Application of PHF20 in preparation of medicine for treating systemic lupus erythematosus, method and medicine

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application and a method of PHF20 in preparation of a medicine for treating systemic lupus erythematosus and the medicine. The invention discloses an application of PHF20 in treatment of systemic lupus erythematosus, a lentivirus with murine PHF20 is selected as a gene therapy tool, an R848 induced lupus mouse model is taken as an experimental subject for gene therapy, and results show that injection of the overexpressed PHF20 lentivirus in a lupus mouse body has the following effects: (1) spleen swelling is improved, and spleen index is significantly reduced; (2) reducing urine protein, urea nitrogen level, anti-dsDNA antibody level and IgG antibody level of the model mouse; and (3) the pathological damage to the kidney of the model mouse is relieved. The research results show that the PHF20 serving as the target provides important reference value for potential application prospects in systemic lupus erythematosus gene therapy and even autoimmune disease gene therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of PHF20 in preparing a drug for treating systemic lupus erythematosus, a method and a drug. Background Art

[0002] Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by multi-organ and multi-system damage, characterized by abnormal activation of T and B lymphocytes and the formation of autoantibodies. It is a common, chronic disease with fatal and disabling consequences. Clinically, immunosuppressants, hormones, B cell-targeted therapies, and stem cell-based therapies are the primary treatment options. However, the limited availability of specific therapeutic agents and the toxic side effects associated with long-term use limit the treatment of SLE patients. Therefore, new therapies to slow the progression of SLE are urgently needed.

[0003] The development and function of pathogenic cells in systemic lupus erythematosus (SLE) are regulated by multiple transcription factors. In-depth exploration of these molecular mechanisms will deepen our understanding of the pathogenesis of autoimmune diseases. Mice induced with the Toll-like receptor 7 (TLR-7) / Toll-like receptor 8 (TLR-8) agonist resiquimod (R848) are a commonly used SLE mouse model. These mice exhibit elevated serum ANA and anti-double-stranded DNA antibody (anti-dsDNA) levels, multiple organ involvement including the kidneys and liver, and increased T and B lymphocyte activation reactivity, demonstrating typical SLE clinical manifestations.

[0004] Previous literature reports indicate that SLE patients have defects in the DNA damage repair mechanism, which may lead to the accumulation of endogenous and exogenous DNA damage. (1) Immune tolerance destruction: Normal DNA repair mechanisms help maintain cell stability and immune tolerance. When the DNA damage response (DDR) function is abnormal, DNA damage in cells cannot be effectively repaired, which may cause the immune system to misidentify self-cells, thereby triggering an autoimmune response. (2) Production of autoantibodies: Failure to effectively repair DNA damage may release nucleic acids and other cellular components. These substances can act as antigens to trigger immune responses and stimulate the production of antibodies against self-components (such as anti-double-stranded DNA antibodies), which is an important feature of SLE. (3) Apoptosis and inflammation: Abnormal DDR may lead to abnormal cell apoptosis, causing more cells to release intracellular components, which may further promote inflammatory responses and enhance autoimmune manifestations. (4) Genomic instability: In SLE patients, defects in the DNA damage response may cause genomic instability, which may not only promote autoimmune responses but also be associated with the risk of cancer. Understanding the role of DDR in SLE provides potential directions for the development of new treatments. Drugs targeting the DNA damage repair pathway may help improve the symptoms of SLE patients. Summary of the Invention

[0005] The purpose of the present invention is to provide an application, method and medicine of PHF20 in the preparation of a drug for treating systemic lupus erythematosus, so as to achieve effective treatment of systemic lupus erythematosus by regulating the expression of PHF20.

[0006] To this end, the present invention provides the following technical solutions.

[0007] The first aspect of the present invention provides any of the following applications: (1) Use of reagents for quantitatively detecting the expression level of the PHF20 gene or its encoded protein in the preparation of products for diagnosing systemic lupus erythematosus; (2) Use of an agent that promotes the expression level of the PHF20 gene or its encoded protein in the preparation of a pharmaceutical composition for treating systemic lupus erythematosus; (3) Application of PHF20 in screening candidate drugs for the treatment of systemic lupus erythematosus; and (4) Application of PHF20 in constructing a computational model for predicting systemic lupus erythematosus.

[0008] A second aspect of the present invention provides a pharmaceutical composition for treating systemic lupus erythematosus, comprising: (i) an agent that promotes the expression level of the PHF20 gene or the protein encoded by it; and (ii) pharmaceutically acceptable carriers and / or excipients.

[0009] Preferably, the agent promoting the expression level of the PHF20 gene or the protein encoded therein promotes the transcription of the PHF20 gene, promotes the expression of the PHF20 gene, promotes the function of the PHF20 protein, and / or inhibits the degradation of the PHF20 protein.

[0010] Preferably, the agent that promotes the expression level of the PHF20 gene or the protein encoded by it includes a vector that overexpresses PHF20 or contains the PHF20 gene, such as a viral vector including but not limited to adenovirus, lentivirus, retrovirus, adeno-associated virus, a non-viral vector including but not limited to a cationic polymer vector, a nanoparticle vector, a liposome or a liposome complex, a ligand-mediated targeting vector, a natural polymer material such as collagen, hyaluronic acid, chitosan or alginate.

[0011] Preferably, the agent for promoting the expression level of the PHF20 gene or the protein encoded thereby is a lentivirus that overexpresses PHF20.

[0012] Preferably, the dosage form of the pharmaceutical composition includes oral preparations such as tablets, powders, granules, capsules, pills, sustained-release pellets, solid dispersions, inclusion compounds, liquid preparations such as suspensions, emulsions, melts, syrups, mixtures, solutions, injectable preparations such as injections, aqueous or oily suspensions, emulsions, liposomes, microcapsules, microspheres, nanoparticles, sustained-release or controlled-release preparations, preferably injectables.

[0013] A third aspect of the present invention provides a product for diagnosing systemic lupus erythematosus, comprising a chip, a kit or a nucleic acid membrane strip capable of quantitatively detecting the expression level of the PHF20 gene or the protein encoded by it in a sample.

[0014] Preferably, the chip comprises a reagent capable of detecting the expression level of the PHF20 gene or the protein encoded by it.

[0015] Preferably, the kit includes reagents for detecting the expression level of the PHF20 gene or the protein encoded by it by RT-PCR, qRT-PCR, biochip detection, Southern blotting, in situ hybridization, or immunoblotting.

[0016] Preferably, the nucleic acid membrane strip comprises a reagent capable of detecting the expression level of the PHF20 gene or the protein encoded by it.

[0017] Preferably, the reagent is selected from an oligonucleotide probe that specifically recognizes the PHF20 gene, a primer that specifically amplifies the PHF20 gene, or a binding agent that specifically binds to a protein encoded by the PHF20 gene in a sample.

[0018] Preferably, the sample is peripheral blood and / or spleen tissue.

[0019] A fourth aspect of the present invention provides a method for screening candidate drugs for treating systemic lupus erythematosus, comprising: treating a culture system expressing or containing the PHF20 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the PHF20 gene or the protein encoded by it in the system; Wherein, when the substance to be screened promotes the expression level or activity of the PHF20 gene or the protein encoded by it, the substance to be screened is a candidate drug for treating systemic lupus erythematosus.

[0020] By means of the above technical solution, the present invention has at least the following advantages: The present invention discloses the application of PHF20 in the treatment of systemic lupus erythematosus. By selecting a lentivirus carrying mouse-derived PHF20 as a gene therapy tool, gene therapy was carried out in an R848-induced lupus mouse model. The results showed that injection of a lentivirus overexpressing PHF20 in lupus mice had the following effects: (1) improvement in spleen enlargement and a significant decrease in spleen index; (2) reduction in urine protein, urea nitrogen levels, anti-dsDNA antibodies, and IgG antibody levels in the model mice; and (3) alleviation of renal pathological damage in the model mice. The above research results suggest that PHF20 as a target has important potential application prospects in gene therapy for systemic lupus erythematosus and even in gene therapy for autoimmune diseases.

[0021] The present invention aims to disclose the application of PHF20 overexpression in the preparation of drugs for the treatment of systemic lupus erythematosus. By constructing a PHF20 overexpression plasmid or lentivirus, the present invention fills the technical gap in regulating the expression of PHF20 to alleviate the condition of lupus mice. It is found that PHF20 can effectively inhibit disease activity in lupus mice and reduce spleen and kidney damage, becoming a potential gene therapy drug for the treatment of systemic lupus erythematosus.

[0022] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The plasmid map of the present invention pLV3-CMV-PHF20 (mouse)-3×FLAG-eGFP is shown; Figure 2 Shown is a flow chart of the in vivo experimental design of the present invention; Figure 3The figure shows the in vivo infection efficiency verification of mice in each treatment group in the embodiment of the present invention; wherein A is the change of PHF20 expression level in PBMCs of mice in each group; B is the flow cytometry detection and statistical graph of PHF20 expression in spleen tissue of mice in each group; C is the change of PHF20 expression level in spleen tissue of mice in each group; D is the WB graph of PHF20 expression in spleen tissue of mice in each group; Figure 4 The figures show the survival rate and weight change of mice in each treatment group in the embodiment of the present invention; wherein A is the weekly change in survival rate of mice in each group; B is the weight change of mice in each group; Figure 5 Schematic diagram showing the disease phenotype changes of mice in each treatment group according to the embodiment of the present invention: wherein A is a statistical diagram of the swelling size of the mouse spleen tissue and the spleen index; B is the mouse plasma ANA level; C is the mouse plasma anti-dsDNA antibody level; D is the mouse plasma anti-IgG level; Figure 6 The figures show the kidney damage of mice in each treatment group in the embodiment of the present invention: A is a direct view of the mouse kidney tissue and a statistical graph of the kidney index; B is the urine protein level of the mouse; C is the urea nitrogen level of the mouse; D is the blood creatinine level of the mouse; Figure 7 Schematic diagram of H&E staining of the kidneys of mice in each treatment group according to the embodiment of the present invention is shown; Figure 8 Schematic diagram of immunofluorescence staining of kidney C3 and IgG in mice in each treatment group according to the embodiment of the present invention is shown; Among them, B6+acetone: n=8, B6+R848: n=7, R848+NC: n=6, R848+OE: n=7; * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, ns indicates no statistical difference. DETAILED DESCRIPTION

[0024] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0025] The present invention is based on many factors, such as the poor specificity of traditional SLE drugs, which, although able to alleviate some symptoms, cannot control the progression of the disease in the long term; the various toxic side effects of traditional drug treatments due to low in vivo targeting; the complex pathogenesis of systemic lupus erythematosus and the current lack of gene therapy methods for treating systemic lupus erythematosus. The present invention provides an application, method and drug of PHF20 in the preparation of a drug for treating systemic lupus erythematosus.

[0026] To this end, in one embodiment of the present invention, an application as described in any one of the following is provided: (1) Use of reagents for quantitatively detecting the expression level of the PHF20 gene or its encoded protein in the preparation of products for diagnosing systemic lupus erythematosus; (2) Use of an agent that promotes the expression level of the PHF20 gene or its encoded protein in the preparation of a pharmaceutical composition for treating systemic lupus erythematosus; (3) Application of PHF20 in screening candidate drugs for the treatment of systemic lupus erythematosus; and (4) Application of PHF20 in constructing a computational model for predicting systemic lupus erythematosus.

[0027] As used herein, PHF20, short for PHD finger protein 20, also known as NZF, TZP, GLEA2, or HCA58, is a 1012-amino acid protein localized in the cell nucleus. It contains a PHD-type zinc finger, a C2H2-type zinc finger, and an A / T hook DNA binding domain. PHF20 is expressed in multiple tissues, including the liver, heart, lung, pancreas, spleen, testis, and placenta. Reports suggest a possible role in the development of cancers including glioblastoma, lung cancer, colon cancer, and ovarian cancer. The PHF20 gene exists in three alternatively spliced ​​isoforms (150 kd, 130 kd, and 120 kd) and is encoded by a gene mapped to human chromosome 20 (22072714). Furthermore, the PHF20 gene is a subunit of the lysine acetyltransferase complex MOF-NSL, which catalyzes the acetylation of histone H4 and non-histone proteins. It specifically recognizes methylated lysines and is essential for cellular reprogramming (doi:10.1016 / j.cell.2013.02.006). Currently, reports suggest its involvement in cancer and metabolic syndrome. However, there are no reports of PHF20 being used to treat systemic lupus erythematosus (SLE), and the mechanism by which this gene regulates SLE progression remains unclear.

[0028] PHF20 includes wild-type, mutant forms, or fragments thereof. The term encompasses full-length, unprocessed PHF20, as well as any form of PHF20 derived from processing in cells. The term encompasses naturally occurring variants of PHF20 (e.g., splice variants or allelic variants). The term encompasses, for example, the PHF20 gene, human PHF20, as well as PHF20 from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats). As a preferred embodiment, in the present invention, PHF20 is a mouse gene, Gene ID: 228829.

[0029] In one embodiment of the present invention, a pharmaceutical composition for treating systemic lupus erythematosus is provided, wherein the pharmaceutical composition comprises an agent for promoting the expression level of the PHF20 gene or the protein encoded thereby, and a pharmaceutically acceptable carrier and / or excipient.

[0030] In some embodiments of the present invention, the agent that promotes the expression level of the PHF20 gene or the protein encoded by it promotes the transcription of the PHF20 gene, promotes the expression of the PHF20 gene, promotes the function of the PHF20 protein, and / or inhibits the degradation of the PHF20 protein.

[0031] In some embodiments of the present invention, the agent that promotes the expression level of the PHF20 gene or its encoded protein refers to any substance that can increase the activity of the PHF20 protein, improve the stability of the PHF20 gene or protein, upregulate the expression of the PHF20 protein, increase the effective action time of the PHF20 protein, or promote the transcription and translation of the PHF20 gene. These substances can be used in the present invention as substances useful for upregulating PHF20, thereby being used to prevent or treat systemic lupus erythematosus. For example, the agent includes a nucleic acid promoter and a protein promoter. The agent includes but is not limited to a vector or construct that overexpresses PHF20, a PHF20 protein, or an active peptide thereof.

[0032] Numerous suitable vectors are known to those skilled in the art of molecular biology, and the choice of vector depends on the desired function. The present invention is not particularly limited to vectors, but can be any vector capable of replicating and / or expressing polynucleotides in eukaryotic or prokaryotic cells, including mammalian cells (e.g., human, monkey, rabbit, rat, hamster, or mouse cells), plant cells, yeast cells, insect cells, and bacterial cells (e.g., E. coli). Preferably, the vector comprises at least one selectable marker operably linked to a suitable promoter, such that the polynucleotide can be expressed in the host cell. For example, the vector can include a polynucleotide introduced into a phage, plasmid, cosmid, minichromosome, virus, or retroviral vector, or other vectors conventionally used, for example, in genetic engineering.

[0033] As an optional aspect of the present invention, the vector is a virus. Viral vectors are used to introduce non-endogenous nucleic acid sequences encoding target-specific polypeptides. Viral vectors can be retroviral vectors or lentiviral vectors. Viral vectors can also include a nucleic acid sequence encoding a transduction marker. Suitable viral vectors include RNA virus-based vectors, such as retroviral vectors, such as Moloney murine leukemia virus (MLV)-derived vectors, and more complex retroviral vectors, such as lentiviral vectors. HIV-1-derived vectors fall into this category.

[0034] Viral vectors include, but are not limited to, retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses (e.g., orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), and double-stranded DNA viruses, including, but not limited to, adenoviruses, herpes viruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus), and pox viruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, but are not limited to, norwalk viruses, togaviruses, flaviviruses, reoviruses, papillomaviruses, hepatitis viruses, and hepaciviruses. Examples of retroviruses include, but are not limited to, avian leukosarcoma, mammalian C, B, and D viruses, the HTLV-BLV groups, lentiviruses, and foamy viruses.

[0035] As an optional aspect of the present invention, the vector is an expression vector. The expression vector according to the present invention can direct the replication and expression of the nucleic acid molecule of the present invention in a host, and thus ensure the expression of the PHF20 of the present invention encoded thereby in the selected host. Non-limiting examples of vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), λgt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027 pCAGKosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (EdgeBiosystems), pTriEx-Hygro (Novagen), and pCINeo (Promega). Non-limiting examples of plasmid vectors suitable for Pichia pastoris include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all Invitrogen). Another vector suitable for expressing proteins in Xenopus embryos, zebrafish embryos, and a variety of mammalian and avian cells is the multi-purpose expression vector pCS2+.

[0036] As a more preferred embodiment, the agent for promoting the expression level of the PHF20 gene or the protein encoded by it is a lentivirus that overexpresses PHF20.

[0037] In the present invention, agents that promote PHF20 expression can be administered via liposomes, which serve to target the drug to specific tissues and increase the drug's half-life. Liposomes include, but are not limited to, emulsifiers, foaming agents, liquid lipids, solid lipids, insoluble monolayers, phospholipid dispersants, and surfactants. The liposomes may also include agents that bind to receptor molecules in targeted cells or other therapeutic or immunogenic compositions.

[0038] In some embodiments of the present invention, the dosage form of the pharmaceutical composition includes oral preparations such as tablets, powders, granules, capsules, pills, sustained-release pellets, solid dispersions, inclusion compounds, liquid preparations such as suspensions, emulsions, melts, syrups, mixtures, solutions, injectable preparations such as injection solutions, aqueous or oily suspensions, emulsions, liposomes, microcapsules, microspheres, nanoparticles, sustained-release or controlled-release preparations, preferably injectable preparations.

[0039] As used herein, the term "expression level" or "gene expression level" refers to the level of mRNA and pre-mRNA nascent transcripts, transcript processing intermediates, mature mRNA and degradation products in a cell or the level of protein encoded by the gene.

[0040] The pharmaceutically acceptable carrier or excipient described in the present invention refers to additives commonly used in the pharmaceutical field other than active ingredients, including but not limited to diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, and disintegrants. Among them, diluents such as lactose, sodium chloride, glucose, urea, starch, water, etc.; binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose, etc.; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc.; wetting agents such as glycerol, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin and bentonite, etc.; lubricants such as zinc stearate, monostearate, etc. Fatty acid glycerides, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium lauryl sulfate, etc.; fillers such as mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugars, coupling sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate and sodium bicarbonate, etc.; disintegrants such as cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharides.

[0041] The pharmaceutical composition of the present invention may further include additives such as stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH control agents, and surfactants. Stabilizers include human serum albumin, L-amino acids, sugars, and cellulose derivatives. L-amino acids may also include any one of glycine, cysteine, and glutamic acid. Carbohydrates include monosaccharides such as glucose, mannose, galactose, and fructose; sugar alcohols such as mannitol, inositol, and xylitol; disaccharides such as sucrose, maltose, and lactose; and polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate, hyaluronic acid, and their derivatives. Cellulose derivatives include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and sodium hydroxymethylcellulose. Surfactants include ionic or nonionic surfactants such as polyoxyethylene alkyl esters, sorbitan monoacyl esters, and fatty acid glycerides. Additive buffers may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid, and their corresponding salts (alkali metal or alkaline rare earth metal salts thereof, such as sodium salts, potassium salts, calcium salts, and magnesium salts). Isotonic agents include potassium chloride, sodium chloride, sugars, and glycerol. Chelating agents include sodium edetate and citric acid.

[0042] The pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. Oral administration or injection is preferred. The pharmaceutical compositions of the present invention may contain any conventional non-toxic pharmaceutically acceptable carrier, excipient, or vehicle.

[0043] The drugs of the present invention can also be used in combination with other drugs for treating systemic lupus erythematosus. The other therapeutic compound can be administered simultaneously with the main active ingredient (e.g., a PHF20 enhancer), or even administered simultaneously in the same composition. The other therapeutic compound can also be administered separately in a separate composition or in a dosage form different from that of the main active ingredient. A portion of the main ingredient (e.g., a PHF20 enhancer) can be administered simultaneously with the other therapeutic compound, while the remaining dose can be administered alone.

[0044] As used herein, the term "treatment" generally refers to treatment and therapy of humans or animals (e.g., in veterinary applications) in which some desired therapeutic effect is achieved, such as inhibiting the progression of a disease, and includes reducing the rate of progression, halting the rate of progression, alleviating symptoms of a disease, ameliorating the disease, and curing the disease. Treatment as a preventative measure (i.e., prophylaxis) is also included. For example, the term "treatment" also includes application to patients who do not yet have a disease but are at risk of developing the disease.

[0045] As used herein, "treatment" (and grammatical variations thereof) refers to clinical intervention intended to alter the natural course of the individual being treated, either for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and eliminating or improving prognosis. In some embodiments of the present invention, agents that promote PHF20 expression are used to delay the development of the disease or slow its progression.

[0046] In one embodiment of the present invention, a product for diagnosing systemic lupus erythematosus is provided, comprising a chip, a kit or a nucleic acid membrane strip capable of quantitatively detecting the expression level of the PHF20 gene or the protein encoded by it in a sample.

[0047] In some embodiments of the present invention, the chip includes a reagent capable of detecting the expression level of the PHF20 gene or the protein encoded by it.

[0048] In some embodiments of the present invention, the kit includes reagents for detecting the expression level of the PHF20 gene or its encoded protein by RT-PCR, qRT-PCR, biochip detection, Southern blotting, in situ hybridization, or immunoblotting.

[0049] In some embodiments of the present invention, the nucleic acid membrane strip includes a reagent capable of detecting the expression level of the PHF20 gene or the protein encoded by it.

[0050] Chips include gene chips and protein chips. The gene chips include oligonucleotide probes targeting the PHF20 gene for detecting PHF20 gene transcription levels, while the protein chips include specific binding agents for the PHF20 protein. Kits include gene detection kits and protein detection kits. The gene detection kits include reagents or chips for detecting PHF20 gene transcription levels, while the protein detection kits include reagents or chips for detecting PHF20 protein expression levels.

[0051] The term "kit" refers to a set of components provided in the context of a system for sequencing nucleotides and / or isolating nucleotide sequences and / or diagnosing a subject with a disease or infection based on the presence, absence, and / or amount of expressed nucleotide sequences from a sample or cell. In some embodiments, the term "kit" refers to a set of components provided in the context of a system for sequencing nucleotides and / or isolating nucleotide sequences and / or diagnosing a subject with a disease or infection based on the spatial location of expressed nucleotide sequences in a sample or cell. Such systems may include, for example, systems that allow for the storage, verification, or delivery of expressed genes from one location to another in one or more cells (e.g., oligonucleotides in appropriate containers, oligonucleotides encoding enzymes, extracellular matrix components) and / or support materials (e.g., buffers, culture media, cells, written instructions for performing the assay). For example, in some embodiments, a kit includes one or more housings (e.g., cassettes) containing the relevant reaction reagents and / or support materials. The term "fragmentation kit" refers to a diagnostic assay comprising two or more separate containers, each containing a subset of the total kit components. The containers can be delivered to the intended recipient together or separately. For example, a first container may contain a solid support or polystyrene plate for a cell culture assay, while a second container may contain cells, such as control cells. As another example, a kit may include a first container containing a solid support, such as a chip or slide, with one or more ligands having affinity for one or more biomarkers disclosed herein, and a second container containing any one or more reagents necessary to detect and / or quantify the amount of lipid-modified oligonucleotide in a sample. The term "fragmentation kit" is intended to encompass, but is not limited to, kits containing analyte-specific reagents (ASRs) as defined under section 520(e) of the Federal Food, Drug, and Cosmetic Act. The term "fragmentation kit" encompasses any delivery system comprising two or more separate containers, each containing a subset of the total kit components. In contrast, a "combination kit" refers to a delivery system containing all components in a single container (e.g., a single cartridge containing each required component). The term "kit" encompasses both fragmented and combined kits.

[0052] In some embodiments of the present invention, the reagent is selected from an oligonucleotide probe that specifically recognizes the PHF20 gene, a primer that specifically amplifies the PHF20 gene, or a binding agent that specifically binds to the protein encoded by the PHF20 gene in a sample. Oligonucleotide probes targeting the PHF20 gene can be DNA, RNA, DNA-RNA chimeras, PNA, or other derivatives. The probe length is not limited; any length is acceptable as long as it achieves specific hybridization and specific binding to the target nucleotide sequence. The probe length can be as short as 25, 20, 15, 13, or 10 base pairs. Similarly, the probe length can be as long as 60, 80, 100, 150, 300 base pairs, or even longer, including the entire gene. Because different probe lengths have varying effects on hybridization efficiency and signal specificity, the probe length is typically at least 14 base pairs and generally no longer than 30 base pairs. A length complementary to the target nucleotide sequence of 15-25 base pairs is optimal. The probe itself preferably contains less than 4 base pairs of complementary sequence to avoid affecting hybridization efficiency.

[0053] The term "binding agent" refers to a naturally occurring or non-naturally occurring molecule that specifically binds to a target. Examples of specific binding agents include, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, and lipids. In certain embodiments, the specific binding agent is an antibody.

[0054] In the present invention, a binding agent that specifically binds to a protein encoded by the PHF20 gene, such as a receptor for the protein PHF20, a lectin that binds to the protein PHF20, an antibody against the protein PHF20, a peptide antibody against the protein PHF20, a bispecific dual binding agent, or a bispecific antibody format. Specific examples of specific binding agents include peptides, peptide mimetics, aptamers, spiegelmers (mirror image oligonucleotides), darpins (designed ankyrin repeat proteins), ankyrin repeat proteins, Kunitz-type domains, antibodies, single-domain antibodies, and monovalent antibody fragments.

[0055] In some embodiments of the present invention, the sample is peripheral blood and / or spleen tissue.

[0056] The chip, kit, or nucleic acid membrane strip described herein can be used to detect the expression levels of multiple genes or proteins, including the PHF20 gene or protein, and their expression products (e.g., multiple genes or proteins associated with systemic lupus erythematosus). Simultaneous detection of multiple markers of systemic lupus erythematosus can significantly improve the accuracy of systemic lupus erythematosus diagnosis.

[0057] The present invention further provides a method for screening candidate drugs for treating systemic lupus erythematosus, comprising: treating a culture system expressing or containing the PHF20 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the PHF20 gene or the protein encoded by it in the system; wherein, when the substance to be screened promotes the expression level or activity of the PHF20 gene or the protein encoded by it, the substance to be screened is a candidate drug for treating systemic lupus erythematosus.

[0058] The culture system includes, but is not limited to, a cell system, a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model, preferably an animal model of a non-human mammal, such as a mouse, rabbit, sheep or monkey).

[0059] In an embodiment of the present invention, the method further comprises: further testing the candidate drug obtained in the above step for its effect of inhibiting systemic lupus erythematosus; if the test compound has a significant inhibitory effect on systemic lupus erythematosus, it indicates that the candidate drug is a candidate drug for treating systemic lupus erythematosus.

[0060] The present invention provides the use of PHF20 in constructing a computational model for predicting systemic lupus erythematosus. As is well known to those skilled in the art, the step of correlating marker levels with a probability or risk can be implemented and realized in various ways. Preferably, the measured concentrations of the marker and one or more other markers are mathematically combined, and the combined value is correlated to the underlying diagnostic question. The measured marker values ​​can be combined using any suitable prior art mathematical method.

[0061] The present invention will be further described in detail below in conjunction with the accompanying drawings and Examples. The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Simple modifications to the present invention made according to the essence of the present invention all fall within the scope of protection claimed in the present invention.

[0062] Example 1: Construction of Lentivirus Overexpressing PHF20 In this example, a PHF20 overexpression lentivirus was constructed, wherein the PHF20 coding sequence (GenBank ID: 228829) was ligated to the lentiviral expression vector pLV3-CMV-3×FLAG-eGFP. The resulting PHF20 overexpression plasmid was named: pLV3-CMV-PHF20 (mouse)-3×FLAG-eGFP. The construction of this recombinant plasmid was completed by Innosilicon (Nanjing) Biotechnology Co., Ltd. The plasmid map is shown in Figure 1 Lentiviral packaging plasmids psPAX2 and pMD2G were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0063] The PHF20 lentiviral overexpression reagent in this example is obtained by plasmid transformation, lentiviral coating and concentration, and lentiviral titration detection. The specific steps are as follows: 1. Plasmid Amplification and Extraction 1) Prepare LB medium: First, add half of the ddH2O to a clean Erlenmeyer flask. Then, add 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride. For solid culture medium, add 15-20 g / L agar. Shake well to the desired volume to obtain LB liquid / solid culture medium. Seal the flask with tin foil, autoclave, and cool to room temperature before use.

[0064] Prepare a 10 cm culture dish. Add ampicillin resistance gene (AmpR) antibiotic (1:1000 dilution) to the LB solid medium cooled to approximately 50°C. Slowly shake to mix well. Quickly invert the plate and wait for it to cool and solidify. Mark the time and resistance on the lid of the dish.

[0065] 2) Take four 1.5 mL Eppendorf tubes and add 30-50 μL of DH5α competent cells (thawed on ice). Then, add 1 μL of each of the pMD2.G / psPAX2 / PHF20 overexpression plasmid and empty control plasmid. Gently tap the tubes to mix thoroughly. Incubate on ice for 45 minutes. After incubating at 42°C in a water bath for 90 seconds, quickly transfer the tubes to ice and incubate for 2 minutes. Do not shake the tubes. Then, add 1 mL of antibiotic-free LB medium to each tube. Mix thoroughly and allow the cells to recover on a shaker at 37°C at 200 rpm for 1 hour. Centrifuge at 5000 rpm for 3 minutes. Discard 800-900 μL of the supernatant, resuspend the cells, and evenly spread the suspension onto a solid culture medium plate using a small ball bearing the suspension. Place the plate upright at 37°C for approximately 30 minutes. Once the suspension has been completely absorbed, invert the plate and incubate overnight at 37°C. Add ampicillin (1:1000) to the LB medium again, take a 10 μL sterile pipette tip, pick up a single colony and throw it into a conical flask. Reseal the flask with tin foil and place it on a shaker at 200 rpm for overnight culture at 37°C for 16 hours. Use and follow the experimental steps of the Kangwei Century GoldHi Endofree Plasimid Maxi Kit to extract the plasmid from the activated and amplified bacterial solution, and store the plasmid at -20°C.

[0066] 2. Lentiviral Packaging 1) Seed well-grown 293T cells into a 100 mm culture dish in advance. When the cell confluence is close to 60-70%, the subsequent transfection step can be performed. 2) Prepare the EZ Trans-DNA complex: Prepare the target plasmid pLV3-CMV-PHF20 (mouse)-3×FLAG-eGFP, the backbone plasmid psPAX2, and the packaging plasmid pMD2G. For each 10 cm dish, add 36 μL of EZ Trans Cell Transfection Reagent (Cat. No. AC04L092, purchased from Heyuan Liji (Shanghai) Biotechnology Co., Ltd.), 6 μg of the target plasmid, 8 μg of psPAX2 plasmid, and 4 μg of pMD2.G plasmid to 1 mL of DMEM high-glucose medium. Calculate the total volume required and dilute the plasmids according to the above ratio into 5 mL of culture medium. Add 360 μL of transfection reagent to another centrifuge tube containing 5 mL of culture medium. Immediately add the diluted transfection reagent to the plasmid solution, mix gently, and incubate at room temperature for 15 minutes to allow EZ Trans to form the EZ Trans-DNA complex. Then, use a dropper to add 1 mL of EZ Trans-DNA complex to each culture dish. Mix the liquid using the cross method and place it in a 37°C constant temperature incubator for culture. Replace the culture medium after 6 to 8 hours. Collect and combine the supernatants 48 hours and 72 hours after replacing the culture medium. Centrifuge at 4°C and 5000 rpm for 20 min. Take the supernatant and filter it through a 0.45 μm filter into a new centrifuge tube to obtain the lentiviral solution.

[0067] 3. Lentivirus Concentration and Purification The lentiviral solution obtained above was concentrated using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100kD. The dissolved virus suspension was then aliquoted into 600 μL EP tubes and stored at -80°C to avoid repeated freezing and thawing that would reduce the titer of the virus solution.

[0068] 4. Virus Titer Detection One day in advance, 293T cells in good growth condition were seeded into 96-well plates, with 1×10 4 / well, place it in a 37 ° C constant temperature incubator for overnight culture. The next day, make a 10-fold gradient dilution in a 1.5 mL EP tube, with 6 consecutive dilutions. The specific dilution method is as follows: prepare 6 EP tubes, add 90 μL of culture medium to each tube, add 10 μL of virus stock solution to the first tube, mix well, and then draw 10 μL and add it to the second tube to mix well. Similarly, make 5 dilutions to obtain 6 different virus dilution concentrations, and add 10 μL of diluted virus solution to the cell well plate that has been replaced with fresh complete culture medium, and make 3 replicate wells for each dilution. Observe the results under a fluorescence microscope after 48-72 hours, select the wells with a fluorescence ratio between 20% and 30% for cell counting, and calculate the virus titer: Titer (TU / mL) = 4 × 10^ 4 ×30%×virus dilution factor (10)×10^ 3 =1.2×10^ 8 TU / mL.

[0069] Example 2: Study on the effect of PHF20 on improving lupus-like phenotype and renal damage in R848-induced lupus mice 1. Experimental Design The overall experimental design process is as follows Figure 2 As shown. Eight-week-old, healthy female C57 / B6 mice were purchased from Nanjing Huimiaoxin Biotechnology Co., Ltd. All animals were maintained under specific pathogen-free conditions at the Laboratory Animal Center of Nanjing University Drum Tower Hospital. All experimental procedures and husbandry methods were approved by the Laboratory Animal Ethics Review Committee of Nanjing University Drum Tower Hospital.

[0070] 2. Experimental methods: (1) Establishment of R848-induced lupus mouse model Eight-week-old female healthy C57 / B6 mice were randomly divided into a B6+acetone control group and a B6+R848 model group. Each group was treated as follows: B6+R848 modeling group: 40 μL of R848 (dissolved in acetone to 2 mg / mL) was applied to the skin of one side of the mouse's auricle every three days for 13 times to establish the model; B6+acetone control group: Apply the same volume of acetone as the model group.

[0071] The disease onset and modeling status of mice were assessed based on changes in body weight and urine protein levels compared to the control group. Modeling was successful when proteinuria was detected in the model group (control group 0.26±0.086 vs. model group 0.70±0.34), along with symptoms such as slow movement and weight loss.

[0072] (2) Grouping of mice and tail vein injection The successfully modeled mice were randomly divided into three groups: an R848 modeling group (B6+R848), a control reagent-treated group (R848+NC), and a treatment group (R848+OE). The B6+acetone control group consisted of mice treated with acetone, i.e., normal mice. Each group received the following treatments: Treatment group (R848+OE): 3×10 PHF20 overexpression virus solution obtained in Example 1 was injected into the tail vein every week. 7 TU / mouse, continuous injection for 4 weeks; Control reagent-treated group (R848+NC): Inject an equal amount of control virus solution (empty plasmid pLV3-CMV-3×FLAG-eGFP) in the same manner as the treatment group for 4 weeks; R848 model group (B6+R848): No treatment was given and the same feeding conditions were maintained until the end of observation; B6+acetone control group (B6+acetone): No treatment was given and the same feeding conditions were maintained until the end of observation.

[0073] At 20 weeks of age, mice in each group were treated and corresponding test specimens were obtained, including: (i) Mouse specimen collection: A. Obtain peripheral blood After anesthesia, the eyeballs of mice in each group were removed and blood was collected into EDTA anticoagulant tubes. The blood was centrifuged at 2000 rpm for 10 min to separate the plasma, and PBMCs were isolated by Ficoll density gradient centrifugation.

[0074] B. Separation of spleen and kidney After sacrifice, mice in each group were placed in the right lateral decubitus position. The abdomen and back of the mice were disinfected with 75% ethanol. The skin was cut open along the edge of the spleen, and the spleen was removed with forceps. The spleen tissue was obtained after rinsing with an appropriate amount of PBS. Some of the spleen tissue was frozen at -80°C, and the rest was soaked in PBS in a culture dish until use. The left kidney of the mouse was retained and frozen at -80°C; the right kidney of the mouse was retained and fixed in 4% paraformaldehyde for H&E staining and immunofluorescence experiments. C. Preparation of spleen single-cell suspension Grind the spleen thoroughly until it becomes a cell suspension. Filter the cell suspension through a 200-mesh sieve into a 15 mL centrifuge tube. Centrifuge at 1800 rpm at room temperature for 5 minutes, discard the supernatant, and obtain a spleen cell pellet. Lyse the cells with 3 times the volume of the pellet using red blood cell lysis buffer and centrifuge for 5 minutes. Discard the supernatant, at which point the cell pellet will turn from red to white. Resuspend the cell pellet in an appropriate volume of PBS, count, and set aside.

[0075] (3) After obtaining the corresponding specimens, relevant indicators are tested, including: (i) Mouse renal function test The Bradford spectrophotometer was used to detect urine protein in mice, the sarcosine oxidase method was used to detect urine creatinine in mice, and the urease method was used to detect urea nitrogen in mice.

[0076] (ii) Mouse plasma antibody detection The anti-dsDNA content in the plasma of each group of mice was detected according to the instructions of the mouse anti-double-stranded DNA antibody enzyme-linked immunosorbent assay kit provided by Jianglai Biological Co., Ltd.; the IgG antibody content in the serum of each group of mice was detected by double antibody sandwich enzyme-linked immunosorbent assay technology according to the mouse IgG detection kit provided by Lianke Biotechnology Co., Ltd.; the ANA content in the serum of each group of mice was detected according to the instructions of the mouse anti-nuclear antibody enzyme-linked immunosorbent assay kit provided by Wuhan Huamei Biological Co., Ltd.

[0077] (iii) Flow cytometry analysis of PHF20 and γH2AX expression in lupus mouse cells Mouse spleen single-cell suspension was washed twice and resuspended in an appropriate amount of PBS. Fixable Viability Dye eFluor 506 was diluted 1:1000 in pre-chilled PBS. 100 μL / well of the diluted antibody was added to the sample tube, mixed, and incubated at 4°C in the dark for 10 min. The supernatant was discarded. Surface antibodies Percp-cy5.5 anti-mouse CD45 (1:300) and PE-CF594 anti-mouse CD19 (1:300) were added and stained at 4°C in the dark for 30 min. An appropriate amount of PBS was added, and the cells were centrifuged at 1650 rpm and the supernatant was discarded. 100 μL / well of membrane permeabilization reagent was added and the cells were incubated at 4°C in the dark for 45 min. After washing, 100 μL / well of diluted APC anti-H2AX-Phosphorylated (Ser139) and PHF20 primary antibodies (catalog number #3934, purchased from Cell Signaling) were added. Technology), both were diluted 1:200, resuspended and incubated at 4°C in the dark for 45 min, the cells were terminated and washed to remove non-specific binding antibodies; 100 μL / well Fab2 Donkey Anti-Rabbit IgG PE (1:200) was added, mixed and incubated at 4°C in the dark for 30 min, 150 μL / well washing solution was added to terminate the reaction, and the supernatant was removed by centrifugation; 200 μL / well PBS was added to resuspend the cells, mixed thoroughly, filtered through a 200-mesh filter membrane into a new centrifuge tube, and detected using a BD LSRFortessa flow cytometer.

[0078] (iv) Real-time fluorescence quantitative PCR detection of gene expression A. Reverse transcription reaction RNA was extracted using the RNA isolater Total RNA Extraction Reagent provided by Nanjing Novozymes; genomic DNA was removed using 4 μL of 4X gDNA wiper mix; and the reverse transcription reaction system was prepared using 4 μL of 5X HiScript II qRT SuperMix II (see Table 1). The reverse transcription reaction procedure was 50°C for 15 min and 85°C for 5 sec. After completion of the reaction, cDNA was obtained for subsequent qPCR reactions. Primer sequences were designed using Primer Preimer 5.0 software and synthesized at GenScript Biotech Co., Ltd. after searching using the Basic Local Alignment Search Tool. The primer sequences are as follows: Mouse GAPDH gene primers: Forward primer: 5'-AGGTCGGTGTGAACGGATTTG-3' Reverse primer: 5'-GGGGTCGTTGATGGCAACA-3' Mouse PHF20 gene primers: Forward primer: 5'-TCTCCACCCAAAACATTTGCT-3' Reverse primer: 5'-ACTCCAAAGAAAGCTCTCGGAA-3' Table 1 Reverse transcription reaction system B. Real-time fluorescence quantitative PCR reaction The prepared cDNA for testing was added to a 384-well plate at 1 μL / well. The above primers were added, followed by 10 μL of SYBR Green Master Mix (ChamQ Universal, Nanjing Novozymes Biotechnology Co., Ltd.) to prepare a fluorescent quantitative PCR reaction system (see Table 2). The reaction system was placed in a QuantStudio™ 6 Flex Real-Time PCR Detection System (Applied Biosystems, USA) for PCR. The amplification reaction program was as follows: initial denaturation at 95°C for 30 seconds, followed by denaturation at 95°C for 10 seconds and 60°C for 30 seconds, for a total of 40 cycles. Each reaction was repeated three times for each gene.

[0079] Table 2 Fluorescence quantitative PCR reaction system C. Data Analysis The data were analyzed using QuantStudio™ Real-time PCR software, and the relative expression levels of target genes were calculated using the 2-ΔΔCt algorithm.

[0080] (vi) H&E staining After mouse kidneys were isolated, they were placed in 4% paraformaldehyde for tissue fixation and then treated with 75% ethanol (4 h), 85% ethanol (2 h), 95% ethanol (1 h), anhydrous ethanol I (30 min), anhydrous ethanol II (30 min), xylene I (10 min), xylene II (10 min), wax I (1 h), wax II (1 h), and wax III (1 h).

[0081] Dehydrated tissue was gently placed in an embedding frame containing melted wax blocks and cooled at -20°C. The blocks were sliced ​​into thin sections using a paraffin microtome, with a thickness of 2-5 μm. The sections were then unfolded in 40°C warm water, mounted on glass slides, and dried in a 60°C oven. Tissue sections were sequentially exposed to xylene solution (10 min), a xylene / ethanol mixture (1:1, 5 min), 100% ethanol (5 min), 95% ethanol (5 min), 85% ethanol (5 min), and 70% ethanol (5 min). After treatment, the sections were rinsed with running water. After washing and drying, the sections were added with an appropriate amount of hematoxylin solution and stained for 5-10 min. After staining, the sections were rinsed with running water and placed in 0.5% hydrochloric acid-alcohol differentiation solution. The staining of the nuclei and intranuclear regions was observed microscopically. The sections were then immersed in ddH2O for 10 min, alkalized by adding saturated lithium carbonate solution, and rinsed with running water. Tissue sections were stained with 0.1% eosin solution for 5 minutes and then dehydrated by immersion in the following solutions: 70% ethanol (2 minutes), 85% ethanol (2 minutes), 95% ethanol (2 minutes), 100% ethanol, and xylene (5 minutes). After the samples were allowed to dry slightly, they were covered with coverslips, mounted with neutral resin, and left to dry overnight in a fume hood. Kidney HE sections were then examined under a microscope.

[0082] (vii) Immunofluorescence staining of mouse kidney Mouse kidneys were isolated and placed in an embedding cassette. An appropriate amount of OCT embedding medium was added and the tissue was cooled to -20°C to solidify. The tissue was then cut into 10 μm sections using a freezing microtome and mounted on glass slides. The samples were fixed with 4% paraformaldehyde for 30 minutes and then washed three times with PBS for 5 minutes each. The samples were placed in a retrieval cassette containing EDTA antigen retrieval buffer and microwaved on high for 3 minutes, then off for 10 minutes, and then on low for 3 minutes. After cooling, the samples were washed three times with PBS for 5 minutes each. After the sections were slightly shaken dry, a circle was drawn around the tissue using a histochemical pen. An appropriate amount of 5% BSA was added and blocked for 30 minutes. Appropriate amounts of diluted C3 antibody (Cat. No. DF13224, purchased from Affinity Biosciences, 1:100 dilution) and IgG antibody (Cat. No. 115-025-062, purchased from Jackson ImmunoResearch, 1:200 dilution) were then added dropwise to the sections and refrigerated overnight at 4°C. The samples were then removed and returned to room temperature. The primary antibodies were recovered and the sections were washed three times with PBST. Appropriate amounts of secondary antibodies (488 goat anti-rabbit, Cat. No. ab150077, purchased from Abcam; Cy3 goat anti-mouse, Cat. No. ab150114, purchased from Abcam, both at 1:400 dilution) were added dropwise to the kidney tissue sections and incubated at room temperature for 1 hour. The sections were washed three times with PBST and allowed to dry briefly. An appropriate amount of anti-fluorescence quencher containing DAPI was added dropwise. The sections were carefully covered with a coverslip, mounted with neutral resin, and allowed to dry overnight in a fume hood. Confocal microscopy was used, and images were taken.

[0083] (4) Data analysis Graphpad Prism 9.0 was used for data analysis and graphs were plotted. For statistical comparisons between the two groups, the unpaired t-test was used to compare normally distributed quantitative data, and the Kruskal-Wallis H test was used to compare data with heterogeneity of variance. P < 0.05 was considered statistically significant.

[0084] 3. Results and Analysis according to Figure 3 The results showed that compared with the B6+acetone group, the expression level of PHF20 in PBMCs and spleen cells of the model group (R848+NC group and B6+R848 group) was significantly reduced. After the tail vein injection of PHF20 overexpressing lentivirus (R848+OE group), the expression level of PHF20 in PBMCs and spleen cells of lupus mice was significantly increased ( Figure 3 AC). WB showed that the PHF20 protein content in the R848+OE group was significantly higher than that in the other groups ( Figure 3D). The above results show that the expression level of PHF20 in PBMCs and spleen cells of mice with systemic lupus erythematosus is significantly reduced, and injection of PHF20-overexpressing lentivirus can significantly increase the expression level of PHF20 in mouse PBMCs and spleen cells to close to normal levels.

[0085] according to Figure 4 The results showed that compared with the B6+acetone group, the survival rate of the lupus mice groups was reduced. At the end of the observation period, the survival rate of the B6+R848 group and the R848+OE group decreased from 100% to 87.5%, while that of the R848+NC group decreased to 75% ( Figure 4 A). In addition, compared with the B6+acetone group, the weight of the other three groups of model mice generally decreased, while the weight of the overexpression group (R848+OE group) began to increase after the second injection, and at the fourth week of injection, the weight was significantly higher than that of the B6+R848 mice: 22.42±0.76 in the R848+OE group vs 19.14±1.35 in the B6+R848 group ( Figure 4 B).

[0086] according to Figure 5 The results showed that compared with the B6+acetone group, the spleen size and weight of mice in the B6+R848 group (0.089±0.016 vs 0.58±0.29) were significantly increased, while the spleen index of mice in the R848+OE group after treatment with PHF20 lentivirus was significantly reduced, basically close to normal levels ( Figure 5 A). In addition, after treatment with PHF20-expressing lentivirus, the anti-dsDNA antibody (R848+NC group 1.99±0.82 vs R848+OE group 1.09±0.61) and anti-IgG levels (R848+NC group 1.63±0.28 vs R848+OE group 1.21±0.22) in the plasma of lupus mice in the R848+OE group were significantly decreased, and the ANA level was also reduced (R848+NC group 19.48±9.01 vs R848+OE group 12.98±2.76), approaching the level of normal mice ( Figure 5 BD).

[0087] according to Figure 6 The results showed that overexpression of PHF20 in vivo could reduce the renal index of lupus-like mice ( Figure 6A); In addition, compared with the B6+acetone group, urine protein, urea nitrogen and blood creatinine of mice in the R848+R848 and R848+NC groups were significantly increased. After tail vein injection of PHF20 overexpressing virus solution (R848+OE group), urine protein (R848+NC group 1.00±0.78 vs R848+OE group 0.51±0.32) and urea nitrogen (R848+NC group 10.27±0.82 vs R848+OE group 6.78±2.39) of lupus-like mice were significantly decreased, and blood creatinine showed a downward trend (R848+NC group 19.75±5.57 vs R848+OE group 17.28±6.23), which was close to the level of normal mice ( Figure 6 BD).

[0088] according to Figure 7 The results showed that compared with the B6+acetone group, mice in the R848+R848 and R848+NC groups showed obvious glomerular inflammatory cell infiltration and morphological changes, while the glomerular inflammatory infiltration in the kidneys of mice in the PHF20 overexpression group (R848+OE group) was improved.

[0089] according to Figure 8 The results showed that compared with the B6+acetone group, the kidneys of mice in the R848+R848 and R848+NC groups showed obvious C3 and IgG deposition, while after tail vein injection of PHF20 overexpressing virus solution (R848+OE group), the C3 and IgG deposition in the kidney tissue of mice was significantly reduced.

[0090] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can make slight changes or modifications to equivalent embodiments of the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Any of the following applications: (1) Use of reagents for quantitatively detecting the expression level of the PHF20 gene or its encoded protein in the preparation of products for diagnosing systemic lupus erythematosus; (2) Use of an agent that promotes the expression level of the PHF20 gene or its encoded protein in the preparation of a pharmaceutical composition for treating systemic lupus erythematosus; (3) Application of PHF20 in screening candidate drugs for the treatment of systemic lupus erythematosus; and (4) Application of PHF20 in constructing a computational model for predicting systemic lupus erythematosus.

2. A pharmaceutical composition for treating systemic lupus erythematosus, characterized in that: The pharmaceutical composition comprises: (i) an agent that promotes the expression level of the PHF20 gene or the protein encoded by it; and (ii) pharmaceutically acceptable carriers and / or excipients.

3. The pharmaceutical composition according to claim 2, characterized in that The agent for promoting the expression level of the PHF20 gene or the protein encoded by it promotes the transcription of the PHF20 gene, promotes the expression of the PHF20 gene, promotes the function of the PHF20 protein, and / or inhibits the degradation of the PHF20 protein.

4. The pharmaceutical composition according to claim 3, characterized in that The agent for promoting the expression level of the PHF20 gene or the protein encoded by it is a lentivirus that overexpresses PHF20.

5. A product for diagnosing systemic lupus erythematosus, characterized in that: The product includes a chip, a kit or a nucleic acid membrane strip that can quantitatively detect the expression level of the PHF20 gene or the protein encoded by it in a sample.

6. The product according to claim 5, characterized in that The chip includes a reagent capable of detecting the expression level of the PHF20 gene or the protein encoded by it.

7. The product according to claim 5, characterized in that The kit includes reagents for detecting the expression level of the PHF20 gene or the protein encoded by it through RT-PCR method, qRT-PCR method, biochip detection method, Southern blotting method, in situ hybridization method, and immunoblotting method.

8. The product according to claim 5, characterized in that The nucleic acid membrane strip includes a reagent capable of detecting the expression level of the PHF20 gene or the protein encoded by it.

9. The product according to any one of claims 6 to 8, characterized in that The reagent is selected from an oligonucleotide probe that specifically recognizes the PHF20 gene, a primer that specifically amplifies the PHF20 gene, or a binding agent that specifically binds to a protein encoded by the PHF20 gene in a sample.

10. A method for screening candidate drugs for treating systemic lupus erythematosus, characterized in that: include: treating a culture system expressing or containing the PHF20 gene or the protein encoded by it with a substance to be screened; and detecting the expression or activity of the PHF20 gene or the protein encoded by it in the system; Wherein, when the substance to be screened promotes the expression level or activity of the PHF20 gene or the protein encoded by it, the substance to be screened is a candidate drug for treating systemic lupus erythematosus.

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