Use of miR-501-3p inhibitor or miR-501-3p antagonist in preparation of a medicament for promoting healing of oral mucosa wound

Abstract

The application belongs to the technical field of biotechnology and pharmaceutical technology. The application provides application of a miR-501-3p inhibitor or a miR-501-3p antagonist in preparation of a drug for promoting oral mucosa wound healing. The application obtains a solution for promoting oral mucosa wound healing based on miRNA technology, and develops application of the miR-501-3p inhibitor or the miR-501-3p antagonist in preparation of the drug for promoting oral mucosa wound healing. The application can provide a basis for research and development of a tissue replacement material for efficiently repairing large-area keratinized mucosa defects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and pharmaceutical technology, and relates to the field of biotechnology and pharmaceutical technology for promoting the healing of oral mucosal wounds. Specifically, it relates to the application of miR-501-3p inhibitors or miR-501-3p antagonists in the preparation of drugs for promoting the healing of oral mucosal wounds. Background Technology

[0002] Repairing extensive keratotic mucosal defects after surgery for oral and maxillofacial trauma, tumors, infections, and congenital malformations is a major challenge in oral and maxillofacial surgery. Autologous soft tissue transplantation offers limited tissue volume and carries the risk of donor site damage, making it unsuitable for repairing extensive keratotic mucosal defects. Tissue replacement material transplantation, involving only one surgical area and with unlimited tissue volume, represents the most promising repair option for extensive keratotic mucosal defects. However, the survival rate of simple scaffold material transplantation varies, and tissue-engineered oral mucosal transplantation carries the risk of immune rejection. The integration of bioactive substances into scaffold materials is currently a hot research topic. However, growth factors have poor stability, exosome extraction is complex, and market supply may fall short of demand. Currently, there is no ideal bioactive substance that can be integrated into scaffold materials for repairing extensive keratotic mucosal defects.

[0003] miRNAs are a class of non-coding RNAs with a length of 20-24 nucleotides. As important regulators of the genome, miRNAs can achieve precise multi-threaded regulation and have almost no side effects on normal tissues, showing great potential in the development of next-generation drugs. Wound healing is a complex process involving many continuous and parallel pathways. Due to their multi-target regulatory capabilities, miRNAs can simultaneously affect multiple signaling pathways and participate in various cellular functions, potentially offering advantages in the regulation of wound healing. Furthermore, miRNAs exhibit significant human-mouse homology; the sequences of many miRNAs are completely identical in human and mouse tissues, which greatly facilitates related research.

[0004] Given the characteristics of miRNAs mentioned above, research has been conducted on the potential mechanisms of miRNAs in wound healing and the selection of specific miRNAs that can promote wound healing. (Wu Minfeng et al.) [1] This paper summarizes the research progress on miRNAs in wound healing, and elucidates the pathophysiological intervention mechanisms of miRNAs at different stages, based on existing research. Regarding the screening research of specific miRNAs, Wu Peng... [2] A study was conducted on the regulation of wound healing by microRNA-96-5p through inhibition of the BNIP3-mediated FAK signaling pathway. The study found that miR-96-5p has a negative regulatory effect on downstream target genes BNIP3 and the FAK pathway, and promotes wound repair. (Tang Huiyi) [3]Research was conducted on the mechanism by which microRNA-200b / c targets Rac1 to regulate skin wound healing. It was found that miR-200b / c directly targets the Rac1 3'UTR, inhibiting Rac1 expression in keratinocytes and thus suppressing skin wound healing. TGF-β1, by slowing down the targeted inhibition of Rac1 by miR-200b / c, regulates the EMT-like process of wound re-epithelialization, thereby maintaining the homeostasis of skin wound healing. Xu Juan et al. [4] The study investigated how miRNA-221-3p promotes skin wound healing in diabetic mice and found that miRNA-221-3p is one of the miRNAs highly expressed in EPC exosomes and can promote skin wound healing in diabetic mice.

[0005] However, research on miRNAs in wound healing has so far focused primarily on skin wound healing, with almost no research on oral mucosal wound healing. Skin and oral mucosa differ significantly at multiple levels, including cellular and tissue levels. The keratinized layer of skin is significantly thicker than that of the mucosa, and the dermis contains skin appendages such as sweat glands and hair follicles. During wound healing, skin often heals with scarring, while oral mucosal scarring is rare. Furthermore, due to the complex environment of the oral cavity, including saliva and bacteria, the healing process of the oral mucosa differs significantly from that of the skin.

[0006] Therefore, how to conduct research on the role of relevant miRNAs in oral mucosal wound healing, taking into account the specific characteristics of oral mucosal trauma, and how to obtain a solution that can promote oral mucosal wound healing are urgent problems to be solved in this field.

[0007] This section cites existing technology reports: [1] Wu Minfeng, Chen Yu, Li Ying, et al. Research progress of miRNA in wound healing [J]. Journal of Clinical and Pathological Research, 2015, 35(07):1408-1412. [2] Wu Peng. Study on the regulation of wound healing by microRNA-96-5p by inhibiting the BNIP3-mediated FAK signaling pathway [D]. Shandong University, 2020. [3] Tang Huiyi. Study on the mechanism of microRNA-200b / c targeting Rac1 to regulate skin wound healing [D]. Southern Medical University, 2022. [4]Juan Summary of the Invention

[0008] In view of the shortcomings of the existing technology and the technical needs in this field, the purpose of this invention is to provide a specific miRNA-related technical solution that can promote the healing of oral mucosal wounds.

[0009] To address the aforementioned technical objectives, the present invention provides the following technical solution: Application of miR-501-3p inhibitors or miR-501-3p antagonists in the preparation of drugs that promote the healing of oral mucosal wounds.

[0010] Specifically, the miR-501-3p inhibitor or miR-501-3p antagonist can downregulate the expression or activity of miR-501-3p.

[0011] Prior to this invention, research on miR-501-3p mainly focused on diseases such as schizophrenia and cancer. [5-8] There are very few reports on miR-501-3p in terms of wound healing, and the few reports that exist only focus on wound healing of the skin. To the inventor's knowledge, there are no reports on miR-501-3p in terms of wound healing of the oral mucosa.

[0012] As shown in the embodiments of the present invention, the inventors found that when miR-501-3p inhibitors or miR-501-3p antagonists are added, downregulating miR-501-3p in HOK cells can promote cell proliferation and accelerate the cell cycle, while also promoting the expression or phosphorylation of cell cycle-related proteins and activating the AKT pathway related to cell proliferation; in a mouse palatal mucosal defect model, downregulating miR-501-3p can accelerate the wound healing rate.

[0013] This phenomenon contrasts with that of some other miRNAs in skin wound healing, such as those observed by Pan Xiaoqiu et al. [9]Studies have found that compared with normal tissue, the expression of miR-99a-5P, miR-99b-3P, and miR-99b-5P in deep second-degree burn skin tissue is downregulated. Therefore, promoting wound healing requires upregulating the expression of the aforementioned miRNAs.

[0014] The beneficial effects of this invention are: This invention provides a solution for promoting oral mucosal wound healing based on miRNA technology, and develops the application of miR-501-3p inhibitors or miR-501-3p antagonists in the preparation of drugs that promote oral mucosal wound healing. This invention can provide a foundation for the development of highly efficient tissue replacement materials for repairing large-scale keratinized mucosal defects.

[0015] This section cites existing technology reports: [5]C. D, J. L, L. Z, et al. Micro RNA-501-3p Functions as a TumorSuppressor in Non-Small Cell Lung Cancer via Downregulating RAP1A[J]. AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, 2020, 201. [6]Dai J, Lu L, Kang L, et al. MiR-501-3p promotes osteosarcoma cellproliferation, migration and invasion by targeting BCL7A[J]. Human Cell, 2021, 34(2). [7]Liujia H, Shiming C, Yufan Y, et al. MicroRNA-501-3p inhibits theproliferation of kidney cancer cells by targeting WTAP.[J]. Cancer medicine, 2021,10(20). [8] Liang Wenquan. Downregulation of miR-501-3p expression associated with schizophrenia leads to social and memory abnormalities in mice through hyperglutamatergic transmission mediated by mGluR5 [D]. Southern Medical University, 2023. DOI:10.27003 / d.cnki.gojyu.2022.000569. [9] Pan Xiaoqiu, Zhai Hongjun, Li Lihui, et al. Study on differential expression of miRNA in deep second-degree burn skin [J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2018, 23(12):1321-1328. Attached Figure Description

[0016] Figure 1 The image shows the miRNA expression changes in a mouse palatal mucosal defect model. (A) miRNA sequencing and Wayne analysis revealed that only miR-501-3p showed significant expression changes on days 2, 4, and 6 after trauma model establishment. (B) miRNA sequencing results showed that miR-501-3p expression significantly increased on days 2, 4, and 6 after palatal mucosal trauma in mice. (C) qPCR verification confirmed that miR-501-3p expression significantly increased on days 2, 4, and 6 after palatal mucosal trauma in mice. Figure 2 The effect of upregulation or downregulation of miR-501-3p on the expression of this miRNA in HOK cells was detected by qPCR.

[0017] Figure 3 The effect of upregulation or downregulation of miR-501-3p on HOK cell proliferation was detected by CCK-8 assay. The experiment showed that miR-501-3p inhibitor had no significant effect on HOK cell proliferation, while miR-501-3p mimics could inhibit HOK cell proliferation.

[0018] Figure 4 To investigate the effects of upregulation or downregulation of miR-501-3p on the HOK cell cycle using flow cytometry, the results showed that miR-501-3p mimics could increase the G0 / G1 phase ratio of HOK cells, indicating cell cycle arrest, while miR-501-3pinhibitor had no statistically significant effect on the cell cycle.

[0019] Figure 5 To investigate the effects of upregulation or downregulation of miR-501-3p on HOK cell cycle proteins using Western blot, the results showed that miR-501-3p mimics significantly reduced the expression of cell cycle-related proteins CDK2 and Cyclin E1, while miR-501-3p inhibitors increased CDK2 expression but had no statistically significant effect on Cyclin E1 expression.

[0020] Figure 6Figure 1 shows the results of miR-501-3p gain-of-function and loss-of-function experiments in a mouse palatal mucosal trauma model. Figure 2 shows the effect of miR-501-3p agomir / antagomir on the healing of palatal mucosal defects in mice; it can be seen that miR-501-3p agomir (with the same effect as mimics) can slow down the healing rate of the mouse palatal mucosa; while miR-501-3pantagomir (with the same effect as inhibitor) can accelerate the healing rate of the mouse palatal mucosa. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention. Example 1

[0022] Experimental materials: 1. miR-501-3p agomir: miR-501-3p agonist Product Information: mirON mmu-miR-501-3p agomir, in vivo, 5nmol, Product No.: miR40003509-4-5, Specification: 5nmol, Manufacturer: Guangzhou Ruibo Biotechnology Co., Ltd.; 2. miR-501-3p antagomir: miR-501-3p antagomir Product Information: mirOFF mmu-miR-501-3p antagomir, in vivo, 5 nmol, Product No.: miR30003509-4-5, Specification: 5 nmol, Manufacturer: Guangzhou Ruibo Biotechnology Co., Ltd. 3. miR-501-3p agomir-NC: Product Information: micrON agomir NC #22, in vivo, 5 nmol, Product No.: miR4N0000001-4-5, Specification: 5 nmol, Guangzhou Ruibo Biotechnology Co., Ltd.; 4.miR-501-3p antagomir-NC: Product Information: mirOFF antagomir NC #22, in vivo, 5 nmol, Product Number: miR3N0000001-4-5, Specification: 5 nmol, Guangzhou Ruibo Biotechnology Co., Ltd.; 5. miR-501-3p mimics: miR-501-3p emulators Product Information: mirON mmu-miR-501-3p mimic, 5nmol, Product Number: miR10003509-1-5, Specification: 5noml, Guangzhou Ruibo Biotechnology Co., Ltd.; 6. miR-501-3p inhibitor: miR-501-3p inhibitor Product Information: mirOFF mmu-miR-501-3p inhibitor, 5 nmol, Product No.: miR20003509-1-5, Specification: 5 nmol, Guangzhou Ruibo Biotechnology Co., Ltd.

[0023] Experimental plan: 1. Preparation of keratinized mucosal lesions in the oral cavity of mice 1) Accurately weigh the mice using an electronic scale. Anesthetize the mice by intraperitoneal injection of 1 w / v% sodium pentobarbital and physiological saline solution at a dose of 50 mg / kg.

[0024] 2) Disinfect the mouse's oral cavity with iodine swabs, and use a 2.0 mm diameter biopsy punch (IntegraMiltex, Integra York PA, Inc. York, PA, USA) to prepare a full-thickness keratinized mucosal wound in the center of the hard palate between the mouse's first molars.

[0025] 3) After the wound is prepared, apply pressure to stop the bleeding.

[0026] 4) Postoperatively, monitor the mice's food and water intake, urine output, and mental state.

[0027] 5) Immediately after euthanizing mice by cervical dislocation on days 2, 4, and 6 following palatal mucosal wound preparation, photographs of the keratinized mucosal wounds were taken using a stereomicroscope (Leica, Germany) at 1.5× magnification and a fixed focal length. A 1mm ruler was used as the reference scale for the images. All measurements were performed by the same examiner.

[0028] 6) After photographing the healing status of the mouse keratotic mucosal wound using a stereomicroscope, the intact keratotic mucosa was immediately excised using a No. 11 scalpel. After fixation, dehydration, clearing, embedding, and sectioning, HE staining was performed. HE-stained images of the mouse wound healing were acquired using an upright microscope and the image capture software CellSens Entry (Olympus, Japan). Images were taken at 10× magnification.

[0029] 2. Experiments on gain-of-function and loss-of-function of miRNAs in a mouse model of keratinization and mucosal defects. 1) Add 250 μL of Nuclease-free water to each tube of miR-501-3p antagomir and miR-501-3p antagomir to prepare a 20 mM storage solution, vortex to mix, centrifuge briefly, aliquot into 200 μL PCR tubes, and store at -80℃. After establishing a keratotic mucosal defect model in the hard palate of mice, 25 μL of 20 mM miR-501-3p agomir and miR-501-3p antagomir were injected into the keratotic mucosal wound margin area. The control group received the same amount of miR-501-3p agomir-NC and miR-501-3p antagomir-NC. The healing of keratinized mucosal defects in mice was observed until the keratinized mucosal lesions in either the experimental group (miR-501-3p agomir and miR-501-3p antagomir) or the control group (miR-501-3p agomir-NC and miR-501-3p antagomir-NC) were completely healed. Mice in both the experimental and control groups were then sacrificed.

[0030] Photographs of keratotic mucosal lesions in mice were taken using a stereomicroscope (Leica, Germany) at 1.5x magnification and a fixed focal length.

[0031] 3. Experiments on gain-of-function and loss-of-function of miRNAs in human oral keratinocytes (HOK) 1) Add 250 μL of Nuclease-free water to each tube of miR-501-3p mimics and miR-501-3p inhibitor to prepare a 20 μM storage solution. Shake to mix, centrifuge briefly, and aliquot into 200 μL PCR tubes. Store at -80°C. 2) Collect cells in the logarithmic growth phase and seed them into 6-well plates. Incubate them in a 37°C constant temperature incubator containing 5% CO2. Transfect the cells when they reach 70-80% confluency. 3) Prepare the reagents by mixing 5 μL miR-501-3p mimics (final concentration 50 nM) or 10 μL miR-501-3p inhibitor (final concentration 100 nM), 5 μL JetPrimer transfection reagent, and 200 μL transfection buffer per well. Mix the three together, vortex to mix, centrifuge briefly, and incubate at room temperature for 10 min. 4) Change the medium in the culture plate, adding 1.8 mL of DMEM complete medium to each well; 5) Add 200 μL of the mixture to each well, shake well, and incubate in a 37°C constant temperature incubator containing 5% CO2 for 6 h. Then replace the medium with serum-free medium. 6) Activation / inhibition effects were verified by Real-time qPCR 24 h after transfection. Cell proliferation was detected by CCK-8 assay, cell cycle was detected by flow cytometry, and expression of cell cycle-related proteins was detected by Western blot.

[0032] Experimental results: miRNAs with significantly altered expression levels on days 2, 3, and 4 of wound healing were screened using miRNA sequencing. The results showed that miR-501-3p was significantly upregulated on days 2, 4, and 6 after trauma to the keratinized mucosa in mice. Further qPCR confirmed the upregulation of miR-501-3p after trauma to the keratinized mucosa in mice (see attached). Figure 1 ).

[0033] Gain-and-loss-of-function experiments were conducted on miRNAs in human oral keratinocytes (HOK) to verify their impact on wound healing-related biological functions. Results showed that upregulation of miR-501-3p inhibited HOK cell proliferation, slowed the cell cycle, and reduced the expression of cell cycle-related proteins CDK2 and Cyclin E1. Downregulation of miR-501-3p had no significant effect on cell proliferation, but increased CDK2 expression at the protein level (see appendix). Figure 5 This result may be related to the fact that the expression level of miR-501-3p in cells is low under normal physiological conditions, so further downregulation of its expression has no significant effect on cell proliferation.

[0034] In a mouse model of palatal mucosal trauma, miRNA gain-and-loss experiments were conducted to verify its effect on oral mucosal wound healing. The results showed that downregulation of miR-501-3p significantly accelerated wound healing, while upregulation of miR-501-3p significantly delayed wound healing. (See appendix) Figure 6 ).

[0035] Based on in vitro cell experiments and animal model experiments, miR-501-3p expression was elevated during oral mucosal wound healing. The inventors initially believed that elevated miR-501-3p was a normal phenomenon during oral mucosal healing and that it might promote healing. However, subsequent in vitro and in vivo experiments yielded the opposite result: elevated miR-501-3p expression inhibited epithelial cell proliferation and arrested cell cycle progression in vitro; in a mouse palatal mucosal defect model, upregulation of miR-501-3p inhibited wound healing, while downregulation promoted it. These experimental results indicate that elevated miR-501-3p expression may be a detrimental factor in wound healing, and downregulation of miR-501-3p can promote wound healing.

Claims

1. The use of miR-501-3p antagonist miR30003509-4-5 in the preparation of a drug for promoting the healing of oral mucosal wounds, wherein the manufacturer of miR-501-3p antagonist miR30003509-4-5 is Guangzhou Ruibo Biotechnology Co., Ltd.

2. The application according to claim 1, characterized in that, miR-501-3p antagonists can downregulate the expression or activity of miR-501-3p.

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