Application of miRNA-211-5p inhibitor in preparation of medicine or kit for preventing and / or treating chronic osteomyelitis bone defect

Abstract

The invention discloses application of a miRNA-211-5p inhibitor in preparation of a medicine or a kit for preventing and / or treating chronic osteomyelitis bone defects, and relates to the field of molecular biology diagnosis and treatment. The invention particularly relates to screening and verification of the influence of miRNA-211-5p on proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). It is found that transfected miRNA-211-5p simulant (mimic) inhibits expression of BMPR1a and inhibits proliferation and differentiation of BMSCs; the miRNA-211-5p inhibitor is transfected to promote the expression of BMPR1a, and a new target and a new way are provided for clinical diagnosis and treatment of infectious bone nonunion.

Description

Technical Field

[0001] The present invention relates to the field of molecular biological diagnosis and treatment, and in particular to the use of a miRNA-211-5p inhibitor in the preparation of a medicine or a kit for preventing and / or treating bone defects in chronic osteomyelitis. Background Art

[0002] Chronic osteomyelitis (CO) is an infectious disease of bone tissue characterized by progressive bone destruction and bone necrosis. The incidence rate is as high as 10% and is on the rise. Due to its easy recurrence, difficult to cure, high incidence of bone defects or nonunions, it has become a difficult disease in clinical orthopedics. At present, the treatment principle of CO is to thoroughly debride the infected site and then reconstruct the bone defect area through bone transplantation, Masquelet technique, etc. However, although the above methods can effectively solve the segmental bone defects after debridement of chronic osteomyelitis, due to the large surgical trauma and medullary cavity clearance, the osteogenic function of the bone defect site at the fracture end can be inhibited, and there are still problems such as difficulty in healing of the bone defect. Delayed bone healing or nonunion will cause serious limb dysfunction and bring heavy economic burden to the patient's family and society. In clinical practice, exogenous growth factors such as basic fibroblast growth factor and bone morphogenetic protein are given to promote bone repair, but these exogenous factors have a short half-life in the body, are easily degraded, are expensive, and have the potential risk of inducing tumors. Therefore, exploring the bone defect repair mechanism and intervention strategy is of great significance for the treatment of bone defects in chronic osteomyelitis.

[0003] Bone defect repair is a regenerative process, including multiple physiological processes such as inflammation, osteogenesis, and chondrogenesis. Under normal physiological conditions, bone trauma initially triggers acute inflammatory activation in the body, and the inflammatory response subsides before entering the bone tissue repair or regeneration stage. However, external factors such as pathogenic bacteria invasion, endotoxins, and exogenous scaffolds at the site of bone injury can cause excessive inflammatory responses in the body, leading to the synthesis and release of multiple inflammatory factors such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α), causing damage to the normal physiological functions of tissues or cells, thereby affecting the bone repair process. Bone marrow mesenchymal stem cells (BMSCs) have the potential for self-renewal and multidirectional differentiation. They participate in bone formation by differentiating into osteoblasts (OBs), and have been proven to play a huge role in bone repair and bone regeneration. Existing studies have shown that excessive inflammatory responses inhibit the differentiation function of BMSCs, which is closely related to the occurrence and development of bone defects in chronic osteomyelitis. TNF-α is the most important inflammatory factor throughout the entire process of CO inflammatory response and is a commonly used inflammatory environment mimicking factor. Studies have found that it can significantly inhibit the production of BMSCs mineralized nodules and osteogenic differentiation factor alkaline phosphatase (ALP), delaying bone healing. This indicates that inflammatory microenvironment-mediated BMSCs osteogenic differentiation dysfunction is an important cause of bone defects in chronic osteomyelitis. Revealing the BMSCs differentiation regulation mechanism under the condition of chronic osteomyelitis bone defects is an important part of developing new therapies.

[0004] MicroRNAs (miRNAs) are RNAs of approximately 22 nucleotides that recognize target mRNAs by interacting with recognition sites in 3′-untranslated regions (UTRs) and inhibit the expression of target genes after transcription. Previous studies have shown that the miRNA profiles of osteomyelitis bone defect patients and model animals are significantly altered, and some miRNAs have been shown to be directly involved in the regulation of bone regeneration and bone repair, and more and more evidence has confirmed that miRNAs-mediated bone repair is closely related to the regulation of BMSCs differentiation. For example, miR-211-5p regulates the osteogenic differentiation of mouse BMSCs through the Smad / β-catenin pathway (Wang H, Shi X, Guo Z, et al. microRNA-211-5p predicts the progression of postmenopausal osteoporosis and attenuates osteogenesis by targeting dual specific phosphatase 6 [J]. Bioengineered. 2022, 1 3 (3): 5709-5723.); the expression of osteogenic differentiation factor Runx2 was significantly reduced in BMSCs overexpressing miR-211-5p ([1] Ju W, Zhang G, Zhang X, et al. Involvement of MiRNA-211-5pan and Arhgap 11a Interaction During Osteogenic Differentiation of MC3T3-E1Cells [J]. Front Surg.2022, 9: 857170.doi: 10.3389 / fsurg.2022.857170.[2]Huang J, Zhao L, Fan Y, et al. The microRNAs miR-204and miR-211maintain joint homeostasisand protect against osteoarthritis progression[J].Nat Commun.2019, 10(1):2876.doi:10.1038 / s41467-019-10753-5.) etc.During the development of bone defects in chronic osteomyelitis, the inflammatory factor TNF-α mediates changes in the miRNAs spectrum, resulting in dysfunctional osteogenic differentiation of BMSCs (Kuang W, Zheng L, Xu X, et al. Dysregulation of the miR-146a-Smad4 axis impairs osteogenesis of bonemesenchymal stem cells under inflammation[J]. Bone Res. 2017, 21(5): 17037. doi: 10.1038 / boneres). Summary of the invention

[0005] To solve the above problems, the applicant's previous study found that the expression of miR-211-5p in the serum of patients with chronic osteomyelitis bone defect and the bone tissue of rats with chronic osteomyelitis bone defect was significantly upregulated. After TNF-α induced BMSCs, the expression of miR-211-5p was significantly increased, and the osteogenic differentiation function was inhibited. MiR-211-5p may be used as an important marker of chronic osteomyelitis bone defect.

[0006] Based on the applicant's previous research, the therapeutic effect of miR-211-5p inhibitor on rats with chronic osteomyelitis bone defects was further studied. Therefore, the present invention provides an application of a miRNA marker miRNA-211-5p, specifically the application of a miRNA-211-5p inhibitor in the preparation of a drug or kit for preventing and / or treating chronic osteomyelitis bone defects. The miRNA data of patients with nonunion and patients with normal bone healing in the GEO database were deeply analyzed to detect differential miRNA expression profiles. Osteomyelitis nonunion rats and TNF-α-induced BMSCs models were prepared, and real-time quantitative PCR was used to verify miRNA-211-5p expression. Further miRNA-211-5p agonists (mimic) and inhibitors (inhibitors) were prepared to study their effects on the osteogenic differentiation ability of BMSCs and the target gene Bmpra. Provide new markers for the clinical diagnosis and treatment of infected nonunion.

[0007] The specific technical solutions of the present invention are as follows:

[0008] The present invention provides the use of a miRNA-211-5p inhibitor in the preparation of a medicine or a kit for preventing and / or treating bone defects in chronic osteomyelitis.

[0009] Preferably, the sequence of the miRNA-211-5p inhibitor is:

[0010] 5'-AGGCAAAGGAUGACAAAGGGAA-3'.

[0011] Specifically, when used, the miRNA-211-5p inhibitor activates the BMP2 signaling pathway and promotes BMPR1a expression in bone nonunion or inflammatory conditions.

[0012] That is, miRNA inhibitors can increase the transcription of BMPR1a gene and protein expression.

[0013] The present invention also provides an application of a composition in the preparation of a drug or a kit for preventing and / or treating bone defects in chronic osteomyelitis, wherein the composition contains an effective amount of a miRNA-211-5p inhibitor and a pharmaceutically acceptable virus, carrier or excipient.

[0014] Preferably, the sequence of the miRNA-211-5p inhibitor is:

[0015] 5'-AGGCAAAGGAUGACAAAGGGAA-3'.

[0016] Specifically, the pharmaceutically acceptable carrier or excipient is selected from one or more of chitosan, cholesterol, liposomes and nanoparticles.

[0017] The present invention also provides an application of a probe or primer for specifically detecting miRNA-211-5p in preparing a kit for diagnosing and / or prognostic evaluation of bone defects in chronic osteomyelitis.

[0018] Specifically, the sequences of the primers are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0019] The present invention specifically involves screening and verifying the effect of miRNA-211-5p on the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). It is found that transfection of miRNA-211-5p mimics inhibits the expression of BMPR1a and inhibits the proliferation and differentiation of BMSCs; transfection of miRNA-211-5p inhibitors promotes the expression of BMPR1a, providing new targets and new approaches for the clinical diagnosis and treatment of infected bone nonunion. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The miRNA-mRNA network diagram of patients with osteomyelitis and nonunion of the present invention is constructed; A represents the volcano plot differential expression of miRNA; B represents the volcano plot differential expression of mRNA; C and D represent the Venn diagram of the intersection of target genes predicted by differential miRNA and differential genes; E and F represent the differential miRNA-mRNA network diagram.

[0021] Figure 2The figure shows the changes of bone tissue pathological histology and structural parameters in rats with chronic osteomyelitis and nonunion; A: bone tissue appearance of each group; B: Micro-CT scan of bone tissue of each group; C: bone tissue pathological histology; D: bone mineral density (BMD); E: bone surface area (BS); F: bone volume (BV); G: bone volume / total volume (BV / TV); H: trabecular number (Tb.N); I: trabecular number separation (Tb.Sp). Compared with the NG group, *p<0.05.

[0022] Figure 3 The imbalance of miR-211-5p / Bmpr1a axis in COD rats; A-I are the gene expressions of miR-211-5p, Bmpr1a, Smad1, Smad3, Smad5, Smad4, Runx2, and Alp in the tibia of COD rats; J-M are the semi-quantitative analysis of protein expression of BMPR1A, RUNX2, ALP, and COL1A1 in the tibia of COD rats; N is the strip chart of protein expression in the tibia of COD rats. Compared with the NG group, #p<0.05, ##p<0.01.

[0023] Figure 4 TNF-α inhibits the osteogenesis of BMSCs by regulating the miR-211-5p / Bmpr1a axis; A: TNF-α-induced ARS staining of BMSCs; B: TNF-α-induced ALP staining of BMSCs; C: TNF-α-induced Bmpr1a mRNA expression in BMSCs; D: TNF-α-induced Smad1 mRNA expression in BMSCs; E: TNF-α-induced Smad3 mRNA expression in BMSCs; F: TNF-α-induced Runx2 mRNA expression in BMSCs; G: TNF-α-induced Alp mRNA expression in BMSCs. Compared with the KB group, #p<0.05, ##p<0.01.

[0024] Figure 5 The effect of miR-211-5p on osteogenic differentiation of BMSCs; A: The effect of miR-211-5p on alkaline phosphatase staining of BMSCs; B: The effect of miR-211-5p on alizarin red staining of BMSCs; C: The effect of miR-211-5p on Bmpr1a mRNA expression in BMSCs; D: The effect of miR-211-5p on Runx2 mRNA expression in BMSCs; E: The effect of miR-211-5p on Alp mRNA expression in BMSCs. Compared with the NC group, #p<0.05, ##p<0.01.

[0025] Figure 6Effects of miR-211-5p antagomir on bone microstructure in COD rats; A is the gross appearance of bone tissue and representative Micro-CT scans of rats in antagomir NC group; B is the gross appearance of bone tissue and representative Micro-CT scans of rats in miR-211-5p antagomir group; C: bone mineral density (BMD); D: trabecular connection (Con); E: trabecular connection density (Con.Dn); F: trabecular number separation (Tb.Sp); * indicates p < 0.05. DETAILED DESCRIPTION

[0026] The following examples will enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0027] Example 1 Screening and verification of differentially expressed miRNAs in chronic osteomyelitis and nonunion

[0028] 1. Database analysis of differentially expressed miRNAs in patients with nonunion

[0029] The GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ) was searched with the term "Osteoarthritis" Homosapiens", and the chips GSE62137 and GSE22529 were downloaded from the GEO database according to the search results. Twenty-six samples were included in the data set, including 20 normal human serum samples and 6 serum samples from patients with chronic osteomyelitis and nonunion. The matrix and platform files of the chip were downloaded. The miRNA differential analysis was performed using R language. All chips were median-normalized and differentially expressed probes were screened. Fold change (FC) and p-value were used to screen the differential expression of the two groups of samples, and differential miRNAs were screened with logFC>1.0 and corrected p-value<0.05. Finally, the differentially expressed miRNAs and predicted targets were analyzed.

[0030] Experimental results: The results are as follows Figure 1 As shown in Figure 1, for the miRNA dataset, 39 differentially expressed microRNAs (DEMs) were screened, 19 miRNAs were significantly upregulated, and 20 miRNAs were significantly downregulated, as shown in Table 1. For the mRNA dataset, 438 differentially expressed genes (DEGs) were identified in samples from patients with chronic osteomyelitis and nonunion compared with healthy control samples, including 173 downregulated DEGs and 265 upregulated DEGs. The differential miRNA and mRNA volcano plots are shown in Figure 1. Figure 1 A and Figure 1As shown in B. The target genes of differential miRNAs were predicted using miRBase. Overlapping genes were obtained between the predicted target genes and differential genes, as shown in Figure 1 C and Figure 1 As shown in D in Figure 2, the results showed that 6 overlapping genes were upregulated, namely PSIP1, LSM3, SOX6, TBCA, GYPA and THEM5. In addition, 17 overlapping differential genes were downregulated, such as ATRX, BMPR1A, BPY2C, C1D, ESR1, etc. The differential miRNA and gene regulatory network was constructed to understand the correspondence between them, see Figure 1 E and Figure 1 F in. hsa-miR-570-3p, hsa-miR-607, and hsa-miR-211-5p are located at the center of the network, regulating three or two target genes, respectively, and hsa-miR-211-5p was found to target bone morphogenetic protein receptor 1A (BMPR1A), see Figure 1 E in.

[0031] Table 1 Differentially expressed miRNAs

[0032] Gene logFC adj.P.Val Gene logFC adj.P.Val hsa-mir-1290 -1.6 6.72E-12 hsa-mir-587 1.0 2.83E-06 hsa-mir-938 -1.6 6.01E-17 hsa-mir-1274b 1.0 1.64E-07 hsa-mir-566 -1.5 2.00E-15 hsa-mir-570 1.1 1.10E-07 hsa-mir-1288 -1.4 2.66E-14 hsa-mir-583 1.1 9.79E-07 hsa-mir-374a -1.4 8.83E-13 hsa-mir-200b 1.1 6.97E-09 hsa-mir-410 -1.4 3.32E-09 hsa-mir-934 1.1 2.01E-07 hsa-mir-626 -1.3 3.51E-14 hsa-mir-220b 1.1 1.60E-09 hsa-mir-380 -1.2 9.14E-16 hsa-mir-211 1.1 9.93E-09 hsa-mir-1272 -1.2 4.48E-15 hsa-mir-455 1.2 5.32E-10 hsa-mir-889 -1.2 2.45E-11 hsa-mir-891b 1.3 5.47E-09 hsa-mir-1267 -1.2 2.75E-16 hsa-mir-147 1.3 3.12E-06 hsa-mir-658 -1.2 1.59E-15 hsa-mir-760 1.4 6.05E-11 hsa-mir-939 -1.1 1.76E-16 hsa-mir-642 1.4 5.53E-10 hsa-mir-1298 -1.1 7.91E-14 hsa-mir-548n 1.4 9.04E-08 hsa-mir-643 -1.1 3.69E-08 hsa-mir-148a 1.4 6.06E-10 hsa-mir-508 -1.0 7.51E-12 hsa-mir-579 1.5 7.93E-08 hsa-mir-650 -1.0 3.04E-13 hsa-mir-607 1.7 2.35E-08 hsa-mir-548j -1.0 8.41E-05 hsa-mir-1269 1.7 2.96E-13 hsa-mir-1323 -1.0 2.38E-11 hsa-mir-449b 1.9 7.51E-12 hsa-mir-555 -1.0 1.75E-11

[0033] 2. Establishment of a rat model of chronic osteomyelitis with bone defect and verification of differential expression of miRNA-211-Sp in bone tissue

[0034] Twelve SD rats were anesthetized with sodium pentobarbital. The hair from the knee to the ankle of the left lower limb was shaved and disinfected with povidone-iodine. A 2 mm bone drill was used to drill a hole into the medullary cavity on the anterior medial side of the upper end of the tibia, and Staphylococcus aureus was injected into the medullary cavity. The skin was sutured to form the modeling group (COD); 6 SD rats only had their skin cut open to form the sham operation group (NG). After 30 days of modeling, the modeling group and the sham operation group underwent debridement surgery and then received vancomycin (administered by intramuscular injection, 40 mg / kg, once a day) from the original incision on the medial side of the left tibia. After expansion and debridement, the wound was sutured. Six weeks later, the healing of the rat bone gap was observed with the naked eye, and bone tissue pathology and gene detection such as miR-211-5p were performed.

[0035] (1) Changes in bone histopathology and structural parameters in COD rats

[0036] After the experiment, sodium pentobarbital anesthesia was performed, blood was collected from the abdominal aorta, and the rat tibia tissue was quickly dissected and fixed in 75% ethanol; the SkyScan 1176 small animal Micro-CT scanning imaging system of Bruker Company of Germany was used for scanning and analysis of bone microstructure parameters. After the scanning, the bone tissue was stained with conventional decalcification agent H&E, and pathological tissue changes were observed under an electron microscope.

[0037] Experimental results: The results are as follows Figure 2 As shown, compared with NG, COD rats had abscesses and osteomyelitis bone defects in the tibia tissue of model rats after 6 weeks ( Figure 2 A in the figure). Micro-CT analysis showed that the model rats had obvious bone defects, and compared with NG, the bone microstructure parameters BMD, BS, BV, BV / TV, and Tb.N of COD rats were significantly reduced, and Tb.Sp was significantly increased (p<0.05) ( Figure 2 B and D~I in the figure); Histopathological results showed that a large number of inflammatory cells infiltrated in the defect ( Figure 2 C).

[0038] (2) Changes in the expression of miR-211-5p and bone formation-related mRNA and protein in COD rats.

[0039] Real-time fluorescence quantitative PCR was used to detect bone tissue miRNA-211-5p (sequence shown in SEQ ID NO.1) and gene expression. The specific operation is as follows:

[0040] a. Sample preparation: Grind the combined tissue with liquid nitrogen to obtain crushed bone tissue;

[0041] b. RNA extraction: Use a professional bone tissue RNA extraction kit (EASYspinPlus Bone Tissue RNA Rapid Extraction Kit, Allied Biotechnology RN54);

[0042] c. RNA concentration detection: After extraction, the total RNA concentration and purity were detected using the thermo scientific NanodropOne instrument;

[0043] d. cDNA synthesis: Shandong Sikoje miRNA reverse transcription kit (AG0502 / AG504) was used, and the specific operation was carried out according to the instruction manual.

[0044] e.qPCR reaction procedure: Design primer sequences, see Table 2 for details. Place the prepared samples in the ABI7500 instrument for amplification. The amplification procedure is: start at 94°C for 2 minutes, then use a two-step PCR cycle, i.e., 94°C for 10 seconds, 60°C for 35 seconds, and 40 cycles. U6 was used as an internal reference for miRNA-211-5p, and β-actin was used as a standard control for the others, and 2 -ΔΔCt Methods The fold change of each target gene was calculated.

[0045] Table 2 Rat miRNA-211-5p and U6 reverse transcription primers

[0046]

[0047] Western-blot method was used to detect the expression of BMPR1A, RUNX2, ALP and COL1A1 proteins in bone tissue. The specific operation is as follows:

[0048] a. Sample preparation: After the bone tissue is taken out of the -80℃ refrigerator, it is broken into pieces with a hammer. Then, a protein lysis buffer containing protease inhibitors and phosphatase inhibitors is added, and ultrasonic extraction is performed in an ice bath for 30 minutes. Then, the lysis is continued in a 4℃ refrigerator for 30 minutes, and the supernatant is collected after centrifugation at 12,000 rpm for 10 minutes.

[0049] b. Concentration determination and protein denaturation: The protein concentration of each sample was detected using a BCA kit (Keygen Biotechnology, KGB2101-1000), the sample concentration was adjusted, and then the loading buffer was added.

[0050] b. SDS-PAGE gel electrophoresis: Gel was prepared using Shanghai Bio-Tech Gel Kit (C631100-0200), electrophoresis was performed using Shanghai Yamei Bio Tris / Glycine / SDS electrophoresis buffer (PS105), and membrane transfer was performed using ice-free rapid transfer buffer (PS201S).

[0051] c. Antibody incubation and development: Add primary antibodies BMPR1A, RUNX2, ALP and COL1A1, respectively, with an antibody dilution ratio of 1:1000, incubate at 4°C overnight, wash the membrane with PBST, add HRP-conjugated Affinipure Goat Anti-RabbitIgG (H+L) secondary antibody (1:10000), and incubate at room temperature for 1 hour. Use Shanghai Yamei Biological Universal Chemiluminescence Detection Kit (SQ203) for development.

[0052] d. Data processing: The grayscale value of each band of the obtained protein band diagram was analyzed using Image J software, and β-actin was used as a standard control to calculate the relative expression level of the protein.

[0053] Experimental results: The results are as follows Figure 3 As shown in the figure, compared with NG, RT-PCR results showed that the expression of miR-211-5p in the tibia tissue of COD rats was significantly increased, and the expression of Bmpr1a, Smad1, Smad3, Smad5, Smad4, Runx2, and Alp genes was significantly decreased (p<0.01). WB results showed that compared with NG, the expression of BMPR1A, RUNX2, ALP, and COL1A1 proteins in the bone tissue of COD rats was significantly decreased (p<0.01).

[0054] 3. Establishment of TNF-α-induced BMSCs model and verification of differential miRNA-211-5p expression

[0055] Rat bone marrow mesenchymal stem cells (BMSCs, purchased from Saiye Biotechnology Co., Ltd., catalog number: RASMX-01101). The cells were seeded in 25 cm 2 The cells were cultured in a flask at 37°C and 5% CO2 incubator. After 2 generations, the cell suspension was collected. 3 The cells were inoculated in 6-well plates with DMEM high-glucose complete medium (Zhejiang Senrui Biotechnology Co., Ltd., batch number: 2022032902), and subsequent experiments were performed when the cell confluence reached about 80-90%.

[0056] Preparation of 50mL of osteogenic induction medium: Take 0.0028g of ascorbic acid (Aladdin, A171447), 0.123055g of sodium β-glycerophosphate (Sigma, BCCD6874), 20μL of dexamethasone solution (Aladdin, B1704084, 1mg dexamethasone powder is dissolved in 1mL of blank medium, 20μL of it is diluted to 1mL, and then 20mL of medium is diluted to 1mL to obtain dexamethasone solution) and add them to 50mL of complete medium. Preparation of osteogenic induction medium with different TNF-α: Take 1μL of antibody stock solution of 1mg / mL TNF-α antibody (Bioss), add 1mL of osteogenic induction solution to obtain 1μg / mL (1ng / μL) TNF-α solution. Take 120μL of 1ng / μL TNF-α solution and dilute it to 6mL with osteogenic induction medium to obtain 20ng / mL TNF-α solution; take 360μL of 1ng / μL TNF-α solution and dilute it to 6mL with osteogenic induction medium to obtain 60ng / mL TNF-α solution. Discard the DMEM high-glucose complete medium in the 6-well plate, and set up KB, TNF-α20ng / mL, and TNF-α60ng / mL groups. The KB group used osteogenic induction medium, and the other two groups were intervened with the corresponding TNF-α solution. The corresponding culture medium was changed every 3 days, and the cells were collected after 6 days to prepare total RNA. The expression of miRNA-211-5p and Bmpr1a mRNA was detected.

[0057] Experimental results: Compared with the KB group, the staining results showed that TNF-α20, 60ng / mL intervention significantly inhibited the production of alkaline phosphatase and mineralized nodules, inhibiting the osteogenic differentiation of BMSCs; after TNF-α20, 60ng / mL intervention, the expression of miRNA-211-5p was significantly upregulated (p<0.01), and the mRNA expression of osteogenic differentiation genes Bmpr1a, Alp and Runx2 was significantly downregulated (p<0.01). Figure 4 .

[0058] Example 2 Effect of miRNA-211-5p on the osteogenic differentiation phenotype of BMSCs

[0059] Rat bone marrow mesenchymal stem cells (BMSCs, purchased from Saiye Biotechnology Co., Ltd., RASMX-01101). The cells were seeded at 25 cm 2 The cells were cultured in a flask at 37°C and 5% CO2 incubator. After 2 generations, the cell suspension was collected. 3 The concentration was inoculated in a 6-well plate with DMEM high-glucose complete medium (Zhejiang Senrui Biotechnology Co., Ltd., batch number: 2022032902), and the subsequent experiments were carried out when the cell confluence reached about 80-90%. Jiangsu Beixin Biotechnology Co., Ltd. was commissioned to synthesize miR-211-5p mimic, Mimic NC, Inhibitors miR-211-5p, and Inhibitors NC. The specific sequences are shown in Table 3. Subsequently, the synthesized powder was prepared into 100 μM with RNase-free water. For subsequent experiments.

[0060] Table 3 Design sequences of miR-211-5p inhibitors and mimics

[0061]

[0062] The preparation table of 100 nM miR-211-5p mimic transfection system is shown in Table 4, taking the total volume of 2 mL as an example.

[0063] Table 4 Transfection reagent preparation combination table

[0064]

[0065] After preparing the V1 and V2 systems, gently blow and mix them, and let them stand for 5 minutes; then absorb the V2 solution into the V1 system and continue to stand for 20 minutes; add the V3 solution to the V1+V2 centrifuge to obtain 2mL of the transfection system solution. Similarly, prepare the miR-211-5p NC transfection system.

[0066] Alizarin red staining to detect mineralized nodules: Rat BMSCs were seeded at 25 cm 2 The cells were cultured in a flask at 37°C and 5% CO2 incubator. After 2 generations, the cell suspension was collected. 3Seed in 6-well plates with DMEM high-glucose complete medium. Subsequent experiments were carried out when the cell confluence reached about 80-90%. Set up miR-211-5p mimic, Mimic NC, Inhibitors miR-211-5p, and Inhibitors NC groups. After successful transfection, osteogenic induction was performed for 7 days, and mineralized nodules were observed by Alizarin red staining. The specific operation was as follows: first discard the supernatant, wash twice with PBS, add 4% paraformaldehyde to fix for 15 minutes; wash twice with PBS, add Alizarin red reagent to stain for 1 hour, add 2mL to each well of the 6-well plate, and then incubate in a 37°C water bath for 1 hour, then rinse twice with purified water, dry and take pictures. Alkaline phosphatase staining to detect osteogenic differentiation phenotype: Rat BMSCs were inoculated in 25cm 2 The cells were cultured in a flask at 37°C and 5% CO2 incubator. After 2 generations, the cell suspension was collected. 3 The cells were inoculated in 6-well plates with DMEM high-glucose complete medium. Subsequent experiments were performed when the cell confluence reached about 80-90%. The miR-211-5p mimic, Mimic NC, Inhibitors miR-211-5p, and Inhibitors NC groups were set up. After successful transfection, osteogenic induction was performed for 7 days, and alkaline phosphatase calcium cobalt method (Keygen Biotechnology: KGA353) staining was performed.

[0067] The specific operation is as follows: first discard the supernatant, wash twice with PBS, add 4% paraformaldehyde to fix for 15 minutes; wash twice with PBS; add incubation working solution to cover the sample at 37 degrees for 180 minutes. Wash with water for 5-10 minutes. Cover the sample with staining solution A and incubate at room temperature for 5 minutes, wash with water for 2 minutes. Cover the sample with staining solution B and incubate at room temperature for 1 minute, wash with water for 5 minutes. Counterstain the cytoplasm with eosin staining solution. Dehydrate with ethanol gradient dehydration, permeabilize with xylene, and seal with mixed sealing agent.

[0068] Effect of osteogenic differentiation-related gene expression: Discard the complete medium in the 6-well plate, rinse with a small amount of Opti, and then dry the solution. Add 2 mL of the prepared transfection system solution to each well. After 6 hours of transfection, discard the transfection reagent, rinse the medium, and then add 2 mL of osteogenic induction medium to each well and continue to culture for 6 days. Change the osteogenic induction medium every 3 days. After 6 days, collect cells, prepare total RNA, and detect the expression of mRNA such as miR-211-5p, Bmprla and Runx2.

[0069] Experimental results: Compared with the Mimic NC group, the overexpression of miR-211-5p significantly reduced the production of alkaline phosphatase and the formation of mineralized nodules; RT-PCR results showed that the expression of Bmpr1a, Runx2, and Alp mRNA in BMSCs was significantly reduced (p<0.01). Compared with the Inhibitors NC group, the low expression of miR-211-5p significantly increased the production of alkaline phosphatase and the formation of mineralized nodules in BMSCs; RT-PCR results showed that the expression of Bmpr1a, Runx2, and Alp mRNA was significantly increased (p<0.01). Figure 5 .

[0070] Example 3 Effect of miRNA-211-5p antagomir on rats with chronic osteomyelitis and bone defect

[0071] 12 SD rats were anesthetized with sodium pentobarbital. The left lower limbs were shaved from the knee joint to the ankle joint and disinfected with povidone-iodine. A 2mm bone drill was used to drill a hole into the medullary cavity on the anterior medial side of the upper end of the tibia, and Staphylococcus aureus was injected into the medullary cavity. The skin was sutured to form the modeling group (COD). After 7 days of modeling, the modeling group and the sham operation group underwent debridement surgery and then gave vancomycin (administered by intramuscular injection, 40 mg / kg, once every two days). rno-miR-211-5p antagomir (i.e., Inhibitors miR-211-5p) and antagomir NC (i.e., Inhibitors NC), the sequences are shown in Table 3, and 1OD is approximately 5nmol.

[0072] Experimental results Six weeks later, the model rats were divided into an antagomir NC group and a miR-211-5p antagomir group. Subsequently, antagomir NC and miR-211-5p antagomir were injected intramuscularly at a dose of 1 OD / rat once a week for four consecutive times, and the bone tissue healing was observed.

[0073] Experimental results: Micro-CT scanning imaging system was used to scan and analyze bone microstructure parameters. Figure 6 The results showed that compared with the antagomir NC control group, the miR-211-5p antagomir group significantly promoted the healing of bone defects in COD rats, and significantly increased BMD, Con, and Con.Dn bone microstructures, and reduced Tb.Sp (p<0.05).

Claims

1. Use of miRNA-211-5p inhibitors in the preparation of drugs or kits for preventing and / or treating bone defects in chronic osteomyelitis.

2. The use according to claim 1, characterized in that: The sequence of the miRNA-211-5p inhibitor is: 5'-AGGCAAAGGAUGACAAAGGGAA-3'.

3. The use according to claim 1, characterized in that: When applied, the miRNA-211-5p inhibitor activates the BMP2 signaling pathway and promotes BMPR1a expression in nonunion or inflammatory conditions.

4. Use of a composition in the preparation of a drug or kit for preventing and / or treating bone defects in chronic osteomyelitis, wherein the composition contains an effective amount of a miRNA-211-5p inhibitor and a pharmaceutically acceptable virus, carrier or excipient.

5. The use according to claim 4, characterized in that: The sequence of the miRNA-211-5p inhibitor is: 5'-AGGCAAAGGAUGACAAAGGGAA-3'.

6. The use according to claim 4, characterized in that: The pharmaceutically acceptable carrier or excipient is selected from one or more of chitosan, cholesterol, liposomes and nanoparticles.

7. Use of a probe or primer for specifically detecting miRNA-211-5p in the preparation of a kit for diagnosing and / or evaluating the prognosis of bone defects in chronic osteomyelitis.

8. The use according to claim 7, characterized in that: The sequences of the primers are shown in SEQ ID NO.2 and SEQ ID NO.3.

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