Application of H2AC19 in the detection and treatment of lung adenocarcinoma

Abstract

This invention belongs to the fields of biomedicine and molecular biology, specifically relating to the application of H2AC19 in the detection and treatment of lung adenocarcinoma. Specifically, this invention demonstrates that H2AC19 is abnormally highly expressed in lung adenocarcinoma patients, and this high expression is significantly associated with poor prognosis. Knockout of H2AC19 inhibits angiogenesis and tumor growth in vitro and in vivo, and reduces the proliferation, migration, and tube formation capabilities of lung adenocarcinoma cells in a co-culture model with human umbilical vein endothelial cells. Furthermore, this invention reveals a novel mechanism by which H2AC19 regulates angiogenesis through the p300 / EGR1 / MMP-1 pathway. This invention not only provides a superior method for the diagnosis and prognostic assessment of lung adenocarcinoma, but also lays an experimental foundation and expands new perspectives for the development of highly effective drugs for treating lung adenocarcinoma, thus possessing significant practical application value.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and molecular biology, specifically relating to the application of H2AC19 in the detection and treatment of lung adenocarcinoma. Background Technology

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

[0003] Lung cancer is one of the most common and deadliest malignant tumors worldwide, with lung adenocarcinoma (LUAD) being its most frequent subtype, accounting for more than half of all lung cancer cases. Despite recent advancements in the diagnosis and treatment of LUAD, patient prognosis remains unsatisfactory, primarily due to tumor angiogenesis promoting tumor growth, invasion, and metastasis. Angiogenesis is a crucial process in the development of solid tumors, regulated by multiple factors. Targeted therapy against angiogenesis has become an important direction in the treatment of LUAD; however, existing anti-angiogenic drugs suffer from problems such as drug resistance and off-target effects. Therefore, there is an urgent need to find new angiogenesis regulatory targets and treatment strategies.

[0004] Histone variants, as an important component of epigenetic regulation, play a crucial role in tumorigenesis and development. Among them, histone H2A variants are the most numerous and sequence-diverse of all histone variants, playing a central role in transcriptional regulation, DNA repair, DNA replication, and chromosome stability. They are closely related to various pathophysiological mechanisms and the occurrence of different diseases, including cancer, embryonic developmental abnormalities, neurological disorders, and metabolic diseases. H2AC19 is a new member of the H2A family located in the 1q21 region of chromosome 1, encoding a 130-amino acid protein in humans. However, its role in disease is currently poorly understood and reported. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the inventors, through long-term technical and practical exploration, have developed an application of H2AC19 in the detection and treatment of lung adenocarcinoma (LUAD). This invention demonstrates that H2AC19 is significantly upregulated in LUAD samples and promotes LUAD progression in both in vitro and in vivo experiments, indicating its potential use as a biomarker. Furthermore, this invention reveals a novel mechanism by which H2AC19 regulates angiogenesis through the p300 / EGR1 / MMP-1 pathway, suggesting its potential as a therapeutic target for LUAD. Based on these research findings, this invention has been completed.

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

[0007] The first aspect of this invention provides the application of a reagent for detecting the expression level of the H2AC19 encoding gene and its expression products in the preparation of products for detecting lung adenocarcinoma. This invention demonstrates through research that H2AC19 is significantly upregulated in LUAD samples and promotes LUAD progression in both in vitro and in vivo experiments. H2AC19 expression levels in LUAD patients are positively correlated with angiogenesis and tumor progression; LUAD patients with high H2AC19 expression have shorter overall survival, indicating that increased H2AC19 expression is a poor prognostic factor for LUAD.

[0008] Therefore, the detection of lung adenocarcinoma includes screening, diagnosis, monitoring, and prediction of lung adenocarcinoma progression.

[0009] A second aspect of the present invention provides a system for screening, diagnosing, monitoring, and predicting the progression of lung adenocarcinoma, the system comprising:

[0010] (a) An analysis module, the analysis module comprising: a detection reagent for determining the expression level of the subject selected from the above-mentioned H2AC19 encoding gene and its expression product;

[0011] (b) An assessment module comprising: assessing the subject’s disease status based on the expression levels of the H2AC19 coding gene and its expression products as determined in (a).

[0012] A third aspect of the invention provides the use of H2AC19 as a target in the preparation and / or screening of drugs for lung adenocarcinoma.

[0013] The lung adenocarcinoma drugs include drugs for the prevention and / or treatment of lung adenocarcinoma.

[0014] A fourth aspect of the present invention provides the use of an H2AC19 inhibitor in any one or more of the following:

[0015] (a) To prepare a product that inhibits the growth of lung adenocarcinoma cells;

[0016] (b) To prepare a product that inhibits the migration of lung adenocarcinoma cells;

[0017] (c) Preparation of products that inhibit angiogenesis in lung adenocarcinoma;

[0018] (d) Preparation of lung adenocarcinoma drugs.

[0019] A fifth aspect of the present invention provides a method for preventing and / or treating lung adenocarcinoma, the method comprising administering the above-mentioned H2AC19 inhibitor to a patient.

[0020] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial effects:

[0021] The above-mentioned technical approach is the first study to demonstrate that H2AC19 is abnormally highly expressed in patients with lung adenocarcinoma, and that high expression of H2AC19 is significantly associated with poor prognosis in these patients. Knockout of H2AC19 inhibits angiogenesis and tumor growth in vitro and in vivo, and reduces the proliferation, migration, and tube formation capabilities of LUAD cells in a co-culture model with human umbilical vein endothelial cells. Mechanistically, H2AC19 interacts with p300 and recruits it to the EGR1 promoter region, enhancing H3K27 acetylation modification, thereby activating the transcriptional process and promoting MMP-1-mediated angiogenesis and LUAD progression.

[0022] The above-mentioned technical solutions not only provide a superior method for the diagnosis and prognostic assessment of lung adenocarcinoma, but also lay an experimental foundation and expand new perspectives for the development of highly effective drugs for the treatment of lung adenocarcinoma, thus having good practical application value. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0024] Figure 1 In this embodiment of the invention, H2AC19 significantly inhibits angiogenesis in lung adenocarcinoma (LUAD). (A) AAG subgroups and clinicopathological and biological characteristics: Two different subtypes of samples were divided by cluster analysis, and the consensus matrix heatmap shows the two clusters and their related regions. (B) Principal component analysis (PCA) showed significant differences in transcriptome between the two subgroups. (C) Volcano plot of angiogenesis and hypoxia-related genes in the TCGA database: red and blue nodes represent upregulated and downregulated genes, respectively. (D) Cross-validation results of LASSO regression model parameter selection. (E) LASSO coefficient distribution. (F) Corresponding coefficients of 12 candidate genes. (G) Schematic diagram of co-culture of lung adenocarcinoma cells and human umbilical vein endothelial cells (HUVEC). (H) Effect of different treatment conditions of culture medium (CM) on the tube formation ability of HUVEC in A549 cells: tube formation experiments were performed after HUVEC was treated with CM for 48 hours, scale bar = 100µm. (I) Two-tailed Student's t-test analysis was used. Data are expressed as mean ± standard error, where n represents the number of independent biological experiments. P<0.05, P<0.01, P<0.001.

[0025] Figure 2This invention illustrates the association between H2AC19 gene upregulation and poor prognosis in LUAD patients. (A) Representative immunohistochemical images of H2AC19, vascular endothelial growth factor α (VEGFA), CD31, and Ki-67 expression in lung tissues of LUAD patients (normal group n=62, stage I n=21, stage II n=20, stage III n=14, stage IV n=7). (B) Quantitative analysis of H2AC19 in lung tissues of different LUAD patients. (C) Quantitative analysis of VEGFA in lung tissues of different LUAD patients. (D) Quantitative analysis of microvessel density (MVD) in normal and tumor tissues; CD31 expression levels were assessed using microvessel density. (E) Quantitative analysis of Ki-67 in normal and tumor tissues. (F) Correlation analysis of H2AC19 and VEGFA expression in all subjects (n=62). (G) Correlation analysis of H2AC19 and CD31 expression in all subjects (n=62). (H) Correlation analysis of H2AC19 and Ki-67 expression in all subjects (n = 62). Data are presented as mean ± standard deviation, where n represents the number of biologically independent experiments. (I) Survival analysis of high and low H2AC19 score groups based on median H2AC19 staining score (calculated using the OS prediction risk score formula). Data are presented as mean ± standard error, where n represents the number of biologically independent experiments. P<0.05, P<0.01, P<0.001.

[0026] Figure 3In this embodiment of the invention, a prognostic model for LUAD patients was constructed based on 12 key genes, and a risk score was calculated. (A) Kaplan-Meier survival curves of overall survival (OS) were used to divide LUAD patients in the TCGA cohort into high-risk and low-risk groups based on the median risk score. (B) Time-dependent ROC curve analysis of the prognostic model at 1, 3, and 5 years in the TCGA-LUAD cohort. (C) Kaplan-Meier survival curves of overall survival (OS) for LUAD patients in the high-risk and low-risk groups in the validation set GSE50081. (D) ROC curve analysis of the prognostic model at 1, 3, and 5 years in the GSE50081 dataset. (E) Kaplan-Meier survival curves of overall survival (OS) for LUAD patients in the high-risk and low-risk groups in the validation set GSE31210. (F) ROC curve analysis of the prognostic model at 1, 3, and 5 years in the GSE31210 dataset. (G) Nonographs for predicting 1-, 3-, and 5-year overall survival probabilities in LUAD patients in the TCGA cohort. (H) Decision curve analysis (DCA) of the nonographs for predicting 1-, 3-, and 5-year overall survival in LUAD patients. (I) qPCR detection of mRNA expression levels of 12 genes in LUAD tissues. (J) qPCR validation of the silencing efficiency of 12 genes in A549 cells. (IJ) Statistical analysis employed a two-tailed unpaired Student's test. P<0.05, P<0.01, P<0.001.

[0027] Figure 4This invention illustrates how H2AC19 gene knockout inhibits LUAD cell growth, migration, and angiogenesis. (A) Schematic diagram of the CRISPR / Cas9 system in LUAD cells. A549 and H1299 cells were transfected with CRISPR plasmids expressing Cas9 and H2AC19 sgRNA for 12 hours, followed by treatment with puromycin (0.5 μg / mL) for 14 days. Single clones were screened based on GFP expression and amplified into single-clonal cell colonies. (B) Western blot analysis verified the knockout efficiency of H2AC19 in A549 and H1299 cells. (C) Typical Western blot analysis and quantitative analysis of the relative levels of VEGFA protein in A549 and H1299 cells in different treatment groups. (D) Effect of A549 and H1299 cell culture medium on the tube-forming ability of human umbilical vein endothelial cells (HUVECs). Tube-forming experiments were performed after treating HUVECs with culture medium for 48 hours, scale bar = 100µm. (E) Representative images of blood vessels formed after treatment with chicken chorioallantoic membrane (CAM) culture medium. (F) Transwell assay showing the effect of different treatment groups of A549 cell culture medium on the migration ability of HUVECs. Scale bar = 100µm. (G) EDU assay showing the proliferation ability of A549 and H1299 cells in different treatment groups. Scale bar = 50µm. (H) Flow cytometry analysis showing the role of H2AC19 in regulating apoptosis in A549 and H1299 cells. Data are expressed as mean ± standard deviation, where n is the number of biologically independent experiments. Two-tailed Student's t-test was used for analysis (CH). P<0.05, P<0.01, P<0.001.

[0028] Figure 5 This invention demonstrates how H2AC19 gene knockout inhibits tumor progression and angiogenesis in vivo. (A) Typical macroscopic images of tumors from autopsies of mice implanted with A549 cells in different treatment groups. (BC) Tumor volume and body weight of mice implanted with tumors were measured at specified time points after cell implantation. (D) Typical immunohistochemical imaging of CD31, VEGFA, and Ki67 expression in formalin-fixed paraffin-embedded sections of xenografts. Scale bar = 20µm. Data are expressed as mean ± standard deviation, where n is the number of biologically independent experiments. Two-tailed Student's t-test was used for analysis (BD). P<0.05, P<0.01, P<0.001.

[0029] Figure 6 In this embodiment of the invention, H2AC19 deletion inhibits angiogenesis by downregulating MMP-1 expression in LUAD. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis based on RNA sequencing analysis of different groups of A549 cells. (BD) Gene set enrichment analysis (GSEA) shows the enrichment of angiogenesis, VEGFA, and HIF-α pathways. (E) Volcano plot showing differentially expressed genes in each group in RNA sequencing analysis. (FG) qPCR and western blot detection of MMP-1 mRNA and protein levels in A549 and H1299 cells under different treatments. (H) Western blot verification of H2AC19 overexpression efficiency in A549 and H1299 cells. (I) Effect of different culture media on tube formation ability of A549 and H1299 cells. Tube formation experiments were performed after treating HUVECs with culture medium for 48 hours, scale bar = 100µm. (J) Representative vascular imaging of CAM experiments after different culture medium treatments. Data are expressed as mean ± standard deviation, where n represents the number of independent biological experiments. A two-tailed Student's t-test (HG) was used. Tukey's multiple comparisons (IJ) were performed after a two-way ANOVA. P<0.05, P<0.01, P<0.001.

[0030] Figure 7 In this embodiment of the invention, H2AC19 deficiency inhibits angiogenesis in vivo by downregulating MMP-1 expression in LUAD. (A) An in situ model of stable H2AC19 overexpression and MMP-1 knockout in A549 cells was established in nude mice. (BC) Tumor volume and body weight were measured in tumor-implanted mice at specified time points. (D) Representative immunohistochemical images of CD31, VEGFA, and Ki67 expression in paraffin-embedded sections. Scale bar = 20µm. Data are expressed as mean ± standard deviation, and n represents the number of biologically independent experiments. P<0.05, P<0.01, P<0.001.

[0031] Figure 8In this embodiment of the invention, H2AC19 promotes angiogenesis in LUAD cells via the p300 / EGR1 / MMP-1 pathway. (A) Western blot experiments and quantitative analysis show the p300 protein levels in A549 cells under different treatment conditions. FLAG rabbit antibody was used for immunoprecipitation of FLAG-H2AC19, and p300 expression in the immune complex was detected by p300-specific antibody. Normal rabbit IgG, an isotype control, was used as a negative control to verify antibody specificity. (B) Western blot experiments and quantitative analysis show the relative protein levels of key acetylation-related molecules in A549 cells under different treatment conditions. (C) Chromatin immunoprecipitation (ChIP) experiments were performed on A549 cells using FLAG-H2AC19 antibody, and the resulting DNA eluent was analyzed by high-throughput sequencing. Schematic diagrams and heatmaps show the intensity distribution and pattern of H2AC19 signal within 1 kb upstream to 1 kb downstream of the transcription start site (TSS). (D) Venn diagrams show the intersection of differentially expressed genes and angiogenesis in RNA sequencing and chromatin immunoprecipitation sequencing, including genes such as EGR1, ECM1, SPNS2, PTAFR, NFE2, ADORA1, and COL1A1. (E) ChIP-seq results show a significant binding peak in the promoter region near the EGR1 transcription start site. (F) Effects of H2AC19 overexpression or A549 cell culture medium treated with A-485 on the tube-forming ability of human umbilical vein endothelial cells (HUVECs). In the experiment, HUVECs were treated with the culture medium for 48 hours before tube formation experiments were performed. Scale bar = 100 µm. (G) Vascular structures formed in representative images from the CAM experiment after different culture medium treatments. Data are expressed as mean ± standard error, where n represents the number of biologically independent experiments. Two-tailed Student's t-test analysis (B). Two-way ANOVA combined with Tukey's post-hoc test (EG). P<0.05, P<0.01, P<0.001.

[0032] Figure 9In this embodiment of the invention, the expression level of H2AC19 in LUAD patients was significantly correlated with acetylated H3K27 and EGR. (A) Representative immunohistochemical images of H3K27ac and EGR1 expression in lung tissues of normal subjects (n = 62), stage I (n = 21), stage II (n = 20), stage III (n = 14), and stage IV (n = 7) LUAD patients. (B) Quantitative analysis results of H3K27ac in lung tissues of different LUAD patients. (C) Quantitative analysis results of EGR1 in lung tissues of different LUAD patients. (D) Correlation between H2AC19 and H3K27ac expression in all subjects (n = 62). (E) Correlation between H2AC19 and EGR1 expression in all subjects (n = 62). Data are expressed as mean ± standard value, where n represents the number of biologically independent experiments. One-way ANOVA combined with Tukey post-hoc test (BC). Spearman correlation coefficient (DE). P<0.05, P<0.01, P<0.001.

[0033] Figure 10 In this embodiment of the invention, the H2AC19Δaa24-88 mutation reversed the effect of H2AC19 on LUAD progression. (A) Analysis of H2AC19-P300 interaction predicted by alpha fold3. (B) Interactive two-dimensional map of the comparison error (PAE) between H2AC19 and P300 predictions. (C) Three different H2AC19 deletion mutants were constructed, respectively deleting the domains predicted by the SMART and Uniprot databases. (D) Representative Western blot and quantitative analysis showing the expression level of P300 in A549 cells under different treatment conditions. H2AC19 was immunoprecipitated using FLAG rabbit anti-FLAG antibody, and immune complexes were detected using P300 antibody. Isotype control normal IgG (rabbit-derived) was used as a negative control to verify antibody specificity. (E) ChIP-qPCR detection showing the enrichment of the EGR1 promoter region in A549 cells. (F) Representative Western blot and quantitative analysis showing the relative protein levels of EGR1 and MMP-1 in A549 cells under different treatment conditions. (G) Effect of cell culture medium overexpressing H2AC19 or simultaneously mutated Δ24-88 amino acids on the tube-forming ability of human umbilical vein endothelial cells (HUVECs). Tube-forming experiments were performed on HUVECs after treatment with CM for 48 hours, scale bar = 100µm. (H) Representative images of blood vessels formed in CAM experiments after different CM treatments.

[0034] Figure 11 In this embodiment of the invention, the H2AC19Δaa24-88 mutation reversed the effect of H2AC19 on LUAD angiogenesis. (A) VEGFA levels in the culture supernatant of A549 cells in different treatment groups were determined by ELISA. (B) Scratch healing assay showed the effect of different CM treatments on the migration ability of HUVECs induced by A549 cells. Scale bar = 100µm. (C) Transwell assay showed the effect of different CM treatments on the migration ability of HUVECs induced by A549 cells. Scale bar = 100µm. (D) CCK-8 assay showed the proliferation of A549 cells in different treatment groups. Data are expressed as mean ± standard deviation, where n is the number of biologically independent experiments. Two-way ANOVA combined with Tukey post-hoc test (AD) was used. P<0.05, P<0.01, P<0.001.

[0035] Figure 12 This invention illustrates how H2AC19 promotes angiogenesis and tumor progression in a LUAD organoid model. (A) Schematic diagram of lung adenocarcinoma organoid construction. (B) Bright-field images and diameter growth curves of organoids at different time points. (C) Representative immunofluorescence images of TTF1 (green) and Napsin A (red) in PDOs, with DAPI (4',6-diamidinyl-2-phenylindole) as the nuclear staining agent. (D) Representative images of the number and diameter of organoids in different groups. (E) Immunofluorescence images and quantitative analysis of CD31, VEGFA, and Ki-67 expression levels in organoids of different groups, with DAPI (4',6-diamidinyl-2-phenylindole) as the nuclear staining agent. (F) Flow cytometry analysis of the effect of H2AC19 on organoid apoptosis regulation. (G) CCK-8 assay for organoid proliferation in different groups. (H) Representative images of the number and diameter of organoids in different groups. Data are expressed as mean ± standard deviation. P<0.05, P<0.01, P<0.001.

[0036] Figure 13In this embodiment of the invention, H2AC19 promotes angiogenesis and tumor progression in a LUAD organoid model by regulating P300. (A) Immunofluorescence images and quantitative analysis of CD31, VEGFA, and Ki-67 expression levels in different groups of organoids, with DAPI (4', 6-diamidinyl-2-phenylindole) used as a nuclear staining agent. (B) Schematic diagram illustrating how H2AC19 promotes LUAD angiogenesis by regulating the p300 / EGR1 / MMP-1 axis. Data are expressed as mean ± standard deviation. P<0.05, P<0.01, P<0.001. Detailed Implementation

[0037] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0038] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0039] In a typical embodiment of the present invention, a reagent for detecting the expression level of the H2AC19 encoding gene and its expression products is provided for the preparation of products for screening, diagnosing, monitoring, and predicting the progression of lung adenocarcinoma. The present invention demonstrates through research that H2AC19 is significantly upregulated in LUAD samples and promotes LUAD progression in both in vitro and in vivo experiments. The expression level of H2AC19 in LUAD patients is positively correlated with angiogenesis and tumor progression; LUAD patients with high H2AC19 expression have shorter overall survival, indicating that increased H2AC19 expression is a poor prognostic factor for LUAD.

[0040] The expression product of the H2AC19 encoding gene can obviously be the H2AC19 protein.

[0041] The reagents may be reagents for detecting the transcription of H2AC19 encoding genes based on RT-PCR, real-time quantitative PCR, in situ hybridization, gene chips and / or gene sequencing; or reagents for detecting H2AC19 protein expression based on immunoassay methods.

[0042] The products may include primers, probes, nucleic acid membrane strips, gene or protein chips, reagent kits, detection devices and equipment, etc.

[0043] In one or more specific embodiments of the present invention, a system for screening, diagnosing, monitoring, and predicting the progression of lung adenocarcinoma is provided, the system comprising:

[0044] (a) An analysis module, the analysis module comprising: a detection reagent for determining the expression level of the subject selected from the above-mentioned H2AC19 encoding gene and its expression product;

[0045] (b) An assessment module comprising: assessing the subject’s disease status based on the expression levels of the H2AC19 coding gene and its expression products as determined in (a).

[0046] The subjects can be humans or non-human mammals (such as rats, mice, guinea pigs, chimpanzees, monkeys, dogs, etc.), with humans being preferred.

[0047] Furthermore, the assessment module includes at least one risk scoring model, the specific calculation formula of which is as follows: Risk Score = (0.00138418 × SLC16A3) + (1.151744665 × VAX1) + (0.013769648 × PLEK2) - (0.001303427 × SLC2A1) + (0.001938428 × R HOV)+(0.22876788×H2AC19)+(0.013187135×AHNAK2)+(1.390758033×PITX3)-(0.247 881445×ADHFE1)+(0.02923751×MELTF)+(0.001451527×DKK1)+(0.62563978×KCNV1).

[0048] Patients were divided into high-risk and low-risk groups based on their median risk score. Patients with a risk score greater than or equal to the median risk score were classified as high-risk, while those with a risk score less than the median risk score were classified as low-risk.

[0049] In this invention, the overall survival rate of patients in the high-risk group is lower, while the overall survival rate of patients in the low-risk group is higher.

[0050] In one or more specific embodiments of the present invention, the use of H2AC19 as a target in the preparation and / or screening of lung adenocarcinoma drugs is provided.

[0051] The lung adenocarcinoma drugs include drugs for the prevention and / or treatment of lung adenocarcinoma.

[0052] The method for screening lung adenocarcinoma drugs includes:

[0053] (1) Treat the system expressing and / or containing the H2AC19 with the candidate substance; set up parallel controls without the candidate substance treatment;

[0054] (2) After completing step (1), detect the expression level of H2AC19 in the system; if the expression level of H2AC19 in the system treated with the candidate substance is significantly reduced compared with the parallel control, the candidate substance can be used as a candidate lung adenocarcinoma drug.

[0055] In another specific embodiment of the present invention, the system may be a cell system (such as lung adenocarcinoma cells), a solution system, a tissue system, an organ system, or an animal system.

[0056] In one or more specific embodiments of the present invention, the use of H2AC19 inhibitors in any one or more of the following is provided:

[0057] (a) To prepare a product that inhibits the growth of lung adenocarcinoma cells;

[0058] (b) To prepare a product that inhibits the migration of lung adenocarcinoma cells;

[0059] (c) Preparation of products that inhibit angiogenesis in lung adenocarcinoma;

[0060] (d) Preparation of lung adenocarcinoma drugs.

[0061] The H2AC19 inhibitors include, but are not limited to, RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, shRNA, siRNA (as shown in SEQ ID NO.4), substances that carry out lentiviral infection or gene knockout (such as CRISPR / Cas9), and specific antibodies against the H2AC19 protein itself or its upstream and downstream molecules, such as anti-H2AC19 antibodies. They may also include compound inhibitors, which are not specifically limited here.

[0062] In application (c), the inhibition of lung adenocarcinoma angiogenesis is achieved by regulating the p300 / EGR1 / MMP-1 signaling pathway.

[0063] The product may be a drug or a general testing reagent for non-medical purposes, and the general testing reagent is intended for use in basic research.

[0064] When the product is a drug, the drug may also include at least one inactive pharmaceutical ingredient.

[0065] The inactive components of the drug can be pharmaceutically commonly used carriers, excipients, and diluents. Furthermore, according to conventional methods, it can be formulated into oral, topical, suppository, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays.

[0066] The non-pharmaceutical active ingredients that may be included, such as carriers, excipients, and diluents, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0067] In another specific embodiment of the present invention, the drug of the present invention can be administered into the body by known means. For example, it can be delivered to the tissue of interest via intravenous systemic delivery or local injection. Administration can be selected via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via single or multiple doses. Those skilled in the art will understand that the actual dose to be administered in the present invention can vary considerably depending on a variety of factors, such as target cells, biological type or tissue, the general condition of the subject to be treated, route of administration, manner of administration, etc.

[0068] In one or more embodiments of the present invention, a method for preventing and / or treating lung adenocarcinoma is provided, the method comprising administering the above-mentioned H2AC19 inhibitor to a patient.

[0069] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. If specific experimental conditions are not specified in the examples, they are generally performed under conventional conditions or as recommended by the selling company. The present invention is not particularly limited and all items are commercially available.

[0070] Example

[0071] I. Research Methods

[0072] 1. Cell Culture

[0073] Human umbilical vein endothelial cell line (HUVEC) was purchased from Pricella (Wuhan, China). HUVECs were cultured in endothelial cell culture medium (ECM, ScienCell, USA). Human lung adenocarcinoma cell lines A549 and NCI-H1299 were purchased from CELL Research (Shanghai, China). A549 and NCI-H1299 were cultured in RPMI-1640 (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA). All cell lines were identified by short tandem repeat (STR) analysis by Hybribio Limited (China). Cells were incubated at 37°C in a humidified environment with 5% carbon dioxide.

[0074] 2. Clinical specimens

[0075] This study collected 62 pairs of fresh LUAD tissue and adjacent normal tissue (ANT) at Qilu Hospital of Shandong University between January and October 2019. None of the patients had received chemotherapy or radiotherapy prior to sample collection. This study was approved by the Ethics Committee of the School of Medicine, Qilu Hospital of Shandong University (Approval No.: KYLL-202411-039-1). All samples were confirmed by histopathological examination, and informed consent was obtained from all patients / participants.

[0076] 3. Culture of LUAD patient-derived organoids (PDO)

[0077] Fresh tissues were processed immediately upon arrival at the laboratory from the operating room. After being minced, the tissues were placed in 5 mL of collagenase B (5 mg / mL; Roche, #11088815001) and DNase I (100 μg / mL; Merck, #6918230) at 37°C and gently shaken for 30 minutes to 1 hour. The suspension was then filtered through a 70 μm nylon cell sieve (Fisher Scientific) and centrifuged at 280 g for 5 minutes. The precipitate was embedded in Matrigel solution and seeded into 24-well plates. After Matrigel solidified, 250 μL of culture medium was added to each well. PDOs were cultured in a humidified incubator at 37°C with 5% CO2, and the culture medium was changed three times a week.

[0078] 4. Animal Research

[0079] All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of Qilu Hospital, Shandong University (Document No.: KYLL-2024(ZM)-1085) and performed in accordance with the National Institutes of Health's "Guidelines for the Care and Use of Laboratory Animals." Groups were randomly assigned, and researchers blinded the group assignments during surgery and outcome assessment. Except for mice with special dietary requirements, all mice (3-5 per cage) were housed under standard laboratory conditions (12-hour light / dark; lights on at 7:00 AM), at a temperature of 22-24°C, with free access to water and standard laboratory feed (Beijing Keoxili Feed Co., Ltd., Beijing, China). Water and cages were autoclaved. Cages were fitted with standard corncob bedding three times a week. Cell suspensions were adjusted to 10... 7 A subcutaneous tumor model was established by subcutaneously injecting 200 µL of cell suspension into the right back of nude mice. Tumor formation was monitored every two days after each mouse was injected, and mouse weight and tumor volume were recorded. The tumor volume was calculated using the formula: ([L×W]²) / 2. Mice were euthanized under deep anesthesia, and the tumors were removed, photographed, and weighed.

[0080] 5. Gene knockout (KO) using the CRISPR / Cas9 system.

[0081] According to the manufacturer's protocol, CRISPR plasmids (SantaCruz Biotechnology, USA) were transfected into A549 and H1299 cells using Santa Cruz transfection reagent. In short, CRISPR plasmids expressing Cas9 and H2AC19, MMP-1, and EGR1 sgRNAs were transfected into A549 and H1299 cells in 6-well plates for 12 hours. Successful transfection of the CRISPR / Cas9 knockout plasmids was confirmed by detecting green fluorescent protein (GFP). Cells were screened with puromycin (0.5 μg / mL) for 14 days. Single clones were picked from 96-well plates based on GFP expression and amplified to generate monoclonal cell colonies. Protein expression levels of H2AC19, MMP-1, and EGR1 were determined by Western blotting.

[0082] 6. RNA interference

[0083] Cells were cultured in antibiotic-free medium. Short interfering RNAs (siRNAs) of AHNAK2, ADHFE1, DKK1, H2AC19, KCNV1, MELTF, PITX3, PLEK2, RHOV, SLC2A1, SLC16A3, and VAX1, along with equivalent scrambled control (or negative control siRNA), were delivered to the cells using Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer's protocol. The sequences of the siRNA oligonucleotides are listed in Table 1.

[0084] Table 1. siRNA oligonucleotide sequence information

[0085] siRNA sequence GTACAACCGTGTTCTTTGA (SEQ ID NO.1) GTGGCTAAGTATCTGAAGA (SEQ ID NO.2) GGATGGGTATTCCAGAAGA (SEQ ID NO.3) CCGTACTGCTCCCTAAGAA (SEQ ID NO.4) CTCTGCGCATGCTAAAGCT (SEQ ID NO.5) GAGAGACCAGTTACTCTGA (SEQ ID NO.6) AGAGGACGGTTCGCTGAAA (SEQ ID NO.7) GAAGGTGCGTCGCTTTGTT (SEQ ID NO.8) GAGGGACGATGTCAACGTA (SEQ ID NO.9) GCATGTGCTCCAGTATGT (SEQ ID NO.10) GGAGCATCATCCAGGTCTA (SEQ ID NO.11) TGCTGAGGATTGTAACAAA (SEQ ID NO.12)

[0086] 7. Construction of lentiviral vectors and viral transduction

[0087] H2AC19 cDNA was integrated into the Ubi-MCS-3FLAGCBh-gcGFP-IRES-puromycin lentiviral vector. Lentiviral particles were then generated in HEK-293T cells using two packaging plasmids (psPAX2 and pMD2G). Subsequently, A549 and H1299 cells were transfected with H2AC19 virus and corresponding control viruses in the presence of polybrene. Following transfection, cells expressing the target construct were selected within one month using 10 mg / mL puromycin.

[0088] 8. Real-time polymerase chain reaction (qPCR)

[0089] Total RNA was extracted from tissues or cells using Trizol reagent (Invitrogen). mRNA expression levels were determined by real-time quantitative reverse transcription polymerase chain reaction using the Bio-Rad iCycler system (Bio-Rad, Hercules, California, USA). The level of the housekeeping gene β-actin was used as an internal control for standardizing RNA levels between samples. The ΔΔCT method was employed according to Equation 2. -ΔΔCT The fold change in gene expression relative to β-actin was calculated. Results are expressed as fold changes relative to the control group.

[0090] 9. Western blot analysis

[0091] The preparation of total cell lysate and Western blot analysis were performed as follows: Cells were collected using RIPA buffer (150 mM sodium chloride, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1% NP40, 1 mM EDTA, and 50 mM Tris, pH 8.0) and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Samples were transferred to PVDF membranes and incubated with a primary antibody containing 5% skim milk. To validate the loading control, the membrane was re-probeged with an anti-housekeeping protein primary antibody. Quantitative analysis was performed using ImageJ software after scanning; band intensities were normalized to appropriate loading controls and presented as relative abundance.

[0092] 10. Immunohistochemistry (IHC)

[0093] First, tissue specimens were dewaxed, repaired with EDTA, and incubated overnight with primary antibody at 4°C. Secondary antibody was then added, and incubation was performed at room temperature for 60 minutes. Diluted diaminobenzidine (DAB) was added to the pathological sections, and inhibition was achieved using hematoxylin (Sigma). Finally, the sections were dehydrated and sealed. Specimens were scored by three independent pathologists who had no knowledge of any prognostic or clinicopathological variables. Staining intensity scoring was as described previously: 0 points, no staining; 1 point, weak cytoplasmic staining; 2 points, moderate cytoplasmic staining; 3 points, strong cytoplasmic staining, and the percentage of stained cancer cells was recorded. The expression levels of H2AC19, VEGFA, EGR1, and H3K27ac were assessed by staining intensity scoring. CD31 expression levels were assessed by microvessel density (MVD).

[0094] 11. Immunofluorescence staining

[0095] Organoid sections (5 µm) were dewaxed and rehydrated with a series of alcohol solutions of decreasing concentrations (5 minutes each, 100%, 95%, 70%, and 50%), and then stored in PBS for 30 minutes before proceeding to the antigen retrieval process. Antigen retrieval was performed in PBS with 10 mM citrate (pH=6) and microwaved (650 W for 7 minutes once, followed by two 5-minute cycles at 350 W). After washing in PBS for 10 minutes, sections were incubated at room temperature (RT) with blocking buffer (PBS containing 4% donkey serum and 0.1% Triton X-100) for 60 minutes. The sections were then incubated overnight at 4°C with primary antibody diluted in blocking buffer. After washing three times in PBS (5 minutes each), slides were incubated at room temperature with secondary antibody diluted in blocking buffer for 60 minutes. Finally, slides were washed three times in PBS (5 minutes each) and mounted with an aqueous mounting medium containing DAPI.

[0096] 12. Tube Formation Test

[0097] Matrigel (250 µL / well, BD) was added to a 24-well plate and polymerized at 37°C for half an hour. HUVECs (2 × 10⁻⁶ per well) were then added. 5 (100 cells), and incubated in conditioned medium for 8 hours. The number of knots in each well was measured and counted. Each measurement was repeated three times.

[0098] 13. Detection of chicken chorioallantoic membrane (CAM)

[0099] CAM detection was performed on day 8 of fertilized chicken embryos. A 1.0 cm diameter hole was made in the eggshell air sac, and the surface of the dermal flap at the bottom of the air sac was removed to expose the CAM. A 0.5 cm diameter filter paper was placed on top of the CAM, and 100 µL of freshly harvested specified lung cancer cell conditioned medium was carefully added to the center of the filter paper and sealed with medical breathable tape. The chicken embryos were then incubated at 37.8°C and 60%–80% humidity for 5 days. Images of the CAM were captured using a digital camera (Canon, Japan) and analyzed using ImageView 3.7 (Jingtong, China). The effect of the conditioned medium on vascular endothelial cell proliferation and migration was assessed by comparing the number of blood vessels with the control group.

[0100] 14. Chromatin Immunoprecipitation and Quantitative PCR (ChIP-qPCR) Detection

[0101] A549 cells were treated with 5 µM Δ9-THC or 5 µM Δ9-THC combined with 50 µM BODIPY-THIF for 6 hours, then cross-linked with 2% formaldehyde for 15 minutes. The cells were collected by scraping with PBS, centrifuged, and lysed with 1 mL of IP buffer containing protease inhibitors (150 mM sodium chloride, 50 mM Tris-hydrochloric acid pH 7.5, 5 mM EDTA, 0.5% Nonidet P-40, and 1% Triton X-100). The resulting nuclear precipitate was resuspended in IP buffer and sonicated. ChIP experiments were performed using control rabbit IgG or Flag antibody. Immunoprecipitated DNA and infused DNA were extracted by incubating the sample with 100 µL of 10% Chelex resin, followed by boiling to reverse cross-linking, centrifugation to remove the Chelex suspension, and then centrifugation to remove the Chelex suspension.

[0102] 15. Immunoprecipitation

[0103] Protein samples were immunoprecipitated using antibodies against negative controls (normal rabbit IgG, H2AC19, or FLAG), and incubated with 30 µl of Protein A&G magnetic beads at room temperature with constant rotation for 1 hour. The magnetic beads and 500 µg of sample lysis buffer were incubated overnight at 4°C. After elution, the immunoprecipitated proteins were detected by Western blotting using p300 antibody. Isotype-matched normal IgG was used as a negative control to verify the antibody's specificity.

[0104] 16. Transwell Detection

[0105] Cell invasion assays were performed using Transwell chambers. The upper chamber membrane was coated with matrix gel. Cells were seeded into the upper chamber of each insert, and 300 µL of serum-free medium was added. Additionally, 500 µL of medium containing 10% FBS was added to the lower chamber. After incubation for 48 hours, the invading cells in the lower chamber were fixed with 10% methanol and stained with 0.1% crystal violet. The invading cells were photographed under a microscope.

[0106] 17. Wound healing test

[0107] Cells were seeded into 6-well culture plates. After a monolayer was formed, a straight line was drawn using a 10 µL pipette tip. The cells were gently washed with PBS and then replenished with serum-free culture medium. Images were taken under a microscope at 0 h and 24 h.

[0108] 18. Cell Counting Kit 8 Detection Method (CCK8)

[0109] Cells were seeded at a density of 1000 cells per well in 96-well plates. At time points 1, 2, 3, or 4, 20 μL of CCK8 solution (5 mg / ml) was added to each well to measure changes in cell viability. The optical density of each well was measured at 450 nm using a scanning porous spectrophotometer.

[0110] After organoids were dissociated into single cells using TrypLE (Thermo Fisher, Massachusetts, USA), they were cultured in medium for 5–10 days. A 1 mg / mL neutral protease solution was added to the culture plate, and the cells were incubated at 37°C for 10 minutes. The cell suspension was then repeatedly aspirated using a 1 mL pipette tip, washed twice with cold PBS, and centrifuged at 1300 rpm for 3 minutes. The organoid pellet was resuspended in medium containing 5% Matrigel and seeded in 96-well ultra-low adhesion plates (2500 organoids per well). The luminescence intensity was measured using a CTG 3D detection reagent.

[0111] 19. Colony formation test

[0112] Cells (2 × 10⁶ per well) 2 Or 5×10 2 (Number of cells) were inoculated into six-well plates and incubated in a humidified incubator at 37°C and 5% CO2 for 10 days. At the end of the experiment, the cultures were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.1% crystal violet for 5 minutes.

[0113] 20. Apoptosis detection

[0114] After collecting the cells, they were resuspended in Annexin V binding buffer to a final concentration of 10. 6 Cells / mL. Cells were then incubated with Alexa Fluor 647 Annexin V at 4°C in the dark for 15 minutes, followed by the addition of PI (propidium iodide). Samples were immediately analyzed by flow cytometry. Organoids on day 7 were dissociated using 1X TrypLE Express enzyme at 37°C for 10 minutes. The reaction was vortexed once per minute and stopped by adding culture medium. Single-cell suspensions were prepared by filtering cells through a 40-micron Flowmi filter. Data were analyzed using FlowJo software (V10.8.0).

[0115] 21. Enzyme-linked immunosorbent assay (ELISA)

[0116] Collect the cell culture supernatant and determine the VEGF concentration according to the ELISA kit instructions.

[0117] 22. EdU detection

[0118] Cell proliferation was measured using an EdU detection kit. EdU staining was performed according to the manufacturer's instructions. Finally, fluorescence images were acquired using a fluorescence microscope.

[0119] 23. Molecular docking

[0120] The crystal structures of H2AC19 and P300 were predicted using AlphaFold3. The hydrogen bond sites interacting with H2AC19 and P300 were analyzed using ChimeraX 1.8 software, and the results were visualized.

[0121] 24. RNA-seq analysis

[0122] A549 cells expressing the control group and sgH2AC19 were lysed with TRIzol reagent, and the resulting samples were sent to Annoroad Gene Technologies for RNA purification and subsequent RNA sequencing. The RNA sequencing analysis workflow included alignment and assembly, quantification, standardization, and differential expression analysis, which was completed using the HISAT2-StringTie-Deseq2 process.

[0123] 25. ChIP-seq analysis

[0124] Cells were washed twice with PBS and then chemically cross-linked with 1% formaldehyde for 10 minutes at room temperature. The cross-linking reaction was then terminated with 0.125 mol / L glycine. After cell collection, the cells were resuspended in SDS buffer (50 mM Tris-HCl pH 8.0, 100 mM sodium chloride, 5 mM EDTA, and 0.5% SDS) supplemented with protease inhibitors. The cell pellet was collected by centrifugation at 1200 rpm for 10 minutes and then resuspended in ice-cold immunoprecipitation (IP) buffer containing 100 mM sodium chloride, 66.67 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), 0.3% SDS, and 1.67% Triton X-100. The suspension was sonicated, and the supernatant was incubated with antibodies overnight at 4°C. Protein A / G magnetic beads (Bimake, Houston, USA) were then added at 4°C and incubated for 4 hours. Magnetic beads were washed three times with Wash Buffer 1 (150 mM sodium chloride, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, and 20 mM Tris-HCl, pH 8.0), followed by one wash with Wash Buffer 2 (1% Triton X-100, 500 mM sodium chloride, 0.1% SDS, 2 mM EDTA, and 20 mM Tris-HCl, pH 8.0). To decrosslink, samples were incubated at 65°C for 6 hours, followed by DNA extraction for deep sequencing (Novgene Ltd., Tianjin, China). After ChIP-seq, clean data was obtained by removing raw data containing adapter sequences, polyadenylated tails, and low-quality reads. Subsequent analyses were based on the high-quality clean data. The reference genome (UCSC hg38) was used to map the clean data to the human genome using Bowtie2 (v 2.4.5). High-confidence mapped reads were sorted, indexed, and converted to BAM format using SAMtools (v1.6). Repetitive sequences were removed using Sambamba (v0.8.2). Large molecular files were computed and normalized to counts per million (CPM) using bamCompare in Deeptools (v3.5.1). A signal visualization around transcription start sites was generated using the computeMatrix function. Peak identification was performed using Macs2 (v2.2.7.1), followed by blacklisting from all peaks using Bedtools (v2.30.0) (https: / / github.com / BoyleLab / Blacklist). Peak annotation was performed using ChIPseeker (v1.30.3).

[0125] 26. Protein-protein interaction networks

[0126] A protein-protein interaction (PPI) network was constructed using a string database (string-db.org). The network was visualized using Cytoscape software (3.10.1, Cytoscape Consortium).

[0127] 27. Acquisition of genes related to hypoxia and angiogenesis

[0128] Genes related to hypoxia and angiogenesis were obtained from GSEA MSigDB (https: / / www.gsea-msigdb.org / gsea / index.jsp). Specifically, the "HALLMARK_HYPOXIA" and "HALLMARK_ANGIOGENESIS" gene sets were downloaded, yielding a total of 233 genes.

[0129] 28. Consensus Clustering

[0130] Unsupervised clustering was performed using the ConsensusClusterPlus R package. After standardization, consensus clustering divided patients in the TCGA-LUAD cohort into two clusters based on 233 genes associated with hypoxia and angiogenesis, achieving a stable clustering structure at k=2. Based on hypoxia and angiogenesis genes, distributed random neighborhood embedding (t-SNE) analysis was employed, and the data were visualized in one and two dimensions using the Rtsne and ggplot2 packages.

[0131] 29. Construction and validation of a 12-gene prognostic model.

[0132] Prognostic models were constructed using UniCox, Lasso, and MultiCox regression. After consensus clustering, differential expression analysis between the two clusters was performed using the limma R software package. By setting the adjusted p-value <0.05 and |log2FC|≥1 criteria, we identified 554 upregulated genes and 1,019 downregulated genes. Univariate Cox analysis using a survival analysis software package preliminarily identified 1573 differentially expressed genes associated with overall survival (OS) in LUAD patients. Subsequently, Lasso regression was used to screen for significant genes, and prognostic features were further constructed using multivariate Cox analysis. The risk score was calculated as follows: Risk Score = ∑ (coefficients) Expression level); the coefficients and expression levels in this formula represent the coefficient index and expression level of the model gene, respectively. GSE31210 and GSE50081 were used as validation cohorts, and they showed good predictive performance.

[0133] 30. Construction of a Predictive nomogram

[0134] The risk score and clinicopathological features (including histological type, age, stage, and sex) are integrated using the RMS software package to generate a nomogram.

[0135] 31. Kaplan-Meier curve, ROC curve, and DCA curve

[0136] Kaplan-Meier (KM) survival curves were plotted using the Survival R software package. A p-value < 0.05 indicated a statistically significant difference in survival outcomes between the high-risk and low-risk groups. Time-dependent receiver operating characteristic (ROC) curves were generated using the timeROC R software package, while DCA curves were plotted using the dcurves R software package.

[0137] 32. Enrichment analysis of KEGG and GSEA

[0138] KEGG pathway enrichment analysis was performed on differentially expressed genes after H2AC19 knockout using the R package clusterProfiler (version 4.2.0) to identify significantly enriched biological processes. Fisher's exact test was used to calculate the significance of each term. The significance threshold for enrichment analysis was p-value < 0.05, and the enrichment results were further visualized using bubble charts. Gene set enrichment analysis (GSEA) was used to identify key pathways after H2AC19 knockout. GSEA analysis was performed using the R packages GSEABase and msigdbr.

[0139] 33. Statistics

[0140] Data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software (version 8.0). Normality of continuous data was assessed using the Kolmogorov-Smirnov test. Comparisons between two groups were performed using the two-tailed Student's t-test (applicable to normally distributed data). Differences among multiple univariate groups were assessed using one-way ANOVA combined with Tukey's post-hoc test, while comparisons among multiple multivariate groups were assessed using two-way ANOVA combined with Tukey's post-hoc test. The correlation between H2AC19 and other variables was assessed using Spearman correlation analysis, with the coefficient (r) and p-value evaluated. Correlation analysis and linear regression were both performed using GraphPad Prism. Bioinformatics correlation statistical analysis was performed using R software (version 4.3.2). The significance criteria were: P<0.05, P<0.01, P<0.001.

[0141] II. Research Results

[0142] 1. Construction of a LUAD prognostic risk scoring model based on 12 core genes

[0143] Based on consensus clustering analysis of 233 hypoxia and angiogenesis-related genes, we identified two stable LUAD subgroups in the TCGA-LUAD cohort, and principal component analysis (PCA) confirmed their clear transcriptomic segregation characteristics. Figure 1 (A to B). Subsequent differential expression analysis revealed 1,573 differentially expressed genes between the two subgroups, including 554 upregulated genes and 1,019 downregulated genes. Figure 1 C). After unicox analysis, LASSO regression was used to screen for 12 significant genes, and multicox analysis was used to calculate the coefficient of each gene in the risk scoring formula. Finally, a 12-gene feature combination was constructed, and the specific calculation formula for the risk score is as follows: Risk score = (0.00138418×SLC16A3) + (1.151744665×VAX1) + (0.013769648×PLEK2) - (0.001303427×SLC2A1) + (0.001938428×RHOV) + ​​(0.22876788×H2AC19) + (0.013187135×AHNAK2) + (1.390758033×PITX3) - (0.247881445×ADHFE1) + (0.02923751×MELTF) + (0.001451527×DKK1) + (0.62563978×KCNV1).

[0144] Patients were divided into high-risk and low-risk groups based on their median risk score (median risk score of 0.827968; scores greater than or equal to 0.827968 were considered high-risk, and scores less than 0.827968 were considered low-risk). Figure 1 (D to F). In the TCGA-LUAD cohort, high-risk patients defined by this combination of characteristics had significantly poorer overall survival, with time-dependent AUC values ​​of 0.699, 0.714, and 0.683 at 1, 3, and 5 years of follow-up, respectively. Figure 3 (A to B). Furthermore, we validated its prognostic predictive performance in two independent cohorts, GSE31210 and GSE50081, where high-risk patients consistently showed poor prognoses (A to B). Figure 3(C to F). Notably, based on a multivariate Cox regression model, we integrated model feature scores with various clinicopathological features (including histological type, patient age, sex, and tumor stage) to construct a nomogram for predicting the 1-, 3-, and 5-year overall survival probability of LUAD patients. This model demonstrated good predictive accuracy and calibration ability for LUAD patient survival (5-year C-index: 0.991, Figure 3 G). Decision curve analysis showed significant clinical benefit over a wide range of thresholds (G). Figure 3 H).

[0145] 2. Upregulation of H2AC19 is associated with poor prognosis in LUAD patients.

[0146] In addition, we examined the expression patterns of 12 core genes in paired tumor and normal tissues. Figure 3 I) confirmed that most genes showed an upregulated trend in LUAD tissue. To verify their function, we conducted gene silencing experiments in A549 cells (I). Figure 3 J, and explored its effect on the formation of matrix capillary structure in HUVECs by establishing a transwell co-culture model (J), and investigated its effect on the formation of matrix capillary structure in HUVECs by establishing a transwell co-culture model (J). Figure 1 G to I). Notably, compared with the control group, H2AC19 gene silencing in A549 cells significantly inhibited the formation of tubular structures in HUVECs. Given the high expression and anti-angiogenic properties of H2AC19 in LUAD patients, we further explored its biological function and molecular mechanism. We found that H2AC19 expression levels in LUAD patients increased significantly with increasing clinical stage, accompanied by enhanced angiogenesis (confirmed by VEGFA and CD31 staining) and accelerated tumor progression (confirmed by Ki67 staining). Figure 2 A to E). H2AC19 expression level and angiogenesis in LUAD patients ( Figure 2 F to G) and tumor progression ( Figure 2 H) was positively correlated. Kaplan-Meier curves showed that LUAD patients with high H2AC19 expression had shorter overall survival. Figure 2 I). Furthermore, univariate and multivariate Cox regression analyses showed that H2AC19 expression was an independent prognostic factor for overall survival in LUAD patients. In conclusion, these results indicate that increased H2AC19 expression is a poor prognostic factor for LUAD.

[0147] 3. H2AC19 gene knockout inhibited the growth, migration, and angiogenesis of LUAD cells.

[0148] To further elucidate the role of H2AC19 in LUAD, we constructed H2AC19-deficient A549 and H1299 cell lines using CRISPR / Cas9 gene editing technology. Figure 4 (A to B). The study found that knocking out the H2AC19 gene in LUAD cells significantly reduced the expression level of vascular endothelial growth factor α (VEGFA). Figure 4 C) and secretion volume. By co-culturing LUAD cells with human umbilical vein endothelial cells (HUVECs), compared with the control group, the tubular formation ability of HUVECs in the H2AC19 knockout LUAD cell group (C) was significantly increased. Figure 4 D to E), cell migration ability ( Figure 4 F) and proliferation activity ( Figure 4 G) all decreased significantly. In addition, apoptosis detection ( Figure 4 H), colony formation assays and scratch healing assays showed that H2AC19 knockout also significantly inhibited the apoptosis, proliferation and migration of LUAD cells.

[0149] To investigate the role of H2AC19 in regulating LUAD in vivo, we subsequently conducted a xenograft tumor formation experiment. The experiment revealed that H2AC19 gene knockout cells exhibited tumor suppression. Figure 5 A), the weight and volume of the tumors from which it originated were significantly smaller than those in the control group ( Figure 5 Immunohistochemical staining showed that Ki67 expression was significantly reduced in the H2AC19 knockout group (B to C). Figure 5 D). Further investigation into the association between angiogenesis and H2AC19 revealed that, compared to the control group, H2AC19 knockout not only reduced VEGFA expression levels but also decreased microvessel density (MVD).

[0150] 4. H2AC19 deficiency inhibits angiogenesis by downregulating MMP-1 expression in LUAD.

[0151] To investigate the mechanism by which H2AC19 promotes LUAD, we performed unbiased RNA sequencing on A549 cells expressing sg-H2AC19 or sg-NC and analyzed differentially expressed mRNAs. Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA), we confirmed the association between H2AC19 and angiogenesis-related signaling pathways. Figure 6 (A to D). Among all differentially expressed genes, matrix metalloproteinase-1 (MMP-1) is particularly noteworthy, as the most significant change in expression occurred after H2AC19 knockout. Figure 6E), and plays a key role in angiogenesis and tumor progression. Further experiments showed that H2AC19 knockout significantly reduced the mRNA and protein levels of MMP-1 in LUAD cells (E). Figure 6 F to G).

[0152] To further investigate whether MMP-1 is a key regulator linking H2AC19 to angiogenesis, we constructed a gene expressing H2AC19 in LUAD cells. Figure 6 H) and MMP-1 knockout A549 and H1299 cell lines. Studies have shown that MMP-1 knockout in LUAD cells significantly inhibits the effects of H2AC19 on VEGFA secretion and tubular formation in human umbilical vein endothelial cells (HUVECs) and LUAD cells. Figure 6 The effects of I to J), migration and proliferation capacity, and overall growth activity were also observed. These phenomena were similarly verified in in vivo experiments. Figure 7 (A to D).

[0153] 5. H2AC19 promotes angiogenesis in LUAD through the p300 / EGR1 / MMP-1 pathway.

[0154] Analysis using the STRING interactive gene search tool indicated that H2AC19 may have a strong interaction with the adenovirus E1A-associated 300kDa protein (p300). p300 is a key histone acetyltransferase, and recent studies have confirmed its important role in oncogenicity. p300 is known to maintain gene expression programs primarily through acetylation of histone lysines 4, 9, 14, 18, and 27 (i.e., H3K4, H3K9, H3K14, H3K18, and H3K27). We subsequently assessed p300-mediated histone acetylation in LUAD cells and found that H2AC19 binds to p300 (…). Figure 8 A), and knocking out H2AC19 preferentially inhibits the acetylation level of H3K27, while having no significant effect on the acetylation levels of other histones. Figure 8 B). Given the histone characteristics of H2AC19, we also performed whole-genome chromatin immunoprecipitation sequencing (ChIP-seq) ( Figure 8 C). By cross-referencing ChIP-seq and RNA-seq data, we identified 65 potential direct downstream target genes of H2AC19, of which 7 genes were significantly associated with angiogenesis (C). Figure 8D). Notably, early growth response factor 1 (EGR1) showed the most significant differential expression among these genes and is also a direct upstream target of MMP-1. Consistent with this, our results showed a distinct H2AC19 binding peak near the transcription start site in the EGR1 promoter region. We then performed ChIP-qPCR analysis to determine whether H2AC19-p300 mediates H3K27 acetylation in the EGR1 gene promoter region. The results indicated that H2AC19 increases H3K27 acetylation in the EGR1 promoter region, while the potent and specific p300 inhibitor A-485 counteracted this effect. Figure 8 E). Furthermore, we successfully reduced the mRNA and protein levels of EGR1 by knocking out the H2AC19 protein. Further investigation revealed that H2AC19 overexpression not only significantly increased the protein levels of EGR1 and MMP-1, but also promoted angiogenesis and tumor progression, all of which could be addressed by A-485 (E). Figure 8 The H2AC19-p300 axis was reversed (F to G). This suggests that the H2AC19-p300 axis may at least partially bind to and activate the EGR1 promoter region by enhancing the modification of H3K27ac. Notably, both H3K27ac and EGR1 levels were significantly elevated in LUAD patients (F to G). Figure 9 AC), and this indicator is positively correlated with H2AC19 expression levels ( Figure 9 (D to E).

[0155] 6. The H2AC19 deletion mutation at amino acids 24-88 negated the effect of H2AC19 on LUAD.

[0156] Structural analysis of the Alpha 3-fold and Chimera X revealed that H2AC19 shares a direct-binding structural motif with p300 and forms a protein-protein interaction interface. Figure 10 A), which is consistent with the conclusions of the PAE analysis. Figure 10 B). To clarify the binding region between H2AC19 and p300, we constructed three truncated mutants based on Uniprot and the Simple Modular Structure Study Tool (SMART) database: Mut1 (deleting amino acid segments 2-22), Mut2 (deleting amino acid segments 24-88), and Mut3 (deleting amino acid segments 92-126). Figure 10 C). The results showed that the Mut2 mutant, which lacks the amino acid sequence from position 24 to 88, could not bind to p300, while there was no significant difference between the wild-type and the other two mutants in the binding of H2AC19 to p300. Figure 10 D). Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) showed that, compared to the H2AC19 overexpression group, H2AC19-Mut2 inhibited the acetylation modification of H3K27 in the EGR1 promoter region (D). Figure 10 E). Western blot analysis showed that the expression levels of EGR1 and MMP-1 in the H2AC19-Mut2 group were lower than those in the wild-type group. Figure 10 F). Furthermore, we confirmed that H2AC19-Mut2 reversed the effects of H2AC19 on angiogenesis, specifically manifested as a decrease in the tubular formation capacity of HUVECs (F). Figure 10 G to H), VEGFA levels decreased ( Figure 11 A) Reduced cell migration ability ( Figure 11 (B to C) and decreased proliferative capacity ( Figure 11 (D), indicating that the amino acid fragment at positions 24-88 of H2AC19 plays a key role in the direct interaction with p300.

[0157] 7. H2AC19 promotes angiogenesis and tumor progression in LUAD organoid models.

[0158] To further validate the role of H2AC19 in clinical angiogenesis of LUAD, we constructed patient-derived organoids (PDOs) to mimic the characteristics of primordial LUAD. Figure 12 A). Morphological evaluation confirmed the accurate construction of PDOs. Figure 12 B). Immunofluorescence analysis showed significant expression of tumor markers such as Napsin A and TTF1. Figure 12 C), successfully verifying the generation of PDO. It is worth noting that H2AC19 knockout leads to a reduction in PDO size ( Figure 12 D) Reduced angiogenesis ( Figure 12 E), Increased apoptosis ( Figure 12 F) and decreased proliferation capacity ( Figure 12 G). Furthermore, H2AC19 overexpression exacerbates angiogenesis and proliferation in PDO, while A-485 treatment reverses this phenomenon (G). Figure 12 H and 13A). In summary, these results confirm that H2AC19 promotes LUAD progression through the p300 / EGR1 / MMP-1 pathway. Figure 13 B).

[0159] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of H2AC19 inhibitors in the preparation of drugs for the prevention and / or treatment of lung adenocarcinoma; The H2AC19 inhibitor is a siRNA targeting H2AC19, and the siRNA is CCGTACTGCTCCCTAAGAA.

2. The application as described in claim 1, characterized in that, The drug in question is a drug that inhibits the growth of lung adenocarcinoma cells.

3. The application as described in claim 1, characterized in that, The drug in question is a drug that inhibits the migration of lung adenocarcinoma cells.

4. The application as described in claim 1, characterized in that, The drug is a drug that inhibits angiogenesis in lung adenocarcinoma.

5. The application as described in claim 4, characterized in that, The inhibition of angiogenesis in lung adenocarcinoma is achieved by regulating the p300 / EGR1 / MMP-1 signaling pathway.

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