CN116440152A - Application of WTAP inhibitor in preparation of medicine for treating atopic dermatitis - Google Patents
Application of WTAP inhibitor in preparation of medicine for treating atopic dermatitis Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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Abstract
The invention discloses application of a WTAP inhibitor in preparation of a medicament for treating atopic dermatitis, wherein the inhibitor is siRNA. The invention also discloses application of the WTAP serving as a molecular marker in preparation of an atopic dermatitis detection kit. Our study results support: the high expression of WTAP in skin lesions promotes the formation of atopic dermatitis skin lesions, and the targeted inhibition of WTAP molecules can provide a new idea for preparing the medicine for treating atopic dermatitis.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of a WTAP inhibitor in preparation of a medicament for treating AD.
Background
Atopic dermatitis (atopic dermatitis, AD) is a chronic recurrent inflammatory skin disease whose clinical features include impaired skin barrier, enhanced itching and chronic skin inflammation, severely affecting the quality of life of about 15% -20% of children and 1% -3% of adults worldwide. The pathogenesis of AD involves barrier dysfunction, abnormal activation of immune cells, igE-mediated hypersensitivity and environmental factors, and the like. Recent studies confirm a key role for epigenetic changes in AD development, including genomic DNA modification and microRNA post-transcriptional regulation.
N6-methyladenine (m) 6 A) Is the most abundant RNA dynamic reversible internal chemical modification in eukaryotic cells, and plays a vital role in various biological functions. m is m 6 The A modification is co-regulated by the "writers", "erasers" and "readers" proteins, thereby affecting transcription, processing, translation and metabolism of RNA. Research shows that m 6 The a modification is closely related to inflammation, cancer, metabolic, neurological and immune system diseases.
It is of great importance to develop a new drug or a new method for treating AD.
Disclosure of Invention
The invention aims at providing a novel target spot which can be used for preparing medicines for treating AD. In this study we studied m 6 A modification has a potential role in the development of AD. We have found that a wilms tumor 1 associated protein (WTAP) can be produced by mediating m 6 The A level increases the expression level of 15-arachidonic acid lipoxygenase (ALOX 15), calprotectin (S100A 9) and serine protease inhibitor B3 (SERPINB 3), and participates in the pathophysiological development of AD.
The technical scheme of the invention is as follows:
use of an inhibitor of WTAP in the manufacture of a medicament for the treatment of atopic dermatitis.
Preferably, the inhibitor of WTAP is an siRNA. Further preferably, the inhibitor of WTAP is shown in SEQ ID No. 17.
The invention also provides application of the WTAP serving as a molecular marker in preparing a kit for diagnosing atopic dermatitis.
The study of the invention shows that in the study, by aiming at m 6 Analysis of A transcriptome chip data we found the difference m in AD 6 A methylated gene and mRNA level Differential Expression Genes (DEGs). We found that all screened DEGs and most of the pathways appeared enriched in either "hypo-down" or "hyper-up" at the same time, suggesting m 6 The a modification is positively correlated with mRNA expression in AD. From the ranking of the enrichment score and Fisher P value, we determined 16 differential pathways of interest, including 10 up-regulation pathways and 6 down-regulation pathways, we analyzed m of DEGs involved in these pathways of interest 6 Ordering of a level and mRNA level. Furthermore, we found that mRNA expression differences for "writer" WTAP were most pronounced in normal controls and AD by sequencing results.
Next, we confirmed by clinical sample validation that mRNA and protein levels of WTAP were significantly up-regulated in AD lesions compared to normal skin. WTAP is highly expressed in epidermal keratinocytes of AD lesions, while it is poorly expressed in normal skin. We also demonstrated that the first three DE' S (ALOX 15, S100A9 and SERPINB 3) most significantly up-regulated in sequencing results compared to normal skin were significantly up-regulated in mRNA and protein levels of S100A9 and SERPINB3 in AD lesions, whereas ALOX15 had only significantly up-regulated protein levels in AD, with no significant difference in mRNA levels compared to normal skin. The fluorescence intensity of WTAP, S100A9 and SERPINB3 in the epidermal lesions of AD patients is obviously higher than that of the normal control group, while ALOX15 is obviously increased in the AD dermal lesions.
Correlation analysis from GSE193309 dataset results showed that expression levels of WTAP correlated positively with S100A9 and SERPINB3, but not significantly with ALOX 15. AD patients had a higher total m than normal controls 6 Level of a methylation. In addition, AD group S100A9 and SERPINB3mRNA m 6 The A level is obviously increased compared with the normal control group. However, we did not obtain m for the mRNA of ALOX15 in the normal control or AD group 6 Level a. These data indicate that ALOX15, S100A9 and SERPINB3 are m 6 Downstream targets for A RNA methylation may be regulated by WTAP. The present invention also demonstrates that WTAP regulates expression of ALOX15, S100A9 and SERPINB3 at the cellular level.
In summary, the present invention describes m 6 The A modification plays a potential role in the pathological development of AD and demonstrates that WTAP passes through m 6 The a modification upregulates the expression of ALOX15, S100A9 and SERPINB3, thus being involved in the pathological development of AD. Our findings support the concept of: the high expression of WTAP in skin lesions promotes the formation of atopic dermatitis skin lesions, and the targeted WTAP molecules can provide a new idea for preparing the medicine for treating atopic dermatitis.
Drawings
FIG. 1 is a diagram of apparent transcriptome sequencing analysis of AD skin lesions and healthy control skin ArrayStar, wherein FIG. 1 (A) is a diagram of classification of differences between AD and normal controls using Principal Component Analysis (PCA); FIG. 1 (B) volcanic diagram shows m between normal control and AD 6 A differential gene map of methylation; FIG. 1 (C) volcanic plot shows the DEGs between normal control and AD; FIG. 1 (D) Venn diagram shows the difference m in AD 6 Overlapping of A modification genes and pathways; FIG. 1 (E) mRNA level and m in AD 6 16 pathways with altered a level; FIG. 1 (F) is a heat map showing m of the DEGs involved in 16 paths 6 Ordering of a level and mRNA level; FIG. 1 (G) is a heat map showing m between two groups 6 A modification of the expression differences of the related enzymes.
FIG. 2 is a WTAP promotion m 6 Modification of a thus resulted in an up-regulation profile of ALOX15, S100A9 and SERPINB3 in AD lesions, where fig. 2 (a-D) is qRT-PCR to detect mRNA expression of WTAP, ALOX15, S100A9 and SERPINB3 in normal controls (n=16) and AD (n=16). Fig. 2 (E) representative images of WTAP, ALOX15, S100A9 and SERPINB3 western blots and quantification in normal controls (n=4) and AD (n=4). Representative immunofluorescent staining (magnification, x 200) of WTAP, ALOX15, S100A9 and SERPINB3 in fig. 2 (F-I) normal controls (n=3) and AD (n=3); FIG. 2 (J) is a photograph of immunofluorescence in skin lesions of normal and AD patients, CD11c is labeled DC cells, ALOX15 is labeled target gene, and Merge is co-expression.
FIG. 3 is a graph of a correlation analysis of WTAP and its potential targets (ALOX 15, S100A9 and SERPINB 3), wherein FIG. 3 (A) is a correlation analysis of WTAP with S100A9, SERPINB3 and ALOX15 for an AD patient; fig. 3 (B) m in AD damaged skin (n=9) and normal skin (n=9) 6 Total level a; FIG. 3 (C-D) MeRIP-qPCR analysis showed m of S100A9 and SERPINB3mRNA in normal controls (n=5) and AD (n=5) 6 Level a.
FIG. 4 is a graph showing the change in expression of ALOX15, S100A9 and SERPINB3 in KCs and DCs after knockdown and over-expression of WTAP by cell experiments; FIG. 4 (A and B) are m of S100A9 and SERPINB3, ALOX15 in KCs and DCs after specific siRNA knockdown WTAP 6 Graph of significant changes in a level; FIG. 4 (C) is a graph of significant down-regulation of mRNA levels of S100A9 and SERPINB3 in KCs after specific siRNA knockdown WTAP; FIG. 4 (D) is a graph of significant upregulation of mRNA levels for S100A9 and SERPINB3 in KCs after adenovirus over-expression of WTAP; FIG. 4 (E) is a graph of significant down-regulation of protein levels of S100A9 and SERPINB3 in KCs after specific siRNA knockdown WTAP; FIG. 4 (F) is a graph of significant upregulation of protein levels of S100A9 and SERPINB3 in KCs after adenovirus over-expression of WTAP; FIG. 4 (G) is a graph of significant downregulation of ALOX15 mRNA levels in DCs following a specific siRNA knockdown WTAP; FIG. 4 (H) is a graph showing significant upregulation of the mRNA level of ALXO15 in DCs following adenovirus over-expression of WTAP; FIG. 4 (I) is a graph of significant downregulation of protein levels of ALOX15 in DCs after specific siRNA knockdown WTAP; FIG. 4 (J) is a graph showing significant upregulation of protein levels of ALXO15 in DCs following adenovirus overexpression of WTAP.
Detailed Description
The invention will be further explained and illustrated with reference to the drawings and experimental data
Table 1: RT-qPCR primer sequences and other sequences in the specification.
1. Materials and methods
qRT-PCR analysis, western Blot (WB) analysis, immunofluorescence (IF) assay, all of which are prior art methods and are not described further herein.
2. Patient tissue specimen
The study used histopathological diagnosis from three Hunan elea hospitals at university of south China as AD patients and normal control patients. 16 AN skin lesion tissue specimens and corresponding normal control skin tissue specimens were immediately stored at-80℃and used for qRT-PCR and/or Western blotting etc.
3、m 6 A mRNA transcriptome chips
Transcriptome chips (Arraystar) are used for preparation of RNA samples and microarray hybridization. Total RNA was extracted from skin tissue of AD group (n=3) or control group (n=3). The threshold was set to be greater than 1.5 times the change and was statistically significant (p<0.05 Comparison of RNA methylation differences between AD group and normal group. By m 6 A mRNA transcriptome microarray database, KEGG and GO enrichment analysis was performed using R-package cluster analysis.
4、m 6 Quantitative A and MeRIP-qPCR
Using colorimetry m 6 RNA methylation was measured at a wavelength of 450nm after binding of the antibody by the RNA methylation quantification kit (EpiGentek). For MeRIP-qPCR, total RNA (10-20. Mu.g) was randomly fragmented into 100 nucleotide (nt) sizes. By co-immunoprecipitation analysis, the reaction mixture was subjected to m 6 The A-modified RNA fragments were pooled and enriched and then quantified by RT-PCR using the MeRIP m6A kit (EpiGentek).
5. Culture of human KCs and mouse DCs
Isolated cultured KCs were isolated from surgically excised prepuce specimens and mouse bone marrow cells were induced to DCs by GM-CSF.
For RNA interference experiments, si-WTAP (5'-CTAAGAGAGTCTGAAGAAA-3') was selected for intervention effect verification, and after confirmation of the intervention effect, formal knockdown and functional phenotype verification was performed: cells were plated in 6-well plates. The siRNA was transfected with lipo3000 (Invitrogen) into KCs and DCs. For the overexpression experiments, adenovirus intervention was used: the CMV-MCS-SV40-EGFP adenovirus vector was inserted into the WTAP (GenBank: NM-019852, target sequence: 5'-GAGGATCCCCGGGTACCGGCGCCACCATGACCAACGAAGAACCTCTTC-3', SEQ IDNO. 16) gene coding sequence. For viral infection, cells were cultured overnight in DMEM containing 10% fetal bovine serum and adenovirus was added to the well plate at optimal infection diversity (MOI). After 8-16 hours of infection, the medium was replaced with complete medium. The expression levels of protein and mRNA were detected 72h after infection. Expression of the knockdown or over-expressed dry prognosis target gene was detected using RT-qPCR and WB.
6. Results
6.1 m differentially expressed in AD lesions 6 A RNA methylation regulatory molecules and potential targets
Normal control (n=3) and AD lesions (n=3) were biopsied m 6 A sequencing analysis AD and normal controls were completely distinguishable by Principal Component Analysis (PCA) (fig. 1A). Through m 6 Analysis of A transcriptome chip data we found 1057 high m in AD 6 A methylation gene and 235 low m 6 A methylation gene (fold changes is more than or equal to 2, P)<0.05; FIG. 1B) we identified 598 up-regulated genes and 432 down-regulated genes by mRNA-seq (fold change. Gtoreq.2 and P)<0.05; fig. 1C). We found that 181 differentially methylated genes (DEGs) occurred simultaneously with hypo-m 6 A and Down-mRNA (hypo-Down), 532 DEGs occur simultaneously with hyper-m 6 A and up-mRNA (hyper-up). Meanwhile, 54 Biological Process (BP) pathways generated "hypo-down",522 BP pathways generated "hyper-up", 2 hsa pathways generated "hypo-down", and 27 hsa pathways generated "hyper-up" (FIG. 1D). Based on the above results, we found that all screened DEGs and most of the pathways appeared to be enriched simultaneously with either "hypo-down" or "hyper-up", suggesting m 6 The a modification is positively correlated with mRNA expression in AD. Based on the enrichment score and the ordering of Fisher P value, we determined 16 differential pathways of interest, including 10 up-regulated pathways and 6 down-regulated pathways (FIG. 1E), with the up-regulated pathways consisting essentially of viral processes, leukocyte migration processes involved in inflammatory reactions, long chain fatty acid metabolic processes, and phosphatidylethanolamine metabolic processes. Whereas down-regulated pathways mainly include establishment of epithelial cell apical/basal polarity, pyrimidine nucleoside triphosphate metabolic processes, etc. The heatmap shows m of the DEGs involved in these pathways of interest 6 Sequences of a level and mRNA level (fig. 1F). Furthermore, we found that mRNA expression differences for "writer" WTAP were most pronounced in normal controls and AD by sequencing (FIG. 1G).
Next, we demonstrated that mRNA and protein levels of WTAP were significantly up-regulated in AD lesions compared to normal skin (fig. 2A, E). We also demonstrated that the first three DE (ALOX 15) most significantly up-regulated compared to normal skin in sequencing results were significantly up-regulated in mRNA and protein levels of S100A9 and SERPINB3 in AD lesions (fig. 2B, C, E), whereas ALOX15 was significantly up-regulated only in AD (fig. 2E) with no significant difference in mRNA levels compared to normal skin (fig. 2D). The fluorescence intensities of WTAP, S100A9 and SERPINB3 were found to be significantly higher in AD patient epidermal lesions than in normal controls (fig. 2F-H), while ALOX15 was significantly elevated in AD dermal lesions (fig. 2I).
Correlation analysis based on GSE193309 dataset results showed that expression levels of WTAP correlated positively with S100A9 and SERPINB3 (r=0.28, p<0.05 Whereas AD patients (n=9) with no significant correlation to ALOX15 (fig. 3A) had a higher total m than normal controls (n=9) 6 Level of a methylation (fig. 3B). In addition, AD group S100A9 and SERPINB3mRNA m 6 The a level was significantly elevated (n=5) compared to the normal control group (fig. 3C and D). However, we did not obtain m for the mRNA of ALOX15 in the normal control or AD group 6 Level a. These data indicate that ALOX15, S100A9 and SERPINB3 are m 6 Downstream targets for A RNA methylation may be regulated by WTAP.
6.2WTAP regulates expression of ALOX15, S100A9 and SERPINB3
In order to verify the potential regulatory role of WTAP in ALOX15, S100A9 and SERPINB3 expression, we verified and determined siRNA sequences expressed by WTAP in KCs and DCs, which have a significant effect on WTAP, followed by further subsequent experiments. Knock-down of WTAP results in m of S100A9 and SERPINB3 in KCs 6 The a level was reduced (fig. 4A) while the mRNA and protein levels of both were also significantly reduced (fig. 4C, E). Furthermore, WTAP knockdown resulted in ALOX15 m in DCs 6 A. mRNA and protein levels were significantly reduced (FIG. 4A, G, I)
Subsequently we over-expressed WTAP by adenovirus in KCs and DCs, showing that over-expression of WTAP results in m of S100A9 and SERPINB3 in KCs 6 A. mRNA and protein levels increased (FIG. 4B, D, F), also resulting in ALOX15 m in DCs 6 A. mRNA and protein levels are significantly increased(FIG. 4B, H, J). Based on these data, we propose a mechanism hypothesis for the progression of AD disease. M of WTAP through mRNA 6 A methylation promotes expression of ALOX15, S100A9 and SERPINB3, thereby enhancing the development of AD.
In summary, the present study showed that 15-arachidonate oxidase (ALOX 15), calprotectin (S100 A9) and serine protease inhibitor B3 (SERPINB 3) expression levels were significantly elevated in atopic dermatitis (Atopic dermatitis, AD) lesions and their expression was regulated by the wilms tumor 1 associated protein (WTAP). These data indicate WTAP-mediated m 6 The A modification up-regulates the expression of ALOX15, S100A9 and SERPINB3, and promotes the occurrence and development of AD. Therefore, the WTAP has the application potential of serving as a target site in preparing the drug for treating the AD, and can be further used as a molecular marker for diagnosing the AD to be applied to a clinical detection kit.
Claims (4)
- Use of an inhibitor of wtap in the manufacture of a medicament for the treatment of atopic dermatitis.
- 2. The use according to claim 1, wherein the inhibitor of WTAP is siRNA.
- 3. The use according to claim 1, wherein the inhibitor of WTAP is as shown in SEQ ID No. 15.
- Application of WTAP as a molecular marker in preparing a kit for diagnosing atopic dermatitis.
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