Use of miR-30a-5p in preparation of product for treating airway stenosis

Abstract

The application discloses application of miR-30a-5p in preparation of a product for treating airway stenosis. The application finds that miR-30a-5p plays an important regulatory role between airway basal stem cells and airway fibroblasts, and through the carrying of extracellular vesicles, miR-30a-5p is transmitted from the airway basal stem cells to the airway fibroblasts, targets and regulates the expression of a key gene FAP, inhibits the activation of the airway fibroblasts, reduces the collagen gel contraction ability of the airway fibroblasts, improves the extracellular matrix remodeling of the airway fibroblasts, can effectively stabilize the tracheal granulation tissue, prevents the recurrence of airway stenosis, and has application value for treating airway stenosis.

Description

[0001] The present application relates to the field of biotechnology, in particular, to the application of miR-30a-5p in the preparation of products for treating airway stenosis. BACKGROUND

[0002] Airway stenosis is a common respiratory disease that can be caused by various factors such as inflammation, tumors, trauma or congenital abnormalities, which seriously affects the respiratory function and quality of life of patients. Traditional surgical treatment is suitable for severe stenosis cases, with high risk and the possibility of recurrence. Drug treatment such as bronchodilators and steroid hormones can alleviate the symptoms of airway stenosis in the short term, but the long-term effect is limited and is accompanied by side effects. New treatment methods for airway stenosis need to be explored.

[0003] It is currently believed that airway stenosis includes three overlapping development stages: inflammatory stage, proliferative stage and mature stage. Initially, airway mucosa damage caused by infection or non-infection factors leads to the release of inflammatory mediators; then, airway fibroblasts (AFs) are activated and proliferate, forming fresh granulation tissue; finally, airway remodeling occurs, with collagen deposition forming scar stenosis. AFs are located in the airway wall and are usually in a resting state, but when stimulated by inflammatory factors or other stimuli, they are activated and rapidly transform into myofibroblasts with higher activity and higher contractility. In addition, AFs can synthesize and secrete extracellular matrix proteins such as collagen and elastin, which are essential for maintaining the integrity of the airway structure. After airway injury, fibroblasts migrate to the damaged area and participate in the repair process. However, abnormal activation of fibroblasts can lead to airway fibrosis, which is an important factor in airway stenosis. Airway stenosis can be caused by chronic inflammation, infection or excessive activity of fibroblasts after injury. This excessive fibrosis process leads to thickening and hardening of the airway wall, ultimately causing airway stenosis, which manifests as symptoms such as difficulty breathing.

[0004] MiRNAs are small non-coding RNAs with a length of about 20-25 nucleotides that inhibit gene expression at the post-transcriptional level by complementary pairing with target mRNAs. Previous studies have found that changes in miRNA expression are closely related to physiological processes such as proliferation, differentiation, apoptosis and ECM synthesis. However, there have been few reports on the functional mechanisms of miRNAs in airway fibroblasts. SUMMARY

[0005] To solve the above problems in the prior art, the present application provides the application of miR-30a-5p in the preparation of products for treating airway stenosis.

[0006] The first object of the present application is to provide the application of miR-30a-5p and / or its mimics in the preparation of products for treating airway stenosis.

[0007] A second object of the present application is to provide the use of miR-30a-5p and / or its mimics in the preparation of a product for inhibiting the activation of airway fibroblasts.

[0008] A third object of the present application is to provide the use of miR-30a-5p and / or its mimics in the preparation of a product for reducing the collagen gel contraction ability of airway fibroblasts.

[0009] A fourth object of the present application is to provide the use of miR-30a-5p and / or its mimics in the preparation of a product for improving the extracellular matrix remodeling of airway fibroblasts.

[0010] A fifth object of the present application is to provide the use of miR-30a-5p and / or its mimics in the preparation of an agent for inhibiting the expression of FAP gene and / or FAP protein.

[0011] A sixth object of the present application is to provide the use of an agent for inhibiting the expression of FAP gene and / or FAP protein in the preparation of a product for treating airway stenosis.

[0012] A seventh object of the present application is to provide the use of an agent for inhibiting the expression of FAP gene and / or FAP protein in the preparation of a product for any one or several of inhibiting the activation of airway fibroblasts, reducing the collagen gel contraction ability of airway fibroblasts, or improving the extracellular matrix remodeling of airway fibroblasts.

[0013] An eighth object of the present application is to provide the use of extracellular vesicles comprising miR-30a-5p and / or its mimics in the preparation of a product for treating airway stenosis.

[0014] A ninth object of the present application is to provide the use of extracellular vesicles comprising miR-30a-5p and / or its mimics in the preparation of a product for any one or several of inhibiting the activation of airway fibroblasts, reducing the collagen gel contraction ability of airway fibroblasts, or improving the extracellular matrix remodeling of airway fibroblasts.

[0015] To achieve the above objects, the present application is implemented by the following scheme:

[0016] The present application discovers that miR-30a-5p is highly enriched in airway basal cell-derived extracellular vesicles, which is delivered to airway fibroblasts by extracellular vesicles to play multiple regulatory roles, inhibits the activation of airway fibroblasts and improves extracellular matrix remodeling, and is an important bioactive molecule for treating airway stenosis.

[0017] Therefore, the present application claims the following:

[0018] Use of miR-30a-5p and / or its mimics in the preparation of a product for treating airway stenosis.

[0019] Use of miR-30a-5p and / or its mimics in the preparation of a product for inhibiting airway fibroblast activation.

[0020] Use of miR-30a-5p and / or its mimics in the preparation of a product for reducing collagen gel contraction ability of airway fibroblasts.

[0021] Use of miR-30a-5p and / or its mimics in the preparation of a product for improving extracellular matrix remodeling of airway fibroblasts.

[0022] Use of miR-30a-5p and / or its mimics in the preparation of an agent for inhibiting FAP gene and / or FAP protein expression.

[0023] The present application also finds that miR-30a-5p targets and regulates the transcription and protein expression of FAP gene. Therefore, the present application also claims the following:

[0024] Use of an agent for inhibiting FAP gene and / or FAP protein expression in the preparation of a product for treating airway stenosis, the agent for inhibiting FAP gene and / or FAP protein expression comprising miR-30a-5p and / or its mimics.

[0025] Use of an agent for inhibiting FAP gene and / or FAP protein expression in the preparation of a product for any one or several of inhibiting airway fibroblast activation, reducing collagen gel contraction ability of airway fibroblasts, or improving extracellular matrix remodeling of airway fibroblasts, the agent for inhibiting FAP gene and / or FAP protein expression comprising miR-30a-5p and / or its mimics.

[0026] Use of extracellular vesicles comprising miR-30a-5p and / or its mimics in the preparation of a product for treating airway stenosis.

[0027] Use of extracellular vesicles comprising miR-30a-5p and / or its mimics in the preparation of a product for any one or several of inhibiting airway fibroblast activation, reducing collagen gel contraction ability of airway fibroblasts, or improving extracellular matrix remodeling of airway fibroblasts.

[0028] As a preferred solution, the nucleotide sequence of miR-30a-5p is shown in SEQ ID NO: 4.

[0029] As a preferred solution, the nucleotide sequence of the miR-30a-5p mimics is shown in SEQ ID NO: 7-8.

[0030] As a preferred solution, the extracellular vesicle is an airway basal cell-derived extracellular vesicle.

[0031] As a more preferred solution, the preparation method of the extracellular vesicle comprises the following steps: collecting a cell supernatant of cultured airway basal stem cells, and isolating the extracellular vesicle.

[0032] As a preferred solution, the airway stenosis is a benign airway stenosis.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] The present application finds that miR-30a-5p plays an important regulatory role between airway basal stem cells and airway fibroblasts, and through the carrying of extracellular vesicles, miR-30a-5p is delivered from airway basal stem cells to airway fibroblasts, targets and regulates the expression of a key gene FAP, inhibits the activation of airway fibroblasts, reduces the collagen gel contraction ability of airway fibroblasts, improves the extracellular matrix remodeling of airway fibroblasts, can effectively stabilize the tracheal granulation tissue, prevent the recurrence of airway stenosis, and has application value for treating airway stenosis. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 HE and fluorescence in situ hybridization were used to detect the localization of miR-30a-5p in human airway tissues, Normal is a normal airway tissue sample, and BTS is a stenotic airway tissue sample.

[0036] Figure 2 BSC-EVs identification results; A is the TEM identification result; B is the NanoFCM identification result; C is the WB identification result, BSC represents the total protein sample of BSCs, and N1-N3 represents the total protein sample of three parallel prepared BSC-EVs.

[0037] Figure 3 RT-qPCR was used to detect the expression of miR-30a-5p in airway fibroblasts of BTS treated with PBS or BSC-EVs, *p<0.05.

[0038] Figure 4 RT-qPCR was used to detect the transfection effect of miR-30a-5p mimics and inhibitors, ***p<0.001.

[0039] Figure 5 The relative luciferase activity analysis results of the control group, the FAP 3'UTR WT group and the FAP 3'UTR MUT group, **p<0.01, ns represents p≥0.05.

[0040] Figure 6The results of immunofluorescence identification of primary airway fibroblasts.

[0041] Figure 7 The flow chart of treatment for BSC-EVs+NC inhibitors group and BSC-EVs+miR-30a-5p inhibitors group.

[0042] Figure 8 The results of FAP cell immunofluorescence detection and collagen contraction experiment of airway fibroblasts in BSC-EVs group, Blank group, BSC-EVs+NC inhibitors group and BSC-EVs+miR-30a-5p inhibitors group; A is the fluorescence staining image and the gel image; B is the statistical result of FAP positive cell rate in A; C is the statistical result of gel area in A.

[0043] Figure 9 The schematic diagram of constructing the co-culture system of airway basal stem cells (BSCs) and airway fibroblasts (AFs).

[0044] Figure 10 The expression amount of miR-30a-5p in airway fibroblasts in NC inhibitors+DMSO group, NC inhibitors+GW4869 group, miR-30a-5p inhibitors+DMSO group and miR-30a-5p inhibitors+GW4869 group detected by qRT-PCR method, *p<0.05, ns represents p≥0.05.

[0045] Figure 11 The relative expression amount of FAP mRNA in airway fibroblasts in NC inhibitors+DMSO group, NC inhibitors+GW4869 group, miR-30a-5p inhibitors+DMSO group and miR-30a-5p inhibitors+GW4869 group detected by qRT-PCR method, *p<0.05, ns represents p≥0.05.

[0046] Figure 12 The results of FAP cell immunofluorescence detection and collagen contraction experiment of airway fibroblasts in NC inhibitors+DMSO group, NC inhibitors+GW4869 group, miR-30a-5p inhibitors+DMSO group and miR-30a-5p inhibitors+GW4869 group.

[0047] Figure 13For qRT-PCR detection of the transcription level of related genes in airway fibroblasts of NC inhibitors+DMSO group, NC inhibitors+GW4869 group, miR-30a-5p inhibitors+DMSO group and miR-30a-5p inhibitors+GW4869 group, A~F are MMP1, MMP2, MMP3, MMP7, MMP9 and COL1A2 in turn, **p<0.01, ***p<0.001, ns represents p≥0.05.

[0048] Figure 14 For WB detection of the protein level of cell activation related proteins (FAP, α-SMA and Collagen I) and extracellular matrix remodeling related proteins (MMP2, MMP7, MMP9, TIMP1, TIMP2 and TIMP3) in airway fibroblasts of NC inhibitors+DMSO group, NC inhibitors+GW4869 group, miR-30a-5p inhibitors+DMSO group and miR-30a-5p inhibitors+GW4869 group.

[0049] Figure 15 Survival analysis results of BSC-EVs intervention on benign airway stenosis animal model.

[0050] Figure 16 Therapeutic effect evaluation of BSC-EVs intervention on benign airway stenosis animal model; from left to right are the results of bronchoscopic performance, HE staining and MASSON staining.

[0051] Figure 17 WB detection results of BSC-EVs intervention on benign airway stenosis animal model, A is the WB banding chart, and B is the protein expression amount statistics results of Collagen I, FAP, MMP2, MMP3, MMP9, TIMP1 and TIMP2. DETAILED DESCRIPTION

[0052] The application will be further described below in conjunction with the drawings and specific embodiments of the present application, which are only used to explain the present application and are not used to limit the scope of the present application. In the following examples, the test methods used are conventional methods unless otherwise specified; and the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0053] Example 1 Positioning and expression of miR-30a-5p in human normal airway and stenosis airway epithelial tissues

[0054] 1. Preparation of airway tissue paraffin sections

[0055] After the informed consent of the subjects, human normal airway tissue samples (Normal) and stenosis airway tissue samples (BTS) were collected from the clinic, embedded with paraffin, sliced, and paraffin sections of normal airway tissue samples (referred to as normal tissue sections) and paraffin sections of stenosis airway tissue samples (referred to as stenosis tissue sections) were obtained.

[0056] 2. Dewaxing and hydration

[0057] The normal tissue sections and stenosis tissue sections were placed in a 65°C oven for 2h, and then taken out while hot, and dewaxed and hydrated according to the following conditions: TO, 10min x 2 times; 100% ethanol, 5min x 2 times; 95% ethanol, 5min x 1 time; 80% ethanol, 5min x 1 time; 75% ethanol, 5min x 1 time; pure water, 5min x 2 times.

[0058] 2. HE staining

[0059] The normal tissue sections and stenosis tissue sections after dewaxing and hydration were subjected to HE staining, and the specific operation steps were as follows: first, the tissue sections were placed in a hematoxylin staining solution (Saville, China) for 5min; the stained tissue sections were rinsed with running water for 5min; the rinsed tissue sections were placed in a hematoxylin differentiation solution (Saville, China) for 5 seconds to selectively decolorize the over-stained nuclei and tissue components that should not be colored; the differentiated tissue sections were rinsed with running water for 1min, then placed in a bluing solution (Saville, China) for 5 seconds and rinsed with running water for 1min again; the sections were placed in an eosin (alcohol solution) staining solution (Saville, China) for 5min; the sections were rinsed with running water for 5min.

[0060] 3. Fluorescence in situ hybridization (FISH)

[0061] The normal tissue sections and stenosis tissue sections after dewaxing and hydration were subjected to digoxin fluorescence in situ hybridization, and the specific operation steps were as follows:

[0062] (1) Digestion: according to the length of time of tissue fixation, the tissue sections were boiled in a repair solution for 10min and naturally cooled; then a pen was used to circle the tissue sections, and according to the characteristics of different tissues and different indicators, proteinase K (20ug / ml) was added to the tissue sections and digested at 37°C for 30min; then the tissue sections were rinsed with pure water and then washed with PBS for 3 times, 5min each time.

[0063] (2) Blocking endogenous peroxidase: 3% methanol-H2O2 was added to the tissue sections, and the slides were incubated at room temperature in the dark for 15min, then placed in PBS (PH 7.4) and shaken on a decolorizing shaker for 3 times, 5min each time.

[0064] (3) Pre-hybridization: pre-hybridization solution was added to the tissue sections, and the slides were incubated at 37°C for 1h.

[0065] (4) Hybridization: The pre-hybridization solution was poured off, and the miR-30a-5p probe hybridization solution was added to the tissue section, and hybridized overnight. The probe sequence was: miR-30a-5p: 5'-CTTCCAGTCGAGGATGTTTACA-3' (SEQ ID NO: 1).

[0066] (5) Post-hybridization washing: The hybridization solution was washed off, and the tissue section was washed with 2xSSC at 37°C for 10 min; then washed with lxSSC at 37°C for 2 times, 5 min each time; finally washed with 0.5xSSC at room temperature for 10 min.

[0067] (6) Blocking: Normal rabbit serum was added to the tissue section, and blocked at room temperature for 30 min.

[0068] (7) Adding anti-DIG-HRP: The blocking solution was poured off, and anti-DIG-HRP was added to the tissue section; incubated at 37°C for 50 min; washed with PBS for 3 times, 5 min each time.

[0069] (8) Adding FITC-TSA: FITC-TSA reagent was added to the tissue section, and reacted in the dark at room temperature for 5 min; washed with TBST for 3 times, 10 min each time; washed with PBS for 5 min.

[0070] (9) DAPI re-staining of the nucleus and mounting: DAPI staining solution was added to the tissue section, and incubated in the dark for 8 min, and then washed and mounted with anti-fluorescence quenching mounting medium.

[0071] 4. Results analysis

[0072] The results of HE staining and FISH staining are shown in Figure 1 , and miR-30a-5p is more widely expressed in normal airway epithelial tissue than in narrow airway tissue.

[0073] Example 2 High expression of miR-30a-5p in an animal model treated with extracellular vesicles of normal human-derived airway basal stem cells

[0074] 1. Sample collection and sequencing

[0075] Six cases of airway basal stem cells (BSCs) derived from healthy people were collected for culture, and extracellular vesicles in the cell culture supernatant were separated by ultracentrifugation, and were recorded as BSC-EVs. After identification, Figure 2BSC-EVs showed typical cup-shaped vesicle structure with an average diameter of about 70 nm, and expressed exosome marker proteins CD9, CD63, CD81, HSP70 and TSG101. Transcriptome sequencing analysis (Guangzhou Kiddo) of BSC-EVs showed that miR-30a-5p was significantly overexpressed in BSC-EVs.

[0076] 2. Construction of a rabbit model of benign airway stenosis (BTS)

[0077] New Zealand white rabbits (male or female, body weight about 3 ± 0.2 kg) were anesthetized and sprayed with lidocaine at the epiglottis. A hard nylon brush (0.2 mm in diameter, 6 mm in outer diameter, 2 cm in length with bristles) was inserted into the tracheal tube, and the brush head was extended outside the tracheal tube. The brush was rotated rapidly to scrape the inner wall of the trachea 10 times to cause airway injury. The brush was then removed, and the injury was observed immediately under a bronchoscope. After the operation, adrenaline was administered to stop bleeding. A BTS was thus constructed. The day on which the above operation was completed was recorded as the first day of modeling.

[0078] 3. Treatment of BTS

[0079] On the 3rd and 5th days of modeling, the BTS was given airway secretion and necrosis removal treatment. On the 7th day of modeling, fresh granulation tissue was observed in the airway of the BTS under a bronchoscope. At this time, 100 μL of a suspension containing 6 × 10 10 After the suspension of particle BSC-EVs was resuspended in 500 μL of normal saline, 600 μL of BSC-EVs injection solution was obtained. The solution was injected into the granulation tissue on the inner wall of the airway of the BTS under a bronchoscope in three doses (i.e., 200 μL each time), with a total of three injection sites. This was recorded as the BSC-EVs treatment group (i.e., the BSC-EVs group). The BTS injected with the same volume of normal saline solution under a bronchoscope was used as the control group (i.e., the PBS group). The two groups of rabbits were then continuously raised until the 42nd day of modeling.

[0080] 4. Real-time fluorescent quantitative PCR (RT-qPCR)

[0081] After the experiment, the airway tissue miRNA of the PBS group and the BSC-EVs group was extracted using a cell / tissue miRNA extraction kit (Yixing, China). Reverse transcription was performed using a tailing method kit (miRNA first strand cDNA synthesis reagent, Shenguo, China). RT-qPCR was then performed using a MicroRNas qPCR kit (Shenguo, China) to detect the relative expression of miR-30a-5p in the two groups of cells. The relative expression of miRNA was standardized using U6, and 2 -ΔΔCtMethod calculation. The sequence of the upstream detection primer of U6 was 5'-CGCTTCGGCAGCACATATAC-3'(SEQ ID NO: 2), and the sequence of the upstream detection primer of hsa-miR-30a-5p was 5'-CGCTGTAAACATCCTCGACTGGAAG-3'(SEQ ID NO: 3). The downstream detection primers of U6 and hsa-miR-30a-5p were the tailing method universal downstream primers provided in the kit.

[0082] 5. Result analysis

[0083] As shown in Figure 3 compared with the PBS group, the expression of miR-30a-5p in the BSC-EVs group was significantly up-regulated. It is proved that the BSC-EVs rich in miR-30a-5p can deliver miR-30a-5p to the airway tissues of animals, causing high expression of miR-30a-5p in the airway tissue samples of BTS after treatment.

[0084] Example 3 Preparation of miR-30a-5p mimics and inhibitors

[0085] 1. Design and synthesis of miR-30a-5p mimics and inhibitors

[0086] According to the nucleotide sequence of human miR-30a-5p (5'-UGUAAACAUCCUCGACUGGAAG-3'(SEQ ID NO: 4)), NC mimics (Sense: 5'-UUCUCCGAACGUGUCACGUTT-3'(SEQ ID NO: 5), Antisense: 5'-ACGUGACACGUUCGGAGAATT(SEQ ID NO: 6)), hsa-miR-30a-5p mimics (Sense: 5'-UGUAAACAUCCUCGACUGGAAG-3'(SEQ ID NO: 7), Antisense: 5'-UCCAGUCGAGGAUGUUUACAUU-3'(SEQ ID NO: 8)), NC inhibitors (5'-CAGUACUUUUGUGUAGUACAA-3'(SEQ ID NO: 9)) and hsa-miR-30a-5p inhibitors (5'-CUUCCAGUCGAGGAUGUUUACA-3'(SEQ ID NO: 10)) were designed and synthesized.

[0087] Explanation of SEQ ID NO: 1-10 in the Sequence Listing of the specification: According to the editing rules of WIPO Sequence software, the nucleotide sequence must only contain the symbols listed in "WIPO ST.26 Annex I Part 1", and the base "t" in the RNA sequence is "u", so the SEQ ID NO: 1-10 in the specification of the present application is substantially the same as the SEQ ID NO: 1-10 in the sequence listing.

[0088] 2. Transfection effect identification

[0089] According to the method of Example 1, BSCs from healthy people were obtained, and when the BSCs grew to 70% confluence, NC mimics, hsa-miR-30a-5p mimics, NC inhibitors and hsa-miR-30a-5p inhibitors were transfected into BSCs respectively using lipo 3000 kit. After 24 hours of transfection, the relative expression amount of miR-30a-5p in BSCs was detected according to the RT-qPCR method in Example 1.

[0090] As shown in Figure 4 compared with BSCs transfected with NC mimics, the expression of miR-30a-5p in BSCs transfected with miR-30a-5p mimics was significantly increased; compared with BSCs transfected with NC inhibitors, the expression of miR-30a-5p in BSCs transfected with miR-30a-5p inhibitors was significantly reduced. It is shown that transfection of hsa-miR-30a-5p mimics successfully overexpresses miR-30a-5p, and transfection of hsa-miR-30a-5p inhibitors successfully inhibits the expression of miR-30a-5p.

[0091] Example 4 miR-30a-5p targets and regulates airway fibroblast activation protein (FAP)

[0092] 1. Construction of dual luciferase reporter vector

[0093] pmirGLO Dual-Luciferase miRNA Target Expression Vector (referred to as pmirGLO Vector) expresses both Firefly Luciferase and Renilla Luciferase, and the miRNA target gene is constructed into the 3' end of the Firefly Luciferase gene. The effective miRNA sequence binds to the seed region of the 3'-UTR of the target gene through incomplete base complementation, thereby causing the mRNA of the target gene to be degraded or directly inhibiting the protein expression and translation process, and the detection is the weakening of the Firefly Luciferase substrate fluorescence.

[0094] pmirGLO Vector as a skeleton carrier, the 3'-UTR sequence of the FAP gene (5'-ttttattttttatgttgtaaaatgctagtataaacaaacaaattaatgttgttctaaaggctgttaaaaaaaagatgaggactcagaagttcaagctaaatatTGTTTACAttttctggtactctgtgaaagaagagaaaagggagtcatgcattttgctttggacacagtgttttatcacctgttcatttgaagaaaaataataaa-3' (SEQ ID NO: 11)) was connected to the pmirGLO Vector to construct the h-FAP-miR-30a-5p-wt vector; the 3'-UTR mutant sequence of the FAP gene (5'-ttttattttttatgttgtaaaatgctagtataaacaaacaaattaatgttgttctaaaggctgttaaaaaaaagatgaggactcagaagttcaagctaaatatACAAATGTttttctggtactctgtgaaagaagagaaaagggagtcatgcattttgctttggacacagtgttttatcacctgttcatttgaagaaaaataataaa-3' (SEQ ID NO: 12)) was connected to the pmirGLO Vector to construct the h-FAP-miR-30a-5p-mut vector.

[0095] 2. Cell transfection

[0096] 293T cells were cultured, and when the cells were in good growth condition and the growth density reached 70%, lipo 3000 reagent kit was used for transient transfection. The NC mimics or hsa-miR-30a-5p mimics in Example 3 were co-transfected into 293T cells with pmirGLO Vector, and the obtained cells were recorded as the control group. The NC mimics or hsa-miR-30a-5p mimics were co-transfected into 293T cells with h-FAP-miR-30a-5p-wt vector, and the obtained cells were recorded as the FAP 3'UTR WT group. The NC mimics or hsa-miR-30a-5p mimics were co-transfected into 293T cells with h-FAP-miR-30a-5p-mut vector, and the obtained cells were recorded as the FAP 3'UTR MUT group.

[0097] 3. Detection analysis

[0098] The transfected cells were detected according to the dual luciferase reporter gene detection kit (Yixing, China).

[0099] As shown in Figure 5 , miR-30a-5p can down-regulate the relative luciferase activity of h-FAP-miR-30a-5p-wt vector, but has no effect on the relative luciferase activity of h-FAP-miR-30a-5p-mut vector. The results show that miR-30a-5p can target the 3'UTR region of FAP, and FAP is a target gene downstream of miR-30a-5p, and miR-30a-5p can inhibit the expression of FAP.

[0100] Example 5 Verification of the function of miR-30a-5p on primary airway fibroblasts at the site of airway stenosis

[0101] 1. Isolation of airway fibroblasts (AFs)

[0102] Airway epithelium of patients with airway stenosis was brushed, and airway fibroblasts (AFs) were obtained by differential centrifugation. The cells were cultured in DMEM complete medium, and when the growth density reached 70%, immunofluorescence identification was performed. The primary antibodies used were a-SMA antibody (ab7817, 1:200) and Ki67 antibody (1:200, Abeam, ab15580). As shown in Figure 6 , a large amount of a-SMA protein was expressed in the cytoplasm, and a large amount of Ki67 protein was expressed in the nucleus, which was consistent with the typical characteristics of airway fibroblasts. It was proved that the airway fibroblasts were successfully isolated.

[0103] 2. Treatment of AFs

[0104] To the AFs with 70% growth density, 2 x 105 4 particles / cell concentration of BSC-EVs were added, recorded as BSC-EVs group, and continued to culture for 30h for subsequent detection. To the AFs with 70% growth density, PBS with the same volume as BSC-EVs was added, recorded as Blank group, and continued to culture for 30h for subsequent detection.

[0105] As shown in Figure 7 , to the AFs with 70% growth density, 2 x 105 4 particles / cell concentration of BSC-EVs were added, and after 6h of continuous culture, NC inhibitors or miR-30a-5p inhibitors were added, and the resulting cells were recorded as BSC-EVs+NC inhibitors group and BSC-EVs+miR-30a-5p inhibitors group, respectively; after 24h of continuous culture, subsequent detection was performed.

[0106] 3. FAP cell immunofluorescence and collagen contraction experiment

[0107] (1) Immunofluorescence

[0108] Immunofluorescence detection was performed on the AFs of BSC-EVs group, Blank group, BSC-EVs+NC inhibitors group and BSC-EVs+miR-30a-5p inhibitors group, and the specific steps were as follows: the cells were washed with PBS for 3 times; the cells were covered with 4% paraformaldehyde and fixed at room temperature for 15min, and washed with PBST for 3 times, each for 5min; the cells were covered with 0.1% Triton X-100 and incubated at room temperature for 20min, and washed with PBST for 3 times, each for 5min; the slices were placed in a wet box, and the samples were completely covered with 5% BSA and incubated at room temperature for 60min; the primary antibody (FAP antibody, CST66562, dilution ratio 1:50) was diluted with 5% BSA blocking solution, mixed well and covered on the samples, and incubated at 4°C overnight; the next day, after rewarming at room temperature, the samples were washed with PBST for 3 times, each for 5min; the anti-rabbit fluorescent secondary antibody (ab150077) was used to incubate the samples at room temperature in the dark for 1h, and the samples were washed with PBST for 3 times, each for 5min; the residual liquid was absorbed with absorbent paper, DAPI / anti-quenching agent two-in-one mounting solution was added dropwise, a cover glass was covered to avoid air bubbles. The staining results were observed under a fluorescence microscope and photographed.

[0109] (2) Collagen contraction experiment

[0110] After digestion, AFs from the BSC-EVs group, Blank group, BSC-EVs+NC inhibitors group, and BSC-EVs+miR-30a-5pinhibitors group were suspended in DMEM medium and mixed with 3 mg / mL rat tail type I collagen (Corning, USA) at a volume ratio of 2:1. The mixture was then cured at 37°C for 2 hours. Subsequently, 1 mL of complete DMEM medium was added to each well, and the mixture was incubated for 8 hours. The formation of collagen gels from airway fibroblasts in each group was observed and photographed. The gel area was measured using ImageJ software, and the formula was used to calculate the area of ​​the collagen gel for each group: Gel area (%) = x / n * 100% (x = area of ​​each group containing cells, n = area of ​​the well).

[0111] (3) Results Analysis

[0112] like Figure 8 As shown in Figures A through C, compared to the Blank group, the expression level of FAP in airway fibroblasts was decreased and the collagen area was increased in the BSC-EVs or BSC-EVs+NC inhibitors groups; however, this effect could be reversed by miR-30a-5p inhibitors. This indicates that BSCs can inhibit the activation of airway fibroblasts, and that miR-30a-5p plays an important role in this anti-fibrotic effect.

[0113] Based on the results of this embodiment, it is shown that miR-30a-5p can regulate the activation of airway fibroblasts, collagen gel contraction, and extracellular matrix remodeling, and plays this regulatory role in the antifibrotic effect mediated by BSC-EVs.

[0114] Example 6: Functional evaluation of primary airway fibroblasts at airway stenosis by extracellular vesicles containing miR-30a-5p human-derived airway basal stem cells.

[0115] 1. Construction of the co-cultivation system

[0116] This embodiment constructs a co-culture system of airway basal stem cells (BSCs) and airway fibroblasts (AFs), the procedure is as follows: Figure 9 As shown, the specific steps are as follows:

[0117] (1) Isolation, culture and transfection of BSCs

[0118] BSCs from healthy people were obtained according to the method of Example 5, inoculated into the upper chamber of a Transwell culture plate (Cat. No. 3470, Corning), and when the BSCs grew to 70% confluence, the NC inhibitors or hsa-miR-30a-5p inhibitors in Example 3 were transfected into the BSCs using the lipo 3000 kit, and the obtained cells were respectively recorded as the NC inhibitors group and the hsa-miR-30a-5p inhibitors group. The culture was continued for 24 h.

[0119] (2) Isolation and culture of AFs

[0120] AFs from the airway stenosis of a patient with airway stenosis were obtained according to the method of Example 1, inoculated into the lower chamber of a Transwell culture plate, and the culture was continued for 6 h.

[0121] (3) Co-culture

[0122] GW4869 (an extracellular vesicle secretion inhibitor) was added to the upper chamber of the NC inhibitors group at a final concentration of 10 μM, and then co-cultured with the lower chamber of the AFs culture, and the obtained cells were recorded as the NC inhibitors+GW4869 group. The same volume of DMSO as GW4869 was added to the upper chamber of the NC inhibitors group, and then co-cultured with the lower chamber of the AFs culture, and the obtained cells were recorded as the NC inhibitors+DMSO group. GW4869 was added to the upper chamber of the miR-30a-5p inhibitors group at a final concentration of 10 μM, and then co-cultured with the lower chamber of the AFs culture, and the obtained cells were recorded as the miR-30a-5p inhibitors+GW4869 group. The same volume of DMSO as GW4869 was added to the upper chamber of the miR-30a-5p inhibitors group, and then co-cultured with the lower chamber of the AFs culture, and the obtained cells were recorded as the miR-30a-5p inhibitors+DMSO group.

[0123] The 4 groups of cells were co-cultured for 24 h, and then subjected to subsequent detection.

[0124] 2. Determination of relative expression amount of miR-30a-5p

[0125] The relative expression amount of miR-30a-5p in the AFs in the lower chamber of each group was detected according to the RT-qPCR method in Example 1, and the results are shown in Table 1. Figure 10As shown, compared with the NC inhibitors + DMSO group, the expression of miR-30a-5p in the AFs of the miR-30a-5p inhibitors + DMSO group was significantly down-regulated, indicating that the expression of miR-30a-5p in the BSCs was successfully inhibited; compared with the NC inhibitors + GW4869 group, the expression of miR-30a-5p in the AFs of the miR-30a-5p inhibitors + GW4869 group was significantly down-regulated and was equivalent to that in the AFs of the miR-30a-5p inhibitors + DMSO group, indicating that inhibiting the release of extracellular vesicles from the BSCs could inhibit the transmission of miR-30a-5p from the BSCs to the AFs.

[0126] 3. Determination of FAP transcription level

[0127] The total RNA of the lower chamber AFs of each group was extracted using a TransZol Up Plus RNA kit (Fullway, China), and the cDNA template was obtained by reverse transcription using a First-Strand cDNA Synthesis Super-Mix Kit. The relative expression of FAP mRNA was detected according to the RT-qPCR method in Example 1, and GAPDH was used for standardization. The upstream detection primer sequence of GAPDH was 5'-GTGGACCTGACCTGCCGTCTAG-3' (SEQ ID NO: 13), and the downstream detection primer sequence was 5'-GAGTGGGTGTCGCTGTTGAAGTC-3' (SEQ ID NO: 14); the upstream detection primer sequence of FAP was 5'-CAAAGGCTGGAGCTAAGAATCC-3' (SEQ ID NO: 15), and the downstream detection primer sequence was 5'-ACTGCAAACATACTCGTTCATCA-3' (SEQ ID NO: 16).

[0128] As shown in Table 2, compared with the NC inhibitors + DMSO group, the FAP transcription level in the lower chamber AFs of the miR-30a-5p inhibitors + DMSO group was significantly increased; compared with the NC inhibitors + GW4869 group, the FAP transcription level in the AFs of the miR-30a-5p inhibitors + GW4869 group was significantly increased and was equivalent to that in the AFs of the miR-30a-5p inhibitors + DMSO group. It was indicated that miR-30a-5p in the BSCs was transmitted to the AFs through extracellular vesicles to inhibit the transcription and expression of FAP. Figure 11

[0129] ​4. FAP cell immunofluorescence and collagen contraction experiment

[0130] The FAP immunofluorescence and collagen contraction of the AFs in each group were detected according to the method in Example 5.

[0131] Figure 12 As shown, compared with the NC inhibitors + DMSO group, the expression of FAP in the AFs of the miR-30a-5p inhibitors + DMSO group was increased, and the collagen gel contraction ability was enhanced; compared with the NC inhibitors + GW4869 group, the expression of FAP in the AFs of the miR-30a-5p inhibitors + GW4869 group was significantly increased, the collagen gel contraction ability was enhanced, and was equivalent to that of the AFs of the miR-30a-5p inhibitors + DMSO group. It is indicated that the miR-30a-5p in the BSCs inhibits the expression of FAP protein in the AFs by extracellular vesicle transfer, inhibits the activation of the AFs, and reduces the collagen gel contraction ability of the AFs.

[0132] 5. Determination of matrix metalloproteinase (MMPs) and type I collagen (COL1A2) transcription levels

[0133] The total RNA of each group of lower chamber AFs was extracted according to the method of the present example, and cDNA template was obtained by reverse transcription, and the relative expression amount of mRNA of MMPs (MMP1, MMP2, MMP3, MMP7, MMP9) and COL1A2 was detected by RT-qPCR method, and GAPDH was used for standardization. The upstream detection primer sequence of MMP1 is: 5'-ACTGCTGCTGCTGCTGTTCTG-3'(SEQ ID NO: 17), and the downstream detection primer sequence is: 5'-TGCCTCCCATCATTCTTCAGGTTG-3'(SEQ ID NO: 18); the upstream detection primer sequence of MMP2 is: 5'-CACTGTTGGTGGGAACTCAGAAGG-3'(SEQ ID NO: 19), and the downstream detection primer sequence is: 5'-GGTCATCATCGTAGTTGGCTGTGG-3'(SEQ ID NO: 20); the upstream detection primer sequence of MMP3 is: 5'-GCATCCCGAAGTGGAGGAAAACC-3'(SEQ ID NO: 21), and the downstream detection primer sequence is: 5'-AGCCTGGAGAATGTGAGTGGAGTC-3'(SEQ ID NO: 22); the upstream detection primer sequence of MMP7 is: 5'-ACCGTGCTGTGTGCTGTGTG-3'(SEQ ID NO: 23), and the downstream detection primer sequence is: 5'-TCCTGAGCCTGTTCCCACTGTAG-3'(SEQ ID NO: 24); the upstream detection primer sequence of MMP9 is: 5'-CCCTGGTCCTGGTGCTCCTG-3'(SEQ ID NO: 25), and the downstream detection primer sequence is: 5'-CTGCCTGTCGGTGAGATTGGTTC-3'(SEQ ID NO: 26); the upstream detection primer sequence of COL1A2 is: 5'-CCGTGGCAGTGATGGAAGTGTG-3'(SEQ ID NO: 27), and the downstream detection primer sequence is: 5'-GCAGGACCAGCGTTACCAACAG-3'(SEQ ID NO: 28).

[0134] As Figure 13The transcription levels of MMP1 and MMP9 in the AFs of the miR-30a-5p inhibitors+DMSO group were significantly down-regulated compared with the NC inhibitors+DMSO group, the transcription levels of MMP1 and MMP9 in the AFs of the miR-30a-5p inhibitors+GW4869 group were significantly down-regulated compared with the NC inhibitors+GW4869 group, and were equivalent to the transcription levels of MMP1 and MMP9 in the AFs of the miR-30a-5p inhibitors+DMSO group; the transcription levels of MMP2 and MMP3 in the AFs of the NC inhibitors+GW4869 group were significantly down-regulated compared with the NC inhibitors+DMSO group, while there was no significant difference in the transcription levels of MMP2 and MMP3 in the AFs of the miR-30a-5p inhibitors+DMSO group and the miR-30a-5p inhibitors+GW4869 group; there was no significant difference in the transcription levels of MMP7 and COL1A2 in the AFs of the four groups.

[0135] 6. Western blotting (WB) was used to detect protein expression

[0136] The AFs of each group were washed once with PBS pre-cooled at 4°C, and then protein samples of the AFs of each group were extracted with a protein lysis solution (RIPA with protease phosphatase inhibitors) and mixed with 5x loading buffer, and then placed at 100°C for 10 minutes and quickly cooled in an ice bath. The protein concentration was determined by a BCA kit, and then 20 μg of protein was loaded per well, 10% SDS-PAGE gel was used for electrophoresis, membrane transfer, blocking, and the primary antibody Collagen I (ab21286, 1:1000), α-SMA (ab7817, 1:1000), FAP (CST66562, 1:1000), and Matrix Remodeling Antibody Sampler Kit (CST, 1:1000) were incubated at 4°C overnight. After washing the membrane with TBST, the secondary antibody was incubated, and then the membrane was washed with TBST and placed in a chemiluminescence imager, and then ECL luminescent liquid was uniformly added, and the instrument was operated for luminescence development and photographing.

[0137] As Figure 14As shown, compared with the NC inhibitors+DMSO group, the miR-30a-5p inhibitors+DMSO group showed increased expression levels of FAP and Collagen I in AFs, while decreased expression levels of MMP2, MMP7, MMP9, TIMP1, TIMP2, and TIMP3. Compared with the miR-30a-5p inhibitors+DMSO group, the miR-30a-5p inhibitors+GW4869 group showed increased expression levels of FAP, Collagen I, α-SMA, MMP7, and TIMP2 in AFs, while decreased expression level of TIMP1. This indicates that miR-30a-5p in BSCs is translocated to AFs via extracellular vesicles, inhibiting their activation and regulating extracellular matrix (ECM) remodeling.

[0138] Based on the results of this embodiment, it is shown that miR-30a-5p can regulate the activation of airway fibroblasts, collagen gel contraction, and extracellular matrix remodeling. miR-30a-5p exerts this regulatory function by being delivered to airway fibroblasts through extracellular vesicles of BSCs.

[0139] Example 7: BSC-EVs for the treatment of benign airway stenosis

[0140] 1. Construction and treatment of BTS

[0141] A BTS model was constructed and BSC-EVs treatment was performed according to the method in Example 2.

[0142] 2. Survival Analysis

[0143] From the first day of modeling, the survival status of the rabbits was monitored daily, and mortality was recorded promptly until the end of the experiment. The survival rates of the BSC-EVs treatment group and the control group were statistically analyzed, and survival curves were plotted. Figure 15 As shown, the survival rate of rabbits in the control group was only 20% on day 14 of modeling, and all rabbits in this group died by day 29. In contrast, the survival rate of rabbits in the BSC-EVs treatment group was as high as 75% on day 14 of modeling, and one rabbit survived until the end of the experiment. This indicates that BSC-EVs treatment significantly prolongs the survival of BTS.

[0144] 3. Bronchoscopy

[0145] The airway stenosis of rabbits in each group was examined under bronchoscopy on days 14, 21, and 28 after modeling, and photographs were taken and recorded. Figure 16 As shown, on day 14, compared with the control group, the treatment group showed reduced granulation tissue growth and improved lumen patency, and the degree of stenosis progressed slowly over time after treatment.

[0146] 4. Histological analysis and evaluation of treatment efficacy

[0147] Following the BTS construction and treatment method of this embodiment, another batch of New Zealand white rabbits were treated in parallel. On days 14, 21, and 28 after modeling, one rabbit from each group was randomly selected to isolate airway tissue, prepare paraffin sections, and then bake them in a 65°C oven for 2 hours. While still hot, the sections were dewaxed and hydrated sequentially with TO and gradient ethanol, followed by HE staining and MASSON staining. HE staining was performed according to the method in Example 1. The specific steps for MASSON staining are as follows: fix the dewaxed sections with Bouin's solution for 1 hour, then rinse with running water for 5 minutes; then stain with iron-containing hematoxylin solution for 5 minutes, then rinse with deionized water; stain with basic fuchsin solution for 5 minutes, then rinse with deionized water; treat with a mixed solution of phosphotungstic acid and phosphomolybdic acid for 5 minutes, avoiding rinsing; stain with aniline blue solution for 5 minutes, then rinse with deionized water; dehydrate using an alcohol gradient, and finally mount with xylene and neutral resin.

[0148] Based on the stenosis index of HE-stained images, the calculation formula is: Stenosis index = [1-(d1+d2) / (D1+D2)]×100%, where d1 and d2 are the major and minor diameter measurements of the stenotic part, respectively, and D1 and D2 are the major and minor diameter measurements of the adjacent non-stenotic part, respectively.

[0149] like Figure 16 As shown, HE staining results revealed abundant proliferating granulation tissue at the narrowed site in the control group. This granulation tissue was mainly composed of newly formed capillaries, fibroblasts, and inflammatory cells. MASSON staining results showed abundant proliferating collagen fibers in the control group, forming large fiber bundles, which contrasted with the less abundant and orderly fiber distribution in the treatment group. Narrowing index analysis showed that the luminal diameter of the narrowed airway in the treatment group was significantly larger than that in the control group. This indicates that compared to the control group, the BSC-EVs treatment group showed significantly less granulation tissue at the narrowed airway site, a more unobstructed lumen, and less collagen at the granulation site.

[0150] 5. Evaluation of treatment efficacy by Western blot analysis (WB)

[0151] For the BTS of the previous step, on day 14 of modeling, 5 rabbits were randomly selected from each group to isolate airway tissue, and WB detection was performed according to the method in Example 2. The primary antibodies used included: Collagen I antibody (ab21286, 1:1000), FAP antibody (CST66562, 1:1000), β-actin antibody (CST4967, 1:1000), and Matrix Remodeling Antibody Sampler Kit (CST73959, 1:1000).

[0152] As shown in Figure 17 Collagen I and FAP expression in the BSC-EVs treatment group decreased significantly, and MMP2 and MMP9 expression increased significantly compared with the control group. This indicates that the degree of fibrosis in the treatment group is reduced, and the remodeling of the extracellular matrix and the repair of the tissue are improved. These changes work together to possibly promote the healing process after airway injury, making tissue repair more effective and orderly, helping to alleviate airway stenosis and improve airway patency.

[0153] The above results of this example show that BSC-EVs enriched in miR-30a-5p have excellent airway stenosis treatment effects, less collagen in the airway wall, improved airway wall remodeling, and inhibition of granulation tissue proliferation, which in turn expands the airway lumen, significantly relieves stenosis symptoms, and prolongs the survival cycle.

[0154] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and do not limit the scope of protection of the present application. For those skilled in the art, based on the above description and ideas, other different forms of changes or modifications can also be made, which do not need or cannot be exhaustively listed here. Any modification, equivalent replacement and improvement made within the spirit and principles of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. Use of miR-30a-5p for the preparation of a product for the treatment of benign airway stenosis, characterized in that, The nucleotide sequence of the miR-30a-5p is shown as SEQ ID NO:

4.

2. Use according to claim 1, characterized in that, The treatment of the benign airway stenosis comprises inhibiting airway fibroblast activation.

3. Use according to claim 1, characterized in that, The treatment of the benign airway stenosis comprises reducing collagen gel contraction ability of airway fibroblasts.

4. Use according to claim 1, characterized in that, The treatment of the benign airway stenosis comprises improving extracellular matrix remodeling of airway fibroblasts.

5. The use of an agent that inhibits the expression of a FAP gene and / or FAP protein for the manufacture of a product for the treatment of benign airway narrowing, characterized in that, The agent for inhibiting FAP gene and / or FAP protein expression is miR-30a-5p, and the nucleotide sequence of the miR-30a-5p is shown as SEQ ID NO:

4.

6. Use according to claim 5, characterized in that, The treatment of the benign airway stenosis comprises any one or several of inhibiting airway fibroblast activation, reducing collagen gel contraction ability of airway fibroblasts, or improving extracellular matrix remodeling of airway fibroblasts.

7. Use of extracellular vesicles comprising miR-30a-5p for the manufacture of a product for the treatment of benign airway narrowing, characterized in that, The extracellular vesicle is an airway basal layer stem cell-derived extracellular vesicle.

8. Use according to claim 7, characterized in that, The treatment of the benign airway stenosis comprises any one or several of inhibiting airway fibroblast activation, reducing collagen gel contraction ability of airway fibroblasts, or improving extracellular matrix remodeling of airway fibroblasts.

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