Cux1 Inhibitors and Their Uses

Abstract

The present application discloses a Cux1 inhibitor and its uses. In the present application, the single-cell transcriptome atlas of a chronic skin dryness mouse model was innovatively studied, and several potential therapeutic targets were discovered through bioinformatics analysis. In the present application, novel skin dryness-induced Cux1+ proliferative basal cells were discovered, the mechanism of action and influence were confirmed through ligand-receptor research, and it was verified through gene knockout experiments that Cux1-positive basal cells are closely related to the epidermal hyperplasia in psoriasis patients. Therefore, Cux1 inhibitors can be widely used to treat epidermal hyperplasia skin diseases. The present application also newly designed an inhibitor for reducing the expression level of Cux1.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to Cux1 inhibitors and their uses. Background Art

[0002] Xerosis can be divided into physiological xerosis and pathological xerosis. Among them, pathological xerosis often appears accompanied by some skin diseases, such as psoriasis, atopic dermatitis, and ichthyosis, etc. Its clinical features are mainly manifested as dry and rough skin, with an appearance similar to a grid or paving stone pattern, showing varying degrees of skin hyperplasia, scaling, and desquamation, often accompanied by erythema, cracks, and itching. There is still no good treatment method for pathological xerosis at present, which has become a thorny medical burden. Therefore, studying the pathogenic mechanism of pathological xerosis, searching for potential therapeutic targets and specific drugs has become a very important research direction. Summary of the Invention

[0003] An object of the present invention is to provide a Cux1 inhibitor.

[0004] Another object of the present invention is to provide the use of a Cux1 inhibitor.

[0005] Another object of the present invention is to provide a method for treating diseases related to Cux1 overexpression.

[0006] Another object of the present invention is to provide a method for reducing the expression level of Cux1 in cells.

[0007] To solve the above technical problems, in a first aspect of the present invention, there is provided the use of a Cux1 inhibitor for preparing a drug or a pharmaceutical composition, wherein the drug or composition is used for:

[0008] (i) reducing the expression level of Slc39a1;

[0009] (ii) reducing the expression level of Cux1;

[0010] (iii) reducing the expression level of Bmpr1a, Bmpr2, and / or Egfr;

[0011] (iv) reducing the expression level of CDK2, CCND1, CCND2, and / or CCNE1;

[0012] (v) reducing the expression level of Krt6a, Hes1, Ctnbp1, Cebpb, Aqp3, and / or Mafb;

[0013] (vi) inhibiting keratinocyte proliferation; and / or

[0014] (vii) preventing and / or treating diseases related to Cux1 overexpression.

[0015] In some preferred embodiments, the application is non-therapeutic in vitro.

[0016] In some preferred embodiments, diseases associated with Cux1 overexpression include pathological xerosis cutis and its complications.

[0017] In some preferred embodiments, the complications of pathological xerosis cutis include epidermal hyperplasia, psoriasis, atopic dermatitis, and ichthyosis.

[0018] In some preferred embodiments, the Cux1 inhibitor is selected from: siRNA, microRNA, shRNA, dsRNA, antibody, compound, or a combination thereof.

[0019] In a second aspect of the present invention, there is provided a Cux1 inhibitor, the Cux1 inhibitor being an siRNA targeting Cux1, and the target sequence of the siRNA being selected from any one of the following:

[0020] (i) a polynucleotide sequence as shown in SEQ ID NO.2; and

[0021] (ii) a polynucleotide sequence having a homology of ≥90%, preferably ≥95%, more preferably ≥98%, and most preferably ≥99% with the polynucleotide sequence shown in SEQ ID NO.2.

[0022] In a third aspect of the present invention, there is provided a Cux1 inhibitor, the Cux1 inhibitor being an shRNA targeting Cux1, and the shRNA comprising a first sequence unit, a second sequence unit complementary to the first sequence, and a stem-loop sequence unit located therebetween.

[0023] The first sequence unit comprises a polynucleotide sequence as shown in SEQ ID NO.2.

[0024] In a fourth aspect of the present invention, there is provided a composition for (i) inhibiting skin hyperplasia, and / or (ii) relieving or improving skin dryness, and / or (iii) relieving or improving skin itching, and / or (iv) relieving or improving skin inflammation, and / or (v) inhibiting keratinocyte proliferation, and / or (vi) treating pathological xerosis cutis and its complications, the composition comprising: a substance that inhibits intracellular Cux1 expression and / or a substance that promotes intracellular Cux1 degradation.

[0025] Preferably, the composition of the present invention can be used in beauty or pharmacy and is particularly useful for dermatological applications.

[0026] In some preferred embodiments, the composition is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0027] In some preferred embodiments, the pharmaceutically acceptable carrier or excipient is selected from: purified distilled water, vegetable oil, sesame oil, tea oil, peach kernel oil, olive oil, peanut oil, ethyl oleate, benzoate, propylene glycol, polyethylene glycol, dimethylacetamide, ethanol, glycerol, sorbitol, mannitol, benzyl alcohol, petrolatum, paraffin wax, lanolin, beeswax, dimethyl silicone oil, sodium dodecyl sulfate, magnesium dodecyl sulfate, polyoxyethylene monostearate magnesium, EDTA, talcum powder, colloidal silica, starch, magnesium stearate, gelatin, dextrin, agar, gum arabic, cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, polyvinylpyrrolidone, and lecithin.

[0028] In some preferred embodiments, the composition is a cosmetic composition, and the cosmetic composition further comprises cosmetically acceptable adjuvants.

[0029] In some preferred embodiments, the cosmetically acceptable adjuvants are selected from solvents, solubilizers, preservatives, antioxidants, pH regulators, penetration enhancers, liposomes, humectants, thickeners, chelating agents, skin feel regulators, surfactants, emulsifiers, propellants / propellants, fragrances, pigments, and other functional additives.

[0030] In some preferred embodiments, the cosmetic composition is selected from any one of the following forms: lotion, emulsion, cream, essence, mask, paste gel, spray, soap, facial cleanser, body wash, shampoo, conditioner, foundation, powder cream, body lotion, or massage cream.

[0031] In a fifth aspect of the present invention, there is provided a method for (i) inhibiting skin hyperplasia, and / or (ii) alleviating or improving skin dryness, and / or (iii) alleviating or improving skin pruritus, and / or (iv) alleviating or improving skin inflammation, and / or (v) inhibiting keratinocyte proliferation, and / or (vi) treating pathological xerosis and its complications, the method comprising the step of administering to a subject at least one substance selected from:

[0032] (i) a substance that inhibits intracellular Cux1 expression;

[0033] (ii) a substance that promotes intracellular Cux1 degradation;

[0034] (iii) Substances that reduce the expression levels of Krt17, Defb6, Ctnnb1, Marcks, Cdk4, Ddit4, Srsf2, Tubb5, Crip1, Ccl2, Smc4, Hist1h1b, Pclaf, Slc39a10, Top2a, Mki67, Hes1, Cd74, Postn, Krt71, ler3, Krt79, Adamts1, Krt14, Krt5, Krt6a, Gja1, Stfa3, Fam162a, Ptgs2, Atp1a1, Lars2, Fgfbp1, ll33, Stfa1, Ctsc, Klf4, Ovol1, Ggct, Dusp1, Krt16, Ccdc711, Fabp5, Mafb, Krt1, Ankfy1, Teddm3, Krt6b, Phlda1, Casp14, Aprr1b, flg, Blmh, Cebpb, Ppif, Klk8, Arc and / or Ccl20 in cells;

[0035] (iv) Substances that increase the expression levels of Thbs1, Sparc, Dst, Krt15, Ccl27a, Txnip, Col1a1, Col1a2, Col3a1, Actg1, Cdkn1a, Tacstd2, Plet1, Fosl1, Pof1b, Slc6a14, Dapl1, Ssfa2, Spink5, Cysrt1, Krt80, Rgcc, Eppk1, Sbsn, Sdc4, Lce1m, Wfdc21, Egr1 and / or Hspb1 in cells.

[0036] In the sixth aspect of the present invention, a method for reducing the expression level of Cux1 in cells is provided. The method includes the step of culturing cells in the presence of the siRNA described in the first aspect of the present invention, thereby reducing the expression level of Cux1 in the cells.

[0037] In some preferred embodiments, a vector containing a polynucleotide sequence of shRNA targeting Cux1 is introduced into cultured cells.

[0038] In some preferred embodiments, the vector is a lentiviral vector.

[0039] The present invention has at least the following advantages over the prior art:

[0040] (1) In the present invention, the single-cell transcriptome atlas of a chronic skin dryness mouse model was innovatively studied, and several potential therapeutic targets for chronic skin dryness were discovered through bioinformatics analysis, laying a foundation for subsequent drug research and development;

[0041] (2) In the present invention, a novel Cux1+ proliferative basal cell induced by skin dryness was creatively discovered. Its action and influence mechanism were confirmed through ligand-receptor research, and gene knockout experiments verified that Cux1-positive basal cells are closely related to the epidermal hyperplasia symptoms of psoriasis patients. Therefore, Cux1 inhibitors can be widely used in the treatment of skin diseases with epidermal hyperplasia.

[0042] (3) A Cux1 inhibitor was also designed in the present invention.

[0043] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] One or more embodiments are illustrated by way of example in the accompanying drawings, and these exemplary illustrations do not limit the embodiments.

[0045] Figure 1 It is a schematic diagram of the scRNA-seq process for mouse skin samples treated with water and AEW according to an embodiment of the present invention;

[0046] Figure 2 It is a UMAP graph according to an embodiment of the present invention, showing the heterogeneity of cells in all samples, that is, the total samples can be divided into eight different cell clusters and are coded with different colors respectively. The name of each cell cluster and the percentage of each cell cluster in the total number of cells are annotated on the right side of the graph;

[0047] Figure 3 It is an image obtained by coding the expression levels of marker genes of the standardized cell clusters with different colors and projecting them on the UMAP graph according to an embodiment of the present invention; [[ID=。。。]] [[ID=。。。]]

[0048] [[ID=。。。]] Figure 4 It is a dot plot showing the average expression levels of representative marker genes of different cell types according to an embodiment of the present invention;

[0049] Figure 5 It is a separate UMAP graph of the data sets of the water and AEW groups according to an embodiment of the present invention, where each point corresponds to a cell and is colored according to the cell type;

[0050] Figure 6 It is a graph showing the average percentage of each cell type from the water and AEW groups in the total number of cells according to an embodiment of the present invention;

[0051] Figure 7It is a comprehensive comparative analysis of the upregulated DEGs (left) and downregulated DEGs (right) in the main cell types of the water group and the AEW group according to the embodiments of the present invention. Upregulated DEGs: upregulated in the AEW group and downregulated in the water group; downregulated DEGs: upregulated in the water group and downregulated in the AEW group;

[0052] Figure 8 It is the representative GO terms and pathways enriched in the upregulated DEGs (left) and downregulated DEGs (right) according to the functional enrichment analysis of different skin cell types in the embodiments of the present invention. "Count" represents the number of genes, and the color scale, from pink to red (left), or from gray to blue (right), represents the range of P values;

[0053] Figure 9 It is the UMAP plot of five KC subpopulations according to the embodiments of the present invention, color-coded in the total sample, and the name of each subpopulation and the percentage of each cell subpopulation in the total number of cells are annotated on the right side of the figure;

[0054] Figure 10 It is the image obtained by coding the expression levels of cell subpopulation marker genes after normalization with different colors and projecting them on the UMAP plot according to the embodiments of the present invention;

[0055] Figure 11 It is the dot plot of the average expression levels of representative marker genes of different cell types according to the embodiments of the present invention;

[0056] Figure 12 It is the separate UMAP plots of the water and AEW datasets according to the embodiments of the present invention, where each point in the figure corresponds to a cell and is colored according to the subpopulation type;

[0057] Figure 13 It is the average cell percentage plot of each subpopulation from the Water and AEW groups according to the embodiments of the present invention;

[0058] Figure 14 It is the schematic diagram of the representative GO terms and pathways enriched in the upregulated DEGs in the functional enrichment analysis of different KC subpopulations according to the embodiments of the present invention. In the figure, "count" represents the number of genes, and the color scale from pink to red represents the range of P values;

[0059] Figure 15 It is the gene set score analysis of different GO biological processes in different KC subpopulations of the water group and the AEW group according to the embodiments of the present invention. The horizontal line represents the median, and the vertical bars extend to the farthest data points within 1.5 times the interquartile range of the maximum quartile. The Wilcoxon rank sum on both sides is used to determine statistical significance, *p<0.05; **p<0.01; ***p<0.001; ns, no statistical difference;

[0060] Figure 16 It is the up-regulated genes of different KC subgroups shared by at least three subgroups between the dot plot display water group and the AEW group according to the embodiments of the present invention. The size of the dots is positively correlated with the percentage of a specific subgroup, and the color scale from gray to red indicates the gene expression level from low to high;

[0061] Figure 17 It is the trajectory reconstruction of the PBC, BC, SC, and GC subgroups of AEW-treated epidermal cells according to the embodiments of the present invention, shown as a linear pseudotime progression, where PBC: proliferative basal cells; BC: basal cells; SC: spinous cells; GC: granular cells;

[0062] Figure 18 It is a schematic diagram of the expression of marker genes of the PBC, BC, SC, and GC subgroups in the entire linear pseudotime trajectory according to the embodiments of the present invention;

[0063] Figure 19 It is a heat map according to the embodiments of the present invention showing that genes (rows) with differential expression along the pseudotime (columns) are hierarchically clustered into four clusters, and the representative pathways of each cluster are annotated on the right side of the figure;

[0064] Figure 20 It is a heat map of the expression levels of 58 intersection genes between the up-regulated DEGs and trajectory-related genes in the KC subgroup of the AEW group according to the embodiments of the present invention;

[0065] Figure 21 It is a heat map of the expression levels of 31 intersection genes between the down-regulated DEGs and trajectory-related genes in the KC subgroup of the AEW group according to the embodiments of the present invention;

[0066] Figure 22 It is a UMAP map of two further subdivided PBC subgroups according to the embodiments of the present invention. Two types of PBC subgroups are coded with different colors in the total sample, and the name and color of each subgroup are annotated on the right side of the figure;

[0067] Figure 23 It is a heat map according to the embodiments of the present invention showing Col17a1 hi PBC or Cux1 hi The expression levels of representative genes highly expressed in PBC, with the color scale from blue to red indicating the gene expression level from low to high, and the pathways related to the gene are annotated on the right side of the figure;

[0068] Figure 24 It is according to the embodiments of the present invention that the expression levels of the standardized marker genes are coded with different colors and projected on the UMAP map to facilitate the identification of two types of PBC;

[0069] Figure 25 is based on Col17a1 in the embodiments of the present invention hi PBC and Cux1 hi Separate UMAP plots of PBC in the Water and AEW datasets, with each point corresponding to a cell and colored according to cell type. The black arrow represents the developmental direction of the cells;

[0070] Figure 26 is based on the RNAScope data in the embodiments of the present invention showing the spatial distribution of Cux1 RNA and Ki67 protein, and Col17a1 RNA and Ki67 protein in the skin treated with water and AEW. The white scale bar represents 50 mm, and the enlarged image of the area within the white box is shown on the right to highlight the details of the positive cells. The scale bar represents 5 mm, and the histogram represents the statistical results of the percentage of positive cells. n = 3 mice in each group, and the data are expressed as mean ± SEM. Student's t-test was used to determine statistical significance, **p < 0.01; ns, no statistical difference;

[0071] Figure 27 is based on Col17a1 in the embodiments of the present invention hi and Cux1 hi Trajectory analysis results of PBC, colored by subpopulation;

[0072] Figure 28 is based on the box plot in the embodiments of the present invention showing the CytoTRACE values of Col17a1 hi and Cux1 hi PBC;

[0073] Figure 29 is based on the heatmap of the incoming communication patterns of two PBC states and other skin cell types in the water group and the AEW group in the embodiments of the present invention. The relative intensity of each signaling pathway is color-coded from gray to blue;

[0074] Figure 30 is based on the heatmap of the outgoing communication patterns of two PBC states and other skin cell types in the water group and the AEW group in the embodiments of the present invention. The relative intensity of each signaling pathway is color-coded from gray to green;

[0075] Figure 31 is based on the sorting plot of all important signaling pathways according to the differences in the overall information flow in the signal network between the water group and the AEW group in the embodiments of the present invention. The signaling pathways marked in red above are more abundant in the AEW group, the signaling pathways marked in black in the middle are equally abundant in both groups, and the signaling pathways marked in green at the bottom are more abundant in the Water group;

[0076] Figure 32Shows the expression levels of the relevant ligand and receptor genes of Tnf, Vegfa, Bmp2, and Hbegf signals between the water group and the AEW group according to the violin plot of the embodiments of the present invention;

[0077] Figure 33 Shows the Tnf-Tnfrsf1a signal transduction network between different cell populations in the AEW group according to the circular plot of the embodiments of the present invention. Each cell type is color-coded differently, and the line width represents the communication probability;

[0078] Figure 34 Is the Vegfa-Flt1 signal network of the AEW group according to the embodiments of the present invention;

[0079] Figure 35 Is the Bmp2-Bmpr1a and Bmp2-Bmpr2 signal networks of the AEW group according to the embodiments of the present invention;

[0080] Figure 36 Is the Hbegf-Egfr signal network in the AEW group according to the embodiments of the present invention;

[0081] Figure 37 Shows the relative CUX1 expression levels of the CUX1 shRNA transfection group and the control group according to the embodiments of the present invention. Each group has n = 3 replicates, and the data are expressed as mean ± SEM. Student's t-test was used to determine statistical significance, **P<0.01;

[0082] Figure 38 Is the CCK8 proliferation experiment of the CUX1 shRNA transfection group and the control group according to the embodiments of the present invention. The data are expressed as mean, and the error is too small to be shown in the figure. Student's t-test was used to determine statistical significance, ****p<0.0001;

[0083] Figure 39 Is the representative graph of the colony formation experiment of the CUX1 shRNA transfection group and the control group according to the embodiments of the present invention;

[0084] Figure 40 Shows the flow cytometry analysis results of the CUX1 shRNA transfection group and the control group according to the embodiments of the present invention. The left side is the representative result graph, and the histogram represents the statistical results of the cell proportion in each cell cycle stage. The data are expressed as mean ± SEM. Student's t-test was used to determine statistical significance, ****p<0.0001; ns, no statistical difference;

[0085] Figure 41According to the embodiments of the present invention, in the QPCR, the expression levels of specific cell cycle-related genes in the group transfected with CUX1 shRNA and the control group were shown, and the data were expressed as mean ± SEM. Student's t-test was used to determine statistical significance, *p<0.05; **p<0.01; ***p<0.001; ns, no statistical difference;

[0086] Figure 42 According to the embodiments of the present invention, in the KC cell subset of the psoriatic model mouse scRNA-seq data (GSE165021), the normalized expression levels of proliferation marker genes such as Ube2c, Top2a, and Mki67 were used to color-code the gene expression levels and project them onto the UMAP plot;

[0087] Figure 43 According to the embodiments of the present invention, in the KC cell subset of the psoriatic patient scRNA-seq data (GSE162183), the normalized expression levels of proliferation marker genes such as UBE2C, TOP2A, and MKI67 were used to color-code the gene expression levels and project them onto the UMAP plot;

[0088] Figure 44 According to the embodiments of the present invention, it is the UMAP plot of PBC and other types of KC in the GSE165021 dataset;

[0089] Figure 45 According to the embodiments of the present invention, it is the average cell percentage plot of PBC and other types of KC in the dataset GSE165021;

[0090] Figure 46 According to the embodiments of the present invention, it is the UMAP plot of PBC and other types of KC in the GSE162183 dataset;

[0091] Figure 47 According to the embodiments of the present invention, it is the average cell percentage plot of PBC and other types of KC in the dataset GSE162183;

[0092] Figure 48 According to the embodiments of the present invention, it is the expression level plot of CUX1 in PBC of the healthy control group and psoriatic patients;

[0093] Figure 49The spatial distributions of CUX1 and Ki67 proteins in skin sections of psoriasis patients and healthy controls are shown according to the immunofluorescence staining results in the embodiments of the present invention. The white scale bar represents 50 μm, and the enlarged image of the area within the white frame is shown on the right to highlight the details of positive cells. The scale bar represents 10 μm. The histogram represents the statistical results of the percentage of positive cells. n = 9 in the healthy group and n = 8 in the psoriasis group. The data are expressed as mean ± SEM, and the student's t-test is used to determine statistical significance, **p < 0.01;

[0094] Figure 50 The HE staining of water- and AEW-treated skin according to the embodiments of the present invention is shown. The black scale bar represents 200 μm, and the enlarged image of the area within the black frame is shown on the right to highlight the details of the epidermal layer. The scale bar represents 20 μm. The histogram represents the statistical results of the epidermal thickness. n = 3 mice in each group. The data are expressed as mean ± SEM, and the student's t-test is used to determine statistical significance, **p < 0.01; ns, no statistical difference;

[0095] Figure 51 The Masson's staining of water- and AEW-treated skin according to the embodiments of the present invention;

[0096] Figure 52 The scatter plot shows the quality control metrics used in the scRNA-seq analysis according to the embodiments of the present invention. The left figure shows the percentage of reads mapped to the mitochondrial genome, and the right figure shows the number of unique genes detected in each cell;

[0097] Figure 53 The violin plot of various quality control metrics of all scRNA-seq datasets generated in this study according to the embodiments of the present invention;

[0098] Figure 54 The UMAP plot of cell heterogeneity according to the embodiments of the present invention. In four separate samples, eight different cell populations are identified and color-coded differently. The name of each cell population is annotated on the right side of the figure;

[0099] Figure 55 The average cell percentage of each cell type obtained from four separate samples according to the embodiments of the present invention;

[0100] Figure 56 The analysis of the scores of inflammatory response gene sets for eight cell types in the water group and the AEW group according to the embodiments of the present invention. The horizontal line represents the median, and the vertical bars extend to the farthest data points within 1.5 times the interquartile range of the maximum quartile. The Wilcoxon rank sum on both sides is used to determine statistical significance, *p < 0.05; **p < 0.01; ***p < 0.001; ns, no statistical difference;

[0101] Figure 57 It is a dot plot of intersection gene points between genes in the "inflammatory response" GO gene set and upregulated DEGs in eight cell populations according to an embodiment of the present invention;

[0102] Figure 58 It is an analysis diagram of gene set scores of eight cell types in the extracellular matrix tissue according to an embodiment of the present invention;

[0103] Figure 59 It is a dot plot of intersection gene points between genes in the "extracellular matrix organization" GO gene set and upregulated DEGs in eight cell populations according to an embodiment of the present invention;

[0104] Figure 60 It is a representative GO term and pathway enriched in downregulated DEGs in the functional enrichment analysis of five KC subgroups according to an embodiment of the present invention. "Count" represents the number of genes. The color scale ranges from gray to blue, indicating the range of P values;

[0105] Figure 61 It is an analysis diagram of gene set scores related to tight junction tissue in five KC subgroups according to an embodiment of the present invention. The horizontal line represents the median, and the vertical bars extend to the farthest data points within 1.5 times the interquartile range of the maximum quartile. The Wilcoxon rank sum on both sides is used to determine statistical significance, *p<0.05; **p<0.01; ***p<0.001; ns, no statistical difference;

[0106] Figure 62 It is an analysis diagram of gene set scores related to tight junction tissue in five KC subgroups according to an embodiment of the present invention;

[0107] Figure 63 It is a dot plot of intersection gene points between genes in the "tight junction tissue" GO gene set and downregulated DEGs in five KC subgroups according to an embodiment of the present invention;

[0108] Figure 64 It is a dot plot of intersection gene points between genes in the "extracellular matrix organization" GO gene set and downregulated DEGs in five KC subgroups according to an embodiment of the present invention;

[0109] Figure 65 It is a box plot of CytoTRACE values of PBC, BC, SC, and GC according to an embodiment of the present invention;

[0110] Figure 66 It is a diagram of a precise epidermal cell development model created by SCORPIUS based on scRNA-seq data according to an embodiment of the present invention;

[0111] Figure 67It is a heatmap of the expression levels of secreted factors in the KC subgroup of the AEW group according to an embodiment of the present invention;

[0112] Figure 68 It is a heatmap of the expression levels of the extracellular matrix in the KC subgroup of the AEW group according to an embodiment of the present invention;

[0113] Figure 69 It is a heatmap of the expression levels of transcription factors in the KC subgroup of the AEW group according to an embodiment of the present invention;

[0114] Figure 70 It is the result of immunofluorescence staining showing the distribution of Ki67 protein in the skin treated with water and AEW. The white scale bar represents 25 μm, and the histogram represents the statistical results of the percentage of positive cells. n = 3 mice in each group. Data are expressed as mean ± SEM, and the student t-test was used to determine statistical significance, **P < 0.01;

[0115] Figure 71 It is a graph of the expression levels of selected marker genes in two groups of PBC according to an embodiment of the present invention;

[0116] Figure 72 It is a result graph of immunofluorescence staining, showing the spatial distribution of Ki67 protein in skin sections of psoriasis patients and healthy controls. The white scale bar represents 50 μm, and the histogram represents the statistical results of the percentage of positive cells. n = 9 in the healthy group and n = 8 in the psoriasis group. Data are expressed as mean ± SEM. The student t-test was used to determine statistical significance, **p < 0.01. Detailed implementation mode

[0117] The present invention independently examines the RNA expression profiles of single cell populations in the AEW model through single cell RNA sequencing analysis and in-situ hybridization techniques, and identifies a novel proliferative basal cell state that specifically expresses the transcription factor CutLike Homeobox 1 (Cux1), and accordingly studies the mechanism of action of Cux1 in the AEW model, providing a targeted potential therapeutic target for skin dryness-related skin diseases. The present invention also designs an RNAi inhibitor targeting Cux1, starting directly from the source, silencing the mRNA of related targets at the gene level so that it cannot express Cux1 protein to achieve the therapeutic purpose.

[0118] Cux1 inhibitor

[0119] The present invention relates to Cux1 inhibitors. As used herein, the term "Cux1" refers to the homeobox CUT-like protein 1, which is a member of the DNA-binding protein homeodomain family. Cux1 has a polynucleotide sequence as shown in SEQ ID NO.1; or a nucleic acid molecule comprising the polynucleotide sequence shown in SEQ ID NO.1; or a polynucleotide sequence obtained by substitution, addition or deletion of any one or more nucleotides in the polynucleotide sequence shown in SEQ ID NO.1; or a polynucleotide sequence having a homology of ≥90%, preferably ≥95%, more preferably ≥98%, most preferably ≥99% with the polynucleotide sequence shown in SEQ ID NO.1.

[0120] SEQ ID NO.1: Human CUX1 number 1523

[0121] In the present invention, the scope of the term "Cux1 inhibitor" includes all substances that can reduce the intracellular Cux1 level, including substances that directly or indirectly (preferably directly acting on Cux1) inhibit the intracellular Cux1 expression and / or promote the intracellular Cux1 degradation and / or inhibit the Cux1 expression or transcription. The inhibitors of Cux1 include (but are not limited to): siRNA, microRNA, compounds, shRNA, dsRNA, antibodies or combinations thereof. The preferred inhibitors of Cux1 are siRNA or shRNA.

[0122] In the present invention, the term "siRNA" or "siNA" or "short interfering nucleic acid molecule" or "short interfering RNA" refers to any nucleic acid molecule that inhibits or down-regulates gene expression or viral replication in a sequence-specific manner by mediating RNA interference ("RNAi") or gene silencing. These nucleic acid molecules may refer to individual nucleic acid molecules, multiple such nucleic acid molecules, or a combination of such nucleic acid molecules. An siRNA may be a double-stranded nucleic acid molecule comprising self-complementary sense and antisense strands, wherein the antisense strand comprises a nucleotide sequence complementary to the nucleotide sequence or a portion thereof in a target nucleic acid molecule, and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. An siRNA may be a polynucleotide having a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence or a portion thereof in a separate target nucleic acid molecule, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. An siRNA may be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence or a portion thereof in a target nucleic acid molecule, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide may be processed in vivo or in vitro to produce an active siRNA molecule capable of mediating RNAi. An siRNA may also comprise a single-stranded polynucleotide having a nucleotide sequence complementary to the nucleotide sequence or a portion thereof in a target nucleic acid molecule (e.g., wherein such an siRNA molecule does not require the presence of a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof within the siRNA molecule), wherein the single-stranded polynucleotide may further comprise terminal phosphate groups, such as a 5'-phosphate (see, e.g., Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568) or a 5',3'-diphosphate.

[0123] In the present invention, the term "target sequence of siRNA" refers to the sense strand of an siRNA.

[0124] In the present invention, the term "microRNA" or "miRNA" has the meaning generally accepted in the art. This term generally refers to small double-stranded RNAs that regulate the expression of target messenger RNAs by mRNA cleavage, translational repression / inhibition, or heterochromatin silencing.

[0125] In the present invention, the terms "shRNA" and "short hairpin RNA" are used interchangeably and refer to a molecule in which siRNAs are linked to form a hairpin or stem-loop structure, including two short inverted repeats, and a LOOP sequence.

[0126] In the present invention, the term "RNA interference" or "RNAi" refers to a biological process of inhibiting or down-regulating gene expression in cells mediated by short interfering nucleic acid molecules, as generally known in the art.

[0127] In a preferred embodiment of the invention, the Cux1 inhibitor is an siRNA targeting Cux1, and the siRNA is selected from any one of the following:

[0128] (i) a polynucleotide sequence as shown in SEQ ID NO.2; and

[0129] (ii) a polynucleotide sequence having a homology of ≥90%, preferably ≥95%, more preferably ≥98%, and most preferably ≥99% with the polynucleotide sequence shown in SEQ ID NO.2.

[0130] SEQ ID NO.2: 5’-AAGAAGAACACTCCAGAGGATTT-3’.

[0131] In a preferred embodiment of the invention, the Cux1 inhibitor is a shRNA targeting Cux1, and the shRNA consists of a first sequence unit, a second sequence unit complementary to the first sequence, and a stem-loop sequence unit located therebetween.

[0132] The first sequence unit includes a polynucleotide sequence as shown in SEQ ID NO.2.

[0133] The embodiments of the present invention also relate to a vector containing cDNA, and the cDNA sequence contains a nucleic acid fragment complementary to the shRNA sequence targeting Cux1, such as a lentiviral vector.

[0134] Use of the Cux1 inhibitor

[0135] The present invention also relates to the use of the Cux1 inhibitor. The uses of the Cux1 inhibitor include being used for preparing a drug or a composition, and the drug or composition is used for:

[0136] (i) reducing the expression level of Slc39a1;

[0137] (ii) reducing the expression level of Cux1;

[0138] (iii) reducing the expression level of Bmpr1a, Bmpr2 and / or Egfr;

[0139] (iv) reducing the expression level of CDK2, CCND1, CCND2 and / or CCNE1;

[0140] (v) Reduce the expression levels of Krt6a, Hes1, Ctnbp1, Cebpb, Aqp3, and / or Mafb;

[0141] (vi) Inhibit keratinocyte proliferation; and / or

[0142] (vii) Prevent and / or treat pathologic xerosis cutis and its complications.

[0143] In some preferred embodiments, the complications of pathologic xerosis cutis include psoriasis, atopic dermatitis, ichthyosis, and the like.

[0144] In some preferred embodiments, the Cux1 inhibitor inhibits keratinocyte proliferation and / or treats pathologic xerosis cutis and its complications by inhibiting the expression of genes related to keratinocyte proliferation, differentiation, and / or migration.

[0145] In some preferred embodiments, the genes related to keratinocyte proliferation, differentiation, and / or migration are selected from at least one of the following: Krt6a, Hes1, Ctnbp1, Cebpb, Aqp3, and / or Mafb.

[0146] In some preferred embodiments, the Cux1 inhibitor inhibits keratinocyte proliferation and / or treats pathologic xerosis cutis and its complications by inhibiting the expression of Bmpr1a, Bmpr2, and / or Egfr.

[0147] In some preferred embodiments, the Cux1 inhibitor inhibits keratinocyte proliferation and / or treats pathologic xerosis cutis and its complications by reducing the mRNA expression levels of CDK2, CCND1, CCND2, and / or CCNE1.

[0148] In some preferred embodiments, the Cux1 inhibitor reduces the number of Cux1 hi status cells in PBC cells to inhibit keratinocyte proliferation and / or treat pathologic xerosis cutis and its complications.

[0149] In some preferred embodiments, the Cux1 inhibitor inhibits keratinocyte proliferation and / or treats pathologic xerosis cutis and its complications by blocking or slowing down the transition of PBC cells from the Col17a1 hi status to the Cux1 hi status.

[0150] Composition

[0151] The present invention also relates to a composition, comprising a substance that inhibits intracellular Cux1 expression and / or a substance that promotes intracellular Cux1 degradation as an active substance, and further comprising other components. The types of other components vary depending on the use of the composition.

[0152] In some embodiments, when the composition is used as a pharmaceutical composition for medical use, in addition to the active substance, the composition further comprises a pharmaceutically acceptable carrier and excipients.

[0153] In the present invention, the pharmaceutically acceptable carrier is non-limitingly selected from: purified distilled water, vegetable oil, sesame oil, tea oil, peach kernel oil, olive oil, peanut oil, ethyl oleate, benzoate, propylene glycol, polyethylene glycol, dimethylacetamide, ethanol, glycerol, sorbitol, mannitol, benzyl alcohol, petrolatum, paraffin wax, lanolin, beeswax, dimethyl silicone oil, sodium lauryl sulfate, magnesium lauryl sulfate, polyoxyethylene monostearate magnesium, EDTA, talc powder, colloidal silica, starch, magnesium stearate, gelatin, dextrin, agar, gum arabic, cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, polyvinylpyrrolidone, lecithin, cationic lipids, cholesterol, polyethylene glycolated phospholipids, sucrose or trehalose, etc. The pharmaceutical composition according to the present invention may further comprise at least one of a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington’s Pharmaceutical Sciences (19th Edition, 1995).

[0154] The pharmaceutical composition of the present invention can be formulated together with the pharmaceutically acceptable carrier and / or medium as described above. Non-limiting examples of the formulations include, but are not limited to, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, topical formulations such as powders, emulsions, ointments, film-forming agents, tinctures, spirits, pastes, oils, lotions, creams, suppositories, liposomes and sterile injections, preferably liposomes.

[0155] In some embodiments, when the composition is used as a cosmetic composition for beauty and other purposes, the cosmetic composition further comprises cosmetically acceptable excipients. As used herein, the term "cosmetic composition" refers to a chemical industrial product or fine chemical product that is applied, sprayed or otherwise dispersed on any part of the human body surface, such as the skin, hair, fingernails, toenails, lips and teeth, etc., for the purpose of cleaning, maintaining, beautifying, modifying and changing the appearance, or correcting the body odor and maintaining a good state. According to a preferred embodiment of the present invention, the cosmetics include: moisturizing cosmetics, anti-inflammatory cosmetics with moisturizing effects, and antipruritic cosmetics with moisturizing effects. In the present invention, the cosmetic composition is selected from any one of the following forms: lotion, emulsion, cream, essence, mask, paste gel, spray, soap, facial cleanser, body wash, shampoo, conditioner, foundation, powder cream, body lotion or massage cream. As used herein, the term "cosmetically acceptable excipients" is selected from solvents, solubilizers, preservatives, antioxidants, pH regulators, penetration enhancers, liposomes, humectants, thickeners, chelating agents, skin feel regulators, surfactants, emulsifiers, propellants / propellants, fragrances, pigments, and other efficacy additives.

[0156] The cosmetic composition should be topically applied regularly to any skin area that requires treatment at a frequency and dosage necessary to obtain the desired result. Preferably, the cosmetic is applied at least once a day, most preferably twice a day. The frequency of treatment depends on the degree of skin damage or deterioration, the reactivity of the user's skin, the concentration of the active ingredient in the cosmetic, the effectiveness of the carrier used to transport the active ingredient into the stratum corneum, the convenience of removing the product by physical contact with clothing or by sweating or other internal or external fluids, and the convenience for the type of user. Based on the total weight of the cosmetic, the typical concentration of a relatively simple bioactive substance such as the cosmetic described herein can range from about 0.01% to about 5.0% (by weight), and the product should be applied at a rate equal to about 1.0 mg / cm 2 skin - about 20.0 mg / cm 2 skin ratio to the skin.

[0157] Indications and treatment methods

[0158] In the present invention, the drug or composition is applicable to (i) inhibiting skin hyperplasia, and / or (ii) relieving or improving skin dryness, and / or (iii) relieving or improving skin itching, and / or (iv) relieving or improving skin inflammation, and / or (v) inhibiting keratinocyte proliferation, and / or (vi) treating pathological xerosis and its complications (psoriasis, atopic dermatitis, ichthyosis, etc.).

[0159] In the present invention, the "subject" has the meaning generally accepted in the art. The subject can be a mammal or mammalian cell, including a human or human cell. The term also refers to the donor or recipient of explanted cells or the organism of the cells themselves.

[0160] In the present invention, a "drug" or "composition" is administered to a subject for preventive, alleviating or therapeutic purposes. The "drug" or "composition" should be formulated into a pharmaceutical form according to the characteristics of the administered subject and the requirements of treatment, including several forms of unit dosage forms and multi-dose forms. Non-limiting examples of such pharmaceutical forms include, but are not limited to, oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, topical preparations such as ointments and creams, suppositories and sterile injections, preferably topical ointment bases, cream bases, gels, liquid coatings. In the present invention, preferably, the "drug" or "composition" is prepared in the form of a topical preparation and administered to the subject, and the preferred route of administration is external application, smearing, subcutaneous injection; for example, contacting the drug or composition containing the active ingredient of the present invention with the skin / scalp / mucosa of the subject, and then it can be washed or not washed. In the present invention, the preferred mode of administration is local administration, for example, contacting the drug or composition containing the active ingredient of the present invention with the local skin / scalp / mucosa of the subject that is sensitive and / or inflamed and / or dry and / or itchy and / or reactive and / or intolerant, and then it can be washed or not washed.

[0161] The present invention also relates to a method for (i) inhibiting skin hyperplasia, and / or (ii) alleviating or improving skin dryness, and / or (iii) alleviating or improving skin itching, and / or (iv) alleviating or improving skin inflammation, and / or (v) inhibiting keratinocyte proliferation, and / or treating pathological xerosis and its complications (such as psoriasis, atopic dermatitis and ichthyosis, etc.), by administering to a subject at least one substance selected from the following:

[0162] (i) A substance that inhibits intracellular Cux1 expression;

[0163] (ii) A substance that promotes intracellular Cux1 degradation;

[0164] (iii) Substances that reduce the expression levels of Krt17, Defb6, Ctnnb1, Marcks, Cdk4, Ddit4, Srsf2, Tubb5, Crip1, Ccl2, Smc4, Hist1h1b, Pclaf, Slc39a10, Top2a, Mki67, Hes1, Cd74, Postn, Krt71, ler3, Krt79, Adamts1, Krt14, Krt5, Krt6a, Gja1, Stfa3, Fam162a, Ptgs2, Atp1a1, Lars2, Fgfbp1, ll33, Stfa1, Ctsc, Klf4, Ovol1, Ggct, Dusp1, Krt16, Ccdc711, Fabp5, Mafb, Krt1, Ankfy1, Teddm3, Krt6b, Phlda1, Casp14, Aprr1b, flg, Blmh, Cebpb, Ppif, Klk8, Arc and / or Ccl20 in cells;

[0165] (iv) Substances that increase the expression levels of Thbs1, Sparc, Dst, Krt15, Ccl27a, Txnip, Col1a1, Col1a2, Col3a1, Actg1, Cdkn1a, Tacstd2, Plet1, Fosl1, Pof1b, Slc6a14, Dapl1, Ssfa2, Spink5, Cysrt1, Krt80, Rgcc, Eppk1, Sbsn, Sdc4, Lce1m, Wfdc21, Egr1 and / or Hspb1 in cells.

[0166] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are by weight percentage and weight parts. The experimental materials and reagents used in the following embodiments can be obtained from commercial channels without special instructions.

[0167] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the technical field to which this application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of this application.

[0168] Example 1: Identification of cell types in dry skin in the AEW mouse model by single-cell RNA sequencing analysis

[0169] Tissue samples were obtained from the dorsal skin of mice treated with water and AEW and stained with HE.

[0170] According to the staining results, compared with the water group, the epidermal thickness in the AEW group was significantly increased. In addition, the epidermis in the AEW group showed obvious hyperplasia and parakeratosis ( Figure 50 ). However, the results of Masson staining showed that there was no significant difference in the collagen density in the dermis between the AEW group and the water group ( Figure 51 ). The above results indicate that AEW treatment mainly damaged the epidermal structure rather than the dermal structure.

[0171] To systematically dissect the transcriptomic differences between homeostasis and chronic dry skin at the single-cell level, the inventors set up two biological replicate samples in each group, with each biological replicate sample containing skin samples from three mice, and performed single-cell RNA sequencing studies on the samples of each group ( Figure 1 ). After quality control, 18,578 cells were obtained in the AEW group and 24,160 cells were obtained in the water group for downstream analysis ( Figure 52 and 53 ). After data preprocessing, sample integration, and principal component analysis, through UMAP analysis, high-quality cells were divided into eight major clusters ( Figure 2 . According to recognized specific cell markers, eight cell clusters were delineated, including fibroblasts (FIB, Dcn+), keratinocytes (KC, Krt10+, and Krt14+), myeloid cells (MYL, Cd74+), T cells (TC, Cd3g+), adipocytes (ADI, Scd1+), endothelial cells (ENDO, Pecam1+), mast cells (MC, Mcpt1+), and vascular smooth muscle cells (vSMC, Acta1+) ( Figure 3 and Figure 4 ). By analyzing the AEW group and the Water group separately, the above eight cell populations were observed, and there was no new cell population in the AEW group during the first-level clustering analysis ( Figure 5 ). Compared with the water group, the average percentage of MYL in the AEW group increased, while the percentages of KC and vSMC decreased ( Figure 6 ). By analyzing four samples of the AEW group and the Water group separately, eight cell populations were still observed, and the average percentage of each cell type was similar to the number observed in the comprehensive analysis ( Figure 54 and 55 ).

[0172] To further explore the pathogenic mechanism at the cellular level, the inventors analyzed the differentially expressed genes (DEGs, |avg log2FC|>0.4 and p adj<0.05) of each cell type ( Figure 7; Table S3). For cell type-specific DEGs, the inventors found that FIB, MYL, and KC were the top three cell types with the most significantly upregulated differential genes, with 100, 70, and 61 DEGs, respectively. In contrast, TC, KC, and MYL were the top three cell types with the most significantly downregulated differential genes, with 34, 32, and 20 DEGs, respectively. GO analysis showed that the upregulated DEGs by AEW treatment were mainly related to regulating inflammatory responses and regulating epithelial cell proliferation, while the downregulated DEGs were mainly related to skin development and extracellular matrix (ECM) organization( Figure 8 ). All cell types in the AEW group highly expressed inflammatory response genes( Figure 56 ). In contrast, except for ENDO, other cell types in the AEW group lowly expressed ECM organization genes( Figure 57 ). For example, the marker gene Cma1 of MC, which is responsible for encoding chymase-1 protein, and the infiltration of MC are closely related to inflammatory skin diseases such as atopic dermatitis. Cma1 was significantly increased in MC of the AEW group( Figure 58 ). Genes involved in ECM protein production, such as Aebp1 and Lrp1, were significantly decreased in FIB of the AEW group( Figure 59 ). These results indicate that DEGs related to skin dryness were mainly found in KC and MYL, highlighting the roles of these two cell types in skin dryness.

[0173] Example 2. Classifying mouse keratinocytes into 5 subpopulations according to their transcriptional characteristics

[0174] Under dehydration conditions, the dysfunction of KC is considered to be an inducer of skin dryness. Therefore, KC was analyzed in more detail next. All KCs were regrouped into five subpopulations, including spinous cells (SC, Krt1+), basal cells (BC, Krt14+), sebaceous gland cells (SGC, Krt79+), proliferative basal cells (PBC, Mki67+), and granular cells (GC, Lor+)( Figures 9 - 11 ). Samples in both the water group and the AEW group contained the above five KC subpopulations( Figure 12 ). By comparing the distributions of each KC subpopulation, the inventors found that compared with the water group, the proportion of PBC increased and the proportions of SC and SGC decreased in the AEW group( Figure 13 ).

[0175] The upregulated DEGs in KC were mainly involved in transepidermal water loss, keratinization, and epidermal development( Figure 14 ). Correspondingly, genes related to transepidermal water loss and keratinization were significantly increased in GC, showing the highest scores at the same time. In addition, genes related to KC proliferation and positive regulation of the cell cycle were significantly increased in PBC, showing the highest scores at the same time( Figure 15)。In addition, the downregulated DEGs in KC were mainly involved in extracellular matrix (ECM) organization and tight junction (TJ) organization ( Figure 60 )。TJ organization-related genes were reduced in SC, BC, and GC, while ECM-related genes were reduced in all types of KC ( Figures 61 - 64 )。

[0176] Further analyzing the specifically KC-upregulated DEGs, the inventors found that the AEW skin dryness model had many similar transcriptional features to other skin diseases. Fabp5 is a fatty acid-binding protein that responds to chemically induced skin inflammation and tumorigenesis. Cd74 is the receptor of MIF, which is enhanced in KC of systemic sclerosis and may be related to the pro-inflammatory pathway. Il33 is a well-known type II inflammatory cytokine that is highly expressed in KC of patients with atopic dermatitis. The above-mentioned significantly increased gene expressions indicate that the skin of AEW model mice is undergoing a severe inflammatory response. Genes related to KC proliferation, differentiation, and migration, including Krt6a, Hes1, Ctnbp1, Cebpb, Aqp3, and Mafb, were also upregulated in the AEW model, revealing the potential for accelerated cell development ( Figure 16 )。

[0177] Example 3. Pseudotime trajectory analysis reveals the dynamics of keratinocyte differentiation in dry skin

[0178] To study whether dry skin conditions would affect epidermal development, the inventors applied pseudotime trajectory analysis to reveal the differentiation pathway of KC. It was experimentally observed that all five subpopulations of KC in the AEW group were arranged in a developmental trajectory, differentiating and developing in sequence from PBC, BC, SC to GC, and the developmental trajectory did not bifurcate ( Figure 17 and 65 )。

[0179] The inventors also performed SCORPIUS, a new analytical method to infer trajectories in a purely data-driven manner, to verify the above KC differentiation trajectory. After analysis with SCORPIUS, a consistent developmental trajectory was obtained ( Figure 66 )。The cell proliferation marker Mki67 was mainly distributed in the starting part of the trajectory, the keratin genes Krt14 and Krt11 were distributed in the middle part, and the involucrin gene Lor was distributed at the end ( Figure 18 )。Then, the inventors identified 327 pseudotime-dependent genes from all KC in the AEW group, and these genes had obvious changes at different developmental stages ( Figure 19)。Genes related to specific biological processes are highly expressed at each transition stage. The PBC state is defined by the first genome, which is related to DNA packaging, the cell's response to DNA damage stimuli, and cell cycle processes; the BC state is defined by the second genome, which is related to skin development, ECM organization, and positive regulation of cell migration; the SC state is defined by the third genome, which is related to skin development, transepidermal water loss, and wound response; the GC state is defined by the fourth genome, which is related to epidermal development, the formation of the cornified envelope, and the establishment of the skin barrier. These pseudotime-related genes include secreted factors ( Figure 67 , extracellular matrix ( Figure 68 ) and transcription factors ( Figure 69 ). The inventors also found that 58 upregulated DEGs and 31 downregulated DEGs in KCs of the AEW group are pseudotime-related genes ( Figure 20 and 21 ). The pseudotime analysis in this example established a basis for exploring the KC differentiation regulation program in the AEW model.

[0180] Example 4. Novel Cux1+ proliferative basal cells in dry skin

[0181] Since the proportion of Ki67+ cells in the epidermis is significantly increased in AEW skin ( Figure ), the inventors further explored the heterogeneity and transcriptional profiles of PBCs in the AEW model. By reanalyzing PBCs, it was found that they could be subdivided into two states (Col17a1hi and Cux1hi) ( ​ ). The Col17a1hi state is characterized by high expression of the Anxa2 gene, a gene encoding cell surface annexins, and high expression of Col17a1, a marker gene for epidermal stem / progenitor cells. The Cux1hi state is characterized by high expression of the zinc ion transmembrane transporter gene Slc39a1 and high expression of the cell cycle-related transcription factor gene Cux1 ( ​ and 24 ; ​ ). Cux1hi PBCs are mainly present in the AEW group ( ​ ). Then we used RNAScope to study the distribution of the two PBC states in skin tissues. The co-staining analysis results of the Col17a1 RNA probe and Ki67 protein showed that approximately 40% of PBCs expressed high levels of Col17a1, but AEW treatment did not affect the cell proportion of Col17a1hi PBCs. The co-staining analysis results of the Cux1 RNA probe and Ki67 protein showed that approximately 28% of PBCs expressed Cux1, but AEW treatment significantly increased the cell proportion of Cux1hi PBCs ( ​)。 Different pseudotime analysis methods (SCORPIUS and CytoTrace) both showed that the developmental order of Col17a1hi PBC was higher than that of Cux1hi PBC, meaning that in the AEW model, the developmental trajectory of PBC was from the Col17a1hi state to the Cux1hi state ( ​ and 28 ). The above data revealed a brand-new Cux1hi PBC state induced by dry skin.

[0182] Example 5. Potential ligand-receptor interactions between PBC and other cells in dry skin

[0183] To investigate the potential cell-cell communication patterns between PBC and other cell types, the inventors performed CellChat analysis on the scRNA-seq dataset. CellChat detected 22 important signaling pathways in both the AEW group and the Water group ( ​ and 30 ). Among them, 8 signaling pathways, including CXCL, IGF, PROS, PTN, GRN, CCL, FGF, and GAS, showed similar information fluxes in the water group and the AEW group, indicating that they played equally important roles in the biological functions of the skin in both treatments. Meanwhile, compared with the water group, the information fluxes of the other 14 signaling pathways changed significantly in the AEW group: the increased pathways were (TNF, VISFATIN, VEGF, IL6, BMP, KIT, COMPLEMENT, MIF, TGFb, EGF, and GALECTIN), and the decreased pathways were (CSF, CALCR, and ANGPTL) ( ​ ). Next, the inventors further analyzed the roles of Col17a1hi PBC and Cux1hi PBC in the TNF, VEGF, BMP, and EGF signaling pathways ( ​ ). Both Col17a1hi PBC and Cux1hi PBC expressed the receptor Tnfrsf1a to receive the Tnf signal from MC. Col17a1hi PBC was the main secretion source of Vegfa, and Flt1 in ENDO was the only recipient of the Vegfa signal. Bmp2 and Hbegf both played roles in an autocrine and paracrine manner. Col17a1hi PBC was both a sender and a recipient, while Cux1hi PBC only played the role of a recipient by expressing Bmpr1a, Bmpr2, and Egfr. Generally speaking, the above results mean that AEW treatment can affect the biological functions of PBC by regulating the cell-cell signal network.

[0184] Example 6. Transcription factor Cux1 can promote keratinocyte proliferation

[0185] To further investigate the molecular function of Cux1 in KCs, the inventors performed knockdown experiments using shRNA (specific shRNA sequences are shown in Table 1 below) in HaCaT cells (an immortalized human KC cell line). QPCR analysis (primer sequences are shown in Table 2) showed that after lentiviral transfection of CUX1-target shRNA and negative control shRNA, the mRNA expression level of CUX1 decreased significantly ( ​ ). Since Cux1hi PBCs showed strong proliferative ability, the effect of CUX1 on KC proliferation was examined by CCK-8 and colony formation assays. The results showed that compared with the negative control group, CUX1 knockout significantly impaired the cell proliferation ability and colony formation ability of HaCaT cells ( ​ and 39 ). We also analyzed the effect of CUX1 on the cell cycle by flow cytometry. The results showed that knockout of CUX1 led to an increase in the proportion of HaCaT cells in G1 phase and a decrease in the proportion of HaCaT cells in S phase ( ​ ). Accordingly, the inventors detected the effect of CUX1 on cell cycle regulatory proteins by QPCR. The results showed that knockout of CUX1 decreased the mRNA expression of CDK2, CCND1, CCND2, and CCNE1, but did not affect CDK4 and CDK6 ( ​ ). In summary, these findings indicate that CUX1 promotes the proliferation of KCs.

[0186] Table 1 shRNA sequences

[0187] ​ ​ ​ ​ ​ ​

[0188] Note: The sequences shown in Table 1 above were combined with other parts of the shRNA designed by software and integrated into the lentiviral vector.

[0189] Table 2. RT-PCR primer sequences

[0190]

[0191]

[0192] Example 7. Increased number of Cux1+ proliferating basal cells in psoriasis patients

[0193] Psoriasis is one of the most common chronic skin diseases in adults, characterized by dry skin and excessive proliferation of KCs. To test the applicability of the research results, the inventors analyzed scRNA-seq data from recently published mouse and human psoriasis studies. Proliferation markers Ube2c, Top2a, and Mki67 were used to distinguish PBCs from other types of KCs ( ​and 43 )。The inventors found that the proportion of PBCs was significantly increased in both psoriasis model mice and patients compared with the control group( ​ )。To further verify the protein expression levels of Cux1 in psoriasis patients and healthy controls, the inventors collected human skin tissue sections from Zhejiang Provincial People's Hospital. The immunofluorescence staining results further showed that the proportion of Ki67+ cells in the epidermis of psoriasis patients was significantly increased( ​ )。In addition, the scRNA data and immunofluorescence staining results indicated that both the mRNA and protein levels of CUX1 were increased in PBCs of psoriasis patients( ​ and 49 )。Taken together, these results suggest that the increase in Cux1+ PBCs may be a common mechanism leading to epidermal hyperplasia in psoriasis patients.

[0194] Those of ordinary skill in the art can understand that the above embodiments are specific examples for implementing the present invention, and in practical applications, various changes can be made in form and details without departing from the spirit and scope of the present invention.

Claims

1. Use of a Cux1 inhibitor in the preparation of a drug or pharmaceutical composition for preventing and / or treating a disease associated with Cux1 overexpression, characterized in that, The diseases related to Cux1 overexpression are pathological xerosis cutis and its complications, and the complications of pathological xerosis cutis are epidermal hyperplasia and psoriasis; The Cux1 inhibitor is siRNA targeting Cux1, and the target sequence of the siRNA is: (i) The polynucleotide sequence shown in SEQ ID NO. 2; or The Cux1 inhibitor is shRNA targeting Cux1, and the shRNA is composed of a first sequence unit, a second sequence unit complementary to the first sequence, and a stem-loop sequence unit located between the two, The first sequence unit is the polynucleotide sequence shown in SEQ ID NO.

2.

2. A composition for treating pathological xerosis cutis and its complications, characterized in that, The composition includes a substance that inhibits Cux1 expression in cells and / or a substance that promotes Cux1 degradation in cells; The substance that inhibits Cux1 expression in cells and / or the substance that promotes Cux1 degradation in cells is siRNA targeting Cux1 or shRNA targeting Cux1; The target sequence of the siRNA is: (i) The polynucleotide sequence shown in SEQ ID NO. 2; The shRNA is composed of a first sequence unit, a second sequence unit complementary to the first sequence, and a stem-loop sequence unit located between the two, The first sequence unit is the polynucleotide sequence shown in SEQ ID NO. 2; The complications of pathological xerosis cutis are epidermal hyperplasia and psoriasis.

3. The composition according to claim 2, wherein The composition is a pharmaceutical composition, and the pharmaceutical composition further includes a pharmaceutically acceptable carrier and excipient.

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