Chromatin modification detection kit and detection method for regulating ATIC expression by PRMT8

Abstract

The invention belongs to the technical field of kit detection, discloses a chromatin modification detection kit for regulating ATIC expression through PRMT8, and discovers the specific expression and cancer promotion function of arginine methyltransferase PRMT8 and purine de novo synthesis key enzyme ATIC in triple negative breast cancer for the first time. The specific biological effect of the PRMT8 in the triple negative breast cancer is researched for the first time, and the PRMT8 promotes ATIC transcriptional activation by mediating phosphorylation of an ATIC promoter region H4R3me2a and an effect protein TDRD3 of the PRMT8. The important function of arginine methyltransferase PRMT8 in a tumor purine metabolic pathway is found by gradually exploring the transcriptional activation process of purine metabolic rate-limiting enzyme ATIC, the post-translational modification function of histone is associated with the activation of the breast cancer purine metabolic pathway, and a new theoretical basis is provided for treating breast cancer by targeting PRMT8.

Description

Technical Field

[0001] The present invention belongs to the technical field of kit detection, and in particular relates to a chromatin modification detection kit and a detection method for PRMT8 regulating ATIC expression. Background Art

[0002] 1) An important factor affecting the poor prognosis of triple-negative breast cancer: activation of the purine biosynthesis pathway

[0003] Triple-negative breast cancer (TNBC) is a molecularly diverse disease, named for the fact that immunohistochemical staining shows less than 1% expression of estrogen and progesterone receptors, and the lack of Her2 receptor expression or amplification. This type of breast cancer accounts for approximately 15%-20% of all breast cancers, but exhibits strong malignant behavior, including rapid tumor progression and early metastasis. Despite more sophisticated screening methods and the popularization of the concept of early diagnosis and early treatment, TNBC remains the molecular subtype with the worst prognosis in breast cancer. The American Society of Oncology statistics show that its 5-year overall survival rate is approximately 78%, which is 8-16% lower than that of the hormone receptor-positive type. Once distant metastasis occurs, patients need to undergo surgery, chemotherapy, radiotherapy, immunotherapy, etc., and the prognosis is extremely poor (the 5-year overall survival rate is 4-20%), which places a huge burden on both the patient and medical resources. Therefore, it is very important to conduct in-depth research on the molecular mechanism of the high malignancy of triple-negative breast cancer.

[0004] Metabolism is the basis of cell survival. Changes in metabolic pathways are one of the hallmarks of malignant tumors. In malignant tumors, in order to adapt to rapid cell proliferation, cancer cells regulate the synthesis of biomacromolecules and energy supply through metabolic reprogramming. Among them, the purine biosynthesis process plays an important role in cancer cell division, DNA damage repair and other processes. Dysregulation of the purine synthesis pathway in malignant tumors has been shown to be related to the occurrence and development of tumors. The applicant and his team also have a certain research foundation in this field.

[0005] 3) Arginine methyltransferase PRMT8 is a key regulator of H4R3me2a in the ATIC promoter region in breast cancer

[0006] The arginine methyltransferase family (PRMT) is a family of enzymes that can methylate arginine residues in proteins, wherein the first type of protein arginine methyltransferase (Type I protein arginine methyltransferases) has the function of catalyzing the asymmetric dimethylation of H4R3 (for example: PRMT1, PRMT8, etc.), and the H4R3 methylation modification mediated by the first type of PRMTs is involved in various cell processes, including DNA repair, RNA transcription, signal transduction, and protein translation. However, existing inhibitors of arginine methyltransferase PRMT1 have failed in clinical studies, mainly because the research on the molecular mechanism has only focused on PRMT1, and the research on the remaining protein family members is very limited. In the PRMTs family, PRMT1 and PRMT8 are both genes differentially expressed in TNBC

Prior Art 3a-b

Prior Art 3c-d

[0007] 4) PRMT8 / FYN / TDRD3 complex promotes H4R3me2a-mediated ATIC transcriptional activation

[0008] Previous studies have shown that PRMT1 and PRMT8 have about 80% sequence similarity. To further explore the functional characteristics of PRMT8 in TNBC and analyze the difference in the effect of PRMT1 and PRMT8 on ATIC transcription in

Prior Art 3

[0009] Many experiments have confirmed that the effector protein of histone modification can be modified by phosphorylation, thereby changing the binding of downstream transcription initiation-related proteins, and then affecting gene transcription; the present invention

Prior Art 5b, d

[0010] Through the above analysis, the problems and defects of the prior art are:

[0011] Breast cancer is a highly heterogeneous malignancy, triple-negative breast cancer lacks estrogen and progesterone receptors and HER2 expression, has significantly different biological characteristics from other molecular subtypes, and the prognosis is poor. Imbalance of purine metabolism is one of the important factors leading to malignant progression of breast cancer, but its specific regulatory mechanism in triple-negative breast cancer still needs to be clarified. SUMMARY

[0012] In view of the problems existing in the prior art, the present application provides a PRMT8 regulated ATIC expression chromatin modification detection kit detection method.

[0013] The present application is realized in that a PRMT8 regulated ATIC expression chromatin modification detection kit detection method comprises:

[0014] Step 1, screening of cell biosynthesis pathways and factors related to poor prognosis of triple-negative breast cancer;

[0015] Step 2, exploring the influence of key enzyme ATIC of purine metabolism pathway of triple-negative breast cancer on malignant phenotype and its transcriptional activation mechanism;

[0016] Step 3, in-depth study of the function of PRMT8, the H4R3me2a modification regulatory enzyme of ATIC promoter region in triple-negative breast cancer;

[0017] Step 4, exploring the influence of PRMT8 on breast cancer cell function in in vivo and in vitro experiments;

[0018] Step 5, in vivo and in vitro functional experiments confirm that PRMT8 can regulate purine metabolism and malignant phenotype of breast cancer through H4R3me2a / ATIC signal axis;

[0019] Step 6, exploring whether PRMT8 promotes the phosphorylation of H4R3me2a effector protein TDRD3 by combining FYN to further promote the transcription of ATIC;

[0020] Step 7, evaluating the expression and clinical value of PRMT8 in triple-negative breast cancer in big data and clinical samples.

[0021] Further, the screening of cell biosynthesis pathways and factors related to poor prognosis of triple-negative breast cancer:

[0022] 1) Obtain the mRNA expression profile of triple-negative breast cancer in TCGA-BRCA, use the metabolic related data set in GO database for ssGSEA analysis, and use Boruta to screen the important predictive factors of metabolic related ssGSEA score for TNBC prognosis;

[0023] 2) Obtain the mass spectrometry analysis of early metastatic triple-negative breast cancer in situ tumor samples in the unit to explore the differences of their metabolites;

[0024] 3) Western blot analysis was used to detect the protein expression levels of ATIC, a key rate-limiting enzyme for IMP production, in each cell line subjected to high glucose-induced activation of purine metabolism and in the aforementioned primary cell lines. Quantitative PCR was used to detect the mRNA levels of ATIC in each cell line to determine the expression of ATIC in each cell line and the level at which it changes.

[0025] Furthermore, the study explored the effect of ATIC, a key enzyme in the purine metabolism pathway of triple-negative breast cancer, on the malignant phenotype and its transcriptional activation mechanism:

[0026] 1) Using a lentiviral transfection system, plasmids were constructed: LV-Luc-ATIC and LV-Luc-shATIC were used to stably overexpress and knockdown ATIC, respectively. Colony formation, softagar and transwell migration and invasion assays, and stem cell sphere formation assays were used to examine the effects of ATIC on breast cancer cell proliferation, invasion, metastasis, and stem cell properties.

[0027] 2) Based on ATIC expression, patients with triple-negative breast cancer in TCGA were divided into high / low expression groups. The ssGSEA method was used to enrich the histone post-translational modification-related pathways in the two groups of patients, and the Wilcox method was used to test the differences between the two groups;

[0028] 3) Collect surgically resected breast tissue from patients with triple-negative breast cancer and prepare tissue microarrays from the tissues. Immunohistochemistry was used to detect histone arginine methylation and analyze its correlation with clinical pathological data.

[0029] 4) Chromosome immunoprecipitation assay was used to detect the signal intensity of the ATIC promoter region sequence bound to methylated histone 4 in breast cancer cell lines with different molecular subtypes, including MCF-7, MDA-MB-453, and MDA-MB-231;

[0030] 5) Treat MCF-7, MDA-MB-453, and MDA-MB-231 breast cancer cell lines of different subtypes with histone arginine methylation inhibitors, and use Western blot and quantitative PCR to detect changes in histone arginine methylation in the ATIC promoter region and its mRNA expression level;

[0031] 6) Site-directed mutagenesis was used to construct a plasmid encoding the third arginine mutation in H4 and then transfected into breast cancer cells to verify the change in ATIC expression.

[0032] Furthermore, the in-depth study of the function of the PRMT8 enzyme regulating the H4R3me2a modification in the ATIC promoter region in triple-negative breast cancer is as follows:

[0033] 1) Co-IP and DNA-pulldown experiments were performed in MDA-MB-231 TNBC breast cancer cell line using anti-PRMT8 and ATIC promoter region DNA probe respectively to verify the binding ability of PRMT8 to ATIC promoter region;

[0034] 2) Western blot experiment was used to detect the protein expression level of PRMT8 in MCF-7, MDA-MB-453, MDA-MB-231 different subtypes of breast cancer cell lines;

[0035] 3) Lentivirus transfection system was used to construct plasmids: LV-Luc-PRMT8 and LV-Luc-shPRMT8 were used to stably overexpress / knock down PRMT8, and Western blot and fluorescent quantitative PCR were used to detect the changes of ATIC expression;

[0036] 4) ChIP method was used to determine the changes of H4R3me2a degree in ATIC promoter region in the above overexpression / knockdown cell lines.

[0037] Further, the effect of PRMT8 on the function of breast cancer cells in in vivo and in vitro experiments was explored:

[0038] 1) MCF-7, MDA-MB-453 and MDA-MB-231 were selected as the main experimental cells; lentivirus transfection system was used to construct plasmids: LV-Luc-PRMT8 and LV-Luc-shPRMT8 were used to stably overexpress / knock down PRMT8; colony formation, soft agar, transwell migration and invasion experiment and stem cell sphere formation experiment were used to detect the effect of PRMT8 on the proliferation, invasion and metastasis and stem cell characteristics of breast cancer cells;

[0039] 2) 13C1-glycine / 15N-labeled glutamine was added to the glycine / glutamine-free medium of the above PRMT8 overexpressed / knocked out tumor cells to detect the generation level of 13C1 / 15N2-labeled IMP, AMP and GMP in PRMT8 knocked down / overexpressed tumor cells, so as to detect the effect of PRMT8 on the purine metabolic pathway of the corresponding tumor cell lines;

[0040] 3) Application of the above normal, transfected LV-Luc-PRMT8 and LV-Luc-shPRMT8 tumor cells breast fat pad injection to construct SCID mouse breast orthotopic tumor model, compare the fluorescence intensity, volume of tumor in experimental and control groups; tail vein injection to establish SCID mouse lung metastasis model, detect the fluorescence intensity, number of lung metastasis nodules in experimental and control groups; the above orthotopic tumor and metastatic tumor tissues are collected and HE or IHC is used to determine the tissue type.

[0041] Further, the in vivo and in vitro functional experiments prove that PRMT8 can regulate breast cancer purine metabolism and malignant phenotype through H4R3me2a / ATIC signal axis:

[0042] 1) The above PRMT8 overexpressing / knocking out cell lines are treated with histone methylation inhibitors and ATIC inhibitors; Western blot and RT-qPCR experiments are used to detect whether PRMT8 has transcriptional regulation function on ATIC;

[0043] 2) The above PRMT8 overexpressing / knocking out cell lines are treated with histone methylation inhibitors and ATIC inhibitors for colony formation, soft agar, transwell migration and invasion experiments, and stem cell sphere formation experiments to detect the influence of the breast cancer cell proliferation, invasion, metastasis and stem cell characteristics;

[0044] 3) The above PRMT8 overexpressing / knocking out tumor cells are treated with histone methylation inhibitors and ATIC inhibitors, 13C1-glycine / 15N-labeled glutamine is added in the glycine / glutamine-free medium, and the generation level of 13C1 / 15N2-labeled IMP, AMP and GMP in tumor cells is detected to judge the change of purine metabolism pathway of the corresponding tumor cell line;

[0045] 4) Application of the above PRMT8 overexpressing / knocking out tumor cells breast fat pad injection to construct SCID mouse breast orthotopic tumor model, orthotopic injection of methylation inhibitor GSK3368715, compare the fluorescence intensity, volume of tumor in experimental and control groups; tail vein injection to establish SCID mouse lung metastasis model, detect the fluorescence intensity, number of lung metastasis nodules in experimental and control groups; the above orthotopic tumor and metastatic tumor tissues are collected and HE or IHC is used to determine the tissue type;

[0046] 5) In H4R3 replacement cell lines, overexpression / knockout PRMT8, use Western blot, RT-qPCR experiments to detect whether there is ATIC transcriptional regulation function of PRMT8; Perform colony formation, soft agar, transwell migration and invasion experiments and stem cell sphere formation experiments to detect the effects of PRMT8 on breast cancer cell proliferation, invasion, metastasis and stem cell properties; Add 13C1-glycine / 15N-labeled glutamine to the glycine / glutamine-free medium to detect the generation of 13C1 / 15N2-labeled IMP, AMP and GMP in tumor cells to determine the changes in the purine metabolic pathway of the corresponding tumor cell lines;

[0047] Explore whether PRMT8 promotes the phosphorylation of H4R3me2a effector protein TDRD3 by binding FYN to further promote the transcription of ATIC;

[0048] 1) According to the 500bp fragment upstream of the ATIC promoter, design a bait DNA, and use 5'-biotin modified primers and unmodified primers to synthesize DNA probes; After the biotin-labeled bait DNA is adsorbed on Dynabeads MyOne Streptavidin T1, use the MDA-MB-231 nuclear lysate overexpressing PRMT1 or PRMT8 to perform DNA affinity purification; The obtained product is detected by LC-MS; Use protein BLAST1 to compare the peptide sequence with human proteins;

[0049] 2) In breast cancer cell lines, use anti-TDRD3 and anti-PRMT8 for ChIP and Co-IP experiments to test the binding ability of TDRD3 to the ATIC promoter region and PRMT8 to FYN;

[0050] 3) After establishing a PRMT8 overexpression cell line using LV-Luc-PRMT8 in the MDA-MB-231 TNBC cell line, knock down TDRD3, use RT-qPCR and WB to detect ATIC expression, and verify the regulation of TDRD3 on ATIC transcription;

[0051] 4) Use biotin-labeled ATIC promoter region DNA sequence probes to purify DNA binding proteins, and use phos-tag SDS electrophoresis and WB experiments to detect the expression of phosphorylated TDRD3;

[0052] 5) DNA-pulldown experiment and custom pTDRD3 antibody are used to detect the phosphorylation level of TDRD3 in the ATIC promoter region in the TNBC cell lines overexpressing PRMT8; ChIP is used to check the H4R3me2a level; RT-qPCR and WB are used to detect the ATIC expression in the above-mentioned cell lines;

[0053] 6) Mass spectrometry is used to detect the phosphorylation sites of TDRD3 after immunoprecipitation (IP) experiment using anti-TDRD3 in the TNBC cell lines overexpressing PRMT8; Co-IP experiment is used to compare the changes in the affinity of TDRD3 phosphorylation to the key protein TOP3B downstream.

[0054] Another object of the present application is to provide a detection kit for detecting the chromatin modification state of PRMT8-regulated ATIC expression, which comprises:

[0055] (1) a specific antibody for recognizing the asymmetric dimethylation of the 3rd arginine of histone H4 (H4R3me2a) modification;

[0056] (2) a pair of DNA primers designed for the ATIC promoter sequence for ChIP-qPCR or DNA-pulldown detection;

[0057] (3) an immunoprecipitation (Co-IP) related reagent group for detecting PRMT8, TDRD3 and their interaction;

[0058] (4) a fluorescence quantitative PCR (RT-qPCR) related reaction component for evaluating the transcription level of ATIC;

[0059] The kit is used for detecting the ATIC transcriptional regulation mechanism mediated by PRMT8 through H4R3me2a modification, and is suitable for molecular mechanism research or preclinical verification of tumor samples such as breast cancer.

[0060] Further, the H4R3me2a antibody is a monoclonal antibody, which can specifically recognize the asymmetric dimethylation modification of the 3rd arginine of human histone H4, and has ChIP-level purity.

[0061] Further, the pair of DNA primers is used to amplify the core regulatory sequence of the region from-1000bp to +100bp upstream of the ATIC promoter, and is suitable for enrichment analysis of various breast cancer cell lines and tissue samples.

[0062] Further, the phos-tag SDS-PAGE electrophoresis reagent and customized p-TDRD3 antibody for detecting the phosphorylation state of TDRD3 are further included to realize accurate specificity and quantitative analysis of the PRMT8-FYN-TDRD3 regulation pathway.

[0063] In combination with the technical solutions and solved technical problems described above, the technical solutions to be protected by the present application have the following advantages and positive effects:

[0064] Firstly, the present application is based on the clinical characteristics of triple-negative breast cancer, and aims to solve the following scientific problems from the aspects of tumor metabolic disorder and histone post-transcriptional modification:

[0065] (1) Explore the important metabolic regulation mechanism related to poor prognosis of triple-negative breast cancer.

[0066] (2) Determine whether ATIC is a key mediator of purine metabolic disorder in triple-negative breast cancer.

[0067] (3) Explore the role of histone methylation (H4R3me2a) in the regulation of ATIC expression in triple-negative breast cancer.

[0068] (4) Determine the important role of arginine methyltransferase PRMT8 in purine metabolic disorder in triple-negative breast cancer and the related regulation network.

[0069] (5) Use a clinical patient cohort to verify the guiding role of PRMT8 expression in patient risk stratification and treatment.

[0070] 4. The features and innovations of the present application are:

[0071] (1) The specific expression and carcinogenic function of arginine methyltransferase PRMT8 and the key enzyme ATIC of purine de novo synthesis in triple-negative breast cancer are discovered for the first time.

[0072] (2) The specific biological role of PRMT8 in triple-negative breast cancer is explored for the first time: PRMT8 promotes ATIC transcriptional activation by mediating H4R3me2a in the ATIC promoter region and the phosphorylation of its effector protein TDRD3.

[0073] The present application discloses a PRMT8-mediated precise regulation mechanism of asymmetric dimethylation of histone H4R3 site (H4R3me2a) in the ATIC promoter region, which lays a solid foundation for the development of small molecule inhibitors, monoclonal antibodies and companion diagnostic kits targeting PRMT8. Based on the in vitro high-throughput screening platform and tumor xenograft (PDX) model established by the present application, the preclinical evaluation and pharmacodynamics detection of new drug candidate compounds can be accelerated, the development cycle can be significantly shortened, the cost can be reduced, and an intellectual property layout covering the whole chain from target discovery to drug marketing can be formed, which can bring considerable economic benefits.

[0074] In terms of market application, the PRMT8 expression level quantitative detection and ATIC transcriptional activity evaluation provided by the present application can be used as important biomarkers for precise diagnosis and risk prediction in view of the high recurrence and poor prognosis of triple-negative breast cancer (TNBC). In addition, the PRMT8 inhibitor screened by the present application can be used in combination with existing chemotherapy regimens, which is expected to improve the efficacy, reduce drug resistance and improve the quality of life of patients. The technical scheme has strong adaptability, can provide authorization to large pharmaceutical companies, and can also expand the downstream industry chain through technology stockholding, cooperative development and other modes, so the market space is wide.

[0075] The present application fills the technical gap in the field of upstream regulation of tumor purine metabolism epigenetics at home and abroad. Existing literature mainly focuses on the functional ATIC enzyme and the targeted inhibition of the downstream metabolic pathway, and the key role of arginine methyltransferase in the transcriptional activation of purine metabolic rate-limiting enzymes has never been reported. The present application first constructs a PRMT8-H4R3me2a-ATIC-TDRD3 phosphorylation molecular interaction network, which provides a new perspective for the cross-regulation mechanism of tumor metabolism-epigenetics, and breaks through the limitations of the previous single target inhibition strategy of metabolic pathways.

[0076] Compared with the prior art, the present application achieves significant technical progress in both basic research and application transformation: on the one hand, chromatin immunoprecipitation (ChIP), high-resolution mass spectrometry and CRISPR / Cas9 gene knockout are used to accurately verify the modification and transcriptional activation of PRMT8 on the ATIC promoter region; on the other hand, through multi-center clinical sample big data analysis, it is confirmed that the expression of PRMT8 and ATIC is highly related to the prognosis of patients, and has excellent diagnostic and predictive value. This systematic and quantifiable research system lays a strong innovative support for future individualized therapy targeting PRMT8. BRIEF DESCRIPTION OF DRAWINGS

[0077] Figure 1 It is a detection method flow chart of the PRMT8 regulation ATIC expression chromatin modification detection kit provided by the embodiment of the present application.

[0078] Figure 2 This is a structural block diagram of a detection system for a chromatin modification detection kit for PRMT8 regulating ATIC expression provided in an embodiment of the present invention.

[0079] Figure 3 This is a classic pathway diagram of purine metabolism provided by an embodiment of the present invention.

[0080] Figure 4 It is a schematic diagram of the subject hypothesis provided by an embodiment of the present invention.

[0081] Figure 5 This is a technical roadmap provided by an embodiment of the present invention.

[0082] Figure 6 The research basis 1 provided by the embodiment of the present invention is to screen out metabolic pathways and driving factor diagrams related to the prognosis of triple-negative breast cancer.

[0083] Figure 7 This is the research basis provided by the embodiments of the present invention. 2H4R3me2a affects the prognosis of triple-negative breast cancer and regulates the ATIC expression level.

[0084] Figure 8 This is the research basis provided by the embodiments of the present invention 3PRMT8 is a key factor diagram mediating H4R3me2a in the ATIC promoter region.

[0085] Figure 9 The research basis provided by the embodiments of the present invention is 4PRMT8 / H4R3me2a / ATIC axis promoting de novo synthesis of purine metabolism and malignant phenotype of breast cancer.

[0086] Figure 10 This is the research basis provided by the embodiments of the present invention 5PRMT8 promotes the phosphorylation of H4R3me2a effector protein TDRD3 to promote ATIC transcription map.

[0087] Figure 11 The research basis provided by the embodiments of the present invention is the expression of 6PRMT8 clinical specimens and the targeted PRMT8 / FYN restricted tumor growth diagram. DETAILED DESCRIPTION

[0088] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0089] like Figure 1 As shown, the embodiment of the present invention provides a chromatin modification detection kit detection method for PRMT8 regulating ATIC expression, comprising the following steps:

[0090] S101, screening of cellular biosynthetic pathways and factors associated with poor prognosis in triple-negative breast cancer;

[0091] S102: Exploring the impact of ATIC, a key enzyme in the purine metabolism pathway, on the malignant phenotype and its transcriptional activation mechanism in triple-negative breast cancer;

[0092] S103: Further study the function of PRMT8, a regulatory enzyme for H4R3me2a modification in the ATIC promoter region, in triple-negative breast cancer;

[0093] S104, to explore the effects of PRMT8 on breast cancer cell function in vivo and in vitro experiments;

[0094] S105, in vivo and in vitro functional experiments confirmed that PRMT8 can regulate purine metabolism and malignant phenotype in breast cancer through the H4R3me2a / ATIC signaling axis;

[0095] S106, explore whether PRMT8 further promotes ATIC transcription by binding to FYN and promoting the phosphorylation of the H4R3me2a effector protein TDRD3;

[0096] S107, Evaluation of PRMT8 expression and clinical value in triple-negative breast cancer using big data and clinical samples.

[0097] like Figure 2 As shown, the embodiment of the present invention provides a chromatin modification detection kit detection system for PRMT8 regulating ATIC expression, which includes:

[0098] A screening module for screening cell biosynthetic pathways and factors associated with poor prognosis in triple-negative breast cancer;

[0099] The Exploration Module is used to explore the impact of ATIC, a key enzyme in the purine metabolism pathway, on the malignant phenotype and its transcriptional activation mechanism in triple-negative breast cancer; to further study the function of PRMT8, an enzyme that regulates H4R3me2a modification in the ATIC promoter region in triple-negative breast cancer; and to explore the impact of PRMT8 on breast cancer cell function in in vitro and in vivo experiments.

[0100] The verification module is used for in vivo and in vitro functional experiments to confirm that PRMT8 can regulate purine metabolism and malignant phenotypes in breast cancer through the H4R3me2a / ATIC signaling axis;

[0101] The judgment module is used to explore whether PRMT8 further promotes ATIC transcription by promoting the phosphorylation of the H4R3me2a effector protein TDRD3 by binding to FYN;

[0102] An evaluation module is used to evaluate the expression and clinical value of PRMT8 in triple-negative breast cancer using big data and clinical samples.

[0103] Another object of the present invention is to provide a computer device, comprising a memory and a processor, wherein the memory stores a computer program, and when the computer program is executed by the processor, the processor executes the steps of the chromatin modification detection kit detection method for PRMT8 regulating ATIC expression.

[0104] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, enables the processor to execute the steps of the chromatin modification detection kit detection method for PRMT8 regulating ATIC expression.

[0105] Another object of the present invention is to provide an information data processing terminal, which is used to implement the chromatin modification detection kit detection system for PRMT8 regulating ATIC expression.

[0106] The present invention is specifically implemented:

[0107] 1) The present invention intends to analyze the molecular mechanism of TNBC malignant progression from the perspective of purine metabolism regulation. As shown in [Preliminary Foundation 1a], the applicant team obtained the relevant gene set of purine synthesis metabolism (classical pathway) from the GeneOntology (GO) database ( Figure 3 The applicants presented some key products and rate-limiting enzymes in this pathway, and used the ssGSEA method to score the purine metabolism pathway in patients in the TCGA-BRCA. Using the Boruta machine learning method for feature selection, the applicants found that the synthesis of IMP (inosinic acid monophosphate) in the classic purine synthesis pathway has a significant impact on the short overall survival (OS) of TNBC patients [Preliminary Foundation 1a-b]. In addition, the applicants used clinical specimens from patients with early recurrent metastatic / non-recurrent metastatic triple-negative breast cancer for metastatic disease to perform metabolite mass spectrometry analysis, and the results demonstrated that IMP is a differential metabolite in patients with early recurrence [Preliminary Foundation 1c].

[0108] By measuring ATIC expression in clinical patient tissues and combining it with TCGA data, the applicants preliminarily confirmed that ATIC mRNA levels were significantly higher in patients with shorter OS than in controls [Preliminary Basic Research 1d-e]. Subsequently, the applicants used basic experiments to preliminarily confirm that overexpression of ATIC significantly promoted IMP synthesis in TNBC, and that knockdown of ATIC significantly inhibited the proliferation, invasion, and migration of TNBC cell lines [Preliminary Basic Research 1f-g]. 2) Asymmetric dimethylation of histone 4 at arginine 3 (H4R3me2a) promotes ATIC transcriptional activation. Previous research results from the present group have demonstrated that histone post-translational modification is a key factor in the transcriptional activation of ATIC in breast cancer. Acetylation of histone 3 at lysine 27 (H3K27ac) in the ATIC promoter region promotes ATIC transcription. Building on these findings, the present group further explored and found that in triple-negative breast cancer cells, ssGSEA scores for H4R3me2a and H3K27ac were positively correlated, with significant differences between high and low ATIC expression groups [Research Basic Research 2a-b]. ChIP-seq results from our group on triple-negative breast cancer cells revealed that H4R3me2a signal peaks are enriched in the ATIC promoter region, suggesting a potential regulatory role for ATIC expression [Research Basis 2c]. To investigate the mechanism of ATIC transcriptional activation in triple-negative breast cancer, we will focus on the impact of H4R3me2a on ATIC transcription and its molecular mechanisms. [Research Basis 2d-e] showed that H4R3me2a signal is generally elevated in triple-negative breast cancer samples from our center and is associated with early recurrence and metastasis. ChIP experiments also confirmed that high glucose-induced H4R3me2a signal intensity in the ATIC promoter region of triple-negative breast cancer cells is significantly increased. Treatment with the H4R3me2a inhibitor (MS023) significantly decreased ATIC mRNA levels in TNBC cell lines [Preliminary Basis 2f-g]. Therefore, the applicants hypothesize that transcriptional activation of ATIC, a key kinase in purine metabolism, in triple-negative breast cancer is closely associated with elevated H4R3me2a signal in the promoter region.

[0109] 3) Arginine methyltransferase PRMT8 is a key regulator of H4R3me2a in the ATIC promoter region in breast cancer

[0110] The arginine methyltransferase (PRMT) family is a class of enzymes that methylate arginine residues in proteins. Type I arginine methyltransferases (e.g., PRMT1 and PRMT8) catalyze asymmetric dimethylation of H4R3. H4R3 methylation mediated by type I PRMTs is involved in numerous cellular processes, including DNA repair, RNA transcription, signal transduction, and protein translation. However, clinical trials targeting PRMT1 inhibitors have failed, primarily due to limited research on their molecular mechanisms focusing on PRMT1 and the limited study of other protein family members. Within the PRMT family, both PRMT1 and PRMT8 are differentially expressed in TNBC [Preliminary Foundation 3a-b]. To validate the regulatory function of PRMT1 / 8 on ATIC, the applicants overexpressed PRMT1 or PRMT8 in TNBC cell lines. The results showed no statistically significant difference in the extent of H4R3me2a in the ATIC promoter region after overexpression of either PRMT1 or PRMT8. However, ChIP-qPCR experiments following knockdown of PRMT1 and PRMT8 revealed that ATIC transcription levels were primarily influenced by PRMT8 [Preliminary References 3c-d]. Traditionally, PRMT8 is considered a membrane-localized protein primarily expressed in the central nervous system under physiological conditions; however, its expression in non-central nervous system systems is relatively limited. The present invention collected tissue from real clinical patients and prepared tissue microarrays. Immunohistochemical staining revealed that PRMT8 is localized in the nucleus of TNBC tissues, suggesting its spatial localization in regulating histone methylation associated with TNBC development [Preliminary References 2e]. Literature review and sequence alignment also confirmed that H4 contains a conserved sequence recognized by PRMT8, and that overexpression of PRMT8 promotes elevated H4R3me2a levels in cells [Preliminary References 3e-f]. Therefore, the applicants further hypothesized: Is the enhanced H4R3me2a signaling in the ATIC promoter region in triple-negative breast cancer related to dysregulated PRMT8 expression? Through a series of basic experiments and bioinformatics validation, they preliminarily demonstrated the positive regulatory effect of PRMT8 on H4R3me2a signaling in the ATIC promoter region [Preliminary Foundation 3]. In vivo and in vitro functional experiments also preliminarily confirmed that PRMT8 can regulate breast cancer purine metabolism through the H4R3me2a / ATIC signaling axis, thereby promoting purine metabolism and related malignant phenotypes in triple-negative breast cancer [Preliminary Foundation 4].

[0111] 4) The PRMT8 / FYN / TDRD3 complex promotes H4R3me2a-mediated ATIC transcriptional activation

[0112] Previous studies suggest that PRMT1 and PRMT8 share approximately 80% sequence similarity. To further explore the functional characteristics of PRMT8 in TNBC and to dissect the differential effects of PRMT1 and PRMT8 on ATIC transcription (Preliminary Foundation 3), the applicants compared the functional structures of the two proteins and discovered that, outside the methylation catalytic domain, PRMT8 contains a unique SH3-containing protein binding site (Preliminary Foundation 5a). DNA affinity chromatography and mass spectrometry analysis of triple-negative breast cancer cell lines overexpressing PRMT1 and PRMT8 revealed that a phosphokinase FYN containing an SH3 domain participates in the formation of a protein complex in the ATIC promoter region (Preliminary Foundation 5b). Therefore, the present invention proposes the scientific hypothesis that PRMT8 recruits the phosphokinase FYN via the SH3-containing protein binding site. Co-IP experiments have preliminarily confirmed this binding (Preliminary Foundation 5c).

[0113] Numerous experiments have demonstrated that histone modification effector proteins can be phosphorylated, thereby altering their binding to downstream transcription initiation proteins and subsequently affecting gene transcription. The present invention [Preliminary studies 5b, d] demonstrated that Tudor Domain Containing 3 (TDRD3), the primary effector protein (reader) of H4R3me2a, binds to the ATIC promoter and mediates the increased ATIC expression induced by PRMT8 overexpression. This study preliminarily confirmed the presence of phosphorylated TDRD3 in TNBC cell lines with high PRMT8 expression, with differential expression levels between high and low PRMT8 expression groups. The FYN inhibitor Saracatinib significantly reduced TDRD3 phosphorylation and suppressed ATIC expression [Preliminary studies 5e-f]. Therefore, the present invention further proposes the hypothesis that PRMT8 recruitment of FYN leads to phosphorylation of the H4R3me2a effector protein TDRD3, promoting ATIC transcription. In a subcutaneous transplant tumor model of triple-negative breast cancer in mice, the combination of the PRMT8 inhibitor GSK3368715 and the FYN inhibitor Saracatinib significantly improved the therapeutic effect of the PRMT8 inhibitor; this further verified the regulatory role of the PRMT8 / FYN / pTDRD3 axis on purine metabolism in triple-negative breast cancer [preliminary foundation 6d].

[0114] 5) Generation of project ideas

[0115] In summary, the applicant analyzed TNBC patients with poor clinical prognosis and combined bioinformatics and machine learning methods to find that the purine synthesis pathway is a potential mechanism causing poor prognosis in TNBC patients; among them, the IMP synthesis pathway is a key pathway. Subsequently, the applicant's team used basic experiments and bioinformatics analysis to confirm that the transcription level of IMP's catalytic enzyme ATIC is increased in TNBC. The applicant's early research project focused on the post-translational modification of histones in the ATIC promoter region and confirmed that histone acetylation (H3K27ac) promotes ATIC transcription; and the present invention further explored the mechanism on this basis and found that H4R3me2a was significantly correlated with H3K27ac and ATIC expression levels. ChIP experiments preliminarily demonstrated that the H4R3me2a signal in the ATIC promoter region was elevated. To find regulatory factors for histone H4 arginine methylation, the applicant targeted the arginine methyltransferase (PRMT) family through basic experiments and bioinformatics analysis. PRMT8 is located in the nucleus of breast cancer cells and has an H4R3 recognition site. Compared with normal tissues, its expression is significantly upregulated. Its effect on ATIC transcriptional activation is far greater than that of PRMT1 in the same family. At the same time, experiments such as protein imprinting, fluorescence quantitative PCR, and ChIP also confirmed that PRMT8 can regulate the TNBC purine metabolism pathway and related malignant phenotypes through the H4R3me2a / ATIC signaling axis. Next, the applicant further explored the bypass molecular mechanism of the strong activation effect of PRMT8. By comparing the protein domains with its important paralog PRMT1 and performing mass spectrometry analysis, we identified the SH3-containing protein binding domain and its target protein FYN. Targeted Co-IP and other experiments preliminarily confirmed that PRMT8 can bind to the tyrosine kinase FYN, thereby phosphorylating the H4R3me2a effector protein TDRD3, further promoting the transcription of ATIC.

[0116] Finally, the applicant proposed the following hypothesis: the expression of arginine methyltransferase PRMT8 is upregulated in triple-negative breast cancer, which promotes the methylation of H4R3 in the promoter region of ATIC, the rate-limiting enzyme in the purine metabolism pathway, and leads to the activation of ATIC transcription and the breast cancer purine metabolism pathway, thereby enhancing the malignant phenotype of triple-negative breast cancer; at the same time, PRMT8 mediates FYN to phosphorylate the H4R3me2a effector protein TDRD3, further synergistically promoting ATIC transcriptional activation, leading to the progression of TNBC ( Figure 4 ).

[0117] (1) Screening of cell biosynthetic pathways and factors associated with poor prognosis of triple-negative breast cancer.

[0118] 1) Obtain the mRNA expression profile of triple-negative breast cancer in TCGA-BRCA, perform ssGSEA analysis using the metabolism-related dataset in the GO database, and use Boruta to screen the important predictors of metabolism-related ssGSEA scores for TNBC prognosis.

[0119] 2) Obtain in situ tumor samples of early metastatic triple-negative breast cancer from our unit for mass spectrometry analysis to explore the differences in their metabolites.

[0120] 3) Western blot analysis was used to detect the protein expression levels of ATIC, a key rate-limiting enzyme for IMP production, in each cell line subjected to high glucose-induced activation of purine metabolism and in the aforementioned primary cell lines. Quantitative PCR was used to detect the mRNA levels of ATIC in each cell line to determine the expression of ATIC in each cell line and the level at which it changes.

[0121] (2) Explore the effect of ATIC, a key enzyme in the purine metabolism pathway of triple-negative breast cancer, on the malignant phenotype and its transcriptional activation mechanism.

[0122] 1) Using a lentiviral transfection system, plasmids were constructed: LV-Luc-ATIC and LV-Luc-shATIC for stable overexpression and knockdown of ATIC, respectively. Colony formation, softagar and transwell migration and invasion assays, and stem cell sphere formation assays were used to examine the effects of ATIC on breast cancer cell proliferation, invasion, metastasis, and stem cell properties.

[0123] 2) Based on ATIC expression, patients with triple-negative breast cancer in TCGA were divided into high / low expression groups. The ssGSEA method was used to enrich the scores of histone post-translational modification-related pathways in the two groups of patients, and the Wilcox method was used to test the differences between the two groups.

[0124] 3) Surgical resection tissue was collected from patients with triple-negative breast cancer and prepared into tissue microarrays. Histone arginine methylation (H4R3me2a) was detected by immunohistochemistry and its correlation with clinical pathological data was analyzed.

[0125] 4) Chromosome immunoprecipitation (ChIP) experiments were used to detect the signal intensity of the ATIC promoter region sequence bound to methylated histone 4 (H4R3me2a) in different molecularly classified breast cancer cell lines such as MCF-7, MDA-MB-453, and MDA-MB-231.

[0126] 5) Application of histone arginine methylation (H4R3me2a) inhibitor to treat MCF-7, MDA-MB-453, MDA-MB-231 and other different subtypes of breast cancer cell lines, and detect the degree of ATIC promoter region histone arginine methylation (H4R3me2a) and its mRNA expression level changes by Western blot and fluorescent quantitative PCR experiments.

[0127] 6) After using site-directed mutagenesis technology to construct a plasmid of the third arginine mutation in H4, transfect breast cancer cells, and verify the expression changes of ATIC.

[0128] (3) In-depth study of the function of ATIC promoter region H4R3me2a modification regulatory enzyme PRMT8 in triple-negative breast cancer.

[0129] 1) Application of anti-PRMT8 and ATIC promoter region DNA probe in MDA-MB-231 and other TNBC breast cancer cell lines for Co-IP and DNA-pulldown experiments to verify the binding ability of PRMT8 and ATIC promoter region.

[0130] 2) Application of Western blot experiment to detect the protein expression level of PRMT8 in MCF-7, MDA-MB-453, MDA-MB-231 and other different subtypes of breast cancer cell lines.

[0131] 3) Application of lentiviral transfection system to construct plasmid: LV-Luc-PRMT8 and LV-Luc-shPRMT8 to stably overexpress / knock down PRMT8, and use Western blot and fluorescent quantitative PCR to detect the expression changes of ATIC.

[0132] 4) In the above overexpression / knockdown cell lines, ChIP method is used to determine the degree of change of ATIC promoter region H4R3me2a.

[0133] (4) Explore the influence of PRMT8 on breast cancer cell function in in vivo and in vitro experiments.

[0134] 1) Select MCF-7, MDA-MB-453 and MDA-MB-231 as the main experimental cells. Application of lentiviral transfection system to construct plasmid: LV-Luc-PRMT8 and LV-Luc-shPRMT8 to stably overexpress / knock down PRMT8. Use colony formation, soft agar, transwell migration and invasion experiments, and stem cell sphere formation experiments to detect the influence of PRMT8 on breast cancer cell proliferation, invasion and metastasis, and stem cell characteristics.

[0135] 2) 13C1-glycine / 15N-labeled glutamine (amide-15N) was added to the glycine / glutamine-free culture medium of the above-mentioned PRMT8-overexpressing / knockout tumor cells, and the levels of 13C1 / 15N2-labeled IMP, AMP, and GMP production in the PRMT8-knockdown / overexpressing tumor cells were measured to examine the effect of PRMT8 on the purine metabolism pathway of the corresponding tumor cell lines.

[0136] 3) Orthotopic mammary tumor models were established in SCID mice by injecting normal cells, LV-Luc-PRMT8-transfected cells, and LV-Luc-shPRMT8-transfected tumor cells into the mammary fat pad. Tumor fluorescence intensity and volume were compared between the experimental and control groups. A lung metastasis model was established in SCID mice by tail vein injection. The fluorescence intensity and number of lung metastatic nodules were measured between the experimental and control groups. Tissues from both the orthotopic tumors and metastases were collected and histologically confirmed by HE or IHC.

[0137] (5) In vivo and in vitro functional experiments confirmed that PRMT8 can regulate purine metabolism and malignant phenotype of breast cancer through the H4R3me2a / ATIC signaling axis.

[0138] 1) The above-mentioned PRMT8-overexpressing / knockout cell lines were treated with a histone methylation inhibitor (GSK3368715) and an ATIC inhibitor (ATIC-IN-1 acetate); Western blot and RT-qPCR experiments were used to detect whether PRMT8 has a transcriptional regulatory function on ATIC.

[0139] 2) The above-mentioned PRMT8 overexpression / knockout cell lines were treated with a histone methylation inhibitor (GSK3368715) and an ATIC inhibitor (ATIC-IN-1 acetate) for colony formation, softagar, transwell migration and invasion assays, and stem cell sphere formation assays to examine their effects on breast cancer cell proliferation, invasion and metastasis, and stem cell characteristics.

[0140] 3) The above-mentioned PRMT8-overexpressing / knockout tumor cells were treated with a histone methylation inhibitor (GSK3368715) and an ATIC inhibitor (ATIC-IN-1 acetate). 13C1-glycine / 15N-labeled glutamine (amide-15N) was added to the glycine / glutamine-free culture medium, and the levels of 13C1 / 15N2-labeled IMP, AMP, and GMP in the tumor cells were measured to determine changes in the purine metabolism pathway in the corresponding tumor cell lines.

[0141] 4) Application of overexpression / knockout PRMT8 tumor cells breast fat pad injection to construct SCID mouse breast orthotopic tumor model, orthotopic injection of methylation inhibitor GSK3368715, compare the fluorescence intensity, volume of tumor in experimental group and control group; tail vein injection to establish SCID mouse lung metastasis model, detect the fluorescence intensity, number of nodules of lung metastasis nodules in experimental group and control group. The above orthotopic tumor and metastatic tumor tissues are collected and HE or IHC is used to determine the tissue type.

[0142] 5) Overexpression / knockout of PRMT8 in H4R3 replacement cell lines, Western blot, RT-qPCR experiments are used to detect whether PRMT8 exists in ATIC transcriptional regulation function; colony formation, soft agar, transwell migration and invasion experiments and stem cell sphere formation experiments are performed to detect the effects of PRMT8 on breast cancer cell proliferation, invasion, metastasis and stem cell characteristics; 13C1-glycine / 15N-labeled glutamine (amide-15N) is added to the glycine / glutamine-free medium to detect the generation level of 13C1 / 15N2-labeled IMP, AMP and GMP in tumor cells, so as to judge the changes of the corresponding tumor cell line purine metabolic pathway.

[0143] (6) Explore whether PRMT8 promotes the phosphorylation of H4R3me2a effector protein TDRD3 to further promote the transcription of ATIC by binding FYN.

[0144] 1) According to the 500bp fragment upstream of the ATIC promoter (relative to the major transcription start site TSS), a bait DNA is designed, and a 5'-biotin modified primer and an unmodified primer are used for PCR synthesis of DNA probe. After the biotin-labeled bait DNA is adsorbed on Dynabeads MyOne Streptavidin T1, DNA affinity purification is performed using MDA-MB-231 nuclear lysate overexpressing PRMT1 or PRMT8; the obtained product is subjected to LC-MS detection; the peptide sequence is compared with human proteins using protein BLAST1.

[0145] 2) ChIP and Co-IP experiments are performed in breast cancer cell lines using anti-TDRD3 and anti-PRMT8, respectively, to test the binding ability of TDRD3 to the ATIC promoter region and PRMT8 to FYN.

[0146] 3) After establishing PRMT8 overexpression cell lines in MDA-MB-231 and other TNBC cell lines using LV-Luc-PRMT8, TDRD3 is knocked down, and RT-qPCR and WB are used to detect ATIC expression to verify the regulation of TDRD3 on ATIC transcription.

[0147] 4) Using biotin-labeled ATIC promoter region DNA sequence probe to purify DNA binding proteins, and using phos-tag SDS electrophoresis and WB experiments to detect the expression of phosphorylated TDRD3.

[0148] 5) Using FYN inhibitor (Saracatinib) in PRMT8 overexpressed TNBC cell lines, DNA-pulldown experiment and custom pTDRD3 antibody are used to detect the phosphorylation level of ATIC promoter region TDRD3; ChIP is used to check H4R3me2a level; RT-qPCR and WB are used to detect ATIC expression in the above cell lines.

[0149] 6) Using anti-TDRD3 for immunoprecipitation (IP) experiment in PRMT8 overexpressed TNBC cell lines, mass spectrometry is used to detect the phosphorylation sites of TDRD3; Co-IP experiment is used to compare the changes of TDRD3 phosphorylation on its affinity with downstream key protein TOP3B in PRMT8 overexpressed cell lines.

[0150] (7) Evaluate the expression and clinical value of PRMT8 in triple-negative breast cancer in big data and clinical samples.

[0151] 1) Obtain the expression profile data and clinical information of triple-negative breast cancer from TCGA, METABRIC and GEO databases. Through survival analysis, predict the influence of PMRT8 mRNA expression level on prognosis.

[0152] 2) Use self-made tissue chip (this study has obtained the ethical approval of the relying institution and the informed consent of all sample providers, and all samples have been diagnosed by two or more professional pathologists) (151 breast cancer samples, 46 normal breast samples) to detect the expression of PRMT8 in each type of breast cancer tissue, and use immunohistochemical H-score to quantify the expression level of PRMT8 in the tissue; analyze the expression differences between normal tissue and each subtype of breast cancer tissue; according to the median expression of PRMT8, all enrolled samples are divided into high and low expression groups, and the influence of PRMT8 on survival (RFS, OS) is analyzed, and its correlation with clinical characteristics (patient age, tumor size, lymph node status, distant metastasis, molecular subtype) is further evaluated.

[0153] 3) Using single factor and multiple factor COX regression analysis, evaluate whether PRMT8 can become an independent prognostic indicator for triple-negative breast cancer among many clinical pathological factors.

[0154] 4) Using clinical triple-negative breast cancer patient tumor primary cell lines, establish mouse subcutaneous tumor models, and compare and verify the inhibitory effect of PRMT8 inhibitor, FYN inhibitor and double inhibitor combination on triple-negative breast cancer.

[0155] Technical route Figure 5

[0156] Research results related to the present invention:

[0157] (1) Through bioinformatics and mass spectrometry analysis, metabolic pathways and driving factors related to the prognosis of triple-negative breast cancer were screened.

[0158] Figure 6 (a) ssGSEA analysis obtained enrichment scores for relevant gene sets and used Boruta (machine learning) for feature selection to identify important features that influence the prognosis of triple-negative breast cancer. (b) Patients with high ssGSEA scores for the IMP synthesis pathway were associated with shorter OS. (c) Mass spectrometry analysis of clinical samples detected products of the de novo purine synthesis pathway. (d) Expression levels of enzymes related to de novo purine synthesis in patients with early recurrence of triple-negative breast cancer. (e) ATIC is highly expressed in patients with poor prognosis. (f) Low ATIC expression significantly impairs breast cancer cell proliferation, invasion, and metastasis. (g) High ATIC expression promotes IMP production in triple-negative breast cancer.

[0159] (2) H4R3me2a affects the prognosis of triple-negative breast cancer and regulates ATIC expression.

[0160] Figure 7 (ab) Differences and correlations in ssGSEA scores of major histone post-transcriptional modifications between high and low ATIC expression groups. (c) Distribution of H4R3me2a binding peaks in the ATIC promoter region in ChIP-seq experiments. (d) COX regression analysis of the prognostic impact of H4R3me2a on triple-negative breast cancer. (e) Differential expression of H4R3me2a in clinical triple-negative breast cancer specimens and normal tissues. (f) ChIP assay to detect the binding strength of H4R3me2a to the ATIC promoter region sequence in triple-negative breast cancer cells induced by high glucose. (g) H4R3me2a inhibition reduces ATIC expression in triple-negative breast cancer cell lines.

[0161] (3) PRMT8 is a key factor mediating H4R3me2a in the ATIC promoter region.

[0162] Figure 8 (a) Bioinformatics and qPCR experiments suggest that PRMT1 / PRMT8 are differentially expressed genes in triple-negative breast cancer. (c) ChIP assay assesses changes in H4R3me2a expression in the ATIC promoter region following overexpression of PRMT1 or PRMT8. (d) Changes in ATIC expression following knockdown of PRMT1 or PRMT8. (e) PRMT8 overexpression leads to upregulation of H4R3me2a levels across the entire triple-negative breast cancer population. (f) Histone 4 intrinsic sequence and PRMT8 recognition sequence.

[0163] (4) PRMT8 / H4R3me2a / ATIC axis promotes de novo purine synthesis and breast cancer malignant phenotype.

[0164] Figure 9 (a) PRMT / H4R3me2 / ATIC axis functional restoration experiment by colony formation experiment. (b) Transwell detection of corresponding cell line invasion ability. (c) Immunofluorescence experiment to detect the effect of PRMT8 on breast cancer cell stemness. (d) In vivo experiment in SCID mice to detect the effect of PRMT / H4R3me2a / ATIC signal axis on breast cancer cell function. (e) Functional restoration experiment to detect the effect of PRMT / H4R3me2a / ATIC signal axis on breast cancer cell line purine metabolism pathway.

[0165] (5) PRMT8 promotes H4R3me2a effector protein TDRD3 phosphorylation to further promote ATIC transcription.

[0166] Figure 10 (a) PRMT1 / PRMT8 protein structure schematic diagram. (b) DNA affinity chromatography mass spectrometry results of PRMT1 / PRMT8 overexpression triple-negative breast cancer cell lines (top five proteins of expression amount). (c) Co-IP experiment confirmed that PRMT8 but not PRMT1 can bind with FYN. (d) TDRD3 low expression inhibits PRMT8 mediated H4R3me2a regulation of ATIC. (e) FYN inhibitor limits TDRD3 phosphorylation in PRMT8 high expression cell lines. (f) FYN inhibitor down-regulates ATIC transcription level in PRMT8 high expression cell lines.

[0167] (6) PRMT8 inhibitor combined with FYN inhibitor significantly limits tumor growth.

[0168] Figure 11 (a) Survival analysis of PRMT8 high and low expression groups of TCGA-BRCA patients. (b-c) PRMT8 expression was detected by IHC staining of patient tissue chip in our unit. (d) PDX model verified the effect of PRMT8 / FYN combined therapy.

[0169] After completing the project, the present group continued to conduct in-depth research on purine metabolism pathways in breast cancer. Focusing on the poor prognosis of triple-negative breast cancer, they combined bioinformatics methods to identify a metabolic pathway associated with this prognosis: the IMP synthesis pathway. Mass spectrometry and cellular functional experiments pinpointed ATIC, a key enzyme in IMP synthesis, as a key factor in the progression of triple-negative breast cancer. Building on previous research findings, the present group planned to further investigate the transcriptional activation mechanisms of ATIC. Using bioinformatics and basic experimental methods, they discovered another histone modification, H4R3me2a, that influences ATIC transcriptional activation in this clinical setting. They then identified its upstream regulatory factor, PRMT8. Through preliminary experiments to validate its function and mechanism, they ultimately proposed a mechanism by which purine metabolism disorders, mediated by the PRMT8 / H4R3me2 / ATIC signaling axis, regulate the malignant phenotype in triple-negative breast cancer. This project not only validates the previous project but also provides a more detailed and in-depth longitudinal study.

[0170] Research Summary: Abnormal activation of the purine metabolism pathway plays a crucial role in breast tumor progression. The role and mechanism of long noncoding RNA in purine metabolism in breast cancer remain unclear. Early data analysis revealed that the expression of the long noncoding RNA TPT1-AS1 (TPT1-AS1) is reduced in breast cancer tissue. Functional experiments also demonstrated that TPT1-AS1 can inhibit malignant phenotypes such as proliferation, metastasis, and stem cell characteristics of breast cancer cells. Mass spectrometry analysis revealed that the purine metabolism pathway was activated in breast tumor cells with TPT1-AS1 knockout, with a significant increase in the expression of the de novo synthesized end product, IMP (inosinic monophosphate). Further mechanistic studies revealed that TPT1-AS1 inhibits the expression of the kinase ATIC, which catalyzes the production of IMP. Binding of TPT1-AS1 to CBP (CREB-binding protein) leads to decreased histone acetylation in the ATIC promoter region, reducing ATIC expression. Ultimately, the tumor purine metabolism pathway is blocked, suppressing the malignant phenotype of breast cancer. This study will verify this hypothesis, reveal the molecular mechanism by which non-coding RNA regulates the biological functions of breast cancer cells in more detail, and provide possible targets for the treatment of breast cancer.

[0171] Example 1

[0172] In this embodiment, the chromatin immunoprecipitation-real-time quantitative PCR (ChIP-qPCR) method is used to detect the enrichment level of PRMT8-mediated H4R3me2a modification in the ATIC promoter region. First, the triple-negative breast cancer cell line MDA-MB-231 is collected, fixed by 1% formaldehyde cross-linking, and the chromatin is broken by ultrasonic to 200-500 bp fragments; then the specific antibody against H4R3me2a and normal IgG are used for immunoprecipitation, respectively, and after elution, reverse cross-linking and DNA extraction; finally, qPCR amplification is performed with ATIC promoter specific primers, and the enrichment fold difference between PRMT8 knockout group and control group is calculated to evaluate the effect of PRMT8 on ATIC transcription activation.

[0173] Example 2

[0174] In this embodiment, an ELISA-based detection kit is designed for quantitative detection of H4R3me2a modification-mediated ATIC expression in cell or tissue samples. The steps include: ① Prepare 96-well plate coated with anti-H4R3me2a antibody; ② Dilute the nuclear protein extracted from triple-negative breast cancer tissue lysate by gradient and add it to the hole for incubation; ③ Add biotin-labeled ATIC promoter DNA probe, and then add horseradish peroxidase (HRP)-coupled streptavidin; ④ Color development with TMB substrate, and determine the OD450 value by enzyme label instrument, and draw the standard curve to quantitatively evaluate the effect of PRMT8 overexpression or inhibition on H4R3me2a / ATIC signal axis.

[0175] Example 3

[0176] In this embodiment, the PRMT8 interaction with FYN and TDRD3 discussed in step 6 is included in the combined immunocomplex detection process. Specifically, after completing ChIP-qPCR, the same samples are subjected to double immunoprecipitation: first, anti-PRMT8 antibody is used for precipitation, and then anti-FYN or anti-TDRD3 antibody is used for secondary precipitation, followed by Western blot detection of H4R3me2a and TDRD3 phosphorylation signals, to further verify the core role of PRMT8 in ATIC transcription activation through FYN-mediated effector protein modification.

[0177] Example 4

[0178] In the present embodiment, combined with big data and clinical sample analysis (step 7), a multivariate regression model of PRMT8 / H4R3me2a and ATIC expression is constructed. RNA and protein are extracted from 50 cases of triple-negative breast cancer patients and 20 cases of normal breast tissue, respectively, and qRT-PCR and ELISA detection are performed on PRMT8, H4R3me2a level and ATIC mRNA; survival analysis and ROC curve evaluation are performed using SPSS software to determine the diagnostic efficiency of PRMT8 expression and H4R3me2a modification level in predicting the progression-free survival (PFS) and overall survival (OS) of patients, and to provide statistical basis for the clinical application of the kit.

[0179] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any modification, equivalent replacement and improvement made by any person skilled in the art within the technical range disclosed by the present application, as long as it is within the spirit and principle of the present application, should be covered within the protection scope of the present application.

Claims

1. A chromatin modification detection kit detection method for PRMT8 regulating ATIC expression, characterized in that: The following steps are involved: Step 1: Screening of cell biosynthetic pathways and factors associated with poor prognosis of triple-negative breast cancer; Step 2: Explore the impact of ATIC, a key enzyme in the purine metabolism pathway, on the malignant phenotype and its transcriptional activation mechanism in triple-negative breast cancer. Step 3: Further study the function of PRMT8, an enzyme that regulates H4R3me2a modification in the ATIC promoter region, in triple-negative breast cancer; Step 4: Explore the impact of PRMT8 on breast cancer cell function in vivo and in vitro experiments; Step 5: In vitro and in vivo functional experiments confirmed that PRMT8 can regulate purine metabolism and malignant phenotype in breast cancer through the H4R3me2a / ATIC signaling axis; Step 6: Explore whether PRMT8 further promotes ATIC transcription by binding to FYN and promoting the phosphorylation of the H4R3me2a effector protein TDRD3; Step 7: Evaluate the expression and clinical value of PRMT8 in triple-negative breast cancer using big data and clinical samples.

2. The detection method of the chromatin modification detection kit for PRMT8 regulating ATIC expression according to claim 1, characterized in that: The screening of cell biosynthetic pathways and factors associated with poor prognosis of triple-negative breast cancer: 1) Obtain the mRNA expression profile of triple-negative breast cancer in TCGA-BRCA, perform ssGSEA analysis using the metabolism-related dataset in the GO database, and use Boruta to screen the important predictors of TNBC prognosis related to the metabolism-related ssGSEA score; 2) Obtain in situ tumor samples of early metastatic triple-negative breast cancer from our unit for mass spectrometry analysis to explore the differences in their metabolites; 3) Western blot analysis was used to detect the protein expression levels of ATIC, a key rate-limiting enzyme for IMP production, in each cell line subjected to high glucose-induced activation of purine metabolism and in the aforementioned primary cell lines. Quantitative PCR was used to detect the mRNA levels of ATIC in each cell line to determine the expression of ATIC in each cell line and the level at which it changes.

3. The detection method of the chromatin modification detection kit for PRMT8 regulating ATIC expression according to claim 1, characterized in that: The study explores the effect of ATIC, a key enzyme in the purine metabolism pathway of triple-negative breast cancer, on the malignant phenotype and its transcriptional activation mechanism: 1) Using a lentiviral transfection system, plasmids were constructed: LV-Luc-ATIC and LV-Luc-shATIC were used to stably overexpress and knockdown ATIC, respectively. Colony formation, softagar and transwell migration and invasion assays, and stem cell sphere formation assays were used to examine the effects of ATIC on breast cancer cell proliferation, invasion, metastasis, and stem cell properties. 2) Based on ATIC expression, patients with triple-negative breast cancer in TCGA were divided into high / low expression groups. The ssGSEA method was used to enrich the histone post-translational modification-related pathways in the two groups of patients, and the Wilcox method was used to test the differences between the two groups; 3) Collect surgically resected breast tissue from patients with triple-negative breast cancer and prepare tissue microarrays from the tissues. Immunohistochemistry was used to detect histone arginine methylation and analyze its correlation with clinical pathological data. 4) Chromosome immunoprecipitation assay was used to detect the signal intensity of the ATIC promoter region sequence bound to methylated histone 4 in breast cancer cell lines with different molecular subtypes, including MCF-7, MDA-MB-453, and MDA-MB-231; 5) Treat MCF-7, MDA-MB-453, and MDA-MB-231 breast cancer cell lines of different subtypes with histone arginine methylation inhibitors, and use Western blot and quantitative PCR to detect changes in histone arginine methylation in the ATIC promoter region and its mRNA expression level; 6) Site-directed mutagenesis was used to construct a plasmid encoding the third arginine mutation in H4 and then transfected into breast cancer cells to verify the change in ATIC expression.

4. The detection method of the chromatin modification detection kit for PRMT8 regulating ATIC expression according to claim 1, characterized in that: The in-depth study of the function of the H4R3me2a modification regulatory enzyme PRMT8 in the ATIC promoter region in triple-negative breast cancer: 1) Co-IP and DNA pulldown experiments were performed in the MDA-MB-231TNBC breast cancer cell line using anti-PRMT8 and ATIC promoter region DNA probes to verify the binding ability of PRMT8 to the ATIC promoter region; 2) Western blot assay was used to detect the protein expression level of PRMT8 in different breast cancer cell lines (MCF-7, MDA-MB-453, and MDA-MB-231); 3) Using a lentiviral transfection system, construct plasmids: LV-Luc-PRMT8 and LV-Luc-shPRMT8 were used to stably overexpress / knockdown PRMT8, respectively, and Western blot and quantitative PCR were used to detect changes in ATIC expression; 4) ChIP was used to determine the changes in the level of H4R3me2a in the ATIC promoter region in the above-mentioned overexpression / knockdown cell lines.

5. The detection method of the chromatin modification detection kit for PRMT8 regulating ATIC expression according to claim 1, characterized in that: The study explored the effects of PRMT8 on breast cancer cell function in vivo and in vitro experiments: 1) MCF-7, MDA-MB-453, and MDA-MB-231 cells were selected as primary experimental cells. A lentiviral transfection system was used to construct plasmids: LV-Luc-PRMT8 and LV-Luc-shPRMT8, respectively, for stable overexpression and knockdown of PRMT8. Colony formation, softagar and transwell migration and invasion assays, and stem cell sphere formation assays were used to examine the effects of PRMT8 on breast cancer cell proliferation, invasion, metastasis, and stem cell properties. 2) Add 13C1-glycine / 15N-labeled glutamine to the glycine / glutamine-free culture medium of the PRMT8-overexpressing / knockout tumor cells, and measure the levels of 13C1 / 15N2-labeled IMP, AMP, and GMP production in the PRMT8-knockdown / overexpressing tumor cells to examine the effect of PRMT8 on the purine metabolism pathway of the corresponding tumor cell lines; 3) Orthotopic mammary tumor models were established in SCID mice by injecting normal, LV-Luc-PRMT8-transfected, and LV-Luc-shPRMT8-transfected tumor cells into the mammary fat pad. The fluorescence intensity and volume of tumors in the experimental and control groups were compared. A lung metastasis model was established in SCID mice by tail vein injection. The fluorescence intensity and number of lung metastatic nodules in the experimental and control groups were measured. The in situ tumor and metastatic tumor tissues were collected and the tissue types were determined by HE or IHC.

6. The detection method of the chromatin modification detection kit for PRMT8 regulating ATIC expression according to claim 1, characterized in that: The in vivo and in vitro functional experiments confirmed that PRMT8 can regulate purine metabolism and malignant phenotypes in breast cancer through the H4R3me2a / ATIC signaling axis: 1) Treat the above-mentioned PRMT8-overexpressing / knockout cell lines with histone methylation inhibitors and ATIC inhibitors; use Western blot and RT-qPCR to detect whether PRMT8 regulates ATIC transcription; 2) The above-mentioned PRMT8 overexpression / knockout cell lines were treated with histone methylation inhibitors and ATIC inhibitors for colony formation, softagar, transwell migration and invasion assays, and stem cell sphere formation assays to examine their effects on breast cancer cell proliferation, invasion and metastasis, and stem cell characteristics; 3) The above-mentioned PRMT8-overexpressing / knockout tumor cells were treated with a histone methylation inhibitor and an ATIC inhibitor. 13C1-glycine / 15N-labeled glutamine was added to the glycine / glutamine-free culture medium. The levels of 13C1 / 15N2-labeled IMP, AMP, and GMP in the tumor cells were measured to determine changes in the purine metabolism pathway in the corresponding tumor cell lines. 4) Orthotopic mammary tumor models were established in SCID mice by injecting PRMT8-overexpressing / knockout tumor cells into the mammary fat pad. The methylation inhibitor GSK3368715 was injected orthotopically, and the fluorescence intensity and volume of tumors in the experimental and control groups were compared. A lung metastasis model was established in SCID mice by tail vein injection, and the fluorescence intensity and number of lung metastatic nodules in the experimental and control groups were measured. Tissues from the above-mentioned orthotopic tumors and metastases were collected and tissue types were determined by HE or IHC. 5) Overexpress / knockout PRMT8 in the H4R3 replacement cell line and use Western blot and RT-qPCR to test whether PRMT8 regulates ATIC transcription. Perform colony formation, softagar, transwell migration and invasion assays, and stem cell sphere formation assays to test its effects on breast cancer cell proliferation, invasion, metastasis, and stem cell properties. Add 13C1-glycine / 15N-labeled glutamine to glycine / glutamine-free culture medium and measure the levels of 13C1 / 15N2-labeled IMP, AMP, and GMP in tumor cells to determine changes in the purine metabolism pathway in the corresponding tumor cell lines. To explore whether PRMT8 further promotes ATIC transcription by binding to FYN and promoting the phosphorylation of the H4R3me2a effector protein TDRD3; 1) Bait DNA was designed based on a 500-bp fragment upstream of the ATIC promoter, and a DNA probe was synthesized by PCR using 5'-biotin-modified and unmodified primers. The biotin-labeled bait DNA was adsorbed onto Dynabeads MyOne StreptavidinT1 and affinity purified using nuclear lysates of MDA-MB-231 cells overexpressing PRMT1 or PRMT8. The resulting products were analyzed by LC-MS. The peptide sequences were compared with the human protein using Protein BLAST1. 2) ChIP and Co-IP experiments were performed in breast cancer cell lines using anti-TDRD3 and anti-PRMT8, respectively, to test the binding of TDRD3 to the ATIC promoter region and PRMT8 to FYN; 3) In the MDA-MB-231TNBC cell line, LV-Luc-PRMT8 was used to establish a PRMT8-overexpressing cell line, followed by knockdown of TDRD3. ATIC expression was detected by RT-qPCR and western blot to verify the regulation of TDRD3 on ATIC transcription. 4) Use a biotin-labeled ATIC promoter region DNA sequence probe to purify DNA-binding proteins, and use phos-tag SDS electrophoresis and Western blotting to detect the expression of phosphorylated TDRD3; 5) In TNBC cell lines overexpressing PRMT8, use the FYN inhibitor to perform DNA pulldown experiments and a custom pTDRD3 antibody to detect the phosphorylation level of TDRD3 in the ATIC promoter region; ChIP to examine H4R3me2a levels; and use RT-qPCR and western blot to detect ATIC expression in the above cell lines; 6) Immunoprecipitation (IP) experiments were performed using anti-TDRD3 in TNBC cell lines overexpressing PRMT8, and mass spectrometry was used to detect the phosphorylation sites of TDRD3. Co-IP experiments were performed in cell lines overexpressing PRMT8 to compare the changes in TDRD3 phosphorylation and its affinity for the key downstream protein TOP3B.

7. A detection kit for detecting the chromatin modification state of PRMT8 regulating ATIC expression, characterized in that: The kit comprises: (1) A specific antibody for recognizing asymmetric dimethylation of histone H4 at arginine 3 (H4R3me2a); (2) DNA primer pairs designed for the ATIC promoter sequence for ChIP-qPCR or DNA-pulldown assays; (3) Co-immunoprecipitation (Co-IP) reagent kit for detecting PRMT8, TDRD3 and their interactions; (4) RT-qPCR reaction components for evaluating ATIC transcription levels; The kit is used to detect the ATIC transcriptional regulatory mechanism mediated by PRMT8 through H4R3me2a modification, and is suitable for molecular mechanism research or preclinical validation of tumor samples such as breast cancer.

8. The kit according to claim 1, wherein The H4R3me2a antibody is a monoclonal antibody that can specifically recognize the asymmetric dimethylation modification of arginine 3 at position 3 of human histone H4 and has ChIP-level purity.

9. The kit according to claim 1, wherein The DNA primer pair is used to amplify the core regulatory sequence in the region from -1000 bp to +100 bp upstream of the ATIC promoter and is suitable for enrichment analysis of various breast cancer cell lines and tissue samples.

10. The kit according to claim 1, wherein It also includes phos-tag SDS-PAGE electrophoresis reagents and customized p-TDRD3 antibodies for detecting the phosphorylation status of TDRD3, enabling precise qualitative and quantitative analysis of the PRMT8-FYN-TDRD3 regulatory pathway.

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