Application of MUC1-C segment in preparation of medicine for preventing and treating chronic obstructive pulmonary disease

Abstract

The invention discloses application of an MUC1-C segment in preparation of a medicine for preventing or treating chronic obstructive pulmonary disease, and belongs to the technical field of protein functions and targeted medicine development. The MUC1-C protein level in the lung tissue and alveolar epithelial cells of a COPD patient is detected, and cell experiments and animal experiments jointly prove that down-regulation of MUC1-C expression is significantly related to the aging level of the lung tissue, the level of differentiation from AT2 cells to AT1 cells, the inflammation level, mitochondrial dysfunction and reduction of lung functions. MUC1-C overexpression can effectively relieve deterioration of the pathological process. The invention can be applied to the fields of research and development of new drugs and new technologies for diagnosing, preventing and treating chronic obstructive pulmonary disease, and the like.

Description

Technical Field

[0001] This invention belongs to the field of protein function and targeted drug development technology, particularly the application of the C-segment domain of MUC1 in the preparation of products for the prevention and treatment of chronic obstructive pulmonary disease. Simply put, the C-segment region can serve as a key functional target for the design of small molecule modulators, protective antibodies, or peptide drugs that specifically bind to this domain. This region can be used to treat COPD by modulating the signaling pathways or protein interactions mediated by this region, thus falling within the realm of precision medicine for respiratory diseases. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a disease characterized by progressive and irreversible decline in lung function caused by airflow obstruction, lung parenchymal destruction, and emphysema. COPD has become a growing public health problem and may become the third leading cause of death worldwide, affecting over 300 million people. Exposure to tobacco smoke (CS) is the primary risk factor, driving disease progression by inducing persistent airway inflammation, airway mucus barrier dysfunction, and alveolar destruction. The membrane-bound mucin MUC1, a key component of the airway mucus barrier, has become a research focus due to its unique transmembrane structure and signaling functions. The extracellular domain of MUC1 (MUC1-N) maintains airway barrier function by trapping pathogens and inhibiting excessive activation of Toll-like receptors. Its intracellular domain (MUC1-C) harbors potential phosphorylation sites that interact with multiple pathway proteins, inhibiting excessive activation of proinflammatory signaling pathways and regulating lower respiratory tract microbiota-host interactions, thereby reducing immune damage caused by pathogen colonization. Previous studies have shown that MUC1-C is downregulated in GOLD II-III patients with corticosteroid-resistant COPD, which can weaken the anti-inflammatory effect of corticosteroids, thereby exacerbating the release of pro-inflammatory factors and neutrophil infiltration. This indicates that there is a defect in MUC1-C expression in COPD patients, which not only destroys the inherent anti-inflammatory barrier function, but also causes the anti-inflammatory effect of corticosteroids to fail. Therefore, studying the specific regulatory mechanism of MUC1-C in COPD and developing new therapeutic strategies targeting MUC1-C are expected to not only break through the treatment bottleneck of existing therapies that are ineffective against corticosteroid resistance and dysbiosis, but also achieve global intervention in the pathological process of COPD through the synergistic action of multiple pathways. Summary of the invention

[0003] To solve the above technical problems, the present invention for the first time provides the application of the C-terminal of MUC1 in the preparation of products related to chronic obstructive pulmonary disease, wherein the related products include products related to the research, diagnosis, prevention and / or treatment of chronic obstructive pulmonary disease, and the product types include drugs, reagents and test kits, etc. The diagnosis includes the screening and detection of the presence / absence, high / low risk, and different stages of chronic obstructive pulmonary disease. The present invention detects the protein level of MUC1-C in the lung tissue and alveolar epithelial cells of COPD patients, and jointly proves through cell experiments and animal experiments that the down-regulation of MUC1-C expression is significantly correlated with the levels of lung tissue aging, the differentiation level of AT2 cells into AT1 cells, the inflammation level, mitochondrial dysfunction, and the decline of lung function. The overexpression of MUC1-C can effectively alleviate the deterioration of the above pathological processes. MUC1-C may become a biomarker for chronic obstructive pulmonary disease or an important target for the development of new drugs. The present invention can be applied to the fields of the research and development of new drugs and new technologies for the diagnosis, prevention, and treatment of chronic obstructive pulmonary disease.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] One object of the present invention is to provide the application of the C-terminal of MUC1 or its coding gene in the preparation of products related to chronic obstructive pulmonary disease, and the products related to chronic obstructive pulmonary disease include products related to the research, diagnosis, prevention and / or treatment of chronic obstructive pulmonary disease.

[0006] Further, the products include drugs, reagents and test kits.

[0007] Further, the products related to the research of chronic obstructive pulmonary disease include products that up-regulate and down-regulate the expression level of the C-terminal of MUC1 or its coding gene.

[0008] Furthermore, the research related to chronic obstructive pulmonary disease includes the construction of a chronic obstructive pulmonary disease model.

[0009] Further, the diagnosis includes the screening and detection of the presence / absence, high / low risk, and different stages of chronic obstructive pulmonary disease.

[0010] Furthermore, the products for the diagnosis of chronic obstructive pulmonary disease include products for detecting the expression level of the C-terminal of MUC1 or its coding gene.

[0011] In the products for the diagnosis of chronic obstructive pulmonary disease, the C-terminal of MUC1 or its coding gene is used as a biomarker, and at this time, the product type can be a test kit.

[0012] Further, the products for the prevention and / or treatment of chronic obstructive pulmonary disease include products that overexpress the C-terminal of MUC1 or its coding gene.

[0013] A second object of the present invention is to provide a product for the research related to chronic obstructive pulmonary disease, and the product includes, in the above application, a product for up-regulating and down-regulating the expression level of the MUC1-C segment or its coding gene in the product related to the research on chronic obstructive pulmonary disease.

[0014] A third object of the present invention is a product for diagnosing chronic obstructive pulmonary disease, and the product includes, in the above application, a product for detecting the expression level of the MUC1-C segment or its coding gene in the product for diagnosing chronic obstructive pulmonary disease.

[0015] A fourth object of the present invention is a product for preventing and / or treating chronic obstructive pulmonary disease, and the product includes, in the above application, a product for overexpressing the MUC1-C segment or its coding gene in the product for preventing and / or treating chronic obstructive pulmonary disease.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The present invention finds that the MUC1-C in the lung tissue and alveolar type II epithelial cells of elderly COPD patients decreases, and the expression of MUC1-C protein in the emphysema area of COPD patients is significantly lower than that of the normal control group, and is related to the lung function indexes, providing a new biomarker for COPD staging.

[0018] (2) The present invention finds that MUC1-C deficiency drives the progression of COPD by inhibiting the differentiation of AT2 cells and aggravating the senescence of lung tissue cells. After silencing MUC1-C in cell experiments, the expression of the alveolar epithelial cell differentiation marker AQP5 is down-regulated, and the cell differentiation morphology is abnormal. At the same time, knocking out MUC1 leads to the loss of nuclear localization of Ki67, the up-regulation of P21 protein, and the increase of SA-β-gal activity, confirming that MUC1-C deficiency exacerbates the alveolar repair disorder by blocking the differentiation process and activating the senescence pathway.

[0019] (3) The present invention finds that MUC1-C deficiency promotes the development of the pathological process of COPD by promoting inflammation and mitochondrial dysfunction. After knocking out MUC1, CSE stimulation leads to an increase in ROS in alveolar epithelial cells, a decrease in mitochondrial membrane potential (ΔΨm), and an up-regulation of IL-6 / IL-8 levels. The silencing of MUC1-C further inhibits the expression of the mitochondrial complex I subunit NDUFS4, indicating that the release of inflammatory factors and the formation of mitochondrial dysfunction jointly drive the formation of COPD inflammation and oxidative stress.

[0020] (4) The present invention found that overexpressing MUC1-C in AT2 cells can reverse COPD pathological damage and demonstrate therapeutic potential. In a CS-exposed mouse model, AAV9-mediated AT2 cell-specific overexpression of Muc1-C restored lung function indicators, reduced alveolar structural damage, and reversed the downregulation of AQP5, an AT1 differentiation marker. Transmission electron microscopy showed intact mitochondrial cristae structure and reduced inflammatory cell infiltration. This provides direct evidence for the development of MUC1-C-based gene therapy or small molecule drugs.

[0021] In summary, this study discovered for the first time the core regulatory role of MUC1-C in chronic obstructive pulmonary disease (COPD), and its downregulation is closely related to worsening lung function, alveolar epithelial aging, and inflammatory activation. Through multi-dimensional verification of clinical samples, cells, and animal models, this study clarified that MUC1-C deficiency promotes the worsening of COPD by triggering alveolar epithelial cell aging and transdifferentiation disorders, mitochondrial oxidative stress, etc. An innovative targeted intervention strategy was proposed: AAV9-mediated AT2 cell-specific MUC1-C overexpression can significantly reverse lung function decline and lung tissue damage, repair mitochondrial structure and function, and reduce inflammatory cell infiltration. This achievement not only provides a new diagnostic marker for COPD, but also opens up the direction of gene therapy and small molecule drug development based on MUC1-C, laying a theoretical and practical foundation for precision medicine for COPD. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Figure 1 shows the downregulation of MUC1-C expression in COPD lung tissue and CSE-induced A549 cells. (A) Immunofluorescence analysis showed significantly lower MUC1-C (green) fluorescence intensity in emphysematous regions of COPD patients compared with the normal control group (Normal). Cell nuclei were labeled with DAPI (blue); n = 8 per group. Scale bars: 5 mm in the upper panel, 250 μm in the lower panel. (B-C) Western blot analysis showed decreased MUC1-C protein levels in A549 cells after stimulation with cigarette smoke extract (CSE, 1%-3%); n = 3 per group. (D-E) Western blot analysis showed decreased MUC1-C protein levels in lung tissue of C57 mice exposed to tobacco smoke (CS) for 3 and 6 months compared with the air control group (Air); n = 6 per group. P < 0.05 indicated statistical significance.

[0023] Figure 2This shows the effects of MUC1 knockout (sgMUC1) / MUC1-C silencing (shMUC1-C) on the senescence level, inflammatory level, and mitochondrial function in Example 1 of the present invention, where: (A-B) Western blot analysis showed that after knocking out MUC1, compared with the control group, cigarette smoke extract (CSE) stimulation significantly upregulated the protein level of the senescence marker P21; n = 6 for each group; (C-D) After knocking out MUC1, the mRNA levels of IL-6 and IL-8 were significantly increased whether or not CSE stimulation was received; n = 4 for each group; (E) Immunofluorescence analysis showed that whether or not CSE stimulation was received, MUC1 knockout led to the loss of nuclear localization of Ki67 (green) in A549 cells, and the cell nuclei were labeled with DAPI (blue); n = 6 for each group; (F-H) After silencing MUC1-C, tobacco smoke tar phase extract (CSTar) stimulation significantly increased the protein level of P21; n = 6 for each group; (I-J) After silencing MUC1-C, the mRNA levels of IL-6 and IL-8 were significantly increased whether or not CSE stimulation was received; n = 4 for each group; (K-L) After knocking out MUC1, CSE stimulation promoted a significant increase in ROS; n = 6 for each group; (M-N) After knocking out MUC1, CSE stimulation promoted a significant decrease in mitochondrial membrane potential (ΔΨm); n = 6 for each group. P < 0.05 was considered statistically significant.

[0024] Figure 3 This shows the effects of MUC1-C on the senescence, differentiation, and protein level of mitochondrial complex I subunit NDUFS4 in human primary alveolar type II epithelial cells (hAT2) in Example 1 of the present invention, where: (A-B) Western blot analysis showed that after silencing MUC1-C, cigarette smoke extract (CSE) stimulation significantly upregulated the protein level of the senescence marker P21 in hAT2 cells; n = 3 for each group; (C) β-galactosidase (SA-β-gal) activity staining showed that the SA-β-gal activity (blue particles) was significantly increased in the MUC1-C silencing group; n = 3 for each group, scale bar: 250 μm; (D) Morphological analysis showed that silencing MUC1-C led to the inhibition of hAT2 cell differentiation and abnormal differentiation structure; n = 3 for each group, scale bar: 275 μm; (E-F) Western blot analysis showed that CSE exposure inhibited the expression of AT1 differentiation markers AQP5 and PDPN, and overexpression of the MUC1-C plasmid could reverse the downregulation of AQP5 and PDPN; n = 6 for each group; (G-H) Western blot analysis showed that knocking out MUC1 promoted a significant decrease in the protein level of mitochondrial complex I subunit NDUFS4 in hAT2 cells, and overexpression of MUC1-C could completely reverse this phenotype; n = 4 for each group. P < 0.05 was considered statistically significant.

[0025] Figure 4 In Example 1 of the present invention, the effects of specific MUC1 gene knockout (Muc1 CKO mice) and overexpression in mouse AT2 cells (mAT2) on lung function and mitochondrial homeostasis after 6 months of mouse exposure to cigarette smoke (CS) were as follows: (A) Immunofluorescence verified the specific knockout of Muc1 (green) in mAT2 cells (SPC, pink), and the cell nuclei were labeled with DAPI (blue); n = 6 for each group, scale bar 100 μm; (B) Western blot confirmed that Muc1 CKO The Muc1-C protein in the lung tissues of mice was significantly reduced; n = 6 for each group; (C-F) Conditional knockout of the Muc1 gene in mAT2 cells exacerbated the decline in lung function after CS exposure in mice. Including functional residual capacity FRC (C), forced vital capacity FVC (D), inspiratory capacity IC (E), ratio of flow at 50% forced vital capacity to forced vital capacity FEV50 / FVC (F), n = 6 for each group; (G) AAV9-mediated overexpression of mAT2-specific Muc1 (green) (SPC, red), and the cell nuclei were labeled with DAPI (blue); n = 8 for each group, scale bar 50 μm; (H) Western blot showed that Muc1-C was significantly upregulated in the lung tissues of the overexpression group (AAV9-AT2-Muc1 group); n = 4 for each group; (I-L) Specific overexpression of Muc1 in mAT2 effectively improved the decline in lung function after CS exposure in mice, including functional residual capacity FRC (I), forced vital capacity FVC (J), inspiratory capacity IC (K), ratio of flow at 50% forced vital capacity to forced vital capacity FEV50 / FVC (L); n = 6 for each group; (M-N) Specific overexpression of Muc1 targeting mAT2 inhibited the aggregation of inflammatory cells in COPD mice to a certain extent; n = 8 for each group, scale bar: 50 μm; (O) Transmission electron microscopy analysis showed that specific overexpression of Muc1 targeting mAT2 significantly inhibited mitochondrial damage and morphological abnormalities caused by CS exposure in mice; n = 3 for each group, scale bar: upper figure 5 μm, lower Figure 1 μm; P < 0.05 was considered statistically significant. Detailed implementation manners

[0026] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all belong to the scope of the present invention. The reagents and instruments used in the following examples can all be obtained commercially, and the methods used in the examples are the same as the commonly used methods unless otherwise specified.

[0027] The technical solutions of the present invention will be further elaborated in detail below in conjunction with the examples.

[0028] Example 1

[0029] 1 Experimental methods

[0030] 1.1 Preparation of cigarette smoke extract (CSE)

[0031] CSE was prepared by the principle of negative pressure suction filtration. 10 ml of RPMI1640 basal medium was placed in 2 bubble collection tubes, and 2 Hongmei brand cigarettes were placed in the smoke generator. The smoke generated by the cigarettes was passed through the medium in the bubble collection tubes by means of vacuum negative pressure suction filtration. The obtained solution was CSE. The pH value was adjusted to 7.0, and it was used within 30 minutes after filtration and sterilization with 0.22 μm filter. The concentration of this CSE was defined as 100%.

[0032] 1.2 Establishment of cell models stimulated by cigarette smoke extract (CSE)

[0033] The in vitro experiments involved in this study were carried out using A549 human non-small cell lung cancer cell line and hAT2 human alveolar type II epithelial cells. The A549 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences, and hAT2 (No. TO482) was purchased from the Chinese agent of ABM Company (Applied Biological Materials Inc.), Zhenjiang Aibimeng Biotechnology Co., Ltd.

[0034] A549 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) and human primary alveolar type II epithelial cells hAT2 cells (purchased from ABM Company [Applied Biological Materials Inc.], the Chinese agent is Zhenjiang Aibimeng Biotechnology Co., Ltd., product number TO482) were routinely resuscitated and cultured in DMEM (GIBCO) medium containing 10% high-quality fetal bovine serum (GIBCO), and placed in a cell culture incubator containing 5% CO2 at 37 °C for culture. When the cell density reached about 80%, subculture was carried out, and the cell morphology was observed at any time. A549 cells and mAT2 were stimulated with different concentration gradients of 1% - 3% CSE to establish an in vitro cell model of COPD.

[0035] 1.3 Mouse model of chronic obstructive pulmonary disease

[0036] Wild-type C57BL / 6J male mice with chronic obstructive pulmonary disease (purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd.) were raised in a specific pathogen-free facility. All experimental protocols were approved by the Animal Protection and Use Committee of Guangzhou Medical University. All methods were carried out in accordance with the guidelines and regulations approved by the Animal Protection and Use Committee of Guangzhou Medical University. The cigarettes used to establish the mouse model of chronic obstructive pulmonary disease were Meizi brand filtered cigarettes (China Tobacco Guangdong Industrial Corporation), and each cigarette contained 11 mg of tar, 1.0 mg of nicotine and 13 mg of carbon monoxide. 6 - 8-week-old Muc1 - / -Mice and their wild-type littermates were exposed to CS using a whole-body exposure system. Briefly, the mice were exposed to CS (9 cigarettes / h, 2 h / session, 2 sessions / day, 6 days / week) for 24 weeks. The control group was only exposed to filtered room air (RA). After the experiment, all mice were subjected to pulmonary function tests and sacrificed.

[0037] 1.4 Conditional knockout of mAT2 (Muc1 CKO ) mouse construction

[0038] Conditional knockout of mAT2 (Muc1 CKO ) mice were purchased from Cyagen Biosciences (Guangzhou). Mouse Muc1 sequence information was obtained from databases such as ENSEMBL, UCSC, and Refseq, and constructed using CRISPR / Cas9 gene knockout technology. By cutting off the Muc1 segment in AT2, mice with selective knockout of the Muc1 segment in mAT2 were constructed, and the successful conditional gene knockout was verified by qRT-PCR and WB.

[0039] 1.5 Bronchoalveolar lavage fluid (BALF) analysis

[0040] 0.6 ml of ice-cold saline was injected into the left lung trachea, 3 times in total. All 1 mL of collected BALF was centrifuged at 4°C for 10 minutes at a mass of 500 g. The cell pellet was resuspended in 1 mL of physiological saline, then stained for differential counting of neutrophils, macrophages, and lymphocytes. In addition, the supernatant was stored at -80°C for cytokine detection.

[0041] 1.6 Measurement of mouse pulmonary function

[0042] Mouse pulmonary function was evaluated using a forced lung movement system (Buxco Research Systems, Wilmington, NC). The core parameters included: functional residual capacity (FRC), forced vital capacity (FVC), inspiratory capacity (IC), and FEV50 / FVC ratio (simultaneously measuring FEV50 and calculating its ratio to FVC). During the experimental procedure, the mice were anesthetized (sodium pentobarbital, 50 mg / kg body weight), tracheally intubated, and then connected to the pulmonary function instrument. The mechanical ventilation parameters were set (respiratory frequency 150 times / min, inhalation / exhalation airflow velocity 0.8 / 0.5 ml / s, pressure limit ±40 cmH2O). The Boyle's law action (measuring FRC), quasi-static pressure-volume action (measuring IC), and rapid flow-volume action (obtaining FVC and FEV50 / FVC ratio) were performed in sequence.

[0043] 1.7 Plasmid construction and transfection

[0044] Construction of AAV9-CMV-hMUC1-C-WPRE overexpression vector: The human recombinant adeno-associated virus vector pDC316-tdTomato (AAV9-hMUC1-C) was purchased from Hanheng Biotechnology (Shanghai) Co., Ltd. Using the hMUC1-C-pcDNA3.1 plasmid as a template, the hMUC1-C gene fragment containing NotI / HindIII restriction sites was amplified by PCR (primer sequences: 5'-GCGGCCGCATGGTGCTGGGC-3' (SEQ ID NO.1), 5'-AAGCTTTCACAGGTGCCCCAG-3' (SEQ ID NO.2)). After separation by 1% agarose gel electrophoresis, the target fragment (1250 bp) was recovered using a Gel Extraction Kit (Qiagen). After double digestion of the PCR product and the shuttle plasmid pDC316-tdTomato with NotI / HindIII (NEB), the recombinant plasmid was constructed by ligation with T4 DNA ligase (Takara) at 16°C for 16 h. The ligation product was transformed into DH5α competent cells (Thermo), and after kanamycin resistance screening and colony PCR verification, the sequence correctness was confirmed by Sanger sequencing (ABI 3730xl). The recombinant plasmid was transfected into HEK293 cells (ATCC CRL-1573) mediated by polyethyleneimine (PEI). After 48 h of transfection, the cell suspension was collected, and after 3 cycles of freezing and thawing at -80°C / 37°C, the virus supernatant was obtained by centrifugation at 12,000×g for 10 min. The virus titer (1.2×10 13 vg / mL) was determined by qPCR (primers targeting the WPRE element). The construction of the mouse AAV9-Muc1-C overexpression vector was carried out according to the above protocol.

[0045] Construction of AAV9-shMUC1-C interference vector: Design the MUC1-C specific shRNA sequence according to the RNAi Consortium (TRC) database: shMUC1-C: 5'-GUUCAGUGCCCAGCUCCUACdTdT-3' (SEQ ID NO.3) (sense strand); Control shRNA: 5'-GUCAGACGUUCGGCGCAUAdTdT-3' (SEQ ID NO.4). Synthesize double-stranded oligonucleotides with AgeI / BamHI sticky ends (Shanghai Sangon), and after denaturation at 95°C for 5 min in annealing buffer (10 mM Tris-HCl, 50 mM NaCl, pH 8.0), gradient cool to 25°C to form a double-stranded structure. Linearize the pDC316-tdTomato-shRNA vector by double digestion with AgeI / BamHI (NEB), and ligate the shRNA insert fragment with the vector backbone using T4 DNA ligase (16°C, 16 h). After the recombinant plasmid is verified by DH5α transformation and Sanger sequencing, use the EndoFree Plasmid Kit (Qiagen) to remove endotoxin. Transfect HEK293 cells with the recombinant plasmid mediated by PEI, and verify the MUC1-C mRNA silencing efficiency by qRT-PCR (TaqMan probe method) 72 h later (82.3 ± 5.1%, n = 3). The construction of mouse AAV9-shMuc1-C was carried out according to the same method.

[0046] 1.8RNA interference

[0047] Cells were seeded in 6-well culture dishes and cultured for 24 h, and transfected with 1.5 μg of siRNA targeting MUC1 mRNA (siMUC1, 5'-GUU CAG UGC CCA GCU CUA C-3' (SEQ ID NO.5)) or non-targeting siRNA (siNC, 5'-GUA GAG CUG GGC ACU GAA C-3' (SEQ ID NO.6)) using Hiperfect transfection reagent (Qiagen, Valencia, CA). Transfection efficiency was detected by Western blot.

[0048] 1.9Western blotting

[0049] Anti-MUC1-C antibody (abcam, ab109185), anti-β-actin antibody (Servicebio, GB15001-100), anti-P21 antibody, anti-PDPN antibody, anti-AQP5 antibody, anti-SFTPC antibody, and anti-NDUFS4 antibody, anti-TOM22 antibody were obtained from ABclonal. Peroxidase-conjugated secondary antibodies were purchased from Sigma-Aldrich (St Louis, MO, USA). The Immun-Star HRP chemiluminescence kit (Bio-Rad, Hercules, CA) was used to detect the signals of the bound antibodies. Western blot images were taken from the Tanon 5200 chemiluminescence imaging system (Shanghai Tanon Science & Technology, Shanghai, China). Image J was used for semi-quantitative analysis of immunoblots.

[0050] 1.10 Histopathology of mouse lung tissue

[0051] Hematoxylin-eosin (HE) staining. The left lungs of mice were fixed with 10% formaldehyde solution for 24 h, then embedded in paraffin, and the paraffin blocks were cut into 4-μm-thick sections. The deparaffinized tissue sections were examined histologically using an HE staining kit.

[0052] 1.11 Flow cytometry

[0053] Cells were seeded in 12-well plates and treated as needed. Thirty minutes before the end of cell treatment, the cells were washed once with PBS, digested with trypsin for 2 minutes, the trypsin was discarded, 500 μl of complete medium was added to each well and transferred to 1.5-ml EP tubes. DCFH-DA (1:1000, C2003S, Beyotime) was added to the cell suspension, and negative and positive controls (ROSup, 1:1000) were set; or JC-1 (1:1000, S0033M, Beyotime) was added to the cell suspension, and negative and positive controls (CCCP, 1:1000) were set. The cells were incubated in a 37 °C cell culture incubator for 20 minutes. The mixture was inverted and mixed every 3-5 minutes. Then, the cells were centrifuged and the cell pellet was washed twice with PBS or JC-1 buffer, and samples were collected using a BD FACSVerse flow cytometer. The gating strategy for all flow cytometry experiments included excluding debris by analyzing FSC-A and SSC-A, and subsequently excluding doublets by analyzing FSC-A and FSC-H. 10,000 cells were obtained for ROS detection in each group, and 50,000 cells were obtained for JC-1 detection in each group. The median mean fluorescence intensity (MFI) of all cells under each condition was calculated using FlowJo v.10.9.0.

[0054] 1.12 Activity staining of cellular β-galactosidase (SA-β-gal)

[0055] Use the cellular β-galactosidase (SA-β-gal) kit (Servicebio, G1073-100T). Seed the cells in a 6-well plate. After the cells are confluent and subjected to the corresponding treatment, add 1 mL of β-galactosidase staining fixation solution and fix at room temperature for 15 min. Prepare the staining working solution according to the kit instructions, incubate overnight at 37 °C, and observe under an ordinary optical microscope.

[0056] 1.13 Immunofluorescence technique

[0057] Paraffin sections of mouse lung tissue are dewaxed with xylene (I, II) in gradient and hydrated step by step with ethanol, then washed with PBS; antigen is repaired by microwave in citrate buffer at pH = 6 (boil at high heat and turn to medium heat to maintain for 20 min), cooled and blocked (5% BSA, 37 °C for 40 min), incubated with the target primary antibody (overnight at 4 °C), horseradish peroxidase (HRP)-labeled secondary antibody (1 h at 37 °C in the dark), and labeled with tyramide signal amplification (TSA) fluorescent dye (10 min at room temperature); for double-label experiments, the antigen repair and primary antibody / secondary antibody / TSA steps need to be repeated (changing to different wavelength fluorescent dyes), and finally the nuclei are stained with DAPI and sealed with an anti-quencher, and imaged with a fluorescence microscope. Cells in confocal dishes are fixed with 4% paraformaldehyde (20 min at room temperature), blocked with 5% BSA, and the incubation with the primary antibody and subsequent steps are the same as those for tissue samples (HRP secondary antibody, TSA labeling, DAPI sealing).

[0058] 1.14 Real-time quantitative PCR

[0059] Total RNA is extracted from cell or tissue samples by the TRIzol method, and the concentration and purity are measured by NanoDrop (A260 / A280 ≥ 1.8), and cDNA is synthesized using a reverse transcription kit; qPCR is performed by the SYBR Green method, and the primers are designed by NCBI Primer-BLAST (IL-6, IL-8, and the internal reference gene GAPDH). The reaction system contains SYBR Premix, primers, and templates. The program is set for pre-denaturation at 95 °C for 30 sec, 40 cycles of amplification (95 °C for 5 sec, 60 °C for 30 sec), and the melting curve is used to verify the specificity; the relative gene expression level is calculated by the ΔΔCt method, and statistical analysis is performed using GraphPad Prism 10.1.

[0060] 1.14 Statistical analysis

[0061] Measurement data conforming to the normal distribution are expressed as (Mean±SD). When the measurement data show a skewed distribution that cannot be corrected, Median(IQR) is used. The t-test is applied for comparing the means between two groups of samples. For multiple groups of samples that conform to the normal distribution and have homogeneous variances, one-way ANOVA is used for comparing the overall means, followed by pairwise comparison using the LSD method. If the samples do not conform to the normal distribution or have heterogeneous variances, the data are transformed (such as sqrt, lg, exp, etc.) and then subjected to normality test and homogeneity of variance test. If they still do not conform to the homogeneity of variance or the normal distribution, non-parametric tests are used. Pearson correlation analysis is performed on measurement data that conform to linearity and the normal distribution, while Spearman's correlation analysis is used for data that do not conform to linearity and the normal distribution. GraphPad Prism 10.1 software is used for statistical analysis, and P<0.05 is considered statistically significant.

[0062] 2 Experimental results

[0063] 2.1 MUC1-C expression was significantly downregulated in elderly COPD patients and the cigarette smoke exposure model

[0064] To investigate the expression of MUC1-C in elderly COPD patients, we analyzed the fluorescence intensity of MUC1-C in the emphysema area of COPD patients by immunofluorescence, stimulated A549 cells with 1%-3% CSE to observe the changes in MUC1-C protein levels, and detected the Muc1-C protein levels in the COPD mouse model under continuous CS exposure for 3 months and 6 months. The results are as Figure 1 shown.

[0065] The specific experimental methods are as follows: Immunofluorescence was used to detect and analyze the changes in MUC1-C fluorescence intensity in lung tissue sections of elderly COPD patients and the normal control group ( Figure 1 A), with n = 8 in each group; Western blot was used to detect the MUC1-C protein level in A549 cells after CSE stimulation ( Figure 1 B- Figure 1 C), with n = 3 in each group; Western blot was used to detect the Muc1-C protein level in the lung tissue of C57 mice after 3 months and 6 months of CS exposure, with n = 6 in each group.

[0066] Immunofluorescence analysis showed that the fluorescence intensity of MUC1-C (green) in the emphysema area of elderly COPD patients was significantly lower than that in the normal control group (Normal) ( Figure 1 A). Western blot analysis showed that after stimulation with cigarette smoke extract (CSE, 1%-3%), the MUC1-C protein level in A549 cells decreased in a concentration-dependent manner ( Figure 1B- Figure 1 C), and compared with the air control group (Air), the protein level of Muc1-C in the lung tissues of C57 mice exposed to cigarette smoke (CS) for 3 months and 6 months was down-regulated ( Figure 1 D- Figure 1 E). In summary, these results indicate that MUC1-C is down-regulated in elderly COPD patients, A549 cell models stimulated by cigarette smoke, and COPD mouse models.

[0067] 2.2 Deletion of MUC1-C exacerbates senescence, inflammation and mitochondrial dysfunction in A549 cells

[0068] To investigate the effects of MUC1-C on the senescence, inflammation level and mitochondrial function of A549 cells, we knocked out MUC1 (sgMUC1) and silenced MUC1-C (shMUC1-C), and stimulated them with cigarette smoke extract (CSE) or cigarette smoke tar phase extract (CSTar), and detected the senescence-related index P21 protein, IL-6 / IL-8, and mitochondrial ROS level and mitochondrial membrane potential level. The results are as Figure 2 shown.

[0069] The specific experimental methods are as follows: In the sgMUC1 knockout model, we used Western blot to detect the protein level of the senescence-related marker P21 in the sgMUC1 group and the control group after CSE stimulation ( Figure 2 A- Figure 2 B), with n = 6 in each group; we used qPCR to detect the mRNA levels of IL-6 and IL-8 ( Figure 2 C- Figure 2 D), with n = 4 in each group; we used immunofluorescence (Ki67 / DAPI double staining) to detect the nuclear localization of Ki67 ( Figure 2 E), with n = 6 in each group; we used DCFH-DA probe combined with JC-1 staining and flow cytometry to detect the mitochondrial ROS level and mitochondrial membrane potential level after CSE stimulation ( Figure 2 K- Figure 2 N), with n = 6 in each group. In the shMUC1-C silencing model: We used Western blot to detect the expression level of P21 protein in the shMUC1-C group and the control group after CSTar stimulation ( Figure 2 F-2H), with n = 6 in each group; we used qPCR to detect the mRNA levels of IL-6 and IL-8 in the shMUC1-C group and the control group under CSE stimulation ( Figure 2 I-J), with n = 4 in each group.

[0070] In the sgMUC1 knockout model, Western blot analysis showed that after knocking out MUC1, compared with the control group, cigarette smoke extract (CSE) stimulation significantly upregulated the protein level of the senescence marker P21 ( Figure 2 A- Figure 2 B). The qPCR results showed that after knocking out MUC1, the mRNA levels of IL-6 and IL-8 were significantly increased regardless of whether CSE stimulation was received ( Figure 2 C- Figure 2 D). Immunofluorescence analysis showed that regardless of whether CSE stimulation was received, MUC1 knockout led to the loss of nuclear localization of Ki67 (green) in A549 cells ( Figure 2 E), suggesting cell proliferation arrest; the results of flow cytometry detection combined with DCFH-DA probe and JC-1 staining showed that CSE stimulation promoted a significant increase in ROS and a significant decrease in mitochondrial membrane potential (ΔΨm) ( Figure 2 K- Figure 2 N). In the shMUC1-C silencing model, Western blot analysis showed that tobacco smoke tar phase extract (CSTar) stimulation significantly increased the protein level of P21 ( Figure 2 F- Figure 2 H). The qPCR results showed that the mRNA levels of IL-6 and IL-8 were significantly increased regardless of whether CSE stimulation was received ( Figure 2 K- Figure 2 L). In summary, MUC1-C deficiency synergistically drives COPD-related pathological phenotypes through activating the senescence pathway, releasing inflammatory factors, and mitochondrial dysfunction.

[0071] 2.3 MUC1-C deletion exacerbates senescence, differentiation inhibition, and mitochondrial dysfunction in human primary alveolar type II epithelial cells (hAT2)

[0072] To investigate the effects of MUC1-C on senescence, differentiation inhibition, and mitochondrial dysfunction in human primary alveolar type II epithelial cells (hAT2), we knocked out MUC1 or silenced MUC1-C, and stimulated with cigarette smoke extract (CSE), and detected the protein level of the senescence marker P21, the activity of β-galactosidase (SA-β-gal), the differentiation of AT2, and the change of the protein level of mitochondrial complex I subunit NDUFS4. We also observed the differentiation of hAT2 under CSE stimulation, and whether overexpressing the MUC1-C plasmid could reverse the differentiation inhibition. The results are as Figure 3 shown.

[0073] The specific experimental methods are as follows: In the shMUC1-C silencing model: We used Western blot to detect the protein level of the senescence marker P21 in hAT2 cells under the stimulation of cigarette smoke extract (CSE)Figure 3 A- Figure 3 B), with n = 3 in each group; we used senescence-associated β-galactosidase (SA-β-gal) activity staining to detect the SA-β-gal activity of hAT2 cells under CSE stimulation ( Figure 3 C), with n = 3 in each group; we used morphological analysis to detect the differentiation of hAT2 cells ( Figure 3 D), with n = 3 in each group. We used Western blot to detect the expression of AQP5 and PDPN, markers of AT1 cell differentiation in hAT2 cells under CSE exposure, and to detect the therapeutic effect of overexpressing MUC1-C on this phenotype ( Figure 3 E- Figure 3 F), with n = 6 in each group. We used Western blot to detect the effect of knocking out MUC1 on the protein level of NDUFS4, a subunit of mitochondrial complex I in hAT2 cells, and to detect the therapeutic effect of overexpressing MUC1-C on this phenotype ( Figure 3 G- Figure 3 H), with n = 4 in each group.

[0074] In the shMUC1-C silencing model, Western blot analysis showed that after silencing MUC1-C, cigarette smoke extract (CSE) stimulation significantly upregulated the protein level of the senescence marker P21 in hAT2 cells ( Figure 3 A- Figure 3 B); the SA-β-gal activity increased significantly ( Figure 3 C); morphological analysis showed inhibition of hAT2 cell differentiation and abnormal differentiation structure ( Figure 3 D). Western blot analysis showed that CSE exposure inhibited the expression of AQP5 and PDPN, markers of AT1 differentiation, and overexpression of the MUC1-C plasmid could reverse the downregulation of AQP5 and PDPN ( Figure 3 E- Figure 3 F); knocking out MUC1 promoted a significant decrease in the protein level of NDUFS4, a subunit of mitochondrial complex I in hAT2 cells, and overexpression of MUC1-C could completely reverse this phenotype ( Figure 3 G- Figure 3 H). In summary, MUC1-C deficiency exacerbated the senescence level, differentiation inhibition, and mitochondrial dysfunction of hAT2 cells, and overexpression of MUC1-C could reverse the differentiation inhibition of hAT2 cells and the decrease in the NDUFS4 protein level.

[0075] 2.4 Overexpression of Muc1-C can reverse the decline in lung function and mitochondrial damage in mice after CS exposure

[0076] To understand the effects of conditional knockout and overexpression of MUC1 gene in mouse AT2 cells (mAT2) on lung function and mitochondrial homeostasis after 6 months of exposure to cigarette smoke (CS) in mice, we constructed a mouse model with conditional knockout of Muc1 gene in mAT2 cells (Muc1 CKO mice). After 6 months of CS exposure, compared with the control group, the changes in several lung function parameters, including FRC, FVC, IC, and FEV50 / FVC, were detected in mice. Subsequently, Muc1 was specifically overexpressed in mAT2 of the mouse model and control mice after 6 months of CS exposure, the changes in the above-mentioned lung function parameters were detected, and the morphological changes of mitochondria were observed by transmission electron microscopy. The results are as Figure 4 shown.

[0077] The specific experimental methods are as follows: Muc1 + / + and Muc1 CKO mice were continuously exposed to CS for 6 months. We used immunofluorescence and Western blot to detect the protein level of Muc1 in lung tissues ( Figure 4 A-4B), with n = 6 in each group. We used a forced lung operating system to measure lung function parameters ( Figure 4 C- Figure 4 F), including FRC (C), FVC (D), IC (E), and FEV50 / FVC (F) values, with n = 6 in each group. At 3 months of CS exposure, AAV9-mSFTPC-promoter-Muc1 was instilled into the airways. After 6 months of CS exposure, samples were taken. We used immunofluorescence and Western blot to detect the protein level of Muc1 in lung tissues ( Figure 4 G-4H), with n = 6 in each group; a forced lung operating system was used to measure lung function parameters ( Figure 4 I- Figure 4 L), including FRC (I), FVC (J), IC (K), and FEV50 / FVC (L) values, with n = 6 in each group; HE staining was used to analyze the level of inflammatory cells in bronchoalveolar lavage fluid ( Figure 4 M- Figure 4 N), with n = 8 in each group; finally, transmission electron microscopy was used to observe the number and morphology of mitochondria ( Figure 4 O), with n = 3 in each group.

[0078] Immunofluorescence and Western blot analysis confirmed that the Muc1-C protein in the lung tissues of Muc1 CKO mice was significantly reduced ( Figure 4 A-4B). Lung function test analysis showed that conditional knockout of Muc1 gene in mAT2 cells exacerbated the decline in lung function in mice after CS exposure ( Figure 4 C- Figure 4F), including FRC (C), FVC (D), IC (E), FEV50 / FVC (F). After airway instillation of AAV9-mSFTPC-promoter-Muc1, immunofluorescence and Western blot were used to confirm that Muc1-C was significantly upregulated in the lung tissue of the overexpression group (AAV9-AT2-Muc1 group) Figure 4 G-4H). Pulmonary function analysis showed that specific overexpression of Muc1 in mAT2 effectively improved the decline in pulmonary function in 12-month-old mice after CS exposure Figure 4 I- Figure 4 L), including FRC (I), FVC (J), IC (K), FEV50 / FVC (L). Targeted specific overexpression of Muc1 in mAT2 inhibited the aggregation of inflammatory cells in COPD mice to a certain extent Figure 4 M- Figure 4 N). Transmission electron microscopy analysis showed that targeted specific overexpression of Muc1 in mAT2 significantly inhibited the mitochondrial damage and morphological abnormalities caused by CS exposure in mice Figure 4 O). In summary, knockout of Muc1-C would further exacerbate the decline in pulmonary function in COPD group mice, while overexpression of Muc1-C could reverse the decline in pulmonary function and mitochondrial damage in mice after CS exposure.

[0079] Note: Since in the NIPOST.26 version, "T" represents uracil in the RNA sequence and thymine in the DNA sequence, so "U" in the above sequences was converted to "T" during production.

[0080] The embodiments described above are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of the MUC1-C segment or its encoding gene in the preparation of products related to chronic obstructive pulmonary disease, characterized in that, The chronic obstructive pulmonary disease-related products include products related to chronic obstructive pulmonary disease research, diagnosis, prevention, and / or treatment.

2. The application according to claim 1, wherein The products include drugs, reagents, and kits.

3. The application according to claim 2, characterized in that, The chronic obstructive pulmonary disease-related research products include products that up-regulate and down-regulate the expression level of the C-terminal of MUC1 or its coding gene.

4. The application according to claim 3, characterized in that The chronic obstructive pulmonary disease-related research includes the construction of chronic obstructive pulmonary disease models.

5. The application according to claim 2, characterized in that, The diagnosis includes screening tests for the presence / absence, high / low risk, and different stages of chronic obstructive pulmonary disease in the population.

6. The application according to claim 5, characterized in that The chronic obstructive pulmonary disease diagnostic products include products that detect the expression level of the C-terminal of MUC1 or its coding gene.

7. The application according to claim 2, characterized in that The chronic obstructive pulmonary disease prevention and / or treatment products include products that overexpress the C-terminal of MUC1 or its coding gene.

8. A product for research related to chronic obstructive pulmonary disease, characterized in that, The products include the products that up-regulate and down-regulate the expression level of the C-terminal of MUC1 or its coding gene in claim 3.

9. A product for diagnosing chronic obstructive pulmonary disease, characterized in that, The products include the products that detect the expression level of the C-terminal of MUC1 or its coding gene in claim 6.

10. A product for preventing and / or treating chronic obstructive pulmonary disease, characterized in that, The products include the products that overexpress the C-terminal of MUC1 or its coding gene in claim 7.

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