Use of circfoxo3 gene expression inhibitor in preparation of medicine for preventing and / or treating periodontitis

Abstract

The application discloses application of a circFOXO3 gene expression inhibitor in preparation of a medicine for preventing and / or treating periodontitis, and belongs to the technical field of medicines.The siRNA of the circFOXO3 is adopted to down-regulate the expression of the circFOXO3 gene, so that the expression amount of ITGB6 is up-regulated, the activation of TGF-beta 1 is promoted, and then the occurrence and development of periodontitis are inhibited.The siRNA of the cholesterol-modified circFOXO3 can increase the stability and in-vivo metabolic time of the siRNA.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of a circFOXO3 gene expression inhibitor in the preparation of drugs for the prevention and / or treatment of periodontitis. Background Technology

[0002] Periodontitis is a common inflammatory disease that chronically and progressively damages and destroys the supporting tissues of the teeth. It is not only a leading cause of tooth loss in adults, but also increases the risk of various systemic diseases such as cardiovascular disease, diabetes, and kidney disease, making it a major threat to oral and overall health.

[0003] Currently, it is believed that the initiating factor of periodontitis is caused by periodontogenic bacteria and their products. These pathogens disrupt the normal oral microbiota, leading to the formation of pathogenic plaque biofilms on the tooth surface, gums, and gingival region. Periodontogenic bacteria do not directly cause periodontal tissue destruction or alveolar bone resorption; rather, the key to periodontal tissue destruction lies in the host's inflammatory immune response to the pathogens and their toxic products after infection. These periodontogenic bacteria are mainly located deep within the periodontal pockets of patients with periodontitis. Lipopolysaccharides and other virulence factors from these periodontal pathogens stimulate host cells, leading to the production of a series of inflammatory cytokines, such as tumor necrosis factor-α, interleukin-1β, and prostaglandin E2. These cytokines stimulate macrophages, fibroblasts, junctional epithelial cells, and neutrophils to produce matrix metalloproteinases, thereby mediating the destruction of periodontal tissues, especially the destruction of collagen fibers in the periodontal ligament. Furthermore, inflammatory factors induce the expression of nuclear factor κB receptor activator ligand in osteoblasts and helper T cells. Subsequently, RANKL interacts with nuclear factor κB receptor activator ligands in osteoclast precursor cells, leading to the formation and maturation of osteoclast precursor cells, which in turn mediates alveolar bone loss.

[0004] The inflammation-related gene ITGB6 is downregulated in periodontitis. Downregulated integrin β6 reduces TGF-β1 activation, leading to the occurrence and progression of periodontal inflammation. However, the molecular mechanism by which ITGB6 expression is regulated to inhibit periodontitis progression is not fully understood. Inflammatory human gingival tissue exhibits different circRNA expression profiles compared to normal human gingival tissue, and the specific mechanisms by which circRNAs function in periodontitis progression remain poorly understood. It is known that circFOXO3 can sponge-adsorb miRNAs to regulate the expression of its parent gene FOXO3, while ITGB6 expression is transcriptionally regulated. The transcription factor FOXO3 is involved in the downregulation of ITGB6 mRNA expression by *Porphyromonas gingivalis*.

[0005] Studies have found that infection with the periodontitis-causing bacterium *P. globosa* induces the activation of transcription factors FOXO1, FOXO3, and FOXO4 in gingival epithelial cells, which are involved in the expression of molecules related to oxidative stress, inflammatory response, and apoptosis. Furthermore, *P. globosa* upregulates the mRNA expression of transcription factors FOXO1 and FOXO3, and downregulates the mRNA expression of the synergists ITGB1, ITGB3, and ITGB6. This suggests that activated transcription factors FOXO1 and FOXO3 may be involved in the downregulation of ITGB6 transcriptional signaling induced by *P. globosa* infection. However, the activated transcription factors involved in the downregulation of ITGB6 gene transcriptional signaling in gingival epithelial cells during periodontitis have not yet been reported.

[0006] Because the specific pathogenic mechanisms of periodontitis are not yet fully understood, its treatment remains limited to traditional mechanical therapies, lacking effective treatments that modulate the host's immune system, and thus cannot completely restore damaged periodontal tissues to normal. Therefore, elucidating the molecular mechanisms of immune regulation in periodontal inflammatory responses will help deepen our understanding of the pathogenesis of periodontal disease and lead to the discovery of new interventions. Summary of the Invention

[0007] The purpose of this invention is to propose the application of a circFOXO3 gene expression inhibitor in the preparation of drugs for the prevention and / or treatment of periodontitis, so as to overcome at least one of the above-mentioned defects in the prior art.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] The application of the circFOXO3 gene expression inhibitor provided by this invention in the preparation of drugs for the prevention and / or treatment of periodontitis.

[0010] Preferably, the circFOXO3 gene expression inhibitor includes circFOXO3 siRNA.

[0011] Preferably, the nucleotide sequence of the siRNA sense strand is shown in SEQ ID NO.1, and the nucleotide sequence of its antisense strand is shown in SEQ ID NO.2.

[0012] Preferably, the siRNA is cholesterol-modified siRNA.

[0013] Preferably, the circFOXO3 gene expression inhibitor downregulates circFOXO3 gene expression, thereby upregulating ITGB6 expression, promoting TGF-β1 activation, and thus inhibiting the occurrence and development of periodontitis.

[0014] Preferably, the circFOXO3 gene expression inhibitor downregulates the expression of the circFOXO3 gene, thereby downregulating the expression of the transcription factor FOXO3, inhibiting the binding of FOXO3 to the ITGB6 promoter, promoting ITGB6 transcription, and upregulating the expression of ITGB6 to promote the activation of TGF-β1, thereby inhibiting the occurrence and development of periodontitis.

[0015] Preferably, the circFOXO3 gene expression inhibitor downregulates the expression of the circFOXO3 gene, thereby upregulating the expression of the transcription factor JunB, promoting the binding of JunB to the ITGB6 promoter, and thus promoting ITGB6 transcription, which in turn upregulates the expression of ITGB6, thereby promoting the activation of TGF-β1 and inhibiting the occurrence and development of periodontitis.

[0016] Preferably, the circFOXO3 gene expression inhibitor downregulates circFOXO3 gene expression, inhibits the miR-141-3p / FOXO3 / JunB signaling pathway, upregulates ITGB6 expression, promotes TGF-β1 activation, and thus inhibits the occurrence and development of periodontitis.

[0017] Preferably, the drug is an injection, powder, granule, pill, oral preparation, tablet, capsule, suppository, spray, or ointment.

[0018] The present invention also provides a pharmaceutical composition for treating periodontitis, the pharmaceutical composition comprising a circFOXO3 gene expression inhibitor.

[0019] The beneficial effects of this invention are as follows:

[0020] This invention relates to the preparation of drugs for the prevention and / or treatment of periodontitis using circFOXO3 gene expression inhibitors. Specifically, circFOXO3 siRNA is used to downregulate circFOXO3 gene expression, thereby upregulating ITGB6 expression and promoting TGF-β1 activation, thus inhibiting the occurrence and development of periodontitis. Cholesterol-modified circFOXO3 siRNA can increase its stability and in vivo metabolic time. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the expression of circFOXO3 in normal gingival epithelial tissue and periodontitis-affected gingival tissue according to the present invention.

[0022] Figure 2 This is a schematic diagram showing the potential binding sites of transcription factors FOXO3 and JunB in the -150 / -3 region of the ITGB6 promoter region of this invention.

[0023] Figure 3This is a diagram showing the changes in the binding of transcription factor FOXO3 to the ITGB6 promoter region after overexpression of circFOXO3 in HaCaT and IHGK cells, as detected by ChIP experiments of this invention.

[0024] Figure 4 This is a diagram showing the changes in the binding of transcription factor JunB to the ITGB6 promoter region after HaCaT and IHGK cells overexpressed with circFOXO3, as detected by ChIP experiments according to this invention.

[0025] Figure 5 This is a schematic diagram showing the potential binding site of transcription factor FOXO3 in the JunB promoter region of this invention.

[0026] Figure 6 These are micro-CT reconstruction images of the molar region of rats in the normal group and the ligated group, based on the present invention.

[0027] Figure 7 This is an animal model of periodontitis caused by circFOXO3 siRNA injection into the gingival sulcus according to the present invention.

[0028] Figure 8 This invention presents micro-CT reconstructions of bone tissue images of normal rats, periodontitis rats, and rats with periodontitis injected with circFOXO3 siRNA in the gingival sulcus, representing the molar region of these rats.

[0029] Figure 9 This is a schematic diagram illustrating the expression of miR-141-3p of the present invention in normal gingival epithelial tissue and periodontal disease-affected gingival tissue.

[0030] Figure 10 This is a schematic diagram illustrating the expression of FOXO3 in normal gingival epithelial tissue and periodontitis-affected gingival tissue according to the present invention.

[0031] Figure 11 This is a schematic diagram illustrating the expression of JunB of the present invention in normal gingival epithelial tissue and periodontal disease gingival tissue.

[0032] Figure 12 This is a schematic diagram illustrating the expression of ITGB6 of the present invention in normal gingival epithelial tissue and periodontal disease gingival tissue.

[0033] Figure 13 This is a diagram showing the changes in FOXO3 protein expression in periodontitis tissue detected by immunohistochemistry according to the present invention.

[0034] Figure 14 This is a diagram showing the changes in JunB protein expression in periodontitis tissue detected by immunohistochemistry according to the present invention.

[0035] Figure 15 This is a diagram showing the changes in ITGB6 protein expression in periodontitis tissue detected by immunohistochemistry according to the present invention.

[0036] Figure 16 This is a diagram showing the changes in p-Smad2 protein expression in periodontitis tissue detected by immunohistochemistry according to the present invention.

[0037] Figure 17 This is a schematic diagram illustrating the progression of periodontitis according to the present invention. Detailed Implementation

[0038] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments.

[0039] This embodiment describes the application of the circFOXO3 gene expression inhibitor in the preparation of a medicament for the prevention and / or treatment of periodontitis. The circFOXO3 gene expression inhibitor of this embodiment comprises circFOXO3 siRNA. Specifically, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.1, and the nucleotide sequence of its antisense strand is shown in SEQ ID NO.2. The siRNA is cholesterol-modified siRNA.

[0040] Specifically, circFOXO3 gene expression inhibitors downregulate circFOXO3 gene expression, leading to upregulation of ITGB6 expression, which in turn promotes TGF-β1 activation and thus inhibits the occurrence and development of periodontitis. Similarly, circFOXO3 gene expression inhibitors downregulate circFOXO3 gene expression, leading to upregulation of the transcription factor FOXO3, which inhibits FOXO3 binding to the ITGB6 promoter, thereby promoting ITGB6 transcription and upregulating ITGB6 expression, which in turn promotes TGF-β1 activation and thus inhibits the occurrence and development of periodontitis. Finally, circFOXO3 gene expression inhibitors downregulate circFOXO3 gene expression, leading to upregulation of the transcription factor JunB, which in turn promotes JunB binding to the ITGB6 promoter, thereby promoting ITGB6 transcription and upregulating ITGB6 expression, which in turn promotes TGF-β1 activation and thus inhibits the occurrence and development of periodontitis. circFOXO3 gene expression inhibitors downregulate circFOXO3 gene expression, inhibit the miR-141-3p / FOXO3 / JunB signaling pathway, and upregulate ITGB6 expression to promote TGF-β1 activation, thereby inhibiting the occurrence and development of periodontitis.

[0041] The drugs include injections, powders, granules, pills, oral preparations, tablets, capsules, suppositories, sprays, or ointments.

[0042] This embodiment also provides a pharmaceutical composition for treating periodontitis, the pharmaceutical composition comprising a circFOXO3 gene expression inhibitor.

[0043] I. Material Preparation

[0044] 1.1 Origin of human gingival tissue:

[0045] Gingival tissue samples were collected from the Stomatological Hospital of Xiamen Medical College. All samples came from patients undergoing orthodontic treatment or tooth extraction for periodontitis during routine oral care. None of the patients received any pre-operative medication, and informed consent was obtained from each patient. Periodontal health was categorized into healthy and inflamed gingival tissue based on symptoms such as periodontal pocket formation, alveolar bone loss, and gingival recession.

[0046] 1.2 Cell source:

[0047] Immortalized human keratinocytes (HaCaT) were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences; immortalized human gingival keratinocytes (IHGK) were purchased from Applied Biological Materials, Canada; and human embryonic kidney epithelial cells (293T) were purchased from the Cell Bank of Wuhan University.

[0048] II. Experimental Methods

[0049] 2.1 Cell Culture:

[0050] 2.1.1 Cell resuscitation:

[0051] 1. Preheat the water bath to 37°C and transfer the complete culture medium stored at 4°C to a 37°C incubator for preheating; 2. Quickly place the cryovials on a float plate and thaw them in a 37°C water bath; 3. Quickly place the cryovials in a low-speed centrifuge at 1200 rpm for 3 minutes; 4. Spray the transferred cryovials with 75% alcohol and transfer them to a biosafety cabinet; 5. Carefully aspirate the supernatant and add 1 mL of the corresponding complete culture medium. 6. Resuspend the cell clumps in culture medium, mix well by pipetting, and transfer to a 6cm cell culture dish. Add 3mL of complete culture medium to bring the total volume to 4mL. 7. Gently mix the cells using a figure-eight motion. 8. Label the culture dish with the cell name, passage number, and time, and transfer the dish to a cell culture incubator. 9. Once the cells have adhered, aspirate the culture medium and rinse with 1mL of sterile PBS. 10. Add 3mL of complete culture medium and place the culture dish in a 37°C, 5% CO2 cell culture incubator.

[0052] 2.1.2 Cell passage:

[0053] 1. HaCaT cells should reach a density of 90%, while IHGK cells should reach 95% before they can be passaged. 2. Discard the culture medium in a biosafety cabinet, then rinse twice with preheated PBS to remove any residual culture medium. 3. Add 1 mL of trypsin to a 6 cm culture dish and 2.5 mL of trypsin to a 10 cm culture dish. Transfer the culture dish with added trypsin to a cell culture incubator for digestion. Digestion time varies depending on the cell type: approximately 5 min for IHGK cells, approximately 7 min for HaCaT cells, and approximately 30 s for 293T cells. 4. After complete cell digestion, add 1 mL of complete culture medium to terminate digestion (F12 / DMEM is used to terminate digestion for IHGK cells), gently blow off the cells, and transfer the cell suspension to a 4 mL centrifuge tube. 5. Centrifuge at 1200 rpm for 3 min at room temperature. 6. Spray the centrifuge tube with 75% alcohol and transfer it to a biosafety cabinet. 7. Discard the supernatant, add 2 mL of the corresponding complete culture medium to resuspend the cell clumps, add 1 mL of cell suspension to each 6 cm culture dish, and then add another 2 mL of complete culture medium to maintain a total volume of 3 mL. Place the dish in a cell culture incubator for further culture.

[0054] 2.1.3 Cell cryopreservation:

[0055] 1. HaCaT cells should reach a density of 90%, while IHGK cells should reach 95% before passage and preservation. 2. Refer to "Cell Passage" for cell digestion and centrifugation procedures. 3. For HaCaT and 293T cells cultured in 6cm dishes, resuspend in 500μL of complete culture medium, add 400μL of serum and 100μL of DMSO, mix thoroughly, and transfer to cryovials. For IHGK cells, cryopreserve at a ratio of complete culture medium to DMSO of 9:1 (total volume 1mL). Cryopreserve two vials in 10cm dishes and one vial in 6cm dishes (label the cryovials with the cell name, passage number, and time of cryopreservation). 4. After transferring the cryovials to the cryopreservation box, quickly transfer the box to a -80°C freezer. 5. For long-term storage, transfer the cryovials to liquid nitrogen after 24 hours of cryopreservation.

[0056] 2.1.4 Cell Counting:

[0057] 1. Observe cell growth under a microscope. If cells are growing well, count them and seed them into a cell counting chamber. 2. Prepare a cell counting chamber, counter, and coverslips in advance. 3. Spray the coverslips and cell counting chamber with 75% alcohol and allow them to dry before use. 4. Refer to "Cell Passage" for cell digestion and centrifugation procedures. 5. Resuspend the cells in 1 mL of complete culture medium. If the cell count is too high, dilute the chamber. Carefully pipette 20 μL of the cell suspension between the cell counting chamber and the coverslip. 6. Count the cells in the four squares of the cell counting chamber, following the principle of "counting the top but not the bottom, counting the left but not the right." Count the cells three times and take the average. Cell count = (total number of cells in four squares / 4) × 10⁴ × dilution factor.

[0058] 2.2 Cell transfection and infection:

[0059] 2.2.1 Plasmid transformation:

[0060] 1. Remove competent DH5α cells from the -80℃ freezer and thaw them on ice. Add 100 ng of plasmid to each competent cell, gently tap the centrifuge tube to mix, and incubate on ice for 30 min. 2. Preheat the water bath to 42℃, then quickly place the competent cells onto a float plate and immediately place them back into the water bath for heat shock for 60-90 s. 3. Place the heat-shocked competent cells on ice for 3 min. 4. Add 100 μL of liquid bacterial culture medium to the centrifuge tube containing the competent cells and incubate at 37℃ and 200 rpm on a shaker for 1 h. 5. Spray the centrifuge tube containing the competent cells with 75% alcohol, transfer it to a clean bench, gently tap the centrifuge tube to mix the competent cells, pipette the liquid onto a pre-prepared bacterial culture dish, spread evenly with a glass rod, and place upright in a 37℃ bacterial incubator for 1 h. After the liquid in the plate has evaporated, invert the plate and incubate for 12-16 h.

[0061] 2.2.2 Bacterial Culture:

[0062] 1. Prepare a sterile 1.5mL EP tube in a clean bench and add 500μL of liquid bacterial culture medium. Pick a single colony from the bacterial culture dish and place it in the bacterial culture medium. Incubate on a shaker at 37℃ and 200rpm for 4 hours. 2. Add approximately 25mL of liquid bacterial culture medium to a 50mL centrifuge tube and add the appropriate proportion of antibiotics. Transfer the bacterial solution from the 1.5mL EP tube to the 50mL centrifuge tube and incubate on a shaker at 37℃ and 200rpm for 12-16 hours. 3. Perform a preservation treatment with glycerol to bacterial solution at a ratio of 1:4. At the same time, take 1mL of bacterial solution for sequencing to detect whether the target plasmid has been successfully transformed. Use the remaining bacterial solution for plasmid extraction.

[0063] 2.2.3 Plasmid extraction:

[0064] Plasmids were extracted using the Migi Low Endotoxin Small-Scale Extraction Kit.

[0065] 1. Centrifuge at 5000 rpm, 4℃ for 10 min; 2. Discard the supernatant and invert on absorbent paper for 3 min to remove residual liquid; 3. Add 600 μL of Buffer E1 containing RNase A and vortex thoroughly until the bacterial precipitate disappears; 4. Transfer the suspension to a 2 mL centrifuge tube, add 600 μL of Buffer E2, and invert 7 times to mix; 5. Incubate at room temperature for 2 min, inverting 2-3 times during this period (the total time for adding Buffer E1 and Buffer E2 should not exceed 4 min); 6. Add 600 μL of Buffer E3 and immediately invert 10 times; 7. Centrifuge at 13000 g, room temperature for 10 min; 8. Transfer the supernatant to a 4 mL centrifuge tube and add 550 μL of Buffer E3. After E4, invert and mix 7 times; 9. Place the adsorption column into the collection tube, pipette 750 μL of supernatant into the column, and centrifuge at 10000g for 1 min at room temperature; 10. Discard the filtrate, return the adsorption column to the collection tube, and repeat the previous operation until all liquid in the 4 mL centrifuge tube has been filtered; 11. Add 650 μL of Buffer E5 to the column, and centrifuge at 10000g for 1 min at room temperature; 12. Discard the filtrate, add 650 μL of Buffer PW2 (diluted with anhydrous ethanol), and let stand for 2 min; 13. Centrifuge at 10000g for 1 min at room temperature; 14. Discard the filtrate, and add 650 μL of Buffer E5 to the adsorption column. PW2 (diluted with anhydrous ethanol); 15. Centrifuge at 10000g for 1 min at room temperature; 16. Discard the filtrate, put the adsorption column back into the collection tube, centrifuge at 10000g for 2 min at room temperature; 17. Put the adsorption column into a sterile 1.5mL centrifuge tube, add 60-100μL Buffer TE, and let stand for 2 min; 18. Centrifuge at 10000g for 2 min at room temperature; 19. Discard the adsorption column, mix the filtrate by pipetting, determine the concentration and purity of the extracted plasmid, and store at -20℃.

[0066] 2.2.4 Instantaneous transfection:

[0067] 1. Prepare cells with a density of 70% and good growth status, and starve them for 24 hours; 2. Taking a 6cm culture dish as an example, aspirate 500μL of serum-free culture medium into a sterile 1.5mL centrifuge tube, then add 6-8μL of the transfection reagent Turbofect and 8μg of the target plasmid, and incubate at room temperature for 15 minutes; 3. Change the medium for the starved cells to 2.5mL of complete culture medium, gently add the medium containing the plasmid-liposome complex to the supernatant of the cells to be transfected, gently shake to mix, and place in a cell culture incubator at 37℃ and 5% CO2; 4. Change the medium 6 hours after transfection; 5. Collect the transfected cells for subsequent experiments 48 hours after transfection.

[0068] 2.2.5 Lentiviral Packaging:

[0069] 1. Prepare 293T cells with a density of 50% and good growth status; 2. Taking a 6cm culture dish as an example, add 500μL of serum-free culture medium to a sterile 1.5mL centrifuge tube; 3. Add the target plasmid, packaging plasmid, and transfection reagent Turbofect, and incubate at room temperature for 15min. The volume of transfection reagent and the amount of relevant plasmids used are shown in Table 1.

[0070] Components Turbofect Target plasmid VSVG PHR OPTI-MEM / DMEM Dosage 12μL 3μg 0.75μg 2.25μg 500μL

[0071] Table 1

[0072] 4. Change the medium for 293T cells, adding 2 mL of complete culture medium; 5. Add the culture medium containing the transfection complex to a culture dish, gently shake to mix, and transfer to a cell culture incubator for incubation; 6. Replace with 4 mL of complete culture medium 6 hours after transfection; 7. Collect the supernatant into a sterile 4 mL centrifuge tube 48 hours after transfection; 8. Centrifuge at 1200 rpm for 3 min at room temperature; 9. Remove dead cells from the supernatant using a 0.45 μm filter, and use the filtered virus solution for subsequent cell infection.

[0073] 2.2.6 Viral infection:

[0074] 1. Prepare cells with a density of 60% and good growth status for infection; for IHGK, the density should reach 70%. 2. Using a 6cm culture dish as an example, replace 2mL of fresh complete culture medium with 2μL of polybreme (1mg / mL) before viral infection, gently shake to mix, and transfer the 6cm culture dish to a cell culture incubator for 30min. 3. Add 2mL of virus solution to the culture dish (virus solution: complete culture medium = 1:1). 4. Replace with 3mL of complete culture medium 6 hours after infection. 5. Add appropriate antibiotics 48 hours after infection for screening. Continue screening for one week to obtain stable transfected cell lines for subsequent experiments.

[0075] 2.2.7 miR-141-3p mimics / inhibitor transfection:

[0076] 1. Prepare cells with a density of 50% and good growth status, and starve them for 24 hours; 2. Using a 6cm culture dish as an example, add 400μL of serum-free culture medium to a sterile 1.5mL RNase-free centrifuge tube; 3. Add 8μL of transfection reagent siRNA-mate and 560ng miR-141 mimics / inhibitor, and incubate at room temperature for 15 minutes; 4. Change the medium for the starved cells to 2mL of complete culture medium, gently add the medium containing the transfection complex to the supernatant of the cells to be transfected, gently shake to mix, and place in a cell culture incubator at 37℃ and 5% CO2; 5. Change the medium 6 hours after transfection; 6. Collect the transfected cells for subsequent experiments 48 hours after transfection.

[0077] 2.3 Real-time quantitative PCR:

[0078] 2.3.1 Total RNA extraction:

[0079] 1. For RNA extraction from gingival tissue, place it in a 1.5 mL centrifuge tube, add 1 mL of TRIzol, and homogenize using cryogenic homogenate. For total RNA extraction from cells, discard the culture medium, wash three times with 1×PBS, and add an appropriate amount of TRIzol. For example, add 1 mL of TRIzol to a 3.5 cm culture dish, lyse on ice for 15 min, and collect in a 1.5 mL RNase-free centrifuge tube. 2. Add 200 μL of chloroform, vortex vigorously to mix, and centrifuge at 13140 rpm at 4°C for 15 min. 3. Collect the colorless aqueous supernatant in a fresh RNase-free centrifuge tube. 1. Add 1.5 mL to a centrifuge tube (avoiding the white protein layer in the middle); 4. Add an equal volume of isopropanol (approximately 550 μL) and incubate on ice for 10 min; 5. Centrifuge at 13140 rpm, 4℃ for 15 min; 6. After centrifugation, a white, blocky RNA precipitate will be visible at the bottom. Discard the supernatant and resuspend the white precipitate in 75% ethanol; 7. Centrifuge at 13140 rpm, 4℃ for 10 min; 8. After discarding the supernatant, briefly remove the tube from the centrifuge tube, carefully aspirate any remaining liquid, and place in a fume hood to dry for 10 min; 9. Add 20-100 μL of DEPC water, mix thoroughly by pipetting, and then determine the RNA concentration and purity.

[0080] 2.3.2 circFOXO3 and mRNA reverse transcription:

[0081] Reverse transcription of circFOXO3 and mRNA was performed using the TaKaRa PrimeScript™ RT reagent Kit.

[0082] 1. Removal of genomic DNA: The reaction system should be prepared on ice, and the reaction conditions are 42°C for 2 minutes. The specific reaction system is shown in Table 2.

[0083] Mix 1 Volume (μL) gDNA Eraser 0.5 5×gDNA Eraser Buffer 1 Total RNA *(Volume corresponding to 500ng) <![CDATA[RNase-free ddH2O]]> up to 5

[0084] Table 2

[0085] 2. Reverse transcription: The reaction system should be prepared on ice. The reaction conditions are 37℃ for 15 min, 85℃ for 5 s, and stored at 4℃. The specific reaction system is shown in Table 3.

[0086] Mix 2 Volume (μL) Mix 1 5 <![CDATA[RNase-free ddH2O]]> 2 5×PrimerScript Buffer 2 2 RT Primer Mix 0.5 PrimerScript RT Enzyne Mix 1 0.5 Total 10

[0087] Table 3

[0088] 2.3.3 miR-141-3p reverse transcription:

[0089] Reverse transcription of miR-141-3p was performed using the Enzyme miRNA 1st Strand cDNA Synthesis Kit.

[0090] 1. Removal of genomic DNA: The reaction system should be prepared on ice, and the reaction conditions are 42°C for 2 minutes. The specific reaction system is shown in Table 4.

[0091]

[0092]

[0093] Table 4

[0094] 2. Reverse transcription: The reaction system should be prepared on ice. The reaction conditions are 25℃ for 5 min, 50℃ for 15 min, 85℃ for 5 min, and stored at 4℃. The specific reaction system is shown in Table 5.

[0095] Mix 2 Volume (μL) Mix 1 5 <![CDATA[RNase-free ddH2O]]> 2.5 10×RT Mix 1 Hiscript Enzyme Mix 1 Stem-loop Primer (2μM) 0.5 Total 10

[0096] Table 5

[0097] 2.3.4 qRT-PCR:

[0098] RT-qPCR experiments were conducted using SuperReal PreMix Plus (SYBR Green) from Tiangen Pharmaceuticals.

[0099] 1. Reaction system preparation: The total reaction volume per well is 20 μL, containing 18 μL Mix and 2 μL cDNA (three replicates are set for each gene to be detected in each sample). The specific reaction system is shown in Table 6.

[0100] reagent components Volume (μL) <![CDATA[RNase-free ddH2O]]> 6.4 2×supereal premix plus 10 50×ROX Reference Dye 0.4 primer F (10μM) 0.6 primer R (10μM) 0.6 cDNA 2 Total 20

[0101] Table 6

[0102] *Note: The amount of cDNA template is usually below 100ng. Because different templates contain different copy numbers of genes, the template can be diluted to ensure the Ct cycle number is between 15-35, reducing error; 2. ABI 7500 Real-Time PCR amplification instrument detection of PCR reaction: A three-step PCR reaction is performed; 3. Data analysis: After the PCR program is completed, the Ct values ​​are exported to Excel, and the ΔΔCt value method is used for data analysis, which is the Ct value. 目的基因 -Ct 内参基因 =ΔCt; ΔCt 处理样本 -ΔCt 对照样本 =ΔΔCt value method, multiple change = 2 -ΔΔCt .

[0103] 2.4 Genomic DNA Extraction:

[0104] 1. Once the cells have reached confluence and are in good growth condition, they can be collected. Using a 3.5cm culture dish as an example, discard the culture medium and rinse three times with pre-cooled 1×PBS; 2. Discard the residual liquid, add approximately 400μL of 1×PBS solution, and collect the PBS solution containing cells into a 1.5mL centrifuge tube; 3. Centrifuge at 5000rpm, 4℃ for 3min; 4. Discard the supernatant, add 500μL Lysis Buffer, 5μL β-mercaptoethanol, and 10μL Proteinase. K. Incubate at 56℃ for 2-16 hours; 5. Add 700 μL of phenol-chloroform-isoamyl alcohol (pH=8.0, 25:24:1), shake vigorously to mix; 6. Centrifuge at 13140 rpm, 4℃ for 15 min; 7. Transfer the supernatant to a new 1.5 mL centrifuge tube, add double the volume of isopropanol, invert to mix, and incubate on ice for 10 min; 8. Centrifuge at 13140 rpm, 4℃ for 15 min; 9. Discard the supernatant, leaving the white precipitate, add 1 mL of pre-cooled 75% ethanol; 10. Centrifuge at 13140 rpm, 4℃ for 5 min; 11. After discarding the supernatant, briefly remove from the centrifuge tube, carefully aspirate any remaining liquid, and place in a fume hood to dry for 10 min; 12. Add 20-100 μL of DEPC water to the centrifuge tube, mix by pipetting, and then determine the DNA concentration and purity.

[0105] 2.5 RNase R treatment and RT-PCR:

[0106] 1. Take 5 mg of total RNA and incubate with RNase R (3 U / mg) at room temperature for 10 min; 2. Refer to the experimental procedure for "circFOXO3 and mRNA reverse transcription" for the reverse transcription steps; 3. Take 2 μL of cDNA template, 6 μL of ddH2O, 1 μL of primer F, 1 μL of primer R and 10 μL of 2×PCR Mix; 4. Mix well and place in a PCR instrument for amplification.

[0107] 2.6 Agarose gel electrophoresis:

[0108] 1. Add an appropriate amount of 1×TAE solution and agarose powder to an Erlenmeyer flask to prepare a 1% agarose gel; 2. Heat the solution in a microwave oven until boiling, remove and shake well, then repeat boiling twice more until the solution is clear, transparent and free of particles; 3. Pour the solution into a gel casting container with a comb inserted, and let it stand for 30 minutes to allow the gel to form; 4. Carefully remove the comb and place the gel in an electrophoresis tank (TAE solution must submerge the gel); 5. Use a pipette to draw 9 μL of DNA sample and 1 μL of 10×Loading Buffer, mix well, and carefully add to the lanes. Add a marker to each lane for reference; 6. Electrophoresis at 110V for 25 minutes; 7. Remove the agarose gel and place it in a gel imaging system for observation and photography.

[0109] 2.7 Western blot for protein immunoblotting:

[0110] 2.7.1 Total protein extraction:

[0111] 1. Absorb the culture medium and wash three times with 1×PBS to remove residual culture medium; 2. Add approximately 400 μL of 1×PBS solution and collect the PBS solution containing cells into a 1.5 mL centrifuge tube; 3. Centrifuge at 5000 rpm and 4°C for 3 min; 4. Aspirate the supernatant and add 100 μL of pre-mixed RIPA lysis buffer (pre-added with 1 μL PIC and 1 μL PMSF); 5. Vortex to mix and lyse on ice for 30 min; 6. Sonicate at 20% power for 7 seconds each time until the solution is clear or the solution is no longer viscous when aspirated; 7. Centrifuge at 13140 rpm and 4°C for 15 min; 8. Carefully aspirate the supernatant to a new 1.5 mL centrifuge tube, being careful not to aspirate the precipitate at the bottom.

[0112] 2.7.2 BCA method for determining protein concentration:

[0113] 1. Prepare 0.5 mg / mL BSA working solution. Prepare BCA working solution at a ratio of solution A: solution B = 50:1. Also prepare 1×PBS. The standard protein curve is shown in Table 7.

[0114] serial number 1 2 3 4 5 6 7 8 PBS 20 19 18 16 12 8 4 0 BSA 0 1 2 4 8 12 16 20 BCA 100 100 100 100 100 100 100 100

[0115] Table 7

[0116] 2. Pipette 2 μL of the protein sample to be tested, 18 μL of 1×PBS, and 100 μL of BCA working solution into the enzyme-linked immunosorbent assay (ELISA) strip and incubate at 37°C for 30 min; 3. Set the wavelength of the ELISA reader to 570 nm and measure the absorbance value of each well; 4. Plot a standard protein curve with BSA amount on the x-axis and absorbance value on the y-axis. R² ≥ 0.99 indicates that the results are relatively reliable; 5. Calculate the loading amount of each sample based on the standard curve.

[0117] 2.7.3 Protein denaturation:

[0118] 1. Add 1 / 4 of the sample volume of 5×SDS Loading Buffer; 2. Boil at 100℃ for 10 min, centrifuge at 5000 rpm for 1 min, vortex to mix, and store at -20℃.

[0119] 2.7.4 SDS-PAGE electrophoresis:

[0120] 1. Assemble the cleaned and dried glass plates and gel casting rack; 2. Add 7.5 mL of the prepared separating gel solution to each side, and add anhydrous ethanol to the edge of the glass plate. Let it stand at room temperature for 50 min; 3. Discard the anhydrous ethanol, and rinse with ultrapure water to remove any residual anhydrous ethanol. Then, blot away excess water with filter paper; 4. Add the prepared stacking gel to each side, ensuring the stacking gel just covers the edge of the glass plate. Quickly insert the comb, and let it stand at room temperature for 25 min before use; 5. Assemble the electrophoresis tank, add 1× electrophoresis buffer, and gently remove the comb from the gel; 6. Remove the protein sample, boil for 5 min, centrifuge at 5000 rpm for 30 s at room temperature, vortex to mix, add the same mass of protein sample to each well, and add 3 μL of protein marker to each lane as a control. Load all lanes with 1× SDS-PAGE. 7. Add buffer to match the total volume; 8. Perform electrophoresis at a constant voltage of 80V. When the marker is clearly separated from the bromophenol blue, increase the voltage to 120V; 9. Stop electrophoresis when the target band is close to the bottom of the separating gel.

[0121] 2.7.5 Immunoblot analysis:

[0122] 1. Wet transfer: Remove the gel and cut it to an appropriate size according to the sample loading situation, then immerse it in the wet transfer solution; 2. Cut a PVDF membrane to an appropriate size and activate it in methanol for 90 seconds; 3. Assemble the gel, PVDF membrane, sponge pad, and wet transfer box according to the electrode sequence, and place the assembled wet transfer box in an ice-water mixture for 1.5 hours of constant voltage wet transfer at 100V; 4. Blocking: Remove the PVDF membrane and add 5% skim milk, incubate at room temperature for 1 hour (phosphorylated proteins are blocked with 5% BSA); 5. Incubation with primary antibody: Place the cut target band into the protective card membrane, add the corresponding primary antibody, and incubate in suspension at 4°C. 6. Washing: After collecting the primary antibody, use tweezers to transfer the target band to the incubation box; 7. Rinse 4 times with 1×TBST, 5 minutes each time; 8. Incubate with secondary antibody: Discard 1×TBST, add 2.5 mL of secondary antibody of the same species as the primary antibody (1×TBST diluted to the corresponding ratio) to each compartment of the incubation box, and incubate at room temperature on a shaker for 2 hours; 9. Washing: Discard the secondary antibody, rinse 4 times with 1×TBST, 5 minutes each time; 10. Development: Take appropriate amounts of ECL luminescent solution A and B, mix them in equal proportions, immerse PVDF in the luminescent solution, use tweezers to remove it and place it on a chemiluminescence imaging instrument for development.

[0123] 2.8 Dual-luciferase reporter gene:

[0124] 2.8.1 Cell transfection:

[0125] 1. Seed 293T cells in 96-well plates (12,000 cells / well). Transfection can be performed when the confluence reaches 60%. 2. Transfer 270 μL of serum-free DMEM to a sterile 500 μL centrifuge tube, add 6 μL of Turbofect transfection reagent, and then add 0.5 μg of each plasmid to be transfected (if the vector does not contain Renilla luciferase fragment, add 2.5 μg of Renilla plasmid to each tube). Mix well and incubate at room temperature for 15 min. 3. After 6 h of transfection, change the medium for 293T cells. Add 100 μL of complete culture medium to each well, and then add 50 μL of the transfection mixture to each well. 4. After culturing for 24-48 h, detect Luciferase activity.

[0126] 2.8.2 Luciferase activity assay:

[0127] 1. Discard the culture medium and rinse each well twice with 100 μL of 1×PBS; 2. Add 23 μL of 1×Lysis Buffer to each well and lyse at room temperature for 15 min; 3. Transfer 20 μL of supernatant to a 1.5 mL centrifuge tube, add 20 μL of LAR solution, mix well, and measure the firefly luciferase level; 4. Add 20 μL of STOP & Glo Buffer and measure the Renilla luciferase level; 5. Calculate the ratio of firefly luciferase to Renilla luciferase in each group and discard values ​​with large deviations. This experiment was conducted in 5 replicates, and the results were repeated at least 3 times.

[0128] 2.9 Chromatin Immunoprecipitation:

[0129] 2.9.1 Formaldehyde cross-linking:

[0130] 1. Seed cells in 10cm culture dishes. Once the cells have reached confluence and are in good growth condition, they can be collected for experiments (approximately 1×10⁷ cells); 2. Reserve 4mL of culture medium in the culture dish, add 108μL of 37% formaldehyde for cross-linking (working concentration 1%), transfer the culture dish to a shaker, and incubate at room temperature for 10min; 3. Add 10×Glycine and incubate at room temperature for 7min; 4. Discard the supernatant, wash three times with 1×PBS, collect the cells in a 1.5mL centrifuge tube, add 1μL PMSF and 1μL PIC, and store at -80℃.

[0131] 2.9.2 Ultrasonic fragmentation:

[0132] 1. Add 200 μL Lysis Buffer (pre-added with 1 μL PIC and 1 μL PMSF), and lyse on ice for 30 min; 2. Sonicate cells at 20% power for 12 seconds per cycle, for a total of 14 cycles; 3. Centrifuge at 13140 rpm at 4℃ for 15 min; 4. Transfer the supernatant to a new 1.5 mL centrifuge tube, add 600 μL Dilution Buffer, mix well, and store at -80℃.

[0133] 2.9.3 Chromatin fragmentation detection:

[0134] 1. Transfer 50 μL from the 800 μL mixture to a 1.5 mL centrifuge tube, and store the remaining 750 μL solution at -80°C; 2. Add 4 μL Proteinase K (working concentration 0.4 mg / mL) and 10 μL 5M NaCl (working concentration 0.2 M), and incubate overnight at 65°C; 3. Centrifuge at 5000 rpm for 30 s at room temperature. After the liquid on the tube wall is collected, add 1 μL RNase A (final concentration 10 μg / μL), and incubate at 37°C for 15 min; 4. Add an equal volume of phenol-chloroform-isoamyl alcohol (pH = 8.0, 25:24:1), and mix vigorously; 5. Centrifuge at 13140 rpm for 15 min at 4°C; 6. Transfer the supernatant to a 1.5 mL centrifuge tube, add 500 μL pre-chilled anhydrous ethanol, 1 μL glycogen, and 20 μL 3M NaCl. 7. Place at -80℃ for 1 hour or at -20℃ overnight; 8. Remove and thaw, centrifuge at 13140 rpm, 4℃ for 15 min; 9. Discard the supernatant, add 75% ethanol to resuspend the precipitate, centrifuge at 13140 rpm, 4℃ for 5 min; 10. Discard the supernatant, place in a fume hood to dry for 10 min, add 12 μL of DEPC aqueous solution, and mix by pipetting; 11. Determine the concentration and purity of the DNA fragment using a spectrophotometer; 12. Detect the size of the sonicated fragment by agarose gel electrophoresis (fragment size of 300-500 bp is more suitable).

[0135] 2.9.4 Chromatin Immunoprecipitation:

[0136] 1. Calculate the amount of chromatin in the remaining 750 μL of the mixture based on the DNA fragments detected, ensuring consistent chromatin content across all IP groups. The chromatin content of the Input group should be 1 / 5 that of the IP group. Divide the experimental samples into three portions: Input, IP, and IgG. 2. Add the corresponding experimental antibodies to the IP and IgG samples, and bring the volume to the same level using Dilution Buffer. Incubate at 4°C for 16 hours. Store the Input group samples at -80°C. 3. Add 20 μL of protein A / G beads to each IP and IgG sample. Transfer an appropriate amount of magnetic beads to a 1.5 mL centrifuge tube, wash twice with 1 mL of Dilution Buffer, and restore to the initial volume with additional Dilution Buffer. 4. Add the equilibrated magnetic beads to the IP and IgG samples and incubate at 4°C for 2 hours. 5. Collect the magnetic beads using a magnetic rack and wash three times with 150 μL of Low Salt Buffer. 6. Collect the magnetic beads using a magnetic rack and add 150 μL of High Salt Buffer. 7. Collect the magnetic beads with a magnetic rack and wash once with 150 μL LiCl Wash Buffer; 8. Collect the magnetic beads with a magnetic rack and wash once with 150 μL TE Buffer.

[0137] 2.9.5 Chromatin decrosslinking and purification:

[0138] 1. Add 150 μL of Elution Buffer, 5 μL of 5M NaCl, and 2 μL of proteinase K to each of the IP and IgG groups (for the Input group, bring the volume to 150 μL with Elution Buffer, then add 5 μL of 5M NaCl and 2 μL of proteinase K); 2. Incubate at 65°C for 2 hours; 3. Use a magnetic rack to collect the liquid into a new 1.5 mL centrifuge tube by adsorbing magnetic beads; 4. Add 150 μL of Elution Buffer to the old tube and incubate at 65°C for 5 minutes; 5. Transfer the supernatant from the old tube to the new tube and incubate with Elution Buffer. 6. Add 500 μL of phenol-chloroform-isoamyl alcohol (pH = 8.0, 25:24:1) to the Buffer to bring the sample volumes of Input, IP, and IgG groups to 500 μL; 7. Centrifuge at 13140 rpm, 4°C for 15 min; 8. Transfer the supernatant (approximately 440 μL) to a new 1.5 mL centrifuge tube, add 200 μL of phenol-chloroform-isoamyl alcohol (pH = 8.0, 25:24:1), and mix vigorously; 9. Centrifuge at 13140 rpm, 4°C for 15 min; 10. Transfer the supernatant (approximately 400 μL) to a new 1.5 mL centrifuge tube, add 10 μL of 0.25% acrylamide and 40 μL of 3M... 11. Add NaAC (pH=5.2) and 1 mL of pre-cooled anhydrous ethanol, and precipitate at -80℃ for 1 h or at -20℃ overnight; 12. Remove and thaw, centrifuge at 13140 rpm, 4℃ for 15 min; 13. Discard the supernatant, add 1 mL of 75% ethanol to resuspend the precipitate, and centrifuge at 13140 rpm, 4℃ for 5 min; 14. Discard the supernatant, and dry in a fume hood for 10 min; 15. Add 20 μL of DEPC water to dissolve the DNA fragment, and store at -20℃.

[0139] 2.9.6 PCR detection:

[0140] 1. Qualitative analysis: conventional PCR; 2. Quantitative analysis: RT-qPCR.

[0141] 2.10 Immunoprecipitation:

[0142] 1. Seed cells in 10cm culture dishes. Once the cells have reached confluence and are in good growth condition, they can be harvested for experiments (approximately 1×10⁷ cells); 2. Discard the culture medium and wash three times with 1×PBS; 3. Add 10μL PMSF and 10μL PIC, then add 1mL mild RIPA lysis buffer; 4. Collect cells in 1.5mL centrifuge tubes and lyse on ice for 30min; 5. Centrifuge at 13140rpm, 4℃ for 15min; 6. Collect the supernatant in 1.5mL centrifuge tubes, mix well, and then aspirate 100μL as the Input group. Divide the remaining 900μL equally into IgG and IP groups; 7. Aspirate 100μL of magnetic beads into 1.5mL centrifuge tubes, wash twice with 1mL RIPA, and then add 100μL of RIPA solution. 8. Add 50 μL of magnetic beads to each of the IgG and IP groups, and incubate in a resuscitator at 4℃ for 2 h; 9. After the magnetic beads are adsorbed by the magnetic rack, aspirate the supernatant into a 1.5 mL centrifuge tube, add the corresponding antibodies to the IgG and IP groups, and incubate in a resuscitator at 4℃ for 16 h; 10. Add 50 μL of protein A / G beads to each of the IP and IgG groups, aspirate an appropriate amount of magnetic beads into a 1.5 mL centrifuge tube, add 1 mL of RIPA and wash twice, then add an appropriate amount of RIPA to restore to the initial volume; 11. Add the equilibrated magnetic beads to the IgG and IP samples, and incubate in a resuscitator at 4℃ for 2 h; 12. Collect the magnetic beads with the magnetic rack, add 500 μL of RIPA and wash three times; 13. Collect the magnetic beads with the magnetic rack, add 30 μL of 2×Loading Buffer, add 25 μL of 5×Loading Buffer to the Input group, boil in boiling water for 10 min for denaturation, and store at -20℃; 14. Perform SDS-PAGE electrophoresis and Western blot analysis.

[0143] 2.11 Immunofluorescence:

[0144] 2.11.1 Cell smear immunofluorescence:

[0145] 1. Place sterilized glass slides in 6-well plates, seed with an appropriate amount of cells (approximately 75% cell density is sufficient for subsequent experiments); 2. Discard the culture medium and wash three times with 1×PBS; 3. Add 500 μL of 4% paraformaldehyde and fix at room temperature for 20 min; 4. Discard the 4% paraformaldehyde and wash three times with 1×PBS, 3 min each time; 5. Add 500 μL of 0.5% Triton X-100 and permeabilize at room temperature for 20 min (this step can be omitted for membrane protein detection); 6. Discard the 0.5% Triton X-100 solution. 7. Wash the slide three times with X-100 and 1×PBS, 3 min each time; 8. Remove the residual liquid from the slide and draw circles with an oil-based pen; 9. Block with 5% goat serum for 20 min; 10. Absorb the blocking solution with absorbent paper, add the corresponding primary antibody directly, and incubate overnight at 4°C; 11. Discard the primary antibody, wash three times with 1×PBS, 3 min each time; 12. Remove the residual liquid from the slide, add diluted fluorescent secondary antibody, and incubate at room temperature for 1 h (this step should be performed under light-protected conditions); 13. Discard the secondary antibody, wash three times with 1×PBS, 3 min each time; 14. Remove the slide, absorb the residual liquid from the slide with absorbent paper, add 10 μL of mounting solution containing DAPI to mount the slide; 15. Acquire images under a fluorescence microscope.

[0146] 2.11.2 Immunofluorescence of paraffin sections:

[0147] 1. Place the cut slides in a 65℃ oven and bake for 2 hours; 2. Perform dewaxing and rehydration according to the following steps: xylene for 15 min × 2, then immerse in 100%, 95%, 85%, and 75% ethanol (3 min each time), followed by 1×PBS for 3 min; 3. Measure 19 mL of 0.1 mol / L sodium citrate solution and 81 mL of 0.1 mol / L citric acid solution, then add 980 mL of double-distilled water to prepare 1 L of 0.01 M sodium citrate buffer (pH = 6.0). Boil the buffer in a microwave oven, then soak the slides in the solution for antigen retrieval for 8 min; 4. After the antigen retrieval solution cools down, wash three times with 1×PBS for 3 min each time; 5. Remove water droplets from the slides, draw circles with an oil-based pen, add endogenous peroxidase, and incubate at room temperature for 25 min; 6. Wash three times with 1×PBS for 3 min each time; 7. Remove water droplets from the slides, draw circles with an oil-based pen, add endogenous peroxidase, and incubate at room temperature for 25 min; 6. Wash three times with 1×PBS for 3 min each time; 7. 8. Remove the liquid from the slide, add 5% goat serum, and incubate at room temperature for 20 min; 9. Remove the blocking solution from the slide, add the corresponding primary antibody directly, and incubate at 4℃ overnight; 10. Discard the primary antibody, wash three times with 1×PBS, 3 min each time; 11. Remove the residual liquid from the slide, add diluted fluorescent secondary antibody, and incubate at room temperature for 1 h (this step should be performed under light-protected conditions); 12. Discard the secondary antibody, wash three times with 1×PBS, 3 min each time; 13. Absorb excess water from the slide with absorbent paper, add 10 μL of mounting solution containing DAPI, and mount the slide; 14. Acquire images under a fluorescence microscope.

[0148] 2.12 Immunoprecipitation of RNA-binding proteins:

[0149] RIP experiments were performed using the Guangzhou Boxin Bio-RNA Immunoprecipitation (RIP) Kit.

[0150] 2.12.1 Cell lysis:

[0151] 1. Seed cells in 10cm culture dishes. After the cells have grown to confluence and are in good condition, proceed with subsequent experiments. This experiment used two 10cm culture dishes (approximately 2 × 10⁷ cells); 2. Transfer 1.7mL of lysis buffer to a 1.5mL centrifuge tube, add 7.5μL of RNase inhibitor and 17μL of protease inhibitor, mix well, and store on ice; 3. Discard the culture medium, wash three times with 1×PBS, collect the cells in a 2mL centrifuge tube, quickly add the mixed polysome lysis buffer, vortex to mix, and freeze at -80℃ for 5min; 4. Remove the sample, thaw at room temperature, and then lyse on ice for 5min.

[0152] 2.12.2 DNA Removal:

[0153] 1. Add 8.5 μL DNase salt stock and 20 μL DNase, and incubate at 37°C for 10 min; 2. Transfer the sample to ice, add 17 μL DTT, 3.6 μL EGTA and 9 μL EDTA, and vortex to mix; 3. Centrifuge at 16100g at 4°C for 15 min; 4. Transfer the supernatant to a new 2 mL RNase-free centrifuge tube.

[0154] 2.12.3 Immunoprecipitation:

[0155] 1. Take 100 μL of sample as Input, and then take 800 μL of sample as IP and IgG respectively; 2. Add the corresponding antibodies to the IP and IgG groups respectively, and incubate in a resuspending apparatus at 4℃ for 16 h; 3. Take 40 μL of magnetic beads into a 1.5 mL centrifuge tube; 4. Collect the magnetic beads with a magnetic rack, remove the magnetic bead preservation solution, add 500 μL of polysome lysis buffer to wash twice, and then add 40 μL of polysome lysis buffer to restore to the initial volume; 5. Add 20 μL of magnetic beads to the IP and IgG groups respectively, and incubate in a resuspending apparatus at 4℃ for 2 h; 6. Adsorb the magnetic beads with a magnetic rack, and discard the supernatant; 7. Add 500 μL of polysome washing buffer 1 and 5 μL of DTT to the IP and IgG groups respectively; 8. Wash 3 times, placing in a resuspending apparatus each time, and incubate at 4℃ for 5 min; 9. Adsorb the magnetic beads with a magnetic rack, and discard the supernatant; 10. Add 500 μL of polysome washing buffer 2 and 5 μL of DTT to the IP and IgG groups respectively. DTT; 11. Wash twice, each time in a resuspender, incubate at 4℃ for 5 min; 12. Adsorb magnetic beads using a magnetic rack, discard the supernatant; 13. Add 200 μL of elution buffer, 2 μL of proteinase K and 2 μL of DTT to the IP and IgG groups respectively to resuspend the magnetic beads; 14. Add 100 μL of elution buffer, 2 μL of proteinase K and 2 μL of DTT to the Input group; 15. Incubate at 55℃ for 1 h; 16. Adsorb magnetic beads using a magnetic rack, collect the supernatant into a new 1.5 mL RNase-free centrifuge tube.

[0156] 2.12.4 RNA Extraction and Purification:

[0157] 1. Add 200 μL of phenol-chloroform-isoamyl alcohol (25:24:1) to each of the Input, IP, and IgG groups, and vortex vigorously to mix. 2. Centrifuge at 13000g, 4℃ for 10 min. 3. Collect the colorless aqueous supernatant into a new 1.5 mL RNase-free centrifuge tube. 4. Add 1 μL of Glycogen, 10 μL of sodium acetate, and 500 μL of pre-chilled anhydrous ethanol to each of the Input, IP, and IgG groups, vortex to mix, and place in a -80℃ freezer for 3 h to precipitate. 5. Centrifuge at 16100g, 4℃ for 30 min. 6. Discard the supernatant, resuspend the precipitate in 1 mL of pre-chilled 75% ethanol, and centrifuge at 16100g, 4℃ for 10 min. 7. Discard the supernatant and dry in a fume hood for 10 min. 8. Add 15 μL of RNase-free water solution to precipitate the RNA and store at -80℃.

[0158] 2.12.5 PCR detection:

[0159] 1. Qualitative analysis: conventional PCR; 2. Quantitative analysis: RT-qPCR.

[0160] 2.13 Animal model of periodontitis:

[0161] 2.13.1 Ligation of rats:

[0162] 1. Twenty-three male SD rats weighing approximately 200g were purchased from the Experimental Animal Center of Xiamen University and subjected to experiments after 4 days of acclimatization. 2. The rats were divided into three groups: unligated + si-nc group (n=7), ligated + si-nc group (n=8), and ligated + si-circFOXO3 group (n=8). The rats were anesthetized with 10% chloral hydrate (dose was 0.4mL / 100g). 3. The rats were fixed to the fixation board with rubber bands. After opening the oral cavity, the SD rats were bilaterally ligated with 5.0 non-absorbable sutures. The ligation area was the second molar in the maxillary molar region. The rats were fed normally for 4 weeks after ligation.

[0163] 2.13.2 Injection of si-circFOXO3 into the gums:

[0164] 1. Both si nc and si-circFOXO3 are modified with cholesterol to increase their stability and in vivo metabolic time, and are prepared with RNase-free physiological saline; the nucleotide sequences of si nc and si-circFOXO3 are shown in Table 8:

[0165]

[0166] Table 8

[0167] 2. After anesthetizing the rats with isoflurane, fix them on a fixation plate, open their mouths, and inject 30 μL of siRNA into the maxillary second molar region on both sides using a 1 ml syringe (siRNA dose is 0.5 nmol / side); 3. After the first injection of siRNA, inject again after two days, for a total of 5 injections of siRNA; 4. Sacrifice the rats by CO2 asphyxiation, dissect the tissue in the maxillary molar region on both sides, and fix it with 4% paraformaldehyde.

[0168] 2.13.3Micro-computed tomography (Micro-CT) scan:

[0169] 1. Use Micro-CT 360° scan to scan the maxillary molar region tissue; 2. Use a high-resolution micro-CT imaging data analysis system to reconstruct the maxillary molar region for subsequent analysis.

[0170] 2.14 Paraffin Embedding:

[0171] 1. Fix fresh tissue in 4% paraformaldehyde solution for 24 hours; 2. Rinse with running water for 3 hours, then immerse the tissue sequentially in 50% ethanol for 30 minutes on a shaker, followed by sequential immersion in 75%, 85%, 95%, and 100% ethanol for 30 minutes each time (the volume of ethanol used for dehydration should be at least 4 times the volume of the embedded tissue); 3. Extract for 1 hour, then melt the tissue in a 65°C water bath using a wax container containing 1 / 2 xylene and 1 / 2 paraffin, a 100% paraffin wax container I, and a 100% paraffin wax container II; 4. Remove the tissue dehydrated with 100% ethanol and immerse it in a solution containing 1 / 2 anhydrous ethanol and 1 / 2 xylene for 15 minutes, followed by immersion in xylene for 30 minutes; 5. Remove the tissue and immerse it in a wax container containing 1 / 2 xylene and 1 / 2 paraffin for 30 minutes; 6. Remove the tissue and immerse it in a solution containing 100% paraformaldehyde and 1 / 2 paraffin. 7. Place the embedding machine in a 100% paraffin wax container I for 40 minutes and in a 100% paraffin wax container II for 1 hour (the water bath must be maintained at 65℃ during the experiment to ensure that the wax in the wax container is completely melted); 8. Turn on the embedding machine in advance. After the wax in the embedding machine melts, place the iron embedding box on the hot stage, add a small amount of paraffin wax, place the tissue at a suitable angle, and continue to add paraffin wax until the iron embedding box is just filled; 9. Transfer the iron paraffin embedding box to the cold stage. After solidification for 10 minutes, the wax block can be removed; 10. Prepare a blade, pencil, tweezers, and brush in advance. Preheat the water bath to 42℃, adjust the microtome parameters to make the section thickness 5mm, and use the brush to gently pick up the section and place it on the water surface for spreading; 11. After the section is fully spread, it can be retrieved with an adhesive slide; 12. Place the cut section in a 55℃ oven and bake overnight. Store the remaining wax block at 4℃.

[0172] 2.15 HE staining:

[0173] 1. Place the cut slides in a 65℃ oven and bake for 2 hours; 2. Dewax and rehydrate according to the following steps: xylene 15 min × 2, then immerse in 100%, 95%, 85%, and 75% ethanol (3 min each time), 1×PBS 3 min; 3. Remove water droplets from the slide, draw circles with an oil-based pen, add hematoxylin staining solution to stain the nucleus for 8 s, rinse with running water to reverse the blue stain; 4. Wash with double-distilled water for 30 s, remove water droplets from the slide, add eosin staining solution, and incubate for 1 min; 5. Rinse off the eosin staining solution with double-distilled water, and dehydrate according to the following steps: 75% ethanol 3 min, 85% ethanol 3 min, 95% ethanol 3 min, 100% ethanol 3 min, xylene 15 min × 2; 6. Mount with neutral resin and air dry in a fume hood; 7. Acquire images under a microscope.

[0174] 2.16 Immunohistochemistry:

[0175] 1. Place the cut sections in a 65℃ oven and bake for 2 hours; 2. Perform dewaxing and rehydration according to the following steps: xylene 15min×2, then immerse in 100%, 95%, 85%, and 75% ethanol (3min each time), followed by 1×PBS. 3 min; 3. Take 19 mL of 0.1 mol / L sodium citrate solution and 81 mL of 0.1 mol / L citric acid solution, then add 980 mL of double-distilled water to prepare 1 L of 0.01 M sodium citrate buffer (pH = 6.0), boil in a microwave oven, then soak the slides and allow antigen retrieval for 8 min; 4. After the antigen retrieval solution cools down, wash 3 times with 1×PBS, 3 min each time; 5. Remove water droplets from the slide, draw circles with an oil-based pen, add endogenous peroxidase, and incubate at room temperature for 25 min; 6. Wash 3 times with 1×PBS, 3 min each time; 7. Remove liquid from the slide, add 5% goat serum, and incubate at room temperature for 20 min; 8. Remove blocking solution from the slide, and directly add the corresponding primary antibody. 9. Incubate overnight at 4℃; 10. Discard the primary antibody, wash 3 times with 1×PBS, 3 min each time; 11. Absorb the residual liquid on the slide with absorbent paper, add the secondary antibody, and incubate at room temperature for 15 min; 12. Discard the secondary antibody, wash 3 times with 1×PBS; 13. Add DAB staining solution, rinse off immediately once a light brown color appears, and wash 3 times with 1×PBS; 14. Absorb the water droplets on the slide with absorbent paper, add hematoxylin staining solution to stain the nucleus for 8 s, rinse with running water to reverse the blue color; 15. Dehydrate according to the following steps: 75% ethanol for 3 min, 85% ethanol for 3 min, 95% ethanol for 3 min, 100% ethanol for 3 min, xylene for 15 min × 2; 16. Mount the slide with neutral resin and place it in a fume hood to dry; 17. Acquire images under a microscope.

[0176] 2.17 Statistical Analysis:

[0177] In this embodiment, Graph Pad Prism 6.0 software was used to perform statistical analysis and graphing of the experimental data. The statistical results are expressed as mean ± standard deviation (mean ± SD) and p-value. The differences between the two groups were analyzed using t-tests, and the comparisons between multiple groups were performed using one-way ANOVA. A p-value < 0.05 was considered statistically significant between the groups.

[0178] III. Experimental Results

[0179] 3.1 High expression of circFOXO3 in human periodontal gingival epithelial tissue:

[0180] To determine whether circFOXO3 is involved in the course of periodontitis, total RNA was first extracted from gingival tissue of 50 patients with periodontitis and 20 healthy individuals. qRT-PCR experiments revealed that the expression level of circFOXO3 in the gingival tissue of patients with periodontitis was approximately 1.7 times that in the gingival tissue of healthy individuals (e.g., ...). Figure 1 As shown, Figure 1 (p<0.0001). Further fluorescence in situ hybridization (FISH) revealed that circFOXO3 was located in the cytoplasm of gingival epithelial tissue, and its fluorescence intensity was significantly stronger in the gingival epithelial tissue of patients with periodontitis than in normal gingival epithelial tissue. These results indicate that circFOXO3 expression is upregulated during the development and progression of periodontitis, and is mainly located in the cytoplasm; therefore, it is speculated that circFOXO3 may be involved in the pathogenesis of periodontitis.

[0181] 3.2 Construction of cicrFOXO3 plasmid and its intracellular circularization verification:

[0182] circFOXO3 is formed by head-to-tail splicing of the second exon of the FOXO3 gene, located at stain 6, and contains 1435 nucleotides. To further determine whether circFOXO3 is involved in the progression of periodontitis, the full-length circFOXO3 sequence was cloned into the lentiviral vector pLV-EGFP-Puro, packaged into lentivirus, and used to infect cells, constructing stable HaCaT and IHGK cell lines overexpressing circFOXO3. RNA was extracted from the cells and RT-PCR was performed using specific transcircular primers. Agarose gel electrophoresis results showed that, compared with the empty vector group, the circFOXO3 overexpression vector in the overexpression group successfully circularized in cells. Sanger sequencing further verified the successful intracellular circularization of the circFOXO3 overexpression vector. It is worth noting that head-to-tail splicing can be generated through trans-splicing or genomic rearrangement. To demonstrate that the intracellular circularization of the circFOXO3 overexpression vector is achieved through trans-splicing, FOXO3 mRNA was further amplified using convergent primers, and circFOXO3 was amplified using divergent primers. The results showed that circFOXO3 was amplified only in cDNA, not in genomic DNA (gDNA), while FOXO3 mRNA was amplified in both cDNA and gDNA by the convergent primers. Furthermore, compared to the control group, treatment with ribonuclease R (RNase R) did not significantly change circFOXO3 expression, but FOXO3 mRNA expression was significantly downregulated, indicating that circFOXO3 is resistant to RNase R treatment due to its closed-circle structure.

[0183] 3.3 circFOXO3 downregulates ITGB6 expression in epithelial cells, mediating TGF-β1 activation:

[0184] To investigate whether circFOXO3 promotes periodontitis progression by inhibiting TGF-β1 activation, we first examined whether circFOXO3 overexpression downregulated ITGB6 expression and Smad2 phosphorylation. The results showed that HaCaT overexpression of circFOXO3 downregulated ITGB6 mRNA expression by 63%, while IHGK overexpression downregulated it by 51%. Simultaneously, circFOXO3 overexpression significantly inhibited ITGB6 protein expression in both types of epithelial cells. Furthermore, circFOXO3 overexpression significantly downregulated p-Smad2 levels. These results suggest that circFOXO3 may promote periodontitis progression by downregulating ITGB6 expression and thus inhibiting TGF-β1 activation.

[0185] 3.4 The ITGB6 promoter region contains binding sites for transcription factors FOXO3 and JunB:

[0186] Using the Jaspar database, it was predicted that the ITGB6 promoter -150 / -3 region contains binding sites for transcription factors FOXO3 and JunB (e.g., ...). Figure 2 (As shown in the image), therefore, it is speculated that FOXO3 and JunB may be involved in the transcription of ITGB6 in epithelial cells. A series of recombinant luciferase reporter gene plasmids containing 5' flanking sequences of the ITGB6 gene of different lengths were co-transfected into 293T cells with pRL-TK and FOXO3 or JunB overexpression plasmids. The results showed that when the 5' flanking fragment of the ITGB6 gene was truncated to between -3 and +208, promoter activity significantly decreased, indicating that the -150 / -3 region may contain binding sites for transcription factors FOXO3 and JunB. To further verify whether FOXO3 and JunB are involved in regulating ITGB6 gene transcription in intracellular chromatin, ChIP technology was used to detect the binding of FOXO3 and JunB to the ITGB6 promoter region in the chromatin of HaCaT and IHGK cells. The results showed that compared with control IgG, FOXO3 and JunB were significantly enriched in the ITGB6 promoter. These results indicate that FOXO3 and JunB can bind to the human ITGB6 promoter region within cells, suggesting their potential involvement in the transcriptional regulation of the human ITGB6 gene.

[0187] 3.5 Transcription factors FOXO3 and JunB mediate the downregulation of ITGB6 expression by circFOXO3:

[0188] To investigate whether transcription factors FOXO3 and JunB mediate the downregulation of ITGB6 expression in HaCaT and IHGK cells by circFOXO3, the effects of circFOXO3 overexpression on FOXO3 and JunB expression in epithelial cells were first examined. The results showed that overexpression of circFOXO3 in HaCaT and IHGK cells significantly upregulated both FOXO3 mRNA and protein expression, while significantly downregulated both JunB mRNA and protein expression. Furthermore, FOXO3 overexpression significantly reduced ITGB6 mRNA and protein levels, while JunB overexpression significantly increased ITGB6 mRNA and protein levels. These results indicate that FOXO3 is negatively correlated with ITGB6 expression, while JunB is positively correlated, suggesting that circFOXO3 may regulate ITGB6 expression by mediating the expression of FOXO3 and JunB. To further clarify whether circFOXO3 regulates ITGB6 expression through FOXO3 and JunB, ChIP experiments were used to investigate the binding of FOXO3 and JunB to the ITGB6 promoter region after overexpression of circFOXO3. ChIP results showed that overexpression of circFOXO3 in both types of epithelial cell lines increased the binding of the transcription factor FOXO3 to the ITGB6 promoter in HaCaT cells by 118% and to the promoter in IHGK cells by 342% (e.g., ...). Figure 3 As shown, Figure 3 In the study, **p<0.01**, the binding of transcription factor JunB to two epithelial ITGB6 promoters decreased by 62% and 86%, respectively (e.g., **p<0.01**). Figure 4 As shown, Figure 4 (**p<0.01). The above results indicate that FOXO3 may act as a transcriptional repressor involved in the downregulation of ITGB6 transcription by circFOXO3, while JunB may act as a transcriptional activator involved in the upregulation of ITGB6 transcription by circFOXO3.

[0189] 3.6 The interaction between transcription factor FOXO3 and JunB mediates the downregulation of ITGB6 by circFOXO3:

[0190] First, an immunoprecipitation assay was used to verify the physical interaction between FOXO3 and JunB, confirming that FOXO3 and JunB do indeed physically bind. Subsequently, an immunofluorescence double staining assay was used to detect the sublocalization of FOXO3 and JunB proteins in cells, showing that FOXO3 and JunB are mainly located in the nucleus of epithelial cells, and that they co-localize. Furthermore, the Jaspar database showed that the JunB gene promoter region contains a binding site for the transcription factor FOXO3 (e.g., ...). Figure 5As shown in the image, the transcription factor FOXO3 may participate in the negative regulation of ITGB6 expression by circFOXO3 in epithelial cells by inhibiting JunB transcription. To further verify this hypothesis, wild-type (WT) and mutant (MUT) sequences of the predicted FOXO3-JunB promoter region binding fragment were inserted into the pmiRGLO reporter gene plasmid vector, and then co-transfected into 293T cells with pCMV6-entry-FOXO3 and its control plasmid, respectively. Dual-luciferase reporter gene assays showed that overexpression of FOXO3 significantly downregulated the activity of pmiRGLO-JunB-WT, indicating that the JunB promoter region contains a binding site for the transcription factor FOXO3. ChIP experiments were then used to verify whether FOXO3 could bind to the JunB promoter in epithelial cells. The results showed that FOXO3 was significantly enriched in the JunB promoter region compared to the control IgG, indicating that FOXO3 can bind to the JunB promoter region. Furthermore, overexpression of FOXO3 significantly inhibited the mRNA and protein expression of JunB. To further demonstrate that FOXO3 participates in regulating the transcriptional activity of the JunB promoter, ChIP experiments were used to verify whether FOXO3 downregulates ITGB6 expression by inhibiting the binding of JunB to the ITGB6 promoter region. The results showed that overexpression of FOXO3 significantly reduced the binding of JunB to the ITGB6 promoter in HaCaT cells. In summary, with increased expression of the transcription factor FOXO3, the FOXO3 protein inhibits JunB transcription by binding to the JunB promoter and by binding to the JunB protein in the cell nucleus, thus preventing JunB from binding to the ITGB6 promoter region and thereby inhibiting ITGB6 transcription.

[0191] 3.7 circFOXO3 sponge adsorption of miR-141-3p modulates the FOXO3 / JunB / ITGB6 signaling pathway:

[0192] The miRanda database predicted a binding site for miR-141-3p in circFOXO3. Furthermore, Target Scan and Starbase databases predicted three binding sequences between miR-141-3p and the 3'-UTR of FOXO3 mRNA, and miR-141 is lowly expressed in periodontitis. Therefore, it was hypothesized that circFOXO3 might regulate the FOXO3 / JunB / ITGB6 signaling pathway by sponge-like adsorption of miR-141-3p. To this end, circFOXO3 and a dual-luciferase reporter gene plasmid (DLC) were constructed. Transfection of miR-141-3p mimics and pmirGLO-circFOXO3-WT into 293T cells resulted in decreased DLC signaling, while transfection of miR-141-3pinhibitor and pmirGLO-circFOXO3-WT enhanced DLC signaling, suggesting that circFOXO3 can target miR-141-3p. Furthermore, transfection of miR-141-3p mimics and pmirGLO-FOXO3-WT into 293T cells resulted in a decrease in dual-luciferase reporter gene signal only at the third binding site. Conversely, transfection of miR-141-3p inhibitor and pmirGLO-FOXO3-WT into 293T cells resulted in an increase in dual-luciferase reporter gene signal only at the third binding site of FOXO3 mRNA. RIP experiments were performed in HaCaT and IHGK cells using the Ago2 antibody to further verify that circFOXO3 regulates FOXO3 expression through sponge adsorption of miR-141-3p. The results showed that circFOXO3, miR-141-3p, and FOXO3 were significantly enriched in the Ago2 group compared to the control IgG group. To further verify the relationship among the three, circFOXO3 was first overexpressed in HaCaT and IHGK cells. The expression level of miR-141-3p was significantly downregulated, indicating that circFOXO3 can inhibit miR-141-3p expression by binding to it. Further experiments showed that transfection of epithelial cells with miR-141-3p significantly inhibited FOXO3 expression, while JunB and ITGB6 mRNA and protein expression were significantly upregulated. Conversely, miR-141-3p inhibitors significantly enhanced FOXO3 mRNA and protein expression in epithelial cells and inhibited JunB and ITGB6 mRNA and protein expression. These results indicate that circFOXO3 regulates the FOXO3 / JunB / ITGB6 signaling pathway by sponging miR-141-3p.

[0193] The effect of 3.8 miR-141-3p on TGF-β1 activation:

[0194] To date, in vitro experimental data strongly suggest that circFOXO3 can regulate the FOXO3 / JunB / ITGB6 signaling pathway through sponge-adsorbed miR-141-3p. To further confirm whether circFOXO3 can influence TGF-β1 activation through the miR-141 / FOXO3 / JunB axis, thereby participating in the periodontitis process, transfection of miR-141-3p pimics into HaCaT and IHGK cells revealed upregulation of p-Smad2 expression, while transfection with miR-141-3p inhibitor downregulated p-Smad2. Furthermore, overexpression of FOXO3 downregulated p-Smad2, while co-transfection with miR-141-3p pimics and FOXO3 restored p-Smad2 expression. Overexpression of JunB upregulated p-Smad2, while overexpression of miR-141-3p inhibitor and JunB reversed p-Smad2 expression. Since overexpression of circFOXO3 can reduce the expression of p-Smad2, the above results demonstrate that circFOXO3 can inhibit the activation of TGF-β1 through the miR-141-3p / FOXO3 / JunB axis, thereby affecting the inflammatory process of periodontitis.

[0195] 3.9 Expression of FOXO3, JunB and ITGB6 in gingival tissue of rats with periodontitis:

[0196] To further verify the expression changes of FOXO3, JunB, and ITGB6 during the pathogenesis of periodontitis in vitro, a periodontitis animal model was established by ligating the maxillary second molars of SD rats. Four weeks later, tissue from the maxillary molar region was harvested, and micro-CT was used to reconstruct the bone tissue of the rat molar region. The experimental results showed that during the formation of periodontitis, severe alveolar bone resorption occurred on both the buccal and lingual sides (e.g., Figure 6 (As shown in the image). Furthermore, HE staining results revealed significant periodontal pocket formation in rats with periodontitis. Immunofluorescence double staining showed co-localization of FOXO3 and JunB in rat gingival tissue, further demonstrating that the transcription factors FOXO3 and JunB may participate in the development and progression of periodontitis through interaction. To further verify the expression of FOXO3, JunB, and ITGB6 in periodontitis, immunohistochemistry was used to detect their expression in rat periodontitis tissue. The results showed that FOXO3 expression was upregulated in periodontitis tissue, while JunB and ITGB6 expression was downregulated, a result consistent with our cell experiments.

[0197] 3.10 Gingival sulcus injection of circFOXO3 siRNA alleviates the progression of periodontitis in rats:

[0198] In vitro experimental data showed that circFOXO3 can downregulate the expression of ITGB6 in epithelial cells, thereby inhibiting the activation of TGF-β1 and promoting the occurrence and development of periodontitis. To further explore the in vivo effects of circFOXO3, a periodontitis animal model was first constructed, and then cholesterol-modified circFOXO3 siRNA was injected into the gingival sulcus of rats (e.g., Figure 7 As shown in the figure), micro-CT results showed that inhibiting circFOXO3 expression significantly alleviated alveolar bone resorption in rats (e.g. Figure 8 (As shown in the image). Secondly, HE staining results showed that inhibition of circFOXO3 expression also significantly alleviated the formation of periodontal pockets in rats. Furthermore, immunohistochemical results indicated that inhibition of circFOXO3 expression significantly reversed the high expression of FOXO3 in periodontitis tissues and led to a recovery in the expression of JunB, ITGB6, and p-Smad2. These studies demonstrate that inhibiting circFOXO3 expression can significantly alleviate the progression of periodontitis.

[0199] 3.11 Expression of miR-141-3p, FOXO3, JunB, ITGB6 and p-Smad2 in human periodontitis tissues:

[0200] To further investigate the expression changes of miR-141-3p, FOXO3, JunB, and ITGB6 in the progression of human periodontitis, and the activation of the anti-inflammatory cytokine TGF-β1 during the pathogenesis of periodontitis, RNA was first extracted from normal human gingival tissue and periodontitis-affected human gingival tissue. The mRNA expression of miR-141-3p, FOXO3, JunB, and ITGB6 was detected using qRT-PCR. The results showed that miR-141-3p was downregulated in periodontitis tissue (e.g., ...). Figure 9 As shown, Figure 9 In the middle, ****p<0.0001), FOXO3 expression was upregulated (e.g. Figure 10 As shown, Figure 10 In the middle, ****p<0.0001), while JunB and ITGB6 expression were downregulated (e.g. Figures 11 to 12 As shown, Figure 11 In the mean, ***p<0.001, Figure 12In the in vitro results (p < 0.0001), this is consistent with our cell experiments. Immunofluorescence double staining showed co-localization of FOXO3 and JunB, demonstrating their interaction in human gingival tissue, consistent with in vitro results. Immunohistochemistry was also used to detect changes in the expression of FOXO3, JunB, ITGB6, and p-Smad2 proteins in human periodontitis tissue specimens. Immunohistochemical results showed that FOXO3 expression was upregulated in periodontitis tissue (e.g., p < 0.0001). Figure 13 As shown), the expression of JunB, ITGB6, and p-Smad2 was downregulated (as shown). Figures 14 to 16 (As shown).

[0201] IV. Conclusion:

[0202] 4.1 circFOXO3 downregulates ITGB6 expression and is involved in the progression of periodontitis:

[0203] Compared with healthy controls, significantly upregulated expression of circMAP3K11 and TLR4 was detected in periodontal ligament tissue during periodontitis, while downregulated expression of miR-511-3p was observed. circMAP3K11 promotes the proliferation, migration, and osteogenic differentiation of PDLSCs through the miR-511-3p / TLR4 axis and reduces PDLSC apoptosis in vitro. Silencing circMAP3K11 expression in vivo can inhibit the progression of periodontitis in mice. circ_0081572 expression was downregulated in periodontitis tissue, and overexpression of circ_0081572 can alleviate LPS-induced PDLSC damage through the circ_0081572 / miR-378h / RORA axis. Furthermore, significantly downregulated expression of circRNA CDR1as in periodontitis tissue inhibits PDLSC proliferation through the miR-7 and ERK / MAPK pathways, suggesting that circRNAs play an important role in the development and progression of periodontitis.

[0204] To further investigate the expression and role of circFOXO3 in periodontal inflammation, qRT-PCR experiments revealed a significant upregulation of circFOXO3 expression in periodontitis tissues. FISH experiments showed that circFOXO3 was mainly located in the cytoplasm, and these results further confirmed the upregulation of circFOXO3 expression in periodontitis. This invention is the first to demonstrate the high expression of circFOXO3 in periodontitis tissues, suggesting that circFOXO3 may act as a pro-inflammatory factor in the development and progression of periodontitis. This is a novel discovery in the field of periodontitis research regarding circFOXO3.

[0205] In this invention, it was found that overexpression of circFOXO3 in epithelial cells significantly downregulated both ITGB6 mRNA and protein expression. To investigate whether circFOXO3 inhibits TGF-β1 activation by downregulating ITGB6 expression, this invention found that p-Smad2 expression was downregulated after overexpression of circFOXO3 in two types of epithelial cell lines. Combined with the downregulation of ITGB6 expression in epithelial cells by circFOXO3, this suggests that circFOXO3 inhibits TGF-β1 activation by downregulating ITGB6 expression in epithelial cells. Reduced activation of TGF-β1 suppresses its anti-inflammatory surveillance function, thereby promoting the occurrence and development of periodontitis. Therefore, it can be concluded that downregulating circFOXO3 gene expression leads to upregulation of ITGB6 expression, thereby promoting TGF-β1 activation and inhibiting the occurrence and development of periodontitis.

[0206] 4.2 The interaction between transcription factor FOXO3 and JunB mediates the downregulation of ITGB6 by circFOXO3:

[0207] This invention, using the Jaspar database, predicted that the -150 / -3 region of the ITGB6 promoter contains a binding site for the transcription factor FOXO3. Since circFOXO3 promotes the transcription and translation of its parent gene FOXO3, this suggests that FOXO3 may be involved in circFOXO3-induced ITGB6 transcriptional regulation. In this invention, experimental results showed that overexpression of circFOXO3 significantly increased FOXO3 expression in epithelial cells and significantly reduced ITGB6 mRNA and protein expression. Furthermore, ChIP experiments showed that circFOXO3 overexpression promoted the binding of FOXO3 to the ITGB6 promoter region. These findings suggest that circFOXO3 promotes FOXO3 binding to the ITGB6 promoter by upregulating FOXO3 expression, and FOXO3, acting as a transcriptional repressor, inhibits ITGB6 gene transcription, thereby downregulating ITGB6 expression.

[0208] This invention predicted, using the Jaspar database, that the transcription factor JunB could bind to the -150 / -3 region of the ITGB6 promoter. We then further validated this prediction using dual-luciferase reporter gene assays and ChIP experiments. Furthermore, overexpression of circFOXO3 in epithelial cells decreased JunB expression, while JunB overexpression promoted ITGB6 expression, suggesting that JunB inhibition is involved in the downregulation of ITGB6 expression in epithelial cells by circFOXO3. ChIP results showed that circFOXO3 inhibited the binding of JunB to the ITGB6 promoter region. Therefore, JunB inhibition participates in the downregulation of ITGB6 expression by circFOXO3 through reduced binding to the ITGB6 promoter region.

[0209] In this invention, it was found that the binding sites of the transcription factor FOXO3 and JunB in the ITGB6 promoter region are also close to each other. Therefore, it is speculated that FOXO3 may interact with JunB and thus participate in the downregulation of ITGB6 expression by circFOXO3. Co-IP experiments confirmed that FOXO3 and JunB also interact in epithelial cells. In addition, immunofluorescence double staining experiments showed that the co-binding of FOXO3 and JunB mainly occurs in the cell nucleus. Therefore, this finding suggests that the transcription factors FOXO3 and JunB interact in the cell nucleus, preventing JunB from binding to the ITGB6 promoter region and inhibiting ITGB6 transcription.

[0210] In this invention, the Jaspar database revealed a binding site for the transcription factor FOXO3 in the promoter region of JunB, suggesting that FOXO3 may be involved in the transcriptional regulation of JunB. Dual-luciferase reporter gene assays and ChIP experiments further confirmed the presence of a FOXO3 binding site in the JunB promoter region. FOXO3 overexpression significantly inhibited JunB mRNA and protein expression in epithelial cells, and FOXO3 overexpression also inhibited JunB binding to the ITGB6 promoter, suggesting that FOXO3 can reduce JunB binding to the ITGB6 promoter by inhibiting JunB expression.

[0211] 4.3 circFOXO3 regulates the progression of periodontitis through miR-141-3p / FOXO3 / JunB / axial negative control:

[0212] This invention further confirmed the low expression of miR-141-3p in periodontitis tissues using qRT-PCR experiments. The miRanda database revealed that circFOXO3 targets miR-141-3p, and this prediction was confirmed by dual-luciferase reporter gene assays. Furthermore, Target Scan and Starbase databases predicted three binding sites for miR-141-3p with the 3'-UTR of FOXO3 mRNA; these were then confirmed by dual-luciferase reporter gene assays to bind only to the third site of the 3'-UTR of FOXO3 mRNA. Previous studies have shown that miR-141-3p can target FOXO3 and negatively regulate its expression in Burkitt's lymphoma cells, but the specific binding sequence was not identified. This invention is the first to confirm the specific binding sequence of miR-141-3p and FOXO3. Simultaneously, RIP experiments further confirmed that circFOXO3 upregulates FOXO3 expression in epithelial cells by sponging miR-141-3p. In summary, circFOXO3 can adsorb various miRNAs to regulate the expression of their parent genes.

[0213] In addition to directly binding to miRNAs, circFOXO3 can also directly target RNA-binding proteins to exert its effects. circFOXO3 can regulate FOXO3 expression by adsorbing miRNAs through a sponge-like structure, and it can also directly bind to FOXO3 proteins to regulate their expression.

[0214] Next, to confirm whether circFOXO3 inhibits TGF-β1 activation through the miR-141-3p / FOXO3 / JunB axis, qRT-PCR experiments were first used to find that circFOXO3 negatively regulates miR-141-3p expression in epithelial cells, while miR-141-3p negatively regulates FOXO3 mRNA and protein expression, and positively regulates JunB and ITGB6 mRNA and protein expression. This suggests that circFOXO3 may downregulate ITGB6 expression in epithelial cells through the miR-141-3p / FOXO3 / JunB axis. However, whether circFOXO3 can mediate TGF-β1 activation through the miR-141-3p / FOXO3 / JunB axis requires further investigation. Therefore, this invention investigated the effects of miR-141-3p, FOXO3, and JunB on TGF-β1 activation in epithelial cells. Experimental results showed that miR-141-3p promoted TGF-β1 activation in epithelial cells, FOXO3 reduced TGF-β1 activation, and JunB increased TGF-β1 activation. Simultaneously, qRT-PCR and immunohistochemical experiments showed that FOXO3 was highly expressed in periodontitis tissues, while JunB and ITGB6 were lowly expressed, and Smad2 phosphorylation levels were decreased in periodontitis. Immunofluorescence results showed that FOXO3 and JunB co-localized in the nuclei of gingival epithelial cells. Notably, in an animal model of periodontitis, this invention utilized gingival sulcus injection of circFOXO3 siRNA, and the results showed that interfering with circFOXO3 expression significantly alleviated the progression of periodontitis in rats. To date, in vitro and in vivo experimental data strongly indicate that circFOXO3 negatively regulates ITGB6 expression in epithelial cells through the miR-141-3p / FOXO3 / JunB axis. Downregulated ITGB6 expression inhibits TGF-β1 activation, leading to reduced activation in periodontal inflamed epithelial tissues and dysregulation of TGF-β1's anti-inflammatory surveillance function, thereby stimulating the occurrence and development of periodontal inflammation. Therefore, it can be concluded that by inhibiting the miR-141-3p / FOXO3 / JunB signaling pathway, ITGB6 expression is upregulated, promoting TGF-β1 activation and thus inhibiting the occurrence and development of periodontitis.

[0215] In summary, circFOXO3 is mainly located in the cytoplasm and is highly expressed in periodontal epithelial tissues. circFOXO3 downregulates the expression of ITGB6 and p-Smad2 in epithelial cells. circFOXO3 upregulates the expression of transcription factor FOXO3 and promotes the binding of FOXO3 to the ITGB6 promoter, thereby inhibiting ITGB6 transcription. Therefore, by downregulating the expression of transcription factor FOXO3, it inhibits the binding of FOXO3 to the ITGB6 promoter, thereby promoting ITGB6 transcription and upregulating ITGB6 expression, thus promoting TGF-β1 activation and inhibiting the occurrence and development of periodontitis. circFOXO3 also downregulates the expression of transcription factor JunB and inhibits the binding of JunB to the ITGB6 promoter, thereby inhibiting ITGB6 transcription. Therefore, by downregulating the expression of transcription factor JunB and inhibiting the binding of JunB to the ITGB6 promoter, it inhibits ITGB6 transcription. JunB expression is upregulated to promote JunB binding to the ITGB6 promoter, thereby promoting ITGB6 transcription and upregulating ITGB6 expression to promote TGF-β1 activation, thus inhibiting the occurrence and development of periodontitis. FOXO3 acts as a transcriptional repressor to inhibit JunB transcription and interacts with the JunB protein. circFOXO3 sponges adsorb miR-141-3p and downregulate p-Smad2 expression in epithelial cells through the miR-141-3p / FOXO3 / JunB axis. FOXO3 and JunB are co-localized in gingival epithelial tissue. miR-141-3p is lowly expressed in periodontitis tissue. FOXO3 is highly expressed in periodontitis tissue. JunB and ITGB6 are lowly expressed in periodontitis tissue. p-Smad2 is lowly expressed in periodontitis tissue. circFOXO3 upregulates FOXO3 expression by adsorbing miR-141-3p through a sponge. FOXO3 can not only bind to the ITGB6 promoter region as a transcriptional repressor, but also form a transcriptional repressor complex with JunB to downregulate the transcriptional signal of ITGB6, thereby reducing the activation of TGF-β1 and promoting the occurrence and progression of periodontal tissue inflammation.

[0216] This invention elucidates the molecular mechanism by which circFOXO3 downregulates ITGB6 transcriptional signaling in gingival epithelial cells, contributing to the development and progression of periodontitis. Specifically, circFOXO3 competitively adsorbs miR-141-3p, inhibiting the formation of the miR-141-3p-FOXO3 mRNA complex, thereby promoting FOXO3 mRNA translation. With increased FOXO3 protein expression, the transcription factor FOXO3 directly binds to the JunB promoter to inhibit its transcription, and also binds to the JunB protein in the cell nucleus, preventing JunB from binding to the ITGB6 promoter region. Furthermore, FOXO3 can also act as a transcriptional repressor, directly binding to the ITGB6 promoter region, inhibiting ITGB6 transcription through a triple action. Downregulation of integrin β6 expression stimulates the occurrence and progression of periodontal inflammation by reducing TGF-β1 activation. Therefore, inhibiting circFOXO3 expression may contribute to increased ITGB6-targeted therapy for periodontitis. circFOXO3 siRNA can effectively inhibit circFOXO3 expression, and cholesterol-modified circFOXO3 siRNA can increase its stability and in vivo metabolic time.

[0217] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of circFOXO3 gene expression inhibitor in the preparation of drugs for the prevention and / or treatment of periodontitis; wherein the circFOXO3 gene expression inhibitor comprises circFOXO3 siRNA; wherein the siRNA is cholesterol-modified siRNA; wherein the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.1, and the nucleotide sequence of its antisense strand is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The circFOXO3 gene expression inhibitor downregulates circFOXO3 gene expression, thereby upregulating ITGB6 expression, promoting TGF-β1 activation, and thus inhibiting the occurrence and development of periodontitis.

3. The application according to claim 2, characterized in that, The circFOXO3 gene expression inhibitor downregulates the expression of the circFOXO3 gene, thereby downregulating the expression of the transcription factor FOXO3, inhibiting the binding of FOXO3 to the ITGB6 promoter, promoting ITGB6 transcription, and upregulating the expression of ITGB6 to promote the activation of TGF-β1, thus inhibiting the occurrence and development of periodontitis.

4. The application according to claim 2, characterized in that, The circFOXO3 gene expression inhibitor downregulates circFOXO3 gene expression, thereby upregulating the expression of transcription factor JunB. This promotes the binding of JunB to the ITGB6 promoter, which in turn promotes ITGB6 transcription and upregulates ITGB6 expression. This, in turn, promotes the activation of TGF-β1 and inhibits the occurrence and development of periodontitis.

5. The application according to claim 2, characterized in that, The circFOXO3 gene expression inhibitor downregulates circFOXO3 gene expression, inhibits the miR-141-3p / FOXO3 / JunB signaling pathway, upregulates ITGB6 expression, promotes TGF-β1 activation, and thus inhibits the occurrence and development of periodontitis.

6. The application according to claim 1, characterized in that, The drug is an injection, powder, granule, pill, oral preparation, tablet, capsule, suppository, spray, or ointment.

Images