[0035] Furthermore, the promoter is a RARRES2 overexpression vector or a RARRES2 protein;

[0036] Furthermore, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier;

[0037] Furthermore, the tumor is selected from breast cancer, melanoma, prostate cancer, acute lymphoblastic leukemia, and adrenocortical carcinoma.

[0038] Furthermore, the tumor is a brain metastasis of breast cancer.

[0039] Another aspect of the present invention provides the use of RARRES2 in screening candidate drugs for treating tumors;

[0040] Furthermore, the method for screening candidate drugs for treating tumors is as follows: treating a culture system expressing or containing the RARRES2 gene or a protein encoded by it with a substance to be screened; detecting the expression or activity of the RARRES2 gene or a protein encoded by it in the system; wherein, when the substance to be screened promotes the expression level or activity of the RARRES2 gene or a protein encoded by it, the substance to be screened is a candidate drug for treating tumors.

[0041] Furthermore, the tumor is selected from breast cancer, melanoma, prostate cancer, acute lymphoblastic leukemia, and adrenocortical carcinoma.

[0042] Furthermore, the tumor is a brain metastasis of breast cancer.

[0043] Another aspect of the present invention provides a method for screening candidate drugs for treating tumors, comprising treating a culture system expressing or containing the RARRES2 gene or a protein encoded by it with a substance to be screened; detecting the expression or activity of the RARRES2 gene or a protein encoded by it in the system; wherein, when the substance to be screened promotes the expression level or activity of the RARRES2 gene or a protein encoded by it, the substance to be screened is a candidate drug for treating tumors;

[0044] Furthermore, the tumors include breast cancer, melanoma, prostate cancer, acute lymphoblastic leukemia, and adrenocortical carcinoma.

[0045] Furthermore, the tumor is a brain metastasis of breast cancer.

[0046] The term "knockdown" in the present invention is equivalent to "low expression", and the two are synonymous and can be replaced with each other. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Display graphs of bioinformatics data analysis results;

[0048] A is a diagram showing the difference in gene expression between the human breast cancer cell line MDA-MB-231 parental cells and MDA-MB-231BrM obtained by analyzing GSE12237;

[0049] B is the difference in gene expression between MDA-MB-231 xenografted into mouse fat pad and mouse brain obtained by analyzing GSE19184;

[0050] C is a graph showing the difference in gene expression between human breast cancer brain metastases and primary breast cancer tumors obtained by analyzing MET500;

[0051] D is a cross-overlap analysis of differentially expressed genes in breast cancer brain metastasis obtained by analyzing GSE12237, GSE19184, and MET500;

[0052] E is the expression difference map of RARRES2 in pre-injected breast cancer parental cells and breast cancer brain metastases obtained by analyzing MetMap;

[0053] F is the expression difference diagram of RARRES2 in breast cancer bone metastasis, breast cancer brain metastasis, and breast cancer lung metastasis obtained by analyzing GSE14020;

[0054] G is the expression difference diagram of RARRES2 in primary breast cancer, breast cancer lung metastasis, breast cancer brain metastasis, and breast cancer liver metastasis obtained by analyzing GSE62598;

[0055] H is the expression difference diagram of RARRES2 in paired primary breast cancer and breast cancer brain metastasis patients obtained by analyzing GSE10534;

[0056] Figure 2 A graph showing the results of single-cell transcriptional sequencing of brain metastasis tumor tissue from a breast cancer patient;

[0057] A is an experimental data diagram of 13 clusters of specific cell types obtained through single-cell transcriptional sequencing of brain metastasis tumor tissues from breast cancer patients;

[0058] B is the expression heat map of cell markers in each cluster;

[0059] C is a graph showing the difference in single-cell transcriptome expression of RARRES2 in primary breast cancer and breast cancer brain metastasis;

[0060] Figure 3 The results of the proliferation and invasion ability of MDA-MB-231 cell lines with knockdown and overexpression of RARRES2 and the fluorescence imaging of brain metastasis of mice overexpressing RARRES2 are shown;

[0061] A is a graph showing changes in proliferation ability of MDA-MB-231 cells after knockdown of RARRES2;

[0062] B is a statistical graph of the invasion ability of MDA-MB-231 cells with knockdown of RARRES2;

[0063] C is a graph showing the invasion ability of MDA-MB-231 cells with RARRES2 knocked down;

[0064] D is a graph showing changes in proliferation ability of MDA-MB-231 cells overexpressing RARRES2;

[0065] E is a statistical graph of the invasion ability of MDA-MB-231 cells overexpressing RARRES2;

[0066] F is a graph showing the invasion ability of MDA-MB-231 cells overexpressing RARRES2;

[0067] G is the statistical diagram of the fluorescence difference between the brain tumors of mice overexpressing RARRES2 and those of the control group

[0068] H is a fluorescence imaging experiment of brain tumors in mice overexpressing RARRES2 and those in the control group. DETAILED DESCRIPTION

[0069] The present invention is described below by means of specific embodiments, but the present invention is not limited thereto.

[0070] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, biological materials, etc. used in the following examples are all commercially available unless otherwise specified.

[0071] Example 1 RARRES2 expression is reduced in breast cancer brain metastasis

[0072] 1. Bioinformatics data analysis

[0073] By analyzing the Gene Expression Omnibus (GEO) dataset, we compared the parental human breast cancer cell line MDA-MB-231 cells with MDA-MB-231BrM (brain metastasis derivative) (GSE12237) ( Figure 1 A) and MDA-MB-231 cells transplanted into mouse fat pads and brain (GSE19184) Figure 1B) The difference in gene expression levels, DLC1, RARRES2, CASK, PUDP, CYP1B1, KDM6A, LSS, CTSB, ADAM12, COL18A1, BMP4, GNG11, CLDN4, RBM47, LITAF, UBA1, CEMIP, CSF1, CTSD, a total of 19 genes were screened out and down-regulated in MDA-MB-231BrM and mouse brain metastases. We used the MET500 dataset ( Figure 1 C) We further analyzed the differentially expressed genes between human breast cancer brain metastases and primary breast cancer tumors, and compared the expression levels of overlapping genes with the above 19 genes. The results showed that in the three datasets of GSE12237, GSE19184 and MET500, the expression level of RARRES2 in breast cancer brain metastases was significantly reduced compared with primary breast tumors ( Figure 1 D). We used MetMap to analyze the difference in RARRES2 expression levels between pre-injected breast cancer parental cells and breast cancer brain metastases (BCBM). The results showed that RARRES2 expression levels in BCBM were significantly downregulated compared with pre-injected breast cancer parental cells ( Figure 1 E). We used the GSE14020 dataset to compare the expression levels of RARRES2 in breast cancer bone metastasis, breast cancer brain metastasis, and breast cancer lung metastasis. The results showed that the expression level of RARRES2 in the brain was significantly lower than that in the bones and lungs ( Figure 1 F). We used the GSE62598 dataset to compare the expression levels of RARRES2 in primary breast cancer, breast cancer lung metastasis, breast cancer brain metastasis, and breast cancer liver metastasis. The results showed that the expression of RARRES2 in liver, bone, and lung metastases was not reduced compared with primary breast tumors ( Figure 1 G), indicating that its function depends on the microenvironment. We further used the GSE100534 dataset to analyze the difference in RARRES2 expression levels between paired primary breast tumors and breast cancer brain metastases in an independent patient population. The results showed that RARRES2 expression levels were also significantly reduced in breast cancer brain metastases (BCBM) compared with primary breast tumors ( Figure 1 H).

[0074] Example 2 Single-cell transcriptome sequencing and analysis based on breast cancer brain metastases

[0075] 1. Case introduction

[0076] The case is a 48-year-old Chinese woman with breast cancer, which is recorded in the clinical trial IMpassion 131 (NCT03125902). The case was randomly treated with atezolizumab and paclitaxel. After the first and second treatments, the examination results showed that breast cancer had metastasized to the lungs and brain, and the patient then ended the treatment. A week later, she began to experience symptoms of headache, vomiting, and blurred vision. A brain MRI showed a 3.1 cm metastatic mass in the left occipital lobe and compression of the left ventricle. The symptoms were relieved after part of the mass was removed. The removed mass was used for scRNA-seq (single-cell RNA sequencing).

[0077] 2. Tumor sample collection and processing

[0078] Tumor samples from brain metastatic lesions were placed in RNAlater (QIAGEN), an RNA storage solution. The stored tumor samples were placed in RPMI-1640 medium (Gibco) and enzymatically digested using the MACS tumor isolation kit (Miltenyi Biotec) for 60 minutes at 37°C. 10% FBS was added to the RPMI-1640 medium (Invitrogen) containing the enzymatically digested tumor samples and filtered using a 40 μm cell strainer (BD) to obtain a uniform cell suspension. The suspended cells were passed through a cell strainer (BD) and centrifuged at 400g for 10 minutes, and the supernatant was discarded to remove red blood cells. After washing twice with 1xPBS (Invitrogen), the cell pellet was resuspended in sorting buffer (PBS supplemented with 1% FBS).

[0079] 3. Single-cell RNA-seq library construction and sequencing

[0080] The kit was Chromium Next GEM Single cell 5'Kit v2 purchased from 10x Genomics. According to the instruction manual, single cells were washed once with PBS containing 0.04% bovine serum albumin (BSA), and single cells of breast cancer brain metastasis were resuspended in PBS containing 0.04% BSA. The number of cells was determined using a Rigel S2 cell counter (Countstar), and the final concentration was 500-1200 cells / μl. A cell suspension containing 10,000 cells was taken to generate nanoliter gel bead emulsions (GEMs). GEMs were reverse transcribed using a C1000Touch Thermal Cycler (Bio-Rad), and the program was 53°C, 45 minutes; 85°C, 5 minutes; 4°C hold. After the program was completed, DynaBeads and SPRIselect reagents (Thermo Fisher Scientific) were added to the reverse transcription product to further separate and purify the cDNA to obtain purified cDNA. Further PCR amplification to synthesize double-stranded cDNA; purify cDNA to remove possible free nucleotides, enzymes, and buffers; repair and purify the cDNA ends; convert the 3' end of the double-stranded cDNA into adenylic acid; connect the adapter to both ends of the double-stranded cDNA; continue to purify and connect the ds cDNA with the end repair of the adapter; use PCR amplification to enrich the library, using the sequence from the adapter as a primer to amplify the existing sequence; purification; library preparation is completed; the library is verified and quality controlled: the single-cell RNA library is sequenced using the Illumina HiSeq X-Ten sequencer, and the sequencing results are 150bp paired-end reads.

[0081] 4. Results

[0082] We screened a total of 5540 cells from the brain metastasis of this breast cancer patient, performed single-cell transcriptome sequencing and result analysis, and screened an average of 3772 genes in each cell for downstream analysis. Based on the cell markers and cancer-related signaling pathways involved in each cell cluster, 13 clusters were identified as specific cell types, and different clusters were marked with different colors ( Figure 2 A). Based on the differential transcriptional signatures of the cell clusters, a signature expression heat map was created ( Figure 2 B). The difference in RARRES2 expression levels between the single-cell transcriptome sequencing data of primary breast cancer in the GSE118389 dataset and the single-cell transcriptome sequencing data of brain metastases of breast cancer patients was analyzed. After the data were standardized, the analysis results showed that compared with the single-cell transcriptome sequencing results of primary breast tumors, the expression level of RARRES2 in brain metastases of breast cancer was significantly downregulated (p<0.001, Figure 2C).

[0083] Example 3 RARRES2 deficiency promotes brain metastasis formation in vitro and in vivo

[0084] 1. Cell culture and animals

[0085] The human breast cancer cell line MDA-MB-231 with brain metastasis potential, the mouse breast cancer cell lines 4T1-luci and 293TN were purchased from the National Cancer Center / Cancer Hospital of the Chinese Academy of Medical Sciences. 10% fetal bovine serum (HyClone) and DMEM (BIOROC, China) were added to MDA-MB-231 and 293TN and cultured in a CO2 standard incubator at 37°C. 10% fetal bovine serum (HyClone) was added to 4T1-luci and cultured in RPMI-1640 medium. BALB / c female mice were purchased from Beijing Weishenghe Experimental Animal Technology Co., Ltd. (Beijing, China) and maintained with a 12-hour light / dark cycle with normal diet and water.

[0086] 2. Construction of a luciferase-labeled RARRES2 overexpression brain breast tumor mouse model

[0087] According to the technical solution provided by Ozawa T, James CD. Establishing intracranial brain tumor xenografts with subsequent analysis of tumor growth and response to therapy using bioluminescence imaging. J Vis Exp. 2010 (41), 3.0×10 4 Mice with 4T1-luci were injected intracranially. The substrate D-luciferin (150 mg / kg) was injected intraperitoneally into mice that had received 4T1-luci intracranially, and the growth of tumor cells was monitored using IVIS LUMINA XRMS (PerkinLemer), and bioluminescent tumor imaging of mice was observed.

[0088] 3. Plasmid and shRNA knockdown

[0089] The full-length human and mouse RARRES2 genes (NM_002889, NM_027852) were cloned into the pCDH-CMV-MCS-EF1-GFP-PURO vector to generate the RARRES2 overexpression plasmid. shRNA1 and shRNA2 were cloned into the PLKO1-TRC plasmid, respectively. The sequence tables of shRNA1 and shRNA2 are shown in Table 1.

[0090] Table 1 shRNA sequence list

[0091]

[0092] 4. Establishment of stable cell lines

[0093] pLKO1 and pCDH were co-transfected with packaging vectors psPAX2 and pMD2 into 293TN cells, respectively. Lentivirus was obtained after 48 and 72 hours. Tumor cells were transduced with lentivirus using 4 μg / ml polybrene for 48 hours. After transduction, puromycin was added to select and obtain stable RARRES2 knockdown MDA-MB-231 cell lines and RARRES2 overexpression MDA-MB-231 cell lines.

[0094] 5. In vitro invasion assay

[0095] 1) Transwell plate chamber operation steps: add 1.0×10 5 MDA-MB-231 cells were suspended in serum-free medium, a 0.5% matrix gel layer (3422, Corning, USA) was added to the upper chamber of the transwell plate, and serum-free medium containing cells was added to the upper chamber of the transwell plate containing the gel.

[0096] 2) Operation steps of the lower chamber of the transwell plate: 600 μl of DMEM medium containing 20% ​​fetal bovine serum was injected into the lower chamber of the 24-well plate.

[0097] 3) The transwell plate was placed in a standard CO2 incubator at 37°C for 24 hours.

[0098] 4) Take out the transwell plate, rinse twice with PBS, carefully wipe off the cells in the upper layer of the microporous membrane of the chamber with a cotton swab, fix with 100% formaldehyde in a 24-well plate for 15 minutes, and stain with 0.5% crystal violet solution for 15 minutes.

[0099] 5) Take photos under an inverted microscope, randomly count 10 fields of view for each sample, take the average value, and perform statistical analysis. Use ImageJ software to calculate the relative area occupied by cells that pass through the membrane. All the above analyses were repeated three times.

[0100] 6. Statistical analysis

[0101] Statistical analysis was performed using GraphPad Prism 8.0. Data are presented as mean ± standard deviation (SD). Student's t-test was used to assess statistical significance. The significance of mean differences between experimental groups is indicated on the graphs as: ns, not significant, *p<0.05, **p<0.01, ***p<0.001.

[0102] 7. Results

[0103] We used shRNA (short hairpin RNA) interference technology to design shRNA1 and shRNA2 sequences and constructed the RARRES2 knockdown MDA-MB-231 cell line. By comparing the normal MDA-MB-231 cell line with the RARRES2 knockdown MDA-MB-231 cell line, we found that knockdown of RARRES2 significantly promoted cell proliferation and invasiveness ( Figure 3 A, 3B, 3C). By comparing the normal MDA-MB-231 cell line with the RARRES2 overexpressing MDA-MB-231 cell line, it was found that RARRES2 overexpression significantly inhibited cell proliferation and invasiveness ( Figure 3 D, 3E, 3F). By constructing a luciferase-labeled RARRES2 overexpression breast cancer brain metastasis mouse model, it was found that RARRES2 overexpression can significantly inhibit the proliferation of breast cancer brain metastases in the brain ( Figure 3 G, 3H).

[0104] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention. Sequence Listing <110> Cancer Hospital of Chinese Academy of Medical Sciences <120> Application of RARRES2 gene in the diagnosis and treatment of brain metastasis of breast cancer <141> 2022-05-18 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 54 <212> DNA <213> Artificial Sequence <400> 1 gcccttccca gctggaatat tctcgagaat attccagctg ggaagggctt tttt 54 <210> 2 <211> 54 <212> DNA <213> Artificial Sequence <400> 2 gcttctactt ccctggacag tctcgagact gtccaggga gtagaagctt tttt

Claims

1. Use of a reagent for detecting the RARRES2 gene in the preparation of a product for diagnosing brain metastases of breast cancer; the application distinguishes brain metastases of breast cancer from primary breast cancer and other breast cancer metastases, wherein the other breast cancer metastases are lung metastases of breast cancer, bone metastases of breast cancer, and liver metastases of breast cancer.

2. The use according to claim 1, wherein the reagent comprises an oligonucleotide probe that specifically recognizes the RARRES2 gene, or a primer that specifically amplifies the RARRES2 gene.

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