Application of TRIM4 as a prognostic and tamoxifen resistance marker for ER-positive breast cancer

Abstract

The present application belongs to the field of medicinal and molecular biology technology, and particularly relates to the application of TRIM4 as a marker for judging the prognosis of ER-positive breast cancer and tamoxifen resistance. The present application finds that the expression level of TRIM4 is related to tamoxifen resistance of breast cancer, and the expression level of TRIM4 in breast cancer cell lines with tamoxifen resistance is significantly reduced. Meanwhile, the prognosis of breast cancer patients with high expression of TRIM4 is significantly higher than that of breast cancer patients with low expression of TRIM4. This shows that TRIM4 can be used for the detection of breast cancer with tamoxifen resistance, and TRIM4 can also be used for predicting the prognosis level of breast cancer patients. In addition, the present application finds that the protein encoded by TRIM4 can degrade SET protein in tumor cells with tamoxifen resistance, which shows that TRIM4 provides a potential target for the treatment of breast cancer in clinic, improves the treatment effect of breast cancer, and has important clinical significance and social benefits.

Description

Technical Field

[0001] This invention belongs to the fields of herbal medicine and molecular biology, specifically involving the application of TRIM4 as a prognostic marker for ER-positive breast cancer and a marker for tamoxifen resistance. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Breast cancer is the most common malignant tumor affecting women's health and a leading cause of morbidity and mortality worldwide. Of all breast cancer subtypes, ER-positive breast cancer accounts for approximately 70%, and ER-α expression is associated with better prognosis and a greater likelihood of patients benefiting from tamoxifen (TAM) treatment. Tamoxifen is currently a widely used endocrine therapy drug in clinical practice, and its application has significantly reduced recurrence and mortality rates; however, tamoxifen resistance remains a major clinical problem. It is well known that ER-α expression levels are a determinant of TAM response, and the absence of ER-α leads to tamoxifen resistance in triple-negative breast cancer (TNBC).

[0004] Because TRIM family proteins can utilize the ubiquitin-proteasome to induce the degradation of specific target substrate proteins, they are involved in a variety of biological processes. Furthermore, TRIM family protein dysregulation is a hallmark of many cancers, viral infections, developmental disorders, and neurodegenerative diseases. TRIM4 has recently been shown to be involved in virus-induced IFN production, oxidative stress-induced cell death, and hepatocellular carcinoma. However, in the field of breast cancer research, there are currently no studies reporting the impact of TRIM4 protein on breast cancer progression and tamoxifen resistance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the inventors, through long-term technical and practical exploration, have developed an application for TRIM4 as a prognostic marker and tamoxifen resistance marker in ER-positive breast cancer. This invention has found that TRIM4 expression is correlated with tamoxifen resistance in breast cancer, with significantly reduced TRIM4 expression in tamoxifen-resistant breast cancer cell lines. Furthermore, breast cancer patients with high TRIM4 expression have a significantly better prognosis than those with low TRIM4 expression. This indicates that TRIM4 can be used to detect tamoxifen-resistant breast cancer and predict its prognosis. Based on these research findings, this invention was completed.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the invention provides the use of substances for detecting the TRIM4 gene and its expression products in the preparation of products for breast cancer prognostic assessment and / or tamoxifen resistance assessment.

[0008] The breast cancer prognostic assessment includes evaluating the survival of patients with (ER-positive) breast cancer.

[0009] The survival period includes overall survival (OS) and recurrence-free survival (PFS).

[0010] Both the TRIM4 gene and its expression product can be of human origin. The TRIM4 gene expression product is obviously the TRIM4 protein. The specific chromosomal location of the TRIM4 gene is 7q22.1 (database source). https: / / www.ncbi.nlm.nih.gov / gene / 89122 The amino acid sequence of the protein is shown in SEQ ID NO.1. This invention has found that the expression level of TRIM4 in tamoxifen-resistant cell lines (MCF7 / TR and T47D / TR) is abnormally downregulated compared to that in ordinary breast cancer cell lines (MCF7 and T47D). Furthermore, TRIM4 expression is significantly associated with prognosis; high TRIM4 expression in overall ER-positive breast cancer patients predicts a better prognosis. Therefore, TRIM4 can serve as a novel prognostic biomarker for ER-positive breast cancer, providing a basis for assessing postoperative survival.

[0011] In a second aspect, the present invention provides a product comprising a substance for detecting the TRIM4 gene and its expression product, the product being used for breast cancer prognostic assessment and / or tamoxifen resistance determination.

[0012] A third aspect of the present invention provides a system for prognostic assessment of breast cancer and / or determination of tamoxifen resistance, the system comprising:

[0013] i) an analysis unit, the analysis unit comprising a detection substance for determining the expression level of the TRIM4 gene and its expression product selected from the above-mentioned TRIM4 gene and its expression product in the subject's test sample;

[0014] ii) An assessment unit comprising determining the subject’s prognosis and / or tamoxifen resistance based on the expression level of the TRIM4 gene and its expression product as determined in i).

[0015] A fourth aspect of the invention provides the use of TRIM4 as a target in the treatment of breast cancer and / or screening of breast cancer drugs.

[0016] In another specific embodiment of the present invention, the method for screening breast cancer drugs includes:

[0017] 1) Treat the system expressing and / or containing the TRIM4 with the candidate drug; set up a parallel control without treatment with the candidate drug;

[0018] 2) After completing step 1), detect the expression level of TRIM4 in the system; if the expression level of TRIM4 in the system treated with the candidate drug is significantly increased compared with the parallel control, the candidate substance can be used as a candidate anti-breast cancer drug.

[0019] The anti-breast cancer drug can be an anti-ER positive breast cancer drug, and more specifically, the anti-breast cancer drug is a drug that reverses tamoxifen resistance.

[0020] A fifth aspect of the invention provides the use of substances that promote the enhancement of TRIM4 and its expression products and / or activity in any one or more of the following:

[0021] (1) Inhibit breast cancer cell proliferation or prepare products that inhibit breast cancer cell proliferation;

[0022] (2) Inhibit breast cancer cell migration or prepare products that inhibit breast cancer cell migration;

[0023] (3) Inhibit breast cancer growth or prepare products that inhibit breast cancer growth;

[0024] (4) Inhibit breast cancer metastasis or prepare products for breast cancer metastasis;

[0025] (5) Reversing tamoxifen resistance or preparing products that reverse tamoxifen resistance;

[0026] (6) Treating breast cancer or preparing products for treating breast cancer.

[0027] The breast cancer mentioned here can be ER-positive breast cancer.

[0028] A sixth aspect of the present invention provides a method for treating breast cancer, the method comprising administering to a subject the aforementioned substance that promotes the expression and / or activity of TRIM4 and its products.

[0029] This invention has found that TRIM4 can target the degradation of SET protein; enhance the ubiquitination of SET protein; and promote the transcription of ESR1 by P53 through binding to SET protein, while also promoting the function of PP2A in stabilizing ESR1 mRNA, thereby inhibiting the occurrence and development of breast cancer and achieving the goal of treating breast cancer.

[0030] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial effects:

[0031] The aforementioned technical approach reveals that TRIM4 and its encoded protein possess the potential to serve as biomarkers for the diagnosis, treatment, and prognosis of breast cancer patients, providing a novel reference factor for predicting the prognosis of breast cancer patients and improving prognostic accuracy. It can also be used to diagnose tamoxifen-resistant breast cancer. Furthermore, the aforementioned technical approach shows that the TRIM4-encoded protein can degrade SET protein in tamoxifen-resistant tumor cells, indicating that TRIM4 provides a potential target for clinical treatment of breast cancer, improving treatment outcomes and possessing significant clinical and social benefits. Attached Figure Description

[0032] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0033] Figure 1 This invention illustrates the changes in TRIM4 expression levels in ER-positive breast cancer tamoxifen-resistant cell lines and the role of TRIM4 in ER-positive breast cancer (proliferation, metastasis, tamoxifen resistance). Specifically, A shows the changes in TRIM4 protein expression levels between the MCF7 and MCF7 / TR breast cancer cell lines. B shows the changes in TRIM4 protein expression levels between the T47D and T47D / TR breast cancer cell lines. C shows the changes in the proliferation rate of the MCF7 cell line after overexpression of TRIM4-flag. D shows the changes in the proliferation rate of the T47D cell line after overexpression of TRIM4-flag. E shows the effect of interfering with TRIM4 expression on the proliferation capacity of the MCF7 cell line. F shows the effect of interfering with TRIM4 expression on the proliferation capacity of the T47D cell line. G is a schematic diagram illustrating the effect of interfering with TRIM4 expression on the migration and invasion abilities of the MCF7 cell line. H is a schematic diagram illustrating the effect of overexpressing TRIM4 on the migration and invasion abilities of the MCF7 cell line. I is a schematic diagram illustrating the effect of interfering with TRIM4 expression on the migration and invasion abilities of the T47D cell line. J is a schematic diagram illustrating the effect of overexpressing TRIM4 on the migration and invasion abilities of the T47D cell line. K is a schematic diagram illustrating the effect of overexpressing TRIM4-flag on the tamoxifen IC50 in the MCF7 cell line. 50 A schematic diagram illustrating the effect of TRIM4 expression on the tamoxifen IC50 value in the MCF7 cell line. L represents the effect of interfering with TRIM4 expression on the tamoxifen IC50 value in this cell line. 50 A schematic diagram illustrating the effect of TRIM4-flag overexpression on the tamoxifen IC50 value in the T47D cell line. M represents the effect of TRIM4-flag overexpression on the tamoxifen IC50 value in the T47D cell line. 50A schematic diagram illustrating the effect of TRIM4 expression on the tamoxifen IC50 value in the T47D cell line. N represents the effect of interfering with TRIM4 expression on the tamoxifen IC50 value in this cell line. 50 A schematic diagram illustrating the effect of TRIM4-flag overexpression on the tamoxifen IC50 value in the MCF7 / TR cell line. O represents the effect of TRIM4-flag overexpression on the tamoxifen IC50 value in the MCF7 / TR cell line. 50 A schematic diagram illustrating the effect of TRIM4-flag overexpression on the tamoxifen IC50 value in the T47D / TR cell line. P represents the effect of TRIM4-flag overexpression on the tamoxifen IC50 value in the T47D / TR cell line. 50 A diagram illustrating the influence of the value.

[0034] Figure 2 The present invention provides that breast cancer patients with high TRIM4 expression have a better prognosis than those with low TRIM4 expression; where A represents overall survival and B represents recurrence-free survival.

[0035] Figure 3 This paper presents the experimental results of TRIM4 promoting SET protein degradation in ER-positive breast cancer cell lines (MCF7 and T47D) and HEK-293T cell line. A shows the effect of interfering with TRIM4 expression on SET protein expression levels in the MCF7 cell line. B shows the effect of overexpressing TRIM4 on SET-myc protein degradation in the HEK-293T cell line. C shows the effect of interfering with TRIM4 expression on SET protein expression levels in the T47D cell line. D shows the effect of interfering with TRIM4 on SET protein degradation in the MCF7 cell line. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0037] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. Experimental methods in the following specific embodiments, unless specific conditions are specified, are generally performed according to conventional methods and conditions in molecular biology within the art, which are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the conditions recommended by the manufacturer.

[0038] As mentioned earlier, ER-positive breast cancer is a subtype of breast cancer and the most common type. Therefore, identifying biomarkers for predicting breast cancer metastasis and prognosis is of significant theoretical and clinical importance for discovering new therapeutic targets, improving treatment efficacy, and enhancing patient outcomes.

[0039] In view of this, in a typical embodiment of the present invention, the application of a substance for detecting the TRIM4 gene and its expression product in the preparation of breast cancer prognostic assessment products and / or tamoxifen resistance assessment products is provided.

[0040] The breast cancer prognostic assessment includes evaluating the survival of patients with (ER-positive) breast cancer.

[0041] The survival period includes overall survival (OS) and recurrence-free survival (PFS).

[0042] Both the TRIM4 gene and its expression product can be of human origin. The TRIM4 gene expression product is obviously the TRIM4 protein. The specific chromosomal location of the TRIM4 gene is 7q22.1 (database source). https: / / www.ncbi.nlm.nih.gov / gene / 89122The amino acid sequence of its protein is as follows: MEAEDIQEELTCPICLDYFQDPVSIECGHNFCRGCLHRNWAPGGGPFPCPECRHPSAPAALRPNWALARLTEKTQRRRLGPVPPGLCGRHWEPLRLFCEDDQRPVCLVCRESQEHQTHAMAPIDEAFESYRTGNFDIHVDEWKRRLIRLLLYHFKQEEKLLKSQRNLVAKMKKVMHLQDVEVKNATQWKDKIKSQRMRISTEFSKLHNFLVEEEDLFLQRLNKEEEETKKKLNENTLKLNQTIAS LKKLILEVGEKSQAPTLELLQNPKEVLTRSEIQDVNYSLEAVKVKTVCQIPLMKEMLKRFQVAVNLAEDTAHPKLVFSQEGRYVKNTASASSWPVFSSAWNYFAGWRNPQKTAFVERFQHLPCVLGKNVFTSGKHYWEVESRDSLEVAVGVCREDVMGITDRSKMSPDVGIWAIYWSAAGYWPLIGFPGTPTQQEPALHRVGVYLDRGTGNVSFYSAVDGVHLHTFSCSSVSRLRPFFWLSPLASLVIPPVTDRK (SEQ ID NO.1). This invention found that TRIM4 expression levels in tamoxifen-resistant cell lines (MCF7 / TR and T47D / TR) were higher than in ordinary breast cancer cell lines (MCF7 and T47D). Furthermore, TRIM4 expression was significantly associated with prognosis; high TRIM4 expression in overall ER-positive breast cancer patients predicted a better prognosis. Therefore, TRIM4 can serve as a novel prognostic biomarker for ER-positive breast cancer, providing a basis for assessing postoperative survival in patients.

[0043] In this invention, the substance for detecting the TRIM4 gene and its expression product is not specifically limited, and includes, but is not limited to, quantitative and / or qualitative detection substances; preferably, the substance for detecting the TRIM4 gene and its expression product is a substance for quantitatively detecting the TRIM4 gene and its expression product in the sample to be tested.

[0044] The sample to be tested can be the subject's breast cancer cells or tissue.

[0045] The subjects were breast cancer patients, specifically ER-positive breast cancer patients.

[0046] In another specific embodiment of the present invention, the substance used to detect the TRIM gene and / or protein level in the sample to be tested is selected from one or more of gene amplification primers, probes, gene chips, antibodies, protein chips, or antibodies.

[0047] In another specific embodiment of the present invention, the sequence of the gene amplification primer is shown in SEQ ID NO.2-3.

[0048] In another specific embodiment of the present invention, a product is provided, the product comprising a substance for detecting the TRIM4 gene and its expression product, the product being used for breast cancer prognostic assessment and / or tamoxifen resistance determination.

[0049] In another specific embodiment of the present invention, the substance for detecting the TRIM4 gene and its expression product is selected from one or more of gene amplification primers, probes, gene chips, antibodies, protein chips, or antibodies.

[0050] The sequences of the gene amplification primers are shown in SEQ ID NO.2-3.

[0051] In another specific embodiment of the present invention, the product may be a detection reagent, a detection kit, or a biosensor, etc., and is not specifically limited thereto.

[0052] In another specific embodiment of the present invention, a system for prognostic assessment of breast cancer and / or determination of tamoxifen resistance is provided, the system comprising:

[0053] i) an analysis unit, the analysis unit comprising a detection substance for determining the expression level of the TRIM4 gene and its expression product selected from the above-mentioned TRIM4 gene and its expression product in the subject's test sample;

[0054] ii) An assessment unit, which includes determining the prognosis and / or tamoxifen resistance of the subject based on the expression level of the TRIM4 gene and its expression product as determined in i);

[0055] The subjects are breast cancer patients, the test samples include but are not limited to breast cancer cells or tissues from breast cancer patients, and the prognostic conditions include but are not limited to assessing the survival of breast cancer patients; the survival includes overall survival (OS) and recurrence-free survival (PFS).

[0056] In another specific embodiment of the present invention, the application of TRIM4 as a target in the treatment of breast cancer and / or screening of breast cancer drugs is provided.

[0057] In another specific embodiment of the present invention, the method for screening breast cancer drugs includes:

[0058] 1) Treat the system expressing and / or containing the TRIM4 with the candidate drug; set up a parallel control without treatment with the candidate drug;

[0059] 2) After completing step 1), detect the expression level of TRIM4 in the system; if the expression level of TRIM4 in the system treated with the candidate drug is significantly increased compared with the parallel control, the candidate substance can be used as a candidate anti-breast cancer drug.

[0060] The anti-breast cancer drug can be an anti-ER positive breast cancer drug, and more specifically, the anti-breast cancer drug is a drug that reverses tamoxifen resistance.

[0061] In another specific embodiment of the present invention, the use of substances that promote the enhancement of TRIM4 and its expression products and / or activity is provided in any one or more of the following:

[0062] (1) Inhibit breast cancer cell proliferation or prepare products that inhibit breast cancer cell proliferation;

[0063] (2) Inhibit breast cancer cell migration or prepare products that inhibit breast cancer cell migration;

[0064] (3) Inhibit breast cancer growth or prepare products that inhibit breast cancer growth;

[0065] (4) Inhibit breast cancer metastasis or prepare products for breast cancer metastasis;

[0066] (5) Reversing tamoxifen resistance or preparing products that reverse tamoxifen resistance;

[0067] (6) Treating breast cancer or preparing products for treating breast cancer.

[0068] In another specific embodiment of the present invention, the breast cancer may be ER-positive breast cancer;

[0069] Substances that promote TRIM4 and its expression products and / or activity include, but are not limited to, substances that upregulate TRIM4 expression and / or promote its activity using gene-specific technologies; such as synthetically produced short hairpin RNA (shRNA) or promoters or lentiviruses that upregulate TRIM4 expression; and also include compound promoters.

[0070] The product may be a drug or an experimental reagent, and the experimental reagent may be used for basic research.

[0071] According to the present invention, when the product is a drug, the drug further includes at least one inactive pharmaceutical ingredient.

[0072] The inactive components of the drug can be pharmaceutically commonly used carriers, excipients, and diluents. Furthermore, according to conventional methods, it can be formulated into oral, topical, suppository, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays.

[0073] The non-pharmaceutical active ingredients that may be included, such as carriers, excipients, and diluents, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0074] In another specific embodiment of the present invention, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate and mineral oil, etc.

[0075] In another specific embodiment of the invention, the drug of the invention can be administered into the body by known means, such as intravenous systemic delivery. Alternatively, it can be administered via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via a single dose or multiple doses. Those skilled in the art will understand that the actual dose to be administered in the invention can vary considerably depending on a variety of factors, such as target cells, biological type or tissue, the general condition of the subject to be treated, route of administration, manner of administration, etc.

[0076] In another specific embodiment of the present invention, the drug can be administered to humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, orangutans, preferably humans.

[0077] In another specific embodiment of the present invention, a method for treating breast cancer is provided, the method comprising administering to a subject the aforementioned substance that promotes the expression and / or activity of TRIM4 and its products.

[0078] This invention has discovered that TRIM4 can target the degradation of SET protein (therefore, the substances that promote TRIM4 and its expression products and / or increase its activity can be used as SET protein inhibitors / degraders); enhance the ubiquitination of SET protein; and promote the transcription of ESR1 by P53 through binding to SET protein, while promoting the function of PP2A in stabilizing ESR1 mRNA, thereby inhibiting the occurrence and development of breast cancer and achieving the purpose of treating breast cancer.

[0079] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Any simple modifications, equivalent variations, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the present invention.

[0080] Unless otherwise specified, all materials, reagents, plasmids, and special kits used in the following examples were obtained commercially.

[0081] Example 1

[0082] 1. Sample collection

[0083] We collected 116 pairs of tumor tissue from patients who underwent breast cancer surgery at Qilu Hospital of Shandong University between 2012 and 2013, and obtained consent from all patients participating in the study.

[0084] 2. Real-time quantitative PCR experiment:

[0085] 2.1 Extraction of total RNA from tissues

[0086] Total RNA was extracted from breast cancer patient tissues using Trizol reagent, and the concentration and purity of the RNA were detected using appropriate equipment.

[0087] 2.2 Reverse transcription of total RNA

[0088] Reverse transcription was performed using a reverse transcription kit from Takara, with an initial RNA volume of 1 μl, following the instructions provided.

[0089] The system is shown in the table below:

[0090]

[0091] Specific reaction program settings within the PCR instrument:

[0092]

[0093] 2.3 Real-time quantitative PCR

[0094] The expression levels of TRIM4 in tumor tissues and adjacent normal control tissues were detected using a real-time quantitative PCR instrument (Roche LightCycler 480).

[0095] The specific reaction system is as follows:

[0096]

[0097] Specific reaction program settings within the PCR instrument:

[0098]

[0099] 2.4 Quantitative PCR Primers

[0100] The TRIM4 provided in this embodiment is a differentially expressed mRNA selected from ER-positive breast cancer drug-resistant cell lines using bioinformatics methods. Its quantification was performed using β-actin as an internal reference gene. Primer sequences are shown in the table below:

[0101]

[0102] Example 2

[0103] The expression level of TRIM4 in sampled ER-positive breast cancer patients was statistically analyzed, and a comprehensive statistical analysis was performed in conjunction with their treatment effects. Figure 2 As shown, ER-positive breast cancer patients with high TRIM4 expression had a better prognosis than ER-positive breast cancer patients with low TRIM4 expression.

[0104] Example 3

[0105] This embodiment designs a detection kit for predicting the prognosis of ER-positive breast cancer. The detection system of the kit includes a reverse transcription reaction system and a qPCR reaction system, as well as conventional reagents for preparing the reverse transcription system and the qPCR reaction system.

[0106] 1. Components of the ER-positive breast cancer tissue diagnostic kit (50 reactions):

[0107] 100ml isopropanol, 100ml chloroform, 50ml Trizol, 10ml DEPC water or enzyme-free water, 10ml double-distilled water, 1ml 5× reverse transcription buffer, 1ml dNTP mixture, 500μl RNase protein inhibitor, 50μl reverse transcriptase solution, 2ml 2× SYBR Premix.

[0108] 200 μl of 10 μM TRIM4-specific PCR primers

[0109] Its forward primer is 5′-AAGGTGAAGACAGTGTGCCA-3′ (SEQ ID NO.2)

[0110] Its reverse primer is 5′-CAGGGAAGCCTATCAAGGGC-3′ (SEQ ID NO.3)

[0111] 200 μl of 10 μM GAPDH-specific PCR primers

[0112] Its forward primer is 5′-GGAGCGAGATCCCTCCAAAAT-3′ (SEQ ID NO.4)

[0113] Its reverse primer is 5′-GGCTGTTGTCATACTTCTCATGG-3′ (SEQ ID NO.5)

[0114] 2. Extraction of total RNA from tissues

[0115] Total RNA was extracted from breast cancer patient tissues using Trizol reagent, and the concentration and purity of the RNA were detected using appropriate equipment.

[0116] 2.1 Reverse transcription of total RNA, the system is shown in the table below:

[0117]

[0118] Specific reaction program settings within the PCR instrument:

[0119]

[0120] 2.2 Real-time quantitative PCR

[0121] The expression levels of TRIM4 in tumor tissues and adjacent normal control tissues were detected using a real-time quantitative PCR instrument (Roche LightCycler 480).

[0122] The specific reaction system is as follows:

[0123]

[0124] Specific reaction program settings within the PCR instrument:

[0125]

[0126] 2.3 Determination of DCT index: DCT refers to the difference between the test TRIM4 and the average Ct value of the internal reference in the same sample. That is, TRIM4 DCT = TRIM4 Mean CT - control Mean CT. In this embodiment, DCT is the difference between the average CT value of TRIM4 and GAPDH. The relative quantitative ΔCT value is obtained and judged.

[0127] Example 4

[0128] Cell transfection assay: Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The shRNA sequence used is shown below:

[0129]

[0130] Transwell assay: After appropriate transfection, cells underwent a 6-hour serum starvation step, and 2×10⁶ cells were then introduced into the cell line. 5 One MCF-7 or T47D cell was suspended in 200 μL of serum-free medium and seeded into the interior of each Transwell compartment (Corning, USA). Simultaneously, 700 μL of medium containing 20% ​​FBS was placed in each well of a 24-well plate. After 72 hours of culture, the infiltrating cells on the lower surface were fixed with methanol and stained with 0.1% crystal violet. Cell invasion assays were performed using the same procedure as cell migration assays, but the interior of each insert was coated with Matrigel (BD Biosciences). Figure 1 As shown in GJ, interference with TRIM4 expression significantly increased the migration and invasion abilities of MCF7 and T47D cell lines. Conversely, overexpression of TRIM4 in MCF7 and T47D cell lines significantly inhibited their migration and invasion abilities.

[0131] CHX and Western Blot Assays: 48 h after transfection, MCF7 or HEK-293T cells were treated with CHX, and proteins were collected at 0, 4, 8, and 12 h for Western Blot experiments. Western Blot Assay: Cells were collected and lysed with Western blotting, IP lysis buffer, and protease inhibitor. The cells were then centrifuged at 12000g for 15 min at 4 °C, and the protein content of the cell lysates was determined using a BCA protein assay kit. The supernatant was transferred to 1.5 ml of EP, 5×SDS was added, and the mixture was boiled for 5 min. Equal volumes of protein were loaded onto SDS-PAGE gels and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk for 1 h, the PVDF membranes were incubated overnight at 4 °C with the corresponding primary antibody, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 2 h. The results were then visualized using an enhanced chemiluminescence detection system. Figure 1 As shown in AB, the protein expression level of TRIM4 was significantly reduced in tamoxifen-resistant cell lines (MCF7 / TR and T47D / TR). Figure 3 As shown in A and 3C: Interference with TRIM4 expression significantly increased SET protein expression levels. Figure 3 As shown in B and 3D: In the MCF7 cell line, interference with TRIM4 expression significantly reduced the level of SET protein degradation. Conversely, in the HEK-293T cell line, overexpression of TRIM4 significantly enhanced the degradation capacity of SET protein.

[0132] MTT cell proliferation assay and IC50 cell viability assay: MTT assay: A single-cell suspension was prepared using culture medium containing 10% fetal calf serum. 1000 cells were seeded into each well of a 96-well plate (100 μL per well). Cells were cultured under the same conditions for 5-7 days. Then, 20 μL of MTT (Sigma) solution was added, and cultured for another 4 hours. The culture was then terminated, and the supernatant was carefully aspirated. 100 μL of DMSO was added to each well, and the plate was shaken for 5 minutes to dissolve any crystals. The absorbance of each well was measured at 490 nm using an ELISA reader. The results were recorded, and a cell growth curve was plotted with time on the x-axis and absorbance on the y-axis. IC50 cell viability assay: 50 Experiment: After transfection, cells were added to 96-well plates (1×10⁶ cells / well). 3 Cells were placed in wells (cells / well). The medium was then replaced with growth medium and supplemented with a range of tamoxifen (TAM) concentrations for 48 hours or a predetermined time. At appropriate time points, cells were washed with PBS, and MTT was added to each well. Cells were then incubated at 37°C for another 4 hours. The medium was then discarded, and formazan crystals were dissolved using dimethyl sulfoxide. Quantification was then performed using an ELISA reader based on absorbance at 490 nm. IC50 50 The value is determined using GraphPad. For example... Figure 1 As shown in Figure C-1F: Interference with TRIM4 expression significantly enhanced the proliferation of MCF7 and T47D cell lines, while overexpression of TRIM4 significantly reduced the proliferation of MCF7 and T47D cell lines. As shown in Figure KN: Overexpression of TRIM4 significantly enhanced the tamoxifen sensitivity of MCF7 and T47D cell lines, while interference with TRIM4 expression significantly reduced the tamoxifen sensitivity of MCF7 and T47D cell lines. Figure 1 As shown in O-1P, overexpression of TRIM4 significantly enhances the tamoxifen sensitivity of MCF7 / TR and T47D / TR cell lines.

[0133] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of substances that detect the TRIM4 gene and its expression products in the preparation of breast cancer prognostic assessment products and / or tamoxifen-resistant breast cancer identification products, wherein the breast cancer prognostic assessment is to evaluate the survival of ER-positive breast cancer patients, and the tamoxifen-resistant breast cancer is ER-positive breast cancer.

2. The application as described in claim 1, characterized in that, The survival period includes overall survival and recurrence-free survival.

3. The application as described in claim 1, characterized in that, The TRIM4 gene and its expression product are both human-derived, and the TRIM4 gene expression product is the TRIM4 protein.

4. The application as described in claim 1, characterized in that, Substances for detecting the TRIM4 gene and its expression products include quantitative and / or qualitative detection substances.

5. The application as described in claim 4, characterized in that, The substance used to detect the TRIM4 gene and its expression product is a substance for quantitative detection of the TRIM4 gene and its expression product in the sample to be tested. The sample to be tested is the subject's breast cancer cells or tissue; The subjects were ER-positive breast cancer patients.

6. The application as described in claim 3, characterized in that, The substance used to detect the TRIM gene and / or protein level in the sample to be tested is selected from one or more of the following: gene amplification primers, probes, gene chips, protein chips, or antibodies.

7. A system for prognostic assessment of breast cancer and / or identification of tamoxifen resistance, characterized in that, The system includes: i) An analysis unit, the analysis unit comprising a detection substance selected from the TRIM4 gene and its expression product in a test sample of a subject; ii) An assessment unit comprising determining the subject’s prognosis and / or tamoxifen resistance based on the expression level of the TRIM4 gene and its expression product as determined in i); The prognostic assessment of breast cancer refers to the evaluation of the survival of patients with ER-positive breast cancer, and the tamoxifen-resistant breast cancer is ER-positive breast cancer.

8. The system as described in claim 7, characterized in that, The test sample includes breast cancer cells or tissues from breast cancer patients; the survival period includes overall survival and recurrence-free survival.

9. The use of substances that promote the enhancement of TRIM4 and its expression products and / or activity in any one or more of the following: (1) Prepare products that inhibit the proliferation of breast cancer cells; (2) To prepare products that inhibit the migration of breast cancer cells; (3) Prepare products that inhibit the growth of breast cancer; (4) Prepare products that inhibit breast cancer metastasis; (5) To prepare products that reverse tamoxifen-resistant breast cancer; (6) Prepare products for the treatment of breast cancer; The breast cancer described is ER-positive breast cancer; The substance that promotes the enhancement of TRIM4 and its expression products and / or activity is a synthetically produced short hairpin RNA or a promoter or lentivirus that upregulates TRIM4 expression; the product is a drug.

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