Application of KMT5A expression inhibitor in preparation of medicine for preventing or treating esophageal squamous carcinoma

Abstract

The invention provides application of an expression inhibitor of KMT5A in preparation of a medicine for preventing or treating esophageal squamous carcinoma. In-vitro experiments prove that ICA can inhibit proliferation of esophageal squamous carcinoma cells in a concentration-dependent manner and remarkably promote cell apoptosis. The action mechanism of the ICA is closely related to the direct targeting KMT5A protein, and experiments show that the ICA can reduce the expression level of the KMT5A and influence the protein stability of the KMT5A. Clinical sample analysis further shows that KMT5A is highly expressed in esophageal cancer tissues, and ICA treatment in animal experiments significantly reduces the tumor volume. The invention provides a new targeted treatment strategy for esophageal squamous carcinoma, and has important clinical transformation value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of KMT5A expression inhibitors in the preparation of drugs for the prevention or treatment of esophageal squamous cell carcinoma. Background Technology

[0002] The role of epigenetic regulation in the development and progression of esophageal cancer is receiving increasing attention. N-lysine methyltransferase (KMT5A) can specifically monomethylate lysine 20 of histone H4 (H4K20me1) and can also directly regulate gene expression through non-histone modifications, playing a crucial role in chromatin structure regulation, DNA replication and repair, and cell cycle control. Dysregulation of KMT5A expression is associated with various pathological conditions, including immune diseases and multiple cancers, but its role in the pathogenesis of esophageal cancer remains unclear.

[0003] Esophageal cancer is one of the most common malignant tumors of the digestive system in my country, ranking 10th in incidence and 6th in mortality among all malignant tumors. Due to factors such as population aging and diverse dietary habits, my country has become a high-incidence area for esophageal cancer worldwide. Early symptoms of esophageal cancer are often subtle, leading to most patients being diagnosed at an advanced stage, with a 5-year survival rate of only 15%-20%. Esophageal squamous cell carcinoma accounts for approximately 80% of esophageal cancer cases globally (and over 90% in China). Normal squamous epithelial dysplasia is an important pathological basis for esophageal squamous cell carcinoma; however, its pathogenesis remains incompletely understood. While treatment methods have improved in recent years, challenges remain, including difficulty in early detection, limited efficacy, and high recurrence rates. Therefore, exploring new therapeutic targets and intervention strategies to prevent and improve the progression of esophageal squamous cell carcinoma has become an urgent research need in this field.

[0004] Currently, existing treatments for esophageal squamous cell carcinoma mainly include surgery, radiotherapy and chemotherapy, immunotherapy, and specific target therapies such as HER2 and VEGF. However, these treatments have limited applicability, limited efficacy, and poor prognosis. Therefore, there is an urgent need to conduct in-depth research on the pathogenesis of esophageal squamous cell carcinoma, identify new therapeutic targets, and fully integrate innovative pharmaceutical development trends to develop novel drugs that can effectively prevent and improve the progression of esophageal squamous cell carcinoma while maintaining high safety. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of KMT5A expression inhibitors in the preparation of drugs for the prevention or treatment of esophageal squamous cell carcinoma. KMT5A expression inhibitors have significant application prospects in the preparation of drugs for the prevention or treatment of esophageal squamous cell carcinoma.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the invention, the use of KMT5A as a target in screening drugs for the prevention or treatment of esophageal squamous cell carcinoma is provided, wherein the screening method includes screening for substances that can inhibit the expression of the KMT5A gene.

[0007] In a second aspect of the invention, the use of an inhibitor of KMT5A expression is provided in the preparation of a medicament for the prevention or treatment of esophageal squamous cell carcinoma.

[0008] Furthermore, the KMT5A expression inhibitor includes at least one of the following components: KMT5A inhibitors; KMT5A knockdown reagent.

[0009] Furthermore, the KMT5A inhibitor is indole-3-carboxylic acid.

[0010] The molecular formula of indole-3-carboxylic acid is C9H7NO2, the CAS number is 771-50-6, and the structural formula is as follows: .

[0011] Furthermore, the KMT5A knockdown reagent comprises: shRNA and / or gRNA and / or siRNA targeting the target gene, wherein the siRNA nucleotide sequence is shown in SEQ ID NO:1.

[0012] In a third aspect of the invention, a pharmaceutical composition for the prevention or treatment of esophageal squamous cell carcinoma is provided, the pharmaceutical composition comprising at least one of a KMT5A inhibitor and a KMT5A knockdown agent.

[0013] The pharmaceutical composition comprises a pharmaceutically acceptable carrier. The choice of carrier depends on the type of KMT5A inhibitor and the route of administration. When the inhibitor is a nucleic acid molecule (such as siRNA), the carrier preferably comprises a delivery carrier, such as lipid nanoparticles (such as Invivofectamine® 3.0 reagent), polymer nanoparticles, cationic liposomes, or viral vectors (such as adeno-associated virus vectors), to facilitate its entry into cells.

[0014] The beneficial effects of the above-described technical solution of the present invention are as follows: This invention provides the application of a KMT5A expression inhibitor in the preparation of a drug for the prevention or treatment of esophageal squamous cell carcinoma. By inhibiting the expression or function of KMT5A, it significantly inhibits the abnormal proliferation of esophageal squamous cell carcinoma cells and promotes their apoptosis, thereby slowing down or improving the disease progression of esophageal squamous cell carcinoma. This provides a new strategy for targeted therapy of esophageal squamous cell carcinoma and holds promise for the development of novel drugs with high safety and efficacy. 1. Significant effects at the in vitro cellular level Inhibition of esophageal squamous cell carcinoma cell proliferation: CCK-8 assay confirmed that ICA significantly inhibited the proliferation activity of esophageal squamous cell carcinoma cell lines KYES30 and KYES150 in a concentration-dependent manner.

[0015] Promoting apoptosis in esophageal squamous cell carcinoma cells: Western blot results showed that ICA treatment significantly upregulated the expression of apoptosis-related protein c-Caspase 3 and downregulated the expression of proliferation-related protein PCNA, demonstrating that ICA can effectively induce apoptosis in cancer cells and inhibit their abnormal proliferation.

[0016] 2. Effects at the molecular mechanism level Directly targeting KMT5A protein: ICA can significantly reduce the protein expression level of KMT5A in KYES30 cells in a concentration-dependent manner.

[0017] Stable molecular binding properties: Molecular docking and kinetic simulations show that the binding energy between ICA and KMT5A is -9.6 kcal / mol. Thermal stability experiments confirm that the binding of ICA can reduce the thermal stability of KMT5A, indicating that there is a stable interaction between the two.

[0018] 3. Validation of clinical relevance: By examining paired cancer-adjacent tissues from patients with primary esophageal cancer, it was found that KMT5A expression was significantly higher in cancerous tissues than in adjacent tissues. This provides clinical evidence for KMT5A as a therapeutic target for esophageal squamous cell carcinoma.

[0019] 4. Validation results in in vivo animal experiments: In a nude mouse subcutaneous tumor model, intragastric administration of 20 mg / kg ICA significantly reduced tumor volume and mass (p<0.05). This confirms the anti-tumor effect of ICA in vivo. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application, the following detailed description of non-limiting embodiments is provided using accompanying drawings, making their features, objectives, and advantages more apparent: Figure 1 CCK8 assays were performed on esophageal cancer cell lines KYES30 and KYES150 under ICA stimulation at 0, 5 μM, 10 μM, and 20 μM to assess cell viability; among them, .

[0021] Figure 2 This study presents the Western blot results and quantitative analysis of c-Caspase 3 (an apoptosis-related protein) and PCNA (a proliferation-related protein) in esophageal cancer cell lines KYES30 and KYES150 after stimulation with 0, 5, 10, and 20 μM ICA. .

[0022] Figure 3 This document presents the Western blot results and quantitative analysis results of KMT5A in the esophageal cancer cell line KYES30 under ICA stimulation at 0, 5 μM, 10 μM, and 20 μM. .

[0023] Figure 4 ICA and KMT5A molecular docking models were developed; the thermal stability of KMT5A under ICA stimulation was assessed; among these, .

[0024] Figure 5 RT-PCR and Western blot results and quantitative analysis results of KMT5A in esophageal cancer tissue and adjacent normal tissue; among which... .

[0025] Figure 6 Tumor tissues were collected from subcutaneously tumor-bearing BALB / c-Nude mice and from BALB / c-Nude mice treated with 20 mg / kg ICA via gavage; among which, . Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings. The invention and its embodiments are described below; however, this description is not restrictive, and actual embodiments are not limited thereto. If those skilled in the art, inspired by this description, design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the scope of protection of the present invention.

[0027] Definition and use of terms Esophageal squamous cell carcinoma refers to a malignant tumor originating from the esophageal mucosal epithelial tissue, diagnosed by endoscopic biopsy or postoperative pathological examination as squamous cell carcinoma, adenocarcinoma or other rare types, excluding metastatic lesions; its diagnosis requires the tumor to have an invasion depth ≥ the submucosa (T1b stage and above), or be accompanied by lymph node metastasis / distant metastasis.

[0028] The core pathological feature of esophageal squamous cell carcinoma is the malignant transformation of the esophageal mucosal epithelium from orderly layering to uncontrolled infiltration. The key lies in the disruption of the cell proliferation / apoptosis balance caused by multi-stage genetic and epigenetic attacks. In the early stages, carcinogenic factors (smoking, alcohol, heat damage, acid reflux, etc.) trigger excessive basal cell proliferation → atypical proliferation, accompanied by mutations in key genes such as p53, p16, and NOTCH1, and abnormal DNA methylation, promoting clonal intraepithelial neoplasia. Subsequently, the basement membrane is penetrated, and atypical epithelial cells (squamous or columnar phenotype) infiltrate the lamina propria, muscularis mucosae, and deeper layers in a finger-like, nested, or single-cell manner, activating CAFs, macrophages, and endothelial cells to form a pro-tumor microenvironment. In the middle and late stages, tumor-associated fibroblasts secrete large amounts of type I / III collagen and fibronectin, leading to stromal fibrosis and a proliferative response of connective tissue. These changes collectively cause progressive thickening of the esophageal wall, narrowing of the lumen, and loss of peristalsis, ultimately affecting swallowing function and leading to distant organ failure through lymphatic-hematogenous metastasis.

[0029] KMT5A inhibitor: In this invention, KMT5A inhibitor refers to ICA, which is an aromatic organic acid containing an indole ring. It has a simple structure but a variety of biological activities. It is produced by tryptophan through intestinal flora metabolism and can circulate throughout the body via the blood.

[0030] Individual: In this invention, the term "individual" refers to a mammal, including but not limited to rats, mice, non-human primates, humans, dogs, cats, horses, cattle, sheep, pigs, and goats. Humans or mice are preferred.

[0031] Prevention and treatment: The term "prevention" in this invention refers to preventing or reducing the occurrence of esophageal squamous cell carcinoma after use in the presence of potential factors that promote its development. The term "treatment" in this invention refers to reducing the degree of proliferation of esophageal squamous cell carcinoma cells or promoting the level of apoptosis in these cells.

[0032] This invention demonstrates through the following examples that inhibiting KMT5A expression in esophageal squamous cell carcinoma cells KYES30 and KYES150 can significantly suppress hypoxia-induced proliferation of KYES30 and KYES150 cells and promote apoptosis. Abnormal proliferation of esophageal squamous epithelial cells is closely related to the occurrence of esophageal squamous cell carcinoma.

[0033] The KYES30 and KYES150 human esophageal squamous cell carcinoma cell lines used in the experiments of this invention were both purchased from ATCC, with catalog numbers CBP60457 and CBP60733, respectively.

[0034] To better explain the present invention, the following detailed description is provided in conjunction with specific embodiments.

[0035] Example 1: Effects of different concentrations of ICA stimulation on the proliferation and apoptosis of esophageal cancer cells; 1. Experimental cells Source: Immortalized esophageal cancer cells KYES30 and KYES150 purchased from the company. 2. Experimental Methods 1) Grouping (n=3 / group) Control group: 0.5% DMSO; ICA group: 5 μM, 10 μM, 20 μM ICA (indole-3-carboxylic acid).

[0036] 2) Drug preparation: Prepare and use fresh complete culture medium immediately.

[0037] 3) Drug administration: KYES30 and KYES150 cells were placed in a cell culture incubator for 48 hours, while the control group was given an equal volume of solvent.

[0038] 3. Experimental Results CCK8: such as Figure 1 The results showed that, compared with the control group, ICA significantly reduced the proliferation activity of KYES30 and KYES150 cells.

[0039] Western blot: such as Figure 2 The results showed that, compared with the control group, ICA significantly increased the expression of c-Caspase3, an apoptosis-related protein in KYES30 and KYES150 cells, and decreased the expression of PCNA, a proliferation-related protein.

[0040] Example 2: Effect of ICA on KMT5A protein levels 1. Experimental cells Source: Immortalized esophageal cancer cells KYES30 and KYES150 purchased from the company. 2. Experimental Methods 1) Grouping (n=3 / group) Control group: 0.5% DMSO; ICA groups: 5uM, 10uM, 20uM ICA.

[0041] 2) Drug preparation: Prepare and use fresh complete culture medium immediately.

[0042] 3) Drug administration: KYES30 and KYES150 cells were placed in a cell culture incubator for 48 hours, while the control group was given an equal volume of solvent.

[0043] 3. Experimental Results Western blot: such as Figure 3 The results showed that ICA significantly reduced the expression of KMT5A in KYES30 cells.

[0044] MMPBSA: such as Figure 4The results show that the binding energy of ICA to KMT5A, as simulated by molecular docking and dynamics, is -9.6 kcal / mol; thermal stability experiments show that the binding of ICA reduces the thermal stability of KMT5A.

[0045] Example 3: High expression of KMT5A in esophageal cancer tissue 1. Experimental Materials Source: Paired cancer-adjacent tissue from primary esophageal cancer patients (T1-3, N0-3, M0); 2. Experimental Methods RNA and protein were extracted from cancer-adjacent tissues, and the expression of KMT5A was detected by RT-PCR and Western blot. The RT-PCR primers used were as follows: 5'-ACCGACGGGGAGAACGTATT-3' (SEQ ID NO.1) and 5'-GCATTCCAGAGCATTTGTTCG-3' (SEQ ID NO.2).

[0046] 3. Experimental Results Western blot: such as Figure 5 The results showed that KMT5A was significantly overexpressed in cancerous tissues when compared with adjacent normal tissues.

[0047] Example 4: ICA inhibits tumor growth 1. Laboratory animals: Source: Nude mice (BALB / c nude mouse) bred in the animal room of Tongji Medical College; Childbearing age: 4-5 weeks; 2. Experimental Methods 1) Grouping (n=5 / group) Control group: KYES30 cells were subcutaneously seeded and administered an equal dose of solvent via gavage.

[0048] ICA group: KYES30 cells were subcutaneously seeded, and 20 mg / kg ICA was administered by gavage.

[0049] 2) Drug preparation ICA (MCE) was added to PBS to prepare a suspension with a stock solution concentration of 2 mg / ml.

[0050] 3) Model construction and drug administration: Tumor-bearing mice were continuously administered the tumor by gavage for one week until the tumor size reached 1.5 cm. The control group was given an equal volume of solvent.

[0051] 3. Experimental Results Tumor assessment: such as Figure 6The results showed that, compared with the control group, the tumors in the ICA treatment group were significantly smaller; the tumor mass was significantly lower than that in the control group; and the tumor volume was significantly smaller than that in the control group ten days after tumor bearing.

[0052] In summary, the data above indicate that inhibiting the KMT5A inhibitor ICA can suppress esophageal cancer cell proliferation and promote apoptosis. Since cancer cell proliferation and apoptosis play crucial roles in the development and progression of esophageal cancer, inhibiting esophageal cancer cell proliferation and promoting apoptosis could slow the progression of esophageal cancer and provide a new therapeutic target. Indole-3-carboxylic acid shows great promise in the preparation of drugs for the prevention or treatment of esophageal squamous cell carcinoma.

[0053] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of KMT5A as a target in screening drugs for the prevention or treatment of esophageal squamous cell carcinoma, characterized in that, The screening method includes screening for substances that can inhibit the expression of the KMT5A gene or the activity of the protein.

2. Application of MT5A expression inhibitors in the preparation of drugs for the prevention or treatment of esophageal squamous cell carcinoma.

3. The application according to claim 2, characterized in that, The KMT5A expression inhibitor includes at least one of the following components: KMT5A inhibitors; Knockdown reagent for KMT5A.

4. The application according to claim 3, characterized in that, The KMT5A inhibitor is indole-3-carboxylic acid.

5. The application according to claim 3, characterized in that, The KMT5A knockdown reagent includes: shRNA and / or gRNA and / or siRNA targeting the target gene.

6. A drug for the prevention or treatment of esophageal squamous cell carcinoma, characterized in that, The drug includes at least one of a KMT5A inhibitor and a KMT5A knockdown agent.

7. A medicament for the prevention or treatment of esophageal squamous cell carcinoma according to claim 6, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

8. A medicament for the prevention or treatment of esophageal squamous cell carcinoma according to claim 7, characterized in that, The pharmaceutically acceptable carriers include one or more of the following: diluents, isotonic agents, pH adjusters, preservatives, thickeners, or sustained-release materials.

9. A medicament for the prevention or treatment of esophageal squamous cell carcinoma according to claim 7, characterized in that, The pharmaceutically acceptable carrier comprises a delivery carrier for delivering the nucleic acid inhibitor, wherein the delivery carrier is one of lipid nanoparticles, polymer nanoparticles, cationic liposomes, or viral vectors.

10. A medicament for the prevention or treatment of esophageal squamous cell carcinoma according to claim 5, characterized in that, The dosage form of the drug includes one of the following: injection, gel, and sustained-release formulation.

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