Use of substances targeting brd4 in the manufacture of a product related to diabetes

Abstract

The present disclosure provides a use of a substance targeting BRD4 in the preparation of a product related to diabetes. In the present disclosure, it is found that the expression of BRD4 in human diabetic beta cells is significantly reduced, and BRD4 plays an important role in maintaining beta cell maturation and differentiation, and long-term and acute BRD4 deficiency can lead to reduced insulin secretion and down-regulation of differentiation markers, highlighting the key role of BRD4 in pancreatic beta cells, providing a target BRD4 that has a significant impact on diabetes, so that substances targeting BRD4 can help to assess the susceptibility of diabetes, screening, prevention, intervention and treatment of diabetes.

Description

Technical Field

[0001] This disclosure relates to the field of biomedical technology, specifically to the application of a substance targeting BRD4 in the preparation of diabetes-related products. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by hyperglycemia, caused by a deficiency in insulin secretion or impaired insulin action. Type 2 diabetes mellitus (T2DM) is the most prevalent type of diabetes, accounting for over 90% of cases. Type 2 diabetes is a chronic disease caused by insufficient insulin secretion and / or insulin resistance (decreased sensitivity to insulin, leading to its ineffective utilization) due to genetic and / or environmental factors, resulting in elevated blood glucose levels. Significant changes in lifestyle and the environment have made T2DM a global health problem.

[0003] Human pancreatic islet cells include pancreatic A cells (α cells) and pancreatic B cells (β cells). α cells account for approximately 20% of pancreatic islet cells and secrete glucagon, which strongly promotes glycogenolysis and gluconeogenesis, significantly raising blood glucose levels. β cells account for 60% to 70% of pancreatic islet cells and secrete insulin, the only hormone in the body capable of lowering blood glucose. Studies have shown that β cell dysfunction and a decrease in the number of β cells are important factors leading to diabetes and its progression.

[0004] BRD4 is a member of the bromodomain and outer terminal (BET) protein family (BRD2, BRD3, BRD4, and BRDt), characterized by two tandem bromodomains (BD1 and BD2). Through interactions with transcription factors, coactivators, and chromatin-modifying enzymes, BRD4 plays a crucial role in embryogenesis and cancer development. Currently, BET inhibitors are commonly used to treat related diseases by inhibiting the proliferation and survival of tumor cells. Studies have shown that JQ1 (a BET inhibitor) increases in vitro insulin secretion and β-cell insulin levels; I-BET151 (another BET inhibitor) promotes β-cell regeneration in NOD mice and induces the proliferation and expression of β-cell differentiation / function-related transcription factor genes.

[0005] However, there is currently no evidence to suggest a relationship between BRD4 and β cells. Therefore, studying and demonstrating the relationship and related mechanisms between BRD4 and β cells will help us understand the development of diabetes and provide a reference for the assessment, screening, prevention, intervention and treatment of diabetes and related diseases. Summary of the Invention

[0006] This disclosure was made in view of the above-mentioned state of the prior art, and its purpose is to provide an application of a substance targeting BRD4 in the preparation of diabetes-related products.

[0007] Therefore, the first aspect of this disclosure provides the application of a substance targeting BRD4 in the preparation of products affecting the proliferation, differentiation, and / or maturation of β cells, with BRD4 serving as a positive regulator of β cell proliferation, differentiation, and / or maturation. This disclosure reveals that BRD4 expression is significantly reduced in human diabetic β cells, and that BRD4 plays a crucial role in maintaining β cell maturation and differentiation. Both long-term and acute BRD4 deficiency lead to decreased insulin secretion and downregulation of differentiation markers. In particular, long-term BRD4 deficiency also affects β cell proliferation, highlighting the key role of BRD4 in pancreatic β cells and its positive effect. This disclosure verifies that BRD4 is a positive regulator of β cell proliferation, differentiation, and maturation, providing a precise target and its impact. Utilizing this principle, a substance targeting BRD4 can be applied to the preparation of products affecting β cell proliferation, differentiation, and / or maturation, which will contribute to advancing research related to β cells.

[0008] In the applications covered by the first aspect of this disclosure, optionally, the substance targeting BRD4 is selected from any one or more of substances targeting the BRD4 gene, substances targeting the BRD4 protein, and substances targeting BRD4 mRNA.

[0009] A second aspect of this disclosure provides the use of a substance targeting BRD4 in the preparation of products for the prevention, intervention, and / or treatment of diabetes. The characteristic feature is that BRD4 influences the progression of diabetes by affecting the proliferation, differentiation, and / or maturation of β-cells, wherein BRD4 is a positive regulator of β-cell proliferation, differentiation, and / or maturation. This disclosure verifies that BRD4 is a positive regulator of β-cell proliferation, differentiation, and / or maturation and a potential target related to diabetes. Utilizing this principle, applying a substance targeting BRD4 in the preparation of products for the prevention, intervention, and / or treatment of diabetes can contribute to the prevention, intervention, or treatment of diabetes.

[0010] A third aspect of this disclosure provides the application of a reagent for detecting BRD4 in a sample in the preparation of products for assessing diabetes susceptibility. This disclosure reveals that BRD4 expression is significantly reduced in human diabetic β-cells, and that BRD4 plays a crucial role in maintaining β-cell maturation and differentiation. Both long-term and acute BRD4 deficiency lead to decreased insulin secretion and downregulation of differentiation markers, highlighting the critical role of BRD4 in pancreatic β-cells and its positive effect; that is, BRD4 is a positive regulator of β-cells. Therefore, by detecting BRD4 in a sample, the β-cell function of a subject can be assessed, thereby evaluating the subject's susceptibility to diabetes.

[0011] A fourth aspect of this disclosure provides a product for assessing the susceptibility of a subject to diabetes, including a reagent for detecting BRD4. In this disclosure, the crucial role of BRD4 in pancreatic β-cells has been verified to have a positive effect; therefore, the product including the reagent for detecting BRD4 can detect BRD4 in a sample, assess the β-cell function of the subject, and thus assess the subject's susceptibility to diabetes.

[0012] The fifth aspect of this disclosure provides the use of a reagent for detecting BRD4 in a sample in the preparation of a product for assisting in the screening of diabetic patients. This disclosure reveals that BRD4 expression is significantly reduced in human diabetic β-cells, and that BRD4 plays a crucial role in maintaining β-cell maturation and differentiation. Both long-term and acute BRD4 deficiency lead to decreased insulin secretion and downregulation of differentiation markers, highlighting the key role of BRD4 in pancreatic β-cells and its positive effect; that is, BRD4 is a positive regulator of β-cells. Therefore, detecting BRD4 in a sample allows for the assessment of β-cell function in subjects, thereby assisting in the screening of diabetic patients.

[0013] A sixth aspect of this disclosure provides a product for assisting in the screening of patients with diabetes, including a reagent for detecting BRD4. In this disclosure, the key role of BRD4 in pancreatic β-cells has been verified, demonstrating a positive effect. Therefore, the product including the reagent for detecting BRD4 can detect BRD4 in a sample, assess the β-cell function of the subject, and thus assist in the screening of patients with diabetes.

[0014] In any of the applications or products involved in the third to sixth aspects of this disclosure, optionally, the reagent for detecting BRD4 is selected from any one or more of reagents for detecting BRD4 deficiency, reagents for detecting BRD4 gene mutations, and reagents for detecting BRD4 protein mutations. This disclosure has verified the adverse effects of BRD4 deficiency on β-cell proliferation, differentiation, and maturation, and that BRD4 mutations may affect downstream BRD4 signaling, leading to β-cell dysfunction. Therefore, by setting these reagents for detecting BRD4, the β-cell function of a subject can be assessed.

[0015] In any of the applications or products involved in the third to sixth aspects of this disclosure, optionally, the BRD4 gene variant includes BRD4 c.2245C->T, and the BRD4 protein variant includes BRD4 p.R749C. In this disclosure, p.R749C has been found to significantly affect downstream BRD4 signaling and may lead to β-cell dysfunction, potentially constituting a pathogenic mutation that contributes to the development of diabetes in patients.

[0016] The seventh aspect of this disclosure provides the use of a specific BRD4 knockout or knockdown animal model in screening or developing drugs for the prevention, intervention or treatment of diabetes.

[0017] According to this disclosure, it is possible to provide the use of a substance targeting BRD4 in the preparation of diabetes-related products. Attached Figure Description

[0018] Figure 1 This is a graph showing the relevant test results of Embodiment 1 involved in this disclosure.

[0019] Figure 2 This is a graph showing the relevant test results of Embodiment 2 involved in this disclosure.

[0020] Figure 3 This is a graph showing the relevant test results of Embodiment 3 involved in this disclosure.

[0021] Figure 4 This is a graph showing the relevant test results of Embodiment 4 involved in this disclosure.

[0022] Figure 5 This is a graph showing the relevant test results of Embodiment 5 involved in this disclosure.

[0023] Figure 6 This is a graph showing the relevant test results of Embodiment 6 involved in this disclosure.

[0024] Figure 7 This is a graph showing the relevant test results of Embodiment 7 involved in this disclosure. Detailed Implementation

[0025] Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same reference numerals are used for the same components, and repeated descriptions are omitted. Furthermore, the drawings are merely schematic diagrams, and the proportions of the components or the shapes of the components may differ from actual figures.

[0026] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0027] It should also be understood that terms such as those defined in general dictionaries should be understood to have meanings consistent with their meanings in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless defined as here.

[0028] This disclosure relates to the use of substances targeting BRD4 in the preparation of diabetes-related products. "Diabetes-related products" include, but are not limited to, products for the assessment, screening, prevention, intervention, and treatment of diabetes. Specifically, it covers the following aspects:

[0029] Application of substances targeting BRD4 in the preparation of products that affect the proliferation, differentiation and / or maturation of β cells;

[0030] The application of substances targeting BRD4 in the preparation of products for the prevention, intervention and / or treatment of diabetes;

[0031] The application of reagents for detecting BRD4 in samples in the preparation of products for assessing susceptibility to diabetes;

[0032] Products used to assess a subject's susceptibility to diabetes, including reagents for detecting BRD4;

[0033] Application of reagents for detecting BRD4 in samples in the preparation of products for assisting in the screening of diabetic patients;

[0034] Products that aid in the screening of diabetic patients, including reagents for detecting BRD4;

[0035] Application of animal models with specific BRD4 knockout or knockdown in screening drugs for diabetes and / or developing drugs for the prevention, intervention and / or treatment of diabetes;

[0036] Application of BRD4 as a target in the assessment, screening, prevention, intervention and / or treatment of diabetes.

[0037] In this disclosure, it was found that the expression of BRD4 was significantly reduced in human diabetic β cells, and that BRD4 plays an important role in maintaining β cell maturation and differentiation. Both long-term and acute BRD4 deficiency lead to reduced insulin secretion and downregulation of differentiation markers. In particular, long-term BRD4 deficiency also affects β cell proliferation, highlighting the key role of BRD4 in pancreatic β cells and its positive effect. In other words, this disclosure verifies that BRD4 is a positive regulator affecting β cell proliferation, differentiation and maturation.

[0038] Therefore, this disclosure can provide accurate targets and their effects. Utilizing this principle, substances targeting BRD4 can be used to prepare products that affect the proliferation, differentiation, and / or maturation of β cells, which will help advance research related to β cells.

[0039] In addition, this disclosure verifies that BRD4 is a positive regulator affecting the proliferation, differentiation and / or maturation of β cells and is a potential target related to diabetes. Utilizing this principle, substances targeting BRD4 can be used to prepare products for the prevention, intervention and / or treatment of diabetes, which can help prevent, intervene or treat diabetes.

[0040] Furthermore, by detecting BRD4 in the sample, the β-cell function of the subject can be assessed, thereby assessing the subject's susceptibility to diabetes and / or assisting in the screening of diabetic patients.

[0041] Regarding the findings in some existing literature concerning the potential of BET inhibitors to increase in vitro insulin secretion and β-cell insulin levels / promote β-cell regeneration in NOD mice, induce β-cell differentiation / proliferate and express function-related transcription factor genes, the applicant has also analyzed these findings and believes they may be attributable to the following factors: 1. A major issue in the field of bromide domain inhibitors is that these inhibitors lack strong selectivity among BRD2 / 3 / 4 isoforms, and the function of all three BRDs may be affected simultaneously during JQ1 / I-BET treatment; 2. In in vitro studies of primary islets, inhibitors target not only β-cells but also α / δ-cells, which may affect paracrine regulation in different cell types; 3. Overall treatment with JQ1 / I-BET may have secondary effects on in vivo β-cell function; 4. The treatment duration and dosage of JQ1 / I-BET may affect the final experimental results.

[0042] The following provides a detailed description of the application of substances targeting BRD4 in the preparation of diabetes-related products.

[0043] In this disclosure, substances targeting BRD4 may include, but are not limited to, substances that detect BRD4, substances that have a direct effect on BRD4, and substances that have an indirect effect on BRD4.

[0044] In this disclosure, diabetes may include type 1 diabetes and / or type 2 diabetes.

[0045] In this disclosure, substances targeting BRD4 can be used to prepare products that affect the proliferation, differentiation, and / or maturation of β cells. This disclosure verifies that BRD4 is a positive regulator affecting the proliferation, differentiation, and / or maturation of β cells, providing a precise target and its effects. Utilizing this principle to apply substances targeting BRD4 to prepare products affecting the proliferation, differentiation, and / or maturation of β cells will help advance research related to β cells.

[0046] In some examples, substances targeting BRD4 can be selected based on actual application needs, choosing substances with positive or negative effects on BRD4. Substances with positive effects on BRD4 can be, for example, substances that inhibit BRD4-related mutations, substances that protect BRD4, or substances that enhance BRD4 activity; substances with negative effects on BRD4 can be, for example, BRD4 inhibitors.

[0047] In some examples, the substance targeting BRD4 may be selected from any one or more of the following: substances targeting the BRD4 gene, substances targeting the BRD4 protein, and substances targeting BRD4 mRNA.

[0048] In this disclosure, substances targeting BRD4 can be used in the preparation of products for the prevention, intervention, and / or treatment of diabetes. BRD4 can influence the progression of diabetes by affecting the proliferation, differentiation, and / or maturation of β-cells. This disclosure verifies that BRD4 is a positive regulator of β-cell proliferation, differentiation, and / or maturation, and a potential target related to diabetes. Utilizing this principle, applying substances targeting BRD4 in the preparation of products for the prevention and / or treatment of diabetes can contribute to the prevention and / or treatment of diabetes.

[0049] In some examples, when substances targeting BRD4 are used in the preparation of products for the prevention, intervention, and / or treatment of diabetes, the substances targeting BRD4 can be those that have a positive effect on BRD4. This can thus promote the proliferation, differentiation, and maturation of β-cells, thereby contributing to the prevention, intervention, and treatment of diabetes.

[0050] In some examples, substances targeting BRD4 can be selected from any one or more of nucleic acid molecules, antibody drugs, and recombinant viruses.

[0051] In this disclosure, products including reagents for detecting BRD4 can detect BRD4 in samples, assess the β-cell function of subjects, thereby assessing the subject's susceptibility to diabetes, and / or assist in screening for diabetic patients.

[0052] In some examples, the presence or absence of BRD4, BRD4 gene mutations, and / or BRD4 protein mutations in the sample can be detected. In some examples, the reagents used to detect BRD4 are selected from any one or more of reagents for detecting BRD4 absence, reagents for detecting BRD4 gene mutations, and reagents for detecting BRD4 protein mutations. In this disclosure, the adverse effects of BRD4 absence on β-cell proliferation, differentiation, and maturation have been verified, and BRD4 mutations may affect downstream BRD4 signaling, leading to β-cell dysfunction. Therefore, by setting up these reagents for detecting BRD4, the β-cell function of the subject can be assessed.

[0053] In some examples, BRD4 gene variants may include BRD4 c.2245C->T, and BRD4 protein variants may include BRD4 p.R749C. Specifically, the BRD4 c.2245C->T mutation refers to the substitution of a C base at position 2245 of the BRD4 gene DNA sequence with a T base, resulting in a mutation at position 749 of the BRD4 protein where arginine (R) is replaced by cysteine ​​(C). In this disclosure, it has been found that BRD4 p.R749C can significantly affect downstream BRD4 signaling and may lead to β-cell dysfunction. It may be a pathogenic mutation leading to the development of diabetes in patients. By detecting whether a sample carries the BRD4 c.2245C->T (BRD4 p.R749C) mutation, the susceptibility to diabetes in subjects can be assessed, and / or it can assist in the screening of diabetic patients.

[0054] In some examples, BRD4 can directly regulate ATF5 (Activating Transcription Factor 5). In this disclosure, we found that ATF5 is a functional target in the BRD4 pathway and a potential target for BRD4 signaling intervention. Specifically, the two isoforms of BRD4 have opposing regulatory effects on ATF5 expression: the long form BRD4L, with its extended C-terminus that can bind to transcriptional cofactors, reduces ATF5 expression; the short form BRD4S, lacking the C-terminal extension characteristic of the long form, increases ATF5 expression.

[0055] In this disclosure, animal models of β-cell dysfunction can be constructed by specific BRD4 knockout or knockdown. In other words, this disclosure provides a method for constructing an animal model of β-cell dysfunction. For example, this disclosure provides a mouse model of β-cell dysfunction. This allows the use of such animal models in screening or developing drugs for the prevention, intervention, or treatment of diabetes. In some examples, animal models of β-cell dysfunction can be constructed by specific BRD4 knockout or knockdown of pancreatic β-cells. In some examples, animal models with long-term or acute BRD4 deficiency can be constructed. This allows for the selection of animal models with different types of β-cell dysfunction as needed. In some examples, specific BRD4 knockdown can be achieved by administering a BRD4 inhibitor.

[0056] In the embodiments of this disclosure, all products involved may be in the form of reagents, reagent kits, reagent kits, or systems comprising testing instruments.

[0057] In some examples, products for assessing diabetes susceptibility or screening for diabetes may also include systems comprising instruments for detecting BRD4. For instance, the product could be a system consisting of PCR reagents and DNA sequencing reagents and a DNA sequencer; or a system consisting of TaqMan probes, PCR primer pairs, a quantitative PCR instrument, a genotyping module, and other reagents required for TaqMan probe technology; or a system consisting of probes, PCR primer pairs, and other reagents and instruments required for ligase detection reactions (LDR); or a system consisting of PCR primer pairs, single-base extension primers, a microarray, a PCR instrument, a genotyping module, and / or other reagents and instruments required for Sequenom MassArray technology. This facilitates the detection of BRD4.

[0058] In some examples, reagents for detecting BRD4 may include primer pairs for amplifying the BRD4 gene and / or probes for detecting variations in the BRD4 gene. Thus, variations in the BRD4 gene can be captured and / or detected using primer pairs and / or probes.

[0059] In some examples, the products disclosed herein for assessing susceptibility to diabetes or screening patients for diabetes may also include reagents for detecting BRD4 mutations other than BRD4 c.2245C->T (BRD4 p.R749C). In some examples, the products disclosed herein for assessing susceptibility to diabetes or screening patients for diabetes may also include detection of all currently known pathogenic and suspected pathogenic mutations in diabetes, excluding BRD4 mutations. Therefore, the ability to detect all relevant sites for diabetes facilitates a more comprehensive and one-time screening for the disease.

[0060] In some examples, the products disclosed herein for assessing susceptibility to diabetes or screening for diabetes may also include reagents for detecting genes, proteins, or mRNAs associated with other diseases. For example, reagents may also be included for detecting genes or proteins associated with fatty liver disease. This allows for simultaneous screening of the tested individual for multiple diseases.

[0061] In some examples, products for the prevention, intervention, and / or treatment of diabetes as described in this disclosure may also include excipients. In some examples, when the product is for the prevention, intervention, and / or treatment of diabetes, the excipients may be pharmaceutically permissible. Thus, the addition of excipients can improve the performance of the drug and make it more suitable for clinical needs. In some examples, the excipients may be pharmaceutically acceptable carriers, excipients, diluents, etc.

[0062] In addition, this disclosure verifies the multifaceted role of BRD4 in β cells. Besides applying this principle to diabetes-related products, it can also be applied to other diseases closely related to β cells.

[0063] To further illustrate this disclosure, the following detailed description of the role and related applications of BRD4 in β cells, in conjunction with embodiments, is provided.

[0064] In the embodiments disclosed herein, unless otherwise specified, all materials, reagents, instruments and software used are commercially available products, and the operating procedures are performed in accordance with the instructions for the reagents, instruments or software.

[0065] Experimental methods

[0066] animal

[0067] Brd4 flox / floxMice were purchased from Cyagen (https: / / www.cyagen.com / us / en / ) and crossed with mice expressing Cre recombinase from the Pdx1-Cre strain (Jackson Laboratory, #024968) induced by the rat insulin promoter (Rip-Cre) to generate BRD4 conditional knockout mice. Tamoxifen (75 mg / kg body weight) was administered intraperitoneally every 24 hours for 7 consecutive days using an ACUC-approved injection procedure to induce recombination.

[0068] The Brd4-flox-forward (TACAAGGAAGGGA TGTTTTAACGC) and reverse (TGCTAACAGGTTGTA TTCACAAGG) primer pairs were used. flox / flox Mice were genotyped. All mice were kept in a pathogen-free environment with a 12-hour light / dark cycle and free access to water and food. All mice used in the experiment were male.

[0069] All animal experiments were conducted in accordance with the "Guide to the Care and Use of Laboratory Animals" published by the National Institutes of Health.

[0070] Mouse pancreatic islet isolation

[0071] Two mL of a 1 mg / mL Sigma-type XI collagenase Hank's buffered saline solution was injected into the pancreas of a mouse model via a bile duct. The pancreas was removed and incubated at 37°C for 17 minutes, then mechanically separated by pipetting. Islets were manually selected under a dissecting microscope. Islets were cultured in RPMI 1640 (Invitrogen) medium supplemented with 10% (v / v) fetal bovine serum (Invitrogen), 100 U / mL penicillin, and 100 μg / mL streptomycin (Invitrogen) in a sterile incubator at 37°C with 5% CO2 infusion and humidified air. Islets were dissociated using trypsin-EDTA solution (Invitrogen) for further sequencing experiments.

[0072] Intermediate Glucose Tolerance Test (IPGTT)

[0073] Mice were fasted for 12 hours prior to the glucose tolerance test. Mice were intraperitoneally injected with glucose at 2 g / kg. Two hours post-injection, glucose levels were measured using a One-Touch Ultra blood glucose meter (LifeScan). Serum insulin levels were measured using a mouse insulin enzyme-linked immunosorbent assay kit (Crystal Chem).

[0074] Statistical analysis

[0075] All data are expressed as mean ± SEM. Differences between groups were assessed using unpaired Student's t-test or one-way ANOVA, followed by Tukey's test using GraphPad Prism 8 software. P < 0.05 was considered statistically significant.

[0076] [Example 1] Study on the expression of BRD4 in human β cells of diabetic and prediabetic individuals

[0077] To investigate the function of BRD4 in human β cells, we analyzed single-nuclear RNA sequencing (snRNA-seq) data from pancreatic islets of non-diabetic, pre-T2D, and type 2 diabetic (T2D) patients.

[0078] Figure 1 This is a graph showing the relevant detection results of Embodiment 1 involved in this disclosure. Among them, Figure 1 Part A in the diagram is a schematic diagram of UMAP dimensionality reduction projection analysis of pancreatic islet samples from non-diabetic, prediabetic, and T2D (type 2 diabetes) cells, colored by cell type. Figure 1 Part B is a schematic diagram showing acinar cells, β cells, α cells, δ cells, PP cells, pancreatic duct cells, ECS stromal cells, and immune cells, stained according to their source, with each point corresponding to an atomic nucleus; Figure 1 Part C in the diagram is a schematic representation of the average proportion of each cell subtype in the non-diabetic, prediabetic, and T2D groups; Figure 1 Part D in the figure is a UMAP visualization showing the clustering of three groups of β cells and the single-cell expression pattern of the BRD4 gene in β cells. The data are colored according to the expression level, and the legend is marked with a logarithmic scale. Figure 1 Part E in the figure is a volcano plot describing significant DEGs (differentially expressed genes) between the prediabetic group and the nondiabetic group, as well as between the T2D group and the nondiabetic group. The data are colored according to the expression level, and the legend is labeled with a logarithmic scale. Figure 1 Part F in the diagram is a bubble graph showing the expression levels of the BRD4 gene in the non-diabetic, prediabetic, and T2D groups. The color depth and size of each bubble are positively correlated with the expression of BRD4.

[0079] Clustering of 121,310 cell nuclei across three groups was performed using uniform manifold approximation and projection (UMAP), and different cell types were identified using cell type markers. Figure 1(Central AC section). Significantly differentially expressed genes (DEGs) were identified between prediabetic and nondiabetic β cells, and between T2D and nondiabetic β cells. At the single-cell level, BRD4 was widely expressed in most islet cells. β cells re-aggregated via UMAP, and BRD4 was significantly reduced in prediabetic cells, with feather plots showing a decrease in T2D β cells (…). Figure 1 (Part D). DEG analysis also showed that BRD4 expression was significantly lower in prediabetic and T2D β cells than in non-diabetic β cells. Figure 1 (The middle EF portion) indicates that BRD4 is associated with human β cell function.

[0080] In summary, BRD4 is downregulated in β cells of people with diabetes and prediabetes.

[0081] [Example 2] Study on β-cell function after BRD4 gene knockout

[0082] To explore the function of BRD4 in β cells, we constructed two BRD4 conditional knockout mouse models. Using the Cre-LoxP recombination system, we generated RIP-Cre... + / BRD4 flox / flox Mice (RBKO) or PDX1-CreER + / BRD4 flox / flox Mouse (PBKO).

[0083] Figure 2 This is a graph showing the relevant detection results of Embodiment 2 involved in this disclosure. Among them, Figure 2 Part A in the diagram is a schematic diagram of the construction and treatment process of the BRD4- / - mouse model; Figure 2 Part B in the image shows representative immunofluorescence (IF) images of BRD4 (green) and insulin (red) in the pancreatic islets of the control and RBKO mice. The arrows indicate BRD4 knockout β cells. Figure 2 Part C in the diagram is a schematic diagram of qPCR analysis of BRD4 gene expression in the control group and RBKO mice; Figure 2 Part D in the diagram is a schematic diagram of blood glucose levels measured in randomly selected control mice and RBKO mice at 4, 8, and 16 weeks. Figure 2 Part E in the figure is a schematic diagram of the results of the glucose tolerance test (IPGTT) for control mice and RBKO mice; Figure 2 The F section in the diagram represents the plasma insulin levels in the control group and RBKO mice during the IPGTT. Figure 2Part G in the diagram illustrates the effects of low (2.8 mM), medium (11.1 mM), and high (25 mM) glucose concentrations on insulin secretion (μg / total insulin) in control and RBKO mice using a glucose-stimulated insulin secretion (GSIS) assay. Values ​​are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001. Scale bar = 20 μm.

[0084] In BRD4, cre-mediated recombination resulted in the deletion of exons 6 and 7. Figure 2 Part A). At the cellular level, immunofluorescence (IF) staining showed decreased BRD4 protein levels in RBKOβ cells ( Figure 2 Part B). PCR analysis further confirmed the decreased BRD4 mRNA level in RBKO mice ( Figure 2 Part C). RBKO mice showed significantly elevated random blood glucose levels at 4, 8, and 16 weeks ( Figure 2 Part D). According to the results of the intraperitoneal glucose tolerance test (IPGTT), the blood glucose levels of RBKO mice at different time points were significantly higher than those of the control group ( Figure 2 In the middle section (E), plasma insulin levels were significantly lower than those in the control group (2F section of the figure). The glucose-stimulated insulin secretion (GSIS) experiment also showed that at higher glucose concentrations, RBKO islets secreted significantly less insulin than control islets. Figure 2 (Part G). These findings further support the phenotype of reduced insulin secretion in RBKO mice and highlight the central role of BRD4 in regulating pancreatic β-cell function.

[0085] In summary, BRD4 gene knockout leads to impaired β-cell function.

[0086] [Example 3] Study on β-cell function after acute BRD4 gene knockout

[0087] To mimic the effects of BRD4 inhibitor treatment, we established an acute or short-term BRD4 conditional knockout (PBKO) model, which was generated by crossing tamoxifen-induced Cre mice with BRD4 flox mice.

[0088] Figure 3 This is a graph showing the relevant detection results of Embodiment 3 involved in this disclosure. Among them, Figure 3 Part A in PDX1-CreER / BRD4 flox / flox A schematic model of tamoxifen-induced BRD4 allele cre-dependent loss in (PBKO) mice; Figure 3Part B in the image depicts representative IF images of BRD4 (green) and insulin, somatostatin (SST), and glucagon (Gcg) (red) in the pancreatic islets of control mice and RBKO mice. Arrows indicate BRD4 expression. Figure 3 Part C in the image depicts representative IF images of Cre (green) and insulin (Insulin), somatostatin (SST), and glucagon (Gcg) (red) in the pancreatic islets of RBKO mice. Arrows indicate Cre expression. Figure 3 Part D in the diagram is a schematic diagram of the random measurement of blood glucose levels in control and RBKO mice at 6 and 8 weeks of age; Figure 3 Part E in the diagram is a schematic diagram of the fasting and refeeding blood glucose levels of the control group and RBKO mice; Figure 3 Part F in the figure is a schematic diagram of plasma insulin levels measured in the control group and PBKO mice. Values ​​are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001. Scale bar = 20 μm.

[0089] This study used PDX1-CreER. Tamoxifen was administered intraperitoneally every 24 hours for 7 consecutive days to induce recombination. Phenotypic results were observed on day 14. Figure 3 Part A of the study showed that BRD4 was knocked out in both β (insulin-positive) and δ (somatostatin-positive) cells, but not in glucagon-positive cells. Figure 3 (Part B). Therefore, IF showed that Cre signals could be detected in β and δ cells, but not in α cells ( Figure 3 (Part C), which explains why BRD4 is deleted only in β and δ cells. Generally, the most impressive phenotypic change in PBKO mice is the elevation of blood glucose levels, including random, fasting, and refeeding blood glucose levels (…). Figure 3 (Middle DE section). Plasma insulin levels in PBKO mice were significantly lower than in control mice ( Figure 3 (Middle F part), indicating that β cell function is impaired after acute BRD4 loss.

[0090] In summary, acute BRD4 gene knockout leads to impaired β-cell function.

[0091] [Example 4] Study on the effects of long-term and acute BRD4 loss on β cells.

[0092] Figure 4 This is a graph showing the relevant detection results of Embodiment 4 involved in this disclosure. Among them, Figure 4Part A in the image shows representative IF images of glucagon (Gcg, red) and DAPI (blue) staining of the islets of RBKO, PBKO, and control mice. Figure 4 Part B in the image shows representative IF images of Ki67 (green), insulin (red), and DAPI (blue) staining in the pancreatic islets of RBKO, PBKO, and control mice. Figure 4 Part C in the diagram is a schematic representation of the percentage (%) of Gcg-positive and Ki67-positive cells shown by the IF analysis of Parts A-B. Figure 4 The DF section in the image contains representative IF images depicting GLUT2 (green), Mafa (green), proinsulin (green), insulin (red), and DAPI (blue) in the pancreatic islets of mice in the control, RBKO, and PBKO groups. Scale bar = 20 μm.

[0093] Compared to PBKO mice, RBKO mice exhibited altered α / β cell ratios and a significantly increased number of glucagon (Gcg)-positive cells. Figure 4 Part A). RBKO mice had significantly fewer Ki67-positive β cells than PBKO and control mice ( Figure 4 (Middle BC section), indicating that long-term loss of BRD4 leads to reduced β-cell proliferation. IF showed that GLUT2 and Mafa expression in both RBKO and PBKO mice were significantly lower than in control mice. Figure 4 The DE portion suggests that BRD4 plays an important role in β-cell differentiation and maturation. Interestingly, the proinsulin level in RBKO β-cells was significantly lower than that in PBKO and control β-cells. Figure 4 (Middle F part), which indicates that after long-term loss of BRD4 in β cells, insulin synthesis rather than processing is impaired.

[0094] In summary, our data indicate that long-term loss of BRD4 affects the proliferation and differentiation of β cells, while acute loss mainly affects differentiation and maturation.

[0095] [Example 5] Study on the effect of BRD4 deficiency on β cell maturation

[0096] To explore the role of BRD4 in β cells and reduce the secondary effects of long-term hyperglycemia, we performed scRNA-seq analysis on PBKO islets.

[0097] Figure 5 This is a graph showing the relevant detection results of Embodiment 5 of this disclosure. Among them, Figure 5Part A in the diagram is a schematic diagram of UMAP analysis of pancreatic islets in 8-week-old large control group (C8) and PBKO group (K8) mice according to cell type, including acinar cells, beta cells, alpha cells, delta cells and PP cells, with each point corresponding to one cell; Figure 5 Part B in the diagram is a schematic diagram of the average proportion of each cell subtype in groups C8 and K8; Figure 5 Part C in the diagram is a bubble chart showing the expression of the top marker gene in each cell cluster; Figure 5 Part D in the figure is a heatmap of the top 20 important DEGs whose expression is increased or decreased in C8 and K8β cells. The data are colored according to the expression level, and the legend is labeled on a logarithmic scale. Figure 5 Part E in the figure consists of volcano plots and bubble plots depicting significant differences between K8 and C8 related to β-cell maturation and function. Data are colored by expression level, and the legend is labeled on a logarithmic scale. Figure 5 Part F in the diagram is a schematic diagram of re-clustering pancreatic islet cells into 21 subtypes using the UMAP diagram; Figure 5 The G section in the diagram is a schematic diagram of the β cell subtypes identified in the UMAP diagrams of C8 and K8, with arrows indicating changes in the C8 and K8 β cell subtypes; Figure 5 The H section contains volcano and bubble plots describing important DEGs related to the maturation and differentiation of K8 subtype 1 (K8_1) and C8 subtype 8 (C8_8) β cells. Data are colored by expression level, and legends are labeled on a logarithmic scale. Figure 5 Part I of the data is a UMAP dimension reduction projection analysis of pancreatic islet cell types in 6-week-old control (C6) and PBKO (K6) mice, including acinar cells, beta cells, alpha cells, delta cells, and PP cells, with each point corresponding to one cell; Figure 5 The J section is a schematic diagram of the β-cell subtypes identified in the UMAP diagrams of C6 and K6.

[0098] 41,121 cells from 8-week-old control (C8) and PBKO (K8) mice were aggregated using UMAP and identified as acinar cells, beta cells, alpha cells, delta cells, and PP cells using specific cell type markers. Figure 5(Middle AC section). Since the main phenotypic change in PBKO mice is decreased insulin secretion, we focused on β cells. The most significant deg in C8 and K8 β cells is shown in the heatmap ( Figure 5 (Part D). At single-cell resolution, BRD4 knockout significantly reduced the expression of differentiation markers such as Chga, Nkx6-1, Mafa, and Ucn3, while transthyretin (Ttr), specifically expressed in α cells, was significantly increased in PBKOβ cells. Figure 5 (Part E). These findings indicate that β-cell maturation is impaired following acute BRD4 loss. Subsequently, islet cells represented by the UMAP diagram were automatically re-clustered into 21 subtypes, providing a comprehensive view of the β-cell landscape. Figure 5 (Middle F section). Differentiating β-cell subtypes in the UMAP plot revealed significant differences between the C8 and K8 groups; compared to C8, K8 showed an increase in subcluster 1 and a decrease in subcluster 8 (…). Figure 5 (Middle G section). DEG analysis showed that the β-cell-specific differentiation markers Chga, Nkx6-1, Mafa, Ucn3, and Slc2a2 in K8 were significantly decreased in subcluster 1, while other pancreatic islet cell markers Sst, Ppy, and Ttr were significantly increased. Figure 5 (Middle H section). These findings indicate that more PBKOβ cells acquired a "progenitor-like" expression profile. We also validated these findings in 6-week-old young control (C6) and PBKO (K6) mice. According to UMAP, K6 and C6 co-aggregated 38,404 cells ( Figure 5 (Part I). DEG analysis showed that Chga, Ucn3, and Slc2a2 were significantly reduced in K6β cells, a phenomenon also observed in K8β cells. Furthermore, 21 subtypes were clustered in the K6 and C6 islet cell populations, similar to the K8 and C6 islet cell populations. Compared to C6β cells, subcluster 1 was significantly increased and subcluster 8 was significantly decreased in K6β cells. Figure 5 (J part), which is consistent with the results of studies on aged PBKO mice.

[0099] In summary, our study indicates that acute BRD4 gene knockout affects β-cell maturation.

[0100] [Example 6] Study on chromatin landscape and epigenetic characteristics of BRD4 knockout in β cells

[0101] Since BRD4 is an important epigenetic regulator, we also applied scATAC-seq to PBKO and control islets to determine whether the chromatin landscape and epigenetic characteristics of β cells changed after BRD4 knockout.

[0102] Figure 6 This is a graph showing the relevant detection results of Embodiment 6 of this disclosure. Among them, Figure 6 Part A in the diagram is a schematic diagram of the chromatin accessibility pattern analysis of transcription factor initiation sites (TFSSs). Pancreatic islet cells are divided into PanSCs (pancreatic stellate cells), MCs (mesothelial cells), ECs (endothelial cells), and epithelial cells, with each point corresponding to one cell. Figure 6 Part B in the diagram is a schematic representation of different epithelial cell aggregations based on the TFSS chromatin accessibility pattern, including α cells, β cells, acinar cells, and δ cells; Figure 6 Part C in the figure is a heatmap of the characteristic peaks of chromatin accessibility in different cell types; Figure 6 Part D in the diagram is a UMAP visualization of the control and PBKO islet cells based on the TFSS intrachromatin accessibility pattern. Figure 6 The E portion represents the ratio of epithelial cells in PBKO and the control group; Figure 6 The F part in the figure is a bar graph of the difference peaks in chromatin accessibility between PBKO and control group β cells, with the main difference located in the promoter region; Figure 6 The G section in the figure is a heatmap of the top 20 degrees of PBKO and control β cells based on the scATAC gene score; Figure 6 The H part in the diagram is a schematic diagram of DEGsGO enrichment analysis based on scATAC gene scores. Figure 6 Part I in the figure is a heatmap of top differentially expressed transcription factors in PBKO and control β cells based on transcription factor activity analysis; Figure 6 The J section is a schematic diagram of IGV visualization of the chromatin accessibility peaks in the Ttc4 and Mafa promoter regions in PBKO and control β cells;

[0103] Based on chromatin accessibility of cell type marker genes, UMAP clustered 22,448 pancreatic islet cells, most of which were identified as epithelial cells. Figure 6 Part A). Furthermore, chromatin accessibility of marker genes based on specific cell types ( Figure 6 In section C, UMAP projection shows acinar cells, β cells, α cells, and δ cells (section 6B in the figure). Notably, the clusters representing PBKO β cells are shifted to the right. Figure 6 The middle D portion indicates that the chromatin landscape was significantly altered after BRD4 loss. As shown by the scA TAC-seq data, UMAP projection also revealed a significant reduction in the number of β cells in PBKO mice. Figure 6 (Part E). Significant differences were found in the position of the chromatin accessibility peak between PBKO and control β cells, with the differences primarily concentrated in the promoter regions of the target genes. Figure 6The middle F part). Based on the scA TAC gene score, deg ( Figure 6 Functional enrichment analysis of the G-section showed that these degs can affect the cell cycle, neurodegeneration, autophagy, and insulin signaling pathways. Figure 6 (Part H). Based on transcription factor activity analysis, differentially expressed transcription factors (DTFs) were analyzed. Figure 6 (Part I) These dtfs affect many important pathways, including cell fate commitment signaling. In particular, the number and height of chromatin accessibility peaks are significantly lower in the promoter regions of TTC4 and Mafa. Figure 6 (Part J in the middle).

[0104] Studies have found that Ttc4 and its family members play important roles in cell cycle and transcriptional regulation and are associated with the development of type 2 diabetes. Therefore, acute BRD4 knockout significantly affects chromatin remodeling and gene accessibility in β cells, highlighting the crucial role of BRD4 in β cells.

[0105] In summary, ScATA C-seq revealed the network of β-cell transcription factor activity and chromatin interaction in BRD4 deficiency.

[0106] [Example 7] Study on downstream targets of the BRD4 pathway

[0107] Figure 7 This is a graph showing the relevant detection results of Embodiment 7 of this disclosure. Among them, Figure 7 Part A in the figure is a volcano plot describing the significant changes in the ATF4 / ATF5 ratio between K8 and C8β cells. Data are colored according to expression level, and the legend is labeled with a logarithmic scale. Figure 7 Part B in the figure is a volcano plot depicting the significant changes in ATF4 / ATF5 expression between K8_1 and C8_8 subtype β cells; Figure 7 Part C in the diagram is a schematic diagram of qPCR analysis of TF4 and TF5 gene expression in the pancreatic islets of control and RBKO mice; Figure 7 Part D in the text is based on ChIP-seq data, which performs IGV visualization analysis on the BRD4 binding peaks in the ATF4 and ATF5 promoter regions of 293 and HepG2 cells. Figure 7 Part E in the image is an IGV visualization showing the chromatin accessibility peaks in the ATF4 and ATF5 promoter regions of PBKO and control β cells; Figure 7 The F part in the diagram is a schematic diagram of the ATF4 and ATF5 luciferase reporter genes; Figure 7 The G part in the diagram is a schematic diagram of the relative luciferase activities of ATF4 and ATF5 expression regulated by BRD4L and BRD4S. Figure 7The H part in the diagram is a schematic diagram of the BRD4 mutation verified by Sanger sequencing; Figure 7 Part I in the diagram is a schematic diagram showing how mutations are mapped to different transcriptional variants of BRD4 and how different functional domains are colored. Figure 7 The J section is a schematic diagram showing a table displaying basic information and functional predictions of BRD4 mutations; Figure 7 The K part in the diagram is a schematic diagram of PCR detection of different BRD4 transcriptomic variations in human pancreatic islets; Figure 7 The L part in the diagram is a schematic diagram showing the effect of mutations on the BRD4-ATF4 / ATF5 pathway as revealed by luciferase assay. The activity of firefly luciferase is normalized to the activity of Renilla luciferase. Figure 7 The M portion of the diagram is a schematic diagram of the BRD4-ATF5 pathway in β cells and a schematic diagram of the different effects caused by long-term and acute loss of BRD4. Values ​​are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001. Con, control. BRD4L, long isoform of BRD4. BRD4S, short isoform of BRD4. TVL, transcriptomorph of long BRD4. TVS, transcriptomorph of short BRD4. TV3, transcriptomorph 3 of BRD4. SIFT, Sorting Intolerant From Tolerant. GERP, genome evolution rate spectrum. ACMG, American College of Medical Genetics and Genomics.

[0108] ATF4 and ATF5 levels in K8β cells were significantly lower than those in C8β cells. Figure 7 (Part A). Similarly, the expression of ATF4 and ATF5 in K8β subcluster 1 cells was significantly lower than that in C8β subcluster 8 cells ( Figure 7 Part B). qPCR results showed that, compared with the control group, both ATF4 and ATF5 in PBKO islets were significantly reduced ( Figure 7 Part C).

[0109] Furthermore, the expression of ATF4 and ATF5 in K6β cells was significantly lower than that in C6β cells. Reanalysis of publicly available ChIP-seq data showed strong BRD4 binding peaks in the promoter regions of ATF4 and ATF5 in both the 293 and HepG2 cell lines. Figure 7 (Part D). Interestingly, after knocking out BRD4 via scA TAC-seq, we did not observe any changes in chromatin accessibility in the ATF4 or ATF5 promoter regions. Figure 7(Part E). Since BRD4 has histone-independent transcriptional function, we used the human promoter regions of ATF4 and ATF5 to generate luciferase reporters to determine whether BRD4 can directly regulate the expression of ATF4 and ATF5. Figure 7 (Middle F part). Luciferase assay showed that the two subtypes of BRD4, BRD4L and BRD4S, could significantly inhibit the expression of ATF4.

[0110] However, BRD4L and BRD4S have opposite regulatory effects on ATF5 expression. ATF5 reporter gene studies show that BRD4L decreases ATF5 expression, while BRD4S increases it. Figure 7 (Part G). Here, our study shows that even after BRD4 knockout, ATF4 expression is regulated by a variety of potential factors that reduce ATF4 expression, and BRD4 appears to be an "additional component" rather than a necessary factor for ATF4 regulation. Meanwhile, ATF5 is directly positively regulated by BRD4S.

[0111] In summary, ATF4 and ATF5 can be directly regulated by BRD4.

[0112] To explore whether BRD4 mutations promote the development of diabetes, we recruited 222 young diabetic patients to screen for BRD4 mutations.

[0113] Finally, four mutations—c.1688G->A (p.S563N), c.2195C->T (p.P732L), c.2204A->G (p.Y735C), and c.2245C->T (p.R749C)—were detected by WES and verified by Sanger sequencing. Figure 7 (Part H in the middle).

[0114] c.1688G->A was mapped to all three transcript variants of BRD4 (TVL, TVS, and TV3), while the other three mutations were specifically mapped to TV3. Figure 7 (Part I). In some tumors, TVL encodes the long isoform of BRD4 (BRD4L), and TVS encodes the short isoform of BRD4 (BRD4S). Studies on TV3 are limited, but it can also be translated into a short isoform 72 amino acid residues longer than the conventional BRD4S. Since screening intolerance (SIFT) predicts the c.1688G->A mutation is unlikely to adversely affect BRD4 function (…),… Figure 7In the J section, we focused on three other mutations (c.2195C->T (p.P732L), c.2204A->G (p.Y735C), and c.2245C->T (p.R749C)). We designed primers targeting specific regions of the three transcript variants. PCR results with different primers showed that only TVL and TV3 were detected in human islets, a finding confirmed by high-fidelity polymerase assays. Figure 7 (Middle K section).

[0115] Wild-type (WT) and mutant TV3, along with either TF4 or TF5 reporter genes, were co-transfected into the INS1 cell line. Luciferase assay showed that c.2245C->T (p.R749C) had the greatest impact on ATF4 and ATF5 expression. Figure 7 (Middle L section). These findings suggest that p.R749C may affect BRD4 function and downstream targets to some extent. Our research indicates that ATF5 is a functional target in the BRD4 pathway and may be a potential target for BRD4 signaling intervention. Figure 7 (M part). In summary, the p.R749C mutation can affect the BRD4-ATF4 / ATF5 signaling pathway.

[0116] In summary, our comprehensive investigation reveals the crucial role of BRD4 in β-cells. BRD4 plays a vital role in maintaining β-cell maturation and differentiation, as both chronic and acute BRD4 deficiency lead to decreased insulin secretion and downregulation of differentiation markers. However, unlike acute BRD4 deficiency, chronic BRD4 deficiency also affects β-cell proliferation.

[0117] In this study, we established long-term and acute conditional knockout mouse models to assess the function of BRD4 in β-cells. Single-cell RNA sequencing (scRNA-seq) and transposase-accessible chromatin single-cell sequencing (scA-TAC-seq) were used to sequence the BRD4 knockout islets to elucidate BRD4 function in β-cells and explore downstream targets of the BRD4 pathway. We also recruited 222 young diabetic patients for whole-exome sequencing (WES) to screen for BRD4 mutations. Our results show that BRD4 expression is significantly reduced in human diabetic β-cells. Long-term BRD4 knockout impaired both β-cell proliferation and maturation, while acute knockout primarily affected β-cell differentiation and maturation. We found that p.R749C significantly affects BRD4 signaling and may be a pathogenic mutation leading to diabetes development in patients. Our study also indicates that ATF5 is a direct target of the BRD4 pathway in β-cells. These findings highlight the crucial role of BRD4 in pancreatic β-cells. Targeting the BRD4-mediated regulatory network may hold promise for developing new therapeutic strategies to restore β-cell function.

[0118] We investigated BRD4 deficiency in a mouse model, gaining important insights into the function of BRD4 in β cells. Specifically, unlike the acute case, long-term conditional BRD4 knockout resulted in reduced β cell proliferation. Given the phenotypic changes in the acute knockout model, this reduction could be a direct effect of BRD4 deficiency in embryonic and adult stages, or a secondary effect of glucose toxicity caused by long-term hyperglycemia. However, we have reason to hypothesize that differentiation or maturation effects are direct regulatory effects of the BRD4 pathway in β cells. Functional maturation and proliferative capacity are mutually exclusive states in β cells, but in the RBKO model, both maturation and proliferative capacity were impaired. RBKO β cells did not revert to a "progenitor-like" state; instead, they transformed into a dedifferentiated or exhausted state, as in the "db / db" diabetic mouse model. ChIP-seq can be used to screen for target genes other than ATF5. As luciferase reporter assays have shown, BRD4 may regulate ATF4 / ATF5 in an epigenetic-independent manner, since chromatin accessibility of the ATF4 / ATF5 promoter regions was not altered in PBKO mice. In fact, the transcriptional function of BRD4 is unrelated to histone binding; for example, some believe that BRD4 helps viral DNA replication without histones.

[0119] Notably, BRD4 expression was not affected in PBKO α cells, suggesting that PBKO is not a suitable model for mimicking BRD4 inhibitor treatment of the entire islet. In mice, early embryonic PDX1-positive cells represent progenitor cells for all mature pancreatic endocrine and exocrine cells; however, as β cells mature, PDX1 expression is limited to β cells during late mouse pancreatic development. In the PBKO model, BRD4 deficiency primarily occurred in β cells. Caution must be exercised when exploring BRD4 function in β cells by treating the entire islet with BRD4 inhibitors, as these inhibitors may also target α cells.

[0120] BRD4S is primarily encoded by TVS, the most well-known transcript variant in tumors. However, we found that TVS is undetectable in human islets, suggesting a cell- or organ-specific expression pattern for the BRD4 isoform. Interestingly, TV3 (NCBI reference sequence: NM_001330384.2), which has been little studied, also encodes a short isoform of BRD4 (here referred to as BRD4S1), which is detectable in human islets. Since BRD4S1 is only 72 amino acids longer than BRD4S, there is reason to believe that these two short isoforms have similar functions. However, subtle functional differences between these two short isoforms warrant further investigation. In young diabetic patients, more mutations were detected in TV3 than in TVL. In particular, the p.R749C mutation can significantly affect BRD4-ATF4 / ATF5 signaling. p.R749C may affect the conformation of BRD4 more significantly than other missense mutations.

[0121] In summary, our study highlights the multifaceted role of BRD4 in β cells. By elucidating the BRD4-regulated regulatory network in pancreatic β cells, targeting this network may offer the potential for developing novel therapeutic strategies to restore β cell function. Our research reveals that therapeutic targeting of the BRD4 pathway is an effective and promising strategy for treating diabetes, and can also provide a reference for the assessment, screening, prevention, intervention, and treatment of diabetes and related diseases.

[0122] In summary, this disclosure provides an application of a substance targeting BRD4 in the preparation of diabetes-related products.

[0123] While the present disclosure has been specifically described above in conjunction with the accompanying drawings and examples, it is to be understood that the foregoing description does not limit the present disclosure in any way. Those skilled in the art can make modifications and variations to the present disclosure as needed without departing from its essential spirit and scope, and all such modifications and variations shall fall within the scope of the present disclosure.

Claims

1. The application of a substance targeting BRD4 in the preparation of products affecting the proliferation, differentiation, and / or maturation of β cells, characterized in that, BRD4 is a positive regulator of β-cell proliferation, differentiation and / or maturation.

2. The application according to claim 1, characterized in that, The substance targeting BRD4 is selected from any one or more of the following: substances targeting the BRD4 gene, substances targeting the BRD4 protein, and substances targeting BRD4 mRNA.

3. The use of a substance targeting BRD4 in the preparation of products for the prevention, intervention, and / or treatment of diabetes, characterized in that, BRD4 influences the progression of diabetes by affecting the proliferation, differentiation, and / or maturation of β cells. BRD4 is a positive regulator of β cell proliferation, differentiation, and / or maturation.

4. The application of a reagent for detecting BRD4 in a sample in the preparation of products for assessing susceptibility to diabetes.

5. A product for assessing the susceptibility to diabetes in subjects, characterized in that, This includes reagents for detecting BRD4.

6. The application of a reagent for detecting BRD4 in a sample in the preparation of a product for assisting in the screening of diabetic patients.

7. A product for assisting in the screening of diabetic patients, characterized in that, This includes reagents for detecting BRD4.

8. The application or product according to any one of claims 4 to 7, characterized in that, The reagent used to detect BRD4 is selected from any one or more of the following: reagents for detecting BRD4 deficiency, reagents for detecting BRD4 gene mutations, and reagents for detecting BRD4 protein mutations.

9. The application or product according to claim 8, characterized in that, The BRD4 gene variant includes BRD4 c.2245C->T, and the BRD4 protein variant includes BRD4 p.R749C.

10. The use of a specific BRD4 knockout or knockdown animal model in screening drugs for diabetes and / or developing drugs for the prevention, intervention and / or treatment of diabetes.

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