Long-chain non-coding RNA and application thereof in regulating osteogenic differentiation of bone marrow mesenchymal stem cells

Abstract

The application discloses a long-chain non-coding RNA and application thereof in regulating osteogenic differentiation of bone marrow mesenchymal stem cells. The application finds that overexpression of a long-chain non-coding RNA (lncRNA Gm20628) with a nucleotide sequence as shown in SEQ ID NO. 1 in bone marrow mesenchymal stem cells can effectively promote osteogenic differentiation of the bone marrow mesenchymal stem cells, and further promote bone defect repair of a skull injury rat. Therefore, the application provides application of an expression promoter of the lncRNA Gm20628 in a preparation for enhancing bone regeneration, bone defect repair, and treating osteoporosis and bone formation disorder related diseases. The application is not only beneficial to treatment of diseases related to bone marrow mesenchymal stem cells, but also beneficial to construction of bone-like organs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of stem cell osteogenic differentiation. More particularly, it relates to a long non-coding RNA and its application in regulating the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). BACKGROUND

[0002] BMSCs have the potential for multi-directional differentiation, which can differentiate into various types of cells, including osteoblasts (OB), chondrocytes, adipocytes (AC), etc. The differentiation of BMSCs is regulated by various factors, especially the specific microenvironment in which it is located plays a decisive role in determining the fate of BMSCs. The BMSCs microenvironment is composed of extracellular matrix, various growth factors, cytokines and chemotactic factors, etc. These factors can activate or inhibit the differentiation of BMSCs, forming a fine-tuned network. Through this network, the differentiation of BMSCs in the osteogenic or adipogenic direction is strictly controlled, thereby maintaining the balance of bone and fat differentiation. When this balance is disturbed, especially in the case of excessive adipogenic differentiation, the osteogenic differentiation of BMSCs is inhibited, ultimately leading to the occurrence of bone metabolic related diseases such as osteoporosis and bone formation disorder related diseases. In addition to the potential for multi-directional differentiation, BMSCs also have low immunogenicity and are easy to isolate and culture, so they have become an important candidate cell for promoting bone regeneration, treating bone metabolic related diseases and bone formation disorder related diseases, etc.

[0003] Osteoporosis is a metabolic bone disease characterized by decreased bone density and damaged bone microstructure, its main features are insufficient bone formation and excessive bone resorption. BMSCs can increase bone density and improve the symptoms of osteoporosis by promoting the generation and function of OBs. Bone formation disorder related diseases are a group of diseases caused by genetic or acquired factors, which are characterized by abnormal bone development or bone metabolic disorders, mainly manifested as decreased bone density, increased bone fragility, skeletal deformity or growth retardation, including osteogenesis imperfecta (brittle bone disease), osteomalacia, rickets, fibrous dysplasia of bone, etc. Kannan Sridharan et al. found that transplantation of BMSCs can significantly improve the bone density and strength of osteogenesis imperfecta model animals and reduce the incidence of bone fractures. Osteopetrosis is a rare genetic bone disease, its main feature is increased bone density but fragile bone structure, prone to bone fractures. BMSCs can improve the symptoms of osteopetrosis and reduce the incidence of bone fractures by regulating the balance between bone resorption and bone formation. It can be seen that promoting the differentiation of BMSCs into OBs is beneficial to the treatment of diseases such as osteoporosis, and finding target points related to the osteogenic differentiation of BMSCs and developing new treatment strategies based on them have important clinical significance.

[0004] Long noncoding RNA (LncRNA) is a special non-coding RNA, usually composed of more than 200 nucleotides, and has no protein coding function. Although lncRNA is not involved in protein synthesis, it plays an important role in gene expression regulation, cell fate determination and regulation of tissue function. Studies have found that LncRNA-ORLNC1 acts as a competitive endogenous RNA (ceRNA) for miR-296, and miR-296 is significantly up-regulated during OB differentiation and promotes osteogenic differentiation by targeting Pten. In addition, lncRNA KCNQ1OT1 promotes osteogenic differentiation of mouse BMSCs by activating the Wnt / β-catenin pathway; lncRNA H19 can promote human MSCs differentiation by inhibiting the expression of transforming growth factor β1 (TGF-β1); and lncRNA AK016739 inhibits OB differentiation, and interference with lncRNA AK016739 can promote bone formation in ovariectomized osteoporotic mouse models. The above studies provide a solid theoretical basis for the importance of lncRNA in regulating osteogenic differentiation. However, most of the existing research on lncRNA is carried out in vitro, and the in vivo research on lncRNA regulating bone formation is relatively limited, which may not fully simulate the actual physiological function and pathological changes of BMSCs in vivo, and is not conducive to the development of bone regeneration drugs and the like. SUMMARY

[0005] The present application aims at the deficiencies in the prior art, and provides a long non-coding RNA, named lncRNA Gm20628, which can be used to regulate osteogenic differentiation of BMSCs.

[0006] The first object of the present application is to provide a long non-coding RNA.

[0007] The second object of the present application is to provide the use of a substance capable of regulating the expression amount of the RNA in regulating osteogenic differentiation of bone marrow mesenchymal stem cells.

[0008] The third object of the present application is to provide the use of a substance capable of regulating the expression amount of the RNA in the preparation of a preparation for regulating osteogenic differentiation of bone marrow mesenchymal stem cells.

[0009] The fourth object of the present application is to provide the use of an expression promoter of the RNA in the preparation of a preparation for enhancing bone regeneration.

[0010] The fifth object of the present application is to provide the use of an expression promoter of the RNA in the preparation of a preparation for bone defect repair.

[0011] A sixth object of the present application is to provide use of the expression promoter of the RNA in the manufacture of a medicament for preventing and / or treating osteoporosis.

[0012] A seventh object of the present application is to provide use of the expression promoter of the RNA in the manufacture of a medicament for treating a disease related to bone formation disorder.

[0013] An eighth object of the present application is to provide use of the expression promoter of the RNA in the manufacture of a medicament for improving symptoms of osteosclerosis.

[0014] A ninth object of the present application is to provide use of the expression promoter of the RNA in the construction of a bone organ or in the manufacture of a preparation for the construction of a bone organ.

[0015] The above objects of the present application are achieved by the following technical solutions.

[0016] The present application discloses a long non-coding RNA, named as lncRNA Gm20628, which nucleotide sequence is shown as SEQ ID NO. 1. - / - ) mice, it was found that ERα gene knockout (ERα - / - ) mice have sparse trabecular bone and present osteoporosis. By comparing and screening the results of transcriptome sequencing (RNA-seq) of wild type (WT) mice and ERα - / - ) mice and performing experiments related to BMSCs osteogenic differentiation, an lncRNA capable of regulating BMSCs osteogenic differentiation was found, named as lncRNA Gm20628, which nucleotide sequence is shown as SEQ ID NO. 1. On this basis, the present application found that a stem cell scaffold constructed by mBMSCs overexpressing the lncRNA Gm20628 can promote the repair of rat calvarial defects by constructing a rat calvarial defect model. That is, the present application confirms the key role of lncRNA Gm20628 in the bone formation process, and also confirms that lncRNA Gm20628 has great potential in promoting BMSCs osteogenic differentiation and enhancing bone regeneration, and can become a new prevention and treatment target for bone metabolism related diseases such as osteoporosis. Therefore, the present application claims protection for lncRNA Gm20628 and its application in regulating BMSCs osteogenic differentiation.

[0017] The present application provides a long non-coding RNA, named as lncRNA Gm20628, which nucleotide sequence is shown as SEQ ID NO. 1.

[0018] In view of the fact that regulating the expression amount of the lncRNA Gm20628 can regulate the osteogenic differentiation of mBMSCs, the present application claims protection for the application of substances capable of regulating the expression amount of the lncRNA Gm20628 in regulating the osteogenic differentiation of mBMSCs.

[0019] The application also claims the use of a substance capable of regulating the expression amount of the lncRNA Gm20628 in the preparation of a preparation for regulating the osteogenic differentiation of mBMSCs.

[0020] Specifically, the expression amount of the lncRNA Gm20628 is increased to promote the osteogenic differentiation of mBMSCs, and the expression amount of the lncRNA Gm20628 is reduced to inhibit the osteogenic differentiation of mBMSCs.

[0021] Alternatively, the substance capable of increasing the expression amount of the lncRNA Gm20628 is an overexpression plasmid of the lncRNA Gm20628, and the substance capable of reducing the expression amount of the lncRNA Gm20628 is a microRNA targeting the lncRNA Gm20628 based on the RNA interference technology.

[0022] In a specific embodiment of the application, the substance capable of reducing the expression amount of the lncRNA Gm20628 is an antisense oligonucleotide targeting the lncRNA Gm20628.

[0023] Alternatively, the antisense oligonucleotide is C*T*A*C*AGTATCTGGTGA*C*T*C*G, and the * in the sequence represents a phosphorothioation modification.

[0024] Alternatively, the antisense oligonucleotide is G*G*A*C*TTCAAGACGGAC*C*T*G*C, and the * in the sequence represents a phosphorothioation modification.

[0025] The application also claims the use of the expression promoter of the lncRNA Gm20628 in the preparation of a preparation for enhancing bone regeneration.

[0026] The application also claims the use of the expression promoter of the lncRNA Gm20628 in the preparation of a preparation for repairing bone defects.

[0027] The application also claims the use of the expression promoter of the lncRNA Gm20628 in the preparation of a drug for preventing and / or treating osteoporosis.

[0028] The application also claims the use of the expression promoter of the lncRNA Gm20628 in the preparation of a drug for treating bone formation disorder-related diseases.

[0029] The bone formation disorder-related disease is caused by inhibition of osteogenic differentiation ability of BMSCs, resulting in insufficient osteoblast generation. Therefore, the expression promoter of the lncRNA Gm20628 can be used to treat related diseases.

[0030] Specifically, the bone formation disorder-related disease is osteogenesis imperfecta.

[0031] The present application also protects the use of the expression promoter of the lncRNA Gm20628 in the preparation of a drug for improving the symptoms of osteosclerosis.

[0032] Specifically, the improvement is to increase bone density and bone strength, and reduce the incidence of bone fracture.

[0033] The present application also protects the use of the expression promoter of the lncRNA Gm20628 in the construction of bone organoids. Similarly, the present application also protects the use of the expression promoter of the lncRNA Gm20628 in the preparation of a preparation for constructing bone organoids.

[0034] Specifically, the expression promoter of the lncRNA Gm20628 promotes the construction of bone organoids by promoting the osteogenic differentiation of stem cells used in the construction of bone organoids.

[0035] Specifically, the expression promoter refers to a substance that can increase the activity and / or expression amount of the lncRNA Gm20628.

[0036] In a specific embodiment of the present application, the expression promoter is an overexpression plasmid of lncRNA Gm20628 or a recombinant lentivirus or recombinant cell containing the overexpression plasmid.

[0037] The present application also provides a drug for preventing and treating osteoporosis or treating bone defects, which contains the expression promoter of the lncRNA Gm20628.

[0038] The present application has the following beneficial effects:

[0039] The present application compares WT mice with ERα - / -The transcriptomic sequencing result of the mouse finds an lncRNA, named lncRNA Gm20628, the nucleotide sequence of which is shown as SEQ ID NO. 1. On this basis, the present application finds that increasing the expression amount of lncRNA Gm20628 can effectively promote the osteogenic differentiation of mBMSCs and further promote the bone defect repair of the rat with skull injury by overexpressing lncRNA Gm20628 in mouse bone marrow mesenchymal stem cells (mBMSCs). Therefore, the present application provides the application of the expression promoter of the lncRNA Gm20628 in the preparation for enhancing bone regeneration, bone defect repair and treating osteoporosis and bone formation disorder related diseases. The present application is not only beneficial to the treatment of diseases related to bone marrow mesenchymal stem cells, but also beneficial to the construction of bone-like organs. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Figure 1 is a volcano plot of 105 differentially expressed lncRNAs found by RNA sequencing and the Real-time PCR detection results of 5 lncRNAs selected from the differentially expressed lncRNAs; A in the figure is a volcano plot; B in the figure is the Real-time PCR detection result; **P<0.01; ***P<0.001; ****P<0.0001 in the figure.

[0041] Figure 2 Figure 2 is the nucleus / cytoplasm localization experiment of lncRNA Gm20628, the alkaline phosphatase (ALP) activity determination in lncRNA Gm20628 overexpression and knockdown cells, the qRT-PCR detection of lncRNA Gm20628 after mBMSCs are cultured in osteogenic induction medium for different times and the methylthiazol tetrazolium detection result of lncRNA Gm20628 overexpression cells; A in the figure is the nucleus / cytoplasm localization experiment result of lncRNA Gm20628; B in the figure is the ALP activity determination result in lncRNA Gm20628 overexpression and knockdown cells; C in the figure is the qRT-PCR detection result of lncRNA Gm20628 after mBMSCs are cultured in osteogenic induction medium for different times; D in the figure is the methylthiazol tetrazolium detection result of lncRNA Gm20628 overexpression cells; ***P<0.001; ****P<0.0001 in the figure.

[0042] Figure 3The results of the influence of overexpression or knockdown of lncRNA Gm20628 on the expression of osteogenesis-related factors ALP, bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX2), and alpha-1 collagen (COL1A1) in mBMSCs; Figure A is the results of Western blot analysis of the osteogenesis-related factors in lncRNA Gm20628 overexpression cells; Figure B is the relative expression of the osteogenesis-related factor proteins in lncRNA Gm20628 overexpression cells; Figure C is the relative expression of the osteogenesis-related factor mRNA in lncRNA Gm20628 knockdown cells; Figure D is the results of Western blot analysis of the osteogenesis-related factors in lncRNA Gm20628 knockdown cells; Figure E is the relative expression of the osteogenesis-related factor proteins in lncRNA Gm20628 knockdown cells; Figure F is the relative expression of the osteogenesis-related factor mRNA in lncRNA Gm20628 overexpression cells; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 in the figure.

[0043] Figure 4 The results of the influence of overexpression of lncRNA Gm20628 on in vivo bone defect repair; Figure A is a three-dimensional reconstruction of the skulls of rats in the lncRNA Gm20628 overexpression group and its corresponding control group; Figure B is the number of bone trabeculae and structural model index of rats in the lncRNA Gm20628 overexpression group and its corresponding control group; Figure C is the hematoxylin and eosin (H&E) staining scanning imaging results of rats in the lncRNA Gm20628 overexpression group and its corresponding control group; the left image is the control group, and the right image is the lncRNA Gm20628 overexpression group; Figure D is the Masson's trichrome staining scanning imaging results of rats in the lncRNA Gm20628 overexpression group and its corresponding control group; the left image is the control group, and the right image is the lncRNA Gm20628 overexpression group. DETAILED DESCRIPTION

[0044] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present application are conventional reagents, methods, and equipment in the technical field.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0046] All animal experiments in the present application are approved by the Animal Protection Committee of Jinan University and conducted in accordance with the Institutional Guidelines of the National Institutes of Health.

[0047] The nucleotide sequence of the lncRNA Gm20628 is shown as SEQ ID NO. 1.

[0048] Example 1 ERa - / - Detection of expression of differential lncRNA in mice

[0049] 1. Experimental method

[0050] (1) Experimental animals

[0051] The experimental animals used in the present application are C57BL / 6J (WT) and ERa - / - mice; ERa - / - The breeding of mice (heterozygous female, heterozygous male, Cat#026176) is from JACKSON laboratory, with C57BL / 6J as background.

[0052] (2) Detection of differential LncRNA by transcriptome sequencing (RNA-seq)

[0053] After the experimental mice (three-month-old female C57BL / 6J WT and ERa - / - ) were anesthetized, the two thighs were separated, the muscle and other tissues were carefully peeled off, the femur was reserved, and the bone surface residual tissues were gently wiped with gauze dipped in normal saline; to prevent degradation, the bone tissue was ground with liquid nitrogen and then RNA extraction was performed using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and total RNA of ERa - / - and WT mice was extracted according to the method provided by the reagent manufacturer; the concentration and purity of the extracted RNA were evaluated using Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA); the qualified RNA was used to construct a complementary deoxyribonucleic acid library from total RNA using Small RNA Sample Prep Kit (Illumina, San Diego, CA, USA); the integrity and size of the cDNA were detected using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA); finally, the qualified cDNA was sequenced on a single-end HiSeq Xten platform (Illumina, San Diego, CA, USA).

[0054] 2. Experimental results

[0055] After RNA sequencing, the present application found that the expression of lncRNA Gm20628 in ERa - / -A total of 105 differentially expressed lncRNAs were found in mice. Among them, 62 lncRNAs were up-regulated, and 43 lncRNAs were down-regulated. The volcano plot of the 105 differentially expressed lncRNAs found by RNA sequencing is shown in FIG. 1A. To verify the lncRNA sequencing results, the present application randomly selected 5 lncRNAs including lncRNA Gm20628 for Real-time PCR detection, and the results are shown in FIG. 1B. Through Real-time PCR detection, it is confirmed that the RNA sequencing results are reliable and that lncRNA Gm20628 is indeed significantly lower in expression in the ERa Figure 1 group than in the WT mice, and further experiments were performed on lncRNA Gm20628. Figure 1 - / - The expression of lncRNA Gm20628 in the femur of mice was indeed significantly lower than that in WT mice, and further experiments were performed on lncRNA Gm20628.

[0056] Example 2 Effect of lncRNA Gm20628 on BMSCs proliferation and differentiation

[0057] 1. Experimental method

[0058] The present application overexpressed lncRNA Gm20628 by constructing an overexpression plasmid, and inhibited its expression by designing an antisense oligonucleotide (ASO) targeting lncRNA Gm20628 to explore the effect of LncRNA Gm20628 on BMSCs proliferation and differentiation. The experiment was divided into 5 groups, namely overexpression control group (sh-NC), LncRNA Gm20628 overexpression group (sh-LncRNA Gm20628), knockdown control group (ASO-NC), knockdown group 1 (ASO1-LncRNA Gm20628) and knockdown group 2 (ASO2-LncRNA Gm20628).

[0059] (1) Cell culture

[0060] The experimental cells used in the present application were mBMSCs purchased from Procell Company, cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) medium supplemented with 15% fetal bovine serum (CellMax) at 37°C in a 5% CO2 environment; when the cell fusion reached 80-90%, the mBMSCs were passaged or cryopreserved with trypsin (Solarbio, Beijing, China), without adding EDTA; only cells from the 3rd to 8th passage were used for experiments.

[0061] (2) Cell transfection

[0062] ​The Shanghai Quanyang Biological Company was commissioned to construct the lncRNA Gm20628 overexpression plasmid and package the lentivirus. The obtained recombinant lentivirus containing the lncRNA Gm20628 overexpression plasmid (CMV-LncRNAGm20628-EF1-copGFP-T2A-Puromycin) was placed in complete culture medium and added to the mBMSCs at a fusion rate of 20-30% for culture (10 9 TU / mL, MOI=10) to construct the lncRNA Gm20628 overexpression group. The recombinant lentivirus containing the control empty plasmid (CMV-EF1-copGFP-T2A-Puromycin) was used to infect the mBMSCs in the same way as described above to construct the overexpression control group. After 16 h of culture, the supernatant was replaced with fresh complete culture medium, and after 72 h, the cells that were not successfully transfected were selected with 20 μg / mL puromycin (Beyotime) for 48 h, after which the cells were collected for RNA and protein extraction.

[0063] The ASO targeting the lncRNA Gm20628 (ASO-Gm20628) and the corresponding negative control were synthesized by RiboBio. The ASO-Gm20628 has two sequences, as shown below (the * in the sequence represents a phosphorothioate modification).

[0064] ASO1: C*T*A*C*AGTATCTGGTGA*C*T*C*G;

[0065] ASO2: G*G*A*C*TTCAAGACGGAC*C*T*G*C;

[0066] The ASO-Gm20628 and the corresponding negative control were dissolved in RNase-free water to a final concentration of 20 μM. 2 μL of the ASO-Gm20628 solution was mixed with 50 μL of Opti-MEM (Invitrogen, USA), and at the same time, 4 μL of Lipofectamine 3000 reagent (Invitrogen, USA) was diluted in 50 μL of Opti-MEM. After 5 min of incubation, the two solutions were mixed and incubated for another 30 min. Subsequently, the mBMSCs were seeded in each well of a 6-well plate at a density of 1.5 x 10 6 The ASO-Gm20628 / Lipofectamine mixture was added to each well, and after 48 h, the cells were collected for RNA and protein extraction.

[0067] (3) LncRNA localization

[0068] To locate the lncRNA Gm20628, the nuclear and cytoplasmic RNA of mBMSCs were isolated and extracted using a nuclear and cytoplasmic separation kit (BestBio), and the expression of lncRNA Gm20628 in the above two parts was detected by qRT-PCR. In the qRT-PCR detection, GAPDH and β-actin were used as cytoplasmic controls, and U6 and MALAT1 were used as nuclear controls. The primers used in the qRT-PCR detection are shown in Table 1, which were designed and synthesized by Shanghai Biotechnology Co., Ltd.

[0069] Table 1 qRT-PCR detection primers

[0070]

[0071]

[0072] (4) Alkaline phosphatase assay (ALP)

[0073] The mBMSCs overexpressing lncRNA Gm20628 (sh-LncRNA Gm20628) and the corresponding control group (sh-NC), and the mBMSCs knocking down lncRNA Gm20628 (ASO1-LncRNA Gm20628 and ASO2-LncRNA Gm20628) and the corresponding control group (ASO-NC) were inoculated into a 6-well plate at a density of 2×10 3 cells / well, and the ALP activity of each group of cells was detected using an ALP kit (A059-2-2, Nanjing Jianjian Biological Engineering Institute) according to the kit instructions, and the absorbance (OD) was measured at a wavelength of 520 nm using a multi-well spectrophotometer (BioTek, Synergy H4).

[0074] (5) Methylthiazol tetrazolium (MTT) test

[0075] The mBMSCs overexpressing lncRNA Gm20628 and the corresponding control group mBMSCs were inoculated into a 96-well plate at a density of 3000 cells / well, and incubated in serum-free medium at 37°C overnight in triplicate; 20 μL of MTT solution (5 mg / mL) (Beinmei) was added to each well, and after incubation for 4 h, the supernatant was carefully discarded; the OD of each well was measured at a wavelength of 490 nm using a microplate reader (Tecan Sunrise, Hombrechtikon, Switzerland).

[0076] (6) RNA extraction and qRT-PCR

[0077] The mBMSCs were cultured in osteogenic induction medium (Guangzhou Peiyu Bioproducts Co., Ltd., Catalog No. AAPR99-F500), and the cells were collected at 0, 3, 7, 10 and 14 days of culture, respectively. The RNA was extracted from the cells using Trizol at 4°C, and the extracted RNA was reverse transcribed into cDNA. Then, qRT-PCR analysis was performed using ABI7500, and the expression of lncRNA Gm20628 was detected by qRT-PCR in triplicate, with GAPDH as an internal reference.

[0078] In addition, the RNA of lncRNA Gm20628 overexpression cells and knockdown cells cultured for 72 h was extracted, respectively. After reverse transcription of the extracted RNA into cDNA, qRT-PCR analysis was performed using ABI7500 fluorescence quantitative PCR instrument (the primers used are shown in Table 1), and the expression of osteogenic differentiation related factors was detected by qRT-PCR in triplicate, with GAPDH as an internal reference. The reaction system was as follows: qPCR Green Master Mix 5 μL, PCR Forward Primer 0.2 μL, PCR Reverse Primer 0.2 μL, template 0.2 μL, DEPC water 4.4 μL; the cycle conditions were as follows: 95°C for 2 min, then 95°C for 15 s and 60°C for 30 s for 40 cycles.

[0079] (7) Western Blotting analysis

[0080] LncRNA Gm20628 overexpression and knockdown cells cultured for 48 h were lysed with RIPA lysis buffer (Shanghai Biodyne Biotech Co., Ltd.) containing 10% PMSF for 15 min, centrifuged at 4°C, 12,500 rpm for 15 min; after removing the supernatant, the protein concentration of each sample was determined using a BCA protein detection kit (Invitrogen, Carlsbad, CA, USA); equal amounts of protein (10-20 μg) in each sample were loaded and separated on a 10% glycine SDS-PAGE, and the separated proteins were electrotransferred to a 0.2 μm polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA) at a constant current of 300 mA; after blocking with 5% (w / v) skim milk in 0.1% Tween 20-containing Tris-buffered saline (TBST) at room temperature for 60 min, the following antibodies were diluted 1:1000 and incubated at 4°C overnight; antibodies: GAPDH (5174S, CST), ALP (PA5-106391.Thermo Fishe), BMP-2 (70583S, CST), RUNX2 (12556S, CST), COL1A1 (91144S, CST); incubation was carried out in Quick Block TM primary antibody dilution buffer for Western Blot (Shanghai Biodyne Biotech Co., Ltd.), and then the membrane was incubated with a secondary antibody appropriately conjugated with horseradish peroxidase; the signal on the membrane was detected using a Bio-Rad Gel Doc XR system (USA Hercules, CA), and the reaction bands were quantified and analyzed using Image J software (version 1.52a). The protein expression level was normalized to GAPDH.

[0081] 2. Experimental results

[0082] The results of the nuclear / cytoplasmic localization experiment of LncRNA Gm20628 are shown in Figure 2 A. From the figure, it can be seen that lncRNA Gm20628 is mainly expressed in the nucleus of mBMSCs cells.

[0083] The results of ALP activity determination in LncRNA Gm20628 overexpression and knockdown cells are shown in Figure 2 B. From the figure, it can be seen that overexpression of lncRNA Gm20628 can significantly promote the osteogenic differentiation of mBMSCs, while knockdown of lncRNA Gm20628 can inhibit this process.

[0084] After mBMSCs were cultured in osteogenic induction medium for different times, the qRT-PCR detection results of lncRNA Gm20628 are shown in Figure 2Figure 6 shows the expression of lncRNA Gm20628 in mBMSCs. Figure 7 shows the effect of lncRNA Gm20628 on the proliferation of mBMSCs. Figure 8 shows the effect of lncRNA Gm20628 on the expression of osteogenic-related factors in mBMSCs. Figure 9 shows the effect of lncRNA Gm20628 on the repair of bone defects in vivo.

[0085] Figure 10 shows the results of MTT detection of lncRNA Gm20628 overexpression cells. Figure 2 Figure 11 shows the effect of lncRNA Gm20628 on the proliferation of mBMSCs. Figure 12 shows the effect of lncRNA Gm20628 on the expression of osteogenic-related factors in mBMSCs. Figure 13 shows the effect of lncRNA Gm20628 on the repair of bone defects in vivo.

[0086] Figure 14 shows the effect of lncRNA Gm20628 on the expression of osteogenic-related factors in mBMSCs. Figure 3 Figure 15 shows the effect of lncRNA Gm20628 on the expression of osteogenic-related factors in mBMSCs. Figure 3 Figure 16 shows the effect of lncRNA Gm20628 on the expression of osteogenic-related factors in mBMSCs. Figure 3 Figure 17 shows the effect of lncRNA Gm20628 on the expression of osteogenic-related factors in mBMSCs. Figure 3 Figure 18 shows the effect of lncRNA Gm20628 on the expression of osteogenic-related factors in mBMSCs.

[0087] Example 3 Effect of lncRNA Gm20628 overexpression on the repair of bone defects in vivo

[0088] 1. Skull defect experiment

[0089] The experimental animals used in this example were 10 female Sprague Dawley rats (body weight 180-220 g) (Bio-science CO, Beijing, China), which were divided into two groups for the experiment, namely the lncRNA Gm20628 overexpression group (sh-Gm20628) and its corresponding control group (sh-NC).

[0090] After complete anesthesia by intraperitoneal injection of 10% chloral hydrate (0.3 mL / 100 g) in rats, the rats were placed in a prone position on the operating table; after shaving and skin disinfection, a median incision of about 2 cm was made; after separating the soft tissue to expose the sagittal suture, a critical bone defect with a diameter of about 4 mm and a depth of about 1 mm was drilled on both sides of the skull using a high-speed skull drill, while paying attention to keep the dura intact. The lncRNA Gm20628 overexpression group and its corresponding control group mBMSCs were respectively injected with 1.0 x 10 7The scaffolds were resuspended at a concentration of [number] cells / mL and inoculated into gelatin (3.5 mm in diameter) at a volume of 200 μL per scaffold. These scaffolds were then implanted into the skull defects of rats, and the skin was sutured with 4-0 sutures. To prevent wound infection, rats were injected intramuscularly with 200,000 units of penicillin within 3 days post-surgery, and then fed under standard conditions. Six weeks after surgery, all rats were euthanized by intraperitoneal injection of a lethal dose of chloral hydrate; skull specimens were collected, fixed in 4% paraformaldehyde (Solarbio, China) for 48 h at room temperature, and then stored in PBS for Micro-CT scanning (Bruker SkyScan 1276, Belgium) and histological analysis.

[0091] 2. Experimental Results

[0092] Three-dimensional reconstruction images of the skulls of rats in the lncRNA Gm20628 overexpression group and their corresponding control group are shown below. Figure 4 As shown in Figure A, bone regeneration in the control group rats was limited, mainly at the defect edges, with no bone bridges forming throughout the defect area. In contrast, the group overexpressing lncRNA Gm20628 showed significantly better bone formation and integration, almost completely filling the effector zone.

[0093] The number of trabeculae (Tb.N) and structural model index (SMI) of rats in the lncRNA Gm20628 overexpression group and its corresponding control group are as follows: Figure 4 As shown in Figure B, the Tb.N of the lncRNA Gm20628 overexpression group was 1.4 times that of the control group (P = 0.039). Furthermore, the SMI of the lncRNA Gm20628 overexpression group was significantly lower than that of the control group (P = 0.021) (2.01 ± 0.16 vs 2.59 ± 0.22), indicating that the lncRNA Gm20628 group formed more trabeculae with a layered structure.

[0094] To understand the specific recovery status, this invention also performed histological analyses such as H&E and Masson trichrome staining, and scanned the results.

[0095] The H&E staining and imaging results of rats in the lncRNA Gm20628 overexpression group and its corresponding control group are as follows: Figure 4The left image in the figure shows the H&E staining scan results of rats in the control group, while the right image shows the H&E staining scan results of rats in the lncRNA Gm20628 overexpression group. In the control group, fibrous connective soft tissue (ST) formed at the defect site, but very limited regenerated bone tissue (RT) was observed at its edge. In contrast, in the lncRNA Gm20628 overexpression group, there was denser RT at both the edge and center of the defect.

[0096] Masson's trichrome staining imaging results of rats in the lncRNA Gm20628 overexpression group and its corresponding control group are as follows: Figure 4 As shown in Figure D; the left image shows the Masson trichrome staining imaging results of the control group rats, and the right image shows the Masson trichrome staining imaging results of the lncRNA Gm20628 overexpression group rats. Consistent with the H&E scanning results, the Masson trichrome staining results show that in the control group, immature bone formation was mainly observed in the center of the defect (marked with triangles), while mature bone formation was rarely observed at the edge of the defect (marked with stars); unlike the control group, the calcium defects in the lncRNA Gm20628 overexpression group were almost filled with mature bone formation.

[0097] The above results indicate that overexpression of lncRNA Gm20628 can significantly promote osteogenic differentiation and bone formation of BMSCs, thereby promoting bone defect repair, and can provide new targets and treatment options for osteoporosis and bone injury-related diseases.

[0098] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of long non-coding RNA lncRNA Gm20628 in the preparation of formulations for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, characterized in that, The nucleotide sequence of the long non-coding RNA is shown in SEQ ID NO.

1.

2. The application of long non-coding RNA lncRNA Gm20628 in the preparation of formulations for enhancing bone regeneration, characterized in that, The nucleotide sequence of the long non-coding RNA is shown in SEQ ID NO.

1.

3. The application of long non-coding RNA lncRNA Gm20628 in the preparation of formulations for bone defect repair, characterized in that, The nucleotide sequence of the long non-coding RNA is shown in SEQ ID NO.

1.

4. The use of long non-coding RNA lncRNA Gm20628 in the construction of bone organoids or in the preparation of formulations for the construction of bone organoids, characterized in that, The nucleotide sequence of the long non-coding RNA is shown in SEQ ID NO.1.

Images