Gene group for breast cancer molecular typing, detection product and application thereof

Abstract

The invention discloses a gene group for breast cancer molecular typing, a detection product and application of the gene group and the detection product, and relates to the technical field of breast cancer tumor gene detection. Comprising the following steps: S1, collecting clinical tissue specimens, including cancer tissues and para-carcinoma tissues, coming from breast cancer patients receiving breast resection, and storing the tissue specimens in liquid nitrogen; s2, cell culture: culturing a breast cancer cell line by using a DMEM culture medium containing 10% of fetal calf serum; the relation between high expression of circACTN4 in breast cancer and clinical pathological characteristics of circACTN4 is proved through multiple methods such as gene chip analysis, qRT-PCR, cell function experiments and animal experiments, and circACTN4 promotes acetylation of histone H4 and increases expression of cell cycle related genes (such as CCNE1 and CDK4) by up-regulating expression of MYC, so that development of breast cancer is promoted; a new molecular marker and a potential treatment target are provided for diagnosis and treatment of breast cancer, and development of new diagnosis products and treatment strategies is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of breast cancer tumor gene detection, and in particular to a gene group, detection product and application thereof for molecular typing of breast cancer. Background Art

[0002] Breast cancer is the most common malignancy in women worldwide, ranks first in the incidence of female cancer, and is the leading cause of cancer death in women worldwide. Although important advances have been made in the diagnosis and treatment of breast cancer in recent decades, its incidence has increased significantly. Breast cancer accounts for approximately 30% of all cancer cases and 15% of cancer deaths in women. Therefore, there is an urgent need to identify new potential molecular targets for cancer diagnosis and treatment;

[0003] Among them, circular RNA (circRNAs) is a type of closed circular single-stranded RNA, which is formed by reverse splicing of linear precursor RNA and does not have a 5' cap structure and a 3' polyadenylic acid tail. Due to its circular structure, circRNAs are more resistant to degradation by nucleases than linear RNAs and are therefore more stable. Increasing evidence shows that some circRNAs are abnormally expressed in a variety of tumors. CircRNAs are associated with tumor occurrence, development and immune escape, suggesting that they may have important functions. CircRNAs can exert their functions by adsorbing microRNAs (miRNAs), interacting with RNA-binding proteins (RBPs), and translating proteins. Studies have reported that some circRNAs play a key role in breast cancer progression. For example, Circ_0000520 promotes triple-negative breast cancer progression by mediating the miR-1296 / ZFX axis. In addition, the EIF6-224 amino acid protein encoded by circ-EIF6 promotes the progression of triple-negative breast cancer by stabilizing myosin heavy chain 9 (MYH9) and activating the Wnt / β-catenin signaling pathway. Furthermore, circ_0001667 promotes breast cancer cell proliferation and angiogenesis by regulating the miR-6838-5p / CXCL10 axis. However, there is currently a lack of methods for using circACTN4 to detect MYC expression and then apply it to cancer diagnosis and treatment. Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a gene group for molecular typing of breast cancer, a detection product and its application.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] An application of a gene group and a detection product for molecular typing of breast cancer comprises the following steps:

[0007] S1: Clinical tissue specimens, including cancerous tissue and adjacent tissue, were collected from breast cancer patients who underwent mastectomy and stored in liquid nitrogen;

[0008] S2: Cell culture, breast cancer cell lines were cultured in DMEM medium containing 10% fetal bovine serum, and normal breast epithelial cell lines were cultured in MEBMBulletKit medium, and cultured at 37°C and 5% carbon dioxide;

[0009] S3: Gene chip analysis, total RNA was extracted from breast cancer tissues and paired normal tissues, circular RNA was enriched, and gene chip analysis was performed to identify the expression of circACTN4;

[0010] S4: Fluorescence in situ hybridization (FISH), using a specific probe for circACTN4 to detect the localization and expression of circACTN4 in breast cancer tissues;

[0011] S5: plasmid construction, RNA interference and transfection, construction of circACTN4 overexpression vector and synthesis of small interfering RNA targeting circACTN4, and cell transfection;

[0012] S6: Cell proliferation assay, using CCK-8 and EdU assay to detect the proliferation ability of breast cancer cells;

[0013] S7: Cell migration and invasion assay, using Transwell chamber to detect the migration and invasion ability of breast cancer cells;

[0014] S8: qRT-PCR detection, extracting total RNA from tissues and cells, and detecting the expression level of circACTN4;

[0015] S9: Cell cycle analysis, using flow cytometry to detect cell cycle distribution;

[0016] S10: Western blotting, detecting the expression levels of related proteins in breast cancer cells;

[0017] S11: Animal experiment, breast cancer cells infected with different lentiviruses were inoculated into nude mice to observe tumor growth;

[0018] S12: Immunohistochemical staining to detect the expression of related proteins in transplanted tumor tissues;

[0019] S13: Histone H4 acetylation detection, using fluorescence method to detect the acetylation level of histone H4 in breast cancer cells;

[0020] S14: Chromatin immunoprecipitation (ChIP) assay to detect the binding of MYC to the promoters of cell cycle-related genes and the acetylation of histone H4;

[0021] S15: Co-immunoprecipitation (co-IP) experiment to detect the binding of MYC and TIP60;

[0022] S16: Statistical analysis: perform data analysis on the experimental results.

[0023] Preferably, the breast cancer tissues and adjacent tissues collected in step S1 are from at least 50 breast cancer patients who underwent mastectomy.

[0024] Further: The breast cancer cell lines used in step S2 include MDA-MB-231 and MDA-MB-453, and the normal breast epithelial cell line is MCF-10A.

[0025] Further: The transfection efficiency of the circACTN4 overexpression vector and the small interfering RNA targeting circACTN4 constructed in step S5 was verified by qRT-PCR.

[0026] As a preferred solution of the present invention: the nude mice used in step S11 are 4-week-old female BALB / c nude mice, and each group contains at least 6 nude mice.

[0027] As a further solution of the present invention: in step S13, the ratio of acetylated histone H4 is proportional to the fluorescence intensity, and the absolute amount of acetylated histone H4 is quantified by standard control.

[0028] As a further solution of the present invention: in step S14, anti-MYC, anti-acetylated histone H4 or IgG antibodies are used to perform ChIP experiments, and the enrichment of the promoter region of the relevant gene is detected by qRT-PCR.

[0029] On the basis of the previous protocol: In step S15, antibodies or IgG against MYC and TIP60 were used for co-immunoprecipitation experiments, and specifically bound proteins were detected by western blotting.

[0030] On the basis of the above scheme: Statistical analysis in step S16, statistical analysis was performed on the data generated in the above steps, expressed as mean ± standard deviation (SD), and differences were compared using two-sided Student's t test, one-way analysis of variance (ANOVA) and chi-square test.

[0031] A gene group for molecular typing of breast cancer, wherein the gene group is circACTN4.

[0032] The beneficial effects of the present invention are:

[0033] 1. A gene group, detection product and application for molecular typing of breast cancer. Through gene chip analysis, qRT-PCR, cell function experiments, animal experiments and other methods, the relationship between the high expression of circACTN4 in breast cancer and its clinical pathological characteristics is proved. circACTN4 promotes the acetylation of histone H4 and increases the expression of cell cycle-related genes (such as CCNE1 and CDK4) by upregulating the expression of MYC, thereby promoting the development of breast cancer. It provides new molecular markers and potential therapeutic targets for the diagnosis and treatment of breast cancer, which is helpful for the development of new diagnostic products and treatment strategies.

[0034] 2. A gene group, detection product and application for molecular typing of breast cancer. Through CCK-8, EdU, Transwell and other cell function experiments and animal experiments, it is proved that circACTN4 can promote the proliferation, migration and invasion of breast cancer cells, indicating that it plays an important role in promoting cancer in the development of breast cancer.

[0035] 3. A gene group, detection product and its application for molecular typing of breast cancer, gene chip analysis, qRT-PCR, cell function experiments, animal experiments, immunohistochemistry, ChIP, Co-IP and other technical means have comprehensively revealed the role and mechanism of circACTN4 in breast cancer, providing multi-dimensional evidence support for the research of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a schematic diagram of a detection process in the application of a gene group and a detection product for molecular typing of breast cancer proposed by the present invention;

[0037] Figure 2 It is a gene group for molecular typing of breast cancer proposed by the present invention, and a correlation diagram of high expression of CircACTN4 and breast cancer tissue with clinical pathological characteristics in the application of detection products;

[0038] Figure 3 The invention proposes a gene group for molecular typing of breast cancer, and an application of a detection product in which CircACTN4 enhances the migration and invasion ability of breast cancer cells and a cell cycle progression diagram;

[0039] Figure 4 It is a gene group for molecular typing of breast cancer proposed by the present invention, and an H4 acetylation map of CircACTN4 promoting MYC expression and MYC target gene in the application of the detection product;

[0040] Figure 5The invention proposes a gene group for molecular typing of breast cancer and an application of a detection product to reverse the upregulation or loss of MYC and induce the knockdown or overexpression of circACTN4. Figure 2 . DETAILED DESCRIPTION

[0041] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0042] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0043] Embodiment 1:

[0044] A gene group, detection product and application thereof for molecular typing of breast cancer, such as Figure 1 As shown, the following steps are included:

[0045] S1: Clinical tissue specimen collection; the collected tissue specimens include cancerous tissue and adjacent tissue, which are from breast cancer patients who have undergone mastectomy. The tissue specimens are immediately stored in liquid nitrogen after surgery until they are taken out for subsequent experiments;

[0046] S2: Cell culture; breast cancer cell lines MDA-MB-231 and MDA-MB-453 from the obtained tissue specimens were cultured in DMEM medium with 10% fetal bovine serum, and normal breast epithelial cell line MCF-10A was cultured in MEBMBulletKit medium. All cell culture media were supplemented with 1% penicillin / streptomycin and cultured at 37°C and 5% carbon dioxide;

[0047] S3: Gene chip analysis; total RNA was extracted from 3 pairs of breast cancer and matched normal tissues using TRIzol reagent, quantified, and linear RNA was removed by RNaseR digestion to enrich circular RNA; then, the Arraystar standard protocol was used for sample preparation and gene chip hybridization, and the Arraystar human circular RNA chip V2 was used for gene chip analysis of circular RNA;

[0048] Genetic analysis showed that circular RNAs (circRNAs) were differentially expressed between breast cancer tissues and paired adjacent paracancerous tissues. According to the circBase database, circACTN4 is located in the exon 2 to exon 7 region of the ACTN4 gene on human chromosome 19 and generates a covalently closed RNA molecule of 571 nucleotides through reverse splicing;

[0049] S4: Fluorescence in situ hybridization (FISH); using a Cy3-labeled specific probe for circACTN4 (5'Cy3-GCCGTGAAGGTCCTCTGCATC-3'Cy3); cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI), and images were acquired using an Olympus BX51 fluorescence microscope;

[0050] Fluorescence in situ hybridization results showed that circACTN4 was highly expressed in breast cancer tissues compared with paired adjacent adjacent tissues, and was mainly distributed in the nuclei of breast cancer cells;

[0051] S5: Plasmid construction, RNA interference and transfection; insert the full-length human circACTN4 into the pLC5-ciR vector to construct a circACTN4 overexpression vector; the control group is an empty vector without circACTN4 sequence;

[0052] Subsequently, small interfering RNAs targeting circACTN4 (si-circ#1, si-circ#2) and negative control small interfering RNAs (NCsiRNAs) were synthesized to knock down circACTN4;

[0053] The human circACTN4 gene was inserted into the lentiviral vector CV146, and si-circ#1 was subcloned and inserted into the lentiviral vector GV344. Each group of lentiviral vectors was transfected into HEK293T cells using Lipofectamine2000. After 72 hours, the viral supernatant was collected for animal experiments. Then, the human MYC gene was inserted into the vector GV658 to construct an overexpression vector, and stably transfected cells were selected with puromycin.

[0054] S6: CCK-8 and EdU experiments; breast cancer cells were inoculated into 96-well plates, with 3 replicate wells and 2.0×10 3 breast cancer cells were incubated with CCK-8 reagent for 1 hour at the specified time, and the absorbance value at 450nm wavelength was recorded at the specified time point; breast cancer cells were then seeded into 24-well plates for 5-ethynyl-2'-deoxyuridine (EdU) experiments, and cells were stained with EdU reagent and 4',6-diamidino-2-phenylindole (DAPI), and cell proliferation signals were captured under a fluorescence microscope;

[0055] S7: Transwell migration and invasion assay: 500 μl of complete culture medium was added to the lower chamber of the Transwell chamber, and then 2×10 4 Breast cancer cells were added to the upper chamber and incubated in an incubator containing 5% carbon dioxide for 24 hours; the cells were then fixed with 75% ethanol and stained with crystal violet solution;

[0056] In the invasion assay, 50 μl of Matrigel diluted with serum-free medium was added to the upper chamber of the Transwell chamber and incubated at 37°C for 1 hour for treatment.

[0057] S8: qRT-PCR detection; total RNA was extracted from tissues and cells using TRIzol reagent and analyzed using PARIS TM Kit was used to isolate RNA from the nucleus and cytoplasm of MCF-7 cells, reverse transcription experiments were performed using the PrimeScriptRT kit, and qRT-PCR was performed using TBgreenpremixEx-Taq. Relative expression levels were calculated by the 2-ΔCT method;

[0058] The expression of circACTN4 in 50 pairs of breast cancer tissues and adjacent tissues was analyzed, indicating that the expression of circACTN4 in breast cancer tissues was significantly upregulated; in addition, qRT-PCR results also showed that the expression of circACTN4 in breast cancer cells was significantly higher than that in the normal breast epithelial cell line MCF-10A;

[0059] S9: Cell cycle analysis; collect 10 6 The cells were fixed with 70% ethanol at 4°C overnight, stained with propidium iodide (PI), and then detected by flow cytometry;

[0060] S10: Western blotting; the content of extracted proteins was determined by 10% SDS-PAGE, and the separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes; after blocking with 6% skim milk powder, the membranes were incubated with primary antibodies against MYC, CDK4, and CCNE1 at 4°C overnight, and then incubated with goat anti-rabbit or goat anti-mouse secondary antibodies at room temperature for 1.5 hours; the bands were detected by enhanced chemiluminescence detection system;

[0061] S11: Animal experiment: 4-week-old female BALB / c nude mice were subcutaneously inoculated with 7 The length and width of the tumor were measured every 7 days, and the volume of each tumor was calculated according to the following formula:

[0062] Length × Width 2 / 2

[0063] After 28 days, the nude mice in each group were sacrificed, and the tumors were collected for immunohistochemistry and pathological analysis;

[0064] S12: Immunohistochemical staining; paraffin-embedded tumor tissue sections were dewaxed with xylene and rehydrated in graded alcohol. Tissue sections were incubated with antibodies against MYC, CCNE1, and CDK4 at 4°C overnight, and then incubated with secondary antibodies at 37°C for 1 hour. Sections were stained with DAB and hematoxylin and photographed under a microscope;

[0065] S13: Histone H4 acetylation detection; MDA-MB-231 cells were lysed and used EpiQuik TM Histones were extracted using a total histone extraction kit;

[0066] EpiQuik Total Histone H4 Acetylation Fluorescence Rapid Detection Kit was used to detect total histone H4 acetylation; the detection steps were as follows: acetylated histone H4 (AcH4) was bound to the detection wells coated with anti-acetylated histone H4 antibodies, and after washing, the labeled detection antibodies were added to the wells, followed by the addition of fluorescent colorimetric reagents; and then the fluorescence was measured at 530EX / 590EMnm using a fluorescent microplate reader;

[0067] The ratio of acetylated histone H4 is proportional to the fluorescence intensity, and the absolute amount of acetylated histone H4 can be quantified by comparing with the standard control;

[0068] S14: Chromatin immunoprecipitation (ChIP) experiment; 1×10 8 MDA-MB-231 cells were lysed and sonicated to produce DNA fragments of 200-1000 bp; the samples were then incubated with anti-MYC (1:100), anti-acetylated histone H4 (1:50) or IgG antibodies at 4°C for 14 hours; ChIP-grade protein A / G magnetic beads were incubated with the above antibody-chromatin complexes at 4°C for 2 hours. DNA was eluted and purified, and then qRT-PCR was performed;

[0069] It was found that in MDA-MB-231 cells transfected with circACTN4 overexpression plasmid and small interfering RNA targeting circACTN4, the expression of circACTN4 had no effect on the binding of MYC to the CDK4 and CCNE1 gene promoters;

[0070] ChIP-qPCR experiments were performed using anti-AcH4 antibodies, and the results showed that upregulation of circACTN4 significantly enhanced the AcH4 binding signal in the promoter regions of cell cycle-related genes CDK4 and CCNE1, while downregulation of circACTN4 significantly reduced the enrichment of AcH4 in the promoter regions of CDK4 and CCNE1;

[0071] Histone H4 acetylation was detected by fluorescence using anti-AcH4 antibody, and fluorescence was measured at 530EX / 590EMnm using a fluorescence microplate reader. The results showed that circACTN4 overexpression significantly increased the relative level of AcH4, while circACTN4 knockdown reduced the expression of AcH4.

[0072] S15: Co-immunoprecipitation (co-IP); Antibodies (1:100) or IgG against MYC and TIP60 were cross-linked to magnetic beads at 4°C for 4 hours; MDA-MB-231 cells were then lysed in IP lysis buffer using the PierceTM Classic Magnetic IP / co-IP Kit. Antibody-coated magnetic beads were mixed with cell lysate at 4°C overnight; proteins specifically bound to the antibody-magnetic bead complex were eluted with sample buffer and then detected by western blotting;

[0073] S16: Statistical analysis; The data generated in the above steps were statistically analyzed using GraphPad Prism 8.0 software and SPSS 21.0 software. The results were expressed as mean ± standard deviation (SD). The differences between two or more groups were compared using two-sided Student's t test, one-way analysis of variance (ANOVA) and chi-square test; P < 0.05 was considered statistically significant.

[0074] Circular RNAs (circRNAs) were differentially expressed between breast cancer tissues and paired paracancerous tissues. Abnormal expression of circACTN4 was observed in breast cancer tissues, and the expression of circACTN4 was significantly upregulated in breast cancer tissues. qRT-PCR results showed that the expression of circACTN4 in breast cancer cells was significantly higher than that in the normal breast epithelial cell line MCF-10A. FISH results showed that circACTN4 was highly expressed in breast cancer tissues compared with paired paracancerous tissues, and was mainly distributed in the nuclei of breast cancer cells. These results indicate that circACTN4 is significantly highly expressed in breast cancer and is a marker for the diagnosis and treatment of breast cancer patients. The data showed that the expression of circACTN4 was positively correlated with T stage (n=50, P=0.023) and TNM stage (n=50, P<0.001).

[0075] In this example, in step S5, a circACTN4 overexpression plasmid and a small interfering RNA targeting circACTN4 were added, and the transfection efficiency was verified by qRT-PCR. The results showed that circACTN4 was significantly knocked down or overexpressed in MDA-MB-231 and MDA-MB-453 cells. Subsequently, after circACTN4 was overexpressed or knocked down, the EdU and CCK-8 experiments in step S6 showed that circACTN4 overexpression significantly increased cell viability, while the loss of circACTN4 significantly inhibited the proliferation ability of breast cancer cells.

[0076] And in the subsequent 1×10 7 .The experimental results of MDA-MB-231 cells infected with circACTN4 shRNA lentivirus, overexpressing circACTN4 lentivirus and control group cells were subcutaneously inoculated into 4-week-old female BALB / c nude mice. The results showed that compared with the control group, circACTN4 overexpression significantly increased the volume and weight of xenograft tumors, while circACTN4 knockdown significantly inhibited tumor growth, indicating that circACTN4 may play a carcinogenic role in the development of breast cancer; indicating that CircACTN4 promotes the proliferation and growth of breast cancer cells both in vitro and in vivo.

[0077] In this example, the results of the Transwell experiment in step S7 showed that, regardless of whether Matrigel was used or not, the upregulation of circACTN4 significantly enhanced the invasion and migration abilities of breast cancer cells, while the downregulation of circACTN4 significantly reduced these abilities. The cell cycle distribution was detected by flow cytometry, and it was found that circACTN4 knockdown resulted in a decrease in S phase cells and an increase in G1 phase cells, indicating that breast cancer cells were arrested in the G1 phase.

[0078] The results of Western blotting in step S15 showed that the levels of cell cycle-related proteins CCNE1 and CDK4 were significantly reduced in breast cancer cells with circACTN4 knockdown;

[0079] In this example, immunohistochemistry (IHC) detected the effect of circACTN4 on the expression of MYC, CCNE1 and CDK4 in transplanted tumor tissues, indicating that upregulation of circACTN4 can increase the expression of MYC, CCNE1 and CDK4, while silencing circACTN4 reduces the expression of these proteins; indicating that CircACTN4 can enhance the migration, invasion ability and cell cycle progression of breast cancer cells;

[0080] At the same time, after knockdown or overexpression of circACTN4, the expression of MYC was detected by qRT-PCR and protein immunoblotting, showing that forced expression of circACTN4 significantly upregulated the expression of MYC, while circACTN4 deficiency produced the opposite effect;

[0081] Co-immunoprecipitation (co-IP) experiments were performed using anti-MYC or anti-TIP60 antibodies, and the results showed that MYC could bind to TIP60 in MDA-MB-231 cells;

[0082] These results suggest that circACTN4 promotes the recruitment of TIP60 to MYC target genes and AcH4 by upregulating MYC expression. MYC may regulate the transcription of cell cycle-related genes CDK4 and CCNE1 by indirectly mediated by TIP60-mediated histone H4 acetylation in target chromatin.

[0083] In this example, knockdown of MYC significantly reversed the promoting effect of circACTN4 upregulation on breast cancer cell proliferation, migration and invasion detected by CCK8, EdU and Transwell assays, while overexpression of MYC in breast cancer cells rescued the inhibitory effect of circACTN4 silencing; protein immunoblotting analysis showed that upregulation or downregulation of MYC could offset the decrease or increase in CCNE1 and CDK4 expression caused by circACTN4 knockdown or overexpression; fluorescence analysis of histone H4 acetylation revealed that MYC overexpression or knockdown reversed the decrease or increase in the relative level of AcH4 mediated by circACTN4 deletion or ectopic expression, respectively;

[0084] These results suggest that circACTN4 promotes breast cancer cell cycle progression and tumorigenesis through MYC-induced histone H4 acetylation, and that upregulation or loss of MYC reverses the biological functions induced by circACTN4 knockdown or overexpression.

[0085] The present invention reveals the relationship between the high expression of circACTN4 in breast cancer and its clinical pathological characteristics through multiple methods such as gene chip analysis, qRT-PCR, cell function experiments, and animal experiments, and explains the mechanism of action of circACTN4 in promoting breast cancer cell proliferation, migration, and invasion. circACTN4 upregulates the expression of MYC, thereby promoting the acetylation of histone H4 and increasing the expression of cell cycle-related genes (such as CCNE1 and CDK4), thereby promoting the development of breast cancer; it provides new molecular markers and potential therapeutic targets for the diagnosis and treatment of breast cancer, and is helpful for the development of new diagnostic products and treatment strategies.

[0086] The above is a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any technician familiar with the field within the technical scope disclosed by the present invention in combination with the prior art or public common sense, within the spirit and principle of the present invention, shall be covered by the protection scope of the present invention.

Claims

1. An application of a gene group and a detection product for molecular typing of breast cancer, characterized in that: The steps include: S1: Clinical tissue specimens, including cancerous tissue and adjacent tissue, were collected from breast cancer patients who underwent mastectomy and stored in liquid nitrogen; S2: Cell culture, breast cancer cell lines were cultured in DMEM medium containing 10% fetal bovine serum, and normal breast epithelial cell lines were cultured in MEBMBulletKit medium, and cultured at 37°C and 5% carbon dioxide; S3: Gene chip analysis, total RNA was extracted from breast cancer tissues and paired normal tissues, circular RNA was enriched, and gene chip analysis was performed to identify the expression of circACTN4; S4: Fluorescence in situ hybridization (FISH), using a specific probe for circACTN4 to detect the localization and expression of circACTN4 in breast cancer tissues; S5: plasmid construction, RNA interference and transfection, construction of circACTN4 overexpression vector and synthesis of small interfering RNA targeting circACTN4, and cell transfection; S6: Cell proliferation assay, using CCK-8 and EdU assay to detect the proliferation ability of breast cancer cells; S7: Cell migration and invasion assay, using Transwell chamber to detect the migration and invasion ability of breast cancer cells; S8: qRT-PCR detection, extracting total RNA from tissues and cells, and detecting the expression level of circACTN4; S9: Cell cycle analysis, using flow cytometry to detect cell cycle distribution; S10: Western blotting, detecting the expression levels of related proteins in breast cancer cells; S11: Animal experiment, breast cancer cells infected with different lentiviruses were inoculated into nude mice to observe tumor growth; S12: Immunohistochemical staining to detect the expression of related proteins in transplanted tumor tissues; S13: Histone H4 acetylation detection, using fluorescence method to detect the acetylation level of histone H4 in breast cancer cells; S14: Chromatin immunoprecipitation (ChIP) assay to detect the binding of MYC to the promoters of cell cycle-related genes and the acetylation of histone H4; S15: Co-immunoprecipitation (co-IP) experiment to detect the binding of MYC and TIP60; S16: Statistical analysis: perform data analysis on the experimental results.

2. The use of a gene group and a detection product for molecular typing of breast cancer according to claim 1, characterized in that: The breast cancer tissues and adjacent cancer tissues collected in step S1 are from at least 50 breast cancer patients who underwent mastectomy.

3. The use of a gene group and a detection product for molecular typing of breast cancer according to claim 1, characterized in that: The breast cancer cell lines used in step S2 include MDA-MB-231 and MDA-MB-453, and the normal breast epithelial cell line is MCF-10A.

4. The use of a gene group and a detection product for molecular typing of breast cancer according to claim 1, characterized in that: The transfection efficiency of the circACTN4 overexpression vector and the small interfering RNA targeting circACTN4 constructed in step S5 was verified by qRT-PCR.

5. The use of a gene group and a detection product for molecular typing of breast cancer according to claim 1, characterized in that: The nude mice used in step S11 are 4-week-old female BALB / c nude mice, and each group contains at least 6 nude mice.

6. The use of a gene group and a detection product for molecular typing of breast cancer according to claim 1, characterized in that: In step S13, the ratio of acetylated histone H4 is proportional to the fluorescence intensity, and the absolute amount of acetylated histone H4 is quantified by standard control.

7. The use of a gene group and a detection product for molecular typing of breast cancer according to claim 1, characterized in that: In step S14, anti-MYC, anti-acetylated histone H4 or IgG antibodies are used to perform ChIP experiments, and the enrichment of the promoter regions of the relevant genes is detected by qRT-PCR.

8. The use of a gene group and a detection product for molecular typing of breast cancer according to claim 1, characterized in that: In step S15, an immunoprecipitation experiment is performed using antibodies or IgG against MYC and TIP60, and the specifically bound proteins are detected by western blotting.

9. The use of a gene group and a detection product for molecular typing of breast cancer according to claim 1, characterized in that: In step S16, statistical analysis is performed on the data generated in the above steps, expressed as mean ± standard deviation (SD), and differences are compared using two-sided Student's t test, one-way analysis of variance (ANOVA) and chi-square test.

10. A gene group for molecular typing of breast cancer according to any one of claims 1 to 9, characterized in that: The gene group is circACTN4.

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