Biomarkers DDX5 and / or sIL-36R for inflammatory skin diseases and their applications

Abstract

The present invention discloses the use of sIL-36R, DDX5, and DDX5 / sIL-36R in the preparation of drugs for inhibiting, alleviating, reducing, and / or treating atopic dermatitis and / or psoriasis. The present invention also discloses the biomarker DDX5 and / or sIL-36R and / or DDX5 / sIL-36R for psoriasis and / or atopic dermatitis and their applications. DDX5 is downregulated in keratinocytes in the skin lesions of psoriasis and / or atopic dermatitis, but the expression of DDX5 returns to normal levels after the skin lesions subside. The low expression of DDX5 leads to changes in the alternative splicing of IL-36R pre-mRNA in keratinocytes, resulting in an increase in IL-36R and a decrease in soluble sIL-36R, thereby promoting the disorder of the IL-36R signaling pathway and inducing skin inflammation; while injecting sIL-36R can effectively inhibit the IL-36R signaling pathway in atopic dermatitis mice or psoriasis mice, and inhibit the expression of cytokines in the skin lesions of atopic dermatitis or psoriasis mice to alleviate atopic dermatitis. The sIL-36R, DDX5, and DDX5 / sIL-36R can be used as targets and biomarkers for the diagnosis and treatment of inflammatory skin diseases.

Description

Technical Field

[0001] The present invention relates to the fields of biochemistry, molecular biology, immunology, dermatology, etc., and relates to the inflammatory skin disease biomarkers DDX5 and / or sIL-36R and their applications. Background Art

[0002] Atopic dermatitis (AD) and psoriasis are two major chronic inflammatory skin diseases. Data from the World Health Organization show that at least 230 million people worldwide suffer from atopic dermatitis [1], and about 125 million people are affected by psoriasis [2]. Among them, the prevalence of atopic dermatitis in children under 7 years old is as high as 25%, and the probability of adults suffering from atopic dermatitis is 7-10%. These two inflammatory skin diseases not only have a high incidence, but also recur frequently, bringing serious troubles to the lives of patients.

[0003] The pathological phenotypes of these two inflammatory diseases, AD and psoriasis, on the skin are mainly caused by the response of keratinocytes to cytokines produced by T cells. However, the T cell subsets that play a pathogenic role in these two diseases are different, so the cytokines acting on keratinocytes also vary greatly [3]. For example, in AD, IL-4 and IL-13 secreted by Th2 cells act on keratinocytes, not only inhibiting the expression of the keratinocyte differentiation gene Flaggerin (FLG) to damage the skin barrier function [4,5], but also inducing keratinocytes to express chemokines such as CCL17 and CCL22 to recruit a large number of inflammatory cells to infiltrate at the lesion site [6]. In psoriasis, cytokines such as IL-17A and IL-17F secreted by Th17 cells act on keratinocytes, inducing keratinocytes to express REG3A to promote epidermal hyperplasia [7] or inducing chemokines such as CXCL1 and CCL20 to recruit more neutrophils and Th17 cell infiltration [8], thus aggravating the skin inflammatory phenotype.

[0004] Although AD and psoriasis are two inflammatory skin diseases with different clinical, histological, and molecular phenotypes, there are also similar mechanisms in their pathogenesis, namely, the response of keratinocytes to external danger signals plays a crucial role in the initiation of inflammation in AD and psoriasis. After skin injury, RNAs released by damaged cells activate TLR3 in keratinocytes to induce the expression of IL-36 [9]. IL-36 activates the signal transduction of the IL-36 receptor (IL-36R) in keratinocytes or dendritic cells to induce the expression of chemokines such as CCL17, CXCL8, CXCL1, and CCL20, recruiting a large number of neutrophil infiltrations or activating cells such as Th17 to secrete more cytokines, thus triggering skin inflammation

[10] . A large number of studies have demonstrated that the high expression of IL-36 family cytokines and the disorder of the IL-36 / IL-36R signaling pathway are important factors in the development of psoriasis (especially pustular psoriasis)

[11] . In AD, the activation of the IL-36 signaling pathway by IL-36 promotes scratching to initiate or maintain AD inflammation

[12] . In addition, Asian AD patients have the pathological phenotypes of both European and American AD and psoriasis. In particular, the high expression of IL-36 in the epidermis of Asian AD is the main factor for them to have these two pathological phenotypes

[13] . In summary, IL-36 family cytokines initiate downstream pro-inflammatory signaling pathways by binding to and activating their receptor IL-36R, inducing the expression of inflammatory factors and chemokines to recruit a large number of inflammatory cell infiltrations, thus triggering AD and psoriasis. Therefore, the IL-36 / IL-36R signaling pathway can be used as a therapeutic target for inflammatory skin diseases such as AD and psoriasis. A recent report of successfully treating 7 patients with pustular psoriasis using an IL-36R neutralizing antibody also confirms this

[14] . In addition to IL-36R neutralizing antibodies, whether there are other factors that can antagonize the IL-36R signaling pathway and whether antagonizing the IL-36R signaling pathway can treat AD remains unknown.

[0005] DDX5 is an ATP-dependent RNA helicase, also known as DEAD box protein 5 or RNA helicase p68

[15] . DDX5 is involved in various pathways including RNA structural alterations, and can act as a transcriptional regulator, a regulator of splicing, and play a role in the processing of non-coding RNAs

[16] . RNA splicing (especially alternative splicing) is one of the important ways of eukaryotic gene expression regulation. Alternative splicing results in the production of multiple transcript variants, and different variants play crucial roles in the occurrence and development of diseases. For example, DDX5 / DDX17 is involved in regulating the alternative splicing of the transcription factor NFAT5 to promote the expression of IL-4 and GATA3, which may promote Th2 immune responses in AD [17, 18]. In addition, existing data show that DDX5 is closely related to flexural, erythrodermic, and chronic plaque psoriasis

[19] , and in the gene analysis of atopic march-related diseases, DDX5 has also been found to be associated with the reduced expression in asthma and the disease process

[20] . Although DDX5 has a certain correlation with AD and psoriasis, how DDX5 is involved in the pathogenesis of AD or psoriasis and whether it can be used as a biomarker for the diagnosis and treatment of inflammatory skin diseases such as AD and psoriasis remains unknown. Summary of the Invention

[0006] The present invention provides the use of sIL-36R, DDX5, and DDX5 / sIL-36R in the preparation of a drug for inhibiting and / or alleviating and / or reducing and / or treating atopic dermatitis (AD) and / or psoriasis.

[0007] The sIL-36R inhibits the IL-36 / IL-36R signaling pathway, including the downstream NFκB and p38MAPK signaling pathways, inhibits the inflammatory response induced by IL-36 family cytokines, and thus plays a role in alleviating and treating AD. Injecting the recombinant sIL-36R of the present invention into MC903-induced AD mice can relieve the pathological symptoms of AD mice and play a role in inhibiting and / or alleviating and / or reducing and / or treating AD.

[0008] The sIL-36R is a soluble receptor of IL-36, which only includes the ligand-binding region of IL-36R; its amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2, and its nucleotide sequence is as shown in SEQ ID NO.3 or SEQ ID NO.4:

[0009] The amino acid sequence of murine sIL-36R is as follows:

[0010] MGVTSLLFCGVFFLLLLFVAADTCEDIFMHNVIISEGQPFPFNCTYPPETNGAVNLTWYKTPSKSPVSNNRHLRVHQDQTWILFLPLTLEDSGIYQCVIRNAHNCYQIAVNLTVLKNHWCDSSMEGSPVNSPDVYQQILPIGKSGSLNCHLYFPESCALDSIKWYKGCEEIKAGKKYSPSGAKLLVNNVAVEDGGSYACSARLTHLGRHFTIRNYIAVNTKEVEYGRRIPNITYPKNNSIEVPLEPMCP。(SEQ ID NO.1)

[0011] The amino acid sequence of human sIL-36R is as follows:

[0012] MWSLLLCGLSIALPLSVTADGCKDIFMKNEILSASQPFAFNCTFPPITSGEVSVTWYKNSSKIPVSKIIQSRIHQDETWILFLPMEWGDSGVYQCVIKTVTRLKGSGSLFWKPGFW(SEQ ID NO.2)

[0013] The nucleotide sequence of mouse sIL-36R is as follows:

[0014] atgggggttacatctttgctcttctgtggggtgtttttcctgcttctgcttttcgtggcagcagatacgtgtgaggacatttttatgcacaatgtgataatttcagagggccagccttttcctttcaactgcacatacccgccagaaacaaacggggcagtaaatctgacatggtacaaaacacctagcaaaagcccagtatctaacaacagacaccttagagttcaccaggaccagacctggatcttgtttcttccattgacactggaggactccggtatctatcagtgtgttataaggaatgcccacaactgctaccaaatagctgtgaacctaaccgttttaaaaaaccactggtgtgactcttccatggaggggagtcccgtaaattcaccagatgtgtaccagcaaatattacccataggaaaatcgggcagtctgaattgtcatctctacttcccagaaagttgtgctttggattcaataaaatggtataagggttgtgaagagattaaagcggggaaaaagtacagcccttcaggagcaaagcttcttgtgaacaacgttgctgtggaggacggcgggagctatgcgtgctcagccagactgactcacttggggagacacttcaccattagaaactacattgctgtgaacaccaaggaagttgagtatggaagaaggatccctaacatcacgtatccaaagaacaactccattgaagttccacttgaaccaatgtgtccttga(SEQ ID NO.3)

[0015] The nucleotide sequence of human sIL-36R is as follows:

[0016] ATGTGGTCCTTGCTGCTCTGCGGGTTGTCCATCGCCCTTCCACTGTCTGTCACAGCAGATGGATGCAAGGACATTTTTATGAAAAATGAGATACTTTCAGCAAGCCAGCCTTTTGCTTTTAATTGTACATTCCCTCCCATAACATCTGGGGAAGTCAGTGTAACATGGTATAAAAATTCTAGCAAAATCCCAGTGTCCAAAATCATACAGTCTAGAATTCACCAGGACGAGACTTGGATTTTGTTTCTCCCCATGGAATGGGGGGACTCAGGAGTCTACCAATGTGTTATAAAGACTGTAACGAGATTAAAGGGGAGCGGTTCACTGTTTTGGAAACCAGGCTTTTGGTGA(SEQ ID NO.4)

[0017] The amino acid sequence of the sIL-36R only contains the ligand-binding region of the full-length receptor and does not have the transmembrane region and intracellular region of the full-length receptor.

[0018] The sIL-36R is obtained by transcribing and translating the IL-36R mRNA lacking the 3rd exon. Since the deletion of the 3rd exon in the IL-36R mRNA causes premature termination of transcription, the translated protein only contains the ligand-binding region and is secreted extracellularly.

[0019] The present invention also provides the use of the sIL-36R in the preparation of a drug for inhibiting the production of cytokines and chemokines induced by IL-36.

[0020] Among them, the cytokines include IL-4, IL-13, TSLP, IL23, IL17a.

[0021] Among them, the chemokines include CCL17, CCL22, CCL20, CXCL1, CXCL2, CCL3, and CCL11.

[0022] Among them, the drug is used to inhibit the inflammatory response of keratinocytes to IL-36.

[0023] The present invention also provides the use of the sIL-36R in the preparation of a drug for inhibiting the IL-36 / IL-36R signaling pathway, including the downstream signaling pathways NFκB and p38 MAPK.

[0024] The present invention also provides the use of the sIL-36R in the preparation of a drug for inhibiting the proliferation of keratinocytes.

[0025] The present invention also provides a biomarker DDX5, which is the RNA helicase DDX5. Its amino acid sequence is as shown in SEQ ID NO.5 or SEQ ID NO.6, and its nucleotide sequence is as shown in SEQ ID NO.7 or SEQ ID NO.8. Its physicochemical characteristics are an ATP-dependent RNA helicase that participates in the transcriptional regulation of genes, is highly expressed in keratinocytes of the normal skin epidermis, and its expression in the skin lesions of AD and psoriasis patients is significantly inhibited.

[0026] The DDX5 is used for diagnosing the onset and / or cure and / or recurrence of atopic dermatitis and / or psoriasis.

[0027] The expression of DDX5 is decreased at the skin lesion sites of atopic dermatitis and psoriasis, and as the skin lesions subside, the expression of DDX5 returns to the normal level.

[0028] The present invention also provides the application of the biomarker DDX5 in diagnosing the onset and / or cure and / or recurrence of inflammatory skin diseases such as atopic dermatitis and / or psoriasis.

[0029] The inflammatory skin diseases include AD, psoriasis, etc.

[0030] The expressions of the biomarker DDX5 and sIL-36R are decreased in the skin lesions of AD and psoriasis, while their expressions are increased in the keratinocytes of skin cancers such as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). The reduction of DDX5 leads to the spontaneous generation of AD-like and psoriasis-like inflammation in the skin, and the deletion of DDX5 under pathological conditions further aggravates the skin inflammation of AD and psoriasis. When the pathological symptoms of AD or psoriasis subside, the expression of DDX5 returns to the normal level.

[0031] The present invention also provides a combined biomarker, which comprises DDX5, sIL-36R, and / or the DDX5 / sIL-36R combination.

[0032] The present invention also provides the application of the DDX5, sIL-36R, and / or DDX5 / sIL-36R combined biomarker in diagnosing the onset and / or cure and / or recurrence of inflammatory skin diseases such as atopic dermatitis or psoriasis.

[0033] The DDX5 regulates the splicing of IL-36R pre-mRNA to generate sIL-36R.

[0034] Reduction of DDX5 leads to an increase in IL-36R, but a decrease in sIL-36R, thus causing disorders in the IL-36 / IL-36R signaling pathway, generating a large number of cytokines and chemokines to trigger skin inflammation. Therefore, the amount of DDX5 is relatively reduced in the skin lesions of AD, psoriasis patients and mice, and the corresponding sIL-36R is also reduced.

[0035] The present invention also provides a detection reagent / kits, which contain the biomarker DDX5, and / or sIL-36R and / or DDX5 / sIL-36R combination.

[0036] The detection reagent / kits are used for diagnosing the onset and / or cure and / or recurrence of inflammatory skin diseases such as atopic dermatitis or psoriasis.

[0037] The present invention also provides a drug or pharmaceutical composition, which contain the biomarker DDX5, sIL-36R and / or DDX5 / sIL-36R combination.

[0038] The present invention also proposes the application of the drug or pharmaceutical composition in the preparation of drugs for inhibiting and / or alleviating and / or reducing and / or treating atopic dermatitis and / or psoriasis.

[0039] The present invention also provides a mouse model with spontaneous dermatitis, that is, a gene knockout mouse with epidermal-specific deletion of DDX5 (Ddx5 Δ / KC ). These mice spontaneously develop atopic dermatitis-like or psoriasis-like dermatitis phenotypes 2 weeks after birth. In addition, in atopic dermatitis mice with keratinocyte-specific deletion of DDX5 induced by MC903, the deletion of DDX5 leads to an increase in cytokines IL-4, IL-13, TSLP and an increase in chemokines CCL11, CCL17 and CCL22. In psoriasis mice with keratinocyte-specific deletion of DDX5 induced by imiquimod, the secretion of cytokines IL-23 and IL-17a and chemokines CCL20 and CXCL1 increases.

[0040] In the present invention, the cytokines include IL-4, IL-13, TSLP, IL23, IL17a. The chemokines include CCL3, CCL11, CCL17, CCL22, CCL20, CXCL1 and CXCL 2.

[0041] The present invention also provides the application of the mouse model with spontaneous dermatitis in inhibiting and / or treating and / or reducing and / or alleviating inflammatory skin diseases such as AD or psoriasis.

[0042] The present invention also provides a method for inhibiting and / or treating and / or alleviating and / or relieving atopic dermatitis or psoriasis, by administering the soluble receptor sIL-36R, DDX5, DDX5 / sIL-36R to an individual subject.

[0043] The present invention also provides a method for detecting the onset, cure and recurrence of inflammatory skin diseases such as AD and psoriasis, by detecting the expression of DDX5 and / or sIL-36R in the skin lesions of an individual subject using the DDX5, sIL-36R and / or DDX5 / sIL-36R reagent or kit.

[0044] The beneficial effects of the present invention include: the soluble receptor sIL-36R provided by the present invention can inhibit the IL-36R signaling pathway activated by IL-36, thereby reducing the inflammatory response of AD and achieving the purpose of treating AD. The present invention also discovers that the combination of DDX5, sIL-36R or DDX5 / sIL-36R can be used as a target and biomarker for diagnosing and treating inflammatory skin diseases, and can effectively inhibit, treat, alleviate and relieve inflammatory skin diseases such as AD and psoriasis. In the present invention, the expression of DDX5 is down-regulated in keratinocytes in the skin lesions of psoriasis and / or atopic dermatitis, but the expression of DDX5 returns to the normal level after the skin lesions subside. The low expression of DDX5 leads to changes in the alternative splicing of IL-36R pre-mRNA in keratinocytes, resulting in an increase in IL-36R and a decrease in soluble sIL-36R, thus promoting the disorder of the IL-36R signaling pathway and inducing skin inflammation; while injecting sIL-36R can effectively inhibit the IL-36R signaling pathway in atopic dermatitis mice or psoriasis mice, and inhibit the expression of cytokines in the skin lesions of atopic dermatitis or psoriasis mice to reduce atopic dermatitis. The sIL-36R, DDX5, DDX5 / sIL-36R can be used as a target and biomarker for diagnosing and treating inflammatory skin diseases. SEQUENCE LISTING <110> East China Normal University <120> Biomarkers DDX5 and / or sIL-36R for Inflammatory Skin Diseases and Their Applications <160> 8 <170> PatentIn version 3.3 <210> 1 <211> 249 <212> PRT <213> Artificial Sequence <400> 1 Met Gly Val Thr Ser Leu Leu Phe Cys Gly Val Phe Phe Leu Leu Leu 1 5 10 15 Leu Phe Val Ala Ala Asp Thr Cys Glu Asp Ile Phe Met His Asn Val 20 25 30 Ile Ile Ser Glu Gly Gln Pro Phe Pro Phe Asn Cys Thr Tyr Pro Pro 35 40 45 Glu Thr Asn Gly Ala Val Asn Leu Thr Trp Tyr Lys Thr Pro Ser Lys 50 55 60 Ser Pro Val Ser Asn Asn Arg His Leu Arg Val His Gln Asp Gln Thr 65 70 75 80 Trp Ile Leu Phe Leu Pro Leu Thr Leu Glu Asp Ser Gly Ile Tyr Gln 85 90 95 Cys Val Ile Arg Asn Ala His Asn Cys Tyr Gln Ile Ala Val Asn Leu 100 105 110 Thr Val Leu Lys Asn His Trp Cys Asp Ser Ser Met Glu Gly Ser Pro 115 120 125 Val Asn Ser Pro Asp Val Tyr Gln Gln Ile Leu Pro Ile Gly Lys Ser 130 135 140 Gly Ser Leu Asn Cys His Leu Tyr Phe Pro Glu Ser Cys Ala Leu Asp 145 150 155 160 Ser Ile Lys Trp Tyr Lys Gly Cys Glu Glu Ile Lys Ala Gly Lys Lys 165 170 175 Tyr Ser Pro Ser Gly Ala Lys Leu Leu Val Asn Asn Val Ala Val Glu 180 185 190 Asp Gly Gly Ser Tyr Ala Cys Ser Ala Arg Leu Thr His Leu Gly Arg 195 200 205 His Phe Thr Ile Arg Asn Tyr Ile Ala Val Asn Thr Lys Glu Val Glu 210 215 220 Tyr Gly Arg Arg Ile Pro Asn Ile Thr Tyr Pro Lys Asn Asn Ser Ile 225 230 235 240 Glu Val Pro Leu Glu Pro Met Cys Pro 245 <210> 2 <211> 116 <212> PRT <213> Artificial Sequence <400> 2 Met Trp Ser Leu Leu Leu Cys Gly Leu Ser Ile Ala Leu Pro Leu Ser 1 5 10 15 Val Thr Ala Asp Gly Cys Lys Asp Ile Phe Met Lys Asn Glu Ile Leu 20 25 30 Ser Ala Ser Gln Pro Phe Ala Phe Asn Cys Thr Phe Pro Pro Ile Thr 35 40 45 Ser Gly Glu Val Ser Val Thr Trp Tyr Lys Asn Ser Ser Lys Ile Pro 50 55 60 Val Ser Lys Ile Ile Gln Ser Arg Ile His Gln Asp Glu Thr Trp Ile 65 70 75 80 Leu Phe Leu Pro Met Glu Trp Gly Asp Ser Gly Val Tyr Gln Cys Val 85 90 95 Ile Lys Thr Val Thr Arg Leu Lys Gly Ser Gly Ser Leu Phe Trp Lys 100 105 110 Pro Gly Phe Trp 115 <210> 3 <211> 750 <212> DNA <213> Artificial sequence <400> 3 atgggggtta catctttgct cttctgtggg gtgtttttcc tgcttctgct tttcgtggca 60 gcagatacgt gtgaggacat ttttatgcac aatgtgataa tttcagaggg ccagcctttt 120 cctttcaact gcacataccc gccagaaaca aacggggcag taaatctgac atggtacaaa 180 acacctagca aaagcccagt atctaacaac agacacctta gagttcacca ggaccagacc 240 tggatcttgt ttcttccatt gacactggag gactccggta tctatcagtg tgttataagg 300 aatgcccaca actgctacca aatagctgtg aacctaaccg ttttaaaaaa ccactggtgt 360 gactcttcca tggaggggag tcccgtaaat tcaccagatg tgtaccagca aatattaccc 420 ataggaaaat cgggcagtct gaattgtcat ctctacttcc cagaaagttg tgctttggat 480 tcaataaaat ggtataaggg ttgtgaagag attaaagcgg ggaaaaagta cagcccttca 540 ggagcaaagc ttcttgtgaa caacgttgct gtggaggacg gcgggagcta tgcgtgctca 600 gccagactga ctcacttggg gagacacttc accattagaa actacattgc tgtgaacacc 660 aaggaagttg agtatggaag aaggatccct aacatcacgt atccaaagaa caactccatt 720 gaagttccac ttgaaccaat gtgtccttga 750 <210> 4 <211> 351 <212> DNA <213> Artificial Sequence <400> 4 atgtggtcct tgctgctctg cgggttgtcc atcgcccttc cactgtctgt cacagcagat 60 ggatgcaagg acatttttat gaaaaatgag atactttcag caagccagcc ttttgctttt 120 aattgtacat tccctcccat aacatctggg gaagtcagtg taacatggta taaaaattct 180 agcaaaatcc cagtgtccaa aatcatacag tctagaattc accaggacga gacttggatt 240 ttgtttctcc ccatggaatg gggggactca ggagtctacc aatgtgttat aaagactgta 300 acgagattaa aggggagcgg ttcactgttt tggaaaccag gcttttggtg a 351 <210> 5 <211> 614 <212> PRT <213> Artificial Sequence <400> 5 Met Ser Gly Tyr Ser Ser Asp Arg Asp Arg Gly Arg Asp Arg Gly Phe 1 5 10 15 Gly Ala Pro Arg Phe Gly Gly Ser Arg Ala Gly Pro Leu Ser Gly Lys 20 25 30 Lys Phe Gly Asn Pro Gly Glu Lys Leu Val Lys Lys Lys Trp Asn Leu 35 40 45 Asp Glu Leu Pro Lys Phe Glu Lys Asn Phe Tyr Gln Glu His Pro Asp 50 55 60 Leu Ala Arg Arg Thr Ala Gln Glu Val Glu Thr Tyr Arg Arg Ser Lys 65 70 75 80 Glu Ile Thr Val Arg Gly His Asn Cys Pro Lys Pro Val Leu Asn Phe 85 90 95 Tyr Glu Ala Asn Phe Pro Ala Asn Val Met Asp Val Ile Ala Arg Gln 100 105 110 Asn Phe Thr Glu Pro Thr Ala Ile Gln Ala Gln Gly Trp Pro Val Ala 115 120 125 Leu Ser Gly Leu Asp Met Val Gly Val Ala Gln Thr Gly Ser Gly Lys 130 135 140 Thr Leu Ser Tyr Leu Leu Pro Ala Ile Val His Ile Asn His Gln Pro 145 150 155 160 Phe Leu Glu Arg Gly Asp Gly Pro Ile Cys Leu Val Leu Ala Pro Thr 165 170 175 Arg Glu Leu Ala Gln Gln Val Gln Gln Val Ala Ala Glu Tyr Cys Arg 180 185 190 Ala Cys Arg Leu Lys Ser Thr Cys Ile Tyr Gly Gly Ala Pro Lys Gly 195 200 205 Pro Gln Ile Arg Asp Leu Glu Arg Gly Val Glu Ile Cys Ile Ala Thr 210 215 220 Pro Gly Arg Leu Ile Asp Phe Leu Glu Cys Gly Lys Thr Asn Leu Arg 225 230 235 240 Arg Thr Thr Tyr Leu Val Leu Asp Glu Ala Asp Arg Met Leu Asp Met 245 250 255 Gly Phe Glu Pro Gln Ile Arg Lys Ile Val Asp Gln Ile Arg Pro Asp 260 265 270 Arg Gln Thr Leu Met Trp Ser Ala Thr Trp Pro Lys Glu Val Arg Gln 275 280 285 Leu Ala Glu Asp Phe Leu Lys Asp Tyr Ile His Ile Asn Ile Gly Ala 290 295 300 Leu Glu Leu Ser Ala Asn His Asn Ile Leu Gln Ile Val Asp Val Cys 305 310 315 320 His Asp Val Glu Lys Asp Glu Lys Leu Ile Arg Leu Met Glu Glu Ile 325 330 335 Met Ser Glu Lys Glu Asn Lys Thr Ile Val Phe Val Glu Thr Lys Arg 340 345 350 Arg Cys Asp Glu Leu Thr Arg Lys Met Arg Arg Asp Gly Trp Pro Ala 355 360 365 Met Gly Ile His Gly Asp Lys Ser Gln Gln Glu Arg Asp Trp Val Leu 370 375 380 Asn Glu Phe Lys His Gly Lys Ala Pro Ile Leu Ile Ala Thr Asp Val 385 390 395 400 Ala Ser Arg Gly Leu Asp Val Glu Asp Val Lys Phe Val Ile Asn Tyr 405 410 415 Asp Tyr Pro Asn Ser Ser Glu Asp Tyr Ile His Arg Ile Gly Arg Thr 420 425 430 Ala Arg Ser Thr Lys Thr Gly Thr Ala Tyr Thr Phe Phe Thr Pro Asn 435 440 445 Asn Ile Lys Gln Val Ser Asp Leu Ile Ser Val Leu Arg Glu Ala Asn 450 455 460 Gln Ala Ile Asn Pro Lys Leu Leu Gln Leu Val Glu Asp Arg Gly Ser 465 470 475 480 Gly Arg Ser Arg Gly Arg Gly Gly Met Lys Asp Asp Arg Arg Asp Arg 485 490 495 Tyr Ser Ala Gly Lys Arg Gly Gly Phe Asn Thr Phe Arg Asp Arg Glu 500 505 510 Asn Tyr Asp Arg Gly Tyr Ser Ser Leu Leu Lys Arg Asp Phe Gly Ala 515 520 525 Lys Thr Gln Asn Gly Val Tyr Ser Ala Ala Asn Tyr Thr Asn Gly Ser 530 535 540 Phe Gly Ser Asn Phe Val Ser Ala Gly Ile Gln Thr Ser Phe Arg Thr 545 550 555 560 Gly Asn Pro Thr Gly Thr Tyr Gln Asn Gly Tyr Asp Ser Thr Gln Gln 565 570 575 Tyr Gly Ser Asn Val Pro Asn Met His Asn Gly Met Asn Gln Gln Ala 580 585 590 Tyr Ala Tyr Pro Ala Thr Ala Ala Ala Pro Met Ile Gly Tyr Pro Met 595 600 605 Pro Thr Gly Tyr Ser Gln 610 <210> 6 <211> 615 <212> PRT <213> Artificial Sequence <400> 6 Met Ser Ser Tyr Ser Ser Asp Arg Asp Arg Gly Arg Asp Arg Gly Phe Gly Ala Pro 1 5 10 15 Arg Phe Gly Gly Ser Arg Thr Gly Pro Leu Ser Gly Lys Lys Phe Gly Asn Pro Gly Glu 20 25 30 35 Lys Leu Val Lys Lys Lys Trp Asn Leu Asp Glu Leu Pro Lys Phe Glu Lys Asn Phe Tyr 40 45 50 55 Gln Glu His Pro Asp Leu Ala Arg Arg Thr Ala Gln Glu Val Asp Thr Tyr Arg Arg Ser 60 65 70 75 Lys Glu Ile Thr Val Arg Gly His Asn Cys Pro Lys Pro Val Leu Asn Phe Tyr Glu Ala 80 85 90 95 Asn Phe Pro Ala Asn Val Met Asp Val Ile Ala Arg Gln Asn Phe Thr Glu Pro Thr Ala 100 105 110 115 Ile Gln Ala Gln Gly Trp Pro Val Ala Leu Ser Gly Leu Asp Met Val Gly Val Ala Gln 120 125 130 135 Thr Gly Ser Gly Lys Thr Leu Ser Tyr Leu Leu Pro Ala Ile Val His Ile Asn His Gln 140 145 150 155 Pro Phe Leu Glu Arg Gly Asp Gly Pro Ile Cys Leu Val Leu Ala Pro Thr Arg Glu Leu 160 165 170 175 Ala Gln Gln Val Gln Gln Val Ala Ala Glu Tyr Cys Arg Ala Cys Arg Leu Lys Ser Thr 180 185 190 195 Cys Ile Tyr Gly Gly Ala Pro Lys Gly Pro Gln Ile Arg Asp Leu Glu Arg Gly Val Glu 200 205 210 215 Ile Cys Ile Ala Thr Pro Gly Arg Leu Ile Asp Phe Leu Glu Cys Gly Lys Thr Asn Leu 220 225 230 235 Arg Arg Thr Thr Tyr Leu Val Leu Asp Glu Ala Asp Arg Met Leu Asp Met Gly Phe 240 245 250 255 Glu Pro Gln Ile Arg Lys Ile Val Asp Gln Ile Arg Pro Asp Arg Gln Thr Leu Met Trp Ser 260 265 270 275 Ala Thr Trp Pro Lys Glu Val Arg Gln Leu Ala Glu Asp Phe Leu Lys Asp Tyr Ile His 280 285 290 295 Ile Asn Ile Gly Ala Leu Glu Leu Ser Ala Asn His Asn Ile Leu Gln Ile Val Asp Val 300 305 310 315 Cys His Asp Val Glu Lys Asp Glu Lys Leu Ile Arg Leu Met Glu Glu Ile Met Ser Glu 320 325 330 335 Lys Glu Asn Lys Thr Ile Val Phe Val Glu Thr Lys Arg Arg Cys Asp Glu Leu Thr Arg 340 345 350 355 Lys Met Arg Arg Asp Gly Trp Pro Ala Met Gly Ile His Gly Asp Lys Ser Gln Gln Glu 360 365 370 375 Arg Asp Trp Val Leu Asn Glu Phe Lys His Gly Lys Ala Pro Ile Leu Ile Ala Thr Asp 380 385 390 395 Val Ala Ser Arg Gly Leu Asp Val Glu Asp Val Lys Phe Val Ile Asn Tyr Asp Tyr Pro 400 405 410 415 Asn Ser Ser Glu Asp Tyr Ile His Arg Ile Gly Arg Thr Ala Arg Ser Thr Lys Thr Gly 420 425 430 435 Thr Ala Tyr Thr Phe Phe Thr Pro Asn Asn Ile Lys Gln Val Ser Asp Leu Ile Ser Val 440 445 450 455 Leu Arg Glu Ala Asn Gln Ala Ile Asn Pro Lys Leu Leu Gln Leu Val Glu Asp Arg Gly 460 465 470 475 Ser Gly Arg Ser Arg Gly Arg Gly Gly Met Lys Asp Asp Arg Arg Asp Arg Tyr Ser Ala 480 485 490 495 Gly Lys Arg Gly Gly Phe Asn Thr Phe Arg Asp Arg Glu Asn Tyr Asp Arg Gly Tyr Ser 500 505 510 515 Asn Leu Leu Lys Arg Asp Phe Gly Ala Lys Thr Gln Asn Gly Val Tyr Ser Ala Ala Asn 520 525 530 535 Tyr Thr Asn Gly Ser Phe Gly Ser Asn Phe Val Ser Ala Gly Ile Gln Thr Ser Phe Arg 540 545 550 555 Thr Gly Asn Pro Thr Gly Thr Tyr Gln Asn Gly Tyr Asp Ser Thr Gln Gln Tyr Gly Ser 560 565 570 575 Asn Val Ala Asn Met His Asn Gly Met Asn Gln Gln Ala Tyr Ala Tyr Pro Ala Thr Ala 580 585 590 595 Ala Ala Ala Pro Met Ile Gly Tyr Pro Met Pro Thr Gly Tyr Ser Gln 600 605 610 615 <210> 7 <211> 1845 <212> DNA <213> Artificial Sequence <400> 7 atgtcgggtt attcgagtga ccgagaccgc ggccgggacc gagggtttgg tgcacctcga 60 tttggaggaa gtagggcagg gcccttatct ggaaagaagt ttggaaaccc tggggagaaa 120 ttagttaaaa agaagtggaa tcttgatgag ctgcctaaat ttgagaagaa tttttatcaa 180 gagcaccctg atttggctag gcgcacagca caagaggtgg aaacatacag aagaagcaag 240 gaaattacag ttagaggtca caactgcccg aagccagttc taaattttta tgaagccaat 300 ttccctgcaa atgtcatgga tgttattgca agacagaatt tcactgaacc cactgctatt 360 caagctcagg gatggccagt tgctctaagt ggattggata tggttggagt ggcacagact 420 ggatctggga aaacattgtc ttatttgctt cctgccattg tccacatcaa tcatcagcca 480 ttcctagaga gaggcgatgg gcctatttgt ttggtgctgg caccaactcg ggaactggcc 540 caacaggtgc agcaagtagc tgctgaatat tgtagagcat gtcgcttgaa gtctacttgt 600 atctacggtg gtgctcctaa gggaccacaa atacgtgatt tggagagagg tgtggaaatc 660 tgtattgcaa cacctggaag actgattgac tttttagagt gtggaaaaac caatctgaga 720 agaacaacct accttgtcct tgatgaagca gatagaatgc ttgatatggg ctttgaaccc 780 caaataagga agattgtgga tcaaataaga cctgataggc aaactctaat gtggagtgcg 840 acttggccaa aagaagtaag acagcttgct gaagatttcc tgaaagacta tattcatata 900 aacattggtg cacttgaact gagtgcaaac cacaacattc ttcagattgt ggatgtgtgt 960 catgacgtag aaaaggatga aaaacttatt cgtctaatgg aagagatcat gagtgagaag 1020 gagaataaaa ccattgtttt tgtggaaacc aaaagaagat gtgatgagct taccagaaaa 1080 atgaggagag atgggtggcc tgccatgggt atccatggtg acaagagtca acaagagcgt 1140 gactgggttc taaatgaatt caaacatgga aaagctccta ttctgattgc tacagatgtg 1200 gcctccagag ggctagatgt ggaagatgtg aaatttgtca tcaattatga ctaccctaac 1260 tcctcagagg attatattca tcgaattgga agaactgctc gcagtaccaa aacaggcaca 1320 gcatacactt tctttacacc taataacata aagcaagtga gcgaccttat ctctgtgctt 1380 cgtgaagcta atcaagcaat taatcccaag ttgcttcagt tggtcgaaga cagaggttca 1440 ggtcgttcca ggggtagagg aggcatgaag gatgaccgtc gggacagata ctctgcgggc 1500 aaaaggggtg gatttaatac ctttagagac agggaaaatt atgacagagg ttactctagc 1560 ctgcttaaaa gagattttgg ggcaaaaact cagaatggtg tttacagtgc tgcaaattac 1620 accaatggga gctttggaag taattttgtg tctgctggta tacagaccag ttttaggact 1680 ggtaatccaa cagggactta ccagaatggt tatgatagca ctcagcaata cggaagtaat 1740 gttccaaata tgcacaatgg tatgaaccaa caggcatatg catatcctgc tactgcagct 1800 gcacctatga ttggttatcc aatgccaaca ggatattccc aataa 1845 <210> 8 <211> 1848 <212> DNA <213> Artificial Sequence <400> 8 atgtcgagtt attctagtga ccgagaccgc ggccgggatc gagggtttgg tgcacctcga 60 Met Ser Glu Leu Ile Leu Ser Asp Pro Glu Thr Arg Gly Gly Ile Glu Gly Phe Gly Cys Thr Ser tttggaggga gtagaacagg acccctctct ggaaagaagt ttggaaatcc tggggagaaa 120 Phe Gly Gly Ser Glu Thr Asp Pro Leu Leu Gly Lys Lys Phe Gly Asn Pro Gly Glu Lys ctagttaaaa agaagtggaa tcttgatgag ctgcccaaat ttgagaagaa tttttatcaa 180 Leu Val Lys Lys Glu Trp Asn Ser Leu Asp Glu Leu Pro Asn Leu Glu Lys Lys Phe Phe Ile Gln gaacaccctg atttggcaag gcgcaccgca caagaggtag atacatacag aagaagcaag 240 Glu His Pro Asp Phe Gly Lys Ala His Ala Gln Arg Arg Tyr Ile Gln Glu Glu Gln Lys gaaattacag ttagaggtca caactgtcca aaacctgttc tgaattttta tgaagcaaac 300 Glu Ile Thr Val Arg Val Thr Thr Ser Lys Thr Val Ser Glu Phe Phe Met Ser Asn tttcctgcga atgtcatgga tgtgattgca aggcagaact ttactgaacc cactgctatt 360 Phe Pro Ala Met Ser Met Cys Ile Ala Arg Gln Thr Phe Thr Glu Pro Thr Ala Ile caagctcagg gctggccagt tgctctcagt ggattggata tggttggagt agctcagact 420 Gln Ala Gln Gly Trp Pro Ser Ala Ser Ser Gly Trp Ile Trp Leu Glu Ser Ala Gln Thr ggatctggga aaacattatc ttatttgctg cctgccattg tacacataaa ccaccagcca 480 Gly Ile Trp Glu Asn Ile Ile Tyr Phe Ala Ala Ala Ile Val Thr Ile Lys Thr Thr Ser Pro ttcctagaga gaggtgatgg gcctatttgc ttggtgctgg caccaactcg agaactggca 540 Phe Pro Arg Glu Gly Met Gly Pro Ile Cys Leu Val Ala Gly Thr Thr Ser Glu Asn Trp Gln cagcaggtgc agcaagtggc tgctgaatat tgtcgagctt gtcgcttgaa gtctacttgc 600 Gln Gln Val Gln Gln Trp Ala Ala Glu Ile Cys Glu Leu Val Ala Glu Ser Tyr Cys atctatggtg gtgctcccaa aggaccacag attcgtgatt tggaaagagg tgtggaaatc 660 Ile Tyr Gly Gly Ala Pro Lys Arg His Arg Ile Arg Val Ile Trp Lys Arg Val Trp Asn Ile tgtattgcaa cacctggaag actgattgac tttttagagt gtgggaaaac caatctgaga 720 Cys Ile Ala Thr Leu Glu Asp Asp Asp Phe Leu Ser Cys Gly Lys Asn Gln Ile Glu agaacaactt accttgtcct tgatgaagct gataggatgc ttgatatggg atttgaaccc 780 cagataagga aaattgtgga tcaaataaga cctgataggc aaacactaat gtggagtgca 840 acttggccaa aagaagtaag acagcttgct gaagatttcc tgaaagacta tattcatatc 900 aatattggtg cactggaact gagtgcaaac cataacattc ttcagattgt ggatgtatgt 960 catgatgtcg aaaaggatga aaagcttatt cgtctgatgg aagaaatcat gagtgagaag 1020 gagaataaaa ctattgtttt tgttgaaacc aaaagaagat gtgatgaact taccagaaaa 1080 atgaggagag atgggtggcc tgccatgggc atccatggtg acaagagtca gcaggaacgt 1140 gactgggttc taaatgaatt caaacatgga aaagctccta ttctgattgc taccgatgtg 1200 gcctccagag ggctagatgt ggaagatgtg aaatttgtca tcaattatga ctaccctaac 1260 tcctcagagg attatattca tcgaattgga agaactgctc gcagtaccaa aacaggcaca 1320 gcatacactt tctttacacc taataacata aagcaagtga gcgaccttat ctctgtgctt 1380 cgggaagcta atcaagcaat taatcccaag ttgcttcagt tggtcgaaga cagaggttca 1440 ggtcgttcca ggggtagagg aggcatgaag gacgatcgtc gtgacagata ctctgcaggc 1500 aaaaggggtg gatttaatac ctttagagac agggaaaact atgacagagg ctactctaat 1560 ctgcttaaga gagattttgg ggctaaaact cagaatggtg tttacagtgc tgcaaattac 1620 accaatggga gctttggaag taattttgta tctgctggca tacagaccag ttttaggact 1680 ggtaatccaa cagggactta ccagaacggt tatgatagca ctcagcaata tggaagtaat 1740 gttgcaaata tgcacaatgg tatgaaccaa caggcatatg catatcctgc taccgcagct 1800 gctgcgccta tgattggcta tcccatgcca acagggtatt ctcaataa 1848 Description of the Drawings

[0045] Figure 1 It shows the mRNA expression of DDX5 in the skin lesions of normal people, psoriasis patients and atopic dermatitis patients in the GEO database.

[0046] Figure 2 It shows that the expression of DDX5 protein is reduced in the skin lesions of psoriasis patients and atopic dermatitis patients. Among them, Figure A shows that the expression of DDX5 protein is reduced in the skin lesions of psoriasis patients, and Figure B shows that the expression of DDX5 protein is reduced in the skin lesions of AD patients.

[0047] Figure 3 It shows that DDX5 is mainly reduced in the expression of keratinocytes in the skin lesions of atopic dermatitis ( Figure 3 A) and psoriasis ( Figure 3 B) patients.

[0048] Figure 4 It shows that the expression of DDX5 is increased in basal cell carcinoma and squamous cell carcinoma.

[0049] Figure 5It is shown that the mRNA and protein levels of DDX5 are decreased in the skin lesions of C57BL / 6 psoriasis mice induced by imiquimod (IMQ) application. Among them, Figure A shows the expression of DDX5 mRNA in the skin lesions of psoriasis mice induced by IMQ for different days. With the extension of the induction days, the expression of DDX5 mRNA is significantly decreased. Figure B shows the expression of DDX5 protein in the skin lesions of psoriasis mice induced by IMQ for different days. With the extension of the induction days, the expression of DDX5 protein is significantly decreased.

[0050] Figure 6 It is shown that the mRNA and protein levels of DDX5 are decreased in the skin lesions of C57BL / 6 atopic dermatitis mice induced by MC903 application. Among them, Figure A shows the expression of DDX5 mRNA in the skin lesions of atopic dermatitis mice induced by MC903 for different days. With the extension of the induction days, the expression of DDX5 mRNA is significantly decreased. Figure B shows the expression of DDX5 protein in the skin lesions of atopic dermatitis mice induced by MC903 for different days. With the extension of the induction days, the expression of DDX5 protein is significantly decreased.

[0051] Figure 7 It is shown that the expression of DDX5 protein is restored in the skin of C57BL / 6 mice during the regression of psoriasis and atopic dermatitis. Among them, Figure A shows the expression of DDX5 protein in normal mouse skin, skin lesions of IMQ-induced psoriasis mice, and the same site skin after psoriasis regression. Figure B shows the expression of DDX5 protein in normal mouse skin, skin lesions of MC903-induced atopic dermatitis mice, and the same site skin after atopic dermatitis regression.

[0052] Figure 8 It is shown that specific knockout of the DDX5 gene (Ddx5 Δ / KC ) in keratinocytes of C57BL / 6 mice spontaneously produces atopic dermatitis and psoriasis-like phenotypes.

[0053] Figure 9 It is shown that the disease condition of imiquimod-induced Ddx5 Δ / KC psoriasis mice is aggravated and the epidermis is thickened. Among them, Figure A shows the back skin photo of mice after IMQ application. Figure B shows the hematoxylin and eosin staining of skin sections of psoriasis mice. Figure C shows the length of epidermal spines.

[0054] Figure 10 It is shown that the imiquimod-induced Ddx5 Δ / KC Compared with control psoriasis mice, the expressions of psoriasis-related cytokines Il-23, Il-17a and chemokines Ccl20, Cxcl1 are significantly increased in psoriasis mice.

[0055] Figure 11 It is shown that the MC903-induced Ddx5Δ / KC In mice with atopic dermatitis, the dermatitis condition worsened, with a significant increase in ear thickness compared to control mice and a significant thickening of the epidermis. Figure A shows a photo of the ears of MC903-induced AD mice, Figure B shows the statistics of ear thickness of AD mice induced by MC903 for different days, and Figure C shows the hematoxylin and eosin staining of ear sections.

[0056] Figure 12 Indicates Ddx5 induced by MC903 Δ / KC Compared with atopic dermatitis and control AD mice, the expressions of AD-related cytokines Il-4, Il-13, and Tslp were significantly increased.

[0057] Figure 13 Indicates that DDX5 regulates the alternative splicing of pre-mRNA of IL-36 receptor in human and murine keratinocytes to produce soluble sIL-36R. Figure A shows the splicing of IL-36R pre-mRNA in human keratinocytes, and Figure B shows the splicing of IL-36R pre-mRNA in murine keratinocytes.

[0058] Figure 14 Indicates that the deletion of DDX5 in keratinocytes leads to an increase in IL-36R expression and a decrease in sIL-36R expression. Figure A shows the splicing of IL-36R pre-mRNA after knocking down DDX5 in human keratinocytes, and Figure B shows the splicing of IL-36R pre-mRNA in keratinocytes of DDX5-deficient mice.

[0059] Figure 15 Indicates that the expression of sIL-36R gradually decreases in imiquimod-induced psoriasis mice, while the expressions of IL-36R and IL-36γ gradually increase. Figure A shows the detection of protein expression by western blot, and Figure B shows the ratio of the gray scale of the statistical protein bands.

[0060] Figure 16 Indicates that the expression of sIL-36R gradually decreases in MC903-induced AD mice, while the expressions of IL-36R and IL-36γ gradually increase. Figure A shows the detection of protein expression by western blot, and Figure B shows the ratio of the gray scale of the statistical protein bands.

[0061] Figure 17 Indicates that sIL-36R inhibits DDX5 mediated by IL-36γ - / - Inflammatory responses in keratinocytes.

[0062] Figure 18 Indicates that injecting recombinant sIL-36R protein can alleviate the pathological symptoms of MC903-induced atopic dermatitis mice.

[0063] Figure 19 It is shown that injecting sIL-36R protein can reduce the expression of cytokines and chemokines in the skin lesions of atopic dermatitis mice induced by MC903.

[0064] Specific experimental methods

[0065] Combined with the following specific embodiments and drawings, the present invention will be further described in detail. The protection scope of the present invention is not limited to the following embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the appended claims are used as the protection scope. The processes, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the specifically mentioned content below, are all common knowledge and well-known common sense in the art, and the present invention has no special restrictions.

[0066] Example 1: Expression of DDX5 in the skin lesions of patients with psoriasis and atopic dermatitis

[0067] First, by analyzing the skin sample data of psoriasis and atopic dermatitis in the GEO database, the results are as Figure 1 shown: Compared with 38 normal human samples, the mRNA level of DDX5 decreased in the skin lesions of 28 patients with psoriasis and 27 patients with atopic dermatitis. The expression of DDX5 mRNA in the skin lesions of psoriasis decreased by about 40%, while the expression of DDX5 mRNA in the skin lesions of atopic dermatitis decreased by about 30%.

[0068] Secondly, the present invention collected the skin of 3 patients with atopic dermatitis, 3 patients with psoriasis and 3 normal humans. The skin tissue was added to a beating tube containing RIPA reagent and beaten at 4°C and 60 Hz for 3 minutes. Then, it was centrifuged at 12,000 rpm for 15 minutes at 4°C, and the upper clear protein sample was collected. After detecting the protein concentration with a BCA kit, the expression of DDX5 in 20 μg of total protein was detected by western blot. The experimental results are as Figure 2 shown: Compared with normal humans, the expression of DDX5 protein decreased by 20% and 60% in the skin lesions of psoriasis and AD patients, respectively.

[0069] Finally, the present invention collected the skin of some normal humans, patients with psoriasis and AD, fixed it with 4% paraformaldehyde for 48 hours, and then carried out a series of dehydrations and paraffin embedding. The embedded wax blocks were cut into 4-μm thin slices and placed on glass slides. After dewaxing the sections, antigen repair was carried out, and 3% BSA was used for blocking to prevent non-specific staining. Then, anti-DDX5 antibody was added and incubated overnight at 4°C. The next day, after incubating the corresponding secondary antibody, the localization and expression of DDX5 were detected by confocal microscopy. The results are as Figure 3Shown as follows: DDX5 is mainly highly expressed in normal human epidermal keratinocytes (shown by green fluorescence), while the green fluorescence is significantly weakened in the epidermal keratinocytes of the skin lesions of AD and psoriasis patients, indicating that the expression of DDX5 in the epidermal keratinocytes of the skin lesions of AD and psoriasis patients is significantly reduced.

[0070] In summary, the results of Western blot and immunofluorescence staining both show that DDX5 is highly expressed in normal human epidermal keratinocytes, while the expression of DDX5 in the epidermal layer of the skin lesions of AD and psoriasis patients is significantly reduced.

[0071] Example 2: Expression of DDX5 in skin cancer

[0072] In this invention, 3 skin sections of basal cell carcinoma and 3 skin sections of squamous cell carcinoma were collected. After dewaxing the sections, antigen retrieval was performed. Blocking was carried out with 3% BSA at room temperature for 1 hour to prevent non-specific staining, and then anti-DDX5 antibody was added and incubated overnight at 4°C. The next day, after incubating the corresponding secondary antibody, the localization and expression of DDX5 were detected by confocal microscopy. The experimental results are as Figure 4 Shown as follows: Green fluorescence characterizes the immunostaining of DDX5. The light intensity of green fluorescence in the keratinocytes of skin cancers such as basal cell carcinoma and squamous cell carcinoma is significantly brighter than that in normal people, indicating that the expression of DDX5 in human skin cancers such as basal cell carcinoma and squamous cell carcinoma is higher than that in normal human skin.

[0073] Example 3: Expression of DDX5 in psoriasis and atopic dermatitis mice

[0074] For the psoriasis mouse model, the back hair of 7-8-week-old wild-type C57BL / 6 mice was removed. Starting from the next day, 62.5 mg of imiquimod (IMQ) cream was continuously applied to the back skin of the mice for 5 days to induce the psoriasis mouse model. One group of mice was sacrificed every day to obtain mouse skin tissues for preparing tissue sections and extracting RNA and proteins. The specific operation method for extracting RNA is as follows: Add tissue the size of a mung bean to 1 ml of Trizol containing 3 mm magnetic beads, beat with a tissue homogenizer at 4°C for 3 minutes, centrifuge for 5 minutes, then aspirate the upper layer of Trizol without aspirating the tissue, then add 1 / 5 volume of chloroform, shake for 10 seconds and let stand at room temperature for 5 minutes, centrifuge at 12,000 rpm at 4°C for 15 minutes, aspirate the upper clear and transparent liquid, add an equal volume of isopropanol, mix well and let stand at room temperature for 10 minutes. Then centrifuge at 12,000 rpm at 4°C for 10 minutes. A milky white RNA precipitate can be seen at the bottom of the tube. Wash it once with 1 ml of 75% pre-cooled ethanol, centrifuge for 5 minutes and then remove the ethanol. After the RNA is dried, add RNase-free water to dissolve the RNA. After detecting the RNA concentration with Nanodrop, 1 μg of total RNA was reverse transcribed into cDNA using Roche reverse transcription reagent. The method for extracting proteins is the same as that in Example 1 of this invention. AsFigure 5 As shown: The mRNA of DDX5 decreased by about 55% on the first day of IMQ induction. As the IMQ induction time extended, the mRNA of DDX5 further decreased, decreasing by about 75% on the 5th day. And the DDX5 protein significantly decreased on the 3rd day of IMQ induction, and decreased by about 75% on the 4th and 5th days compared with that before induction, indicating that the expressions of DDX5 mRNA and protein decreased with the aggravation of the disease in psoriasis lesions.

[0075] For the atopic dermatitis mouse model, 1 nm MC903 was applied to the ears of wild-type C57BL / 6 mice at 7 - 8 weeks old every day to induce the mouse AD model. The ear thickness of the mice was measured with a vernier caliper every day, and a group of mice was sacrificed every 2 days to sample and extract RNA and protein to detect the expression of DDX5. The experimental results are as Figure 6 shown: The mRNA of DDX5 decreased by about 50% on the 4th day of MC903 administration and application, and then remained at a decreased level. The protein of DDX5 decreased by 50% on the 8th day of MC903 administration and application, decreased by about 80% on the 12th day, and was hardly detectable on the 16th day, indicating that the expressions of DDX5 mRNA and protein decreased with the aggravation of the disease in AD lesions.

[0076] In summary, these experimental results indicate that the expressions of DDX5 mRNA and protein are significantly decreased during the pathogenic processes of AD and psoriasis. In addition, as Figure 7 shown: The expression of DDX5 not only decreases with the aggravation of the disease process, but also its expression returns to the normal level with the regression of the disease. Figure A shows that in psoriasis mice induced by IMQ, the expression of DDX5 protein significantly decreased (decreased by about 70%) in the skin lesions of psoriasis mice induced for 5 days, while the expression of DDX5 recovered in the skin after 8 days of drug withdrawal when psoriasis regressed; Figure B shows that the expression of DDX5 decreased by about 50% in the ear samples induced by MC903 for 8 days and returned to the normal level after 10 days of drug withdrawal when the inflammation regressed.

[0077] Example 4: Mice with epidermal-specific deletion of the DDX5 gene (Ddx5 Δ / KC ) spontaneously develop atopic dermatitis and psoriasis-like phenotypes

[0078] DDX5 fl / fl mice were constructed by the cre-loxP technology. After crossing with K14-cre mice, mice with epidermal-specific deletion of DDX5 (Ddx5 Δ / KC ) were born. As Figure 8 shown: In Ddx5 Δ / KC mice, at 2 weeks old, dandruff appeared on the inner side of the elbow or the root of the thigh, and skin rashes appeared on the back skin. The spontaneous rate of the mice was counted. The results are asFigure 8 The spontaneous probability shown in the table is 72.7%; it indicates that mice lacking Ddx5 in keratinocytes can spontaneously develop atopic dermatitis and psoriasis.

[0079] Example 5: Epidermis-specific deletion of DDX5 exacerbates the pathology of atopic dermatitis and psoriasis in mice

[0080] As described in Example 3 of the present invention, psoriasis and AD mouse models were induced with IMQ and MC903 respectively, and Ddx5 Δ / KC The pathological phenotype of psoriasis mice was exacerbated, mainly manifested as: as Figure 9 shown in A, Ddx5 Δ / KC The dandruff on the back of psoriasis mice increased, as Figure 9 shown in B, the skin samples were made into sections for hematoxylin and eosin staining, showing that the epidermal layer of the skin lesion was thickened, and the statistical results of the spines in the epidermal layer showed that the spines increased significantly ( Figure 9 C).

[0081] Figure 10 ELISA was used to detect the expression of cytokines and chemokines in psoriasis skin lesions, and it was found that the psoriasis-related cytokine Il-23 increased from 200 pg / mg to about 600 pg / mg, Il-17a increased from 150 pg / mg to about 300 pg / mg, and the chemokine Ccl20 increased from about 200 pg / mg to 300 pg / mg, and cxcl1 increased from 400 pg / mg to about 700 pg / mg.

[0082] Similarly, Ddx5 Δ / KC The pathological phenotype of AD mice was exacerbated, mainly manifested as: as Figure 11 shown in A, Ddx5 Δ / KC The dandruff on the ears of AD mice increased significantly, Figure 11 shown in B, the ear thickness increased, Figure 11 shown in C, the epidermal layer became thicker and the keratinocytes became spongy.

[0083] RT-PCR was used to detect the expression of cytokines and chemokines in atopic dermatitis skin lesions, and the results were as Figure 12 shown, Ddx5 Δ / KC In the skin lesions of AD mice, the atopic dermatitis-related cytokine Il-4 was upregulated by 2-fold, Il-13 was upregulated by 5-fold, Tslp was upregulated by 1-fold, and the chemokines Ccl11 was upregulated by 1-fold, Ccl17 was upregulated by 3-fold, and Ccl22 was upregulated by 0.8-fold.

[0084] In summary, these results indicate that epidermis-specific deletion of DDX5 will exacerbate atopic dermatitis and psoriasis.

[0085] Example 6: DDX5 regulates the alternative splicing of IL-36R pre-mRNA

[0086] There are different variants of IL-36R in keratinocytes. In human keratinocytes, hsIL-36R is produced by deleting exon 3 from the full-length IL-36R, while in mouse keratinocytes, msIL-36R is produced by deleting exon 6 from the full-length IL-36R. To determine that DDX5 can regulate the splicing of IL-36R pre-mRNA, the present invention designed PCR primers that can distinguish full-length IL-36R and sIL-36R according to the deleted sequences. First, we knocked out DDX5 in the human keratinocyte cell line HaCat using the CRISPR-Cas9 technology to obtain DDX5 – / – HaCat cells. Then, PCR was used to detect the expression of IL-36R and sIL-36R mRNA in WTDDX5 – / – HaCat cells. The experimental results are as Figure 13 shown in A: When DDX5 in human keratinocytes was knocked out, the expression of full-length IL-36R mRNA increased while the expression of sIL-36R mRNA decreased.

[0087] In addition, the present invention also isolated DDX5 fl / fl and Ddx5 Δ / KC from keratinocytes of 2-3-day-old neonatal mice, and used DNA-PAGE to detect the expression of IL-36R and sIL-36R mRNA in keratinocytes of WT and Ddx5 Δ / KC mice, as Figure 13 shown in B: Consistent with human IL-36R, when Ddx5 in keratinocytes was deleted, compared with the control DDX5 fl / fl the expression of IL-36R increased and the expression of sIL-36R decreased.

[0088] These results indicate that DDX5 is involved in regulating the alternative splicing of IL-36R pre-mRNA, and the deletion of DDX5 leads to an increase in the expression of IL-36R and a decrease in the expression of sIL-36R.

[0089] Example 7: Deletion of DDX5 in keratinocytes increases IL-36R and decreases sIL-36R

[0090] Verify the expression of IL-36R and sIL-36R proteins in keratinocytes by Western blot. First, culture human neonatal primary keratinocytes (NHEK) in a 6-well plate. When the cell density reaches 80%, transfer 100 pmol siRNA into NHEK cells using Lipofectamin 2000 reagent. After 4 hours, replace the medium with fresh complete medium. After 36 hours, lyse the cells with RIPA and break them by sonication. After centrifuging at 4°C for 15 minutes, collect the supernatant. After measuring the protein concentration in the supernatant by the BCA method, 20 μg of total protein is used for Western blot. The experimental results are as Figure 14 shown in A; after DDX5 was knocked down, the expression of IL-36R protein in keratinocytes increased by 2.5-fold, and the expression of sIL-36R protein decreased by about 60%.

[0091] In addition, the present invention also isolated keratinocytes (MKC) from Ddx5 f / f and Ddx5 Δ / KC from neonatal mice. The cells were seeded onto a 6-well plate. When the cell density reached 95%, proteins were extracted with RIPA as in NHEK, and the expressions of IL-36R and sIL-36R were detected by Western blot. The results are as Figure 14 shown in B: compared with normal DDX5 fl / fl , after DDX5 was knocked out, the expression of IL-36R in mouse keratinocytes increased by 1.5-fold, and the expression of sIL-36R was almost reduced to undetectable.

[0092] In addition, the present invention also detected in the psoriasis and AD mouse models constructed on different days as in Example 3 by Western blot that with the increase of the induction days, the expressions of IL-36R and IL-36γ in the skin lesions of psoriasis and AD mice showed an increasing trend, while the expression of sIL-36R decreased with the extension of time. The specific expression in psoriasis can be seen in Figure 15 where the expressions of IL-36γ and IL-36R increased on the 1st day and the 2nd day respectively, and the expression of IL-36R was the highest on the 4th day, up to 35-fold; while the expression of sIL-36R increased first on the 1st and 2nd days and then decreased on the 3rd day, continuing until the 5th day. The specific expression in AD can be seen in Figure 16 : the expressions of IL-36γ and IL-36R increased on the 4th day and the 8th day respectively, while the expression of sIL-36R had a slight increase on the 4th day and started to decrease on the 8th day, and the expression of sIL-36R decreased by about 80% until the 16th day.

[0093] In summary, these results indicate that DDX5 can regulate the alternative splicing of IL-36R pre-mRNA, and the reduction or deletion of DDX5 leads to an increase in IL-36R expression and a decrease in sIL-36R protein expression in keratinocytes. Under the pathological conditions of psoriasis and AD, due to the reduction of DDX5, IL-36R in the skin lesions increases while sIL-36R protein decreases, further exacerbating psoriasis and AD.

[0094] Example 8: sIL-36R inhibits the inflammatory response of keratinocytes

[0095] DDX5 – / – HaCat cells were seeded into 24-well plates. When the cell density reached 85%, DDX5 – / – HaCat cells were transfected with 500 ng of sIL-36R plasmid. After 36 hours, WT, DDX5 – / – and DDX5 – / – +sIL-36R cells were stimulated with 100 ng / ml of IL-36γ for 6 hours. After 6 hours, cell RNA was extracted with Trizol, and 1 μg of total RNA was reverse-transcribed. The obtained cDNA was diluted 10-fold with water and then subjected to fluorescence quantitative PCR detection. The experimental results are as Figure 17 shown: After knocking out DDX5 in keratinocytes, CCL20, CXCL1, CXCL2, CCL3, CCL17, and CCL22 induced by IL-36γ were upregulated by 40-fold, 10-fold, 50-fold, 6-fold, 4-fold, and 4-fold, respectively. While overexpressing sIL-36R in DDX5 – / – keratinocytes, the chemokines CCL20, CXCL1, CXCL2, CCL3, CCL17, and CCL22 induced by IL-36γ were all inhibited. These results indicate that sIL-36R can inhibit the inflammatory response induced by IL-36γ.

[0096] Example 9: Injection of recombinant sIL-36R protein can alleviate the pathological symptoms of atopic dermatitis mice

[0097] Twenty microliters of PBS was injected into the left ear of wild-type mice, and 1 μg of sIL-36R protein (20 μl) was injected into the right ear. Then, 1 nM MMC903 was applied to the mouse ears for 15 consecutive days to induce atopic dermatitis. During the induction process, the ear thickness was measured with a vernier caliper every day, and the mouse ears were photographed. On the 16th day, the mice were sacrificed, and ear proteins, RNA, and tissues were collected for section preparation. As Figure 18 and Figure 19 shown, recombinant sIL-36R can significantly inhibit the onset of AD, manifested as: as Figure 18Significant reduction in dandruff was observed in the ears of the mice, and the ear thickness and the thickness of the epidermal layer of the skin lesions became thinner; the expression of cytokines and chemokines in the skin lesions was detected by RT-PCR as shown in Figure 19 follows: sIL-36R could significantly inhibit the expression of cytokines and chemokines in the skin lesions of AD mice. Specifically, Il-4 was inhibited by 75%, Il-13 was inhibited by 90%, Tslp was inhibited by 80%, and the chemokine Ccl11 was inhibited by 70%, and both Ccl17 and Ccl22 were inhibited by 50%.

[0098] These results indicate that the recombinant sIL-36R protein can alleviate the pathological symptoms of atopic dermatitis and play a therapeutic role in atopic dermatitis.

[0099] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, the changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the scope of protection is defined by the appended claims.

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Claims

1. Use of biomarker DDX5 in the preparation of a reagent or kit for diagnosing the onset of atopic dermatitis.

2. Use of a reagent for detecting biomarker DDX5 in the preparation of a kit for diagnosing the onset of atopic dermatitis.

3. The use according to claim 1 or 2, characterized in that the biomarker DDX5 is RNA helicase DDX5, and its amino acid sequence is as shown in SEQ ID NO.5 or SEQ ID NO.6, and its nucleotide sequence is as shown in SEQ ID NO.7 or SEQ ID NO.8.

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