Use of an RBM15 inhibitor in a drug for dry age-related macular degeneration

Abstract

The present invention relates to the field of biomedical technologies, and discloses the application of an RBM15 inhibitor in a drug for dry age-related macular degeneration. The target sequence of the RBM15 inhibitor is SEQ ID NO:3. The RBM15 inhibitor provided by the present invention can successfully treat dry AMD.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of RBM15 inhibitors in drugs for dry age-related macular degeneration. Background Art

[0002] Age-related macular degeneration (AMD) is a common progressive retinal disease in the elderly and is also one of the main causes of irreversible vision loss in the elderly worldwide. Due to the global aging population, its prevalence is increasing. AMD mainly includes two forms: dry and wet macular degeneration, with dry macular degeneration accounting for about 80-90% of all cases. In the early stage of dry AMD, it is characterized by soft drusen. In the late stage, the disease can progress to geographic atrophy (GA) or wet (exudative / neovascular) AMD. With the invention of ranibizumab in 2006, a drug reagent that inhibits the vascular endothelial growth factor (VEGF) signaling pathway, it has been breakthroughly applied clinically, greatly improving the prognosis of patients with wet AMD. However, there is currently no effective treatment method for dry AMD or GA. The pathogenesis of dry AMD has not been fully elucidated yet. Some risk factors, including aging, genetic susceptibility, and inflammation, etc., lead to the disruption of the normal cell homeostasis of the retina and are closely related to the occurrence of dry AMD. The main strategy for treating dry AMD is to reduce the death of retinal pigment epithelium (RPE) and photoreceptors caused by various pathological factors. Currently, except for antioxidant supplementation, there is no approved treatment method for early / mid-stage dry AMD. However, antioxidant vitamin and mineral therapy can only delay the disease progression in 20%-25% of the affected eyes. Therefore, it is urgent to explore the pathogenesis of dry AMD and find new treatment methods to improve the current clinical treatment status of this disease. Summary of the Invention

[0003] In order to solve the above technical problems, the present invention provides the application of RBM15 inhibitors in drugs for dry age-related macular degeneration. The RBM15 inhibitors provided by the present invention can successfully treat dry AMD.

[0004] The present invention provides the application of RBM15 inhibitors in drugs for dry age-related macular degeneration, and the target sequence of the RBM15 inhibitor is SEQ ID NO:3.

[0005] Further, the RBM15 inhibitor includes antisense nucleic acid, siRNA, miRNA or shRNA,

[0006] The target sequence of the antisense nucleic acid, siRNA, miRNA or shRNA is SEQ ID NO:3.

[0007] Further, the sense strand of the siRNA sequence is as shown in SEQ ID NO: 1, and the antisense strand of the siRNA sequence is as shown in SEQ ID NO: 2.

[0008] Further, the shRNA is obtained by constructing siRNA targeting RBM15. The sense strand of the siRNA sequence is as shown in SEQ ID NO: 1, and the antisense strand of the siRNA sequence is as shown in SEQ ID NO: 2.

[0009] Further, the sequence SEQ ID NO: 1 is GGGUUCAACUGGAGGCAAA.

[0010] Further, the sequence SEQ ID NO: 2 is UUUGCCUCCAGUUGAACCC.

[0011] The present invention also provides a pharmaceutical composition for treating dry age-related macular degeneration, which comprises: an RBM15 inhibitor, and a pharmaceutically acceptable carrier;

[0012] The sequence of the RBM15 is as shown in SEQ ID NO: 3;

[0013] The RBM15 inhibitor refers to a molecule having an inhibitory effect on RBM15. The RBM15 inhibitor is selected from antisense nucleic acid, siRNA, miRNA or shRNA, and the target sequence of the antisense nucleic acid, siRNA, miRNA or shRNA is as shown in SEQ ID NO: 3.

[0014] Further, the pharmaceutically acceptable carrier includes lentivirus.

[0015] Further, the SEQ ID NO: 3 is CACATGGTGATAGTTATAGTA.

[0016] The embodiments of the present invention have the following technical effects:

[0017] 1. The entry point is close to clinical practice. The purpose of the present invention is clear, aiming to solve the actual problems that plague the treatment of dry age-related macular degeneration clinically. Starting from the deficiencies of current treatment methods, a new potential treatment method has been invented. If it can be put into clinical practice one day, it can specifically improve the current situation and thus benefit the majority of patients.

[0018] 2. The guiding concept is novel. The present invention combines the latest and most advanced basic research results at home and abroad, proposes a creative hypothesis, and proves the practical operability of the hypothesis on the basis of sufficient experiments.

[0019] 3. It has great economic potential. The present invention focuses on the clinical practical problems with broad market demands. Currently, there are numerous patients with dry age-related macular degeneration, and the treatment methods urgently need to be improved. Therefore, once the present invention is successfully transformed, it will obtain good market benefits.

[0020] 4. In the present invention, dry age-related macular degeneration can be successfully treated by an RBM15 inhibitor. Specifically, the RBM15 inhibitor of the present invention inhibits RBM15, thereby reducing the expression of the m6A methyltransferase, ultimately reducing the methylation of related proteins, and reducing the decline and death of retinal pigment epithelial cells, achieving the purpose of treating dry age-related macular degeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 It is a data graph of animal experiments provided by the present invention, where Figure 1 a in it is the HE staining of the blank group, Figure 1 b in it is the HE staining of the control group, Figure 1 c in it is the TUNEL staining of the blank group, Figure 1 d in it is the TUNEL staining of the control group, Figure 1 e in it is the m6A methylation level.

[0023] Figure 2 It is the relative expression levels of mRNA of the blank group and the control group provided by the present invention.

[0024] Figure 3 It is a data graph of the protein expression levels of the blank group and the control group provided by the present invention, where Figure 3 a in it is the electrophoresis results of the blank group and the control group, Figure 3 b in it is the relative expression level of the METTL3 protein, Figure 3 c in it is the relative expression level of the METTL14 protein, Figure 3 d in it is the relative expression level of the WTAP protein, Figure 3 e in it is the relative expression level of the ZC3H13 protein, Figure 3 f in it is the relative expression level of the KIAA1429 protein, Figure 3 g in it is the relative expression level of the ALKBH5 protein, Figure 3 h in it is the relative expression level of the RBM15 protein, Figure 3Where i is the relative expression level of the FTO protein, Figure 3 where j is the relative expression level of the YTHDF2 protein, Figure 3 where k is the relative expression level of the YTHDC1 protein.

[0025] Figure 4 These are the results of establishing the cell model provided by the present invention.

[0026] Figure 5 These are the detection results of the cell experiments provided by the present invention, where Figure 5 a is the electrophoresis data of the blank group and the control group, Figure 5 b is the relative expression level of the RBM15 protein in the blank group and the control group, Figure 5 c is the relative expression level of the FTO protein in the blank group and the control group.

[0027] Figure 6 These are the expression conditions of the EPR cell senescence genes at the protein level provided by the present invention, where Figure 6 a is the electrophoresis data of the blank group and the control group, Figure 6 b is the relative expression level of the p-ERK1 / 2 protein, Figure 6 c is the relative expression level of the P53 protein, Figure 6 d is the relative expression level of the ERK1 / 2 protein, Figure 6 e is the relative expression level of the P21 protein, Figure 6 f is the relative expression level of the P16 protein, Figure 6 g is the relative expression level of the γ-H2AX protein.

[0028] Figure 7 These are the animal experiment data provided by the present invention, where Figure 7 a is the fundus image of the mouse, Figure 7 b is the OCT of the mice in the Cont group, Figure 7 c is the OCT of the mice in the SI group, Figure 7 d is the OCT of the mice in the SI+si-NC group, Figure 7 e is the OCT of the mice in the SI+si-RBM15 group.

[0029] Figure 8 These are the staining results of the animal experiments provided by the present invention, where Figure 8 a is the HE staining of the Cont group, Figure 8 b is the HE staining of the SI group, Figure 8 c is the HE staining of the SI+siNC group, Figure 8 d is the HE staining of the SI+siRBM15 group, Figure 8 e is the TUNEL staining of the Cont group, Figure 8 f is the TUNEL staining of the SI group,Figure 8 In g, it is the TUNEL staining of the SI + siNC group. Figure 8 In h, it is the TUNEL staining of the SI + siRBM15 group.

[0030] Figure 9 It shows the mitochondrial conditions of the blank group and the control group in the cell experiment. Among them, Figure 9 In a, it is the fluorescence dots of the nuclear dye DAPI in the SI + si-NC group. Figure 9 In b, it is the fluorescence dots of mitochondria in the SI + si-NC group. Figure 9 In c, it is the fluorescence dots of all fluorescence channels of the nucleus in the SI + si-NC group. Figure 9 In d, it is the fluorescence dots of the nuclear dye DAPI in the SI + si-RBM15 group. Figure 9 In e, it is the fluorescence dots of mitochondria in the SI + si-RBM15 group. Figure 9 In f, it is the fluorescence dots of all fluorescence channels of the nucleus in the SI + si-RBM15 group.

[0031] Figure 10 It shows the detection of the cell senescence marker Aβ1-42 in the blank group and the control group of the cell experiment. Among them, Figure 10 In a, it is the fluorescence dots of the nuclear dye DAPI in the SI + si-NC group. Figure 10 In b, it is the fluorescence dots of Aβ1-42 cells in the SI + si-NC group. Figure 10 In c, it is the fluorescence dots of all fluorescence channels of the nucleus in the SI + si-NC group. Figure 10 In d, it is the fluorescence dots of the nuclear dye DAPI in the SI + si-RBM15 group. Figure 10 In e, it is the fluorescence dots of Aβ1-42 cells in the SI + si-RBM15 group. Figure 10 In f, it is the fluorescence dots of all fluorescence channels of the nucleus in the SI + si-RBM15 group.

[0032] Figure 11 It shows the expression of the EPR cell senescence gene at the mRNA level provided by the present invention. Among them, Figure 11 In a, it is the relative mRNA expression level of the P53 protein. Figure 11 In b, it is the relative mRNA expression level of the P16 protein. Figure 11 In c, it is the relative mRNA expression level of the P21 protein.

[0033] Figure 12 It shows the SA-β-gal staining results provided by the present invention. Among them, Figure 12 In a, it is the staining result of the blank group. Figure 12 In b, it is the staining result of the control group.

[0034] Figure 13It is an indicator of ROS oxidative stress loss in animal experiments provided by the present invention, where Figure 13 in a, a is the fluorescence point of dye DAPI in the Cont group, Figure 13 in b, b is the fluorescence point of ROS in the Cont group, Figure 13 in c, c is the fluorescence point of all fluorescence channels in the Cont group, Figure 13 in d, d is the fluorescence point of dye DAPI in the SI group, Figure 13 in e, e is the fluorescence point of ROS in the SI group, Figure 13 in f, f is the fluorescence point of all fluorescence channels in the SI group, Figure 13 in g, g is the fluorescence point of dye DAPI in the SI+siNC group, Figure 13 in h, h is the fluorescence point of ROS in the SI+siNC group, Figure 13 in i, i is the fluorescence point of all fluorescence channels in the SI+siNC group, Figure 13 in j, j is the fluorescence point of dye DAPI in the SI+siRBM15 group, Figure 13 in k, k is the fluorescence point of ROS in the SI+siRBM15 group, Figure 13 in l, l is the fluorescence point of all fluorescence channels in the SSI+siRBM15 group.

[0035] Figure 14 It is the staining condition of γ-H2AX in animal experiments provided by the present invention, where Figure 14 in a, a is the fluorescence point of dye DAPI in the Cont group, Figure 14 in b, b is the fluorescence point of γ-H2AX in the Cont group, Figure 14 in c, c is the fluorescence point of all fluorescence channels in the Cont group, Figure 14 in d, d is the fluorescence point of dye DAPI in the SI group, Figure 14 in e, e is the fluorescence point of γ-H2AX in the SI group, Figure 14 in f, f is the fluorescence point of all fluorescence channels in the SI group, Figure 14 in g, g is the fluorescence point of dye DAPI in the SI+siNC group, Figure 14 in h, h is the fluorescence point of γ-H2AX in the SI+siNC group, Figure 14 in i, i is the fluorescence point of all fluorescence channels in the SI+siNC group, Figure 14 in j, j is the fluorescence point of dye DAPI in the SI+siRBM15 group, Figure 14 in k, k is the fluorescence point of γ-H2AX in the SI+siRBM15 group, Figure 14 in l, l is the fluorescence point of all fluorescence channels in the SI+siRBM15 group.

[0036] Figure 15 It is a data graph of cell senescence in animal experiments of the present invention, where Figure 15 in a, a is the experimental result of the Cont group,Figure 15 where b is the experimental result of the SI group, Figure 15 where c is the experimental result of the SI+siNC group, Figure 15 where d is the experimental result of the SI+siRBM15 group. Specific implementation manners

[0037] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described clearly and completely below. Apparently, the described embodiments are only some embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope protected by the present invention.

[0038] In a first aspect, in some embodiments of the present invention, there is provided an application of an RBM15 inhibitor in a drug for dry age-related macular degeneration, and the target sequence of the RBM15 inhibitor is SEQ ID NO:3.

[0039] In some embodiments, the RBM15 inhibitor includes antisense nucleic acid, siRNA, miRNA or shRNA,

[0040] and the target sequence of the antisense nucleic acid, siRNA, miRNA or shRNA is SEQ ID NO:3.

[0041] In some embodiments, the sense strand of the siRNA sequence is as shown in SEQ ID NO:1, and the antisense strand of the siRNA sequence is as shown in SEQ ID NO:2.

[0042] In some embodiments, the shRNA is obtained by constructing an siRNA targeting RBM15, the sense strand of the siRNA sequence is as shown in SEQ ID NO:1, and the antisense strand of the siRNA sequence is as shown in SEQ ID NO:2.

[0043] In some embodiments, the sequence SEQ ID NO:1 is GGGUUCAACUGGAGGCAAA.

[0044] In some embodiments, the sequence SEQ ID NO:2 is UUUGCCUCCAGUUGAACCC.

[0045] In the present invention, the directions of the sequences of the sense strand and the antisense strand are both 5'-3'.

[0046] In a second aspect, some embodiments of the present invention further provide a pharmaceutical composition for treating dry age-related macular degeneration, the pharmaceutical composition comprising: an RBM15 inhibitor, and a pharmaceutically acceptable carrier;

[0047] The sequence of the RBM15 is as shown in SEQ ID NO:3;

[0048] The RBM15 inhibitor refers to a molecule having an inhibitory effect on RBM15, and the RBM15 inhibitor is selected from antisense nucleic acids, siRNA, miRNA or shRNA, and the target sequence of the antisense nucleic acid, siRNA, miRNA or shRNA is as shown in SEQ ID NO:3.

[0049] In some embodiments, the pharmaceutically acceptable carrier includes lentivirus.

[0050] In some embodiments, the SEQ ID NO:3 is CACATGGTGATAGTTATAGTA.

[0051] The following is a detailed description in combination with specific examples and comparative examples:

[0052] (1) Design and synthesis of siRNA and shRNA:

[0053] According to the mRNA sequence of the RBM15 protein, siRNA targeting RBM15 was designed by ourselves. The sense strand sequence of the siRNA is as follows: GGGUUCAACUGGAGGCAAA. Then, General Biosystems Co., Ltd. was commissioned to synthesize the siRNA and prepare a working solution with a concentration of 50 nmol for standby. And the siRNA sequence was inserted into pLK0.1puro by gene cloning method to construct shRNA targeting RBM15, named sh-RBM15. At the same time, Hanheng Biotechnology Co., Ltd. was commissioned to package it into lentivirus and lentivirus without siRNA sequence for standby (virus titer 10 11 viruses / mL).

[0054] (2) Cell culture:

[0055] The RPE cells (retinal pigment epithelial cells) were cultured in DMEM high-glucose medium containing 10% fetal bovine serum in a constant temperature incubator at 37 °C and 5% CO2.

[0056] (3) Construction of cell model by treating RPE cells with sodium iodate:

[0057] The collected RPE cells after digestion were counted and seeded into 6-well plates at 1.2×10 5 cells / well, and placed in a constant temperature incubator at 37 °C and 5% CO2 for culture.

[0058] On the next day, after observing cell adhesion, discard the old culture medium. After treating RPE cells with 0 mM, 1 mM, 2 mM, 3 mM, 10 mM, 20 mM, 30 mM, and 40 mM sodium iodate for 24 h and 48 h respectively, digest the cells and centrifuge (1000 rpm, 3 min). The RPE cells treated with sodium iodate are denoted as SI, and the RPE cells not treated with sodium iodate are denoted as control.

[0059] (4) Cell experiment:

[0060] After treating RPE cells with 20 mM sodium iodate for 24 h in (3) above, change the RPE cells to DMEM high-glucose culture medium containing 10% fetal bovine serum for culture; take out 2 1.5 ml EP tubes, add 100 μL of DMEM high-glucose culture medium without fetal bovine serum to each tube for culture. Add 3 μL of Lip3000 transfection reagent to one tube, and add 5 μL of the RBM15 inhibitor (siRNA working solution) obtained in (1) to the other tube. The group without the RBM15 inhibitor is the blank group (SI + si-NC), and the other tube with the RBM15 inhibitor is the control group (SI + si-RBM15). Mix the two tubes separately and place them at room temperature for 15 min. Slowly add the liquid in the above tubes to the cells (discard the original culture medium in the cells and add 1.8 mL of fresh culture medium), and continue to culture in the cell culture incubator for 24 h.

[0061] (5) Establish a dry AMD mouse model:

[0062] Twelve 6-8-month-old male C57B / L6 nude mice are randomly divided into two groups, with 6 mice in each group. The two groups are: the cont group (i.e., 0 mg / mL NaIO3) and the control group (i.e., SI, 35 mg / mL NaIO3). By means of tail vein injection, inject the mice in the control group every day, and inject the same volume of normal saline into the cont group as that in the control group. According to the body weight of the mice, the volume of each tail vein injection should meet 35 mg / kg of sodium iodate, and inject continuously for 7 days. Then, perform HE, TUNEL staining on the eyeballs of the mice, detect the total RNA m6A methylation level in the retinal tissue, and detect the mRNA and protein expression changes of the main m6A methyltransferases (METTL3, METTL14, WTAP, RBM15, ZC3H13, KIAA1429, YTHDF2, and YTHDC1), demethylases (FTO and ALKBH5) by qRT-PCR and Western blot.

[0063] (6) Animal experiment to verify that the RBM15 inhibitor can treat dry AMD:

[0064] Twenty-four male C57B / L6 nude mice aged 6 - 8 months were randomly divided into four groups of 6 mice each. One group was the blank group (i.e., the Cont group), and the mice in one group were injected with the same volume of normal saline into the tail vein as the second group; the second group was injected with NaIO3 into the tail vein (i.e., the SI group); the third group was injected with NaIO3 into the tail vein and the RBM15 inhibitor (5 μL) was injected into the mouse eyeball, where the RBM15 inhibitor was the lentivirus obtained in (1) (i.e., the SI + siRBM15 group); the fourth group was injected with NaIO3 into the tail vein and NC vitreous was injected into the mouse eyeball, where 5 μL of the lentivirus without the siRNA sequence was injected into the mouse eyeball (i.e., the SI + siNC group).

[0065] (7)Western blot test for protein concentration:

[0066] After collecting the cells in (4) above, 100 μL of 1× loading buffer was added respectively, and boiled at 95℃ for 5 min to prepare for Western blot:

[0067] 1) Prepare 10% SDS denaturing polyacrylamide gel (lower separating gel, single-sided): 780 μL of 1% methylene, 2 mL of 30% acrylamide, 1.5 mL of 1.5 M tris(hydroxymethyl)aminomethane (pH 8.8), 30 μL of 20% SDS, 40 μL of 10% ammonium persulfate, N,N,N ' ,N ' -tetramethylethylenediamine (TEMED) 4.0 μL. After mixing, quickly pour the gel to about 2 / 3 of the total height of the glass plate, and then add 1 mL of water-saturated n-butanol above the gel to ensure the flatness of the upper layer of the gel, and let it stand until the gel solidifies.

[0068] 2) Prepare 4% SDS denaturing polyacrylamide gel (upper stacking gel, single-sided): 200 μL of 1% methylene, 270 μL of 30% acrylamide, 500 μL of 1.0 M tris(hydroxymethyl)aminomethane (pH 6.8), 30 μL of 20% SDS, 40 μL of 10% ammonium persulfate, N,N,N ' ,N ' -tetramethylethylenediamine 4 μL. After mixing, quickly pour the gel to fill the glass plate, insert the comb, and let it stand until the gel solidifies. Before electrophoresis, remove the comb, place the gel in 1× Tris-glycine electrophoresis buffer, and blow clean the loading wells with a syringe needle.

[0069] 3) Take 20 μL of protein samples for loading respectively, and perform 10% SDS denaturing polyacrylamide gel electrophoresis (SDS-PAGE) until the target protein is effectively separated and then stop electrophoresis.

[0070] 4) After electrophoresis, take out the gel and place it in a sandwich clip dedicated for membrane transfer. Place the gel at the negative electrode and the PVDF membrane at the positive electrode. Transfer the membrane at a constant current of 200 mA at 4 °C for 1 h in the transfer buffer to transfer the proteins in the gel onto the PVDF membrane to form an imprint.

[0071] 5) Place the membrane in the blocking solution and shake it at room temperature for half an hour for blocking.

[0072] 6) Cut the membrane according to the imprint position of the protein, place it in the antibody dilution solution containing the corresponding primary antibody CALB2 (diluted 1:500), and shake it at 4 °C overnight.

[0073] 7) Place the membrane in 1×TBST buffer and shake it for rinsing for 5 min, three times in total.

[0074] 8) Place the membrane in TBST containing the corresponding secondary antibody (goat anti-rabbit HRP-labeled 1:10000) and incubate it at room temperature for 1 h.

[0075] 9) Place the membrane in 1×TBST solution and shake it for rinsing for 5 min, three times in total.

[0076] 10) Place the membrane in Super ECL Plus hypersensitive luminescent solution for 2 min.

[0077] 11) Immediately place the membrane in an exposure cassette, expose the photosensitive film in a darkroom, and then perform development and fixation.

[0078] (8) qRT-PCR (real-time fluorescence quantitative PCR):

[0079] First, extract the total RNA of the cells to be processed, then reverse-transcribe it into cDNA, and finally design corresponding primers according to different genes for qRT-PCR amplification reaction. The primer sequences are as follows:

[0080] Table 1 Primer sequences

[0081]

[0082] Results and analysis:

[0083] Dry AMD is a common eye disease in the elderly, usually manifested as central vision loss. An important pathological feature of this disease is the functional decline or death of RPE cells, resulting in retinal pigmentation, retinal degenerative changes, and the formation of new blood vessels. Figure 1 (a)- Figure 1 (b) Using NaIO3 to construct a dry AMD mouse model, it can be found that when injecting 35 mg / kg via the tail vein, metaplasia of retinal pigment epithelial cells occurred. Figure 1 (c)- Figure 1(d), it can be found that under a microscope with a magnification of 400 times, obvious apoptosis of retinal pigment epithelial cells was observed in the control group of mice. It can be seen that in the present invention, an AMD mouse model was successfully constructed by injecting NaIO3.

[0084] Figure 1 (e), the m6A methylation levels of the mice in the blank group and the control group were tested. The test results showed that the m6A methylation level in the control group was significantly increased. It was speculated that the increase in the m6A methylation level might cause the decline and death of retinal pigment epithelial cells.

[0085] The present invention further studied the mRNA expression results of m6A methyltransferases related to the m6A methylation level, as Figure 2 shown. In Figure 2 , it was found that the mRNA expressions of METTL3, METTL14, ZC3H13, ALBKH5, and FTO were significantly decreased, while the mRNA expressions of WTAP, RBM15, and YTHDF2 were significantly increased, and there were no obvious differences in others. In Figure 3 , compared with the Control group, the protein expressions of METTL14 and FTO were significantly decreased, and the protein expression of RBM15 was significantly increased, and there were no obvious differences in others. Since the results of RBM15 and FTO were consistent with the results of the total m6A RNA methylation level, these 2 targets of RBM15 and FTO were selected for cell experiments.

[0086] In Figure 4 , after 24 h and 48 h, compared with the 0 mM group, the viability of RPE cells in the 1 mM, 2 mM, 3 mM, 10 mM, 20 mM, 30 mM, and 40 mM groups was significantly decreased. Among them, the cell viability of 2 mM sodium iodate at 24 h was 48.75%, which was about half of the cell viability of 0 mM sodium iodate at 24 h. Therefore, a cell model was successfully established with 2 mM sodium iodate for 24 h. The retinal pigment epithelial cells were cultured with 2 mM sodium iodate for 24 h to construct a cell model for subsequent experiments. Figure 5 , in the cell model, compared with the Control group, the protein expression of RBM15 in the SI group was significantly increased, while the protein expression of FTO had no obvious difference. It can be seen that the result of RBM15 was consistent with the m6A RNA methylation level. It was speculated that RBM15 might mediate the regulation of RPE cell senescence and thus participate in the pathogenesis of AMD.

[0087] Figure 6 , in the two dry AMD cell models formed by stimulating with sodium iodate, in one of the cell models, the RBM15 of the cells was down-regulated (SI + si-RBM15) and compared with the blank group (SI + si-NC). Figure 6The results showed that the expression levels of γ-H2AX, p-ERK1 / 2, P53 protein, P21 protein, P16 protein, and ERK1 / 2 in cells could be used to evaluate the senescence and proliferation of retinal pigment epithelial cells. When the RBM15 of cells was down-regulated, compared with the blank group, the expression levels of γ-H2AX, p-ERK1 / 2, P53 protein, P21 protein, and P16 protein in the SI+si-RBM15 group decreased significantly, and there was no significant difference in the expression level of ERK1 / 2. It can be seen that by inhibiting the expression of RBM15 in cells, the senescence of retinal pigment epithelial cells can be reduced and the proliferation of retinal pigment epithelial cells can be promoted, thereby achieving the effect of reducing the incidence of AMD and treating dry AMD.

[0088] In Figure 11 it was evaluated the senescence and proliferation of cells at the mRNA level. The results showed that compared with the control group, and compared with the SI+si-NC group, the expression levels of P53 protein, P21 protein, and P16 protein in the SI+si-RBM15 group decreased significantly, further verifying that by inhibiting the expression of RBM15 in cells, the senescence of retinal pigment epithelial cells can be reduced and the proliferation of retinal pigment epithelial cells can be promoted.

[0089] Figure 9 it was found that compared with the blank group control group, the number of mitochondria in the SI+si-NC group was small and aggregated to one side, while the number of mitochondria in the SI+si-RBM15 group recovered. In cells, mitochondrial damage will cause cell death. In Figure 9 the results showed that inhibiting RBM15 could reduce mitochondrial death, was beneficial to mitochondrial recovery, and thus reduced the death of retinal pigment epithelial cells.

[0090] In Figure 10 it was detected the Aβ1-42 cells of cell senescence markers and found that compared with the control group, the senescence of Aβ1-42 cells in the SI-si-NC group was significantly weakened. It can be seen that inhibiting RBM15 can also relieve cell senescence.

[0091] Figure 12 it was performed SA-β-gal staining in cell experiments. SA-β-gal is a kind of β-galactosidase, and its activity will increase during cell senescence, which is a commonly used marker of cell senescence. In Figure 12 the results showed that the number of positive cells stained by SA-β-gal in the control group decreased compared with the blank group. It can be seen that inhibiting RBM15 can relieve cell senescence.

[0092] In Figure 7As shown in [reference], by comparing normal animals and the sodium iodate-induced dry age-related macular degeneration animal model, it was found that the eyes of mice in the SI group developed lesions of dry AMD, resulting in changes in the RPE layer. However, in the SI+siRBM15 group of mice, the injection of the RBM15 inhibitor achieved the effect of treating dry AMD, and there was little difference in the RPE layer between the SI+siRBM15 group and the Cont group.

[0093] In Figure 8 further verified by case indicators, it was found in HE staining that metaplasia occurred in the RPE layer of the SI group. In the SI+siRBM15 group, it was found that the RPE layer was treated and no metaplasia occurred. Moreover, in other layer structures of the eyeball, there was also a certain therapeutic effect. In TUNEL staining, the detection of apoptosis showed that the apoptotic cells in the SI+siRBM15 group were significantly reduced compared with those in the SI group and the SI+siNC group.

[0094] In Figure 13 , ROS is an indicator of oxidative stress damage. When cells and tissues are damaged, the ROS indicator can be used for evaluation. In Figure 13 the results showed that after adding the RBM15 inhibitor (SI+siRBM15), the oxidative stress damage of cells and tissues could be reduced.

[0095] In Figure 14 , γ-H2AX is a histone and a member of the histone H2A family. Histones are one of the main structural proteins of chromatin, participating in the regulation of gene transcription and DNA packaging, and playing an important role in maintaining genomic stability and DNA damage repair. In addition to its role in DNA damage repair, γ-H2AX is also involved in cell processes such as apoptosis, cell cycle regulation, chromatin remodeling, and transcriptional regulation. Therefore, abnormal or defective functions of the γ-H2AX protein may lead to cell death. In Figure 14 the results showed that after adding the RBM15 inhibitor, the abnormality of γ-H2AX could be reduced, thereby reducing cell death caused by DNA damage.

[0096] Figure 15 is the result of cell senescence. It can be found that after adding the RBM15 inhibitor, the senescence of RPE cells can be reduced.

[0097] In summary, the RBM15 inhibitor in the present invention can mediate the regulation of RPE cell senescence, thereby participating in the treatment of dry AMD.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. Use of an RBM15 inhibitor in the preparation of a drug for dry age-related macular degeneration, characterized in that, The target sequence of the RBM15 inhibitor is SEQ ID NO: 3; The RBM15 inhibitor is siRNA or shRNA; The sense strand of the siRNA sequence is SEQ ID NO: 1, and the antisense strand of the siRNA sequence is SEQ ID NO: 2; The shRNA is obtained by constructing siRNA targeting RBM15.

2. A pharmaceutical composition for treating dry age-related macular degeneration, characterized in that, The pharmaceutical composition comprises: an RBM15 inhibitor, and a pharmaceutically acceptable carrier; The sequence of the RBM15 is SEQ ID NO: 3; The RBM15 inhibitor refers to a molecule having an inhibitory effect on RBM15, and the RBM15 inhibitor is siRNA or shRNA; The sense strand of the siRNA sequence is SEQ ID NO: 1, and the antisense strand of the siRNA sequence is SEQ ID NO: 2; The shRNA is obtained by constructing siRNA targeting RBM15.

3. The pharmaceutical composition for treating dry age-related macular degeneration according to claim 2, wherein, The pharmaceutically acceptable carrier includes lentivirus.

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