Application of circEIF4B in the preparation of reagents to promote healing of tooth extraction wounds in patients with diabetes

Abstract

The present invention discloses the use of circEIF4B to prepare an agent for promoting healing of tooth extraction wounds in diabetic patients. The use of circEIF4B in the preparation of a preparation or medical device for improving delayed or impaired healing of tooth extraction wounds in diabetic patients. The use of bone marrow mesenchymal stem cells overexpressing circEIF4B in the preparation of a preparation for preserving tooth extraction sites in diabetic patients. BMSCs overexpressing circEIF4B are injected around the extraction socket or implanted into the extraction socket after incubation with a collagen scaffold, for preserving the extraction site of diabetic patients and promoting healing of the extraction socket.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to the application of circEIF4B in preparing a reagent for promoting healing of tooth extraction wounds in diabetic patients. Background Art

[0002] For implant treatment, it is of great significance to maintain the width and height of the alveolar ridge at the extraction site after tooth extraction. After tooth extraction, the new bone in the alveolar socket often fails to reach the original height, and the absorption of the labial and buccal bone plates is particularly severe, which leads to a decrease in the height and width of the alveolar ridge. Diabetes is a group of metabolic diseases characterized by hyperglycemia[1]. In the past decade, the incidence of diabetes has risen sharply and has become the leading chronic disease facing global public health[2]. Chronic hyperglycemia is associated with a series of metabolic disorders, endangering the health of patients[3]. Due to the impact of the high sugar microenvironment on the alveolar bone, diabetic patients often show poor socket healing and more severe alveolar ridge absorption than healthy people. Studies have shown that diabetes interferes with the osteogenic capacity of bone marrow mesenchymal stem cells (BMSCs) and interferes with the healing of the extraction socket, leading to a delay in the first-stage oral implant surgery[4].

[0003] Site preservation refers to a series of clinical treatment methods taken during or after tooth extraction to reduce alveolar bone resorption and promote new bone formation in the alveolar socket. There are two classic extraction site preservation techniques: one is to implant artificial bone powder particles in the extraction socket, and cover the surface with absorbable collagen membrane to repair the alveolar bone defect; the other is to place collagen in the extraction socket and use soft tissue circumcision technology to cut the palatal mucosa to close the extraction site. Site preservation can slow down the horizontal and vertical absorption of the alveolar ridge, maintain the stability of the alveolar socket after tooth extraction, and reduce the risk of later treatment caused by insufficient bone volume at the extraction site. Clinically proven, site preservation can not only reduce the reduction in the width and height of the alveolar ridge, but also accelerate the healing of the alveolar socket, providing better conditions for later implantation. BMSCs are derived from the mesoderm and have multidirectional differentiation potential. They can differentiate into bone, cartilage, fat, tendon, ligament, muscle, nerve and other tissues. They are rich in sources, easy to obtain, simple to culture, low in immunogenicity, easy to transfect and express foreign genes for a long time, and have excellent osteogenic differentiation ability. They are widely used in bone tissue engineering. Various biomaterials used in site preservation can provide a framework for the colonization of BMSCs in the alveolar socket, promote the proliferation and osteogenic differentiation of BMSCs, and accelerate the healing of the alveolar socket after tooth extraction.

[0004] Circular RNAs (circRNAs) are a class of non-coding RNAs with covalently closed loop structures. To date, some evidence has shown that circRNAs can regulate mRNA expression by acting as miRNA sponges, thereby participating in the regulation of bone-related diseases. For example, circDAB1 can sponge miR-1270 and miR-944, upregulate RBPJ and induce DAB1 expression, thereby regulating the proliferation and osteogenesis of BMSCs [5]. Chen et al. found that in steroid-induced femoral head necrosis, the circCDR1as / miR-7-5p / WNT5B axis plays a key role in the adipogenic / osteogenic differentiation disorder of BMSCs [6]. In addition, BMSCs extracellular vesicles carrying circ_0072464 upregulated the expression of NRF2 by absorbing miR-98, thus providing a potential target for the treatment of intervertebral disc degeneration [7]. On the other hand, given their unique biological structure, circRNAs can also act as scaffolds to interact with RNA-binding proteins (RBPs) and play a vital role in the occurrence and development of diseases. For example, a recent study demonstrated that circStag1 promoted bone regeneration through HuR / Wnt signaling, thereby reducing bone loss in ovariectomized rats [8]. Summary of the invention

[0005] The purpose of the present invention is to provide a circRNA for alleviating delayed or impaired healing of tooth extraction wounds in diabetic patients, promoting preservation of extraction sites, preventing alveolar ridge resorption, and accelerating healing of tooth extraction wounds in view of the above-mentioned deficiencies in the prior art.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] Application of circEIF4B in the preparation of a preparation or medical device for improving delayed or impaired healing of tooth extraction wounds in diabetic patients. The nucleotide sequence of circEIF4B is shown in SEQ ID NO.1.

[0008] Application of circEIF4B in the preparation of preparations for preservation of tooth extraction sites in diabetic patients.

[0009] Application of circEIF4B in the preparation of a preparation for increasing the osteogenic ability of BMSCs. circEIF4B binds to the RNA-binding protein IGF2BP3, thereby increasing the osteogenic ability of BMSCs.

[0010] Bone marrow mesenchymal stem cells overexpressing circEIF4B.

[0011] As a preferred embodiment of the present invention, the bone marrow mesenchymal stem cells overexpressing circEIF4B are obtained by transfecting bone marrow mesenchymal stem cells with a virus overexpressing circEIF4B.

[0012] Use of the bone marrow mesenchymal stem cells overexpressing circEIF4B of the present invention in the preparation of a preparation or medical device for improving delayed or impaired healing of tooth extraction wounds in diabetic patients.

[0013] Use of the bone marrow mesenchymal stem cells overexpressing circEIF4B of the present invention in preparing a preparation for preserving tooth extraction sites in diabetic patients.

[0014] Use of the bone marrow mesenchymal stem cells overexpressing circEIF4B of the present invention in preparing a preparation for promoting healing of tooth extraction sockets in diabetic patients.

[0015] Use of substances that overexpress circEIF4B or increase the activity of circEIF4B in the preparation of preparations or medical devices for improving delayed or impaired healing of tooth extraction wounds in patients with diabetes.

[0016] Use of substances that overexpress circEIF4B or increase the activity of circEIF4B in the preparation of preparations for preserving tooth extraction sites in diabetic patients.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides an application of circEIF4B in a tooth extraction site preservation technology for diabetic patients, which provides a new approach for treating delayed or impaired healing of tooth extraction wounds in diabetic patients. BMSCs overexpressing circEIF4B are injected around the tooth extraction socket for site preservation in diabetic patients after tooth extraction. BMSCs overexpressing circEIF4B are incubated with a collagen scaffold and then implanted into the tooth extraction socket for site preservation in diabetic patients after tooth extraction. This application promotes the expression of circEIF4B or enhances the active function of circEIF4B; strengthens the adsorption of miR-186-5p by the circEIF4B sponge, thereby recruiting FOXO1 to improve the osteogenic differentiation of BMSCs; and promotes the binding of circEIF4B to the RNA binding protein IGF2BP3, thereby increasing the osteogenic capacity of BMSCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 circEIF4B is downregulated in BMSCs of diabetic patients

[0020] Figure A shows that circEIF4B (hsa_circ_0026506) is composed of exon 3 to exon 6 of the EIF4B gene; Sanger sequencing verified the splicing site of circEIF4B; Figure B shows that qRT-PCR detected the downregulation of circEIF4B expression after BMSCs were stimulated with 30mM glucose.

[0021] Figure 2 circEIF4B significantly promotes osteogenic differentiation of BMSCs under high glucose conditions

[0022] Figure AB shows the overexpression and knockout efficiency of circEIF4B in BMSCs with overexpression and knockout of circEIF4B verified by qRT-PCR; Figure CD shows that after circEIF4B overexpression, qRT-PCR and Western blot detected upregulation of osteogenesis-related indexes; Figure E shows that after circEIF4B overexpression, ARS staining deepened and calcium deposition increased; Figure F shows that after circEIF4B overexpression, ALP staining deepened and ALP activity increased.

[0023] Figure 3 CircEIF4B sponge adsorbs miR-186-5p to recruit FOXO1 to improve osteogenic differentiation of high-glucose BMSCs. Figure AB shows that qRT-PCR and Western blot experiments confirmed that miR-186-5p inhibitor reversed the inhibitory effect of circEIF4B knockdown on osteogenic-related indicators and FOXO1 expression; Figure C shows ALP staining and ALP activity confirmed that miR-186-5pinhibitor reversed the inhibitory effect of circEIF4B knockdown on ALP expression.

[0024] Figure 4 circEIF4B binds to the RNA-binding protein IGF2BP3 to promote osteogenic differentiation of high-glucose BMSCs. Figure A shows that the pull-down experiment confirmed that IGF2BP3 was significantly enriched in the circEIF4B probe; Figure B shows that IF and FISH experiments showed that circEIF4B and IGF2BP3 were co-localized in the cytoplasm; Figures C and D show that after knocking down IGF2BP3, qRT-PCR and Western blot detected downregulated expression of osteogenic-related indicators; Figure E shows that after knocking down IGF2BP3, ALP staining became lighter and ALP activity decreased.

[0025] Figure 5:circEIF4B promotes the healing of tooth extraction wounds in diabetic mice and skull defects in diabetic rats. Figure A shows that the injection of BMSCs overexpressing circEIF4B is beneficial to the healing of tooth extraction sockets in diabetic mice; Figure B shows that BMSCs overexpressing circEIF4B were incubated with collagen scaffolds and then implanted into skull defects in diabetic rats, which promoted the formation of new bone in the defect. DETAILED DESCRIPTION

[0026] In order to make the present invention more clearly understood, the following embodiments are described in detail:

[0027] The technical solution adopted by the present invention is to provide an application of the circRNA marker circEIF4B in tooth extraction site preservation technology to improve delayed or impaired healing of tooth extraction wounds in diabetic patients; the present invention provides BMSCs overexpressing circEIF4B, which are injected around the tooth extraction socket or implanted into the tooth extraction socket after incubation with a collagen scaffold, and are used for tooth extraction site preservation in diabetic patients, which is beneficial to slow down alveolar ridge absorption and promote extraction socket healing; the application is to promote the expression of circEIF4B or enhance the active function of circEIF4B; the application is to strengthen the adsorption of miR-186-5p by circEIF4B sponge to recruit FOXO1 to improve the osteogenic differentiation of BMSCs; the application is to promote the binding of circEIF4B with RNA binding protein IGF2BP3, thereby increasing the osteogenic ability of BMSCs; and it brings a new approach to the treatment of delayed or impaired healing of tooth extraction wounds in diabetic patients.

[0028] Example 1

[0029] 1. Materials and Methods

[0030] 1.1 Cell culture and transfection:

[0031] Young patients (18-22 years old) who were admitted to the Affiliated Stomatological Hospital of Nanjing Medical University were given cancellous bone from their jaws during orthognathic surgery after signing informed consent. The cancellous bone was cut into pieces and spread on the bottom of the dish. After about 7 days, primary BMSCs (P0) crawled out from the bone tissue. The cells were passaged to 3-5 generations in α-MEM medium (gibco, 10% FBS and 1% PS) for subsequent experiments. 30mM glucose was used in this paper to simulate the hyperglycemic environment of diabetes.

[0032] The overexpression and knockdown viruses of circEIF4B (ov-circEIF4B and sh-circEIF4B) were constructed in Guangzhou Jikai Company, and the stable transfected strains were constructed after transfection of BMSCs for 72 hours. The results of verifying the overexpression and knockdown efficiency are shown in Figure 2AB. At the same time, miR-186-5p mimics (mimics NC) and inhibitor (inhibitor NC), as well as IGF2BP3 siRNA and corresponding si-NC (all from Shanghai Ruibo Company) were customized and transfected using lipo 3000 (Thermo Fisher Scientific) for conventional procedures.

[0033] 1.2 RNA extraction, reverse transcription and real-time fluorescence quantitative PCR (RT-qPCR):

[0034] Total RNA was extracted from cells using Trizol reagent (Takara), and linear and circular RNA were converted into cDNA using a reverse transcription kit (Novozyme). GAPDH was used as an endogenous reference, and the expression of the target gene was detected using the QuantStudio7 system. The miRNA was specifically reverse transcribed using a tailing kit (Acori), and U6 was selected as an endogenous reference.

[0035] 1.3 Western blot:

[0036] After lysing the cells with RIPA (Biyuntian) for 15 minutes, the supernatant was collected by centrifugation. The concentration of the extracted protein was determined by the BCA kit (Biyuntian), and the protein samples were collected after adding the loading buffer (Xin Saimei) and boiling. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Yisheng) and transferred to poly (vinylidene fluoride) (PVDF, Millipore) membrane. Blocked with 5% skim milk (Biyuntian) on a shaker for 2 hours, and then the diluted primary antibody (1:1000, proteintech) was added and incubated on a shaker at 4°C overnight. The next day, after the membrane returned to room temperature, it was rinsed with TBST 3 times, each time for 10 minutes. Subsequently, the secondary antibody (1:2500, Jackson) was incubated for 1 hour and rinsed with TBST for 10 minutes*3 times. Finally, the protein signal was detected by ECL luminescent liquid (Xin Saimei).

[0037] 1.4 Alizarin red staining and calcium deposition:

[0038] BMSCs were seeded into 6-well plates (2×10 5 Cells / well) were added, and when the confluence reached 70-80%, the osteogenic differentiation medium (10% FBS, 1% PS, 10mM β-GP, 300μM Aa and 0.1μM dexamethasone) was replaced. After 14 days of culture, the cells were fixed, and the calcium nodules in the extracellular matrix were stained with Alizarin Red S (ARS, Regen), and the images were taken under an inverted microscope. After the photo was taken, 10% hexadecylpyridinium chloride (CPC, Aladdin) was used to shake and elute Alizarin Red for 1h at room temperature. The eluate was collected, and the absorbance was measured at a wavelength of 562nm and quantitatively analyzed.

[0039] 1.5 ALP staining and ALP activity:

[0040] After 4 days of osteogenic induction culture, BMSCs were fixed with 4% paraformaldehyde for 30 minutes. According to the instructions of the ALP staining kit (Biyuntian), BCIP / NBT working solution was prepared to stain the cells. ALP activity in BMSCs samples was detected using a kit (Nanjing Jiancheng): RIPA was used to extract cell protein, mixed with the reagent, and incubated in a 37°C water bath for 15 minutes. After adding the color developer, the absorbance at a wavelength of 520 nm was measured, and the ALP activity (King's unit / gprot) was calculated according to the instructions.

[0041] 1.6 RNA pull-down experiment:

[0042] The pull-down kit (Thermo Fisher Scientific) was used to detect proteins interacting with circEIF4B. The biotin-labeled circEIF4B probe was designed and produced by Shanghai Ruibo Company. 7 After washing with PBS, add pull-down lysis buffer and lyse on ice for 10 minutes. After repeated freezing and thawing in liquid nitrogen for 3 times, centrifuge, take the supernatant and add RNase inhibitor. Subsequently, add biotin-labeled circular RNA probe and shake at 37°C for 4 hours. After washing with magnetic beads, add cell lysis buffer and rotate at 4°C 10r / min overnight. The next day, remove the supernatant and wash the magnetic beads. Next, add elution buffer and shake at 37°C for 30 minutes. Add protein loading buffer and boil for subsequent Western blot verification.

[0043] 1.7 Immunofluorescence (IF) and FISH staining:

[0044] In order to detect the localization of circEIF4B in cells, a FISH probe for circular RNA was designed (Shanghai Ruibo). The cells were inoculated on the slides, fixed with 4% paraformaldehyde for 30 minutes after 24 hours, and permeabilized with Trition X-100 for 10 minutes. According to the instructions of the kit, the pre-hybridization solution was added and placed at 37 degrees for 30 minutes, and the hybridization solution was preheated at 37 degrees. The FISH probe was added to the hybridization solution and incubated at 37°C overnight in the dark. The next day, the cells were washed with SSC wash solution of different concentrations at 42°C to reduce the background signal. The primary antibody was diluted (1:200, proteintech) and incubated with the cells and placed in a wet box at 4°C overnight. On the third day, the slides were washed with PBS, and the fluorescent secondary antibody (1:50, proteintech) was added and incubated at 37°C for 1 hour in the dark to mark the target protein. After washing with PBS, the anti-fluorescence quencher containing DAPI was added to the slides, and the images were observed and taken under a laser confocal microscope.

[0045] 1.8 Establishment of tooth extraction injury model in diabetic mice:

[0046] 8-week-old C57BL / 6N mice (male SPF grade) were fed a high-sugar and high-fat diet for 4 weeks, and then injected with 0.25% streptozotocin (STZ) at a dose of 25 mg / kg, and continued to be fed a high-sugar and high-fat diet for 4 weeks. Typical diabetic symptoms of polydipsia, polyphagia, polyuria, and weight loss were observed in the mice. After intraperitoneal injection of anesthetics, the minimally invasive tooth extraction elevator was wedged into the distal buccal bone plate of the maxillary first molar of the mouse. After loosening the maxillary first molar, it was clamped with toothed forceps to the root bifurcation, and the maxillary first molar was shaken buccal-lingually. After loosening, it was completely extracted by buccal dislocation. BMSCs were treated differently, namely the control group (BMSCs were not treated) and the circEIF4B group (BMSCs overexpressed circEIF4B), and the cells were collected after culturing in mineralization induction solution for 21 days. 10 7 The cells were mixed with PBS and injected at multiple points around the tooth extraction socket of mice. The mice were fed for 10 days after surgery and then sacrificed. The jaws were fixed in 4% paraformaldehyde for subsequent histological analysis.

[0047] 1.9 Establishment of skull defect model in diabetic rats:

[0048] 8-week-old SD rats (male SPF grade) were fed a high-sugar and high-fat diet for 4 weeks, and then injected with 0.25% streptozotocin (STZ) at a dose of 25 mg / kg, and continued to be fed a high-sugar and high-fat diet for 4 weeks. Typical diabetic symptoms of polydipsia, polyphagia, polyuria, and weight loss were observed in the rats. After intraperitoneal injection of anesthetics, a model of critical bone injury in the rat skull was established (bilateral circular defects with a diameter of 5 mm). BMSCs were treated differently, namely the control group (BMSCs were not treated) and the circEIF4B group (BMSCs overexpressed circEIF4B), and the cells were collected after culturing in mineralization induction solution for 21 days. 10 7 The cells were loaded onto the collagen scaffold, incubated at 37°C for half an hour, and then placed in the skull defect of the rats. The rats were fed for 12 weeks after surgery and then killed. The skull defect was fixed in 4% paraformaldehyde for subsequent histological analysis.

[0049] 1.10 H&E staining

[0050] After decalcification and dehydration of the tissue, paraffin sections (5 μm thickness) were made. The sections were dewaxed to water, and H&E staining was performed according to the instructions of the kit (Nanjing Jiancheng) to observe the morphological structure of the new bone in the tooth extraction socket and bone defect area. Among them, the cell nucleus was stained bright blue by hematoxylin, and the cytoplasm was stained pink with different shades by eosin. 2. Experimental results:

[0051] 2.1circEIF4B expression is downregulated in BMSCs of diabetic patients

[0052] According to the circular RNA interactome database, we determined that the genomic length of circEIF4B was 3830 bp and the splicing length was 516 bp. The Circbank database showed that circEIF4B originated from the EIF4B gene located on chromosome 12 (e.g. Figure 1 We further examined the structure of circEIF4B and found that it was formed by reverse splicing of exons 3 to 6 of the linear gene EIF4B. The splicing structure of circEIF4B was confirmed by Sanger sequencing of the amplified product. In addition, compared with the control group, the level of circEIF4B was significantly reduced in high-glucose medium, suggesting that circEIF4B is associated with high-glucose BMSCs dysfunction (e.g. Figure 1 (Fig. B).

[0053] 2.2circEIF4B significantly promotes osteogenic differentiation of BMSCs under high glucose conditions

[0054] To further evaluate the role of circEIF4B in osteogenic differentiation of BMSCs, we constructed circEIF4B overexpression and knockout viruses and verified the overexpression and knockout efficiency of circEIF4B in BMSCs by qRT-PCR (Fig. Figure 2 In high-glucose osteogenic induction medium, circEIF4B overexpression significantly increased the mRNA and protein levels of BMSCs osteogenic-related indicators (e.g. Figure 2 In addition, the results of ARS staining, calcium deposition, ALP staining and ALP activity showed that the increased expression of circEIF4B contributed to the enhanced osteogenesis of BMSCs (e.g. Figure 2 (Fig. EF in middle).

[0055] 2.3circEIF4B sponge adsorbs miR-186-5p to recruit FOXO1 to improve osteogenic differentiation of high-glucose BMSCs

[0056] In this part, we further explored the mechanism of circEIF4B in promoting osteogenesis in BMSCs. Given that circRNA can act as a miRNA sponge to regulate the transcription of downstream mRNA. qRT-PCR and Western blot results showed that osteogenesis-related indicators and FOXO1 were downregulated in the circEIF4B knockdown group, while miR-186-5p inhibitor significantly reversed the inhibitory effect of sh-circEIF4B on osteogenesis and FOXO1 expression (e.g. Figure 3ALP staining and ALP activity showed similar results (Fig. Figure 3 The above observations suggest that the interaction between circEIF4B and miR-186-5p may recruit the target gene FOXO1, thereby regulating the osteogenic differentiation of BMSCs under high glucose microenvironment.

[0057] 2.4circEIF4B binds to RNA-binding protein IGF2BP3 to promote osteogenic differentiation of high-glucose BMSCs

[0058] We identified RBPs that interact with circEIF4B using the RBPDB, circular RNA interactome, and circatlas databases. Next, we applied specific biotin-labeled circEIF4B to RNA pull-down analysis, and we found that IGF2BP3 was significantly enriched in the circEIF4B probe compared with the IgG group (e.g. Figure 4 Subsequently, we confirmed the colocalization of circEIF4B and IGF2BP3 in the cytoplasm by IF and FISH analysis (Fig. Figure 4 Next, we used small interfering RNA (siRNA) to knock down IGF2BP3 for loss-of-function studies. qRT-PCR and Western blot results showed that after IGF2BP3 knockdown, the expression of osteogenic-related markers was downregulated (e.g. Figure 4 ALP staining and ALP activity results also confirmed the positive effect of IGF2BP3 on the osteogenic differentiation of high glucose BMSCs (Fig. Figure 4 (Fig. E).

[0059] 2.5circEIF4B promotes healing of tooth extraction wounds in diabetic mice and skull defects in diabetic rats

[0060] H&E staining results showed that after injection of BMSCs overexpressing circEIF4B, the new bone in the tooth extraction socket of diabetic mice increased compared with the control group (e.g. Figure 5 In the diabetic rat skull defect model, implantation of collagen scaffolds with BMSCs overexpressing circEIF4B significantly promoted new bone formation at the defect site (Fig. Figure 5 The above results verified the positive role of circEIF4B in the diabetic bone regeneration model in vivo.

[0061] Conclusion: Studies have shown that circRNAs are involved in regulating the function and differentiation of osteoblasts, osteoclasts and mesenchymal stem cells [10-12]. However, the functions of most circRNAs remain to be explored, and only limited experimental evidence has revealed the role of certain circRNAs in the regulation of bone metabolism. In the present invention, circEIF4B was found to be significantly downregulated in a high-glucose culture environment, suggesting that circEIF4B is associated with the dysfunction of high-glucose BMSCs. In addition, in vitro experiments further confirmed that circEIF4B overexpression significantly enhanced the osteogenic differentiation of BMSCs.

[0062] The present invention demonstrated that miR-186-5p inhibitor reversed the inhibitory effect of sh-circEIF4B on osteogenic-related indicators and FOXO1 through qRT-PCR and Western blot experiments, confirming that circEIF4B can sponge miR-186-5p and then recruit FOXO1 to improve the osteogenic differentiation of high-glucose BMSCs. On the other hand, we found that circEIF4B interacted with IGF2BP3, and the protein level of IGF2BP3 was positively correlated with the expression of circEIF4B. Yang et al. reported that Gegenqinglian Decoction (GQD, a drug for long-term treatment of T2DM) can play a protective role in type 2 diabetic osteoporosis (T2DOP) by upregulating the expression of IGF2BP3

[13] . In addition, IGFB2P3 in serum may also be a new biomarker for the treatment of T2DOP

[13] . Consistently, we used siRNA targeting IGF2BP3 and confirmed that it regulates the osteogenic differentiation of BMSCs in a positive manner.

[0063] Finally, the tooth extraction socket wound healing model of diabetic mice and the skull defect experiment of diabetic rats showed that compared with the control group, circEIF4B overexpression significantly promoted the healing of tooth extraction wounds in mice and the formation of new bone in the skull defect of rats, confirming that circEIF4B helps promote the osteogenic differentiation of BMSCs in a high-glucose microenvironment and has a positive effect on bone regeneration in diabetic animal models.

[0064] In summary, we first provided evidence that promoting the expression of circEIF4B or enhancing its activity can improve the osteogenic ability of BMSCs in a high-glucose microenvironment. Mechanistically, circEIF4B absorbs miR-186-5p to activate FOXO1 transcription and interacts with IGF2BP3 to promote the osteogenic differentiation of high-glucose BMSCs. Therefore, circEIF4B may serve as a new intervention target for diabetic bone regeneration, bringing new prospects for the treatment of delayed or impaired healing of tooth extraction wounds in diabetic patients.

[0065] References:

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Claims

1. Use of circEIF4B in the preparation of a drug or preparation for improving delayed or impaired healing of tooth extraction wounds in patients with diabetes, characterized in that: The nucleotide sequence of circEIF4B is shown in SEQ ID NO.

1.

2. Use of circEIF4B in the preparation of a preparation for preserving tooth extraction sites in diabetic patients, characterized in that: The nucleotide sequence of circEIF4B is shown in SEQ ID NO.

1.

3. Bone marrow mesenchymal stem cells overexpressing circEIF4B shown in SEQ ID NO.1, characterized in that: It is obtained by transfecting bone marrow mesenchymal stem cells with a virus that overexpresses circEIF4B.

4. Use of the bone marrow mesenchymal stem cells according to claim 3 in the preparation of a medicine or preparation for improving delayed or impaired healing of tooth extraction wounds in diabetic patients.

5. Use of the bone marrow mesenchymal stem cells according to claim 3 in preparing a preparation for preserving tooth extraction sites in diabetic patients.

6. Use of the bone marrow mesenchymal stem cells according to claim 3 in the preparation of a preparation for promoting healing of tooth extraction sockets in diabetic patients.

Images