Application of SIX1 expression inhibitor in preparation of medicine for treating chronic obstructive pulmonary disease

Abstract

The invention belongs to the technical field of biological pharmacy, and discloses application of an SIX1 expression inhibitor in preparation of a medicine for treating chronic obstructive pulmonary disease. Targeted knock-down SIX1 inhibits high secretion of airway mucus and airway inflammation of the chronic obstructive pulmonary disease by improving lung functions, inhibiting SPDEF expression, inhibiting transformation of epithelial Clara cells to goblet cells and reducing production of mucoprotein MUC5AC and secretion of inflammatory factors IL6 and IL8, supplements are made for pathogenesis of the chronic obstructive pulmonary disease, and the targeted knock-down SIX1 has a good application prospect. And a new strategy is provided for targeted therapy.

Description

Technical Field

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

[0002] Chronic obstructive pulmonary disease (COPD) is characterized by a 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, which imposes enormous financial and social pressures on society. Excessive airway mucus secretion is a prominent feature of COPD. 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 airways. Goblet cell metaplasia refers to the process in which cells with differentiation potential (Clara cells) in the airways differentiate into terminal goblet cells. Goblet cells are the main cells that secrete mucus. Smoking is the main cause of COPD. Smoking can induce goblet cell metaplasia, thereby promoting airway mucus hypersecretion and exacerbating inflammation along the airways. At present, the pathological mechanism of goblet cell metaplasia induced by cigarette smoke has not been determined. Therefore, it is necessary to explore the basic processes by which smoking regulates goblet cell differentiation in order to develop new treatment options for COPD-associated airway mucus hypersecretion and airway inflammation.

[0003] Sine oculis homobox homolog 1 (SIX1) is a member of the homobox transcription factor family and is essential for the expansion and differentiation of progenitor cell populations during development and organogenesis. SIX1 has a relatively conserved DNA-binding sequence and regulates a variety of genes related to cell differentiation, proliferation, and inflammatory responses. SIX1 is essential for the proliferation and differentiation of pneumocytes, as evidenced by abnormal lung morphogenesis, septal and arterial remodeling in SIX1- / - mice. Existing literature has shown that processes such as lung disease pathogenesis are affected by upregulation of SIX1. For example, SIX1 promotes lung cancer invasion, and inhibition of SIX1 reduces allergic airway inflammation and remodeling, while attenuating pulmonary fibrosis. However, the role of SIX1 in airway epithelial differentiation and GCM in COPD is still unclear. Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide an application of an expression inhibitor of SIX1 in the preparation of a drug for the treatment of chronic obstructive pulmonary disease. Targeted knockdown of SIX1 improves lung function and inhibits airway mucus hypersecretion and airway inflammation in chronic obstructive pulmonary disease, providing a new strategy for the targeted treatment of chronic obstructive pulmonary disease, and has good clinical application value.

[0005] In order to achieve the above purpose, this application adopts the following technical solutions: In a first aspect, the present invention provides use of an expression inhibitor of SIX1 in the preparation of a medicament for treating chronic obstructive pulmonary disease.

[0006] In the above technical solution, the drug is a drug that inhibits airway mucus hypersecretion or a drug that inhibits airway inflammation.

[0007] In the above technical scheme, 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.

[0008] In the above technical solution, the SIX1 expression inhibitor is a reagent that targets silencing SIX1 or downregulating the expression level of SIX1.

[0009] In the above technical solution, the reagent is siRNA of SIX1 or a virus interfering with the expression of SIX1, and the nucleotide sequence of the siRNA is shown in SEQ ID NO: 1, which is GCCAGGAGCTCAAACTATT.

[0010] In the above technical solution, the virus that interferes with the expression of SIX1 is AAV-sh-SIX1.

[0011] In a second aspect, the present invention provides a pharmaceutical composition for treating chronic obstructive pulmonary disease, wherein the pharmaceutical composition comprises a component that targets and knocks down SIX1 expression in lung tissue as an active ingredient, and comprises a pharmaceutically acceptable carrier.

[0012] In the above technical solution, the component for targeted knockdown of SIX1 expression in lung tissue is siRNA or a virus interfering with SIX1 expression, and the nucleotide sequence of the siRNA is shown in SEQ ID NO: 1, which is GCCAGGAGCTCAAACTATT.

[0013] In the above technical solution, the dosage form of the pharmaceutical composition is an external dosage form or an internal dosage form.

[0014] In the above technical scheme, the dosage form of the pharmaceutical composition is a patch, paste, ointment, gel, film coating, cataplasm, spray, capsule, granule, tablet, pill, oral liquid or injection.

[0015] The beneficial effects of the present invention are: targeted knockdown of SIX1 improves lung function, inhibits SPDEF expression, inhibits the transformation of epithelial Clara cells to goblet cells, reduces the production of mucin MUC5AC and the secretion of inflammatory factors IL6 and IL8, thereby inhibiting the hypersecretion of airway mucus and airway inflammation in chronic obstructive pulmonary disease, supplements the pathogenesis of chronic obstructive pulmonary disease, and provides a new strategy for its targeted treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Knockdown of SIX1 using AAV-sh-SIX1 alleviates smoking-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) Determination of lung function (FEV0.1 / FVC). (D) Classification and counting of inflammatory cells in bronchoalveolar lavage fluid (BALF). (E-G) Hematoxylin-eosin (H&E) staining showing airway inflammation (E), mucus Alcian blue and periodic acid-Schiff (AB-PAS) staining (E-F) and MUC5AC immunostaining and its semi-quantification (E, G). (HI) Expression of inflammatory cytokines IL-6 and KC in BALF was calculated by ELISA. CS: cigarette smoke. Data are shown as mean ± SEM. , , One-way analysis of variance was used, followed by the Newman-Keuls test.

[0017] Figure 2 (A) Western blot analysis and semi-quantification of SIX1 protein after siRNA transfection. (B) RT-qPCR detection of MUC5AC mRNA expression. (CD) MUC5AC immunostaining and semi-quantification. (EF) ELISA method to calculate the expression of inflammatory factors IL-6 and IL-8 in cell culture supernatant. CSE: cigarette smoke extract. Contra: control. Lv: lentivirus. NC: negative control. Data are shown as mean ± SEM. , , One-way analysis of variance was used, followed by the Newman-Keuls test.

[0018] Figure 3(AB) Western blot analysis and semi-quantification of SPDEF after SIX1 overexpression or knockdown. (C) Modified sequence of SIX1, (D) Predicted binding site of SIX1 and SPDEF. (EF) CHIP-qPCR validation of SIX1 binding on SPDEF. CSE: cigarette smoke extract. Lv: lentivirus. NC: negative control. CHIP: chromatin immunoprecipitation Data are shown as mean ± SEM. , One-way analysis of variance was used, followed by the Newman-Keuls test. DETAILED DESCRIPTION

[0019] In order to better illustrate the purpose, 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 being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0020] The "treatment" mentioned in the present invention refers to the alleviation of lung function decline and the occurrence of airway mucus hypersecretion and airway inflammation in chronic obstructive pulmonary disease after use in the presence of possible factors that promote the development of lung function decline and chronic obstructive pulmonary disease (smoking). The present invention confirms through the following examples that targeted knockdown of SIX1 improves lung function, inhibits SPDEF expression, inhibits the transformation of epithelial Clara cells to goblet cells, reduces the production of mucin MUC5AC and the secretion of inflammatory factors IL6 and IL8, thereby inhibiting airway mucus hypersecretion and airway inflammation in chronic obstructive pulmonary disease. The relevant content supplements the pathogenesis of chronic obstructive pulmonary disease and provides a new strategy for its targeted treatment. In order to better explain the present invention, it is described in detail below in conjunction with specific examples.

[0021] Example 1 Knockdown of SIX1 in mouse lung tissue using AAV-sh-SIX1 alleviates cigarette smoke-induced lung function impairment, airway mucus hypersecretion and airway inflammation Experimental animals and materials: 1. Experimental animals: Source, strain, and line: Wild-type mice (WT, C57BL / 6) were ordered from the Hubei Branch of Jiangsu Jicui Yaokang Experimental Animal Technology Co., Ltd.; Reproductive age: 8-10 weeks; 2. Experimental materials: Cigarettes: purchased from Wuhan Promoter Biological Co., Ltd.

[0022] Isoflurane: purchased from Wuhan Promoter Biological Co., Ltd.

[0023] AAV-sh-SIX1: purchased from Shanghai Heyuan Biotechnology.

[0024] 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, forming a hairpin structure controlled by the polⅢ promoter. Then 5-6 Ts are connected as the transcription terminator of RNA polymerase Ⅲ.

[0025] AAV refers to adeno-associated virus (AAV for short). 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. AAV has extremely low immunogenicity and almost no pathogenicity in vivo, making AAV an ideal tool for many animal studies.

[0026] The shRNA interference fragment targeting SIX1 was designed and constructed into an adeno-associated virus vector through molecular biological methods. This vector can interfere with the SIX1 gene. In addition to being able to directly transiently transfect cells for interference with the SIX1 gene, this vector can also package adeno-associated viruses to interfere with the expression of the SIX1 gene at the animal level. After mice were infected by airway injection of AAV-sh-SIX1, the expression of SIX1 in mouse lung tissues was reduced.

[0027] 3. Experimental methods: 3.1 Mouse treatment Wild-type mice were anesthetized by isoflurane inhalation 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 hours a day (1.5 hours in the morning and 1.5 hours in the afternoon), and continued for 6 months ( Figure 1 A-1B). After modeling, the lung function, airway mucus secretion and airway inflammation of each group of mice were evaluated.

[0028] 3.2 Immunofluorescence staining (1) Preparation before the experiment: prepare a slide rack, antigen repair box, histochemical pen, and wet box, and preheat the oven to 60°C. Prepare 1x EDTA antigen repair solution (PH=9), 1x PBS phosphate buffer (add 2 L of pure water to a packet of PBS powder, and make sure to fully dissolve and mix) and 10% donkey serum. Make sure there are enough reagents in various reagent dishes for dewaxing in the fume hood.

[0029] (2) Dewaxing: Place the paraffin sections of human lung tissue on a slide rack, dry-bake the slide rack in a preheated oven for 45–60 min, quickly remove the slide rack and place it in a container containing xylene I in a fume hood, and dewax the slides in the following order: xylene I (15 min) – xylene II (15 min) – anhydrous ethanol I (5 min) – anhydrous ethanol II (5 min) – 85% alcohol (5 min) – 75% alcohol (5 min). After dewaxing, take out the slide rack and rinse it indirectly in flowing distilled water outside the fume hood for 5 min.

[0030] (3) Antigen repair: Add 250 mL of 1 x antigen repair solution to the antigen repair box, and place the slides rinsed with distilled water into the antigen repair box. Place the antigen repair box in a microwave oven for microwave repair. The repair process is: medium-high heat for 5 minutes, stop the heat for 5 minutes, medium heat for 5 minutes, stop the heat for 5 minutes, medium-low heat for 5 minutes, stop the heat for 5 minutes, and then let the slides cool naturally to room temperature in the repair solution (note that water evaporates during the microwave process, and be careful to avoid drying the slides during the whole process).

[0031] (4) Draw circles with a histochemical pen to block water: Discard the antigen retrieval solution that has cooled to room temperature, and wash the slides three times with PBS phosphate buffer on a shaker at room temperature, 5 minutes each time. Take the slide and shake off excess water as much as possible, and use a histochemical pen to draw circles around the tissue block on the slide (be careful to prevent the slide from drying out).

[0032] (5) Blocking: Add 10% donkey serum into the tissue circle (to ensure that the tissue block is completely covered) and place the slide in a humidified box and incubate at room temperature for 45 to 60 minutes.

[0033] (6) Primary antibody incubation: According to the antibody instructions, use the primary antibody diluent to prepare a primary antibody mixture with MUC5AC antibody and CC10 antibody. After the slide is sealed, shake off the sealing solution and add an appropriate amount of diluted primary antibody mixture to the tissue block (to ensure that the tissue block is completely covered). Place the slide in a humidified box and incubate overnight at 4°C.

[0034] (7) Secondary antibody incubation: On the second day, remove the wet box and rewarm at room temperature for 30 min. Shake off the primary antibody, place the slide in the antigen retrieval box and wash again with PBS phosphate buffer on a shaker at room temperature three times, 5 min each time. Prepare a mixture of anti-rabbit fluorescent secondary antibody (1:200) and anti-mouse fluorescent secondary antibody (1:200) with PBS, add the prepared mixture to the tissue block (make sure the tissue block is completely covered) and incubate in the wet box at room temperature in the dark for 1 h. After the incubation, wash the slide three times with PBS phosphate buffer on a shaker at room temperature in the dark for 5 min each time.

[0035] (8) DAPI nuclear staining: Take the slide and shake off excess water as much as possible, add ready-to-use DAPI staining solution in the tissue circle (to ensure that the tissue block is completely covered), and incubate the slide in a humidified box at room temperature and away from light for 5 to 10 minutes. After the incubation, wash the slide three times with PBS phosphate buffer on a shaker at room temperature and away from light, each time for 5 minutes.

[0036] (9) Sealing: Take the slide and shake off excess water as much as possible. Add an appropriate amount of anti-fluorescence quenching agent into the tissue circle and seal the slide with a coverslip.

[0037] (10) Observe the slices under a fluorescence microscope, collect images and perform data analysis.

[0038] 3.3 Alcian blue-periodic acid Schiff (AB-PAS) staining (1) Dewaxing of paraffin sections: The sections were dewaxed in xylene for 10 min, then replaced with fresh xylene for 10 min, dewaxed in anhydrous ethanol for 5 min, fresh anhydrous ethanol for 5 min, 90% ethanol for 5 min, 75% ethanol for 5 min, and washed with tap water.

[0039] (2) Immerse the slides in AB-PAS stain solution C for 8-10 min and then wash with tap water until the water on the slides turns colorless.

[0040] (3) The sections were immersed in AB-PAS dye solution B for 10-15 min and then rinsed in pure water three times, each time for about 10 s.

[0041] (4) Immerse the sections in AB-PAS staining solution A (restored to room temperature in advance) and cover in a dark place for 25-30 min. Rinse with running water for 5 min.

[0042] (5) Dehydrate the sections in three cylinders of anhydrous ethanol for 5 min each time. Clear them in xylene for 5 min, replace with fresh xylene for another 5 min, and then seal the sections with neutral gum.

[0043] 3.4 Enzyme-linked immunosorbent assay (ELISA) (1) Preparation before the experiment: According to the instructions of the mouse IL-6 and KC ELISA kits, the capture antibody, detection antibody and standard in the kit were fully dissolved and mixed, and then aliquoted. The aliquoted reagents were stored in a -80 ℃ refrigerator for later use. Take out the mouse BALF supernatant to be tested and place it at room temperature to thaw and rewarm.

[0044] (2) Pre-coated plates: Dilute the capture antibody storage solution into working solution with coating buffer, add it to the ELISA-specific 96-well plate with a dispenser, seal the 96-well plate with a sealing film and centrifuge it instantly, and incubate the 96-well plate in a 37°C incubator overnight.

[0045] (3) Blocking: The next day, take out the ELISA auxiliary kit and rewarm it at room temperature for 1 hour. 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 with a spray gun, and wash at room temperature for 2 minutes. Wash 3 times in total (be sure to tap off 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 ℃ incubator for 1 hour.

[0046] (4) Preparation of standard curve ①Preparation before the experiment: Rewarm the standard stock solution, prepare 1.5 mL EP tubes, numbered 1 to 6, and add 200 μL (if preparing a KC standard curve, add 500 μL) of Assay Diluent solution to each tube.

[0047] ② Preparation of mouse IL-6 standard curve: Take the frozen IL-6 standard storage solution, thaw it and centrifuge it, pipette 200 μL of the storage solution into EP tube 1, and dilute the standard in multiple ratios as shown in the figure below.

[0048] ③ Preparation of mouse KC standard curve: Take the frozen KC standard storage solution, thaw it and centrifuge it, pipette 500 μL of the storage solution into EP tube 1, and dilute the standard according to the instructions.

[0049] (5) Sample addition: After the 96-well plate is blocked, remove the sealing film, discard the blocking solution, and use a spray gun to add 300 μL of washing solution to the 96-well plate. Let it stand at room temperature for 2 min, and wash it 3 times in total (be sure to tap off all the washing solution in the well each time). Mix the sample and standard solution and centrifuge it. Add the sample and standard to the 96-well plate that has been tapped dry. After a brief centrifugation, incubate the 96-well plate in a constant temperature incubator at 37 °C for 1 h.

[0050] (6) Detection antibody incubation: Take out the detection antibody storage solution, melt it, and centrifuge it briefly. Dilute the detection antibody into a working solution with 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 dispenser, and wash it at room temperature for 2 min. Wash it 3 times in total (be sure to tap off the washing solution in the well each time). Add 100 μL of detection antibody working solution to the 96-well plate with a dispenser, centrifuge it briefly, and incubate the 96-well plate in a 37°C incubator for 1 h.

[0051] (7) HRP incubation: Dilute 40x HRP to 1x with Assay Diluent solution (all operations after this step should be performed 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 dispenser, and wash at room temperature for 2 min. Wash 3 times in total (be sure to tap off all the washing solution in the well each time). Add 100 μL of 1x HRP working solution to the 96-well plate with a dispenser, centrifuge briefly, and incubate the 96-well plate in a 37°C incubator for 30 min.

[0052] (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 dispenser, and wash at room temperature for 2 min. Wash 3 times in total (note to tap off the washing solution in the wells each time). Mix the Color A and Color B solutions in the auxiliary kit in a 1:1 ratio, and add 100 μL of the mixed solution to the 96-well plate with a dispenser. After instant centrifugation, incubate the 96-well plate in a 37°C incubator for 15-20 min. During this period, pay attention to the color changes in each well. When the wells with a standard curve concentration of 31.3 pg / mL or 62.5 pg / mL show obvious blue color visible to the naked eye, the reaction can be terminated.

[0053] (9) Reaction termination and reading: Turn on the full-wavelength microplate reader to preheat in advance, use a dispenser to draw 50 μL of the reaction stop solution and add it to the 96-well plate to terminate the color development reaction. Place the 96-well plate in the microplate reader and read the results three times at wavelengths of 450 nm and 630 nm respectively (before reading, make sure there are no bubbles in the 96-well plate).

[0054] (10) Draw a standard curve, calculate the concentration of IL-6 or KC in each sample based on the standard curve, and analyze the data.

[0055] 3.5 Collection of mouse bronchoalveolar lavage fluid (BALF) (1) Preparation before the experiment: prepare ice, 1 mL syringe (with heparin-moistened tube), cotton pads and cotton balls, 1.5 mL EP tube (with 40 μl heparin-moistened tube) (2) Blood collection from the inferior vena cava: Remove the mouse from the mouse pulmonary function instrument, place the mouse in a supine position on a foam board, fix the limbs with tape, and secure the head with a rubber band around the teeth. Lift the abdominal skin with tweezers, cut the abdominal skin longitudinally with scissors to fully expose the internal organs, find the inferior vena cava of the mouse, and use a 1 mL syringe to draw as much blood from the inferior vena cava as possible. The drawn blood is placed in a 1.5 mL EP tube and centrifuged at 4 °C at 980 rpm for 5 min. Use a new EP tube to collect the upper plasma, and freeze the plasma for later use.

[0056] (3) Cardiac perfusion: After blood collection, cut the inferior vena cava with scissors, cut open the chest cavity, and fully expose the heart and lungs. Draw 4 mL of normal saline with a 5 mL syringe and flush as much pulmonary circulating blood as possible through the mouse heart.

[0057] (4) Bronchoalveolar lavage: Use a 1 mL syringe to draw up saline solution, and inject the saline solution into the mouse lungs through the tracheal needle. After the liquid stays in the lungs for 1 min, withdraw the liquid and recover it. The saline solution was 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 recovered liquid from the first lavage was retained in an EP tube A. The lavage fluid from the next two lavages was retained in the same EP tube B. The two EP tubes were centrifuged at 4 °C, 980 rpm, for 8 min. The liquid in EP tube A was recovered, which was the alveolar lavage fluid of the mouse. The liquid in EP tube B was discarded. The cells at the bottom of EP tubes A and B were collected for slides and Liu staining.

[0058] 3.6 Collection and processing of mouse lung tissue specimens (1) Preparation before the experiment: Take a 4 mL EP tube, number it and add 3 mL of tissue fixative. Prepare tissue grinding tube A, tissue grinding tube B and cryotubes and number them in advance.

[0059] (2) Collection of mouse lung tissue specimens: After bronchoalveolar lavage, the lingula and three lobes of the right lung (upper lobe, middle lobe, and lower lobe) were ligated with sutures. 400 μL of tissue fixative was drawn into a 1 mL syringe and injected into the left lung through the tracheal needle. The left lung was removed and placed in an EP tube filled with tissue fixative, and the tube mouth was sealed with cotton. The lingula and right lung were cut off. The lingula and right lower lobe were placed in a cryopreservation tube. The right upper lobe was stored in grinding tube A, and the right middle lobe was in grinding tube B. The fixed mouse lung tissue was embedded and sliced, and the specific steps were the same as those for embedding and slicing human lung tissue. The lung tissue in grinding tube A was used for mouse lung tissue RNA extraction. The lung tissue in grinding tube B was used for mouse lung protein extraction.

[0060] 3.7 BALF cell slides and Liu staining (1) Preparation before the experiment: prepare ice and number the slides (2) Lyse red blood cells: Take 300 μL of red blood cell lysis solution and add it to the above EP tube B, and mix it by blowing. Pipette the liquid in EP tube B into EP tube A and mix it by blowing. 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 4 ℃ centrifuge 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 culture medium to grinding tube A and mix it by blowing.

[0061] (3) Slide: Turn on the microscope and count the cells in the cell suspension in EP tube A under the microscope. Use complete culture medium to adjust the cell density of the cell suspension in EP tube A to 250 cells / μL. Take a glass slide, number it and install it on the slide holder. Add 80 μL of cell suspension to the slide holder. Put the slide holder into the slide machine and spin at 980 rpm for 5 min.

[0062] (4) Liu's staining: Take out the shaken cell slide and use a rubber-tipped dropper to add 3-4 drops of Liu's stain A to the location of cell aggregation on the slide. Stain for 3-4 seconds. Then add 10-12 drops of Liu's stain B to the location of cell aggregation and stain for 10 seconds. Rinse the slide under running water for 30 seconds. After drying the slide, add neutral resin to seal the slide.

[0063] (5) BALF cell slides and Liu's staining The sealed cell slides with Liu's staining were photographed under an optical microscope (200x, 12 non-overlapping fields of view were randomly selected for each slide). After the photography was completed, the cells were classified, counted and analyzed.

[0064] 3.8 Western blot (WB) (1) Preparation before the experiment: prepare ice, pipettes and pipette tips of different ranges, gel tip droppers, 50 mL centrifuge tubes, clean and dried gel combs, etc. Take out the samples and protein markers and thaw them on the ice. Prepare sufficient long and short glass plates, gel clips, and gel racks according to the specific needs of the experiment.

[0065] (2) Leak detection before making glue: Clean the long and short glass plates for making glue, clamp the long and short glass plates with glue clamps and fix them on the glue rack. Fill the gap between the long and short glass plates with pure water, let it stand for 10 to 15 minutes, and then observe the downward movement of the liquid level. After confirming that the liquid level has not moved downward significantly, you can proceed to the next step.

[0066] (3) Preparation of WB gel: Pour out and absorb the pure water between the glass plates with clean filter paper, prepare the stacking gel and separation gel in a 50 mL centrifuge tube according to the reagents in the rapid gel kit, use a rubber-tipped dropper to first add the mixed separation gel to the gap between the glass plates to a suitable height, then use another rubber-tipped dropper to gently and slowly add the mixed stacking gel at a uniform speed until the gap between the glass plates is filled, then slowly insert the comb teeth into the stacking gel. Let it stand for 15-20 minutes to wait for the separation gel and stacking gel to solidify.

[0067] (4) Preparation of electrophoresis fluid: Prepare sufficient amount of electrophoresis fluid according to specific experimental requirements, and make sure that all electrolytes are fully dissolved and mixed.

[0068] (5) Sample loading: Carefully remove the comb teeth on the concentrated gel, remove the long and short glass plates and install them on the electrophoresis device, add an appropriate amount of electrophoresis solution to the electrophoresis tank, remove the foam on the surface of the electrophoresis tank, and fully expose the sample loading slots for easy sample loading. Vortex the melted sample and protein marker on a vortex oscillator to mix well and centrifuge for sample loading. The principle of calculating the sample loading volume is to ensure that the total mass of each sample loaded is the same. If the sample loading volume in different slots of the same gel plate is 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 slots on the left and right sides of the sample respectively to serve as an indicator. Connect the electrophoresis tank, electrophoresis cover and electrophoresis instrument, turn on the power, and start electrophoresis (pay attention to the direction of electrode connection, the initial electrophoresis voltage is 70~80 V, and after the protein marker bands are clearly separated, adjust the voltage to 110~120 V).

[0069] (6) Preparation of transfer solution: Prepare a sufficient amount of transfer solution according to the specific experimental requirements, and make sure that all electrolytes are fully dissolved and mixed (methanol is volatile and should be added before the transfer solution is needed).

[0070] (7) Preparation before transfer: Prepare the items required for transfer (transfer plate, transfer clip, sponge pad, filter paper, rubber-tipped pipette, large and small steel rulers, glass seesaw, exhaust roller and PVDF membrane, etc.). Add an appropriate amount of transfer solution to the transfer plate, place the transfer clip on the transfer plate (black side of the transfer clip facing down, white side facing up), place a layer of sponge and 2-3 layers of filter paper on the black and white sides of the transfer clip respectively (the amount of filter paper can be increased or decreased according to actual conditions, based on the appropriate tightness of the black and white sides of the transfer clip after clamping), suck the transfer solution with a pipette to wet the sponge pad and filter paper, and use the exhaust roller to remove bubbles between the sponge pad and filter paper and between the filter paper.

[0071] (8) Transfer: After the electrophoresis is completed, remove the glass plate from the electrophoresis tank, separate the long and short glass plates with a glass seesaw (make sure the gel remains on the short glass plate to facilitate subsequent gel cutting), use a small steel ruler to cut off the part that is larger than the gel (retain a molecular weight of 25-180 kD), measure the size of the cut gel and soak the gel in the transfer solution to prevent it from drying out, then remove the PVDF membrane, cut a PVDF membrane of appropriate size according to the measured gel size and soak it in methanol for activation (immersion time is greater than 10 s). Next, transfer the gel to the filter paper of the black-sided transfer clamp, then transfer the activated PVDF membrane to the gel (marks 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 of the white-sided transfer clamp on the PVDF membrane in turn, and finally align the black and white sides of the transfer clamp and fix them. Note that after each stacking, use an exhaust roller to exhaust air to avoid bubbles between the filter paper and gel, between the gel and PVDF membrane, and between the PVDF membrane and the filter paper. Place the transfer clip into the transfer tank, add cooling ice cubes into the transfer tank, and connect the transfer tank, transfer cover and transfer instrument. Turn on the power and start the transfer (Note: Make sure the electrode connection direction is correct, adjust the transfer current to 200~250 mA, maintain the voltage at 90~120 V, and the transfer process lasts for 100~120 min. The entire transfer process is carried out at low temperature. You can make your own transfer ice tank and put the transfer tank into the ice tank, or transfer directly in a 4 ℃ laboratory. Pay attention to replace the cooling ice cubes in the transfer tank halfway through the transfer. Pay attention to check the voltage and current during the entire transfer process to avoid transfer failures caused by water entering the transfer tank and poor power contact.

[0072] (9) Solution preparation: Prepare TBST solution in advance (add one bag of TBS buffer powder and 1 mL of Tween 20 to 2 L of pure water) and 5% skim milk (add 100 mL of TBST solution to every 5 g of skim milk powder to make 100 mL of 5% skim milk, and make sure to fully dissolve and mix). According to the instructions for use of anti-SXI1 antibody and anti-ß-ACTIN antibody, use primary antibody diluent to dilute SXI1 antibody (1:2000) and ß-ACTIN antibody (1:4000) to working solution. Pour the prepared primary antibody solution into different small compartments of the primary antibody incubation box and mark them. For regular antibody incubation boxes, add 4 mL of primary antibody solution to each small compartment.

[0073] (10) Blocking: After the transfer is completed, remove and open the transfer clip, quickly place the PVDF membrane (the surface in contact with the gel is the protein side of the PVDF membrane) in 5% skim milk, and block on a shaker at room temperature for 1 h.

[0074] (11) Primary antibody incubation: Wash the blocked PVDF membrane three times with TBST solution, shaking for 5-10 min each time. Cut the PVDF membrane according to the molecular weight of the target, and place the cut PVDF membrane into the small compartment of the primary antibody incubation box corresponding to the target. Then place the primary antibody incubation box on a shaker at 4 °C overnight.

[0075] (12) Secondary antibody incubation: On the second day, recover the primary antibody solution and wash the PVDF membrane three times with TBST solution at room temperature, shaking for 5-10 min each time. Then add 4 mL of 5% skim milk to the incubation box with the PVDF membrane, and add 1 μL of the corresponding HRP goat anti-rabbit or anti-mouse secondary antibody to the incubation box according to the species of the primary antibody. Continue to incubate the antibody incubation box at room temperature on a shaker for 1 h. After incubation, shake again with TBST solution three times, shaking for 5-10 min each time.

[0076] (13) Band development: Preheat the band developer in advance and set the filter of the developer to the filter-free mode. According to the instructions for preparing the chemiluminescent solution, prepare the luminescent solution in a light-proof environment with a ratio of 1:1 between reagent A and reagent B, and make sure to mix it thoroughly. Next, drop the luminescent solution on the luminescent plate, and then place the PVDF strip in the luminescent solution on the luminescent plate (make sure the PVDF membrane protein side faces up). After the strip has been incubated for 1 to 2 minutes, place the luminescent plate in the developer and adjust the position. Select immunoblotting in the computer software, adjust the exposure time, and then expose. Be sure to save the experimental data.

[0077] 4. Experimental results: Figure 1 A shows the time points for airway injection of AAV-sh-SIX1 or control virus and systemic cigarette smoke exposure modeling. Airway injection of AAV-sh-SIX1 virus can significantly reduce the expression of SIX1 in mouse lung tissue, indicating that the expression of SIX1 in mouse lung tissue has been successfully reduced. Cigarette smoke intervention induces decreased lung function in mice, and SIX1 knockdown can alleviate the decline in lung function caused by cigarette smoke ( Figure 1 C); Cigarette smoke intervention induced 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 the number of macrophages and neutrophils ( Figure 1 D) H&E staining showed that cigarette smoke intervention induced the accumulation of inflammatory cells around the airways, while knockdown of lung SIX1 prevented the infiltration of inflammatory cells into the airways ( Figure 1 E). In addition, Alcian blue-periodic acid Schiff (AB-PAS) staining indicated that cigarette smoke intervention induced increased secretion of airway epithelial mucus (AB-PAS staining was blue or purple) in mice, and knockdown of lung SIX1 reduced epithelial mucus secretion ( Figure 1 E). Immunofluorescence staining showed that cigarette smoke intervention induced an increase in the expression of MUC5AC in the airway epithelium of mice (red), and knockdown of lung SIX1 reduced the expression of 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 SIX1 knockdown also reduced the expression of KC in the BALF ( Figure 1 H-1I).

[0078] Example 2 Knockdown of SIX1 in airway epithelium by siRNA alleviates the expression of epithelial mucin MUC5AC and the secretion of inflammatory factors IL6 and IL8 induced by cigarette smoke extract To further explore the factors that affect the expression of airway epithelial mucins and the secretion of inflammatory factors by knocking down SIX1, we designed an in vitro experiment as follows: Experimental cells and materials: 1. Experimental cells: 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 and a CO2 concentration of 5%.

[0079] 2. Experimental materials: Cigarettes: purchased from Wuhan Promoter Biological Co., Ltd.

[0080] Cell culture medium: purchased from Wuhan Promoter Biological Co., Ltd.

[0081] Cell culture dishes: purchased from Wuhan Promoter Biological Co., Ltd.

[0082] Transfection reagent: purchased from Invitrogen.

[0083] siRNA (SIX1): purchased from RiboBio.

[0084] 3. Experimental methods: 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 SIX1 knockdown on the production of MUC5AC and the secretion of inflammatory factors IL6 and IL8 in airway epithelial cells induced by cigarette smoke extract were analyzed.

[0085] 3.1 Cell culture (1) Culture of airway epithelial cell line - 16 HBE cell line ①Preparation before the experiment: prepare different ranges of pipette tips and pipettes, 15 mL sterile centrifuge tubes, 50 mL sterile centrifuge tubes, cell cryopreservation tubes, T25 cell culture flasks, and 12-well cell culture plates, and sterilize them by ultraviolet irradiation in a clean bench. Take out fetal bovine serum, 1640 RMPI culture medium, PBS phosphate buffer, and trypsin and rewarm them at room temperature. Adjust the constant temperature water bath to 37 ℃.

[0086] ② Cell recovery: Add 5 mL of fetal bovine serum and 1640 RMPI medium to a 50 mL centrifuge tube to prepare a complete medium with a serum concentration of 10%. Take 10 mL of complete medium to a 15 mL sterile centrifuge tube for later use. Take out the frozen cells from the liquid nitrogen tank and shake the cryopreserved tube in a 37 °C water bath until the liquid is completely melted. In the clean bench, aspirate the cell suspension in the cryopreserved tube into a 15 mL centrifuge tube. 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 clean bench, add 3 mL of complete medium to the tube, and use a 1 mL pipette tip to blow and mix the cells. 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.

[0087] ③ Cell replacement and digestion and subculture: On the second day of cell recovery, observe the cell adhesion and growth. Discard the supernatant in the culture flask in the clean bench, 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 of cell recovery, perform cell subculture and plating. First, discard the cell supernatant in the clean bench and wash the cells twice with 1 mL of PBS. Take 1 mL of trypsin and add it to the culture flask and let it fully contact the cell surface. Place the culture flask in the incubator for 6~8 min. During the placement, pay attention to the morphology of the cells and the cells falling off from the bottom of the culture flask. When the cells become round and begin to fall off from the bottom, tap the culture flask. When most of the cells are observed to fall off from the bottom of the cells under the microscope, add 2 mL of cell culture medium to the culture flask in the clean bench to stop digestion. Use a 1 mL pipette tip to blow the bottom of the cell flask to cause the cells to fall off completely. Collect the cell suspension after blowing in a 15 mL centrifuge tube, and centrifuge the 15 mL centrifuge tube at 980rpm for 5 min in a normal temperature centrifuge. After centrifugation in a 15 mL centrifuge tube in a clean bench, discard the supernatant. The cells at the bottom of the tube can be used for passaging, freezing, or plating. If the cells need to be passaged, 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 culture medium and 1 mL of cell suspension. If the cells need to be frozen and plated, the specific steps are as follows.

[0088] ④ Cell freezing: prepare cell freezing solution (each 1 mL of cell freezing solution consists of 100 μL of DMSO and 900 μL of fetal bovine serum), add 1 mL of cell freezing solution to the 15 mL centrifuge tube from which the supernatant has been discarded, resuspend the cells, place the cell suspension into cryotubes, place the cryotubes in a cryopreservation box and store them in a -80 ℃ refrigerator (the cryopreservation box can ensure that the cryopreservation tubes are cooled in a gradient manner. After the cryopreservation box has been stored in a -80 ℃ refrigerator for 24 h, the cryopreservation tubes must be stored in a liquid nitrogen tank in a timely manner).

[0089] ⑤Cell plating and intervention: Add 4 mL of cell culture medium to the 15 mL centrifuge tube from which the supernatant was discarded to resuspend the cells, and aspirate 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 of the 12-well plate is 80,000, and the 12-well culture system is 1 mL). After plating, pay attention to observe the cell state and adhesion, and change the cell medium 12 hours after plating. When the cell growth confluence reaches 70%~80%, cigarette smoke extract intervention can be given.

[0090] 3.2 Formulation of cigarette smoke extract (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.

[0091] (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.

[0092] (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).

[0093] 3.3 Collection of cell supernatant (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.

[0094] (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.

[0095] 3.4 Cell protein extraction, concentration determination and WB analysis (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.

[0096] (2) Preparation of cell lysis buffer (3) Cell lysis and protein scraping: Wash the cell culture plate after collecting the cell supernatant three times with 1 mL of pre-cooled PBS. Make sure to drain as much PBS solution as possible in the last wash. Add 80 μL of prepared cell lysis solution to each well of the culture plate and place on ice for 30 min. After sufficient lysis, use a protein scraper to scrape the protein from the wells of the culture plate. Collect the scraped cells and cell lysis solution into a 1.5 mL EP tube.

[0097] (4) Ultrasonication, concentration determination and WB analysis of protein solution: same as above.

[0098] 3.5 Cell RNA extraction, concentration determination and RT-PCR analysis: (1) Preparation before the experiment: prepare ice, prepare 1.5 mL enzyme-free EP tubes and number them, and pre-cool chloroform, anhydrous ethanol, and isopropanol.

[0099] (2) Collect RNA: Wash the cell culture plate from which the cell supernatant has been collected three times with 1 mL of pre-cooled PBS. Be careful to aspirate as much PBS solution as possible the last time. Add 1 mL of Trizol solution to each well of the culture plate in a biosafety cabinet and let it stand on ice for 10-20 min. Use a 1 mL pipette to blow repeatedly to blow the cells off the culture plate, and collect the solution after blowing 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 manually for 30 s and let it stand on ice for 15 min. After standing, centrifuge at 12,000 rpm in a low-temperature centrifuge for 15 min.

[0100] (3) Subsequent steps include extraction of RNA from human lung tissue, concentration determination, reverse transcription, and RT-PCR analysis.

[0101] 3.6 Cell transfection with small interfering RNA (1) Preparation before the experiment: UV irradiate the guns, gun tips and 1.5 mL sterile EP tubes of various ranges in the cell ultra-clean bench. Prepare ice and melt the transfection reagent and small interfering RNA (siRNA) on ice. Rewarm the complete cell culture medium and Optim culture medium at room temperature.

[0102] (2) siRNA transfection: ① Cell preparation: Prepare 16 HBE cells in good growth state for subculturing and plating (12-well plate). Transfection can be performed when the cell growth density reaches 30%~45%.

[0103] ② Preparation of transfection reagent: First, dissolve the powdered siRNA in ddH2O to a storage solution concentration of 20 μM. Prepare the transfection reagent for a single well of a 12-well plate according to the siRNA concentration gradient set below.

[0104] ③ Selection of siRNA concentration: siRNA was purchased from Ribo Biotech Co., Ltd., where SIX1 siRNA is referred to as si-SIX1 below, and control siRNA is referred to as si-Con. When siRNA transfection was performed for the first time, three siRNA concentration gradients of 20 nM, 40 nM, and 80 nM were set to transfect the cells. The cells were replaced with 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. The siRNA concentration that has no obvious effect on the cell growth state and can significantly reduce the expression of SIX1 was selected for the next experiment (the siRNA concentration selected in this experiment is 80 nM).

[0105] ④ Cell transfection: Aspirate the culture medium from the 12-well plate with cell growth density at 30%~45%, and wash the cells twice with PBS. Add 450 μL of complete culture medium to each well of cells. Add 50 μL of the above-prepared transfection reagent (prepared according to the siRNA concentration of 80 nM) to each well of cells.

[0106] 3.7 WB experimental procedures and methods Same as Example 1.

[0107] 4. Experimental results: Knockdown of SIX1 alleviated the increase in airway epithelial mucin MUC5AC and the increased secretion of inflammatory factors IL6 and IL8 induced by cigarette smoke extract.

[0108] Example 3 Inhibition of SIX1 inhibits SPEDF expression through transcription, reduces epithelial inflammation and epithelial mucin secretion To further explore the mechanism of the effect of knocking down SIX1 on airway epithelial mucin expression and inflammatory factor secretion, we conducted further in vitro experiments as follows: Experimental cells and materials: 1. Experimental cells: 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 and a CO2 concentration of 5%.

[0109] 2. Experimental materials: Cigarettes: purchased from Wuhan Promoter Biological Co., Ltd.

[0110] Cell culture medium: purchased from Wuhan Promoter Biological Co., Ltd.

[0111] Cell culture dishes: purchased from Wuhan Promoter Biological Co., Ltd.

[0112] Transfection reagent: purchased from Invitrogen.

[0113] siRNA (SIX1): purchased from RiboBio.

[0114] SIX1 lentivirus (Lv-SIX1): purchased from Shanghai Heyuan Biotechnology.

[0115] Immunoprecipitation kit: purchased from CST 3. Experimental methods: Airway epithelial cells were cultured in vitro. SIX1 expression in airway epithelial cells was knocked down by transfection of siRNA. SIX1 expression in airway epithelial cells was promoted by transfection of Lv-SIX1. The effects of knockdown and overexpression of SIX1 on the expression of transcription factor SPDEF in airway epithelium induced by cigarette smoke extract were analyzed respectively. The mechanism of SIX1 regulating SPDEF expression was also explored.

[0116] 3.1 Cell culture Same as Example 2.

[0117] 3.2 Cigarette smoke production Same as Example 2.

[0118] 3.3 Cell transfection with small interfering RNA Same as Example 2.

[0119] 3.4 WB experimental procedures and methods Same as Example 1.

[0120] 3.5 Cell lentiviral transfection (1) Preparation before the experiment: UV irradiation guns and gun tips of various ranges in the cell clean bench.

[0121] (2) Cell preparation: The cell suspension after cell digestion is inoculated into a 6-well plate, with approximately 100,000 cells inoculated per well. On the second day, the 6-well plate is observed and the medium is changed. When the cell confluency reaches about 70%, the cells can be transfected with viruses.

[0122] (3) Lentivirus transfection pre-experiment: Lentivirus was purchased from Shanghai Heyuan Biotechnology. The SIX1 overexpression virus is hereinafter referred to as Lv-SIX1, and the control virus is referred to as Lv-con. For the first viral transfection, a virus concentration gradient of 0 MOI, 20 MOI, 40 MOI and 80 MOI was set for virus transfection. The medium was changed 12-24 h after viral transfection according to the cell status. 48 h after viral transfection, the fluorescence intensity can be observed under a fluorescence microscope to preliminarily determine the viral transfection efficiency. 72 h after viral transfection, RNA or protein can be extracted from the cells transfected with the virus, and the viral transfection efficiency can be analyzed by RT-PCR or WB. The viral concentration that has no obvious effect on the cell growth state and can significantly overexpress SXI1 was selected for the next experiment (the viral concentration selected in this experiment was 40 MOI).

[0123] (4) Virus transfection and establishment of SXI1 overexpression stable transfectant strains: The virus was transfected in a 6-well plate at a concentration of 40 MOI. The cells were replaced with a culture medium 12 h after transfection. The culture medium was replaced 48 h after transfection and complete culture medium containing 4 μg / mL puromycin was added to the 6-well plate. The cells were replaced with a culture medium 72 h after transfection. The cells after the culture were the cells that stably overexpressed SXI1 and the control cells.

[0124] 3.6 Chromatin Immunoprecipitation (ChIP) (1) Cell preparation ①Preparation before the experiment: 15 cm culture dish, 16HBE cells in good growth condition.

[0125] ②Cell transfection: Digest the 16HBE cells and inoculate the cell suspension into two different 15 cm culture dishes (add 20 mL complete medium for culture), and perform Lv-SIX1 (transfected in culture dish A) and Lv-con (transfected in culture dish B) virus transfection. The specific transfection method is the same as above. When the cell confluence in the culture dish reaches 100%, the cell sample can be cross-linked.

[0126] (2) Cross-linking of cell samples ①Preparation before the experiment: prepare ice and 15 mL centrifuge tubes. Take out the 100x protease inhibitor cocktail (PIC) and 1x glycine solution from the ChIP kit and rewarm them at room temperature (make sure the PIC is completely dissolved). Wash and dry the protein scraping. Pre-cool PBS phosphate buffer.

[0127] ② Preparation of reagents required for the experiment: Take 4 mL of PBS and add 20 μL of 200x PIC.

[0128] ③Cell 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, mix the formaldehyde and culture medium, and place 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 PBS containing PIC prepared above to the culture dish, scrape the cells from the culture dish with a cell scraper, and collect the scraped cells in 15 mL centrifuge tubes (recorded as centrifuge tubes A and B respectively). Adjust the centrifuge to 4°C and the centrifugal force to 2,000 g, centrifuge tubes A and B for 5 min to collect the cells, remove the supernatant after centrifugation, and proceed to the next step.

[0129] (3) Nuclear processing and chromatin shearing ①Preparation before the experiment: Take out the DTT reagent in the ChIP kit and add 1.12 mL dH2O to fully dissolve and mix. Take out 10x ChIP buffer and rewarm it at room temperature. Adjust the water bath to 37°C.

[0130] ② Preparation of reagents required for the experiment: a. Prepare 6 mL of 1x Buffer A (1.5 mL of 4x Buffer A in 4.5 mL of water) and add 3 µL of DTT and 30 µL of 200x Protease Inhibitor Cocktail (PIC) and chill on ice.

[0131] b. Prepare 6.6 mL of 1x Buffer B (1.65 mL of 4x Buffer B plus 4.95 mL of water) and add 3.3 µL of DTT and chill on ice.

[0132] c. Prepare 600 µl 1x ChIP buffer (60 µL 10x ChIP buffer plus 540 µL water) and add 3 µL PIC and pre-cool on ice.

[0133] ③Cell nuclear processing a. Add 3 mL of 1x buffer A to centrifuge tubes A and B respectively, incubate on ice for 10 min, and mix by inverting the tubes every 3 min.

[0134] 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.

[0135] ④Chromatin shearing 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.

[0136] b. After incubation, add 30 µL of 0.5 M EDTA to EP tubes A and B to terminate digestion.

[0137] c. Centrifuge EP tubes A and B at 4°C, 16,000 g for 1 min and discard the supernatant.

[0138] d. Resuspend the nuclei in 300 µL 1x ChIP buffer and incubate on ice for 10 min.

[0139] 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.) 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.

[0140] 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.

[0141] (4) Analysis of chromatin concentration and enzyme digestion: 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.

[0142] ②After incubation, add 2 μL of proteinase K to the sample digested with RNase A. Vortex to mix and incubate at 65°C for 2 h.

[0143] ③ DNA purification by centrifugal column a. Add 750 µL DNA Binding Buffer to EP tubes C and D respectively and vortex briefly.

[0144] b. Insert the DNA spin column into the collection tube and mark it (centrifuge tube C and D). First, draw 450 µL of liquid from EP tube C and D and add it to spin column C and D respectively.

[0145] c. Centrifuge tubes C and D at 18,500 g for 30 s at room temperature.

[0146] d. Take out centrifuge columns C and D from collection tubes C and D respectively, discard the waste liquid and then insert the centrifuge columns back into the collection tubes.

[0147] e. Transfer the remaining 450 µL of sample in EP tubes C and D to the corresponding spin columns and repeat steps 3 and 4. Discard EP tubes C and D at this time.

[0148] f. Add 750 µL of DNA Wash Buffer to spin columns C and D.

[0149] g. Centrifuge columns C and D at 18,500 g for 30 s at room temperature.

[0150] h. Remove the centrifuge column from the collection tube, discard the waste liquid, and insert the centrifuge column back into the collection tube.

[0151] i. Centrifuge columns C and D at 18,500 g for 30 s at room temperature.

[0152] j. Remove spin columns C and D from collection tubes C and D respectively, and discard the collection tubes and waste liquid. Take two new enzyme-free 1.5 mL EP tubes and re-label them as EP tubes C and D, and insert spin columns C and D into EP tubes C and D respectively.

[0153] k. Add 50 µL of DNA elution buffer to each of the spin columns C and D and incubate at room temperature for 2 min.

[0154] l. Place the centrifuge column and EP tube together in a centrifuge and centrifuge at 18,500 g for 2 min to elute the DNA.

[0155] m. Discard the centrifugal column. The eluates in EP tubes C and D are the purified DNA from Lv-SIX1 transfected cells and Lv-Con transfected cells, respectively.

[0156] ④ DNA concentration determination: First, ensure that Nanodrop 2000 is connected to the computer power supply, open the computer software Nanodrop 2000, and select nucleic acid-DNA; then draw 1 μL of eluent water to clean the instrument probe twice, and then use 1 μL of eluent to zero the instrument. Finally, draw 1 μL of the mixed DNA sample in the EP tube for concentration detection, and be sure to record and save the DNA concentration of all samples (generally between 50~200 μg / mL).

[0157] ⑤ Identification of enzyme digestion effect: After the DNA samples are purified, take 5 µL of samples from EP tubes C and D respectively and mix them with 5 μL of 2x DNA loading buffer. Then use the mixed samples to perform nucleic acid gel electrophoresis (using 2% agarose gel, the specific configuration method is the same as mentioned above; 100 bp DNA ladder is used as Marker) to determine the size of DNA fragments.

[0158] (5) Chromatin immunoprecipitation: ①Preparation before the experiment: Prepare ice, take out the sheared chromatin, that is, melt the new EP tubes A and B on ice. Take out the 100x protease inhibitor cocktail (PIC) in the ChIP kit (make sure the PIC is completely dissolved). Preheat the 10x ChIP buffer and make sure the SDS is completely dissolved.

[0159] ②Solution preparation a. Prepare low-salt wash buffer: Prepare 18 mL 1x ChIP buffer (1.8 mL 10x ChIP buffer plus 2.7 mL water) and store at room temperature before use.

[0160] b. Prepare high salt wash buffer: Prepare 6 mL 1x ChIP buffer (100 µL 10x ChIP buffer plus 900 µL water) and add 70 µL 5M NaCl and store at room temperature before use.

[0161] c. Prepare 1x ChIP buffer: Prepare 2.4 mL 1x ChIP buffer (240 µL 10x ChIP buffer plus 2.16 mL water), add 12 µL PIC, mix well and place on ice.

[0162] ③Co-immunoprecipitation a. Take three EP tubes and label them as A1, A2 and A3, and add 8 μg of chromatin from EP tube A to EP tubes A1, A2 and A3 respectively (the amount is calculated based on the measured chromatin concentration). Take three EP tubes and label them as B1, B2 and B3, and add 8 μg of chromatin from EP tube B to EP tubes B1, B2 and B3 respectively (the amount is calculated based on the measured chromatin concentration). Fill EP tubes A1, A2, A3, B1, B2 and B3 with diluted 1x ChIP buffer to 500 μL (the volume to be added is 500 μL minus the amount of chromatin), and add an appropriate amount of PIC, mix well and pre-cool on ice.

[0163] b. Take 10 µL of sample from each of EP tubes A1, A2, and A3 and store it in EP tube G; take 10 µL of sample from each of EP tubes B1, B2, and B3 and store it in EP tube H. In subsequent experiments, 10 µL of sample from EP tube G or H can be taken as 2% sample input control (Input) for Lv-SIX1 group and Lv-Con group, respectively. EP tubes G and H can be stored at -20°C before subsequent use.

[0164] 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 antibodies with sealing glue and incubate overnight on a rotor at 4°C (each EP tube is one immunoprecipitation reaction).

[0165] d. Gently resuspend and mix ChIP protein magnetic beads G, add 30 μL protein magnetic beads G to EP tubes A1, A2, A3, B1, B2 and B3 respectively, seal the tube mouth with sealing glue and place the EP tube on a rotor at 4°C for incubation for 2 h.

[0166] e. Place all EP tubes on the magnetic separation rack and adsorb the protein magnetic beads G to the tube wall. After the solution is clarified for 1-2 minutes, carefully remove the supernatant. Keep the supernatant.

[0167] f. Add 1 mL of low-salt rinse solution, remove all EP tubes from the magnetic separation rack, rotate and incubate on a 4°C rotor for 5 min. Repeat steps e and f above 2 more times, i.e., wash with low-salt rinse solution 3 times in total.

[0168] g. Add 1 mL of high salt rinse solution, remove all EP tubes from the magnetic separation rack, rotate and incubate on a 4°C rotor for 5 min. Then return to the magnetic separation rack, wait for 1-2 min for the solution to become clear, carefully remove the supernatant, and immediately proceed to part (6).

[0169] (6) Elute chromosomes from antibody / protein G beads and reverse cross-links ① Preparation before the experiment: Prepare ice, take out EP tubes G and H and melt them on ice. Preheat 2x ChIP elution buffer in a 37°C water bath and make sure SDS is completely dissolved. Adjust the water bath temperature to 65°C.

[0170] ② Preparation of 1x ChIP elution buffer: Prepare 1.2 mL of 1x ChIP elution buffer (600 µL 2xChIP elution buffer plus 600 µL water) ③Experimental steps: a. Add 10 µL of sample from 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 EP tubes I and L. Leave at room temperature until step 6.

[0171] b. Add 150 μL of 1X ChIP elution buffer to EP tubes A1, A2, A3, B1, B2, and B3 in (5).

[0172] c. All EP tubes were gently shaken (1200 rpm) with a vortex mixer and incubated in a 65°C water bath for 30 min to elute the chromatin from the antibody / protein G beads.

[0173] d. Place all EP tubes on a magnetic separation rack to adsorb protein G beads and wait for 1 to 2 minutes for the solution to become clear.

[0174] e. Carefully transfer the eluted chromatin supernatant from each sample tube into a new centrifuge tube and label them separately.

[0175] f. Add 6 µL 5 M NaCl and 2 µL Proteinase K to EP tubes A1, A2, A3, B1, B2, B3, G, and H and incubate at 65°C for 2 h. After incubation, proceed to the next step immediately.

[0176] (7) Purification of DNA using centrifugal columns: The specific method is the same as above.

[0177] (8) RT-PCR evaluation of enrichment efficiency ①Preparation before the experiment: prepare ice, take out the centrifuged purified DNA and rewarm it at room temperature. Prepare sterile enzyme-free ddH2O, high-pressure sterilized pipette tips, pipettes of various ranges, RT-PCR reagents and primers.

[0178] ② Dilute the immunoprecipitated DNA 5-fold with ddH2O.

[0179] ③RT-PCR analysis method is the same as above (same as Example 1).

[0180] 4. Experimental results: Knockdown of SIX1 reduced the expression of transcription factor SPDEF in airway epithelium induced by cigarette smoke extract. Overexpression of SIX1 promoted the expression of transcription factor SPDEF in airway epithelium induced by cigarette smoke extract. SIX1 promoted its transcription and translation by binding to the promoter region of SPDEF.

[0181] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled 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 methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Application of SIX1 expression inhibitor in the preparation of drugs for treating chronic obstructive pulmonary disease.

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. The application according to claim 1, characterized in that: The SIX1 expression inhibitor is a reagent that targets silencing SIX1 or downregulating the expression level of SIX1.

5. The application according to claim 4, characterized in that: The reagent is siRNA of SIX1 or a virus interfering with the expression of SIX1. The nucleotide sequence of the siRNA is shown in SEQ ID NO: 1, which is GCCAGGAGCTCAAACTATT.

6. The application according to claim 5, characterized in that: The virus that interferes with SIX1 expression is AAV-sh-SIX1.

7. 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 SIX1 expression in lung tissue as an active ingredient, and contains a pharmaceutically acceptable carrier.

8. The use according to claim 7, characterized in that: The component for targeted knockdown of SIX1 expression in lung tissue is siRNA or a virus interfering with SIX1 expression. The nucleotide sequence of the siRNA is shown in SEQ ID NO: 1, which is GCCAGGAGCTCAAACTATT.

9. The use according to claim 7, characterized in that: The dosage form of the pharmaceutical composition is an external dosage form or an internal dosage form.

10. The use according to claim 7, characterized in that: The dosage form of the pharmaceutical composition is a patch, paste, ointment, gel, film coating, cataplasm, spray, capsule, granule, tablet, pill, oral liquid or injection.

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