A small interfering RNA and its application

Abstract

The present invention provides a small interfering RNA and its application, belonging to the technical field of targeted drugs. The small interfering RNA is a PARP10 inhibitor, including any one or both of PARP10 siRNA-1 and PARP10 siRNA-2. The small interfering RNA provided by the present invention is a PARP10 inhibitor, which can target the PARP10 gene of CD4+ T cells of patients with Vogt-Koyanagi-Harada syndrome, thereby down-regulating the expression of the PARP10 gene, inhibiting the proportion of Th1 cells in CD4+ T cells of patients with Vogt-Koyanagi-Harada syndrome, reducing the ocular inflammatory factors of the patients, approaching the intraocular state of normal people, realizing the improvement or treatment of the ocular inflammation of patients with Vogt-Koyanagi-Harada syndrome, and thus achieving the purpose of saving the patients' vision.

Description

Technical Field

[0001] The present invention belongs to the technical field of targeted drugs, and particularly relates to a small interfering RNA and its application. Background Art

[0002] Uveitis is a type of inflammatory disease that seriously affects vision and involves ocular structures such as the uvea, retina, retinal blood vessels, and vitreous. This type of disease is one of the main causes of blindness globally. In different countries and regions, blindness caused by uveitis accounts for 10%-25% of all causes of blindness. Therefore, uveitis poses a major threat to public health globally. Due to its complex etiology and diverse types, the treatment and management of uveitis have always been one of the key areas of ophthalmic research.

[0003] Existing treatment methods mainly focus on controlling the inflammatory response and preventing the occurrence of complications. Among them, Vogt-Koyanagi-Harada (VKH) syndrome is a common type of uveitis, accounting for 13.5% of all cases. The typical features of VKH syndrome include bilateral granulomatous uveitis and may be accompanied by involvement of the central nervous system, auditory system, and skin. If not treated promptly or correctly, the patient's vision will severely decline and even lead to blindness. According to statistics, at the initial diagnosis, the proportions of VKH syndrome patients with vision lower than 0.1 in the posterior uveitis stage, anterior uveal involvement stage, and recurrent anterior uveitis stage are 45%, 33.7%, and 38.0% respectively. In addition, VKH syndrome can also cause complications such as secondary glaucoma, optic nerve atrophy, and retinal atrophy, further exacerbating vision impairment.

[0004] Although there have been a large number of advances in the research on VKH syndrome, its pathogenesis is still not fully understood, especially the specific role of the imbalance between Th1 and Th17 responses in the disease remains controversial. This cognitive limitation hinders the development of more effective treatment methods. Existing treatments mostly rely on immunosuppressive agents and glucocorticoid drugs, but the long-term use of these drugs may cause serious side effects. In addition, the existing treatment methods are not targeted enough to effectively cope with the multi-system effects and complex pathological mechanisms of VKH syndrome. Therefore, finding new treatment targets and strategies has become an urgent problem to be solved.

[0005] Despite the continuous in - depth research on VKH syndrome, its exact pathogenesis remains not fully understood. Current studies suggest that VKH syndrome may be related to autoimmune reactions, especially the immune attack against melanocytes. However, the specific pathogenic process and key molecules have not been fully revealed. This limitation in understanding directly affects the development of new treatment methods. Existing treatment means mainly rely on doctors' experience and clinical observation, lacking targeted treatment for specific pathological mechanisms. In addition, since VKH syndrome involves multiple system involvements, a single treatment strategy is difficult to comprehensively control the disease progression and the occurrence of complications.

[0006] In summary, the existing treatment methods for VKH syndrome have obvious deficiencies, mainly reflected in the following aspects: First, the existing treatment lacks specificity and cannot effectively deal with the complex pathological mechanisms of the disease; Second, the side effects brought by long - term use of immunosuppressants and glucocorticoid drugs are serious, affecting the quality of life of patients; Finally, due to the lack of understanding of the key molecules and mechanisms of the disease occurrence, it limits the development of new targeted therapies. Therefore, in - depth study of the pathogenesis of VKH syndrome and revealing its key pathogenic factors are the key steps for developing more effective treatment methods in the future. This will provide safer and more effective treatment options for patients and help reduce the visual impairment and the decline in quality of life caused by the disease. Summary of the Invention

[0007] To solve the above problems, the present invention provides a small interfering RNA, which is a PARP10 inhibitor, including any one or both of PARP10 siRNA - 1 and PARP10 siRNA - 2.

[0008] Preferably, the nucleotide sequence of the small interfering RNA PARP10 siRNA - 1 is as shown in SEQ ID NO.1.

[0009] Preferably, the nucleotide sequence of the small interfering RNA PARP10 siRNA - 2 is as shown in SEQ ID NO.2.

[0010] Preferably, it also includes the nucleotide sequences of the chemically modified PARP10 siRNA - 1 and PARP10 siRNA - 2.

[0011] In addition, to solve the above - mentioned technical problems, the present invention also provides an application of the small interfering RNA as described above in the preparation of a drug targeting the PARP10 gene.

[0012] In addition, to solve the above - mentioned technical problems, the present invention also provides an application of the small interfering RNA as described above in the preparation of a drug for treating Vogt - Koyanagi - Harada syndrome.

[0013] Preferably, the dosage forms of the drug include: eye drops, eye ointments, injections, implants, microneedles, liposomes, nanoparticles, polymer controlled release systems, colloidal preparations, and suspension preparations.

[0014] In addition, to solve the above technical problems, the present invention also provides an application of the small interfering RNA as described above in the preparation of a drug for treating uveitis.

[0015] The present invention provides a small interfering RNA and its application, relating to the technical field of targeted drugs. The small interfering RNA is a PARP10 inhibitor, including any one or both of PARP10 siRNA-1 and PARP10 siRNA-2. The small interfering RNA provided by the present invention is a PARP10 inhibitor, which can target the PARP10 gene of CD4 + T cells of VKH syndrome patients, thereby downregulating the expression of the PARP10 gene, inhibiting the proportion of Th1 cells in CD4 + T cells of VKH syndrome patients, reducing the ocular inflammatory factors in patients, approaching the intraocular state of normal people, achieving the improvement or treatment of ocular inflammation in VKH syndrome patients, and thus achieving the purpose of saving the patients' vision. Description of the Drawings

[0016] Figure 1 It is the volcano plot of differential genes of CD4 + T cells of VKH syndrome patients in Example 1 of the present invention;

[0017] Figure 2 It is the volcano plot of differential proteins of CD4 + T cells of VKH syndrome patients in Example 2 of the present invention;

[0018] Figure 3 It is the Venn diagram of the combined analysis of differential genes and differential proteins in Example 3 of the present invention;

[0019] Figure 4 It is the schematic diagram of the results of verifying the expression level of PARP10 by real-time fluorescence quantitative PCR reaction in Example 4 of the present invention;

[0020] Figure 5 It is the schematic diagram of the PARP10 gene expression results after treatment with PARP10-si1 group, PARP10-si2 group, and PARP10-si1+si2 group in Example 6 of the present invention;

[0021] Figure 6 It is the flow cytometry scatter plot of investigating the effect of PARP10 interference on the proportion of Th1 cells in CD4 + T cells of VKH syndrome patients in Example 7 of the present invention. Detailed Embodiments

[0022] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the 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 protection scope of the present invention.

[0023] The present invention provides a small interfering RNA, which is a PARP10 inhibitor and includes any one or both of PARP10 siRNA-1 and PARP10 siRNA-2.

[0024] It should be noted that in the present invention, through experiments, it is proved that by using transcriptomics combined with proteomics techniques and in vitro molecular experiments, it is found that PARP10 (poly ADP-ribose polymerase family member 10) has a significantly increased expression level in CD4 + T cells of patients with active VKH syndrome. Inhibiting PARP10 can reduce the proportion of Th1 cells in CD4 + T cells. These results suggest that PARP10 may be a pathogenic molecule of VKH syndrome.

[0025] PARP10 is a member of the poly ADP-ribose polymerase family and plays an important role in DNA damage repair and apoptosis. Research shows that PARP inhibitors enhance the effect of immunotherapy from the perspective of the tumor microenvironment by inhibiting tumor cell-specific DNA damage repair and increasing the release of neoantigens.

[0026] Currently, there are few studies on PARP10. There are no reports at home and abroad on its research in inflammation and autoimmune diseases, and its function and mechanism in patients with VKH syndrome are also unclear. Therefore, the research on the PARP10 gene can provide a new target for the occurrence of VKH syndrome and also provide an exploratory treatment strategy for patients with VKH syndrome.

[0027] Small interfering RNA (siRNA) is a double-stranded RNA molecule composed of 21 to 25 nucleotides. siRNA is very important in the fields of biology and medicine because they are involved in a biological process called RNA interference (RNAi). RNAi is a mechanism for regulating gene expression in cells, and it can specifically reduce the expression of specific genes at the post-transcriptional level.

[0028] The main characteristics and functions of siRNA include:

[0029] Gene silencing: siRNA can specifically degrade the target mRNA complementary to its sequence through the RNAi mechanism, thereby preventing the translation of this mRNA and reducing the production of specific proteins.

[0030] High specificity: The pairing of siRNA molecules with target mRNA has a high degree of sequence specificity, which enables siRNA to precisely regulate specific genes.

[0031] Processing within cells: After being taken up by cells, siRNA is processed by the RNA-induced silencing complex (RISC). In RISC, one strand (the guide strand) of siRNA is retained, while the other strand (the passenger strand) is degraded.

[0032] Involvement in immune responses: siRNA is also involved in the cell's defense mechanism against virus invasion. They can target and degrade viral RNA, thereby inhibiting viral replication.

[0033] Therapeutic applications: Due to the high specificity of siRNA, they show great potential in gene therapy and the treatment of certain diseases, including cancer, genetic diseases, and certain viral infections.

[0034] Drug development: siRNA is being developed as a new type of drug for treating diseases that are difficult to target with traditional small molecules or biopharmaceuticals.

[0035] Research tool: siRNA is also widely used as a tool for studying gene function. By knocking down the expression of specific genes, researchers can explore the roles of these genes in cell physiology and diseases.

[0036] In summary, the development of siRNA technology provides new possibilities for gene regulation and drug development.

[0037] Small interfering RNA (siRNA) technology is a gene silencing strategy that reduces the expression of specific genes in cells by using specific double-stranded RNA molecules. In the context of Vogt-Koyanagi-Harada (VKH) syndrome, siRNA can be designed to target the PARP10 gene in CD4 + T cells.

[0038] VKH syndrome is an autoimmune disease that affects multiple organ systems, including the eyes. In this disease, the immune system mistakenly attacks the body's own tissues, resulting in inflammation and tissue damage. In particular, CD4 + T cells play an important role in the pathogenesis of VKH syndrome.

[0039] CD4 + T cells are a type of T cells that participate in immune responses. They can differentiate into various types of T helper cells (Th cells), including Th1 and Th2 cells. In VKH syndrome, Th1 cells are associated with the inflammatory response.

[0040] Th1 cells secrete an inflammatory factor called interferon-γ (IFN-γ), which plays a key role in promoting the inflammatory response. In patients with VKH syndrome, the overactivation of Th1 cells may lead to ocular inflammation.

[0041] PARP10 (poly(ADP-ribose) polymerase 10) is an enzyme related to DNA repair and cell death. In certain cases, PARP10 may be involved in the activation of immune cells and the inflammatory response.

[0042] In the present invention, specific siRNAs are designed to target the PARP10 gene, which can reduce the expression of PARP10 in CD4 + T cells. By reducing the level of PARP10, the differentiation and activation of Th1 cells can be reduced, thereby reducing the production of inflammatory factors such as IFN-γ.

[0043] By inhibiting the expression of the PARP10 gene with siRNAs, the proportion of Th1 cells in the eyes of patients with VKH syndrome and the secretion of IFN-γ can be reduced, resulting in a decrease in the level of ocular inflammatory factors. This helps to restore the intraocular environment of the patient to a state close to normal, reduce ocular inflammation, and thus helps to improve or treat the ocular inflammation of patients with VKH syndrome and may save or protect the patient's vision.

[0044] The potential of siRNA therapy lies in its high specificity and the advantage of being able to reduce the side effects of systemic drugs. siRNAs can directly act on key molecules in the pathological process, providing a new possibility for the treatment of autoimmune diseases such as VKH syndrome.

[0045] In summary, the small interfering RNA provided by the present invention is a PARP10 inhibitor, which can target the PARP10 gene of CD4 + T cells in patients with VKH syndrome, thereby downregulating the expression of the PARP10 gene, inhibiting the proportion of Th1 cells in CD4 + T cells in patients with VKH syndrome, reducing the ocular inflammatory factors in the patient, approaching the intraocular state of normal people, achieving the improvement or treatment of the ocular inflammation of patients with VKH syndrome, and thus achieving the purpose of saving the patient's vision.

[0046] It should be noted that the small interfering RNA provided by the present invention is a PARP10 inhibitor, including the following several:

[0047] (1) PARP10 siRNA-1;

[0048] (2) PARP10 siRNA-2;

[0049] (3) The combination of PARP10 siRNA-1 and PARP10 siRNA-2.

[0050] Furthermore, the nucleotide sequence of the small interfering RNA PARP10 siRNA-1 is shown in SEQ ID NO.1.

[0051] Furthermore, the nucleotide sequence of the small interfering RNA PARP10 siRNA-2 is shown in SEQ ID NO.2.

[0052] Table 1. Small interfering RNA and nucleotide sequence

[0053] NO. Small interfering RNA Nucleotide sequence 1 PARP10 siRNA-1 5’-CGAGCTGCTCACTCTCTACTT-3’ 2 PARP10 siRNA-2 5’-CTGGAGTTGTACCTGGAGAAT-3’

[0054] It should be noted that according to the WIPO's STANDARD ST.26 rule, U in the RNA sequence is represented by T.

[0055] Furthermore, it also includes the nucleotide sequences of the chemically modified PARP10 siRNA-1 and PARP10 siRNA-2.

[0056] Furthermore, the chemical modification methods include phosphate backbone modification, ribose modification, base modification, end-cap structure modification, Poly(A) tail modification, pseudouridine modification, RNA editing, chemical synthesis modification, peptide nucleic acid modification, locked nucleic acid modification, methoxy- or ethoxy modification, and modification.

[0057] Small interfering RNA (siRNA) is a double-stranded RNA molecule that can direct specific gene silencing, usually 20 - 25 nucleotides in length. siRNA binds to the corresponding mRNA, triggering the RNA-induced silencing complex (RISC), which results in the degradation of the target mRNA and prevents its translation into protein. This mechanism is applied in gene function research and therapeutic gene silencing.

[0058] To change or improve siRNA performance, such as stability and reducing its immunogenicity, various chemical modifications can be performed on small interfering RNA through different chemical modification methods. Such as ribosome modification, phosphodiester backbone modification, and terminal modification. These chemical modifications may change the pharmacokinetic properties of siRNA, which can include but are not limited to its stability, cellular uptake efficiency, and in vivo distribution.

[0059] In addition, the present invention also provides an application of the small interfering RNA as described above in the preparation of a drug targeting the PARP10 gene.

[0060] The small interfering RNA provided by the present invention can target the PARP10 gene of CD4 + T cells in VKH syndrome, thereby downregulating the expression of the PARP10 gene and inhibiting the proportion of Th1 cells in CD4+ T cells of VKH syndrome.

[0061] In addition, the present invention also provides an application of the small interfering RNA as described above in the preparation of a drug for treating Vogt-Koyanagi-Harada syndrome.

[0062] Furthermore, the dosage forms of the drug include: eye drops, eye ointments, injections, implants, microneedles, liposomes, nanoparticles, polymer controlled release systems, colloidal preparations, and suspension preparations.

[0063] In addition, it can also be prepared into other dosage forms for drug administration. It should be noted that for the applications mentioned in the present invention, the drugs prepared therein can all adopt the above different dosage forms.

[0064] In addition, the present invention also provides an application of the small interfering RNA as described above in the preparation of a drug for treating uveitis.

[0065] The present invention will be further described below through specific examples. However, it should be understood that these examples are only for more detailed description and should not be construed as limiting the present invention in any form.

[0066] Example 1: Analysis of differential gene expression in CD4 + T cells of patients with VKH syndrome

[0067] In this example, transcriptomic experiments were carried out on CD4 + T cells isolated from patients with VKH syndrome.

[0068] Experimental method:

[0069] (1) Sample collection and preliminary treatment:

[0070] Collect about 20 mL of peripheral blood from active VKH syndrome patients and healthy controls into heparinized anticoagulant tubes;

[0071] Irradiate the ultra-clean bench with ultraviolet light for about 30 minutes, and first isolate peripheral blood mononuclear cells (PBMCs) from the collected blood samples;

[0072] (2) Isolation of peripheral blood mononuclear cells (PBMCs):

[0073] Dilute the blood sample with PBS buffer, pour the blood sample and PBS into a 50 mL centrifuge tube and mix well;

[0074] Add human peripheral blood lymphocyte separation solution to 1 / 3 of the glass tube, and slowly add the diluted blood sample to the upper layer of the lymphocyte separation solution along the wall with a sterilized Pasteur pipette;

[0075] Centrifuge by Ficoll-Hypaque density gradient centrifugation method at 4 °C, 800 g, with an acceleration of 4 and a deceleration of 0 for 30 minutes;

[0076] Take out the centrifuged sample, suck out the middle cell layer with a Pasteur pipette, transfer it to a 15 mL centrifuge tube, add PBS buffer to blow and wash the cells, and then fill the PBS to the 15 mL centrifuge tube;

[0077] (3) PBMCs recovery and washing:

[0078] Centrifuge with a low-speed centrifuge at room temperature and 1450 rpm for 10 minutes, discard the supernatant, and the precipitate at this time is PBMCs;

[0079] Under the condition of avoiding light in the laminar flow hood, add magnetic bead buffer and CD4 magnetic beads according to the recommended ratio in the instruction manual, gently blow and mix well, and incubate in the dark in a 4 °C refrigerator for about 20 minutes;

[0080] Take out the above sample, add 1 mL of magnetic bead buffer to the incubated cells, centrifuge at 1450 rpm for 10 minutes, discard the supernatant, then add 500 μl of magnetic bead buffer, blow and resuspend the cells;

[0081] (4) CD4 + T cell separation:

[0082] Install an MS column on the MACS separation rack, first add 500 μl of magnetic bead buffer to moisten the column, slowly add the cell suspension blown well in the previous step to the MS column, after the cell suspension has dropped, add 500 μl of magnetic bead buffer to rinse the centrifuge tube, and add the rinsed liquid to the column as well. Repeat the operation three times;

[0083] After all the liquid in the MS column has flowed out, use forceps to clamp the MS column and transfer it to a new 15 mL centrifuge tube. Add 1 mL of magnetic bead buffer to the MS column, and push the liquid into the centrifuge tube with the matching column core, and repeat once;

[0084] Centrifuge the cells collected in the previous step at 1450 rpm for 10 minutes, discard the supernatant, and the precipitate in the tube at this time is CD4 + T cells;

[0085] (5) CD4+ T cell preservation:

[0086] Resuspend the isolated CD4 + T cells in 1 mL of PBS, divide the cells into two equal parts, transfer them into two 1.5 mL Eppendorf tubes respectively, centrifuge at 5000 rpm for 10 minutes at 4 °C, discard the supernatant, freeze the obtained cell pellet at -80 °C for later use, and send it for transcriptomics and proteomics detection.

[0087] (6) Construction and quality control of transcriptome sample libraries

[0088] ① Preparation of starting RNA for library construction:

[0089] The starting RNA for library construction is total RNA with a total amount ≥ 1 μg.

[0090] The library construction kit used in library construction is Illumina's ΜLtraTM RNA LibraryPrep Kit;

[0091] ② mRNA enrichment and fragmentation:

[0092] Use magnetic beads to enrich mRNA with polyA tails;

[0093] Subsequently, randomly fragment the enriched mRNA with divalent cations to obtain fragmented mRNA;

[0094] ③ cDNA synthesis:

[0095] Using the fragmented mRNA as a template and oligonucleotides as primers, synthesize the first strand of cDNA;

[0096] Subsequently, degrade the RNA strand with enzyme-free water;

[0097] And under the system conditions of DNA polymerase I, use dNTPs as raw materials to synthesize the second strand of cDNA;

[0098] ④ Double-stranded cDNA processing and library construction:

[0099] Process the above first strand and second strand, that is, double-stranded cDNA, through end repair, adding a polyA tail and ligating sequencing adapters, then screen cDNA of about 200 bp, perform PCR amplification, and use AM Pure XP beads to purify the PCR product again to obtain the required library;

[0100] ⑤ Library quality detection:

[0101] After the required library is constructed, dilute the library to 1.5 ng / μL, and then use the Agilent 2100 bioanalyzer to detect the insert size of the library;

[0102] After the above detection is completed, use RT-qPCR to accurately quantify the concentration to ensure its quality.

[0103] (7) Sequencing of transcriptome samples on the machine

[0104] ① Perform Illumina sequencing on the successfully constructed and quality-controlled libraries above, and generate 150 bp paired-end reads;

[0105] ② Add four fluorescently labeled dNTPs, DNA polymerase, and adapter primers to the flow cell for Illumina sequencing for amplification, and the sequencer captures fluorescent signals during the amplification process;

[0106] ③ Process the captured fluorescent signals through software to convert them into sequencing peaks, thereby obtaining the sequence information of the fragment to be tested.

[0107] (8) Quantification of gene expression levels and differential gene expression analysis.

[0108] ① Quantification of gene expression levels:

[0109] Feature Counts is used to calculate the reads mapped to each gene, and calculate its FPKM according to the gene length. FPKM refers to the expected number of fragments per kilobase of transcript sequence fragments sequenced per million base pairs;

[0110] At the same time, the influence of sequencing depth and gene length on read counts is considered, and it is the most commonly used method for estimating gene expression levels currently.

[0111] ② Differential gene expression analysis:

[0112] Use R software (1.16.1) to perform differential expression analysis between two comparison groups (three biological replicates in each group). DESeq2 is used to determine differential expression in digital gene expression data using a model based on the negative binomial distribution;

[0113] Use the method of Benjamini and Hochberg to adjust the obtained p-values to control the false discovery rate. Genes with adjusted p-values < 0.05 found by DESeq2 are assigned as differentially expressed;

[0114] For each sequencing library, adjust the read counts through a proportional normalization factor using the edgeR package. Differential expression analysis of two conditions is performed using the edgeR R package (3.18.1);

[0115] Adjust the p-values using the Benjamini & Hochberg method. The adjusted p-values and |log2 fold change| are used as the thresholds for significantly differentially expressed genes.

[0116] The results of Example 1 are shown in Figure 1 (The left arrow ↓ represents genes with downregulated expression in CD4 + T cells of VKH syndrome patients. There are 1196 such genes. The right arrow ↑ represents genes with upregulated expression in CD4 + T cells of VKH syndrome patients. There are 1172 such genes).

[0117] Example 2: Analysis of differential protein expression in CD4 + T cells of VKH syndrome patients

[0118] (1) Protein extraction and mass spectrometry analysis of CD4 + T cells

[0119] 1. Protein extraction and quantification:

[0120] (1) Add another prepared sample of CD4 + T cells to SDT (4% (w / v) SDS, 100 mM Tris / HCl pH 7.6, 0.1 M DTT) lysis buffer to extract proteins. After adding the lysis buffer and sonicating, boil for 15 minutes, centrifuge at 14000 g for 40 minutes, and take the supernatant;

[0121] (2) Perform protein quantification using the BCA method;

[0122] 2. Protease digestion and desalting:

[0123] (1) Take an appropriate amount of protein from each sample and perform trypsin digestion using the Filter aided proteome preparation (FAPP) method;

[0124] (2) Desalt the digested peptide fragments using a C18 Cartridge. After freeze-drying the peptide fragments, dissolve them in 40 μL of Dissolutionbuffer and quantify the peptide fragments (OD280).

[0125] 3. iTRAQ labeling and SCX chromatography fractionation:

[0126] (1) Label the samples according to the instructions of the iTRAQ labeling kit;

[0127] (2) Mix the peptide segments after each group of labeling and perform fractionation. Buffer A is 10 mM KH2PO4, 25% CAN, pH 3.0, and buffer B is 10 mM KH2PO4, 500 mM KCl, 25% CAN, pH 3.0;

[0128] (3) Equilibrate the chromatographic column with buffer A, load the sample onto the chromatographic column by an injector for separation, and the flow rate is 1 mL / min. (The liquid phase gradient is as follows: 0% buffer B for 25 min, linear gradient of buffer B from 0% - 10%; 25 min - 32 min, linear gradient of buffer B from 10% - 20%; 32 min - 42 min, linear gradient of buffer B from 20% - 45%; 42 min - 47 min, linear gradient of buffer B from 45% - 100%; 47 min - 52 min, buffer B is maintained at 100% from 52 - 60 min; after 60 min, buffer B is reset to 0%);

[0129] (4) Monitor the absorbance at 214 nm, collect the eluted components every 1 minute, freeze-dry them respectively, and desalt them with a C18 Cartridge.

[0130] 4. LC-MS / MS Data Acquisition and Analysis:

[0131] (1) Use the HPLC liquid phase system Easy nLC to separate the fractionated samples. Use 0.1% formic acid aqueous solution buffer as buffer A and 0.1% formic acid acetonitrile aqueous solution as buffer B;

[0132] (2) Equilibrate the chromatographic column with 95% buffer A, load the sample onto the loading column (Thermo Scientific Acclaim PepMap100, 100 um * 2 cm, nanoViper C18) by an autosampler, and separate it through an analytical column (Thermo scientific EASY column, 10 cm, ID 75 um, 3 um, C18 - A2), and the flow rate is 300 nL / min;

[0133] (3) After the chromatographic separation is completed, perform mass spectrometry analysis using a Q Exactive mass spectrometer. The detection mode is positive ion, the parent ion scanning range is 300 - 1800 m / z, the resolution of the first-stage mass spectrometry is 70,000 at 200 m / z, the Automatic gain control target is 1e6, the Maximum IT is 50 ms, and the dynamic exclusion time is 60.0 s;

[0134] (4) Collect the mass-to-charge ratios of polypeptides and polypeptide fragments according to the following method: After each full scan (fμLl scan), collect 20 fragment spectra (MS2 scan). The MS2 Activation Type is HCD, the Isolation window is 2 m / z, the secondary mass spectrometry resolution is 17,500 at 200 m / z, the Normalized Collision Energy is 30 eV, and the Underfill is 0.1%.

[0135] Protein identification and quantitative analysis

[0136] Use the software Mascot 2.2 and Proteome Discoverer 1.4 for database search identification and quantitative analysis

[0137] The results of Example 2 are shown in Figure 2 (The left arrow↓ represents the proteins with down-regulated expression in the CD4 + T cells of VKH syndrome patients. There are 112 such proteins. The right arrow↑ represents the proteins with up-regulated expression in the CD4 + T cells of VKH syndrome patients. There are 159 such proteins).

[0138] Example 3: Integrated analysis of transcriptomics and proteomics

[0139] (1) Integrate and analyze the differential molecules of the two omics, and it is found that there are 43 molecules with differential expression at both the transcriptional and protein levels. See Figure 3 .

[0140] (2) Perform bioinformatics clustering analysis on the above 43 molecules, and screen out 13 molecules with consistent expression trends at the transcriptional and protein levels and related to immune and inflammatory pathways. Among them, there are 7 molecules with up-regulated expression and 6 molecules with down-regulated expression in the CD4+ T cells of active VKH syndrome patients. See the following table.

[0141] Table 2. Screening results of clustering analysis

[0142]

[0143] Example 4: Verify the above candidate molecules by real-time fluorescence quantitative PCR reaction

[0144] In this example, a real-time fluorescence quantitative PCR reaction (RT-qPCR) was performed.

[0145] 1. Reverse transcription reaction (convert RNA into cDNA)

[0146] (1) gDNA digestion treatment: In an RNase-free PCR tube, prepare the reaction solution according to the following components. The operation of preparing the reaction solution is carried out on ice, pipette and mix well, and incubate at 42 °C for 2 mins:

[0147] Table 3. Reaction solution components

[0148] Reagent 10 μL system 5×g DNA digester Buffer 2 μL g DNA digester 1 μL Total RNA / mRNA 5 ng - 5 μg / 5 ng - 500 ng <![CDATA[RNase-Free H2O]]> To 10 μL

[0149] (2) Preparation of the reverse transcription reaction system (20 μL system): Directly add 2×Super RTMix to the PCR tube in step (1), and then gently pipette and mix well.

[0150] Table 4. Reaction system

[0151] Reagent 20 μL system Reaction solution of Step 1 10 μL 2×Super RT Mix 10 μL

[0152] (3) Reverse transcription program setting: After mixing the Mix prepared in step 2 evenly, place it in a PCR instrument for reaction. The specific program is as follows:

[0153] Table 5. Reaction program

[0154] Temperature Time 25℃ 5 mins 42℃ 30 - 60 mins 85℃ 4 mins

[0155] (4) After the reaction, place it on ice to cool. The synthesized cDNA can be immediately used for subsequent PCR or qPCR reactions, or stored at -20 °C for short-term or at -80 °C for long-term, and try to avoid repeated freezing and thawing.

[0156] 2. Fluorescent quantitative PCR (qPCR) reaction

[0157] (1) Prepare the PCR reaction system according to the following components on ice:

[0158] Table 6. PCR reaction system

[0159] Reagent 20 μL system Final concentration SYBR Green Master Mix(2×)(Low ROX) 10 μL 1× PCR Forward Primer(10 μM) 0.4 μL 0.2 μM PCR Reverse Primer(10 μM) 0.4 μL 0.2 μM DNA template 2 μL / <![CDATA[ddH2O (double distilled water)]]> 7.2 μL / Total 20 μL /

[0160] (2) Perform Real Time PCR reaction. The PCR reaction is as follows:

[0161] Table 7. Reaction program

[0162]

[0163] The results of Example 4 are shown in Figure 4 , and the above-mentioned candidate molecules were verified by real-time fluorescence quantitative PCR reaction. Only PARP10 showed expression differences (the other 12 genes without expression differences are not shown), suggesting that PARP10 is a key pathogenic molecule for VKH syndrome.

[0164] Example 5: Parallel reaction monitoring (PRM) to verify the expression of PARP10 at the protein level

[0165] In this example, a parallel reaction monitoring (PRM) experiment was performed.

[0166] 1. Sample preparation:

[0167] A. Acetone precipitation:

[0168] (1) Mix the corresponding samples according to their concentrations;

[0169] (2) Acetone was precooled to -20°C, and 5 times the volume of acetone was added to the sample and shaken to mix, and the protein was precipitated at -40°C overnight;

[0170] (3) Centrifuge at 12,000 rpm, 4°C for 10 min and carefully remove the supernatant;

[0171] (4) Add 200 μL of pre-cooled 80% acetone to rinse the precipitate twice, centrifuge at 12,000 rpm, and carefully remove the supernatant. Repeat once.

[0172] B. Protein resolubilization, reduction and alkylation:

[0173] (1) Add 80 μL of protein reconstitution solution and sonicate in a water bath for 5 min to dissolve the protein precipitate;

[0174] (2) Add TCEP to 5 mM and incubate at 55 °C with shaking for 10 min to reduce disulfide bonds;

[0175] (3) Cool the sample to room temperature, add IAA to 10 mM and react in the dark for 15 min to alkylate the reduced disulfide bonds.

[0176] C. Proteolysis:

[0177] (1) Dissolve Trypsin in resuspension buffer to 0.5 μg / μL and incubate at room temperature for 5 min.

[0178] (2) Mix Trypsin and sample thoroughly at a ratio of Trypsin:protein = 1:50;

[0179] (3) After brief centrifugation, incubate at 37°C with shaking at 1000 rpm overnight.

[0180] D. Remove SDC:

[0181] (1) Add TFA to the mixed sample (final concentration 2%, pH < 2), and mix well to precipitate SDC;

[0182] (2) Centrifuge at high speed for 10 min, and transfer the supernatant to a new EP tube;

[0183] (3) Add 100 μL of 2% TFA, mix well, and centrifuge at 13,000 rpm for 10 min to extract the coprecipitated polypeptides (repeat the extraction 2 times);

[0184] (4) Combine the supernatants from the 3 extractions, centrifuge at high speed for 10 min, and transfer the supernatant to a new EP tube. Randomly select one sample from each group, take 10 μg, mix them, and desalt.

[0185] E. Polypeptide desalting:

[0186] (1) Add 1000 μL of Buffer C to activate the C18 desalting column;

[0187] (2) Add 1 mL of Buffer A to wash away the residual ACN and equilibrate the desalting column;

[0188] (3) Add the sample to the desalting column, and let the sample slowly flow through the desalting column to collect the eluate A;

[0189] (4) Wash the column once with 1 mL of Buffer A to remove the residual salts;

[0190] (5) Add 400 μL of Buffer B to elute the polypeptide and collect the eluate B;

[0191] (6) Repeat the entire desalting process once with the collected eluate A;

[0192] (7) Combine the eluates B from the two times and dry by vacuum centrifugation.

[0193] 2. nanoLC-MS / MS detection

[0194] (1) Approximately 1 μg of total peptides from each sample was separated by the nano-UPLC liquid phase system EASY-nLC 1200 and then coupled with a mass spectrometer (Q-Exactive HFX) equipped with a nano-ion source for data acquisition. Chromatographic separation was performed using a 100 μM ID × 15 cm reversed-phase chromatographic column (ReprosilPur 120 C18-AQ, 1.9 μM, Dr. Math). The mobile phase used was an acetonitrile-water-formic acid system, where mobile phase A was 0.1% formic acid - 98% aqueous solution (2% acetonitrile), and phase B was 0.1% formic acid - 80% acetonitrile solution (20% water). After the chromatographic column was equilibrated with 100% of phase A, the sample was directly loaded onto the chromatographic column by an autosampler and then gradient separated by the chromatographic column at a flow rate of 300 nL / min for a gradient duration of 90 min. The proportion of mobile phase B: 2 - 5% for 2 min, 5 - 22% for 68 min, 22 - 45% for 16 min, 45 - 95% for 2 min, 95% for 2 min.

[0195] (2) Mass spectrometry analysis used the parallel reaction monitoring (PRM) method and adopted the positive ion detection mode. The quadrupole isolation window was 0.7 m / z, the normalized collision energy (NCE) was 27%, and the second-stage scanning resolution was 15 k.

[0196] 3. Data analysis:

[0197] A. Database searching for identification and protein quantification: In this experiment, two methods were used to generate the Spectra library: (1) The classical DDA data acquisition mode, combined with database searching software for identification; (2) For target polypeptides or proteins that could not be identified by DDA, a deep learning model (Prosit) was used for prediction.

[0198] B. Construction of the PRM method and quantitative analysis: The construction of the PRM method was completed using Skyline software, and the general process was as follows:

[0199] (1) The target polypeptide sequence was imported into Skyline software. After setting information such as polypeptide charge and heavy isotope labeling, the inclusion list was exported to Xcalibar software to construct the PRM method for collecting actual samples;

[0200] (2) The PRM raw data was imported into Skyline software. The chromatographic peak boundaries of the target polypeptides were determined according to the spectra and RT and other characteristics in the Spectra library. When selecting the secondary spectrum information, the DDA database search results were given priority;

[0201] (3) For each target polypeptide, a maximum of 5 fragment ions were selected for quantification.

[0202] The results of Example 5 are shown in the following table. Through PRM verification, it was found that the expression at the protein level of PARP10 was consistent with that at the transcriptional level, that is, PARP10 was significantly highly expressed in patients with VKH syndrome, which was 1.65 times that of normal controls.

[0203] Table 8. PRM verification results

[0204] Protein ID Name PRM fold change P value of PRM ENSP00000432733 PARP10 1.654066507 0.0028

[0205] Example 6: Establishment and identification of PARP10 siRNA interference model

[0206] In this example, a PARP10 siRNA interference model was established and identified.

[0207] (1) Establishment of interference model:

[0208] Peripheral blood from 10 patients with active VKH syndrome was collected, and CD4 + T cells were separated by Ficoll-Hypaque density gradient centrifugation. The cells were randomly divided into 4 groups: siNC group, PARP10-si1 group, PARP10-si2 group, and PARP10-si1+si2 group.

[0209] siNC was the negative control group, with sequence design and product;

[0210] The sequence of PARP10-si1 was: 5’-CGAGCTGCTCACTCTCTACTT-3’;

[0211] The sequence of PARP10-si2 was: 5’-CTGGAGTTGTACCTGGAGAAT-3’.

[0212] Then, according to the instructions for adenovirus and lip3000 plasmid transfection, PARP10 interference treatment was carried out, and in vitro culture was performed. CD3 / 28 was added to stimulate the activation of CD4 + T cells. The cell precipitate was collected after culturing the treated cells for 3 days.

[0213] (2) Identification: Identification was carried out by RT-qPCR method as described above. Protein immunoblot analysis (Western blot) method was used for identification. Western blot detection: 100 μl of RIPA protein lysate and protease inhibitor were added to each culture dish and stored at -80 °C for lysis. The next day, the samples were centrifuged at 12,000 rpm at 4 °C for 15 min, and the supernatant, that is, the protein lysate, was collected, the concentration was measured, and Western blot protein immunoblot analysis was performed.

[0214] Statistical analysis was performed using GraphPad Prism V.7.0.0. The Shapiro-Wilk normality test was used to test whether the data was normally distributed. For normally distributed data, independent sample t-tests and one-way analysis of variance (ANOVA) were used to test the variance between groups, and the statistical results were presented as Mean±SD; for data that was not normally distributed, the Mann-Whitney test and Kruskal-Wallis test were used for the variance analysis between groups, and the statistical results were presented as the median Median. All statistical results were judged by p<0.05 as the criterion for statistically significant differences.

[0215] Results reference of Example 6 Figure 5 , all three PARP10 gene interference regimens were effective and could achieve targeted interference of PARP10. Compared with the si-NC group, the expression of the PARP10 gene in the PARP10-si1 group, PARP10-si2 group, and PARP10-si1+si2 group was significantly downregulated.

[0216] Example 7: Detection of the change in the proportion of Th1 cells after PARP10 siRNA interference

[0217] Results reference of Example 7 Figure 6 , after PARP10 interference, it could inhibit the proportion of Th1 cells in CD4 + T cells in VKH syndrome patients, reaching the same proportion as Th1 cells in normal people, and significantly improving the intraocular inflammation of the patients.

[0218] Phenotypic verification of PARP10 found that inhibiting PARP10 expression could reduce the proportion of Th1 cells in CD4 + T cells, that is, compared with the si-NC group, the proportion of Th1 cells (secreting inflammatory factor IFN-γ) in CD4 + T cells in the PARP10-si1 group, PARP10-si2 group, and PARP10-si1+si2 group decreased significantly, from 22.4% to 13.3%, 15.4%, and 11.8% respectively;

[0219] At the same time, the proportion of Th1 cells in CD4 + T cells in normal people was detected to be 12.6%. Therefore, the results of this study suggest that after the inhibition of PARP10 expression, the proportion of Th1 cells decreased significantly, reaching the same state as that of normal people, indicating that after PARP10 interference, the purpose of improving ocular inflammation or treating diseases can be achieved.

[0220] The above are the preferred embodiments and corresponding examples of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, including but not limited to adjustments in proportion, process, dosage, and reaction vessel, and these all fall within the protection scope of the present invention.

Claims

1. A small interfering RNA, characterized in that The small interfering RNA is a PARP10 inhibitor, and the small interfering RNA is any one or two small interfering RNAs of PARP10 siRNA-1 and PARP10 siRNA-2; The nucleotide sequence of the small interfering RNA PARP10 siRNA-1 is shown in SEQ ID NO.1; The nucleotide sequence of the small interfering RNA PARP10 siRNA-2 is shown in SEQ ID NO.2; The small interfering RNA can target CD4 + PARP10 gene of T cells, thereby downregulating the expression of PARP10 gene and inhibiting CD4 + The proportion of Th1 cells in T cells.

2. Use of the small interfering RNA as claimed in claim 1 in the preparation of a drug for treating Vogt-Koyanagi-Harada syndrome.

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