Use of tbx family transcription factor as target in preparation of medicine for treating eye diseases

Abstract

The application provides application of a TBX family transcription factor as a target point in preparation of a drug for treating eye diseases. The application develops or screens or prepares the drug for treating eye diseases by taking the TBX family transcription factor as a target point, the drug can target the TBX family transcription factor, significantly reduces choroidal neovascularization proliferation, inflammation and fibrosis of the eye fundus, and has important significance for clinical treatment of eye diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of TBX family transcription factors as targets in the preparation of drugs for treating eye diseases. Background Technology

[0002] Age-related macular degeneration (AMD) is an age-related, blinding eye disease that primarily affects people over 60 years of age and is a leading cause of irreversible blindness in the elderly. AMD is mainly divided into dry and wet types, with wet AMD (wAMD) being the leading cause of blindness due to AMD, accounting for over 90% of AMD-related blindness cases.

[0003] The key pathology of wAMD is choroidal neovascularization in the fundus, leading to subretinal vascular leakage, sclerosis, traction, and scarring, which damages the retinal structure and causes visual impairment or blindness. Neovascularization in wAMD is a process involving the synergistic action of multiple factors and cells. Existing research shows that with increasing age, oxidative stress damage associated with aging leads to macular hypoxia and induces upregulation of vascular endothelial growth factor A (VEGF-A), a key angiogenesis factor. This promotes endothelial cell proliferation and migration, inducing new blood vessels to sprout from existing vessels. VEGF-A-induced new blood vessels are structurally disordered, highly unstable, and have a low survival rate. Meanwhile, Notch, Wnt, and antioxidant pathways promote endothelial cell proliferation and migration and regulate endothelial cell functional specialization, cell polarity, and intercellular connections, enabling the survival and continuous growth of new blood vessels. PDGF signaling stabilizes new blood vessels by recruiting pericytes and smooth muscle cells to encapsulate them.

[0004] Anti-angiogenesis is key to the treatment of wAMD and is the main method used clinically. Currently, wAMD is mainly treated clinically by intravitreal injection of VEGF-A neutralizing antibodies (such as ranibizumab), but the effect is not ideal. Summary of the Invention

[0005] This invention provides the application of TBX family transcription factors as targets in the preparation of drugs for treating eye diseases. By using TBX family transcription factors as targets for the development, screening, or preparation of drugs for treating eye diseases, these drugs can target TBX family transcription factors and significantly reduce neovascularization, inflammation, and fibrosis of the choroidal fundus, which is of great significance for the clinical treatment of eye diseases.

[0006] According to a first aspect of the invention, the use of TBX family transcription factors as targets in the preparation of medicaments for treating eye diseases is provided.

[0007] This invention develops, screens, or prepares drugs for treating eye diseases by targeting TBX family transcription factors. These drugs can significantly reduce neovascularization, inflammation, and fibrosis of the choroidal fundus, which is of great significance for the clinical treatment of eye diseases.

[0008] Preferably, the aforementioned eye diseases include macular degeneration.

[0009] Preferably, the macular degeneration described above is age-related macular degeneration (AMD).

[0010] Preferably, the age-related macular degeneration described above is wet age-related macular degeneration (wAMD).

[0011] The pathogenesis of wAMD is complex, involving multiple factors and cells. Inhibition of a single factor often leads to drug ineffectiveness or drug resistance due to compensatory effects from other factors. Although several factors have been shown to play important roles in the pathological process of wAMD, simultaneous inhibition of multiple factors is difficult, resulting in the inability to completely suppress angiogenesis in wAMD patients. This study discovered the mechanism of action of TBX family transcription factors in angiogenesis, which can serve as targets for the prevention and / or treatment of macular degeneration. Therefore, this study uses TBX family transcription factors as targets to develop, screen, or prepare drugs for the treatment of wAMD, which can significantly inhibit choroidal angiogenesis, inflammatory cell infiltration, and fibrosis, which is of great significance for the clinical treatment of wAMD.

[0012] Preferably, the above-mentioned drugs can inhibit the activity of TBX family transcription factors and / or downregulate the expression level of TBX family transcription factors.

[0013] In the applications covered by this protocol, drugs used to treat eye diseases can significantly reduce choroidal neovascularization, inflammation, and fibrosis by inhibiting the activity of TBX family transcription factors and / or downregulating the expression levels of TBX family transcription factors, which is of great significance for the prevention and clinical treatment of eye diseases.

[0014] Preferably, the TBX family transcription factor is TBX20.

[0015] TBX20, as a key transcription factor, plays multiple functions under physiological and pathological conditions. Existing research indicates that TBX20 plays an important role in cardiac development and the maintenance of normal adult cardiac function. The inventors of this application have discovered that TBX20 promotes angiogenesis, primarily by regulating key components of VEGF, PDGF, Notch, Wnt, HIF, and antioxidant pathways. In addition to promoting angiogenesis, the inventors have also found that TBX20 can promote inflammatory cell infiltration and exacerbate macular degeneration. Therefore, this application targets TBX20 for the development, screening, or preparation of drugs for treating macular degeneration. Using these drugs in macular degeneration can significantly reduce choroidal angiogenesis, inflammation, and fibrosis in the fundus, providing theoretical guidance for the clinical treatment of macular degeneration.

[0016] Preferably, the aforementioned drug includes RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, shRNA, and substances for delivering adeno-associated virus or plasmids or for gene knockout targeting TBX20.

[0017] According to a second aspect of the invention, a pharmaceutical composition for treating an eye disease is provided, the pharmaceutical composition comprising a substance capable of inhibiting the activity of TBX family transcription factors and / or downregulating the expression level of TBX family transcription factors.

[0018] This approach applies substances that can inhibit the activity of TBX family transcription factors and / or downregulate the expression levels of TBX family transcription factors to the preparation of drugs for treating eye diseases. It can significantly inhibit choroidal neovascularization, inflammatory cell infiltration, and fibrosis, providing some theoretical guidance for the prevention and clinical treatment of eye diseases.

[0019] Preferably, the pharmaceutical composition comprises at least one of the following: an RNA interference molecule or antisense oligonucleotide targeting TBX20, a small molecule inhibitor, siRNA, shRNA, and a substance for delivering adeno-associated virus or plasmid, or for gene knockout.

[0020] Preferably, the aforementioned eye diseases include macular degeneration.

[0021] Preferably, the macular degeneration described above is age-related macular degeneration (AMD).

[0022] Preferably, the age-related macular degeneration described above is wet age-related macular degeneration (wAMD).

[0023] Preferably, the TBX family transcription factor is TBX20.

[0024] The pharmaceutical composition provided in this scheme for treating eye diseases contains substances that can inhibit the activity of TBX family transcription factors and / or downregulate the expression level of TBX family transcription factors. It can significantly inhibit choroidal neovascularization, inflammatory cell infiltration, and fibrosis, providing certain theoretical guidance for the prevention and clinical treatment of macular degeneration.

[0025] According to a third aspect of the invention, the use of a substance for detecting TBX family transcription factors in the preparation of diagnostic reagents or auxiliary diagnostic reagents for eye diseases is provided.

[0026] Preferably, the aforementioned eye diseases include macular degeneration.

[0027] Preferably, the macular degeneration described above is age-related macular degeneration (AMD).

[0028] Preferably, the age-related macular degeneration described above is wet age-related macular degeneration (wAMD).

[0029] Preferably, the TBX family transcription factor is TBX20.

[0030] The inventors of this application discovered the mechanism of action of TBX20 in neovascularization through research, and found that, compared with healthy individuals, the expression of TBX20 in the choroid and retina of wAMD patients was significantly upregulated. Therefore, this method uses TBX20 as a detection indicator to prepare a substance that can be used to detect TBX20 and apply it to the preparation of diagnostic reagents or auxiliary diagnostic reagents for wAMD. This allows for the diagnosis of wAMD patients and the monitoring of TBX20 expression levels as a prognostic indicator, demonstrating good clinical application value. Attached Figure Description

[0031] Figure 1 This image shows the expression of TBX20 in the retina and choroid of normal individuals and wAMD patients, obtained through biological analysis using the human eye transcriptome database.

[0032] Figure 2 The diagram shows the expression of TBX20 in smooth muscle cells (HUVSMC) under various pathological conditions provided by this invention.

[0033] Figure 3 The graph shows the expression of TBX20 in pericytes (HBVP) under various pathological conditions provided by this invention.

[0034] Figure 4 The graph shows the expression of TBX20 in endothelial cells (HREC) under various pathological conditions provided by this invention.

[0035] Figure 5The graph shows the expression of TBX20 in various angiogenesis-related cells provided by this invention.

[0036] Figure 6 This figure shows the results of the analysis of TBX20 expression levels in the choroid of mice in the CNV model using real-time quantitative PCR.

[0037] Figure 7 This is a graph showing the results of the analysis of TBX20 expression levels in the choroid of mice in the CNV model using Western blot.

[0038] Figure 8 This is a graph showing the results of using qPCR technology to detect the expression level of TBX20 in the mouse choroid of Example 5.

[0039] Figure 9 This is a graph showing the results of the Western blot experiment used in this invention to detect the expression level of TBX20 in the mouse choroid of Example 5.

[0040] Figure 10 This figure shows the results of the present invention using qPCR technology to detect the expression levels of multiple genes related to angiogenesis, inflammation and fibrosis in the mouse choroid of Example 5.

[0041] Figure 11 This is an immunofluorescence staining image of mouse choroidal neovascularization (IB4) and inflammatory cells (F4 / 80+ / Ibal+) in Example 5 of the present invention.

[0042] Figure 12 This is a statistical diagram of the staining area of ​​neovascularization (IB4) and inflammatory cells (F4 / 80+ / Ibal+) in the mouse choroid of Example 5 of the present invention.

[0043] Figure 13 This is an immunofluorescence staining image of pericytes, smooth muscle cells (α-SMA+), and fibrosis (CollⅠ+) in the mouse choroid of Example 5 of the present invention.

[0044] Figure 14 This diagram illustrates the proliferation of endothelial cells after siRNA-targeted knockdown of TBX20 expression in endothelial cells, as described in this invention.

[0045] Figure 15 This diagram illustrates the migration of endothelial cells after siRNA-targeted knockdown of TBX20 expression in endothelial cells, as described in this invention.

[0046] Figure 16 This diagram illustrates the tube formation of endothelial cells after siRNA-targeted knockdown of TBX20 expression in endothelial cells, as described in this invention.

[0047] Figure 17 This diagram illustrates the inflammatory response of endothelial cells after siRNA-targeted knockdown of TBX20 expression in endothelial cells, as described in this invention.

[0048] Figure 18 This is a GO / KEGG analysis result of the present invention after siRNA targeting and knocking down the expression of TBX20 in endothelial cells.

[0049] Figure 19 This is an image showing the RNA-Seq sequencing results after TBX20 expression was knocked down in endothelial cells using siRNA, as described in this invention.

[0050] Figure 20 This is a Western blot result of the present invention after siRNA targeting and knocking down the expression of TBX20 in endothelial cells. Detailed Implementation

[0051] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] Example 1: Expression of TBX20 in the retina and choroid of healthy individuals and patients with wAMD

[0053] The purpose of this embodiment is to investigate the expression of transcription factor TBX20 in the retina and choroid of patients with wet age-related macular degeneration (wAMD). This embodiment utilizes an existing database (human eye transcriptome database) for bioinformatics analysis to analyze the expression of TBX20 in the retina and choroid of normal individuals and wAMD patients. The results are as follows: Figure 1 As shown.

[0054] Depend on Figure 1 The results show that TBX20 is significantly upregulated in the choroid and retina of wAMD patients compared to healthy individuals. These findings indicate that the transcription factor TBX20 plays a crucial role in wAMD patients, promoting retinal angiogenesis, leading to retinal vascular leakage, hemorrhage, traction, and scarring, ultimately causing visual impairment or blindness in wAMD patients.

[0055] Example 2: Expression of TBX20 in various angiogenesis-related cells under multiple pathological conditions

[0056] The purpose of this embodiment is to investigate the expression of TBX20 in various angiogenesis-related cells (endothelial cells, pericytes, and smooth muscle cells) under multiple pathological conditions, including hypoxia (5%), oxidative stress (H2O2), inflammation (LPS), and serum-free conditions, using real-time quantitative PCR (qPCR) and / or Western blot assays. The expression of TBX20 in HUVSMCs under various pathological conditions is as follows: Figure 2 As shown, the expression of TBX20 in pericytes (HBVP) is as follows: Figure 3 As shown, the expression of TBX20 in endothelial cells (HREC) is as follows: Figure 4 As shown, in the experimental results under 0.1% FBS conditions, the control group indicates that the cells were treated with an equal amount of solvent used to prepare 0.1% FBS; in the experimental results under oxidative stress (H2O2) conditions, the control group indicates that the cells were treated with an equal amount of solvent used to prepare H2O2; in the experimental results under hypoxia (5%) conditions, the control group indicates that the cells were under normoxic conditions; and in the experimental results under inflammation (LPS) conditions, the control group indicates that the cells were treated with an equal amount of solvent used to prepare LPS solution.

[0057] Depend on Figure 2 , 3 As shown in section 4, the transcription factor TBX20 is upregulated in endothelial cells, pericytes, and smooth muscle cells under various pathological conditions, including hypoxia (5%), oxidative stress (H2O2), inflammation (LPS), and serum-free conditions.

[0058] Example 3: Expression of TBX20 in endothelial cells and other related cells

[0059] The purpose of this embodiment is to detect the expression of TBX20 in various angiogenesis-related cells using real-time quantitative PCR (qPCR) technology. The results are as follows: Figure 5 As shown. By Figure 5 It was found that the expression level of transcription factor TBX20 in endothelial cells (HREC) was significantly higher than that in other cell types (HUVSMC, HBVP, and THP1).

[0060] Example 4

[0061] To further verify the expression of transcription factor TBX20 in wAMD patients, this embodiment used a laser-induced mouse choroidal neovascularization (CNV) model to detect TBX20 expression. The specific experimental procedures are as follows:

[0062] 1. Constructing a laser-induced mouse choroidal neovascularization (CNV) model

[0063] Adult C57BL / 6 mice were selected and anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg body weight). Mydriasis was achieved with compound tropicamide eye drops, followed by ocular surface anesthesia with tetracaine hydrochloride eye drops and corneal moistening with hydroxypropyl methylcellulose eye drops. The parameters of the laser machine were adjusted (spot diameter 75 μm, energy 90 mW, duration 75 ms, centered on the optic disc). Laser photocoagulation was performed once each at 3, 6, 9, and 12 o'clock as the experimental group. The choroid of the mice was collected on days 3 and 7 after laser-induced CNV for downstream experiments.

[0064] 2. Downstream molecular validation of TBX20

[0065] (1) Real-time quantitative PCR detection

[0066] Choroid membranes were collected from mice in the CNV model. Total RNA was extracted from the choroid membranes using TRNzol reagent (TIANGEN, CAT: DP424), and then cDNA was synthesized using a reverse transcription kit (TIANGEN, CAT: KR116). A 10 μL PCR reaction system was prepared and the plate was placed in a Quantstudio 6K Flex System (Life Technologies) PCR instrument for real-time quantitative polymerase chain reaction. The results were then analyzed using Quantstudio 12K Flex v1.2.2 software (Thermo Fisher Science). The results are shown below. Figure 6 As shown.

[0067] Depend on Figure 6 It was found that TBX20 was significantly upregulated in the choroid of laser-induced mice (experimental group) compared with untreated healthy mice (control group). This further illustrates that the transcription factor TBX20 plays an important role in wAMD, promoting retinal angiogenesis and inducing retinal vascular leakage, hemorrhage, traction, and scar formation, thereby regulating the progression of wAMD.

[0068] (2) Western blot assay

[0069] Choroid membranes were collected from mice in the CNV model. Proteins were extracted from the choroid using RIPA lysis buffer (Thermo Fisher Science, CAT: A32961) containing protease and phosphatase inhibitors to obtain lysis products. The lysis products were separated by SDS-PAGE under reducing conditions and transferred to a PVDF membrane. The PVDF membrane was then blocked with 5% skim milk powder and incubated overnight at 4°C with primary antibody. Following incubation with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 hour, the PVDF membrane was finally exposed and detected using a Syngene Gbox / Chemi-XT16 instrument. The results are shown below. Figure 7 As shown.

[0070] The antibodies used in the Western blot were as follows: anti-TBX20 (sigma, CAT: AV33177), anti-F4 / 80 (ebioscience, CAT: 11-4801-85), anti-Iba1 (wako, CAT: 019-19741), Donkey anti-Rabbit IgG (H+L) Secondary Antibody (APExBio, CAT: A31572), Donkey anti-rat Cross-Adsorbed Secondary Antibody, and Alexa Fluor 488 (Life, CAT: A21208).

[0071] Depend on Figure 7 It was found that TBX20 was significantly upregulated in the choroid of laser-induced mice compared with normal mice. This further illustrates that the transcription factor TBX20 plays an important role in wAMD, promoting retinal angiogenesis and inducing retinal vascular leakage, hemorrhage, traction, and scar formation, thereby regulating the progression of wAMD.

[0072] The above results indicate that, compared with normal individuals, transcription factor TBX20 is significantly overexpressed in retinal and choroidal neovascularization in patients with wAMD. TBX20 regulates the progression of wAMD by promoting neovascularization in patients. Therefore, based on the above research results, the inventors of this application have used TBX20 as a therapeutic target for wAMD and applied TBX20 inhibitors to the preparation of drugs for the treatment of wAMD, which has important guiding significance for the clinical treatment of wAMD.

[0073] Example 5: Knockdown of TBX20 expression in mouse choroid using adeno-associated virus or plasmid

[0074] The purpose of this embodiment is to use adeno-associated virus or plasmid to knock down TBX20 expression in the mouse choroid to verify the effects of targeting TBX20 on retinal and choroidal angiogenesis, inflammation, and fibrosis in wAMD patients. The specific experimental procedures are as follows:

[0075] (1) Constructing adeno-associated virus (AAV) with endothelial cell-specific knockdown.

[0076] The CDH5 gene transcription start site upstream of -598bp to +378bp was used as an endothelial cell-specific initiator. A vector was constructed using shRNA targeting TBX20 and packaged into serum type 8 AAV, which was stored at -80℃ for subsequent injection experiments in the mouse retina or vitreous cavity.

[0077] The shRNA sequence (shTBX20) targeting TBX20 used in this embodiment is shown in Table 1.

[0078] Table 1. shRNA sequences targeting TBX20

[0079]

[0080] (2) Subretinal or intravitreal injection of AAV or plasmid into mice

[0081] Adult C57BL / 6 mice were selected. The mice were mydriatic in the choroid using compound tropicamide eye drops, anesthetized by intraperitoneal injection of 4% sodium pentobarbital (50 mg / kg body weight), and then surface anesthetized by procainamide eye drops. Sodium carboxymethyl cellulose eye drops were used to maintain corneal moisture and prevent dryness. Using a sterile 5 μL syringe (Hamilton, CAT: 7633-01) and a 33-gauge needle (Hamilton, CAT: 7803-05, 33 / 15 mm / 3), AAV-GFP or AAV-shTBX20 (5 × 10¹³ vg / mL) was injected subretinally through a posterior limbus puncture. The injection volume was 1 μL per eye. Successful subretinal injection was indicated by a semi-circular retinal detachment around the injection site visible under a microscope or through fundus imaging. Follow-up examinations were performed 4 weeks later.

[0082] Example 6

[0083] The expression level of TBX20 in the mouse choroid of Example 5 was detected using qPCR and Western blot experiments. The qPCR results are as follows: Figure 8 As shown, the experimental results of the Western bolt are as follows: Figure 9 As shown. By Figure 8 and Figure 9 It can be seen that, compared with mice treated with AAV-GFP (control group), subretinal injection of AAV-shTBX20 via posterior limbal puncture in mice (experimental group) can significantly reduce the expression of TBX20 in the choroid of mice.

[0084] In addition, this embodiment also used qPCR technology to detect the expression levels of multiple genes related to angiogenesis, inflammation, and fibrosis in the mouse choroid of Example 5, and the results are as follows. Figure 10 As shown. By Figure 10 It is known that subretinal injection of AAV-shTBX20 via posterior limbal puncture in mice can significantly inhibit the expression of multiple genes related to angiogenesis, inflammation, and fibrosis.

[0085] Example 7

[0086] Immunofluorescence staining was performed on the mouse choroidal neovascularization (IB4) and inflammatory cells (F4 / 80+ / Ibal+) from Example 5, and the results are as follows: Figure 11 As shown; simultaneously, statistical analysis was performed on the staining areas of neovascularization (IB4) and inflammatory cells (F4 / 80+ / Ibal+), and the results are as follows. Figure 12 As shown, where, Figure 12 In inner diagram A, the CNV infiltration area represents the infiltration of macrophages / microglia into neovascularization. F4 / 80+ and Iba1+ are both markers for macrophages / microglia; therefore, the areas of F4 / 80+ and Iba1+ both indicate the severity of macrophage and microglia infiltration into the CNV. Figure 11 and Figure 12 It was found that, compared with AAV-GFP treated mice (control group), subretinal injection of AAV-shTBX20 via posterior limbal puncture in mice (experimental group) significantly reduced TBX20 expression in the choroid of mice and significantly inhibited choroidal neovascularization (IB4) and inflammatory cells (F4 / 80). + / Ibal + )infiltration.

[0087] Example 8

[0088] Pericytes and smooth muscle cells (α-SMA) in the mouse choroid of Example 5 + ) and fibrosis (CollⅠ + Immunofluorescence staining was performed on the affected area, and the results were as follows: Figure 13 As shown, α-SMA + and CollⅠ + The number of positive cells indicates the severity of subretinal fibrosis. Figure 13It can be seen that, compared with mice treated with AAV-GFP (control group), subretinal injection of AAV-shTBX20 via posterior limbal puncture in mice (experimental group) can increase the number of peripheral cells and smooth muscle cells (α-SMA) in the choroid of mice. + The number of choroid fibrosis was significantly reduced, and the degree of fibrosis in the mouse choroid was also significantly reduced.

[0089] Example 9: Using siRNA to target and knock down TBX20 expression in endothelial cells

[0090] The siRNA sequence (siTBX20) targeting TBX20 used in this embodiment is shown in Table 2.

[0091] Table 2. siRNA sequences targeting TBX20

[0092]

[0093]

[0094] Note: The last two bases TT in SEQ ID:3 and SEQ ID:4 above are modifications of siRNA, both representing thymine. In the sequence listing, n represents uracil (U).

[0095] To investigate the cellular mechanisms by which the transcription factor TBX20 functions in angiogenesis, this study used siRNA to target and knock down TBX20 expression in endothelial cells. The expression level of TBX20, as well as endothelial cell proliferation, migration, tube formation, and inflammatory responses, were assessed using CCK8 and EdU insertion assays. Endothelial cell proliferation was observed as follows. Figure 14 As shown, the migration situation is as follows Figure 15 As shown, the pipe formation is as follows Figure 16 As shown, the inflammatory response is as follows: Figure 17 As shown, the control group was treated with siCtrl (SiCtrl refers to disordered non-specific interfering RNA), and the experimental group was treated with siRNA targeting TBX20 to knock it down.

[0096] Depend on Figure 14 It is evident that targeting TBX20 with siRNA to knock down its expression in endothelial cells significantly inhibits endothelial cell proliferation. Figure 15 It is evident that targeting TBX20 with siRNA to knock down its expression in endothelial cells significantly inhibits endothelial cell migration. Figure 16 It can be seen that knocking down TBX20 expression in endothelial cells using siRNA can significantly inhibit endothelial cell tube formation; Figure 17It is known that targeting TBX20 with siRNA to knock down its expression in endothelial cells can significantly inhibit the inflammatory response of endothelial cells, that is, inhibit immune cell adhesion. Figure 17 (Internal diagram A) and migration across endothelial cells ( Figure 17 (See Figure B). The above results demonstrate that targeting TBX20 with siRNA to knock down its expression in endothelial cells can significantly inhibit endothelial cell proliferation, migration, tube formation, and inflammatory response, thereby suppressing the in vitro angiogenesis function of endothelial cells.

[0097] Example 10

[0098] The purpose of this embodiment is to target and knock down TBX20 expression in endothelial cells using siRNA, while simultaneously performing RNA-Seq and GO / KEGG analyses to investigate the molecular mechanism of TBX20's function and the signaling pathways it regulates. The GO / KEGG analysis results are as follows: Figure 18 As shown, the experimental results of RNA-Seq are as follows: Figure 19 As shown in the figure. Furthermore, Western blot experiments were used to verify the accuracy of the above results, as shown in the figure. Figure 20 As shown in the figure. The control group represents siCtrl treatment (SiCtrl refers to disordered, non-specific interfering RNA), while the experimental group represents siRNA targeting TBX20 for knockdown.

[0099] Depend on Figure 18 GO / KEGG analysis revealed that TBX20 can regulate multiple angiogenesis and inflammation-related signaling pathways. Knocking down TBX20 expression in endothelial cells using siRNA significantly inhibited these signaling pathways. Figure 19 RNA-Seq results show that TBX20 can regulate not only key molecules in the VEGF / PDGF / NOCTH / HIF / WNT signaling pathway, but also other signaling pathways, including CXCL / IL and cell adhesion family molecules. Figure 20 The results of the Western blot experiment were consistent with those of RNA-Seq and GO / KEGG analysis, indicating that the results for the above signaling pathways were accurate. These results demonstrate that TBX20 can regulate multiple angiogenesis-related signaling pathways, thereby promoting angiogenesis and inflammation. Targeting TBX20 with siRNA to knock down its expression in endothelial cells can significantly inhibit these signaling pathways, thereby suppressing the in vitro angiogenesis function of endothelial cells.

[0100] In summary, the inventors of this application have discovered through research that TBX20 can promote inflammation, angiogenesis, and subsequent fibrosis. TBX20 can serve as a therapeutic target for wet age-related macular degeneration (wAMD). Targeting TBX20 can inhibit its activity and / or downregulate its expression level, thereby reducing angiogenesis, inflammation, and fibrosis, providing a new target for the clinical treatment of wAMD. Therefore, TBX20 can be used as a target in the development, screening, or preparation of drugs for the treatment of wAMD. These drugs can inhibit TBX20 activity and / or downregulate its expression level, significantly reducing retinal and choroidal angiogenesis, inflammation, and fibrosis in wAMD patients, providing a new strategy for the clinical treatment of wAMD.

[0101] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. The application of siRNA or shRNA targeting TBX20 in the preparation of drugs for treating wet age-related macular degeneration in mice, characterized in that: The nucleotide sequences of the forward and reverse primers of the siRNA are shown in SEQ ID:3 and SEQ ID:4, respectively, and the nucleotide sequences of the upstream and downstream primers of the shRNA are shown in SEQ ID:1 and SEQ ID:2, respectively.

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