siRNAs that target and inhibit ZEB1 gene expression and their applications

Abstract

This invention discloses siRNAs that target and inhibit ZEB1 gene expression and their applications, belonging to the field of biomedical technology. The siRNAs designed in this invention that target and inhibit ZEB1 gene expression include siRNA1-2913, siRNA2-652, and siRNA3-1073. The sense and antisense strands of siRNA1-2913 are shown in SEQ ID NO:1-2, the sense and antisense strands of siRNA2-652 are shown in SEQ ID NO:3-4, and the sense and antisense strands of siRNA3-1073 are shown in SEQ ID NO:5-6. These siRNAs can efficiently inhibit ZEB1 gene expression, inhibit the proliferation and migration of vascular endothelial cells, and intervene in the formation of pathological corneal neovascularization. They can be used for research on the pathogenesis and intervention of corneal neovascularization, showing very promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to siRNA that targets and inhibits the expression of the ZEB1 gene and its applications. Background Technology

[0002] Normal angiogenesis maintains tissue homeostasis, facilitates wound repair, and supports organ regeneration. However, imbalances in angiogenesis regulation can trigger various pathological diseases. For example, abnormally activated angiogenesis provides oxygen and nutrients for tumor growth, serving as a core driver of tumor proliferation, invasion, and distant metastasis. In corneal tissue, abnormal infiltration of new blood vessels disrupts the inherent avascular nature of the cornea, leading to decreased corneal transparency and potentially causing visual impairment or even blindness. Anti-vascular endothelial growth factor (VEGF) therapy is currently the mainstream clinical approach for tumors and pathological angiogenesis in the eye. However, this therapy has significant limitations: some patients exhibit VEGF-independent angiogenesis mechanisms, resulting in poor treatment response; furthermore, ocular surface drops have low bioavailability in corneal angiogenesis, making it difficult to effectively reverse angiogenesis in advanced stages of the disease, ultimately requiring physical interventions such as laser therapy or surgery, which increases the burden of treatment for patients. Therefore, identifying the core regulatory factors of pathological angiogenesis and developing novel treatment strategies with stronger targeting and longer-lasting efficacy has become an urgent clinical need.

[0003] The regulatory role of the zinc finger E-box binding homeobox 1 (ZEB1) in angiogenesis is gradually being revealed. In tumor angiogenesis research, ZEB1 has been confirmed as a key pro-angiogenic transcription factor. In corneal angiogenesis research, corneal angiogenesis was inhibited in ZEB1 knockdown mice, and ZEB1 regulates angiogenesis independently of the VEGF pathway. Specifically, ZEB1 forms a complex with C-terminal binding protein (CTBP). When this complex is intact, ZEB1 can recruit histone deacetylases to inhibit the expression of angiogenesis target genes. When the stability of the complex is lost, ZEB1 expression in endothelial cells decreases, thereby inhibiting corneal angiogenesis. This discovery reveals the regulatory pathway of VEGF-independent corneal angiogenesis, providing a new target direction for clinical treatment. RNAi technology can specifically degrade target gene mRNA through small interfering RNA (siRNA), efficiently blocking gene expression. It has been widely used in gene function exploration and gene therapy for major diseases. siRNA targeted delivery technology provides a new direction for the precision treatment of corneal angiogenesis. Currently, there are few studies on targeting ZEB1 with RNAi technology to intervene in pathological angiogenesis. Developing RNAi treatment strategies that target ZEB1, especially ocular surface preparations, is an ideal choice to solve the clinical treatment dilemma. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a siRNA that targets and inhibits the expression of the ZEB1 gene and its application. The siRNA that targets and inhibits the expression of the human ZEB1 gene can efficiently inhibit the expression of the ZEB1 gene, inhibit the proliferation and migration of vascular endothelial cells, and intervene in the formation of pathological neovascularization of the cornea.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides an siRNA that targets and inhibits the expression of the ZEB1 gene, comprising siRNA1-2913, siRNA2-652 and siRNA3-1073, wherein the sense and antisense strands of siRNA1-2913 are shown in SEQ ID NO:1-2, the sense and antisense strands of siRNA2-652 are shown in SEQ ID NO:3-4, and the sense and antisense strands of siRNA3-1073 are shown in SEQ ID NO:5-6.

[0007] Secondly, the present invention provides the encoding gene of the siRNA that targets and inhibits the expression of the ZEB1 gene.

[0008] Thirdly, the present invention provides a recombinant expression vector into which the above-mentioned coding gene is inserted.

[0009] Fourthly, the present invention provides an LNP-siRNA that targets and inhibits the expression of the ZEB1 gene, comprising the above-mentioned siRNA that targets and inhibits the expression of the ZEB1 gene and lipid nanoparticles.

[0010] Based on the above technical solution, the raw materials for the lipid nanoparticles further include Lipofectamine 2000 and SM102 lipid materials.

[0011] Fifthly, the present invention provides the application of the above-mentioned siRNA, encoding gene, recombinant expression vector, and LNP-siRNA that target and inhibit ZEB1 gene expression in the preparation of drugs that inhibit angiogenesis.

[0012] Based on the above technical solution, further applications are made in the preparation of drugs that inhibit corneal neovascularization.

[0013] Based on the above technical solution, the drug further includes pharmaceutically acceptable excipients.

[0014] Based on the above technical solution, the pharmaceutically acceptable excipients further include fillers, binders, disintegrants, and emulsifiers.

[0015] Based on the above technical solution, the dosage form of the drug further includes tablets, oral liquid preparations, drops, injectable preparations, and capsule preparations.

[0016] Sixthly, the present invention provides the application of the above-mentioned siRNA, encoding gene, recombinant expression vector, and LNP-siRNA targeting and inhibiting ZEB1 gene expression in the study of the pathogenesis of corneal neovascularization.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The siRNA1-2913, siRNA2-652, and siRNA3-1073 designed in this invention can all inhibit the expression of the ZEB1 gene in human umbilical vein endothelial cells. The screened siRNA3-1073 can be used for intervention and treatment of rat corneal neovascularization model, which has good application prospects and further illustrates the potential application of this target in the precision treatment of corneal neovascularization. Attached Figure Description

[0018] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0019] Figure 1 The image shows the effect of Western blotting on the knockdown of ZEB1 protein by ZEB1 siRNA in Example 1. In the image, A is the electrophoresis image of ZEB1 protein and B is the result of the relative expression level of ZEB1 protein.

[0020] Figure 2 The results of the real-time quantitative PCR detection of the inhibitory effect on ZEB1 mRNA expression in Example 2 are shown.

[0021] Figure 3 This is an example of EdU staining to detect the effect of ZEB1 knockdown on the proliferation of human umbilical vein endothelial cells. In Example 3, A is a fluorescence image of EdU cell proliferation staining, and B is a quantitative analysis of the relative cell proliferation rate.

[0022] Figure 4 The figure shows the effect of ZEB1 siRNA on the migration function of human umbilical vein endothelial cells in Example 4. In the figure, A is the cell migration trend at 0h, 6h and 12h after ZEB1 knockdown, and B is the cell migration rate at 6h and 12h after ZEB1 knockdown.

[0023] Figure 5 This is a diagram illustrating the intervention effect of ZEB1siRNA as a gene drug on corneal neovascularization in mice in Example 5.

[0024] Figure 6In Example 5, a quantitative graph shows the effect of ZEB1siRNA as a gene drug on corneal neovascularization in mice. In the graph, A represents the change in blood vessel length within 14 days after corneal neovascularization intervention, B represents the change in blood vessel length within 14 days after corneal neovascularization intervention, and C represents the change in blood vessel area within 14 days after corneal neovascularization intervention. Detailed Implementation

[0025] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0026] Example 1: Detection of ZEB1 siRNA sequence knockdown effect by Western blotting Synthesis of siRNA that inhibits ZEB1 gene expression The RefSeq mRNA sequence of the human ZEB1 gene was obtained from the NCBI Gene database. Conserved fragments in the CDS region were screened, and siRNAs were designed. Primers were designed using NCBI Primer-BLAST. After verifying primer specificity, three siRNA sequences capable of inhibiting ZEB1 gene expression were selected: siRNA1, siRNA2, and siRNA3. A random sequence was used as a negative control. In the ZEB1 siRNA sequence, A, G, C, and U represent adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, and uracil ribonucleotide, respectively, and T represents thymine deoxyribonucleotide. Both the ZEB1 siRNA sequence and the negative control were synthesized by General Biotech. The three pairs of synthesized siRNAs are as follows: siRNA1-2913: positive strand 5'-AGGAAGAGGAGGAGGAUAATT-3' (SEQ ID NO:1); Antisense chain 5'- UUAUCCUCCUCCUCUUCCUTT-3' (SEQ ID NO:2); siRNA2-652: positive strand 5'-ACACAUAAGCAGUAAGAAATT-3' (SEQ ID NO:3); Antisense chain 5'- UUUCUUACUGCUUAUGUGUTT-3' (SEQ ID NO:4); siRNA3-1073: positive strand 5'-GGCAAGUGUUGGAGAAUAATT-3' (SEQ ID NO:5); Antisense chain 5'- UUAUUCUCCAACACUUGCCTT-3' (SEQ ID NO:6); Negative control: positive chain 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO:7); Antisense chain 5'-ACUGUGACACGUUCGGAGAATT-3' (SEQ ID NO:8).

[0027] Human umbilical vein endothelial cells (HUVECs) in logarithmic growth phase were divided into groups of 5 × 10⁻⁶. 5Cells were seeded at a density of 1 cell per well in 6-well plates and incubated in a 37°C, 5% CO2 saturated humidity incubator for 24 hours. When the cell confluence reached 60%, ZEB1 siRNA transfection was performed (the Control group was treated with PBS as a control group, and the NC group was treated with blank vector as a negative control). Premix 5 μL of blank vector and the three ZEB1 siRNAs mentioned above in 250 μL of serum-free Opti-MEM medium, with a siRNA transfection concentration of 50 nM. Premix 5 μL of lipofectamine 2000 (1 mg / mL) transfection reagent in 250 μL of serum-free Opti-MEM medium and incubate at room temperature for 5 min. Mix the premixed siRNAs and transfection reagents separately and incubate at room temperature for 20 min. Add 500 μL of the transfection complex dropwise to each well of the plate, bringing the total volume to 2000 μL. After incubating for 6 hours, replace the medium with DMEM containing 10% fetal bovine serum. When the transfection efficiency is confirmed to be as high as 80% using an inverted fluorescence microscope, add 250 μL of cell lysis buffer (RIPA lysis buffer) and 100× cocktail protease inhibitor to each well of a six-well plate, according to the usage volume. Mix 1 mL of PIPA lysis buffer with 10 μL of 100× cocktail protease inhibitor. Prepare a mixed lysis buffer using cocktail protease inhibitors, extract cell samples using a sterile cell scraper, collect cell samples in centrifuge tubes, incubate on ice for 30 min, vortex every 10 min for complete lysis, centrifuge at 12000g for 5 min at 4℃, and the supernatant in the centrifuge tube is the protein sample. The absorbance of each protein sample was measured using an ELISA reader, and the protein concentration was obtained through a standard curve. An appropriate volume of 5× Loading buffer was added to each sample (30 μg loading). The samples were boiled in a 100℃ metal bath for 10 min, cooled to room temperature, and then electrophoresed and transferred using pre-prepared 7.5% SDS-PAGE gel, electrophoresis buffer, transfer buffer, and TBST buffer. Each protein sample was loaded into the lane at 30 μg, and the differential volume was filled with 1× Loading buffer. Electrophoresis was performed at 90 V for 30 min, then at 120 V for 60 min. Electrophoresis was stopped once the target protein was completely separated. PVDF membranes of appropriate size were cut, activated with methanol, and then transferred using a "sandwich" type transfer clamp immersed in transfer buffer. The current was 250 mA, and the transfer was performed at a constant current for 30 min. The PVDF membrane was washed with TBST 5 min / time, rinsed 5 times, blocked with 5% skim milk powder on a shaker at room temperature for 2 h, and incubated overnight at 4℃ with primary antibody. The membrane was washed with TBST 5 times. Wash the membrane 5 times per wash, incubate the secondary antibody on a horizontal shaker at room temperature for 1 hour, wash the membrane with TBST for 5 minutes per wash, rinse 5 times, develop with ECL chemiluminescence solution, take pictures with a gel electrophoresis analysis system, calculate the gray value of the bands using Image-J software, and finally perform relative quantification of the target gene according to the ratio of target gene to internal reference gene.

[0028] The results are as follows Figure 1 As shown, Figure 1 A represents the protein band development results. Figure 1 B represents the quantitative analysis of protein expression. Compared with the control group, the expression levels of the protein products encoded by the genes in the ZEB1siRNA1, ZEB1siRNA2, and ZEB1siRNA3 groups were 19.59±0.09%, 13.97±0.06%, and 20.77±0.05%, respectively, with a gene silencing efficiency of approximately 80%, indicating that ZEB1siRNA effectively inhibited the expression of the ZEB1 gene at the protein level.

[0029] Example 2: Real-time quantitative PCR detection of ZEB1 siRNA knockdown efficiency at the transcriptional level ZEB1 siRNA transfection was performed according to the transfection procedure in Example 1. After transfection, the cells were incubated in an incubator for 6 hours, and then the medium was replaced with DMEM containing 10% fetal bovine serum. When the transfection efficiency was confirmed to be as high as 80% under an inverted fluorescence microscope, Ezol lysis buffer from the OMEGA Cell Tissue Total RNA Purification Kit (USA) was added to the wells of a 6-well plate according to the reagent usage. After pipetting and aspirating, the cell suspension was transferred to an enzyme-free 1.5 mL centrifuge tube and allowed to stand for 5 min. Then, 1 / 5 volume of chloroform substitute was added, and the mixture was vigorously shaken for 15 s and allowed to stand for 3 min. The cells were then centrifuged at 12000 g for 20 min at 4°C. After centrifugation, the upper aqueous phase was transferred to a 2 mL enzyme-free centrifuge tube, and 1.5 volume of anhydrous ethanol was added and mixed by pipetting. The mixture was added to the purification column in the collection tube and allowed to stand at room temperature for 2 min. Then, the cells were centrifuged at 10000 g for 1 min at 4°C. The liquid in the collection tube was discarded. An appropriate amount of washing buffer was added to the purification column and the cells were centrifuged at 10000 g for 1 min at 4°C. Centrifuge at 10000 g for 1 min, discard the liquid in the collection tube; repeat the above washing steps; centrifuge the purification column at 10000 g for 2 min at 4℃; transfer the purification column to a new 1.5 mL enzyme-free centrifuge tube, add an appropriate amount of elution buffer, and let stand at room temperature for 2 min; centrifuge at 10000 g for 1 min at 4℃; discard the purification column, the liquid in the tube is the RNA sample. Detect the absorbance (OD) of the total RNA sample at 260 nm and 280 nm using a UV spectrophotometer. Sample purity: OD 260 / 280= 1.8~2.2. cDNA amplification of RNA samples was performed using the Hifair reverse transcription kit, as follows: Residual genomic DNA was removed from the RNA sample according to the components in Table 1. After mixing by pipetting, the sample was incubated at 42°C for 2 min. Then, a 20 μL reverse transcription reaction system was prepared according to the components in Table 2. The reverse transcription program was set according to the conditions in Table 3. After reverse transcription, the cDNA sample was placed on ice, and qPCR was performed using the Hieff UNICON real-time quantitative PCR kit. The qPCR reaction system is shown in Table 4, the primer sequences are shown in Table 5, and the qPCR amplification program is shown in Table 6.

[0030] Table 1. Components of residual genomic DNA removed from RNA samples

[0031] Table 2 Reverse transcription reaction system

[0032] Table 3. Reverse transcription procedure

[0033] Table 4. qPCR reaction system

[0034] Table 5 Primer Sequences

[0035] Table 6. Amplification program for qPCR

[0036] Use 2 ΔΔCT The computational method quantitatively analyzed the CT value of RNA, using the ΔCT of the control group RNA detection result as a reference standard. The experiment was repeated three times, and the relative gene expression levels of all groups were calculated using a 2T method. ΔΔCT express.

[0037] The results are as follows Figure 2 As shown, compared with the control group, the ZEB1 mRNA expression levels of the ZEB1siRNA1, ZEB1siRNA2, and ZEB1siRNA3 groups were 39.54±0.09%, 24.74±0.10%, and 37.65±0.14%, respectively, with gene silencing efficiencies of approximately 60%–80%. Figure 2NC was the negative control, with an inhibition rate of 0, indicating that ZEB1 siRNA could inhibit the expression of the ZEB1 gene at the transcriptional level. The knockdown effect of all three sequences was higher than 50%. Considering that complete knockdown of the ZEB1 gene would affect cell function, and based on the protein inhibition efficiency, sequence 3, which had a moderate knockdown efficiency, was selected as the research object for subsequent examples.

[0038] Example 3: EdU staining to detect the effect of ZEB1 knockdown on the proliferation of human umbilical vein endothelial cells Human umbilical vein endothelial cells (HUVECs) were in logarithmic growth phase and then fed at a rate of 1×10⁻⁶. 5 Cells were seeded per well in 24-well plates and transfected with ZEB1 siRNA3 according to the procedure described in Example 1. After transfection, the cells were incubated in an incubator for 6 hours, and then the medium was replaced with DMEM containing 10% fetal bovine serum. When the transfection efficiency was confirmed to be as high as 80% using an inverted fluorescence microscope, EdU working solution (20 μM) was prepared according to the 24-well plate specifications. The cells were stained according to the EdU kit (Beyotime China). After covering the cells, the cells were incubated at 37°C and 5% CO2 for 2 hours. A blank control group and an NC negative control group were set up. Discard the EdU working solution, wash the cells, fix them with paraformaldehyde, and permeabilize them with 0.5% Triton X-100. Prepare the Click reaction solution according to the EdU kit instructions (add buffer, CuSO4, and azide fluorescent dye sequentially, mix gently, and avoid vigorous shaking to prevent air bubbles). Place the permeabilized sample in a light-protected humidified chamber, add the Click reaction solution to completely cover the cells, and incubate at room temperature in the dark for 30 minutes to allow the fluorescent dye to covalently bind to the alkyne group of EdU and label the proliferating cells. Add DAPI and anti-fluorescence quencher premix, mount the sample directly, and observe under a fluorescence microscope: EdU-positive cells show corresponding fluorescence (FITC-green), and all cell nuclei labeled with DAPI appear blue. Randomly select 5-10 fields of view for counting, and calculate the EdU positivity rate = (number of EdU-positive cells / total number of cell nuclei) × 100%.

[0039] The results are as follows Figure 3 As shown, Figure 3 Under the inverted fluorescence microscope shown in Figure A, the cell nuclei appear blue after DAPI staining, while cells in the proliferating state show EdU-specific green fluorescence. Figure 3 As shown in Figure B, the EdU-positive cell rates in the control group, NC group, and ZEB1siRNA3 group were 33.75%±0.71%, 32.16%±5.04%, and 16.22%±2.74%, respectively. The EdU-positive rate in the ZEB1siRNA3 group was significantly lower than that in the control group and the negative control group, and the difference between the groups was statistically significant. P<0.05). The above results indicate that ZEB1 gene knockout can significantly inhibit the in vitro proliferation capacity of vascular endothelial cells.

[0040] Example 4: Effects of ZEB1 siRNA on the migration function of human umbilical vein endothelial cells HUVECs in the logarithmic growth phase were divided into 5 × 10 5 Cells were seeded at a density of 1 cell / well in 6-well plates and transfected with ZEB1 siRNA3 according to the transfection procedure described in Example 1. After transfection, the cells were incubated in an incubator for 6 hours, and then the medium was replaced with DMEM containing 10% fetal bovine serum. When the transfection efficiency was confirmed to be as high as 80% by using an inverted fluorescence microscope, a straight line was drawn vertically along the pre-drawn horizontal line on the bottom of the plate at a uniform speed using a 200 μL pipette tip. 1-2 parallel scratches were made in each well. Cells were washed 3 times with PBS buffer, and then cultured in serum-free / low-serum DMEM medium and photographed. Photographs were taken at 0h, 6h, and 12h, and the scratches were photographed at three positions: the center, left, and right. The scratch area was calculated using Image-J software. The siRNA group was transfected at the same time, while the Control group was treated with PBS buffer as a control group.

[0041] The results are as follows Figure 4 As shown, Figure 4 A showed that after ZEB1 gene knockdown, the number of cells in the scratch region migrating to the scratch center was significantly less than that in the control group; Figure 4 B showed that the scratch healing rate at 6 hours was 8.39±3.74% in the control group and 8.09±0.21% in the ZEB1siRNA3 group. At 12 hours, the scratch healing rate was 71.67±2.77% in the control group and 8.39±4.84% in the ZEB1siRNA3 group. The healing rate in the ZEB1siRNA3 group was significantly lower than that in the control group. P The value was <0.05, suggesting that ZEB1 gene knockdown as an intervention method can inhibit the migration ability of vascular endothelial cells.

[0042] Example 5: The intervention effect of ZEB1siRNA as a gene therapy in a mouse corneal neovascularization model After one week of acclimatization feeding, C57BL / 6 mice were randomly divided into 4 groups of 4 mice each. The following treatments were performed: ① PBS negative control group; ② Free siRNA treatment group (ZEB1siRNA3); ③ SM102-siRNA treatment group (SM102 lipid material was dissolved in ethanol (organic phase), ZEB1siRNA3 was dissolved in citrate buffer (aqueous phase), and the aqueous phase was rapidly added to the organic phase at a N / P ratio of 6:1 at room temperature. After vortexing for 30 s, the organic solvent was removed by dialysis with PBS buffer to obtain the SM102-ZEB1siRNA3 nanocomplex); ④ Lipo2000-siRNA treatment group (4 μL of Lipofectamine 2000 reagent and ZEB1siRNA3 were diluted with Opti-MEM medium, respectively, and mixed to a total volume of 2 mL after standing at room temperature for 5 min. The mixture was gently blown and incubated at room temperature for 20 min to form the Lipo2000-ZEB1siRNA3 complex). All mice were modeled with corneal alkali burn using 0.1 mol / L NaOH solution. Twenty-four hours after modeling, mice in each group received topical ocular administration of the drug to their right eye, while the left eye was left untreated. Before administration, the conjunctival sac of the mouse's right eye was gently rinsed with sterile saline to remove residual secretions and contaminants. After the ocular surface dried, 5 μL of the corresponding drug (siRNA concentration of 4 μM in each group) was instilled, and the eyelid was gently pinched for 10-15 seconds to ensure full contact and absorption of the drug on the corneal surface. The administration regimen was 14 consecutive days, twice daily (morning and evening), with strict consistency in dosage and frequency. During this period, the mice's mental state, ocular surface infection (e.g., increased secretions, worsening corneal opacity), and systemic adverse reactions were closely monitored. Any abnormalities were recorded and addressed promptly. On days 1, 3, 5, 7, and 14 after administration, the right cornea of ​​the mice was photographed using a handheld slit-lamp microscope. The mouse's head and body position were fixed during photography to ensure consistent viewing angle and focal length for each image, clearly capturing the growth range, density, and branching of corneal neovascularization. The length and area of ​​corneal neovascularization were quantitatively analyzed using ImageJ software.

[0043] The results are as follows Figure 5 and Figure 6As shown, on day 14 after drug administration, the longest vessel length in the PBS negative control group was 1.37±0.17 mm, and the area was 8.39±0.41 mm²; the longest vessel length in the free siRNA treatment group was 1.20±0.07 mm, and the area was 8.26±0.19 mm²; the longest vessel length in the Lipo2000-siRNA treatment group was 0.88±0.05 mm, and the area was 6.79±0.28 mm²; and the longest vessel length in the SM102-siRNA treatment group was 1.01±0.08 mm, and the area was 7.54±0.36 mm². The longest vessel length and area in the SM102-siRNA treatment group and the Lipo2000-siRNA treatment group were significantly lower than those in the PBS control group, and the differences were statistically significant. P The value was <0.05, suggesting that ZEB1siRNA, as a gene therapy drug, can effectively inhibit corneal neovascularization.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A siRNA that targets and inhibits the expression of the ZEB1 gene, characterized in that, The siRNAs include siRNA1-2913, siRNA2-652, and siRNA3-1073. The sense and antisense strands of siRNA1-2913 are shown in SEQ ID NO:1-2, the sense and antisense strands of siRNA2-652 are shown in SEQ ID NO:3-4, and the sense and antisense strands of siRNA3-1073 are shown in SEQ ID NO:5-6.

2. The encoding gene of the siRNA that targets and inhibits ZEB1 gene expression as described in claim 1.

3. A recombinant expression vector into which the encoding gene of claim 2 is inserted.

4. An LNP-siRNA that targets and inhibits ZEB1 gene expression, characterized in that, It comprises siRNA and lipid nanoparticles that target and inhibit ZEB1 gene expression as described in claim 1.

5. The LNP-siRNA according to claim 4, characterized in that, The raw materials for the lipid nanoparticles include Lipofectamine 2000 and SM102 lipid materials.

6. The use of the siRNA of claim 1 that targets and inhibits the expression of the ZEB1 gene, the encoding gene of claim 2, the recombinant expression vector of claim 3, and the LNP-siRNA of claim 4 in the preparation of a drug for inhibiting angiogenesis.

7. The application according to claim 6, characterized in that, The drug includes pharmaceutically acceptable excipients.

8. The application according to claim 7, characterized in that, The pharmaceutically acceptable excipients include fillers, binders, disintegrants, and emulsifiers.

9. The application according to claim 6, characterized in that, The dosage forms of the drugs include tablets, oral liquid preparations, drops, injectable preparations, and capsule preparations.

10. The application of the siRNA of claim 1 that targets and inhibits ZEB1 gene expression, the encoding gene of claim 2, the recombinant expression vector of claim 3, and the LNP-siRNA of claim 4 in the study of the pathogenesis of corneal neovascularization.

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