circubxn4-shb axis as a target spot in the preparation of drugs for treating asthma

Abstract

The application discloses application of circUBXN4-SHB axis as a target in preparation of a drug for treating asthma, and finds that in vitro, knockdown of circUBXN4 can inhibit expression of eosinophil chemotactic factor by inhibiting a JAK1-STAT6 signal path; in an allergen-induced mouse model, knockdown of circUBXN4 can reduce airway eosinophil inflammation and mucous cell metaplasia.Mechanically, circUBXN4 is combined with an adapter protein SHB, enhances SHB-JAK1 combination, and enhances JAK1 and STAT6 phosphorylation.In addition, SHB protein is up-regulated in expression in asthma airway epithelial cells.Knockdown of SHB can reduce airway eosinophil inflammation and mucous cell metaplasia.The research result of the application shows that circUBXN4 and SHB are promising treatment targets for asthma, provide a direction for treatment of asthma, and have great clinical significance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of the circUBXN4-SHB axis as a target in the preparation of drugs for treating asthma. Background Technology

[0002] Asthma is a common chronic airway disease characterized by airway eosinophilic inflammation, mucinous metaplasia, and airway hyperresponsiveness. Airway epithelial cells, as the first line of defense and core regulators of the immune response, play a crucial role in the pathogenesis of asthma. IL-13, a type II cytokine, acts on airway epithelial cells in a Janus tyrosine kinase 1 (JAK1)-signal transducer and activator of transcription 6 (STAT6)-dependent manner, inducing eosinophil chemokine secretion and goblet cell metaplasia. IL-13 binds to the type II IL-4 receptor, which is primarily expressed on airway epithelial cells and consists of two subunits: IL-4Rα and IL-13Rα1. Subsequently, JAK1 is activated, leading to phosphorylation of tyrosine residues on IL-4Rα. STAT6 binds to phosphorylated IL-4Rα and is then phosphorylated by JAK1. Phosphorylated STAT6 can form dimers and translocate into the cell nucleus, initiating the transcription of effector genes, including MUC5AC and eosinophil chemokines CCL11, CCL24, and CCL26. Activation of phosphorylated STAT6 can drive massive goblet cell metaplasia, leading to excessive mucus secretion and airway obstruction; it also releases chemokines, recruits eosinophils, and initiates Th2 immune responses; long-term activation also promotes epithelial-mesenchymal transition, causing airway fibrosis and remodeling, exacerbating the pathogenesis of asthma at multiple stages.

[0003] SHB (SH2 domain-containing adapter protein B) is an adaptor protein B containing the SH2 domain encoded by the SHB gene. This protein binds to phosphorylated tyrosine residues and supports signal transduction. It acts as a molecular adaptor in pathways related to hematopoietic stem cell proliferation, immune responses, and oocyte maturation. This protein is located in the cytosol, cytoplasm, nucleoplasm, and cytoplasmic ribonucleoprotein particles, placing it in a compartment that coordinates signal transduction and regulatory interactions. Previous reports have shown that SHB regulates multiple signaling cascades by linking activated receptors to downstream signaling components, including focal adhesion kinase, vascular endothelial growth factor receptor-2, and platelet-derived growth factor receptor. However, the presence of SHB in asthma has not yet been reported.

[0004] Circular RNAs (circRNAs) are a class of covalently closed RNA molecules produced through backsplicing. This structure endows them with extremely high stability, supporting their role as functional molecules rather than transcriptional byproducts. CircRNAs exhibit a variety of regulatory functions, particularly acting as miRNA sponges to influence mRNA translation, and as scaffolds or decoys to regulate protein-protein interactions and signaling pathways. In asthma, multiple studies have shown that epithelial cell circRNAs, acting as miRNA sponges, play a role in regulating airway inflammation and remodeling. For example, airway epithelial cell circZNF652 upregulates JAK2 expression by sponging miR-452-5p, promoting goblet cell metaplasia. Circ-0001454 has been reported to alleviate airway inflammation and remodeling by isolating miR-770-5p, while downregulation of circ-0001875, which binds to miR-31-5p, is associated with macrophage polarization in severe asthma. Hsa_circ_0002981 is formed by backsplicing exons 2-6 of the UBXN4 precursor mRNA located on chromosome 2. In this invention, hsa_circ_0002981 is named circUBXN4. Currently, there are no reports of circUBXN4 being present in asthma.

[0005] Small interfering RNA (siRNA) is a double-stranded RNA composed of 19-25 nucleotide pairs and is a key component in RNA interference technology. After transfection into cells, siRNA binds to the RNA-induced silencing complex (RISC). Subsequently, one sense strand of the siRNA degrades, and the remaining antisense strand guides the RISC to bind to the target mRNA according to the base pairing principle, ultimately inducing the degradation of the mRNA and thus inhibiting the expression of the target gene. Designing siRNAs with high specificity and high interference efficiency will play an important role in the targeted therapy of asthma. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the circUBXN4-SHB axis as a target in the preparation of drugs for treating asthma. Epithelial circUBXN4 binds to SHB and enhances the SHB-JAK1 interaction, thereby amplifying IL-13-activated JAK1-STAT6 signaling and promoting eosinophilic inflammation of the airways in asthma.

[0007] The results of this invention indicate that CircUBXN4 expression is upregulated in asthmatic airway epithelium and is positively correlated with type 2 marker gene expression and exhaled nitric oxide levels. In BEAS-2B cells, knockdown or overexpression of circUBXN4 promotes or inhibits IL-13-induced eosinophil chemokine expression by enhancing or inhibiting JAK1 and STAT6 phosphorylation, respectively. In ovalbumin-sensitized and challenged mice, knockdown of circUBXN4 expression in airway epithelial cells alleviates airway eosinophilic inflammation, mucinous metaplasia, and STAT6 phosphorylation. Mechanistically, circUBXN4 binds to the adaptor protein SHB. Overexpression of CircUBXN4 enhances the interaction between SHB and JAK1 and promotes JAK1 and STAT6 phosphorylation. Furthermore, SHB protein expression levels are upregulated in airway epithelial cells of asthmatic patients. In an asthmatic mouse model, knockdown of SHB alleviates airway eosinophilic inflammation and mucinous metaplasia.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of the circUBXN4-SHB axis as a target in the preparation of a drug for treating asthma. The drug targets the circUBXN4-SHB axis and weakens the JAK1-STAT6 signaling pathway by specifically interfering with and inhibiting the circUBXN4-SHB axis, thereby alleviating airway eosinophilic inflammation and mucinous metaplasia in asthma.

[0009] In the above technical solutions, the drug is siRNA, shRNA, microRNA, or a circUBXN4-SHB axis chemical inhibitor.

[0010] Secondly, the present invention provides the use of circUBXN4 inhibitors in the preparation of medicaments for treating asthma, wherein the circUBXN4 inhibitors are used to knock down the expression level of circUBXN4 in airway epithelial cells.

[0011] In the above technical solution, the circUBXN4 inhibitor is siRNA, and its target sequence is shown in SEQ ID NO. 1, which is AAAGGTGATGATGAACAGT.

[0012] Thirdly, the present invention provides the use of SHB inhibitors in the preparation of drugs for treating asthma, wherein the SHB inhibitors are used to knock down the expression level of SHB in airway epithelial cells.

[0013] In the above technical solution, the SHB inhibitor is siRNA, and its target sequence is shown in SEQ ID NO. 3, which is CAAGGAGTGTAGCTACCTT.

[0014] Fourthly, the present invention provides a medicament for treating asthma, the medicament comprising a circUBXN4-SHB axis inhibitor.

[0015] In the above technical solutions, the drug contains siRNA that specifically knocks down the expression of circUBXN4 in airway epithelial cells and / or siRNA that specifically knocks down the expression of SHB in airway epithelial cells.

[0016] In the above technical solutions, the target sequence of the siRNA that specifically knocks down the expression of circUBXN4 in airway epithelial cells is shown in SEQ ID NO. 1, which is AAAGGTGATGATGAACAGT, and the target sequence of the siRNA that specifically knocks down the expression of SHB in airway epithelial cells is shown in SEQ ID NO. 3, which is CAAGGAGTGTAGCTACCTT.

[0017] The beneficial effects of this invention are as follows: This invention analyzed the expression profile of circRNAs in epithelial cells from bronchial brushing samples of asthma patients and control subjects, and found that circUBXN4 is a significantly upregulated circRNA in asthmatic airway epithelial cells. In vitro, knockdown of circUBXN4 inhibited eosinophil chemokine expression by suppressing the JAK1-STAT6 signaling pathway; in an allergen-induced mouse model, knockdown of circUBXN4 alleviated airway eosinophilic inflammation and mucinous metaplasia. Mechanistically, circUBXN4 binds to the adaptor protein SHB, enhancing SHB-JAK1 binding and enhancing JAK1 and STAT6 phosphorylation. Furthermore, SHB protein expression is upregulated in asthmatic airway epithelial cells. In the mouse model, SHB knockdown also alleviated airway eosinophilic inflammation and mucinous metaplasia. The results of this invention indicate that circUBXN4 and SHB are promising therapeutic targets for asthma, providing direction for asthma treatment and having significant clinical implications. Attached Figure Description

[0018] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that the accompanying drawings are merely schematic illustrations, used to help illustrate the technical solutions and preferred embodiments of the present invention, and do not constitute a limitation on the scope of protection of the technical solutions of the present invention. Within the scope defined by the claims of the present invention, any equivalent transformations or modifications based on the principles of the present invention should be considered to fall within the protection scope of the present invention.

[0019] Figure 1: Experimental diagram of upregulated expression of CircUBXN4 in airway epithelial cells of asthma patients. A, Volcano diagram of circRNA profiles in airway brushing samples from control group (n=5) and asthma patients (n=8). B, Schematic diagram of circUBXN4 generation via exon 2-6 circularization of UBXN4 precursor mRNA. Sanger sequencing verified the reverse splicing site. C, RT-PCR confirms the presence of circUBXN4 in cDNA but not in genomic DNA in BEAS-2B cells. D, RT-PCR and agarose gel electrophoresis verify the circular structure of circUBXN4 in BEAS-2B cells under RNase R treatment or no treatment. E, qRT-PCR analysis of the stability of circUBXN4 and UBXN4 in BEAS-2B cells after treatment with actinomycin D (2 μg / ml). F, qRT-PCR detection of circUBXN4 expression in airway brushing samples from control group (n=8) and asthma patients (n=31). GK, Correlation analysis of circUBXN4 expression in epithelial cells with FeNO (G), mean values ​​of the three genes (H), CLCA1 (I), POSTN (J), and SERPINB2 (K). L, RNA-FISH detection of circUBXN4 expression in airway biopsy tissues from controls and asthma patients. CircUBXN4 probe was labeled with Cy-3. Cell nuclei were stained with DAPI. M, Quantification of relative mean fluorescence intensity of circUBXN4 using ImageJ. All data are expressed as mean ± SD. ****P<0.0001, **P<0.01.

[0020] Figure 2Figure 1: Experimental findings showing the enhancement of eosinophil chemokines and epithelial type 2 inflammatory markers by CircUBXN4. A. RNA-FISH showing circUBXN4 signaling in BEAS-2B cells stimulated with 20 ng / ml IL-13 for 48 hours. The CircUBXN4 probe was labeled with Cy-3. Cell nuclei were stained with DAPI. B. Quantification of mean fluorescence intensity of circUBXN4 using ImageJ. C. qRT-PCR analysis of circUBXN4 expression in BEAS-2B cells stimulated with 20 ng / ml IL-13 for 12, 24, and 48 hours. GUSB was used as an internal control. D. qRT-PCR detection of circUBXN4 expression in BEAS-2B cells transfected with control or circUBXN4-targeting siRNA. E. qRT-PCR detection of circUBXN4 mRNA levels in BEAS-2B cells treated with control or circUBXN4 siRNA and 20 ng / ml IL-13 for 24 hours. FK, qRT-PCR analysis was performed on the expression of eosinophil chemokines CCL11(F), CCL24(G), CCL26(H) and epithelial type 2 inflammatory markers SERPINB2(I), CLCA1(J), and POSTN(K) in BEAS-2B cells treated with 20 ng / ml IL-13 for 24 hours, with or without circUBXN4 knockdown. LN, ELISA was used to detect the protein levels of CCL11(L), CCL24(M), and CCL26(N) in BEAS-2B cell culture medium. All data are expressed as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0021] Figure 3Experimental diagram showing the role of CircUBXN4 in JAK1-STAT6 signaling. A: BEAS-2B cells were transfected with either control or circUBXN4 siRNA, then stimulated with 20 ng / ml IL-13 for 5, 15, or 30 minutes. Western blot analysis was performed to detect JAK1, p-JAK1, STAT6, and p-STAT6, with GAPDH as an internal control. B: Relative optical density quantification was performed using ImageJ. Statistical analysis of the p-JAK1 to total JAK1 ratio. C: Statistical analysis of the p-STAT6 to total STAT6 ratio. Data represent the mean of four independent experiments. D: BEAS-2B cells were transfected with either control or circUBXN4 overexpression plasmid, then stimulated with 20 ng / ml IL-13 for 5, 15, or 30 minutes. Western blot analysis was performed to detect JAK1, p-JAK1, STAT6, and p-STAT6. E: Statistical analysis of the pJAK1 to JAK1 ratio. F: Statistical analysis of the pSTAT6 to STAT6 ratio. Data represent the mean of three independent experiments. G, Immunofluorescence analysis of phosphorylated STAT6 in BEAS-2B cells stimulated with IL-13 (30 min) with or without circUBXN4 knockdown. Cell nuclei were stained with DAPI. H, Quantification of mean fluorescence intensity of p-STAT6 using ImageJ. I, BEAS-2B cells transfected with control or circUBXN4 plasmid were pretreated with AS1517499 (STAT6 inhibitor, 100 nM) or PBS for 1 h, followed by stimulation with IL-13 (20 ng / ml, 24 h). circUBXN4 expression was detected by qRT-PCR and normalized to GUSB. J and K, CCL11 and CCL26 protein levels in culture medium were detected by ELISA. All data are expressed as mean ± SD. *P<0.05,**P<0.01, ***P<0.001, ****P<0.0001.

[0022] Figure 4: In vivo knockdown of CircUbxn4 inhibits type 2 airway inflammation and mucus secretion via JAK1-STAT6 signaling. A, Schematic diagram of the OVA-induced mouse asthma model. in, nasal drop; ip, intraperitoneal injection. B, Representative FISH image of circUbxn4 in mouse lung tissue sections. CircUbxn4 is labeled with Cy3. Cell nuclei are stained with DAPI. C, Classification and counting of inflammatory cells in bronchoalveolar lavage fluid. D, Representative H&E staining image of mouse lung tissue sections. Inflammation score calculated. E, Representative PAS staining image of mouse lung tissue sections. At 200x magnification, the number of PAS-positive cells in six fields of view for each lung section was counted. F, ELISA detection of Ccl24 protein levels in BALF. GI, qRT-PCR analysis of Ccl24 (G), Ccl11 (H), and Muc5ac (I) expression in lung tissue of OVA model mice in each group. Data are normalized to Gapdh. J. Western blot analysis of p-STAT6 and STAT6 in mouse lung tissue. K. Statistical analysis of the p-STAT6 to STAT6 ratio. Each group contained four independent samples. All data are expressed as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0023] Figure 5Figure 1: CircUBXN4 and SHB interaction assay. A) RNA pull-down and mass spectrometry analysis in BEAS-2B cells using either the control probe LacZ or the circUBXN4 probe. Scatter plot shows the identified proteins. B) Venn plot showing the overlap between proteins interacting with JAK1 or STAT6 in the BioGRID database and the top 100 proteins identified by circUBXN4 pull-down mass spectrometry. C) qRT-PCR detection of SHB mRNA levels in BEAS-2B cells after IL-13 (20 ng / ml) stimulation for 24 and 48 hours. D) Western blot detection and quantification of SHB protein levels after IL-13 stimulation for 24 and 48 hours. E) Western blot of the RNA pull-down assay, validating the interaction between SHB protein and circUBXN4 in BEAS-2B cells. F. RNA immunoprecipitation assay showing the binding of SHB to circUBXN4 in BEAS-2B cells transfected with the SHB plasmid (with the HA tag), regardless of IL-13 treatment. G. Western blot analysis of SHB protein levels in BEAS-2B cells after circUBXN4 knockdown. H. Statistical analysis of SHB protein levels. I. qRT-PCR detection of Shb expression in lung tissue. Data are normalized to GAPDH. J. Western blot detection of Shb protein levels in lung tissue. K. Statistical analysis of SHB protein levels. All data are expressed as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0024] Figure 6: Experimental diagram of circUBXN4 promoting the JAK1-STAT6 pathway by binding to SHB. A. Western blot analysis of JAK1, p-JAK1, STAT6, and p-STAT6 expression levels in BEAS-2B cells stimulated with IL-13 for 5, 15, and 30 minutes with or without SHB knockdown. B. Statistical analysis of the p-JAK1 to JAK1 ratio. C. Statistical analysis of the p-STAT6 to STAT6 ratio. D. BEAS-2B cells co-transfected with Flag-JAK1 and HA-SHB plasmids. Immunoprecipitation was performed using HA or Flag antibodies, followed by Western blot. E. BEAS-2B cells transfected with Flag-JAK1 and HA-SHB plasmids (with or without circUBXN4 overexpression) were immunoprecipitated with Flag or HA antibodies, followed by Western blot. F. Schematic diagram of the SHB protein mutation strategy. G. Western blot analysis of SHB protein levels after transfection of three mutant plasmids into BEAS-2B cells. H. RNA immunoprecipitation assay showing the binding of circUBXN4 to the three mutant SHB proteins. I. Co-transfection of circUBXN4 overexpression plasmid with SHB or MUT1-SHB plasmid into BEAS-2B cells, followed by stimulation with IL-13 for 15 or 30 minutes. Phosphorylation of STAT6 was detected by Western blot. All data are expressed as mean ± SD. *P<0.05, **P<0.01, ***P<0.001.

[0025] Figure 7: An in vivo experiment showing how knocking down SHB inhibits type 2 airway inflammation and mucus secretion via JAK1-STAT6 signaling. A, qRT-PCR detection of SHB expression in airway brushing samples from control groups (n=20) and asthma patients (n=57), normalized to ACTB. B, Immunohistochemical analysis of SHB expression in paraffin sections of airway biopsies. C, Quantitative analysis of the ratio of SHB IOD to tissue area in airway biopsy sections from 3 controls and 3 asthma patients (200x magnification). IOD: Integrated optical density. D, Schematic diagram of an OVA-induced mouse asthma model. in, nasal drop; ip, intraperitoneal injection. E, Classification and counting of inflammatory cells in bronchoalveolar lavage fluid. F, Representative H&E staining images of mouse lung tissue sections. Inflammation scores were calculated for lung sections. G, Representative PAS staining images of mouse lung tissue sections. PAS-positive cells were counted in six fields of view for each lung section at 200x magnification. H, Western blot analysis of STAT6 phosphorylation and SHB expression in mouse lung tissue. I, Statistical analysis of SHB expression. J, Statistical analysis of the p-STAT6 to STAT6 ratio. KM, qRT-PCR analysis of Ccl11 (K), Ccl24 (L), and Muc5ac (M) expression in lung tissue. Data are normalized to Actb. All data are expressed as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0026] Figure 8 : Experimental diagram showing the effect of circUBXN4 overexpression on the expression of eosinophil chemokines and epithelial type 2 inflammatory markers. A, B: BEAS-2B cells were transfected with the control plasmid or the circUBXN4 overexpression plasmid, and then treated with IL-13 for 24 hours. The expression of circUBXN4 and UBXN4 was detected by qRT-PCR. CH: In BEAS-2B cells with or without circUBXN4 overexpression, the expression of eosinophil chemokines CCL11 (C), CCL24 (D), CCL26 (E) and epithelial type 2 inflammatory markers SERPINB2 (F), CLCA1 (G), and POSTN (H) was analyzed by qRT-PCR after treatment with 20 ng / ml IL-13 for 24 hours. All data are expressed as mean ± standard deviation. P<0.05, P<0.01, P<0.001, ****P<0.0001.

[0027] Figure 9: An experimental diagram showing the regulation of eosinophil chemokines and epithelial type 2 inflammatory markers by circUBXN4 through the JAK1-STAT6 signaling pathway. AF, BEAS-2B cells were transfected with either the control plasmid or the circUBXN4 overexpression plasmid, pretreated with AS1517499 (STAT6 inhibitor, 100 nM) or PBS for 1 hour, followed by stimulation with IL-13 (20 ng / ml, 24 hours). qRT-PCR was used to detect the transcriptional levels of CCL11(A), CCL24(B), CCL26(C), SERPINB2(D), POSTN(E), and CLCA1(F). Data were normalized using GUSB as an internal control. All data are expressed as mean ± standard deviation. P < 0.05, P < 0.01, P < 0.001, ****P < 0.0001.

[0028] Figure 10 Knockdown of circUbxn4 inhibits the expression of eosinophil chemokines and mucus secretion. A, Sequence similarity analysis of human circUBXN4 and mouse circUbxn4. B, Schematic diagram of the circUbxn4 backsplicing pattern; Sanger sequencing confirmed the splicing site. C, Transfection of 3T3 cells with three siRNAs targeting circUbxn4, qRT-PCR verification of knockdown efficiency, data normalized with GAPDH as internal control. D and E, ELISA analysis of IL-13 and IL-4 protein levels in BALF. F, qRT-PCR analysis of Ubxn4 expression in lung tissues of OVA model mice in each group, data normalized with GAPDH as internal control. All data are expressed as mean ± standard deviation. *P<0.05, **P<0.01, ****P<0.0001.

[0029] Figure 11 Figure 1: No significant change in the transcriptional levels of SFPQ, IFI16, and CTDSPL2 in BEAS-2B cells under IL-13 stimulation. AC: Transcriptional levels of SFPQ (A), IFI16 (B), and CTDSPL2 (C) in BEAS-2B cells after 24 and 48 hours of IL-13 (20 ng / ml) stimulation by qRT-PCR. D: SHB mRNA expression was detected by qRT-PCR after transfection of BEAS-2B cells with control siRNA or SHB-targeting siRNA. All data are expressed as mean ± standard deviation. ***P < 0.001. Detailed Implementation

[0030] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0031] This invention provides the application of the circUBXN4-SHB axis as a target in the preparation of drugs for treating asthma, adding new insights into the study of circRNAs in asthma. Previous studies have mainly focused on circRNAs in BAL cells or immune cells. For example, circZNF652 upregulates JAK2 in BAL cells of children with asthma through sponge-like adsorption of miR-452-5p. In macrophages, circS100A11 promotes STAT6 transcription through the protein S100A11 and the transcription factor SP3. This invention differs from previous reports in both cellular background and mechanism, focusing on airway epithelial cells and identifying the direct protein scaffold role of circUBXN4 in SHB-JAK1 binding. This highlights the functional diversity of circRNAs, which can exert cell-type-specific effects in the same disease through different molecular patterns.

[0032] This invention newly discovers the role of the adaptor protein SHB as a regulator of JAK1 phosphorylation. While SHB has previously been reported to participate in signaling of various receptor tyrosine kinases and cytokine receptors, and its deficiency has been shown to promote T cell polarization towards Th2 and enhance Th2 inflammatory responses in vivo, its function in airway epithelial cell IL-13 signaling remains unclear. This invention demonstrates that SHB binds to JAK1 and is essential for the complete activation of JAK1-STAT6 induced by IL-13 in bronchial epithelial cells. Although circUBXN4 does not alter SHB expression, it acts as a molecular scaffold to enhance SHB-JAK1 interaction and promote JAK1 phosphorylation. In vivo data further solidify the functional relevance of SHB, showing that SHB knockdown significantly attenuates OVA-induced STAT6 activation, airway eosinophilic inflammation, and mucinous metaplasia, similar to the effects of circUBXN4 knockdown. This localizes airway epithelial cell SHB within the circUBXN4-SHB complex as a positive regulator of type 2 inflammation, enriching the existing paradigm for the role of SHB in immunity.

[0033] Upregulation of circUBXN4 in asthmatic airway epithelial cells makes it a potential biomarker for type 2 hyperasthma. Furthermore, the protein scaffold function of circUBXN4 offers a unique therapeutic opportunity. Disruption of the specific interface between circUBXN4 and SHB more precisely attenuates JAK1-STAT6 signaling compared to broad-spectrum JAK inhibitors.

[0034] In summary, this invention depicts a novel epithelial cell circRNA-driven pathway in which circUBXN4 binds to SHB to enhance JAK1 activation and STAT6 signaling, thereby amplifying type 2 inflammation. These findings deepen our understanding of the JAK1-STAT6 signaling regulatory mechanisms and reveal the circUBXN4-SHB interface as a potential therapeutic target for asthma.

[0035] I. Experimental Methods RNA sequencing was used to analyze the expression profile of epithelial circular RNA, and the results were validated in bronchial brushing samples from asthma patients and control subjects. circUBXN4 was knocked down or overexpressed in vitro and in allergen-induced mouse models, and changes in JAK1 and STAT6 phosphorylation and eosinophil chemokine expression were observed. The mechanism by which circUBXN4 regulates the JAK1-STAT6 signaling pathway was investigated using RNA pull-down and mass spectrometry analysis. The binding of SHB to circUBXN4 and its binding site were demonstrated using RIP experiments. By knocking down SHB expression in in vitro cell lines and allergen-induced mouse models, changes in JAK1 and STAT6 phosphorylation levels and eosinophil chemokine expression were observed; airway inflammation and mucus secretion levels in mouse models were also observed.

[0036] II. Statistical Analysis All statistical analyses were performed using GraphPad Prism 10.3.0 software. Data distribution was assessed using the Shapiro-Wilk test. For normally distributed continuous variables, differences between two groups were analyzed using independent samples t-tests; differences among multiple groups were analyzed using one-way or two-way ANOVA, followed by Tukey's test. For non-normally distributed data, we calculated the median and interquartile range and used nonparametric tests (Kruskal-Wallis test and Dunn's multiple comparison test or Mann-Whitney test). Correlation analysis was performed using Spearman's rank correlation analysis. Results are expressed as mean ± standard deviation. P < 0.05 was considered statistically significant.

[0037] III. Experimental Results 1. In asthma patients, the expression of circUBXN4 in epithelial cells is upregulated and correlated with type 2 inflammatory markers. To investigate the role of circRNAs in the pathogenesis of asthma, RNA sequencing was performed on airway brushing samples from 5 controls and 8 asthma patients to analyze circRNA expression profiles. Differential expression analysis identified 93 differentially expressed circRNAs (fold change > 2 and P < 0.05), of which 25 were significantly upregulated and 68 were significantly downregulated. Figure 1 (A). Of the first five upregulated circRNAs, hsa_circ_0002981 was selected for further investigation because it is annotated in circBase and has a conserved mouse homology, mmu_circ_0008151. Hsa_circ_0002981 is formed by backsplicing exons 2-6 of the UBXN4 precursor mRNA located on chromosome 2, and was validated by PCR and Sanger sequencing using divergent primers. Figure 1 Based on the host gene, this invention names hsa_circ_0002981 as circUBXN4. Next, using divergent primers, it was confirmed that circUBXN4 amplifies only from cDNA and not from genomic DNA. Figure 1 Furthermore, circUBXN4 (rather than nonlinear UBXN4 mRNA) is resistant to RNase R digestion. Figure 1 (D). After treatment with the transcription inhibitor actinomycin D, linear UBXN4 mRNA was rapidly degraded, while circUBXN4 showed higher stability ( Figure 1 E).

[0038] To validate the expression of circUBXN4 in airway epithelial cells, airway brushing and biopsy samples were analyzed. Compared with the control group (n = 8), circUBXN4 was significantly upregulated in asthma patients (n = 31). Figure 1 Correlation analysis showed that circUBXN4 expression was positively correlated with exhaled nitric oxide fraction (an indicator of airway eosinophilic inflammation). Figure 1 In addition, CircUBXN4 was positively correlated with the expression of type 2 epithelial cell characteristic genes SERPINB2, POSTN, CLCA1, and their average values ​​(the average of the three genes). Figure 1 (HK). RNA fluorescence in situ hybridization analysis showed that circUBXN4 was upregulated in asthma epithelial cells ( Figure 1 (L and M). Data from this invention indicate that elevated circUBXN4 expression in epithelial cells is associated with inflammation in type 2 asthma.

[0039] 2. CircUBXN4 promotes the expression of eosinophil chemokines and epithelial type 2 characteristic genes. Next, we investigated the role of circUBXN4 in type 2 inflammation. In BEAS-2B cells, RNA-FISH analysis showed a significant increase in circUBXN4 expression after IL-13 stimulation. Figure 2 Similarly, circUBXN4 expression was upregulated at 12, 24, and 48 hours (A and B). Figure 2 To explore its function, siRNA (target sequence AAAGGTGATGATGAACAGT, SEQ ID NO. 1) was used to effectively knock down circUBXN4 without altering UBXN4 mRNA levels. Figure 2 (D and E). Knockdown of circUBXN4 inhibited the upregulation of IL-13-induced transcriptional levels of eosinophil chemokines CCL11, CCL24, and CCL26. Figure 2 FH), and upregulation of epithelial type 2 characteristic genes SERPIB2, CLCA1 and POSTN ( Figure 2 Similarly, IL-13-induced secretion of CCL11, CCL24, and CCL26 proteins was also significantly inhibited. Figure 2 ,LN).

[0040] Conversely, circUBXN4 levels were significantly increased in BEAS-2B cells transfected with the circUBXN4 overexpression plasmid, without affecting UBXN4 expression. Figure 8 (A and B). CircUBXN4 overexpression upregulated basal and IL-13-induced transcriptional levels of CCL11, CCL24, CCL26, as well as SERPINB2, CLCA1, and POSTN. Figure 8 In summary, these results suggest that circUBXN4 may promote type 2 inflammation by regulating the IL-13 signaling pathway through enhancing the expression of eosinophil chemokines.

[0041] 3. CircUBXN4 functions through the JAK1-STAT6 signaling pathway. IL-13 regulates the expression of effector molecules in airway epithelial cells by binding to the type II IL-4 receptor and activating the Janus tyrosine kinase-signal transducer and activator of transcription 6 (JAK-STAT6) cascade, leading to eosinophilic inflammation and mucus hypersecretion. Based on this, we investigated whether circUBXN4 regulates this pathway. In BEAS-2B cells, knockdown of circUBXN4 significantly inhibited IL-13-induced phosphorylation of JAK1 and STAT6. Figure 3 Conversely, circUBXN4 overexpression potently promoted JAK1 and STAT6 phosphorylation within 5 minutes of IL-13 stimulation. Figure 3Immunofluorescence of p-STAT6 showed that knockdown of circUBXN4 inhibited IL-13-induced p-STAT6 expression and significantly attenuated its nuclear aggregation. Figure 3 (G and H). This indicates that circUBXN4 is a positive modulator of the JAK1-STAT6 signal.

[0042] To determine whether circUBXN4 upregulates eosinophil chemokines by promoting JAK1-STAT6 signaling, BEAS-2B cells overexpressing circUBXN4 were pretreated with the STAT6 phosphorylation inhibitor AS1517499 before IL-13 stimulation. We observed that AS1517499 did not affect basal or IL-13-induced circUBXN4 expression. Figure 3 The results indicate that IL-13-induced circUBXN4 production is independent of STAT6 phosphorylation. Notably, circUBXN4 overexpression increases both basal and IL-13-induced CCL11 protein levels (I), indicating that IL-13-induced circUBXN4 production is independent of STAT6 phosphorylation. Figure 3 ,J), and IL-13-induced CCL26 secretion ( Figure 3 AS1517499 treatment significantly inhibited IL-13-induced CCL11 and CCL26 secretion, as well as circUBXN4-enhanced IL-13-induced CCL11 and CCL26 secretion. Similarly, AS1517499 reduced circUBXN4-enhanced IL-13-induced CCL11, CCL24, CCL26, and SERPINB2, POSTN, and CLCA1 mRNA expression. Figure 9 These data indicate that circUBXN4 regulates the expression of eosinophil chemokines and epithelial type 2 characteristic genes through the JAK1-STAT6 pathway.

[0043] 4. CircUBXN4 promotes allergen-induced type 2 airway inflammation and excessive mucus production in mice via JAK1-STAT6 signaling. Next, we will explore the function of circUBXN4 in vivo. The sequence homology between human circUBXN4 and its mouse homolog circUbxn4 is approximately 84.8% ( Figure 10 A divergent primer targeting the circUbxn4 backsplicing site was designed, and its specificity was confirmed by Sanger sequencing. Figure 10 B). To knock down circUbxn4 in vivo, an siRNA (target sequence GAAAGGGGATGATGAGCAA, SEQ ID NO. 2) that effectively inhibits circUbxn4 expression was selected for shRNA construction and AAV packaging. Figure 10One week prior to OVA sensitization, mice were administered AAV expressing either control or circUbxn4-shRNA via intranasal drops. Figure 4 RNA-FISH showed that, compared with the control group, OVA-sensitized and challenged mice had increased circUbxn4 expression in airway epithelial cells, while shRNA-mediated circUbxn4 knockdown effectively inhibited its expression without affecting host UBXN4 mRNA levels. Figure 4 B and Figure 10 In mice treated with AAV shRNA circUbxn4, the OVA-induced eosinophil and lymphocyte counts in BAL fluid were significantly reduced (F). Figure 4 Consistently, H&E staining showed that circUbxn4 knockdown significantly inhibited OVA-induced peribronchial inflammatory cell infiltration (as reflected in inflammation score analysis). Figure 4 Furthermore, OVA stimulation increased the protein levels of Ccl24, Il-13, and Il-4 in BAL solution, while circUbxn4 knockdown significantly decreased these levels (D). Figure 4 F and Figure 10 (D and E). Similarly, the transcriptional levels of Ccl24 and Ccl11 were also suppressed ( Figure 4 Furthermore, circUbxn4 knockdown significantly reduced OVA-induced mucinous metaplasia (PAS staining showed) (G and H). Figure 4 E), and inhibited OVA-induced Muc5ac expression ( Figure 4 To investigate whether circUbxn4 promotes airway inflammation and mucinous metaplasia via STAT6 signaling, this invention assessed the phosphorylation level of STAT6 in lung tissue and found that circUbxn4 knockdown reduced OVA-induced STAT6 phosphorylation (I). Figure 4 (J and K). Data indicate that circUBXN4 promotes airway eosinophilic inflammation and excessive mucus production by upregulating STAT6 signaling.

[0044] 5. CircUBXN4 combines with SHB to promote SHB-JAK1 binding and upregulate the JAK1-STAT6 signal. To elucidate the mechanism by which circUBXN4 regulates JAK1-STAT6 signaling, proteins interacting with circUBXN4 were identified using RNA pull-down and mass spectrometry analysis. This invention compared the proteomic profiles of the circUBXN4 group and the negative control group. Figure 5 (A), and focused analysis was performed by intersecting the top 100 candidate proteins with known JAK1 or STAT6 interacting proteins in the BioGRID database. Figure 5Four candidate molecules were identified, including the adaptor protein B (SHB) containing the SH2 domain. However, it remains unclear whether SHB is involved in regulating JAK1 phosphorylation or JAK-STAT6 signaling. Upon IL-13 stimulation, SHB protein expression was upregulated, although its transcriptional levels decreased simultaneously. Figure 5 (C and D). In contrast, the expression of other candidate molecules was largely unaffected by IL-13 treatment ( Figure 11 AC).

[0045] Next, the physical interaction between SHB and circUBXN4 was verified. An RNA pull-down binding immunoblotting assay was performed in BEAS-2B cells to verify the binding of circUBXN4 to SHB. Figure 5 Furthermore, RNA immunoprecipitation experiments showed that IL-13 stimulation enhanced the interaction between SHB and circUBXN4 (E). Figure 5 Subsequent studies ruled out the possibility that circUBXN4 regulates SHB expression, because its knockdown did not alter the expression of SHB / Shb (SHB refers to human genes or proteins, and Shb refers to mouse genes or proteins) either in vitro or in vivo. Figure 5 Despite decreased mRNA levels, IL-13 stimulation and OVA activation upregulated SHB protein, suggesting that SHB is involved in IL-13 signaling and asthma pathogenesis. Figure 5 (D and IK).

[0046] To investigate the role of SHB in JAK1-STAT6 signaling, SHB was knocked down using siRNA (target sequence CAAGGAGTGTAGCTACCTT, SEQ ID NO. 3). Figure 11 Furthermore, SHB knockdown significantly inhibited the phosphorylation of JAK1 and STAT6 under IL-13 stimulation (D). Figure 6 The presence of α, β, and β (AC) indicates that SHB is essential for pathway activation. Through co-immunoprecipitation experiments, we demonstrated the interaction between SHB and JAK1 (AC). Figure 6 Importantly, circUBXN4 overexpression enhanced the interaction between SHB and JAK1. Figure 6 To locate the binding region, we used the catRAPID database to predict its potential binding sites and constructed three SHB mutants (E). Figure 6 F and G). RIP experiments showed that the mutation in the amino acid 366-386 region (MUT1) eliminated the SHB-circUBXN4 interaction ( Figure 6Consistently, functional analysis showed that, unlike wild-type SHB, the MUT1-SHB mutant, when co-expressed with circUBXN4, did not promote IL-13-induced STAT6 phosphorylation (H). Figure 6 In summary, the results indicate that circUBXN4 binds to SHB and promotes its binding to JAK1, thereby enhancing the JAK1-STAT6 signal.

[0047] 6. SHB expression is upregulated in airway epithelial cells in asthma patients and promotes airway eosinophilic inflammation and mucinous metaplasia in an allergen-induced mouse model. This invention further evaluated the expression of SHB in human airway epithelial cells. Compared with the control group (n = 20), the SHB transcription level was reduced in bronchial brushing samples from asthmatic patients (n = 57). Figure 7 Conversely, immunohistochemical analysis showed a significant increase in SHB protein expression in airway epithelial cells of asthma patients (A). Figure 7 (B and C). This inconsistent pattern of mRNA and protein expression is consistent with our observations in mouse models. To investigate the function of SHB in allergic airway inflammation, AAV vectors carrying either short hairpin RNA of shb or control shRNA were used in OVA-sensitized and challenged mice. Figure 7 D). Knockdown of Shb significantly reduced the eosinophil count in BAL fluid ( Figure 7 E), inhibited peribronchial inflammatory cell infiltration and mucinous cell metaplasia ( Figure 7 Furthermore, Shb knockdown inhibited OVA-induced Stat6 phosphorylation (F and G). Figure 7 HJ), and downregulated the transcriptional levels of Stat6 target genes Ccl11, Ccl24 and Muc5ac ( Figure 7 These data suggest that SHB promotes airway eosinophilic inflammation and mucinous metaplasia by activating STAT6 signaling.

[0048] In summary, this invention reveals a previously unknown mechanism by which airway epithelial cell circRNAs upregulate the JAK1-STAT6 signaling pathway in asthmatic patients. It demonstrates that circUBXN4 is upregulated in asthmatic bronchial epithelial cells, acting as a molecular scaffold to directly bind the adaptor protein SHB. This interaction enhances SHB-JAK1 binding, upregulates JAK1 and STAT6 phosphorylation, and ultimately drives the expression of key eosinophil chemokines CCL11, CCL24, and CCL26. The functional significance of the circUBXN4-SHB axis was confirmed in an allergen-induced mouse model; knockdown of circUBXN4 or SHB alleviated airway eosinophilic inflammation and mucinous metaplasia. These findings collectively establish the circUBXN4-SHB axis as a key upstream regulator of airway eosinophilic inflammation and mucinous metaplasia in asthma.

[0049] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of SHB inhibitors in the preparation of drugs for treating asthma, characterized in that: The SHB inhibitor is used to knock down the expression level of SHB in airway epithelial cells. The SHB inhibitor is an siRNA, and its target sequence is shown in SEQ ID NO.

3.

2. A drug for treating asthma, characterized in that: The drug contains a circUBXN4-SHB axis inhibitor, which contains siRNA that specifically knocks down the expression of circUBXN4 in airway epithelial cells and siRNA that specifically knocks down the expression of SHB in airway epithelial cells. The target sequences of siRNAs that specifically knock down circUBXN4 expression in airway epithelial cells are shown in SEQ ID NO. 1; the target sequences of siRNAs that specifically knock down SHB expression in airway epithelial cells are shown in SEQ ID NO. 3.

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