Use of an inhibitor of SIX1 expression in the preparation of a medicament for treating chronic obstructive pulmonary disease

Abstract

The present invention belongs to the technical field of biopharmaceuticals, and discloses the application of an expression inhibitor of SIX1 in the preparation of a medicament for treating chronic obstructive pulmonary disease. Targeted knockdown of SIX1 inhibits airway mucus hypersecretion and airway inflammation in chronic obstructive pulmonary disease by improving lung function, inhibiting the expression of SPDEF, inhibiting the transformation of epithelial Clara cells into goblet cells, reducing the production of mucin MUC5AC and the secretion of inflammatory factors IL6 and IL8, thus making a supplement to the pathogenesis of chronic obstructive pulmonary disease and providing a new strategy for its targeted treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to the application of an inhibitor of SIX1 expression in the preparation of a medicament for treating chronic obstructive pulmonary disease. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is characterized by progressive decline in lung function and irreversible limitation of airflow. It is the third leading cause of death in the world, with high morbidity and mortality, imposing a huge financial and social burden on society. Excessive airway mucus secretion is a prominent feature of COPD. The high COPD mortality is associated with increased airway mucus and impaired mucociliary clearance, which increases bacterial colonization and exacerbates disease progression. Goblet cell metaplasia is the main cause of the increase in the number of goblet cells in the airway. Goblet cell metaplasia refers to the process by which airway cells with differentiation potential (Clara cells) differentiate into terminal goblet cells. Among them, goblet cells are the main cells secreting mucus. Smoking is the main inducer of COPD. Smoking can induce goblet cell metaplasia, thereby promoting airway mucus hypersecretion and exacerbating airway inflammation. At present, the pathological mechanism of cigarette smoke-induced goblet cell metaplasia has not been determined. Therefore, it is necessary to explore the basic process of smoking regulating goblet cell differentiation to develop new treatment regimens for COPD-related airway mucus hypersecretion and airway inflammation.

[0003] Sine oculis homobox homolog 1 (SIX1) is a member of the homobox transcription factor family and is crucial for the expansion and differentiation of progenitor cell populations during development and organogenesis. SIX1 has a relatively conserved DNA binding sequence and can regulate multiple genes related to cell differentiation, proliferation, and inflammatory responses. SIX1 is crucial for the proliferation and differentiation of lung cells, as demonstrated by the abnormal lung morphogenesis, lung septum, and arterial remodeling in SIX1 - / - mice. Existing literature has shown that the pathogenesis of lung diseases such as those is affected by the upregulation of SIX1. For example, SIX1 promotes the invasion of lung cancer, and inhibiting SIX1 reduces allergic airway inflammation and remodeling while attenuating pulmonary fibrosis. However, the role of SIX1 in COPD airway epithelial differentiation and GCM is currently unclear. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide the application of an inhibitor of SIX1 expression in the preparation of a medicament for treating chronic obstructive pulmonary disease. Targeted knockdown of SIX1 provides a new strategy for its targeted treatment of chronic obstructive pulmonary disease by improving lung function and inhibiting airway mucus hypersecretion and airway inflammation in chronic obstructive pulmonary disease, and has good clinical application value.

[0005] To achieve the above object, the present application adopts the following technical solutions:

[0006] In a first aspect, the present invention provides the use of an inhibitor of SIX1 expression in the preparation of a medicament for treating chronic obstructive pulmonary disease.

[0007] In the above technical solution, the medicament is a medicament for inhibiting airway mucus hypersecretion or a medicament for inhibiting airway inflammation.

[0008] In the above technical solution, the inhibitor of SIX1 expression inhibits the transformation of epithelial Clara cells into goblet cells by inhibiting SPDEF expression, reduces the production of mucin MUC5AC and the secretion of inflammatory factors IL6 and IL8, thereby inhibiting airway mucus hypersecretion and airway inflammation.

[0009] In the above technical solution, the inhibitor of SIX1 expression is a reagent for targeting and silencing SIX1 or down-regulating the expression level of SIX1.

[0010] In the above technical solution, the reagent is siRNA of SIX1 or a virus interfering with SIX1 expression, and the nucleotide sequence of the siRNA is as shown in SEQ ID NO: 1, which is GCCAGGAGCTCAAACTATT.

[0011] In the above technical solution, the virus interfering with SIX1 expression is AAV-sh-SIX1.

[0012] In a second aspect, the present invention provides a pharmaceutical composition for treating chronic obstructive pulmonary disease, the pharmaceutical composition taking the component targeting and knocking down SIX1 expression in lung tissue as the active ingredient and containing a pharmaceutically acceptable carrier.

[0013] In the above technical solution, the component targeting and knocking down SIX1 expression in lung tissue is siRNA or a virus interfering with SIX1 expression, and the nucleotide sequence of the siRNA is as shown in SEQ ID NO: 1, which is GCCAGGAGCTCAAACTATT.

[0014] In the above technical solution, the dosage form of the pharmaceutical composition is an external dosage form or an internal dosage form.

[0015] In the above technical solution, the dosage form of the pharmaceutical composition is a patch, a paste, an ointment, a gel, a film-forming agent, a cataplasm, a spray, a capsule, a granule, a tablet, a pill, an oral liquid or an injection.

[0016] The beneficial effects of the present invention are as follows: Targeted knockdown of SIX1 inhibits airway mucus hypersecretion and airway inflammation in chronic obstructive pulmonary disease by improving lung function, inhibiting the expression of SPDEF, inhibiting the transformation of epithelial Clara cells into goblet cells, reducing the production of mucin MUC5AC and the secretion of inflammatory factors IL6 and IL8, thus making a supplement to the pathogenesis of chronic obstructive pulmonary disease and providing a new strategy for its targeted treatment. Brief Description of the Drawings

[0017] Figure 1 SIX1 knockout by AAV-sh-SIX1 alleviates cigarette smoke-induced airway mucus hypersecretion and airway inflammation in mice. (A) Intratracheal injection of AAV-sh-SIX1 increases the expression of SIX1 in the lung. Mice injected with AAV-sh-SIX1 or AAV-sh-scramble were exposed to cigarette smoke for three months. (B) Western blot analysis and semi-quantification of SIX1. (C) Pulmonary function (FEV0.1 / FVC) was measured. (D) Classification and counting of inflammatory cells in bronchoalveolar lavage fluid (BALF). (E-G) Hematoxylin-eosin (H&E) staining shows airway inflammation (E), mucin alcian blue and periodic acid-Schiff (AB-PAS) staining (E-F), and MUC5AC immunostaining and its semi-quantification (E, G). (H-I) ELISA was used to calculate the expression of inflammatory cytokines IL-6 and KC in BALF. CS: Cigarette smoke. Data are shown as mean ± SEM. , , One-way analysis of variance was used, and Newman-Keuls test was performed.

[0018] Figure 2 (A) Western blot analysis and semi-quantification of SIX1 protein after siRNA transfection. (B) RT-qPCR was used to detect the expression of MUC5AC mRNA. (C-D) MUC5AC immunostaining and semi-quantification. (E-F) ELISA was used to calculate the expression of inflammatory factors IL-6 and IL-8 in cell culture supernatant. CSE: Cigarette smoke extract. Con: Control. Lv: Lentivirus. NC: Negative control. Data are shown as mean ± SEM. , , One-way analysis of variance was used, and Newman-Keuls test was performed.

[0019] Figure 3(A-B) Western blot analysis and semi-quantification of SPDEF after overexpression or knockdown of SIX1. (C) Modified sequence of SIX1, (D) predicted binding site of SIX1 to SPDEF. (E-F) CHIP-qPCR verification of the binding of SIX1 to SPDEF. CSE: cigarette smoke extract. Lv: lentivirus. NC: negative control. CHIP: chromatin immunoprecipitation data are shown as mean ± SEM. , One-way ANOVA was used and Newman-Keuls test was performed. Detailed implementation manners

[0020] To better illustrate the objectives, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the present invention to those skilled in the art. The present invention will be defined only by the claims.

[0021] As used herein, "treatment" means that in the presence of factors (smoking) that may promote the decline of lung function and the occurrence and development of chronic obstructive pulmonary disease, the use can relieve the decline of lung function and the occurrence of airway mucus hypersecretion and airway inflammation in chronic obstructive pulmonary disease. The present invention has confirmed that targeted knockdown of SIX1 can inhibit the expression of SPDEF, inhibit the transformation of epithelial Clara cells into goblet cells, reduce the production of mucin MUC5AC and the secretion of inflammatory factors IL6 and IL8 by improving lung function, thereby inhibiting airway mucus hypersecretion and airway inflammation in chronic obstructive pulmonary disease. The relevant content makes a supplement to the pathogenesis of chronic obstructive pulmonary disease and also provides a new strategy for its targeted treatment. To better explain the present invention, the following will be described in detail in conjunction with specific embodiments.

[0022] Example 1 Using AAV-sh-SIX1 to knockdown SIX1 in mouse lung tissue to relieve cigarette smoke-induced lung function decline, airway mucus hypersecretion and airway inflammation

[0023] Experimental animals and materials:

[0024] 1. Experimental animals:

[0025] Source, strain, line: wild-type mice (WT, C57BL / 6) ordered from Hubei Branch of Jiangsu Jicui Yakang Experimental Animal Technology Co., Ltd.;

[0026] Reproductive age: 8-10 weeks old;

[0027] 2. Experimental materials:

[0028] Cigarettes: Purchased from Wuhan Promoter Biotechnology Co., Ltd.

[0029] Isoflurane: Purchased from Wuhan Promoter Biotechnology Co., Ltd.

[0030] AAV-sh-SIX1: Purchased from Shanghai Hiyuan Biotechnology Co., Ltd.

[0031] It should be noted that sh refers to short hairpin RNA, which includes two short inverted repeat sequences. The shRNA cloned into the shRNA expression vector includes two short inverted repeat sequences separated by a stem-loop sequence in the middle, forming a hairpin structure, which is controlled by the polⅢ promoter. Subsequently, 5-6 Ts are connected as the transcription terminator of RNA polymerase Ⅲ.

[0032] AAV refers to adeno-associated virus. The AAV-shRNA interference vector system is a very efficient vector tool that can stably interfere with the expression of target genes in various mammalian cells in vivo and in vitro. The immunogenicity of AAV is extremely low and it has almost no pathogenicity in vivo, making AAV an ideal tool for many animal studies.

[0033] Design an shRNA interference fragment targeting SIX1, and construct this interference fragment into an adeno-associated virus vector by molecular biological means. This vector can interfere with the SIX1 gene. In addition to directly transiently transfecting cells for SIX1 gene interference, this vector can also package adeno-associated virus to interfere with the expression of the SIX1 gene at the animal level. After infecting mice by airway injection of AAV-sh-SIX1, the expression of SIX1 in the lung tissue of mice can be reduced.

[0034] 3. Experimental methods:

[0035] 3.1 Mouse treatment

[0036] Wild-type mice were anesthetized by inhaling isoflurane, and then injected with AAV-sh-SIX1 virus or control virus through the airway. Two weeks later, whole-body smoke exposure was started, 5 days a week, 3 h per day (1.5 h in the morning and 1.5 h in the afternoon), and the exposure continued for six months ( Figure 1 A-1B). After the model was established, the lung function, airway mucus secretion and airway inflammation of each group of mice were evaluated.

[0037] 3.2 Immunofluorescence staining

[0038] (1)Preparation before experiment: Prepare a slide rack, an antigen retrieval box, a histochemistry pen, and a humid box, and preheat the oven to 60 °C. Prepare 1x EDTA antigen retrieval solution (pH = 9), 1x PBS phosphate buffer (add 2 L of pure water to one packet of PBS powder, and pay attention to fully dissolve and mix evenly), and 10% donkey serum. Ensure that there is enough reagent in each reagent dish for dewaxing in the fume hood.

[0039] (2)Dewaxing: Place the paraffin sections of human lung tissue on the slide rack, dry-bake the slide rack in the preheated oven for 45 - 60 min, quickly take out the slide rack and place it in the dish containing xylene I in the fume hood, and dewax the slides in the following order: xylene I (15 min) - xylene II (15 min) - absolute ethanol I (5 min) - absolute ethanol II (5 min) - 85% alcohol (5 min) - 75% alcohol (5 min). After that, take out the slide rack to the flowing distilled water outside the fume hood and rinse indirectly for 5 min.

[0040] (3)Antigen retrieval: Add 250 mL of 1x antigen retrieval solution to the antigen retrieval box, and place the slides rinsed with distilled water into the antigen retrieval box. Place the antigen retrieval box in the microwave oven for microwave retrieval. The retrieval process is as follows: medium-high heat for 5 min, stop for 5 min, medium heat for 5 min, stop for 5 min, medium-low heat for 5 min, stop for 5 min, and then let the slides cool naturally to room temperature in the retrieval solution (note that there is water evaporation during the microwave process, and avoid dry slides throughout the process).

[0041] (4)Blocking water with a histochemistry pen: Discard the antigen retrieval solution cooled to room temperature, wash the slides three times with PBS phosphate buffer on a shaker at room temperature, 5 min each time. Take out the slides and try to shake off the excess water, and draw a circle around the tissue block on the slides with a histochemistry pen (pay attention to preventing dry slides).

[0042] (5)Blocking: Drop 10% donkey serum into the tissue circle (ensure that the tissue block is completely covered), and place the slides in a humid box and incubate at room temperature for 45 - 60 min.

[0043] (6)Primary antibody incubation: According to the antibody instruction manual, prepare a primary antibody mixture of MUC5AC antibody and CC10 antibody with a primary antibody diluent. After the slides are blocked, shake off the blocking solution, and drop an appropriate amount of the diluted primary antibody mixture onto the tissue block (ensure that the tissue block is completely covered). Place the slides in a humid box and incubate at 4 °C overnight.

[0044] (7)Secondary antibody incubation: On the second day, take out the humid box and warm it to room temperature for 30 min. Discard the primary antibody, place the slides in an antigen retrieval box and wash them three times with PBS (phosphate buffered saline) on a shaker at room temperature for 5 min each time. Prepare a mixed solution of anti-rabbit fluorescent secondary antibody (1:200) and anti-mouse fluorescent secondary antibody (1:200) with PBS, drop the prepared mixed solution on the tissue sections (ensure complete coverage of the tissue sections) and incubate them in the humid box at room temperature in the dark for 1 h. After incubation, wash the slides three times with PBS on a shaker in the dark at room temperature for 5 min each time.

[0045] (8)DAPI nuclear staining: Take the slides and try to shake off the excess moisture, drop the ready-to-use DAPI staining solution in the tissue circle (ensure complete coverage of the tissue sections), place the slides in the humid box and incubate them at room temperature in the dark for 5 - 10 min. After incubation, wash the slides three times with PBS on a shaker in the dark at room temperature for 5 min each time.

[0046] (9)Cover slipping: Take the slides and try to shake off the excess moisture, drop an appropriate amount of anti-fluorescence quenching agent in the tissue circle and cover slip the slides.

[0047] (10)Observe the sections under a fluorescence microscope, collect images and perform data analysis.

[0048] 3.3 Alcian blue - Periodic acid - Schiff (AB - PAS) staining

[0049] (1)Deparaffinize the paraffin sections to water: The sections are successively deparaffinized with xylene for 10 min, replaced with fresh xylene for 10 min, absolute ethanol for 5 min, fresh absolute ethanol for 5 min, 90% ethanol for 5 min, 75% ethanol for 5 min, and then washed with tap water.

[0050] (2)Immerse the sections in AB - PAS stain C for 8 - 10 min, and wash with tap water until the running water on the slides is colorless.

[0051] (3)Immerse the sections in AB - PAS stain B for 10 - 15 min, and wash with pure water 3 times, about 10 s each time.

[0052] (4)Immerse the sections in AB - PAS stain A (pre - warmed to room temperature in advance) with a cover in the dark for 25 - 30 min, and rinse with running water for 5 min.

[0053] (5)Dehydrate the sections successively in three cylinders of absolute ethanol for 5 min each; clear with xylene for 5 min, replace with fresh xylene and clear for another 5 min, and then seal with neutral gum.

[0054] 3.4 Enzyme - linked immunosorbent assay (ELISA)

[0055] (1)Preparation before experiment: According to the instructions of the ELISA kits for mouse IL-6 and KC, dissolve, mix well and aliquot the capture antibody, detection antibody and standard in the kits, and store the aliquoted reagents in a -80 °C refrigerator for later use. Take out the supernatant of mouse BALF to be detected and thaw it at room temperature.

[0056] (2)Pre-coating the plate: Dilute the capture antibody stock solution into a working solution with Coating buffer, add it to a 96-well ELISA plate using a multi-channel pipette, seal the 96-well plate with a sealing film and centrifuge briefly, then incubate the 96-well plate overnight in a 37 °C incubator.

[0057] (3)Blocking: The next day, take out the ELISA auxiliary kit and thaw it at room temperature for 1 h. Prepare the blocking solution and washing solution according to the kit instructions. Take out the 96-well plate from the incubator, discard the liquid in the wells, add 300 μL of washing solution to the 96-well plate using a multi-channel pipette, let it stand at room temperature for 2 min, and wash 3 times in total (note to drain the washing solution in the wells each time). Add 300 μL of blocking solution to the 96-well plate, seal it with a sealing film, and incubate it in a 37 °C incubator for 1 h.

[0058] (4)Preparation of standard curve

[0059] ① Preparation before experiment: Thaw the standard stock solution, prepare 1.5 mL EP tubes numbered 1 - 6, and add 200 μL (if preparing the KC standard curve, add 500 μL) of Assay Diluent solution to each tube.

[0060] ② Preparation of mouse IL-6 standard curve: Take out the frozen IL-6 standard stock solution, thaw it, centrifuge it, and pipette 200 μL of the stock solution into EP tube 1, and serially dilute the standard as shown in the following figure.

[0061] ③ Preparation of mouse KC standard curve: Take out the frozen KC standard stock solution, thaw it, centrifuge it, and pipette 500 μL of the stock solution into EP tube 1, and serially dilute the standard according to the instructions.

[0062] (5)Sample addition: After the blocking of the 96-well plate is completed, remove the sealing film, discard the blocking solution, add 300 μL of washing solution to the 96-well plate using a multi-channel pipette, let it stand at room temperature for 2 min, and wash 3 times in total (note to drain the washing solution in the wells each time). Mix the sample and standard solution and centrifuge, then add the sample and standard to the dried 96-well plate in sequence, centrifuge briefly, and incubate the 96-well plate in a 37 °C incubator for 1 h.

[0063] (6) Detection antibody incubation: Take out the detection antibody storage solution, melt it and centrifuge briefly. Dilute the detection antibody into a working solution with the Assay Diluent solution. Take out the 96-well plate from the incubator, remove the sealing film, discard the blocking solution, add 300 μL of washing solution to the 96-well plate with a multichannel pipette, let it stand at room temperature for 2 min, and wash 3 times in total (note to drain the washing solution in the wells each time). Add 100 μL of the detection antibody working solution to the 96-well plate with a multichannel pipette, centrifuge briefly, and incubate the 96-well plate in a 37 °C constant temperature incubator for 1 h.

[0064] (7) HRP incubation: Dilute 40x HRP to 1x with the Assay Diluent solution (all operations after this step are carried out in a light-proof environment). Take out the 96-well plate from the incubator, remove the sealing film, discard the blocking solution, add 300 μL of washing solution to the 96-well plate with a multichannel pipette, let it stand at room temperature for 2 min, and wash 3 times in total (note to drain the washing solution in the wells each time). Add 100 μL of the 1x HRP working solution to the 96-well plate with a multichannel pipette, centrifuge briefly, and incubate the 96-well plate in a 37 °C constant temperature incubator for 30 min.

[0065] (8) Color reaction: Take out the 96-well plate from the incubator, remove the sealing film, discard the blocking solution, add 300 μL of washing solution to the 96-well plate with a multichannel pipette, let it stand at room temperature for 2 min, and wash 3 times in total (note to drain the washing solution in the wells each time). Mix Color A and Color B in the auxiliary kit at a ratio of 1:1, add 100 μL of the mixed solution to the 96-well plate with a multichannel pipette, centrifuge briefly, and incubate the 96-well plate in a 37 °C constant temperature incubator for 15 - 20 min. During this period, pay attention to observing the color changes in each well. When obvious blue color visible to the naked eye appears in the wells with a standard curve concentration of 31.3 pg / mL or 62.5 pg / mL, the reaction can be terminated.

[0066] (9) Reaction termination and reading: Turn on the full-wavelength microplate reader in advance and preheat it. Aspirate 50 μL of the reaction termination solution with a multichannel pipette and add it to the 96-well plate to terminate the color reaction. Place the 96-well plate in the microplate reader and read 3 times at wavelengths of 450 nm and 630 nm respectively (note to ensure there are no bubbles in the 96-well plate before reading).

[0067] (10) Draw a standard curve, calculate the concentration of IL-6 or KC in each sample according to the standard curve, and analyze the data.

[0068] 3.5 Collection of mouse bronchoalveolar lavage fluid (BALF)

[0069] (1)Preparation before experiment: Prepare ice, 1 mL syringe (lubricated with heparin), cotton pads and cotton balls, 1.5 mL EP tubes (lubricated with 40 μl heparin).

[0070] (2)Blood collection from the inferior vena cava: Remove the mouse from the mouse pulmonary function instrument, place the mouse supine on a foam board, fix the four limbs with tape, and fix the head with a rubber band around the teeth. Lift the abdominal skin with forceps and longitudinally cut the abdominal skin with scissors to fully expose the internal organs. Locate the inferior vena cava of the mouse and draw as much blood from the inferior vena cava as possible with a 1 mL syringe. The drawn blood is placed in a 1.5 mL EP tube and centrifuged at 980 rpm for 5 min in a 4 °C centrifuge. Take a new EP tube to collect the upper plasma, and freeze the plasma for later use.

[0071] (3)Heart perfusion: After blood collection, cut the inferior vena cava with scissors, cut open the thoracic cavity to fully expose the heart and lungs. Draw 4 mL of normal saline with a 5 mL syringe and flush the blood in the pulmonary circulation through the mouse heart as much as possible.

[0072] (4)Bronchoalveolar lavage: Draw normal saline with a 1 mL syringe and inject the normal saline into the mouse lung through the tracheal needle. Then wait for 1 min for the liquid to stay in the lungs and draw the liquid back. The normal saline is lavaged three times in total, 700 μL for the first time, 800 μL for the second time, and 800 μL for the third time. The liquid recovered from the first lavage is retained in an EP tube A. The liquid from the latter two lavages is retained in the same EP tube B. Centrifuge the two EP tubes at 980 rpm in a 4 °C centrifuge for 8 min. Recover the liquid in EP tube A, which is the bronchoalveolar lavage fluid of the mouse. Discard the liquid in EP tube B. Collect the cells at the bottom of EP tubes A and B for smear and Liu's staining.

[0073] 3.6 Collection and processing of mouse lung tissue specimens

[0074] (1)Preparation before experiment: Label 4 mL EP tubes and add 3 mL of tissue fixative. Prepare tissue grinding tubes A, B and cryopreservation tubes and pre-label them.

[0075] (2)Collection of mouse lung tissue specimens: After bronchoalveolar lavage, ligate the lingula of the lung and the right lung of the three lobes (upper lobe, middle lobe, lower lobe) with sutures. Draw 400 μL of tissue fixative with a 1 mL syringe and inject it into the left lung through the tracheal needle. Remove the left lung and place it in an EP tube containing tissue fixative, and seal the tube mouth with cotton. Cut off the lingula and the right lung. Among them, the lingula and the lower right lobe are placed in cryopreservation tubes. The upper right lobe is stored in grinding tube A, and the right middle lobe is stored in grinding tube B. The fixed mouse lung tissue is embedded and sectioned, and the specific steps are the same as those for human lung tissue embedding and sectioning. Among them, the lung tissue in grinding tube A is used for RNA extraction of mouse lung tissue. The lung tissue in grinding tube B is used for protein extraction of mouse lung.

[0076] 3.7 BALF Cell Cytospin and Liu's Staining

[0077] (1)Preparation before experiment: Prepare ice and number the glass slides.

[0078] (2)Lysis of red blood cells: Add 300 μL of red blood cell lysis buffer to EP tube B above, pipette and mix well. Aspirate the liquid in EP tube B into EP tube A and pipette and mix well. Place EP tube A on ice and incubate for 3 min. Then add 300 μL of PBS phosphate buffer to EP tube A to terminate the reaction. Place EP tube A in a centrifuge at 4 °C and centrifuge at 980 rpm for 5 min. After centrifugation, take out EP tube A, discard the supernatant, and add 80 - 240 μL of complete medium to grinding tube A and pipette and mix well.

[0079] (3)Cytospin: Turn on the microscope and count the cells in the cell suspension in EP tube A under the microscope. Adjust the cell density of the cell suspension in EP tube A to 250 cells / μL with complete medium. Number the glass slides and install them on the cytospin holder. Add 80 μL of the cell suspension to the cytospin holder, and place the cytospin holder in the cytospin centrifuge and spin at 980 rpm for 5 min.

[0080] (4)Liu's staining: Take out the spun cell glass slide, use a dropper to add 3 - 4 drops of Liu's stain A to the cell aggregation position on the slide and stain for 3 - 4 s. Then add 10 - 12 drops of Liu's stain B to the cell aggregation position and stain for 10 s. Place the slide under running water and rinse for 30 s. After air-drying the slide, add neutral resin to cover the slide.

[0081] (5)BALF Cell Cytospin and Liu's Staining: Take pictures of the sealed cell Liu's stain glass slides under an optical microscope (200x, randomly select and photograph 12 non-overlapping fields for each slide). After photographing, classify and count the cells for analysis.

[0082] 3.8 Western Blot (WB)

[0083] (1)Preparation before experiment: Prepare ice, pipettes with different ranges and tips, droppers, 50 mL centrifuge tubes, clean and air-dried gel combs, etc. Take out the samples and protein Marker and thaw them at room temperature on ice. Prepare sufficient long and short glass plates for gel preparation, gel clamps, and gel racks according to the specific experimental requirements.

[0084] (2)Leak detection before gel preparation: Clean the long and short glass plates for gel preparation. Clamp one long and one short glass plate with a gel clamp and fix them on the gel rack. Fill the gap between the long and short glass plates with pure water, let it stand for 10 - 15 min, and observe the liquid level drop. After determining that the liquid level does not drop significantly, proceed to the next step.

[0085] (3) Preparation of WB gel: Pour pure water between the glass plates and suck it dry with a clean filter paper. Prepare the stacking gel and separating gel in a 50 mL centrifuge tube according to the reagents in the rapid gel kit. First, use a pipette to add the mixed separating gel to an appropriate height between the glass plates, and then use another pipette to gently, slowly and evenly add the mixed stacking gel to fill the gap between the glass plates. Next, slowly insert the comb into the stacking gel. Let it stand for 15 - 20 min and wait for the separating gel and stacking gel to solidify.

[0086] (4) Preparation of electrophoresis buffer: Prepare a sufficient amount of electrophoresis buffer according to the specific experimental requirements, and pay attention to ensuring that various electrolytes are fully dissolved and mixed evenly.

[0087] (5) Loading samples: Carefully remove the comb from the stacking gel, remove the long and short glass plates and install them on the electrophoresis device. Add an appropriate amount of electrophoresis buffer to the electrophoresis tank, remove the foam on the surface of the electrophoresis tank, and fully expose the sample loading wells for convenient sample loading. After melting the samples and protein Marker and mixing them evenly on a vortex oscillator, centrifuge them for sample loading. The principle for calculating the loading volume is to ensure that the total mass of each sample loaded is the same. When the loading volumes in different wells of the same gel plate are different, use 1x loading buffer diluted with RIPA lysis buffer to make up to the same volume. At the same time, add 4 μL and 1 μL of protein Marker to the empty wells on the left and right sides of the samples respectively for indication. Connect the electrophoresis tank, electrophoresis cover and electrophoresis instrument, turn on the power supply, and start electrophoresis (note the connection direction of the electrodes. The starting electrophoresis voltage is 70 - 80 V. After the protein Marker bands are clearly separated, adjust the voltage to 110 - 120 V).

[0088] (6) Preparation of transfer buffer: Prepare a sufficient amount of transfer buffer according to the specific experimental requirements, and pay attention to ensuring that various electrolytes are fully dissolved and mixed evenly (methanol is volatile and should be added before using the transfer buffer).

[0089] (7) Preparation before transfer: Prepare the items required for transfer (transfer tray, transfer clip, sponge pad, filter paper, pipette, large and small steel rulers, glass spatula, exhaust roller and PVDF membrane, etc.). Add an appropriate amount of transfer buffer to the transfer tray, place the transfer clip in the transfer tray (the black side of the transfer clip is facing down and the white side is facing up), place a layer of sponge on each of the black and white sides of the transfer clip in turn, and 2 - 3 layers of filter paper (the amount of filter paper can be increased or decreased according to the actual situation, and it should be appropriate when the black and white sides of the transfer clip are clamped together). Use a pipette to suck the transfer buffer to moisten the sponge pad and filter paper, and use an exhaust roller to remove the air bubbles between the sponge pad and filter paper and between the filter papers.

[0090] (8)Transfer membrane: After electrophoresis, take out the glass plate from the electrophoresis tank, and use a glass lever to separate the long glass plate and the short glass plate (ensure that the gel remains on the short glass plate for convenient subsequent gel cutting). Use a small steel ruler to cut off the excess gel part (retain the molecular weight of 25 - 180 kD). After measuring the size of the cut gel, soak the gel in the transfer buffer to prevent it from drying out. Next, take out the PVDF membrane, cut a PVDF membrane of appropriate size according to the measured gel size, and soak it in methanol for activation (soaking time is greater than 10 s). Then, transfer the gel to the filter paper on the black side of the transfer cassette in sequence, and then transfer the activated PVDF membrane to the gel (a mark can be made on the non-gel contact surface of the PVDF membrane to ensure the distinction between the protein side and the non-protein side of the PVDF membrane). Then, place the filter paper and sponge pad on the white side of the transfer cassette on the PVDF membrane in sequence. Finally, close and fix the black and white sides of the transfer cassette. Note that each time after stacking, use an exhaust roller to exhaust air to avoid air bubbles between the filter paper and the gel, between the gel and the PVDF membrane, and between the PVDF membrane and the filter paper. Place the transfer cassette into the transfer tank, add cooling ice cubes to the transfer tank, and connect the transfer tank, transfer cover, and transfer instrument. Turn on the power and start the transfer (note: ensure that the electrode connection direction is correct, adjust the transfer current to 200 - 250 mA, and it is appropriate to maintain the voltage at 90 - 120 V. The transfer process lasts for 100 - 120 min. The entire transfer process is carried out at low temperature. You can make a self-made transfer ice tank, place the transfer tank in the ice tank, or directly carry out the transfer in a 4 °C laboratory. Pay attention to replacing the cooling ice cubes in the transfer tank halfway through the transfer. Check the voltage and current during the entire transfer process to avoid transfer failure caused by water ingress into the transfer tank, poor power contact, etc.).

[0091] (9)Solution preparation: Prepare TBST solution in advance (add a bag of TBS buffer dry powder and 1 mL of Tween 20 to 2 L of pure water) and 5% skim milk (add 5 g of skim milk powder to 100 mL of TBST solution to prepare 100 mL of 5% skim milk, note that it should be fully dissolved and mixed evenly). According to the instructions of anti-SXI1 antibody and anti-β-ACTIN antibody, dilute the SXI1 antibody (1:2000) and β-ACTIN antibody (1:4000) to the working solution with the primary antibody diluent. Pour the prepared primary antibody solutions into different small grids of the primary antibody incubation box respectively, and make marks. Add 4 mL of the primary antibody solution to each small grid of the conventional antibody incubation box.

[0092] (10)Blocking: After the transfer is completed, take out and open the transfer cassette, quickly put the PVDF membrane (the contact surface with the gel is the protein side of the PVDF membrane) into 5% skim milk, and block it on a shaker at room temperature for 1 h.

[0093] (11)Primary antibody incubation: The well-blocked PVDF membrane was washed three times with TBST solution on a shaker, with each wash lasting 5 - 10 minutes. The PVDF membrane was cut according to the target molecular weight, and the cut PVDF membrane was placed in a small compartment of the primary antibody incubation box corresponding to the target. Then, the primary antibody incubation box was placed on a shaker at 4 °C and incubated overnight.

[0094] (12)Secondary antibody incubation: On the second day, the primary antibody solution was recovered, and the PVDF membrane was washed three times with TBST solution at room temperature on a shaker, with each wash lasting 5 - 10 minutes. Then, 4 mL of 5% skim milk was added to the small compartment of the incubation box with the PVDF membrane, and 1 μL of the corresponding HRP-conjugated goat anti-rabbit or anti-mouse secondary antibody was added to the incubation compartment according to the species source of the primary antibody. The antibody incubation box was continued to be incubated on a shaker at room temperature for 1 hour. After incubation, it was washed three times again with TBST solution, with each wash lasting 5 - 10 minutes.

[0095] (13)Band development: The band developer was turned on in advance to preheat, and the filter of the developer was adjusted to the no-filter mode. According to the chemical luminescence solution preparation instructions, the luminescence solution was prepared at a ratio of reagent A to reagent B of 1:1 in a light-proof environment, and attention was paid to thorough mixing. Next, the luminescence solution was dropped on the luminescence plate, and then the PVDF strip was placed in the luminescence solution on the luminescence plate (note to ensure that the protein side of the PVDF membrane is facing up). After the strip was incubated for 1 - 2 minutes, the luminescence plate was placed in the developer and the position was adjusted. Immunoblotting was selected in the computer software, and after adjusting the exposure time, exposure was performed. Note to save the experimental data.

[0096] 4. Experimental results:

[0097] Figure 1 A shows the time points for intratracheal injection of AAV-sh-SIX1 or control virus and whole-body cigarette smoke exposure for modeling. Intratracheal injection of AAV-sh-SIX1 virus significantly reduced the expression of SIX1 in mouse lung tissue, indicating successful reduction of SIX1 expression in mouse lung tissue. Cigarette smoke intervention induced a decline in mouse lung function, and SIX1 knockdown could alleviate the lung function decline caused by cigarette smoke ( Figure 1 C); cigarette smoke intervention induced an increase in the total number of inflammatory cells and the number of various inflammatory cells (macrophages, neutrophils, and lymphocytes) in bronchoalveolar lavage fluid (BALF). Knockdown of lung SIX1 reduced the total number of inflammatory cells and macrophages and neutrophils in BALF ( Figure 1 D), and H&E staining showed that cigarette smoke intervention induced the aggregation of inflammatory cells around the airway, while knockdown of lung SIX1 prevented the infiltration of inflammatory cells into the airway ( Figure 1E). In addition, Alcian blue-periodic acid Schiff (AB-PAS) staining indicated that cigarette smoke intervention induced an increase in the secretion of airway epithelial mucus in mice (stained blue or bluish-purple by AB-PAS staining), and knockdown of lung SIX1 reduced epithelial mucus secretion ( Figure 1 E). Immunofluorescence staining indicated that cigarette smoke intervention induced an increase in the expression of epithelial mucin MUC5AC in mice (red), and knockdown of lung SIX1 reduced the expression of epithelial mucin MUC5AC ( Figure 1 G). Enzyme-linked immunosorbent assay (ELISA) found that cigarette smoke intervention induced a significant increase in the inflammatory factors KC and IL6 in the BALF of mice, and knockdown of SIX1 also reduced the expression of KC in the BALF ( Figure 1 H-1I).

[0098] Example 2 Using siRNA to knockdown SIX1 in airway epithelium alleviates cigarette smoke extract-induced epithelial mucin MUC5AC expression and inflammatory factor IL6 and IL8 secretion

[0099] To further explore the factors of knockdown of SIX1 on airway epithelial mucin expression and inflammatory factor secretion. We designed in vitro experiments as follows:

[0100] Experimental cells and materials:

[0101] 1. Experimental cells:

[0102] Bronchial epithelial cell line: The bronchial epithelial cell line used in this experiment was 16 HBE cells, purchased from ATCC, USA. 16 HBE cells were cultured in an incubator at 37 °C with a CO2 concentration of 5%.

[0103] 2. Experimental materials:

[0104] Cigarettes: Purchased from Wuhan Promoter Biotechnology Co., Ltd.

[0105] Cell culture medium: Purchased from Wuhan Promoter Biotechnology Co., Ltd.

[0106] Cell culture dishes: Purchased from Wuhan Promoter Biotechnology Co., Ltd.

[0107] Transfection reagent: Purchased from Invitrogen.

[0108] siRNA (SIX1): Purchased from Ribobio.

[0109] 3. Experimental methods:

[0110] Airway epithelial cells were cultured in vitro. The expression of SIX1 in airway epithelial cells was knocked down by transfection with siRNA. The effects of knockdown of SIX1 on cigarette smoke extract-induced airway epithelial mucin MUC5AC production and inflammatory factor IL6 and IL8 secretion were analyzed.

[0111] 3.1 Cell culture

[0112] (1) Culture of airway epithelial cell line - 16HBE cell line

[0113] ① Preparation before experiment: Prepare pipette tips and pipettes with different measuring ranges, 15 mL sterile centrifuge tubes, 50 mL sterile centrifuge tubes, cell cryopreservation tubes, T25 cell culture flasks, 12-well cell culture plates, and disinfect them by ultraviolet irradiation in the laminar flow hood. Take out fetal bovine serum, 1640 RPMI medium, PBS phosphate buffer, and trypsin, and warm them to room temperature. Set the constant temperature water bath to 37 °C.

[0114] ② Cell resuscitation: Add 5 mL of fetal bovine serum and 1640 RPMI medium to a 50 mL centrifuge tube to prepare a complete medium with a serum concentration of 10%. Take 10 mL of the complete medium and transfer it to a 15 mL sterile centrifuge tube for standby. Take out the cells cryopreserved in the liquid nitrogen tank, and keep shaking the cryopreservation tube in a 37 °C water bath until all the liquid has melted. Aspirate the cell suspension in the cryopreservation tube into a 15 mL centrifuge tube in the laminar flow hood. Centrifuge the 15 mL centrifuge tube at 980 rpm for 5 min in a normal temperature centrifuge. Discard the supernatant after centrifugation in the 15 mL centrifuge tube in the laminar flow hood, add 3 mL of complete medium to the tube, and pipette and mix the cells with a 1 mL pipette tip. Then, re-inoculate the mixed cell suspension into a culture flask (T25 culture flask) for culture, and place the culture flask in an incubator for culture.

[0115] ③Cell medium replacement and digestion for subculture: On the second day after cell resuscitation, observe the cell attachment and growth. Discard the supernatant in the culture flask in the laminar flow hood, wash the cells twice with 1 mL of PBS, and then add 3 mL of fresh complete medium to the culture flask. On the third day after cell resuscitation, perform cell subculture and plating. First, discard the cell supernatant in the laminar flow hood and wash the cells twice with 1 mL of PBS. Add 1 mL of trypsin to the culture flask and let it fully contact the cell surface. Place the culture flask in the incubator for 6 - 8 minutes. During this period, pay attention to observing the cell morphology and the detachment of cells from the bottom of the culture flask. When the cells become round and start to detach from the bottom, gently tap the culture flask. When most cells are observed to detach from the bottom of the cell under the microscope, add 2 mL of cell medium to the culture flask in the laminar flow hood to terminate the digestion. Use a 1 mL pipette tip to pipette the bottom of the cell flask to promote complete cell detachment. Collect the pipetted cell suspension into a 15 mL centrifuge tube, and centrifuge the 15 mL centrifuge tube at 980 rpm for 5 minutes in a room temperature centrifuge. Discard the supernatant after centrifugation in the 15 mL centrifuge tube in the laminar flow hood. The cells obtained at the bottom of the tube can be used for subculture, cryopreservation, or plating. If the cells need to be subcultured, add 4 mL of complete medium to the 15 mL centrifuge tube to resuspend the cells. Take 4 T25 cell culture flasks and add 2 mL of cell medium and 1 mL of cell suspension. If the cells need to be cryopreserved and plated, the specific steps are as follows.

[0116] ④Cell cryopreservation: Prepare cell cryopreservation solution (the composition of each 1 mL of cell cryopreservation solution is 100 μL of DMSO and 900 μL of fetal bovine serum). Add 1 mL of cell cryopreservation solution to the above 15 mL centrifuge tube with the supernatant discarded to resuspend the cells. Put the cell suspension into a cryopreservation tube, place the cryopreservation tube in a cryopreservation box and store it in an -80 °C refrigerator (the cryopreservation box can ensure gradient cooling of the cryopreservation tubes. After the cryopreservation box is stored in the -80 °C refrigerator for 24 hours, the cryopreservation tubes need to be stored in a liquid nitrogen tank in a timely manner).

[0117] ⑤Cell plating and intervention: Add 4 mL of cell medium to the above 15 mL centrifuge tube with the supernatant discarded to resuspend the cells. Pipette 100 μL of the cell suspension for microscopic cell counting. According to the counting results, add an appropriate amount of cell suspension to the cell culture plate (the total number of cells added to each well in a 12-well plate is 80,000, and the culture system for 12 wells is 1 mL). After plating, pay attention to observing the cell state and attachment. Replace the cell medium 12 hours after plating. When the cell growth confluence reaches 70% - 80%, cigarette smoke extract intervention treatment can be carried out.

[0118] 3.2 Preparation of cigarette smoke extract

[0119] (1) Preparation before the experiment: Dispense 20 mL of 1640 RMPI medium into a 50 mL centrifuge tube. Sterilize the high-pressure brown EP tube, 1 mL pipette tip, pipette, 0.22 μm sterile filter, 50 mL syringe and 50 mL centrifuge tube in a clean bench under ultraviolet light. Make a self-made negative pressure cigarette smoke suction device using a negative pressure suction machine, glass catheter and rubber tube, and connect the 3R4F standard cigarette and 15 mL centrifuge tube to the negative pressure cigarette smoke suction device.

[0120] (2) Preparation of cigarette smoke extract: Turn on the vacuum aspirator and light a cigarette. Use the vacuum aspirator to draw the smoke from the lit 3R4F cigarette into the 1640 RMPI medium in a 50 mL centrifuge tube. Allow the smoke to pass through the solution. Pay attention to the speed at which the smoke passes through the solution. Adjust the pressure to ensure that the bubbles are discharged from the liquid surface at a uniform speed. Note: Two cigarettes need to be dissolved in every 20 mL of 1640 RMPI medium. The solution sample obtained at this time is defined as a 100% cigarette smoke extract.

[0121] (3) Filtration, packaging and storage of cigarette smoke extract: In the cell clean bench, inhale the cigarette smoke extract with a 50 mL syringe, connect the syringe to a 0.22 μm sterile filter, and push the syringe to filter the cigarette smoke extract through the sterile filter. The filtered solution is packaged into brown EP tubes (the whole process must be kept away from light, and the packaged solution must be used as soon as possible).

[0122] 3.3 Collection of cell supernatant

[0123] (1) Preparation before the experiment: pre-cool the centrifuge, prepare and number 1.5 mL EP tubes, 1 mL pipette tips and pipettes, prepare ice and pre-cool PBS phosphate buffer on ice.

[0124] (2) Supernatant collection: Take out the cell culture plate from the incubator, pipette the culture fluid from each well of the culture plate into a 1.5 mL EP tube, centrifuge the EP tube at 980 rpm for 5 min, and collect 700 μL of supernatant into a new 1.5 mL EP tube after centrifugation. Store the collected supernatant in a -80 ℃ refrigerator for later use.

[0125] 3.4 Cell protein extraction, concentration determination and WB analysis

[0126] (1) Preparation before the experiment: prepare ice, prepare 1.5 mL EP tubes and protein tubes and number them, wash the egg whites, scrape them and bake them dry.

[0127] (2) Preparation of cell lysis buffer

[0128] (3)Cell lysis and protein scraping: Wash the cell culture plate from which the cell supernatant has been collected three times with 1 mL of pre-cooled PBS. For the last wash, try to aspirate as much PBS solution as possible. Add 80 μL of the prepared cell lysis solution to each well of the culture plate and let it stand on ice for 30 min for lysis. After sufficient lysis, use a protein scraper to scrape the protein from the wells in the culture plate and collect the scraped cells and cell lysis solution into a 1.5 mL EP tube.

[0129] (4)Protein solution sonication, concentration determination and WB analysis: The same as described above.

[0130] 3.5 Cell RNA extraction, concentration determination and RT-PCR analysis:

[0131] (1)Preparation before experiment: Prepare ice, prepare 1.5 mL enzyme-free EP tubes and number them, and pre-cool chloroform, absolute ethanol and isopropanol.

[0132] (2)RNA collection: Wash the cell culture plate from which the cell supernatant has been collected three times with 1 mL of pre-cooled PBS. For the last wash, try to aspirate as much PBS solution as possible. Add 1 mL of Trizol solution to each well of the culture plate in the biosafety cabinet and let it stand on ice for 10 - 20 min. Use a 1 mL pipette to pipette up and down repeatedly to blow the cells off the culture plate, and collect the pipetted solution into a 1.5 mL enzyme-free EP tube. Add 200 μL of pre-cooled chloroform to each EP tube. After sealing the tube, shake it vigorously by hand for 30 s and let it stand on ice for 15 min. After standing, centrifuge at 12000 rpm in a low-temperature centrifuge for 15 min.

[0133] (3)The subsequent steps are the same as those for human lung tissue RNA extraction, concentration determination, reverse transcription and RT-PCR analysis.

[0134] 3.6 Cell small interfering RNA transfection

[0135] (1)Preparation before experiment: Irradiate the pipettes, pipette tips of each range and 1.5 mL sterile EP tubes with ultraviolet light in the cell clean bench. Prepare ice, and melt the transfection reagent and small interfering RNA (siRNA) on ice. Warm the cell complete medium and Optim medium to room temperature.

[0136] (2)siRNA transfection:

[0137] ①Cell preparation: Prepare 16 HBE cells with good growth status for passage and plating (12-well plate). Transfection can be carried out when the cell growth density is 30% - 45%.

[0138] ② Preparation of transfection reagent: First, dissolve the powdered siRNA in ddH2O to a storage solution concentration of 20 μM. Prepare the single-well dosage of the transfection reagent for a 12-well plate according to the siRNA concentration gradient set below.

[0139] ③ Selection of siRNA concentration: The siRNA was purchased from Ribobio Co., Ltd. The siRNA of SIX1 is hereinafter referred to as si-SIX1, and the control siRNA is called si-Con. When performing siRNA transfection for the first time, set three siRNA concentration gradients of 20 nM, 40 nM, and 80 nM to transfect the cells. Replace the cell culture medium 24 h after siRNA transfection. RNA or protein of the transfected cells can be extracted 72 h after transfection, and the transfection efficiency of siRNA can be analyzed by RT-PCR or WB. Select the siRNA concentration that has no obvious effect on cell growth status and can significantly reduce the expression of SIX1 for the next experiment (the siRNA concentration selected in this experiment is 80 nM).

[0140] ④ Cell transfection: Aspirate the culture medium from a 12-well plate with a cell growth density of 30% - 45%, and wash the cells 2 times with PBS. Add 450 μL of complete culture medium to each well of the cells. Add 50 μL of the above-prepared transfection reagent (prepared according to the siRNA concentration of 80 nM) to each well of the cells.

[0141] 3.7 WB experimental procedure

[0142] Same as Example 1.

[0143] 4. Experimental results:

[0144] Knockdown of SIX1 alleviates the increase in airway epithelial mucin MUC5AC and the increase in the secretion of inflammatory factors IL6 and IL8 induced by cigarette smoke extract.

[0145] Example 3 Inhibition of SIX1 reduces epithelial inflammation and epithelial mucin secretion by transcriptional inhibition of SPEDF expression

[0146] To further explore the mechanism by which knockdown of SIX1 affects airway epithelial mucin expression and inflammatory factor secretion. We conducted further in vitro experiments as follows:

[0147] Experimental cells and materials:

[0148] 1. Experimental cells:

[0149] Bronchial epithelial cell line: The bronchial epithelial cell line used in this experiment is 16HBE cells, purchased from ATCC, USA. 16HBE cells are cultured in an incubator at 37 °C with a CO2 concentration of 5%.

[0150] 2. Experimental materials:

[0151] Cigarettes: Purchased from Wuhan Promoter Biotechnology Co., Ltd.

[0152] Cell culture medium: Purchased from Wuhan Promoter Biotechnology Co., Ltd.

[0153] Cell culture dishes: Purchased from Wuhan Promoter Biotechnology Co., Ltd.

[0154] Transfection reagent: Purchased from Invitrogen.

[0155] siRNA (SIX1): Purchased from Ribobio.

[0156] SIX1 lentivirus (Lv-SIX1): Purchased from Shanghai Genechem Co., Ltd.

[0157] Co-immunoprecipitation kit: Purchased from CST

[0158] 3. Experimental methods:

[0159] Airway epithelial cells were cultured in vitro. The expression of SIX1 in airway epithelial cells was knocked down by transfection with siRNA. The expression of SIX1 in airway epithelial cells was promoted by transfection with Lv-SIX1. The effects of knocking down and overexpressing SIX1 on the expression of the transcription factor SPDEF in airway epithelium induced by cigarette smoke extract were analyzed respectively. Meanwhile, the mechanism by which SIX1 regulates the expression of SPDEF was explored.

[0160] 3.1 Cell culture

[0161] Same as Example 2.

[0162] 3.2 Preparation of cigarette smoke

[0163] Same as Example 2.

[0164] 3.3 Transfection of small interfering RNA into cells

[0165] Same as Example 2.

[0166] 3.4 WB experimental procedure

[0167] Same as Example 1.

[0168] 3.5 Transfection of lentivirus into cells

[0169] (1) Preparation before experiment: The guns and gun tips of each range were irradiated with ultraviolet light in the cell ultra-clean bench.

[0170] (2) Cell preparation: The cell suspension after digestion was inoculated into a 6-well plate, and the number of cells inoculated in each well was about 100,000. The 6-well plate was observed and the medium was changed on the second day. When the cell confluence reached about 70%, the cells could be transfected with the virus.

[0171] (3) Lentivirus transfection pre-experiment: The lentivirus was purchased from Shanghai Heyuan Biotechnology. The virus for overexpressing SIX1 is hereinafter referred to as Lv-SIX1, and the control virus is called Lv-con. When performing the first virus transfection, virus transfection was carried out with virus concentration gradients of 0 MOI, 20 MOI, 40 MOI, and 80 MOI. The medium was changed according to the cell status 12 - 24 h after virus transfection. The fluorescence intensity could be observed under a fluorescence microscope 48 h after virus transfection to preliminarily determine the virus transfection efficiency. The RNA or protein of the cells transfected with the virus could be extracted 72 h after virus transfection, and the virus transfection efficiency was analyzed by RT-PCR or WB. The virus concentration that had no obvious effect on cell growth status and could significantly overexpress SXI1 was selected for the next experiment (the virus concentration selected in this experiment was 40 MOI).

[0172] (4) Virus transfection and establishment of SXI1 overexpressing stable cell line: The virus was transfected in a 6-well plate at a concentration of 40 MOI. The medium was changed 12 h after transfection. The medium was changed 48 h after transfection, and complete medium containing 4 μg / mL puromycin was added to the 6-well plate. The cells were passaged 72 h after transfection. The passaged cells were the cells stably overexpressing SXI1 and their control cells.

[0173] 3.6 Chromatin Immunoprecipitation (ChIP)

[0174] (1) Cell preparation

[0175] ① Preparation before experiment: 15 cm culture dish, 16HBE cells in good growth state.

[0176] ② Cell transfection: Digest the 16HBE cells, and inoculate the cell suspension into two different 15 cm culture dishes (added with 20 mL of complete medium for culture), and perform virus transfection of Lv-SIX1 (transfected into culture dish A) and Lv-con (transfected into culture dish B). The specific transfection method is the same as described above. When the cell confluence in the culture dish reaches 100%, cell sample crosslinking can be carried out.

[0177] (2) Cell sample crosslinking

[0178] ① Preparation before experiment: Prepare ice, 15 mL centrifuge tubes. Take out the 100x protease inhibitor cocktail (PIC) and 1x glycine solution in the ChIP kit and warm them to room temperature (note to ensure that the PIC is completely dissolved). Wash and dry the protein scraper. Pre-cool the PBS phosphate buffer.

[0179] ② Preparation of reagents required for the experiment: Add 20 μL of 200x PIC to 4 mL of PBS.

[0180] ③ Cell cross-linking: Take out two 15-cm culture dishes from the incubator, add 540 μL of 37% formaldehyde solution to each culture dish, rotate the culture medium to mix the formaldehyde with the culture medium, and let it stand at room temperature for 10 min. Then add 2 mL of glycine to each culture dish, rotate the culture dish to mix the solution, and incubate at room temperature for 5 min to terminate the reaction. After discarding the liquid in the culture dish, rinse the cells twice with 20 mL of pre-cooled PBS. Add 2 mL of the prepared PBS containing PIC to the culture dish, scrape the cells from the culture dish with a cell scraper, and collect the scraped cells in a 15-mL centrifuge tube (denoted as centrifuge tubes A and B respectively). Adjust the centrifuge to 4°C and the centrifugal force to 2000 g, centrifuge centrifuge tubes A and B for 5 min to collect the cells, remove the supernatant after centrifugation, and proceed to the next step.

[0181] (3)Nucleus treatment and chromatin shearing

[0182] ① Preparation before the experiment: Take out the DTT reagent in the ChIP kit, add 1.12 mL of dH2O to it, and dissolve and mix it well. Take out the 10x ChIP buffer and warm it to room temperature. Adjust the water bath to 37°C.

[0183] ② Preparation of reagents required for the experiment:

[0184] a. Prepare 6 mL of 1x buffer A (add 1.5 mL of 4x buffer A to 4.5 mL of water), and add 3 μL of DTT and 30 μL of 200x protease inhibitor cocktail (PIC), and pre-cool on ice.

[0185] b. Prepare 6.6 mL of 1x buffer B (add 1.65 mL of 4x buffer B to 4.95 mL of water), and add 3.3 μL of DTT, and pre-cool on ice.

[0186] c. Prepare 600 μL of 1x ChIP buffer (add 60 μL of 10x ChIP buffer to 540 μL of water), and add 3 μL of PIC, and pre-cool on ice.

[0187] ③ Nucleus treatment

[0188] a. Add 3 mL of 1x buffer A to centrifuge tubes A and B respectively, incubate on ice for 10 min, and invert the centrifuge tubes every 3 min for mixing.

[0189] b. Centrifuge tubes A and B at 4°C, 2000 g for 5 min to pellet the nuclei. Discard the supernatant and add 3 mL of buffer B to each tube to resuspend the pellet. Centrifuge again and discard the supernatant. Resuspend the pellet in 300 µL of buffer B and transfer the resuspended solution to 15 mL EP tubes A and B.

[0190] ④Chromatin shearing

[0191] a. Add 1.2 µL of micrococcal nuclease to EP tubes A and B, invert to mix, and incubate at 37°C for 20 min, inverting to mix every 3-5 min.

[0192] b. After incubation, add 30 µL of 0.5 M EDTA to EP tubes A and B to terminate digestion.

[0193] c. Centrifuge EP tubes A and B at 4°C, 16,000 g for 1 min and discard the supernatant.

[0194] d. Resuspend the nuclei in 300 µL 1x ChIP buffer and incubate on ice for 10 min.

[0195] e. Fill the volume of the solution in EP tubes A and B to 500 μL with 1x ChIP buffer, then insert EP tubes A and B into ice and sonicate the samples (the probe is 3 mm, the power is 30 W, each sonication is 30 s, a total of 3 times, each time with an interval of 2 min.)

[0196] f. After the ultrasound is completed, centrifuge EP tubes A and B at 4°C at 9,400 g for 10 min to remove the cell nuclear fragments in the sample.

[0197] g. Transfer the supernatant in EP tubes A and B to new EP tubes A and B. The supernatant is the cross-linked chromatin fragment sample. Take 50 µL of chromatin sample from new EP tubes A and B and place it in EP tubes C and D. This part of the sample is used to analyze the enzyme digestion of DNA and determine its concentration. The remaining samples in the new EP tubes A and B are stored at -80°C.

[0198] (4) Analysis of chromatin concentration and enzyme digestion:

[0199] ①Add 100 µL DEPC water, 6 μL 5 M NaCl solution and 2 μL RNase A to EP tubes C and D respectively. Vortex and mix well, then incubate at 37°C for 30 min. After incubation, adjust the water bath to 65°C.

[0200] ② After incubation, add 2 μL of Proteinase K to the sample digested with RNase A. After vortexing to mix evenly, incubate at 65°C for 2 h.

[0201] ③ Purify DNA using a centrifugal column

[0202] a. Add 750 μL of DNA binding buffer to EP tubes C and D respectively, and gently shake with a vortex mixer.

[0203] b. Insert the DNA centrifugal column into the collection tube and label it (centrifugal tubes C and D). First, aspirate 450 μL of the liquid from EP tubes C and D respectively and add it to centrifugal columns C and D.

[0204] c. Centrifuge centrifugal tubes C and D at 18,500 g for 30 s at room temperature.

[0205] d. Remove centrifugal columns C and D from collection tubes C and D respectively. After discarding the waste liquid, insert the centrifugal columns back into the collection tubes.

[0206] e. Transfer the remaining 450 μL of the sample in EP tubes C and D to the corresponding centrifugal columns, and repeat steps 3 and 4. At this time, discard EP tubes C and D.

[0207] f. Add 750 μL of DNA wash buffer to centrifugal columns C and D.

[0208] g. Centrifuge centrifugal columns C and D at 18,500 g for 30 s at room temperature.

[0209] h. Remove the centrifugal column from the collection tube and discard the waste liquid. Insert the centrifugal column back into the collection tube.

[0210] i. Centrifuge centrifugal columns C and D at 18,500 g for 30 s at room temperature.

[0211] j. Remove centrifugal columns C and D from collection tubes C and D respectively. Discard the collection tubes and waste liquid. Take two new 1.5 mL enzyme-free EP tubes and re-label them as EP tubes C and D. Insert centrifugal columns C and D into EP tubes C and D respectively.

[0212] k. Add 50 μL of DNA elution buffer to each of centrifugal columns C and D, and let it stand at room temperature for 2 min.

[0213] l. Place the centrifugal column and the EP tube together in the centrifuge and centrifuge at 18,500 g for 2 min to elute the DNA.

[0214] m. Discard the centrifugal column. The eluates in EP tubes C and D are the purified DNA of Lv-SIX1 transfected cells and Lv-Con transfected cells respectively.

[0215] ④DNA concentration determination: First, ensure that the Nanodrop 2000 is connected to the computer power supply. Turn on the computer software Nanodrop 2000 and select Nucleic Acid - DNA. Then, aspirate 1 μL of the eluate to wash the instrument probe 2 times, and then use 1 μL of the eluate to zero the instrument. Finally, aspirate 1 μL of the mixed DNA sample in an EP tube for concentration detection, and note and save the DNA concentrations of all samples (generally between 50 and 200 μg / mL).

[0216] ⑤Identification of digestion effect: After the DNA samples are purified, take 5 μL of the samples from EP tubes C and D respectively and mix them with 5 μL of 2x DNA loading buffer. Then, perform nucleic acid gel electrophoresis on the mixed samples (using a 2% agarose gel, the specific preparation method is the same as described above; 100 bp DNA ladder is used as a Marker) to determine the DNA fragment size.

[0217] (5)Chromatin immunoprecipitation:

[0218] ①Preparation before experiment: Prepare ice, take out the sheared chromatin, that is, the new EP tubes A and B above, and melt them on ice. Take out the 100x protease inhibitor cocktail (PIC) in the ChIP kit (note to ensure that the PIC is completely dissolved). Preheat the 10x ChIP buffer to ensure that the SDS in it is completely dissolved.

[0219] ②Solution preparation

[0220] a. Prepare low-salt wash buffer: Prepare 18 mL of 1x ChIP buffer (1.8 mL of 10x ChIP buffer plus 2.7 mL of water), and store it at room temperature before use.

[0221] b. Prepare high-salt wash buffer: Prepare 6 mL of 1x ChIP buffer (100 μL of 10x ChIP buffer plus 900 μL of water) and add 70 μL of 5M NaCl, and store it at room temperature before use.

[0222] c. Prepare 1x ChIP buffer: Prepare 2.4 mL of 1x ChIP buffer (240 μL of 10x ChIP buffer plus 2.16 mL of water), and add 12 μL of PIC, mix well and place it on ice.

[0223] ③Immunoprecipitation

[0224] a. Take three EP tubes labeled A1, A2, and A3, and add 8 μg of chromatin from tube A to each of the EP tubes A1, A2, and A3 (calculate the dosage according to the measured chromatin concentration). Take three EP tubes labeled B1, B2, and B3, and add 8 μg of chromatin from tube B to each of the EP tubes B1, B2, and B3 (calculate the dosage according to the measured chromatin concentration). Make up the volume of EP tubes A1, A2, A3, B1, B2, and B3 to 500 μL with diluted 1x ChIP buffer (the volume to be added is 500 μL minus the chromatin dosage). Appropriate PIC can be added, and mix well and pre-cool on ice.

[0225] b. Pipette 10 μL of sample from each of the EP tubes A1, A2, and A3 and store it in EP tube G; pipette 10 μL of sample from each of the EP tubes B1, B2, and B3 and store it in EP tube H. In subsequent experiments, 10 μL of the sample in EP tube G or H can be taken as the 2% sample input control (Input) for the Lv-SIX1 group and the Lv-Con group, respectively. EP tubes G and H can be stored at -20°C before subsequent use.

[0226] c. Add 10 μL of anti-SIX1 antibody to EP tubes A1 and B1; add 10 μL of anti-histone H3 antibody to EP tubes A2 and B2; add 1 μL of anti-IgG antibody to EP tubes A3 and B3. Seal the reaction tubes with antibody added with sealing film and incubate overnight on a rotor at 4°C (each EP tube is an immunoprecipitation reaction).

[0227] d. Gently resuspend and mix well the ChIP protein magnetic beads G, and add 30 μL of protein magnetic beads G to each of the EP tubes A1, A2, A3, B1, B2, and B3. Seal the tube mouths with sealing film and incubate the EP tubes on a rotor at 4°C for 2 h.

[0228] e. Place all EP tubes on a magnetic separation rack to adsorb the protein magnetic beads G to the tube walls. After waiting for 1 - 2 min until the solution is clear, carefully aspirate the supernatant. Retain the supernatant.

[0229] f. Add 1 mL of low-salt wash buffer, and remove all EP tubes from the magnetic separation rack. Rotate and incubate on a rotor at 4°C for 5 min. Repeat steps e and f above 2 more times, that is, wash a total of 3 times with low-salt wash buffer.

[0230] g. Add 1 mL of high-salt wash buffer, and remove all EP tubes from the magnetic separation rack. Rotate and incubate on a rotor at 4°C for 5 min. Then place it back on the magnetic separation rack. After waiting for 1 - 2 min until the solution is clear, carefully aspirate the supernatant, and then immediately proceed to part (6).

[0231] (6) Elute the chromosomes from the antibody / protein G beads and reverse crosslink

[0232] ① Preparation before the experiment: Prepare ice, take out EP tubes G and H and let them thaw on ice. Preheat 2x ChIP elution buffer in a 37°C water bath and confirm that SDS has completely dissolved. Set the water bath temperature to 65°C.

[0233] ② Preparation of 1x ChIP elution buffer: Prepare 1.2 mL of 1x ChIP elution buffer (add 600 µL of water to 600 µL of 2x ChIP elution buffer).

[0234] ③ Experimental steps:

[0235] a. Transfer 10 µL of the samples in EP tubes G and H to EP tubes I and L respectively as 2% sample input control (2% Input). Add 150 µL of 1x ChIP elution buffer to each of EP tubes I and L. Let stand at room temperature until step 6.

[0236] b. Add 150 μL of 1 X ChIP elution buffer to EP tubes A1, A2, A3, B1, B2, and B3 in (5) respectively.

[0237] c. Gently shake all EP tubes with a vortex mixer (1200 rpm) and then incubate in a 65°C water bath for 30 min to elute chromatin from the antibody / protein G microspheres.

[0238] d. Place all EP tubes on a magnetic separation rack to adsorb protein G magnetic beads and wait for 1 - 2 min until the solution is clear.

[0239] e. Carefully transfer the eluted chromatin supernatant in each sample tube to a new centrifuge tube and label them respectively.

[0240] f. Add 6 µL of 5 M NaCl and 2 µL of proteinase K to EP tubes A1, A2, A3, B1, B2, B3, G, and H and incubate at 65°C for 2 h. Proceed to the next step immediately after incubation of the samples.

[0241] (7)Purification of DNA by centrifugal column: The specific method is the same as described above.

[0242] (8)Evaluation of enrichment efficiency by RT-PCR

[0243] ① Preparation before the experiment: Prepare ice, take out the centrifugally purified DNA and let it warm up to room temperature. Prepare sterile and enzyme-free ddH2O, autoclaved pipette tips, pipettes of various ranges, RT-PCR reagents, and primers.

[0244] ② Dilute the DNA obtained by immunoprecipitation 5-fold with ddH2O.

[0245] ③The RT-PCR analysis method was the same as described above (the same as in Example 1).

[0246] 4. Experimental results:

[0247] Knockdown of SIX1 reduced the expression of the transcription factor SPDEF in airway epithelium induced by cigarette smoke extract. Overexpression of SIX1 promoted the expression of the transcription factor SPDEF in airway epithelium induced by cigarette smoke extract. SXI1 promoted its transcription and translation by binding to the SPDEF promoter region.

[0248] Obviously, the above embodiments are only examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. Use of an expression inhibitor of SIX1 in the preparation of a drug for treating chronic obstructive pulmonary disease, wherein the expression inhibitor of SIX1 is an agent that targets silencing SIX1 or downregulating the expression of SIX1; The reagent is siRNA of SIX1, and the nucleotide sequence of the siRNA is shown in SEQ ID NO: 1, which is GCCAGGAGCTCAAACTATT.

2. The application according to claim 1, characterized in that: The drug is a drug for inhibiting airway mucus hypersecretion or a drug for inhibiting airway inflammation.

3. The application according to claim 1, characterized in that: The SIX1 expression inhibitor inhibits the transformation of epithelial Clara cells into goblet cells by inhibiting the expression of SPDEF, reducing the production of mucin MUC5AC and the secretion of inflammatory factors IL6 and IL8, thereby inhibiting airway mucus hypersecretion and airway inflammation.

4. Use of a pharmaceutical composition in the preparation of a drug for treating chronic obstructive pulmonary disease, characterized in that: The pharmaceutical composition has a component that targets and knocks down the expression of SIX1 in lung tissue as an active ingredient, and comprises a pharmaceutically acceptable carrier; The component for targeted knockdown of SIX1 expression in lung tissue is siRNA, and the nucleotide sequence of the siRNA is shown in SEQ ID NO: 1, which is GCCAGGAGCTCAAACTATT.

5. The application according to claim 4, characterized in that: The dosage form of the pharmaceutical composition is an external dosage form or an internal dosage form.

6. The application according to claim 4, characterized in that: The dosage form of the pharmaceutical composition is a patch, paste, ointment, gel, film coating, spray, capsule, granule, tablet, pill, oral liquid or injection.

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