Application of inhibiting RUNX3 gene expression in preparation of medicine for preventing or treating silicosis

Abstract

The invention discloses application of inhibition of RUNX3 gene expression in preparation of medicines for preventing or treating silicosis, and belongs to the technical field of biological medicines. In-vivo and in-vitro silicosis models prove that the RUNX3 has high specificity expression in the silicosis process and is a key factor for driving disease progression. By inhibiting the expression of RUNX3 through a gene knock-down technology, silica-induced lung inflammation infiltration and collagenous fiber deposition can be significantly reduced, and the expression levels of key inflammatory factors (IL-1beta, IL-6 and TNF-alpha) and fibrosis indexes (Collagen I, Collagen III and Fibronectin) can be effectively down-regulated. Therefore, any substance capable of specifically inhibiting the expression of the RUNX3 gene, including but not limited to siRNA, shRNA, antisense oligonucleotide, ribozyme, microRNA and an inhibitor targeting the gene promoter thereof, can be used for developing the pharmaceutical composition for preventing or treating silicosis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of inhibiting RUNX3 gene expression in the preparation of drugs for the prevention or treatment of silicosis. Background Technology

[0002] Silicosis is an irreversible, progressive fibrotic lung disease caused by long-term inhalation of crystalline silica (CS) dust. It is one of the most common and serious occupational diseases. This disease is prevalent in industries such as mining, construction, ceramics, casting, and emerging industries like sandblasting and denim processing, threatening tens of millions of workers worldwide. Patients gradually develop chronic inflammation in their lungs, fibroblast activation, and excessive deposition of extracellular matrix, ultimately leading to lung structural destruction and respiratory failure. Currently, there is no cure for silicosis. Commonly used drugs such as pirfenidone and nintedanib can slow disease progression to some extent, but their efficacy is limited and they have significant side effects. Lung transplantation is an option for end-stage patients, but donor scarcity, high surgical risks, and immune rejection limit its widespread application.

[0003] RUNX3 (Runt-related transcription factor 3), a member of the Runt domain transcription factor family, plays a crucial role in various biological processes, including regulating cell proliferation, apoptosis, immune responses, and tumor suppression. Recent studies have shown that RUNX3 has a regulatory function in lung diseases such as asthma and lung cancer. Chinese invention patent 201610611681.4 discloses a saRNA that activates RUNX3 expression in lung cancer cells and its application. This saRNA targets the RUNX3 promoter region, achieving multiple inhibitory effects on lung cancer cells by specifically activating the expression of this tumor suppressor gene. However, its specific role in the pathogenesis of silicosis, especially its function and mechanism in the development of pulmonary fibrosis, has not yet been systematically studied or clearly reported. Therefore, there is an urgent need in this field to discover new key targets for the pathogenesis of silicosis and to develop more efficient and safer therapeutic strategies based on these targets. Summary of the Invention

[0004] The purpose of this invention is to provide an application of inhibiting RUNX3 gene expression in the preparation of drugs for the prevention or treatment of silicosis, to use the transcription factor RUNX3 as a therapeutic target for silicosis, and to develop various drugs for the prevention or treatment of silicosis, such as nucleic acid drugs and small molecule drugs, by using substances that inhibit RUNX3 gene expression.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention is the first to discover and confirm that the RUNX3 gene is specifically highly expressed in the peripheral blood of silicosis patients and in the lung tissue of silicosis model animals, and that its expression level is positively correlated with disease severity. In the cell model, silica stimulation induces upregulation of RUNX3 expression in macrophages, primarily located in the cell nucleus.

[0006] This invention proposes that any substance capable of specifically inhibiting RUNX3 gene expression can be used to develop drugs for the prevention or treatment of silicosis. Such substances include, but are not limited to, interfering molecules (such as siRNA, shRNA, antisense oligonucleotides, ribozymes, and microRNA) that target the RUNX3 gene or mRNA, as well as promoter inhibitors capable of inhibiting its gene transcription.

[0007] Furthermore, pharmaceutical compositions containing the above-mentioned active substances can be formulated into dosage forms suitable for local pulmonary administration, such as inhaled powders, aerosols, nebulized solutions, etc., to enhance targeting and reduce systemic side effects.

[0008] The beneficial effects of this invention are as follows: (1) Using peripheral blood from pneumoconiosis patients, mouse silicosis models, and macrophage dust-infected models, the key pathogenic role of transcription factor RUNX3 in the development and progression of silicosis was revealed and confirmed for the first time. The study found that RUNX3 is specifically highly expressed in the silicosis process, and its expression level is positively correlated with the severity of the disease. RUNX3 was established for the first time as an original and highly effective target for intervening in the silicosis process, laying a solid theoretical foundation for the development of drugs with novel mechanisms of action.

[0009] (2) Gene knockdown technology demonstrated that inhibiting RUNX3 expression produces a clear therapeutic effect. During the inflammatory phase (1 week of dust exposure), it significantly reduced silica-induced pulmonary inflammatory cell infiltration and effectively downregulated the expression of key pro-inflammatory factors (IL-1β, IL-6, TNF-α), thus inhibiting inflammation. During the fibrotic phase (12 weeks of dust exposure), it significantly reduced collagen deposition in lung tissue, improved the pathological structure of pulmonary fibrosis, and significantly reduced the expression of fibrosis markers (Collagen I, Collagen III, Fibronectin). The RUNX3-targeted treatment strategy covers the core pathological process of silicosis from acute inflammation to chronic fibrosis, offering more comprehensive therapeutic advantages and broader clinical application potential.

[0010] (3) Knocking down RUNX3 in macrophages can inhibit the secretion of silica-induced inflammatory factors, enhance the credibility of the treatment strategy, and effectively block the disease process.

[0011] (4) Based on the inhibition of RUNX3 expression or activity, this lays the foundation for the subsequent development of various drug forms such as nucleic acid drugs and small molecule drugs. Any substance that can inhibit RUNX3 expression or activity can be a candidate drug, including but not limited to: siRNA, shRNA, antisense oligonucleotides that specifically target RUNX3, which have high specificity and designability; antibodies or fragments that can neutralize the function of RUNX3 protein; and small molecule inhibitors that can be developed targeting the structure of RUNX3 protein. This reduces the blind spots in subsequent drug development and shortens the research and development cycle. Attached Figure Description

[0012] Figure 1 The expression level of RUNX3 in the peripheral blood of patients with pneumoconiosis.

[0013] Figure 2 Pathological staining of lung tissue from a silicosis mouse model. A refers to H&E staining of lung tissue from silicosis mice; B refers to Masson staining of lung tissue from silicosis mice; C refers to immunohistochemical staining of RUNX3 in lung tissue from silicosis mice.

[0014] Figure 3 To detect RUNX3 mRNA in lung tissue of a silicosis mouse model.

[0015] Figure 4 To detect RUNX3 protein in lung tissue of a silicosis mouse model.

[0016] Figure 5 The mRNA and protein expression levels of RUNX3 in a multi-dose silica-induced macrophage dust-stained model were determined.

[0017] Figure 6 The mRNA and protein expression levels of RUNX3 in a silica-induced macrophage dust-stained model at multiple time points were determined.

[0018] Figure 7 The expression levels of RUNX3 in the cytoplasm and nucleus of macrophages in a silica-induced dust-stained model.

[0019] Figure 8 Immunofluorescence staining of RUNX3 in a silica-induced macrophage dust-stained model.

[0020] Figure 9 To establish a 1-week RUNX3 knockdown silicosis mouse model. A refers to the mRNA expression level of RUNX3 in the lung tissue of each group of mice; B refers to the immunohistochemical staining of RUNX3 in the lung tissue of each group of mice; C refers to the protein expression level of RUNX3 in the lung tissue of each group of mice; D refers to the semi-quantitative statistical analysis of RUNX3 protein in the lung tissue of each group of mice.

[0021] Figure 10To establish a 12-week RUNX3 knockdown silicosis mouse model. A refers to the mRNA expression level of RUNX3 in the lung tissue of each group of mice; B refers to the immunohistochemical staining of RUNX3 in the lung tissue of each group of mice; C refers to the protein expression level of RUNX3 in the lung tissue of each group of mice; D refers to the semi-quantitative statistical analysis of RUNX3 protein in the lung tissue of each group of mice.

[0022] Figure 11 Pathological staining of RUNX3 in a 1-week RUNX3 knockdown mouse model of silicosis. A and B refer to H&E staining and inflammation scores in the lung tissue of each group of mice; C and D refer to Masson staining and collagen area percentage (%) in the lung tissue of each group of mice.

[0023] Figure 12 Pathological staining of RUNX3 in a 12-week-old RUNX3 knockdown silicosis mouse model. A and B refer to H&E staining and inflammation scores in the lung tissue of each group of mice; C and D refer to Masson staining and collagen area percentage (%) in the lung tissue of each group of mice.

[0024] Figure 13 The mRNA expression levels of inflammatory factors in lung tissue of a 1-week RUNX3 knockdown silicosis mouse model are shown. A represents the expression level of Il1b mRNA in the lung tissue of each group of mice; B represents the expression level of Il6 mRNA in the lung tissue of each group of mice; and C represents the expression level of Tnf mRNA in the lung tissue of each group of mice.

[0025] Figure 14 The protein expression levels of inflammatory factors (IL1β, IL-6, TNF-α) in lung tissue of a 1-week RUNX3 knockdown silicosis mouse model.

[0026] Figure 15 The mRNA expression levels of fibrosis markers in lung tissue of a 1-week RUNX3 knockdown silicosis mouse model are shown. A represents the expression level of Col1a1 mRNA in the lung tissue of each group of mice; B represents the expression level of Col3a1 mRNA in the lung tissue of each group of mice; and C represents the expression level of Fn1 mRNA in the lung tissue of each group of mice.

[0027] Figure 16 Protein expression levels of inflammatory factors (Collagen I, Collagen III, Fibronectin) in lung tissue of a 1-week RUNX3 knockdown silicosis mouse model.

[0028] Figure 17The mRNA expression levels of inflammatory factors in lung tissue of a 12-week-old RUNX3 knockdown silicosis mouse model. A represents the expression level of Il1b mRNA in the lung tissue of each group of mice; B represents the expression level of Il6 mRNA in the lung tissue of each group of mice; C represents the expression level of Tnf mRNA in the lung tissue of each group of mice.

[0029] Figure 18 Protein expression levels of inflammatory factors (IL1β, IL-6, TNF-α) in lung tissue of a 12-week-old RUNX3-knockdown silicosis mouse model.

[0030] Figure 19 The mRNA expression levels of fibrosis markers in lung tissue of a 12-week-old RUNX3-knockdown silicosis mouse model. A represents the expression level of Col1a1 mRNA in the lung tissue of each group of mice; B represents the expression level of Col3a1 mRNA in the lung tissue of each group of mice; C represents the expression level of Fn1 mRNA in the lung tissue of each group of mice.

[0031] Figure 20 Protein expression levels of inflammatory factors (Collagen I, Collagen III, Fibronectin) in lung tissue of a 12-week-old RUNX3-knockdown silicosis mouse model.

[0032] Figure 21 Construction of a stable RUNX3 knockdown macrophage (THP-1) cell line. A refers to the RUNX3 mRNA expression level in the stable RUNX3 knockdown macrophage (THP-1) cell line; B and C refer to the RUNX3 protein expression level and semi-quantitative statistics in the stable RUNX3 knockdown macrophage (THP-1) cell line.

[0033] Figure 22 This study describes the construction of a silica-induced stable cell model of RUNX3 knockdown macrophages (THP-1). A represents the mRNA expression level of RUNX3 in the silica-induced stable cell model; B represents the protein expression level of RUNX3 in the stable RUNX3 knockdown macrophage (THP-1) cell line.

[0034] Figure 23 To reduce the protein expression levels of inflammatory factors (IL1β, IL-6, TNF-α) in a silica-induced RUNX3 knockdown macrophage (THP-1) stable cell model. Detailed Implementation

[0035] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0036] I. Validation of RUNX3 upregulation in silicosis patients and disease models 1. Detection of RUNX3 expression level in patients with pneumoconiosis This embodiment uses real-time quantitative polymerase chain reaction (qPCR) to detect the expression level of RUNX3 mRNA in the peripheral blood of silicosis patients.

[0037] Specific procedures: Peripheral blood was collected from healthy individuals and patients with silicosis at different stages (stage I, II, and III). Red blood cells were lysed using erythrocyte lysis buffer, and total RNA was extracted using Trizol. The RNA was reverse transcribed into template DNA using a reverse transcription kit. Human RUNX3 detection primers were added to the template DNA, followed by the addition of SYBR and DEPC water. PCR detection was then performed in a PCR amplification instrument.

[0038] Human RUNX3 detection primers: Forward primer: 5'-GATGGCAGGCAATGACGAG-3'.

[0039] Reverse primer: 5'-AACGGCTTGGTCTGGTCCT-3'.

[0040] Detection results: Using β-actin as an internal reference gene, 2 -ΔΔct Calculate the relative expression levels of each gene. For example... Figure 1 As shown, compared with the normal population, the mRNA expression of RUNX3 in the peripheral blood of silicosis patients was significantly increased.

[0041] 2. Establishment of a dust-induced mouse model of pulmonary fibrosis (2-1) A mouse model of silicosis was established using male C57BL / 6 mice.

[0042] Specific procedures: Crystalline silica particles (3 μm, Forsmann) were dry-heat sterilized at 200℃ for 2 h, and then added to sterile physiological saline to prepare a silica suspension of 50 mg / mL. Mice were anesthetized and fixed on the operating table. The mouse's tongue was gently removed with forceps. A mercury lamp was used to illuminate the mouse's neck, and the tracheal opening was visually located. The point that flickered with the mouse's breathing was the tracheal opening. A catheter was inserted into the tracheal opening, and the silica suspension was quickly injected. After removing the catheter, the mouse was gently shaken to distribute the silica evenly in the lung tissue. The mouse's tongue was then removed to prevent choking. Once the mouse's breathing stabilized, it was returned to its cage, and vital signs were observed until the mouse regained consciousness.

[0043] This embodiment sets up a control group and a silica model group.

[0044] (2-2) Mice were euthanized after exposure to silica for 1 week, 4 weeks, and 12 weeks, and lung tissue was harvested. H&E and Masson staining and immunohistochemical IHC staining were used to evaluate the lung tissue damage and the distribution of RUNX3 in the lung tissue of silicosis mouse models.

[0045] Fresh lung tissue was fixed in 4% paraformaldehyde for 48 h, then dehydrated, permeabilized, and embedded in paraffin to form paraffin blocks. These blocks were then prepared into 6 μm lung tissue pathological sections using a microtome. After dewaxing and rehydration, the sections were ready for use. Pathological staining was performed using H&E staining kits and Masson staining kits.

[0046] Sections were autoclaved with antigen retrieval solution for 3 minutes to expose the target site, incubated with anti-RUNX3 antibody (9647, CST) overnight at 4°C, washed with PBS, incubated with secondary antibody, and the target protein was visualized using a colorimetric system. Finally, the nuclear structure was counterstained with hematoxylin, dehydrated and cleared, mounted, and analyzed under an optical microscope.

[0047] (2-3) The expression level of Runx3 mRNA in mouse lung tissue was detected by Q-PCR technology. The specific operation was the same as that for detecting RUNX3 mRNA in human peripheral blood in 1.

[0048] Mouse Runx3 detection primers: Forward primer: 5'-ATCGCCTCTTTCTCATCCCT-3'.

[0049] Reverse primer: 5'-GACCCTTTTCCTTTCATTTT-3'.

[0050] (2-4) The expression of RUNX3 protein in lung tissue was detected by Western blotting (WB).

[0051] The specific procedure was as follows: Mouse lung tissue was collected, RIPA lysis buffer was added, and the tissue was thoroughly ground using a tissue homogenizer. The tissue was then sonicated to extract proteins. Using a BCA protein assay kit, the protein concentration of each sample was adjusted to the same level, and an appropriate amount of loading buffer was added followed by boiling. An SDS-PAGE gel was prepared, and an appropriate amount of sample was added to the gel. Electrophoresis was performed at 150 V for 1 h. After electrophoresis, a PVDF membrane was fitted to the gel, transfer buffer was added, and a constant current of 250 mA was applied for transfer for 1 h to transfer the proteins onto the PVDF membrane. The transferred PVDF membrane was incubated in rapid blocking buffer for half an hour. The membrane was cut according to the protein marker instructions and incubated with the corresponding primary antibodies (anti-RUNX3 antibody, 9647, CST; anti-β-actin antibody, R1207-1, huabio) overnight at 4°C. After thorough washing with TBST, the membrane was incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 1 h. After thorough cleaning with TBST, ECL luminescent solution was added, and the mixture was developed in a luminescence analyzer.

[0052] (2-5) Test results.

[0053] IHC pathological staining results of lung tissue from a mouse model of silicosis at different time points, such as Figure 2 As shown, the expression level of RUNX3 protein was significantly upregulated in the silicosis model, and increased with the duration of dust exposure. RUNX3 showed nuclear positive staining, and based on cell morphology and tissue localization, it was preliminarily determined that it was highly expressed in lymphocytes, plasma cells, and epithelial cells.

[0054] Using Q-PCR and Western blotting techniques, this example confirmed, at both the mRNA and protein levels, that RUNX3 expression was significantly elevated in the lung tissue of a silicosis mouse model. Figure 3 and Figure 4 As shown, this is consistent with the IHC results.

[0055] 3. Establishment of a dust-contaminated macrophage model in in vitro experiments.

[0056] A dust-contaminated cell model was constructed using human mononuclear cells (THP-1) (Wuhan Pronosei Life Science Technology Co., Ltd.), and the expression levels of RUNX3 mRNA and protein were verified.

[0057] The specific procedure is as follows: Crystalline silica particles are dry-heat sterilized at 200℃ for 2 h, and a silica suspension of 100 mg / mL is prepared using sterile physiological saline. THP-1 cells are cultured in 1640 medium containing 10% FBS at a concentration of 1×10⁻⁶ cells / mL. 6Six-well plates were densely packed. Silica suspensions were prepared using complete culture medium and adjusted to concentrations of 0, 100, 200, and 400 μg / mL. After stimulation for 24 h, cell mRNA and protein were extracted. Silica suspensions at 200 μg / mL were prepared using complete culture medium and stimulated for 3, 6, 12, 24, and 48 h, respectively, and cell mRNA and protein were extracted.

[0058] The expression of RUNX3 mRNA and protein in cells was detected using Q-PCR and Western blotting. A nucleo-cytoplasmic separation kit was used to detect the expression levels of RUNX3 in the nucleus and cytoplasm of dust-contaminated macrophages. Cells were fixed with 4% paraformaldehyde, punched with Triton X, incubated with primary and secondary antibodies, and the fluorescence distribution was observed under a fluorescence fiber microscope.

[0059] In vitro, this embodiment constructed a dust-contaminated model with multiple concentration gradients and time points. The results showed that the expression level of RUNX3 in dust-contaminated macrophages was significantly upregulated, exhibiting a clear dose- and time-dependent effect. Figure 5 and Figure 6 .

[0060] Nuclear-cytoplasmic separation experiments and immunofluorescence techniques further confirmed that dust-induced RUNX3 was mainly enriched in the cell nucleus, such as... Figure 7 and Figure 8 This suggests that it may participate in gene regulation as a transcription factor.

[0061] In summary, RUNX3 was significantly overexpressed in humans, animals, and cells under silicosis / silica dust stimulation.

[0062] II. Evaluation of the therapeutic effect of in vivo RUNX3 knockdown on silicosis 1. Construct RUNX3 knockdown mice.

[0063] To demonstrate the key role of RUNX3 in silicosis, this embodiment constructs shRNA based on lentivirus-mediated gene knockdown technology, uses viral packaging to concentrate the viral suspension containing shRNA, and targets the lungs to knock down the RUNX3 gene via non-exposed tracheal infusion.

[0064] The operation steps are as follows: (1) Based on the pLKO.1 vector, shRNA sequences targeting the human and mouse RUNX3 gene were designed using databases such as NCBI. Multiple pairs of shRNA sequences were designed (Table 1) for subsequent experiments, and scrambled shRNA was synthesized as a negative control. The vector was linearized by double digestion with AgeI and EcoRI, purified by gel extraction, ligated with the annealed shRNA double-stranded oligo, and transformed into competent E. coli. Positive clones were screened using ampicillin resistance, and the sequence correctness was verified by colony PCR and sequencing (Sangon Biotech). High-purity plasmids were amplified and extracted in large quantities for subsequent virus packaging.

[0065] Table 1 shRNA sequence listing

[0066] (2) A three-plasmid system (packaging plasmid psPAX2, envelope plasmid pMD2.G, and target plasmid) was used to package the virus in 293FT cells using Lipofectamine 3000 transfection reagent. The medium was replaced with complete medium 8 h after transfection, and the viral supernatant was collected at 48 h and 72 h post-transfection. The supernatant was centrifuged at low speed (1250 rpm, 5 min) and filtered through a 0.45 μm filter to remove cell debris. The virus-containing medium used for cell transfection could be aliquoted and stored at -80℃. The virus used for animal infection needed to be concentrated using a commercial virus concentration kit (Beyotime) to ultimately increase the viral titer to 10⁸-10⁹ TU / mL, and then aliquoted and stored at -80℃.

[0067] (3) After anesthetizing the mice, fix them on the operating table, gently remove the mouse's tongue with forceps, and illuminate the mouse's neck with a mercury lamp light source. Visually observe and locate the tracheal opening. Find the point that flashes with the mouse's breathing, which is the tracheal opening. Insert the indwelling needle tubing into the tracheal opening and quickly deliver 70 μL of concentrated virus solution. After removing the indwelling needle, mix the solution to ensure the virus is evenly distributed in the mouse's lungs. Subsequently, one week after viral infection, infuse silica suspension again to construct a mouse pulmonary fibrosis model.

[0068] This embodiment includes a control group (tracheal infusion of physiological saline), a model group (tracheal infusion of silica), a shNC-SiO2 group (tracheal infusion of silica + lentivirus-encapsulated nonsense sequence shRNA), and a shRUNX3-SiO2 group (tracheal infusion of silica + lentivirus-encapsulated RUNX3 shRNA).

[0069] 2. Detection of RUNX3 mRNA and protein levels.

[0070] (2-1) After 1 week and 12 weeks of silica exposure, mice were euthanized, lung tissue was dissected, and the knockdown efficiency of RUNX3 in lung tissue was verified at the tissue localization, transcriptional and translational levels by H&E staining, Masson staining, IHC, qRT-PCR and Western blot techniques.

[0071] (2-2) To investigate the specific role of RUNX3 in the pathogenesis of silicosis, the mRNA and protein expression levels of inflammatory and fibrotic markers in the lung tissue of a RUNX3-knockdown silicosis mouse model were detected.

[0072] 3. Knocking down RUNX3 improves inflammation and fibrosis in silicosis mice.

[0073] To investigate the specific role of RUNX3 in the pathogenesis of silicosis, this study examined the mRNA and protein expression levels of inflammatory markers (IL-1β, IL-6, TNF-α) and fibrotic markers (Collagen I, Collagen III, Fibronectin) in the lung tissue of a RUNX3-knockdown silicosis mouse model.

[0074] Operating steps: (1) Mouse lung lobes were taken, and mRNA was extracted from mouse lung tissue. The mRNA was then reverse transcribed, followed by Q-PCR. The primers used for the PCR experiment are shown in Table 2. 2 -ΔΔct Calculate the relative expression levels of each gene.

[0075] Table 2 PCR primers

[0076] (2) Mouse lung lobes were collected, and total protein was extracted from the mouse lung tissue. After protein sample preparation, electrophoresis, membrane transfer, and blocking, the tissue was incubated with primary antibodies (anti-β-actin antibody (R1207-1, HuaBio); anti-IL1β antibody (Abcam, ab254360); anti-IL-6 antibody (Abcam, ab290735; anti-TNF-α antibody (Abcam, ab183218); anti-Collagen I antibody (Abcam, AB88147-1001); anti-Collagen III antibody (Abcam, AB184993-1001); anti-Fibronectin antibody (Abcam, AB45688-1001)) and corresponding secondary antibodies. ECL chemiluminescence solution was added, and the tissue was developed in a chemiluminescence analyzer. ImageJ was used to calculate the grayscale value, and the relative expression level of each protein was calculated with β-actin as an internal reference.

[0077] 4. Test results.

[0078] The results of Q-PCR and WB experiments indicated that the RUNX3 knockdown mouse model was successfully constructed. Figure 9 and Figure 10 As shown, H&E and Masson staining results indicated that knocking down RUNX3 effectively reduced silica-induced pulmonary inflammatory infiltration and collagen fiber deposition at both 1 week (inflammatory phase) and 12 weeks (fibrosis progression phase) of dust exposure. Figure 11 and Figure 12 As shown.

[0079] The expression levels of inflammatory factors (IL1β, IL-6, TNF-α) and fibrotic proteins (Collagen I, Collagen III, Fibronectin) in lung tissue of a silicosis mouse model were detected using qRT-PCR and Western blotting. The results showed that knocking down RUNX3 effectively reduced the expression levels of inflammatory factors and fibrotic proteins in the silicosis mouse model. Figure 13-16 As shown and Figure 17-20 As shown.

[0080] In summary, lentivirus-mediated knockdown of RUNX3 in lung tissue significantly improved lung inflammation and fibrosis in silicosis model mice and downregulated related molecular markers, demonstrating its therapeutic efficacy.

[0081] III. The inhibitory effect of RUNX3 knockdown in macrophages on silica dust-induced inflammation 1. Based on the lentiviruses shNC and shRUNX3 obtained in previous studies, a stable cell line of shRUNX3 (THP-1) was constructed. THP-1 cells in the logarithmic growth phase were used to construct a stable cell line of shRUNX3 (THP-1) at a concentration of 1×10⁻⁶ cells / mL. 6 Cells were seeded at a density of [number] cells / mL into 6-well plates and induced to adhere as macrophages by adding PMA for 24 hours. Complete culture medium containing 200 μg / mL silica was added, and the cells were stimulated for 24 hours. Total RNA and total protein were extracted from the cells.

[0082] The experiment was set up with shNC group, shRUNX3 group, shNC-CS group and shRUNX3-CS group.

[0083] 2. The expression of RUNX3, IL-1β, IL-6 and TNF-α proteins was detected by Western blotting, following the same experimental procedure as above.

[0084] 3. Test results.

[0085] like Figure 21 As shown, compared with the shNC group, the expression levels of RUNX3 mRNA and protein in the shRUNX3 group were significantly reduced, indicating that the stable cell line was successfully established. Figure 22The results showed that RUNX3 expression was significantly increased in the shNC group after silica treatment, while the expression level was decreased in the shRUNX3-CS group. Furthermore, the increased expression levels of inflammatory factors in the shNC-SiO2 group suggested that silica induced inflammation in dust-contaminated macrophages. Figure 23 The results showed that knocking down RUNX3 reduced the expression levels of IL-1β, IL-6, and TNF-α in macrophages, consistent with the results in RUNX3 knockdown mice, thus confirming that inhibiting RUNX3 expression can alleviate silicosis. These results not only validate the core role of RUNX3 in macrophages, a key effector cell, but also indicate that intervention strategies targeting RUNX3 can directly alleviate the inflammatory cascade response induced by silica dust at its source.

[0086] In addition, we constructed a transient knockdown cell model using siRNA knockdown, and the RUNX3 gene was effectively knocked down using multiple pairs of siRNA sequences (Table 3).

[0087] Table 3 siRNA Sequence Listing

[0088] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The use of a substance that inhibits the expression or activity of the RUNX3 gene in the preparation of a medicament for the prevention and / or treatment of silicosis.

2. The application according to claim 1, characterized in that, The substance is a nucleic acid molecule that can specifically inhibit the expression of the RUNX3 gene or a promoter inhibitor that can inhibit the transcription of the RUNX3 gene; The nucleic acid molecule is siRNA, shRNA, antisense oligonucleotide, microRNA, or ribozyme.

3. The application according to claim 2, characterized in that, The siRNA comprises at least one sequence selected from the sequences shown in SEQ ID NO.1 to SEQ ID NO.6, or a variant that has at least 80% sequence identity with the sequence and retains the RUNX3 gene silencing function.

4. The application according to claim 2, characterized in that, The shRNA comprises at least one sequence selected from the sequences shown in SEQ ID NO.7 to SEQ ID NO.12, or a variant that has at least 80% sequence identity with the sequence and retains the RUNX3 gene silencing function.

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