Use of agents inhibiting m6a methyltransferase writer complex in the preparation of a medicament for treating diseases associated with intraocular fibrosis

Abstract

This invention discloses the application of a reagent that inhibits the m6A methyltransferase Writer complex in the preparation of drugs for treating ocular fibrosis-related diseases, belonging to the field of biomedical technology. The m6A methyltransferase Writer complex includes METTL3, METTL14, and WTAP. This invention reveals that the "pathological over-assembly and stabilization" of the m6A Writer complex is a key upstream switch for the initiation of ocular fibrosis, breaking through the limitations of traditional research focusing on the "overall level of m6A modification," providing a new molecular target dimension for fibrosis intervention, rather than relying on downstream signal blocking. It fundamentally maintains cellular autophagy homeostasis and inhibits the initiation of fibrosis, rather than merely alleviating existing pathological phenotypes, resulting in more precise intervention and longer-lasting efficacy. Overcoming the current scarcity of targets, it enables the development of various types of drugs, including small molecule compounds, biologics, and nucleic acid compositions.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a reagent that inhibits the m6A methyltransferase Writer complex in the preparation of drugs for treating ocular fibrosis-related diseases. Background Technology

[0002] Existing research indicates that intraocular fibrosis is a crucial pathological basis for various blinding eye diseases, such as glaucoma and postoperative scarring. Its occurrence is closely related to trabecular meshwork cell dysfunction, epithelial-mesenchymal transition (EMT), and abnormal extracellular matrix deposition. In recent years, intervention strategies targeting the fibrotic process have gradually shifted from traditional anti-inflammatory and intraocular pressure-lowering approaches to targeted regulation of cell fate determination mechanisms. Among these, autophagy, as a key process for maintaining cellular homeostasis, has been shown to be involved in the initiation of fibrosis due to dysfunction.

[0003] However, existing technologies mostly focus on the inhibition of downstream effector molecules or specific pathways, and have not yet systematically addressed the issue from the perspective of RNA epigenetic regulation to maintain the autophagy capacity of ocular tissue cells and block the initiation of fibrosis at its source. In other words, although existing technologies have made some progress in the field of combating intraocular fibrosis, at the mechanistic level they mostly remain at the regulation of downstream signaling pathways or terminal effector molecules, lacking in-depth analysis of the causal relationship between the maintenance of autophagy capacity and the initiation of fibrosis, and have not introduced the novel regulatory dimension of the m6A methylated writer complex assembly state.

[0004] For example, existing patent CN120346247A discloses the application of ethyl acetate fraction of Sophora flavescens and its active ingredients in the anti-fibrotic effect of trabecular meshwork in glaucoma. This technology, through intervention of the fibrotic process of trabecular meshwork cells by natural product extracts, exhibits a certain anti-fibrotic effect. However, this type of technology does not clarify whether its action involves the regulation of autophagy, nor does it demonstrate its influence on upstream initiation mechanisms of fibrosis (such as EMT or RNA modification imbalance). Its mechanism of action is relatively general, lacking precise intervention design for the autophagy-fibrosis axis, making it difficult to achieve a systematic blockade of the source of intraocular fibrosis.

[0005] Another existing patent, CN121401275A, discloses the application of NSA compounds in the preparation of drugs for treating trabecular meshwork fibrosis in glaucoma. It demonstrates that NSA can improve trabecular meshwork structure and aqueous humor outflow function by inhibiting necroptosis pathways and downregulating key fibrosis factors. However, its core mechanism remains focused on the regulation of cell death pathways, without involving the maintenance or restoration of autophagy function, nor is it related to epigenetic transcriptional regulatory networks such as RNA m6A methylation. Therefore, this method represents an indirect intervention in the progression of fibrosis and fails to block the initial triggering step of fibrosis from the perspective of maintaining cellular homeostasis.

[0006] Therefore, the application strategy of universal targeted therapy based on the m6A methylation regulation pathway has important scientific value and clinical significance for breaking through the treatment bottlenecks of various ophthalmic diseases and improving the clinical prevention and treatment effects. Summary of the Invention

[0007] To address the technical problems existing in the prior art, this invention provides the application of a reagent that inhibits the m6A methyltransferase Writer complex in the preparation of drugs for treating intraocular fibrosis-related diseases. The technical solution is as follows:

[0008] The use of reagents that inhibit the m6A methyltransferase Writer complex in the preparation of medicaments for treating ocular fibrosis-related diseases, wherein the m6A methyltransferase Writer complex includes METTL3, METTL14 and WTAP.

[0009] Optionally, the reagent targets the assembly state of the m6A methyltransferase Writer complex, and restores the normal m6A methylation modification level in ocular tissue by inhibiting the pathological over-assembly or abnormal stabilization of the complex, thereby maintaining the autophagy homeostasis of ocular tissue cells and blocking the initiation of ocular fibrosis.

[0010] Optionally, the reagent that inhibits the m6A methyltransferase Writer complex includes at least one of the following reagents:

[0011] (1) Small molecule compounds that inhibit the pathological assembly of the m6A methyltransferase Writer complex;

[0012] (2) Biological agents that interfere with the stability of WTAP-mediated complexes;

[0013] (3) Nucleic acids that regulate the miR-497-5p / FGF7 axis.

[0014] Optionally, the small molecule compound is selected from at least one of ciclopirox olamine, STM2457, UZH1a, remibrutinib, and RG7834;

[0015] And / or, the biological agent is selected from at least one of anti-WTAP monoclonal antibodies, WTAP-binding peptides, or WTAP functional domain protein fragments;

[0016] And / or, the nucleic acid is selected from at least one of miR-497-5p mimics, miR-497-5p inhibitors, or siRNAs targeting FGF7.

[0017] Optionally, the intraocular fibrosis-related disease is selected from at least one of cataracts, thyroid-associated ophthalmopathy, and diabetic retinopathy.

[0018] A pharmaceutical composition for treating intraocular fibrosis-related diseases, the pharmaceutical composition comprising: an inhibitor of the m6A methyltransferase Writer complex and a pharmaceutically acceptable carrier.

[0019] Optionally, the reagent that inhibits the m6A methyltransferase Writer complex includes at least one of the following reagents:

[0020] (1) Small molecule compounds that inhibit the pathological assembly of the m6A methyltransferase Writer complex;

[0021] (2) Biological agents that interfere with the stability of WTAP-mediated complexes;

[0022] (3) Nucleic acids or combinations thereof that regulate the miR-497-5p / FGF7 axis.

[0023] Optionally, the small molecule compound is selected from at least one of ciclopirox olamine, STM2457, UZH1a, remibrutinib, and RG7834;

[0024] And / or, the biological agent is selected from at least one of anti-WTAP monoclonal antibodies, WTAP-binding peptides, or WTAP functional domain protein fragments;

[0025] And / or, the nucleic acid is selected from at least one of miR-497-5p mimics, miR-497-5p inhibitors, or siRNAs targeting FGF7.

[0026] Optionally, the intraocular fibrosis-related disease is selected from at least one of cataracts, thyroid-associated ophthalmopathy, and diabetic retinopathy.

[0027] The beneficial effects of the technical solutions provided in the embodiments of the present invention include at least the following:

[0028] 1. This invention reveals that the "pathological over-assembly and stabilization" of the m6A Writer complex (METTL3 / METTL14 / WTAP) is a key upstream switch for the initiation of intraocular fibrosis, clarifying its core pathogenic pathway by inhibiting autophagy and triggering EMT, thus addressing the mechanistic blind spot in existing research where "single signaling pathway / single gene intervention cannot explain similar fibrotic phenotypes." Breaking through the limitations of traditional research focusing on the "overall level of m6A modification," this invention discovers that the "physical assembly state" of the Writer complex can serve as an independent functional regulatory layer, providing a novel molecular target dimension for fibrosis intervention, rather than relying on downstream signal blocking.

[0029] 2. This invention intervenes at the source of fibrosis initiation, targeting the core upstream node (abnormal complex assembly), fundamentally maintaining cellular autophagy homeostasis and inhibiting fibrosis initiation, rather than merely alleviating existing pathological phenotypes. This approach offers more precise intervention and longer-lasting efficacy. It clearly defines the miR-497-5p / FGF7 axis as an upstream regulatory valve for complex assembly, forming a complete regulatory chain of "upstream molecular axis - complex assembly - autophagy - fibrosis," providing a basis for multi-target synergistic intervention and avoiding the limitations of single-target intervention.

[0030] 3. This invention identifies "Writer complex assembly interface, WTAP-mediated complex stabilization process, and miR-497-5p / FGF7 axis-related molecules" as novel targets for ophthalmic fibrosis drugs, overcoming the current scarcity of targets and enabling the development of various types of drugs, including small molecule compounds, biologics, and nucleic acid compositions. The drugs can be used in conjunction with ocular targeted delivery carriers, balancing "targeting" and "biocompatibility," reducing systemic side effects, and meeting the clinical needs of local treatment for ophthalmic diseases.

[0031] 4. Based on the universality of the m6A methylation pathway, the intervention strategy of this invention is applicable to intraocular fibrosis-related diseases (such as cataracts, for example, posterior cataracts, thyroid-associated ophthalmopathy, diabetic retinopathy, etc.). Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 This is an experimental result diagram provided in Embodiment 1 of the present invention, wherein... Figure 1 In the left panel, A and B show the results of cell migration detected by Transwell assay and scratch assay, respectively, with a scale bar of 100 μm. The statistical chart on the right is a quantitative analysis of the number of migrating cells and the migration area shown in the left panel.

[0034] Figure 2 This is an experimental result diagram provided in Embodiment 2 of the present invention, wherein... Figure 2 In the middle section, A to F are the m6A Dot Blot detection results of changes in methylation level and their corresponding quantitative statistical graphs; Figure 2 The figure in G is a graph showing the protein binding and interaction between siWTAP and OE-FGF7 after transfection of HLE-B3 cell line with WTAP antibody through immunoprecipitation.

[0035] Figure 3 This is an experimental result diagram provided in Embodiment 3 of the present invention, wherein... Figure 3 Figure A shows the changes in the expression levels of m6A methylated protein, autophagy, and EMT-related proteins after transfection using protein immunoblotting assays. Figure 3 B is the correct answer. Figure 3 Gray-scale statistical analysis of A in the middle; Figure 3 Figure C is a graph showing the binding efficiency of TNFα and OFF miR-497-5p to three predicted interaction binding sites of FGF7 in the m6ARIP product after HLE-B3 transfection with miR-497-5p by MeRIP RT-PCR.

[0036] Figure 4 This is an experimental result diagram provided in Embodiment 4 of the present invention, wherein... Figure 4 Image A shows slit-lamp images of the anterior segment of the rabbit eye at 1, 2, 3, and 4 weeks post-phacoemulsification and implantation of a modified intraocular lens. Figure 4 B is Figure 4 Quantitative statistical turbidity rating analysis chart for A; Figure 4 In the middle C is the Miyake-apple diagram and the corresponding H&E staining diagram. The black arrows represent fibroblast clusters proliferating in the posterior capsule. Scale bar = 100 μm. Figure 4 The image in D is a H&E staining map of the cornea, optic nerve, retina, iris, and ciliary body after IOL implantation, with a scale bar of 100 μm. Detailed Implementation

[0037] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0038] In embodiments of the present invention, words such as "exemplarily," "for example," etc., are used to indicate that something is an example, illustration, or description. Any embodiment or design described as "exemplary" in the present invention should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of the word "exemplary" is intended to present the concept in a concrete manner. Furthermore, in embodiments of the present invention, the meaning expressed by "and / or" can be both, or either one.

[0039] In this invention, the m6A methyltransferase Writer complex is a core protein complex that catalyzes the N6-methyladenosine (m6A) modification of eukaryotic mRNA, and is mainly composed of subunits such as METTL3, METTL14 and WTAP.

[0040] In this invention, METTL3 is the catalytic subunit of the m6A methyltransferase complex, which is an RNA methyltransferase.

[0041] In this invention, METTL14 is the regulatory subunit of the m6A methyltransferase complex. It forms a functional heterodimer with METTL3, assisting METTL3 in recognizing and binding target RNA substrates and participating in the site selection for m6A methylation modification, but it does not possess independent catalytic activity. METTL14 plays a crucial role in maintaining the structural stability of the complex, nuclear localization, and substrate-specific recognition.

[0042] In this invention, WTAP (Wilms Tumor 1 Associating Protein) is a Wilms tumor 1 companion protein and a key scaffold protein of the Writer complex, responsible for maintaining the stability and localization of the complex. Abnormal expression of WTAP can lead to disordered m6A modification levels.

[0043] In this invention, miR-497-5p is a microRNA that binds to the 3' untranslated region of a target gene (mRNA) through the principle of complementary base pairing. After binding, it can cause the target mRNA to be degraded or unable to be translated into protein, thereby inhibiting the expression of the target gene.

[0044] In this invention, FGF7 is Fibroblast Growth Factor 7, a signaling protein that can promote cell growth, migration and differentiation.

[0045] In this invention, the miR-497-5p / FGF7 axis refers to the functional regulatory axis formed by miR-497-5p negatively regulating FGF7 expression by targeting and binding to the 3' untranslated region of FGF7 mRNA, thereby affecting downstream signaling pathways of FGF7 / FGFR2 (such as PI3K / AKT, MAPK / ERK).

[0046] This invention provides an application for maintaining the autophagy capacity of ocular tissue cells and blocking the initiation of intraocular fibrosis. The application targets the assembly state of the m6A methyltransferase Writer complex. By inhibiting the pathological over-assembly or abnormal stabilization of the Writer complex, the normal m6A methylation modification level in ocular tissue is restored, thereby maintaining the autophagy homeostasis of ocular tissue cells and blocking the initiation of intraocular fibrosis.

[0047] This invention discovers that the m6A methyltransferase Writer complex includes METTL3, METTL14, and WTAP, wherein METTL3 and METTL14 are catalytic subunits, and WTAP is an assembly regulatory subunit. The three work together to maintain the m6A methylation homeostasis in ocular tissues. Their assembly state directly regulates the m6A methylation modification level in ocular tissues, thereby maintaining autophagy homeostasis and effectively intervening in the process of ocular fibrosis.

[0048] The physical assembly state of the Writer complex in this invention is a functional regulatory layer independent of the overall m6A modification level. This assembly state directly determines the complex's recognition and methylation efficiency of specific target gene (such as FGF7, HMGA2, PTEN) mRNA, thereby affecting downstream autophagy pathways and EMT processes. By intervening at this assembly interface, specific transcripts related to fibrosis can be precisely regulated without globally inhibiting m6A modification.

[0049] In some embodiments, the modulator is a small molecule compound that can be delivered to ocular tissue, the small molecule compound being selected from at least one of ciclopirox olamine, STM2457, UZH1a, remibrutinib, or RG7834, the small molecule compound being formulated into an ophthalmic formulation in combination with a pharmaceutically acceptable carrier.

[0050] In some embodiments, the ciclopirox olamine is dissolved in an ophthalmic gel matrix at a mass concentration of 0.01-0.1%, the ophthalmic gel matrix comprising carbomer 940, glycerin, triethanolamine, and water for injection, with the pH adjusted to 6.5-7.5. Preferably, the ophthalmic gel matrix comprises 0.2-0.5% carbomer 940, 5-10% glycerin, 0.1-0.3% triethanolamine, and the balance being water for injection, with the pH adjusted to 6.5-7.5 and a viscosity of 2000-5000 mPa·s (25°C), for use as eye drops 2-4 times daily.

[0051] In some embodiments, the STM2457 is loaded into a fluoropolymer carrier G4F7 at a concentration of 0.5-5 μmol / L to form a nanoparticle dispersion with a particle size of 80-200 nm, for intraocular injection or coating on the surface of an intraocular lens. Preferably, the G4F7 is copolymerized from tetrafluoroethylene and perfluoropropyl vinyl ether, with a drug loading of 8-15% and an encapsulation efficiency of 75-88%. The dispersion medium is a phosphate buffer solution (pH 7.2) containing 0.5% poloxamer 188, for intraocular injection or coating on the surface of an intraocular lens to form a drug-release coating.

[0052] In some embodiments, the UZH1a is encapsulated in cationic liposomes at a concentration of 1-10 μmol / L. The cationic liposomes are composed of DOTAP, cholesterol, and DSPE-PEG2000 in a molar ratio of 40:50:10, with an average particle size of 100-150 nm, a zeta potential of +30-45 mV, and an encapsulation efficiency of 80-92%. They are dispersed in water for injection containing 5% glucose and are used for subretinal intracavitary injection.

[0053] In some embodiments, the modulator is a biological agent that interferes with the stability of the WTAP-mediated complex. The biological agent is selected from at least one of anti-WTAP monoclonal antibodies, WTAP-binding peptides, or WTAP functional domain protein fragments. The biological agent is co-formulated with a pharmaceutically acceptable carrier to form an intraocular sustained-release formulation.

[0054] In some embodiments, the anti-WTAP monoclonal antibody is dissolved in 10 mmol / L phosphate buffer (pH 7.4) at a concentration of 0.1-1 mg / mL, sodium alginate is added to a final concentration of 1-3%, and cross-linked to form microspheres with a particle size of 20-50 μm and a drug loading of 15-25% by being added dropwise to 50 mmol / L calcium chloride solution, for subconjunctival implantation.

[0055] In some embodiments, the regulator is a nucleic acid or a combination thereof that regulates the miR-497-5p / FGF7 axis, wherein the nucleic acid is selected from at least one of miR-497-5p mimics, miR-497-5p inhibitors, or siRNAs targeting FGF7, and the nucleic acid is chemically modified and then compounded with a delivery vector to form a nucleic acid composition.

[0056] In some embodiments, there is provided an application in a medicament for the prevention or treatment of m6A methylation-related eye diseases, the medicament comprising a modifier and a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier is an excipient for intraocular injection comprising sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, disodium edetate, and water for injection, with a pH of 7.0-7.4.

[0057] The present invention provides a system for maintaining the autophagy capacity of ocular tissue cells and blocking the initiation of intraocular fibrosis, the system being used in any embodiment of the method, comprising a drug delivery unit, an ocular positioning unit, and a release control unit.

[0058] In some embodiments, the drug delivery unit is a modified intraocular lens whose surface is treated with plasma to introduce carboxyl groups, and then activated by EDC / NHS before covalently grafting PLGA nanoparticles. The PLGA nanoparticles contain STM2457, have a drug loading of 5-20%, an encapsulation rate of 70-90%, and the drug release conforms to the Higuchi model.

[0059] In some embodiments, the eye positioning unit is a magnetically guided microneedle array made of polylactic acid, with a needle length of 300-500 μm and a needle tip loaded with magnetic nanoparticles (Fe3O4@SiO2) containing UZH1a. The core diameter of the magnetic nanoparticles is 10-20 nm, the thickness of the silica shell is 5-10 nm, and the drug loading is 8-15%. Under the guidance of an external magnetic field (strength 0.2-0.5T), precise drug delivery to the ciliary body region is achieved, and the microneedle penetration depth can be controlled within 200-400 μm.

[0060] In some embodiments, the release control unit is a thermosensitive hydrogel, which is copolymerized from N-isopropylacrylamide and acrylic acid in a mass ratio of 90:10, has a low critical dissolution temperature of 32-35°C, and undergoes a phase transition at body temperature to form a gel depot, continuously releasing miR-497-5p mimics for 7-14 days.

[0061] This invention provides an application for maintaining the autophagy capacity of ocular tissue cells and blocking the initiation of intraocular fibrosis in eye diseases, wherein the eye diseases are m6A methylation-related eye diseases.

[0062] In some embodiments, the m6A methylation-related eye disease is age-related macular degeneration, specifically wet AMD. The method inhibits SNAIL transcriptional activation and RPE cell EMT by suppressing excessive assembly of the Writer complex, downregulating the m6A modification level of HMGA2 mRNA, reducing its binding efficiency with YTHDF1, and decreasing HMGA2 protein translation.

[0063] In some embodiments, the m6A methylation modification-related eye disease is cataract, including age-related cataract and diabetic cataract. The method reduces m6A modification of key genes ACSL4 and TFRC in the ferroptosis pathway by regulating the assembly state of the Writer complex, prolonging their mRNA half-life, maintaining iron homeostasis of lens epithelial cells, and inhibiting apoptosis.

[0064] In some embodiments, the m6A methylation-related eye disease is diabetic retinopathy. The method inhibits EndoMT by remodeling the Writer complex assembly homeostasis, upregulating the m6A modification of lncRNA SNHG7, promoting its recognition and degradation by YTHDF2, and releasing the adsorption of miR-199a-5p.

[0065] In some embodiments, the m6A methylation-related eye disease is glaucoma, including pseudoexfoliative glaucoma and postoperative filtration scarring. The method reduces the m6A modification level of Smad3 mRNA by inhibiting excessive assembly of the Writer complex, thereby reducing its protein expression and inhibiting TGF-β1-induced ECM deposition in trabecular meshwork cells.

[0066] In some embodiments, the m6A methylation-related eye disease is uveitis. The method upregulates m6A modification of SIRT1 mRNA by regulating the assembly state of the Writer complex, thereby enhancing its translation efficiency, inhibiting STAT3 phosphorylation, and reducing microglia M1 polarization and IL-6 and TNF-α secretion.

[0067] In some embodiments, the m6A methylation modification-related eye disease is uveal melanoma or retinoblastoma. The method upregulates the m6A modification of the tumor suppressor gene HINT2 by regulating the assembly state of the Writer complex, promoting its mRNA stability, inhibiting the activity of the PI3K / AKT / mTOR pathway, and slowing down the proliferation and migration of tumor cells.

[0068] In some embodiments, the m6A methylation-related eye disease is thyroid-associated ophthalmopathy, and the method reduces m6A modification of IL-17RA mRNA in fibroblasts by interfering with WTAP-mediated Writer complex stabilization, thereby reducing its protein expression and weakening Th17 cell-mediated inflammatory responses.

[0069] In some embodiments, the m6A methylation-related eye disease is myopia, particularly high myopia combined with posterior staphyloma. The method regulates the assembly state of the Writer complex, upregulates the expression of demethylases ALKBH5 and FTO, reduces the m6A modification level of collagen synthesis genes COL1A1 and COL3A1, and inhibits scleral fibroblast activation.

[0070] To better verify the effectiveness of this application, this embodiment uses the small molecule inhibitor STM2457 of m6A methyltransferase METTL3 and miR-497-5p / FGF7 axis regulation as targets to inhibit the pathological process of epithelial-mesenchymal transition in posterior cataracts, and verifies the therapeutic effect of STM2457 on various ophthalmic diseases.

[0071] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0072] Unless otherwise specified, the experimental methods described in the following embodiments are conventional experimental methods well known to those skilled in the art, and are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Where specific conditions are not specified in the experimental methods, they are generally operated under conventional conditions.

[0073] Unless otherwise specified, all materials and reagents described in the following examples are commercially available.

[0074] Example 1:

[0075] HLE-B3 cells are an immortalized human lens epithelial cell line. Because they retain some characteristics of lens epithelial cells, HLE-B3 cells are a classic in vitro model for studying intraocular fibrosis diseases such as secondary cataracts. EMT (Epithelial-Mesenchymal Transition) refers to the epithelial-mesenchymal transition, a biological process in which epithelial cells lose polarity and intercellular connections, transforming into a mesenchymal cell phenotype with migratory capabilities. In ophthalmic diseases, this process is a key initiating step in the development of intraocular fibrosis (such as secondary cataracts).

[0076] 1. Cell Culture

[0077] The human lens epithelial cell line HLE-B3 (purchased from the American Type Culture Collection, ATCC) was placed in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and cultured routinely in a constant temperature incubator at 37°C and 5% CO2.

[0078] 2. Experimental grouping and cell transfection

[0079] To comprehensively test the interaction among miR-497-5p, FGF7, and WTAP, this embodiment employs a 2×2×2 factorial design. HLE-B3 cells were divided into the following eight treatment groups based on whether a miR-497-5p inhibitor was added, whether FGF7 overexpression plasmid (OE-FGF7) was transfected, and whether si-WTAP was transfected:

[0080] Among them, the miR-497-5p inhibitor (OFF miR-497-5p) is a miR-497-5p-specific antagomir. Its sequence design is completely complementary to the mature miR-497-5p (5'-CAGCAGCACACUGUGGUUUGA-3' (SEQ ID No. 1)), with the sequence 5'-UCAAACCACAGUGUGCUGCUG-3' (SEQ ID No. 2). It is produced by full-chain 2'-O-methylation, modification of the phosphate thioate backbone, and 3'-terminal cholesterol conjugation. It was purchased from RiboBio (Guangzhou), miR-497-5p inhibitor (catalog number: miR20000764).

[0081] The siWTAP knockdown of WTAP was performed using an shRNA plasmid targeting the human siWTAP gene. The vector element sequence was U6-MCS-Ubc-Cherry-IRES-puromycin. This knockdown plasmid was constructed by GKG.

[0082] OE-FGF7 is an FGF7 overexpression plasmid with the human FGF7 gene (transcript NM_002009.4) as the target gene. The vector element sequence is Ubc-MCS-3FLAG-CBh-gcGFP-IRES-puromycin. This overexpression plasmid was constructed by Jikai Gene.

[0083] Table 1 Experimental Groups

[0084]

[0085] Note: "+" indicates that the treatment factor is added, and "-" indicates that it is not added, but an equal amount of corresponding negative control is added to maintain a consistent total transfection rate.

[0086] All transfection procedures were performed according to the instructions for use of Lipofectamine 3000 (Invitrogen). The final concentrations of each nucleic acid molecule used were: miR-497-5p inhibitor 50 nM, FGF7 overexpression plasmid 2 μg / mL, and si-WTAP plasmid 2 μg / mL. Fresh complete culture medium was used 6 hours after transfection, and cells were collected after further culturing for subsequent assays.

[0087] 3. Transferability Detection

[0088] A. Transwell assay to detect transferability

[0089] Forty-eight hours after transfection, cells were collected and resuspended in serum-free medium. 200 μL of the cell suspension was added to the upper chamber of a Transwell chamber, and 600 μL of medium containing 20% ​​FBS was added to the lower chamber. After 24 hours of culture, unmigrated cells from the upper chamber were wiped off, the cells were fixed and stained with crystal violet, and the number of cells that had migrated through the membrane was counted by randomly selecting corresponding fields of view under a microscope.

[0090] B. Cell scratch assay to detect migration ability

[0091] Forty-eight hours after transfection, streaks were performed vertically along the bottom of a 6-well plate using a sterile pipette tip. Cells were washed with PBS to remove detached cells, and serum-free medium was added for further culture. Microscopic images were taken at 0 and 24 hours to measure the streak width and calculate the healing rate.

[0092] 4. Experimental Results

[0093] See the experimental results. Figure 1 .from Figure 1 As can be seen from the analysis, the migration ability of cells in each group (scratch healing rate and number of Transwell cells) was compared:

[0094] Inhibition of miR-497-5p or overexpression of FGF7 can significantly promote the migration of HLE-B3 cells, while knockdown of WTAP inhibits cell migration, suggesting that all three are involved in regulating the cell migration process.

[0095] Knocking down WTAP on the basis of inhibiting miR-497-5p or overexpressing FGF7 can completely reverse the cell migration promotion induced by upstream signal activation, indicating that WTAP is a key effector molecule downstream of the miR-497-5p / FGF7 axis.

[0096] The combined treatment of the three factors further confirmed that even under the dual activation of upstream signals by miR-497-5p inhibition and FGF7 overexpression, WTAP knockdown could still effectively block cell migration, indicating that WTAP is a core node in this signaling pathway.

[0097] The above experimental results indicate that WTAP is located downstream of the miR-497-5p / FGF7 axis and is a core effector molecule in the regulation of cell migration in this signaling pathway. Targeting and inhibiting WTAP can block cell migration induced by the activation of this axis, suggesting that WTAP can serve as a key target for regulating the biological effects of this pathway.

[0098] Example 2:

[0099] TNFα (tumor necrosis factor-α) is an important inflammatory cytokine that plays a central role in inflammatory responses and immune regulation. In this embodiment, TNFα treatment of cells was used to simulate the local inflammatory microenvironment of ocular tissue, inducing a fibrosis-related phenotype. Autophagy-related genes are a series of genes involved in regulating the autophagy process, such as Beclin-1 (involved in autophagy initiation and autophagosome nucleation), ATG5 (involved in autophagosome elongation and expansion), and p62 / SQSTM1 (as an autophagy substrate protein, whose expression level is negatively correlated with autophagy flux activity and also involved in signal regulation).

[0100] 1. Cell Culture

[0101] The human lens epithelial cell line HLE-B3 (purchased from the American Type Culture Collection, ATCC) was placed in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and cultured routinely in a constant temperature incubator at 37°C and 5% CO2.

[0102] 2. Experimental grouping and cell transfection

[0103] To investigate the regulatory role of the miR-497-5p / FGF7 axis on m6A modification under inflammatory stimulation, this study first treated cells with TNFα (purchased from MCE). HLE-B3 cells in logarithmic growth phase were seeded in 6-well plates and cultured until approximately 70% confluence. The culture medium was then replaced with fresh medium containing TNFα (10 ng / mL). After 48 hours of treatment, cells were collected, and total RNA was extracted for subsequent analysis.

[0104] To further verify the roles of miR-497-5p, FGF7, and WTAP in TNFα-induced m6A modification activation, the following transfection treatment groups were set up in this embodiment: siFGF7, OFF miR-NC, OFF miR-497-5p, si-NC, si-WTAP, and OE-FGF7. Specific information for siFGF7, OFF miR-NC, OFF miR-497-5p, si-NC, si-WTAP, and OE-FGF7 is as follows:

[0105] siFGF7 is a human FGF7 gene siRNA, which was chemically synthesized by RiboBio (Guangzhou, catalog number: siG00000205).

[0106] si-NC is a negative control siRNA, provided by the above-mentioned supplier along with the target siRNA;

[0107] OFF miR-NC is a negative control for miRNA inhibitors and was purchased from RiboBio antagomir NC (catalog number: miR20000000).

[0108] The miR-497-5p inhibitor (OFF miR-497-5p) is a miR-497-5p-specific antagomir. Its sequence design is completely complementary to the mature miR-497-5p (5'-CAGCAGCACACUGUGGUUUGA-3' (SEQ ID No. 3)), with the sequence 5'-UCAAACCACAGUGUGCUGCUG-3' (SEQ ID No. 4). It has undergone full-chain 2'-O-methylation, phosphate thiocyanate backbone modification, and 3'-terminal cholesterol conjugation. It was purchased from RiboBio (Guangzhou), miR-497-5p inhibitor (catalog number: miR20000764).

[0109] The siWTAP knockdown of WTAP was performed using an shRNA plasmid targeting the human siWTAP gene. The vector element sequence was U6-MCS-Ubc-Cherry-IRES-puromycin. This knockdown plasmid was constructed by GKG.

[0110] OE-FGF7 is an FGF7 overexpression plasmid with the human FGF7 gene (transcript NM_002009.4) as the target gene. The vector element sequence is Ubc-MCS-3FLAG-CBh-gcGFP-IRES-puromycin. This overexpression plasmid was constructed by GKG.

[0111] Table 2 Experimental Grouping for Normal Culture and TNFα Treatment

[0112]

[0113] Table 3. Experimental groups for normal culture and treatment with TNFα, si-NC, siFGF7, OFF miR-NC, and OFF miR-497-5p

[0114]

[0115] Table 4. Experimental groups for normal culture and OFF miR-497-5p, si-WTAP, and OE-FGF7 treatment.

[0116]

[0117] The protein-protein interactions between WTAP, m6A methyltransferase complex, and FGF7 were detected by co-immunoprecipitation (CoIP) assay.

[0118] Table 5. Validation of Immunoprecipitation

[0119]

[0120] 3. Experimental Results

[0121] See the experimental results. Figure 2 .

[0122] Figure 2 Figures A and D show that m6A methylation modification regulates the stability and translation efficiency of autophagy-related genes (Beclin-1, ATG5, p62) by targeting them, thereby regulating autophagy activity and ultimately indirectly affecting the proliferation, apoptosis, and epithelial-mesenchymal transition (EMT) of lens epithelial cells.

[0123] Figure 2 Figures B and E show that after transfection with siFGF7 to knock down FGF7 expression, the TNFα-induced m6A methylation activation effect was significantly weakened; after further transfection with OFF miR-497-5p to inhibit miR-497-5p function, m6A methylation modification was reactivated; that is, the miR-497-5p / FGF7 axis is located at the upstream regulatory node of TNFα-induced m6A methylation modification.

[0124] Figure 2 Figures C and F show that during m6A methylation modification, methyltransferases METTL3 and METTL14 form a stable complex with WTAP. The formation of the METTL3-METTL14 heterodimer is the core catalytic unit of the complex, while WTAP, as a regulatory subunit, can recruit the METTL3-METTL14 heterodimer to a specific region of the target RNA through protein-protein interactions, thereby promoting the stability of the complex. Therefore, WTAP is a key binding site between the methyltransferase complex and the target RNA.

[0125] Under the same transfection conditions, m6A Dot Blot results showed that OFF miR-497-5p treatment promoted m6A methylation activation; overexpression of FGF7 promoted m6A methylation activation; the combined treatment of the two had a synergistic effect; and knockdown of WTAP significantly inhibited m6A methylation modification; that is, WTAP is a key downstream effector molecule that regulates m6A methylation modification along the miR-497-5p / FGF7 axis.

[0126] Figure 2 The results from the G study showed that the immunoprecipitation assay verified that overexpression of FGF7 can enhance the interaction and binding between siWTAP and METTL3, METTL14, and FGF7.

[0127] The above experimental results indicate that the miR-497-5p / FGF7 axis enhances m6A methylation modification by upregulating WTAP to recruit the METTL3 / METTL14 complex, thereby targeting and regulating autophagy gene expression, and ultimately mediating the proliferation, apoptosis, and EMT process of lens epithelial cells.

[0128] Example 3:

[0129] 1. Experimental Materials

[0130] Human lens epithelial cell line HLE-B3 (purchased from the American Type Culture Collection, ATCC) was placed in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and cultured routinely in a 37°C, 5% CO2 incubator. Specific information regarding the miR-497-5p inhibitor (OFF miR-497-5p), FGF7 overexpression vector, and siWTAP knockdown of WTAP is as follows:

[0131] The miR-497-5p inhibitor (OFF miR-497-5p) is a miR-497-5p-specific antagomir. Its sequence design is completely complementary to the mature miR-497-5p (5'-CAGCAGCACACUGUGGUUUGA-3' (SEQ ID No. 5)), with the sequence 5'-UCAAACCACAGUGUGCUGCUG-3' (SEQ ID No. 6). It has undergone full-chain 2'-O-methylation, phosphate thiocyanate backbone modification, and 3'-terminal cholesterol conjugation. It was purchased from RiboBio (Guangzhou), miR-497-5p inhibitor (catalog number: miR20000764).

[0132] OE-FGF7 is an FGF7 overexpression plasmid with the human FGF7 gene (transcript NM_002009.4) as the target gene. The vector element sequence is Ubc-MCS-3FLAG-CBh-gcGFP-IRES-puromycin. This overexpression plasmid was constructed by Jikai Gene.

[0133] The siWTAP knockdown of WTAP was performed using an shRNA plasmid targeting the human siWTAP gene, with the vector element sequence being U6-MCS-Ubc-Cherry-IRES-puromycin; this knockdown plasmid was constructed by GKG.

[0134] 2. Experimental Methods

[0135] HLE-B3 cells were seeded in 6-well plates at a density of 60-70%, transfected according to the Lipofectamine instructions, and samples were collected 48 hours later.

[0136] The grouping experiments for the samples are shown in Table 6 below:

[0137] Table 6

[0138]

[0139] 3. Experimental Results

[0140] See the experimental results. Figure 3 .

[0141] Western blot results showed that inhibiting miR-497-5p (OFF) or overexpressing FGF7 upregulated the expression of METTL3, METTL14, and WTAP proteins, indicating that m6A methylation was activated; this was accompanied by autophagy inhibition (increased P62, decreased LC3-β) and EMT enhancement (increased Vimentin and α-SMA). Transfection with siWTAP to reduce m6A methylation levels reversed these effects, i.e., it activated autophagy and resisted EMT.

[0142] MeRIP RT-PCR results showed that, under TNFα and OFF miR-497-5p treatment, the predicted binding sites 1 and 2 of FGF7 mRNA had high binding efficiency with the m6A RIP product.

[0143] 4. Experimental Conclusions

[0144] In lens epithelial cells, inhibiting miR-497-5p or overexpressing FGF7 can suppress autophagy and promote EMT by activating m6A methylation (upregulating METTL3 / METTL14 / WTAP); while knocking down WTAP reduces m6A levels, thereby activating autophagy and inhibiting EMT. FGF7 is a potential target gene in this regulatory pathway affected by m6A modification.

[0145] The above experimental results indicate that the miR-497-5p / FGF7 / WTAP axis affects the autophagy and EMT processes of lens epithelial cells by regulating m6A methylation modification, and WTAP is the core effector molecule of this pathway.

[0146] Example 4:

[0147] 1. Experimental materials: New Zealand white rabbits, modified intraocular lenses with STM2457 encapsulated in PLGA (experimental group), IOLs with only PLGA loading (PLGA@IOL group), and unmodified IOLs (Ctrl@IOL group).

[0148] 2. Experimental procedure:

[0149] Phacoemulsification was performed on the rabbit's eye, and a corresponding artificial lens was implanted.

[0150] At 1, 2, 3, and 4 weeks post-surgery, the anterior segment was observed and photographed using a slit lamp.

[0151] At the postoperative endpoint, the eyeball was removed for tissue sampling. The Miyake-Apple method, tissue paraffin embedding, and HE staining were used to assess the proliferation of posterior capsule cells.

[0152] H&E staining was performed on key tissues of the eyeball (cornea, optic nerve, retina, iris, ciliary body) to observe changes in tissue structure.

[0153] 3. Experimental Results:

[0154] See the experimental results. Figure 4 .

[0155] Figure 4 Figures A and B show that slit-lamp observation and turbidity rating indicate that the PLGA-encapsulated STM2457-loaded IOL group can significantly inhibit PCO. Quantitative statistical turbidity rating analysis shows that the experimental group mainly has mild and moderate turbidity ratings, which are reduced compared to the PLGA@IOL and Ctrl@IOL groups.

[0156] Figure 4 The results showed that Miyake-Apple observation and H&E staining of the posterior capsule revealed very little proliferation of fibroblast clusters in the posterior capsule region of the intraocular lens in the experimental group, and the proliferation of lens epithelial cells was significantly inhibited.

[0157] Figure 4 The results showed that H&E staining of ocular tissues indicated that the key tissue structures of the cornea, retina, and iris in the experimental group were intact, with no obvious pathological changes, proving that the modified intraocular lens has good in vivo implantation safety.

[0158] The above experimental results demonstrate that the miR-497-5p / FGF7 axis promotes lens epithelial cell EMT by upregulating WTAP to enhance m6A methylation modification and inhibiting autophagy. FGF7, as a target of m6A modification, participates in regulation, confirming the existence of the miR-497-5p / FGF7 / WTAP / m6A / autophagy signaling axis.

[0159] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Use of an agent that inhibits the m6A methyltransferase Writer complex in the manufacture of a medicament for the treatment of an ocular fibrosis-related disease, characterized in that, The m6A methyltransferase Writer complex includes METTL3, METTL14, and WTAP.

2. Use according to claim 1, characterized in that, The reagent targets the assembly state of the m6A methyltransferase Writer complex, and restores the normal m6A methylation modification level in ocular tissue by inhibiting the pathological over-assembly or abnormal stabilization of the complex, thereby maintaining the autophagy homeostasis of ocular tissue cells and blocking the initiation of ocular fibrosis.

3. Use according to claim 1, characterized in that, The reagent that inhibits the m6A methyltransferase Writer complex includes at least one of the following reagents: (1) Small molecule compounds that inhibit the pathological assembly of the m6A methyltransferase Writer complex; (2) Biological agents that interfere with the stability of WTAP-mediated complexes; (3) Nucleic acids that regulate the miR-497-5p / FGF7 axis.

4. The application according to claim 3, characterized in that, The small molecule compound is selected from at least one of ciclopirox olamine, STM2457, UZH1a, remibrutinib, and RG7834; And / or, the biological agent is selected from at least one of anti-WTAP monoclonal antibodies, WTAP-binding peptides, or WTAP functional domain protein fragments; And / or, the nucleic acid is selected from at least one of miR-497-5p mimics, miR-497-5p inhibitors, or siRNAs targeting FGF7.

5. The application according to any one of claims 1-4, characterized in that, The intraocular fibrosis-related diseases are selected from at least one of cataracts, thyroid-associated ophthalmopathy, and diabetic retinopathy.

6. A pharmaceutical composition for treating intraocular fibrosis-related diseases, characterized in that, The pharmaceutical composition comprises: an inhibitor of the m6A methyltransferase Writer complex and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, The reagent that inhibits the m6A methyltransferase Writer complex includes at least one of the following reagents: (1) Small molecule compounds that inhibit the pathological assembly of the m6A methyltransferase Writer complex; (2) Biological agents that interfere with the stability of WTAP-mediated complexes; (3) Nucleic acids or combinations thereof that regulate the miR-497-5p / FGF7 axis.

8. The pharmaceutical composition according to claim 7, characterized in that, The small molecule compound is selected from at least one of ciclopirox olamine, STM2457, UZH1a, remibrutinib, and RG7834; And / or, the biological agent is selected from at least one of anti-WTAP monoclonal antibodies, WTAP-binding peptides, or WTAP functional domain protein fragments; And / or, the nucleic acid is selected from at least one of miR-497-5p mimics, miR-497-5p inhibitors, or siRNAs targeting FGF7.

9. The pharmaceutical composition according to claim 6, characterized in that, The intraocular fibrosis-related diseases are selected from at least one of cataracts, thyroid-associated ophthalmopathy, and diabetic retinopathy.

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