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

Abstract

The invention discloses a long-chain non-coding RNA (Ribonucleic Acid) and application thereof in regulating osteogenic differentiation of bone marrow mesenchymal stem cells. It is found that overexpression of long-chain non-coding RNA (lncRNA Gm20628) with the nucleotide sequence 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 then bone defect repair of a mouse with skull injury is promoted. Therefore, the invention provides application of the expression promoter of the lncRNA Gm20628 in preparation for enhancing bone regeneration, repairing bone defects and treating osteoporosis and bone formation disorder related diseases. The method is not only beneficial to treatment of diseases related to the bone marrow mesenchymal stem cells, but also beneficial to construction of bone organs.

Description

Technical Field

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

[0002] BMSCs have multi-directional differentiation potential and can differentiate into various types of cells, including osteoblasts (OBs), chondrocytes, adipocytes (ACs), etc. The differentiation of BMSCs is regulated by multiple factors, especially the specific microenvironment in which they are located plays a decisive role in determining the fate of BMSCs. The BMSCs microenvironment consists of extracellular matrix, various growth factors, cytokines, and chemokines, etc. These factors can activate or inhibit the differentiation of BMSCs, forming a finely regulated network. Through this network, the differentiation of BMSCs in the osteogenic or adipogenic direction is strictly controlled, thus maintaining the balance of osteogenic and adipogenic differentiation. When this balance is disrupted, especially in the case of excessive adipogenic differentiation, the osteogenic differentiation of BMSCs is inhibited, ultimately leading to the occurrence of bone metabolism-related diseases such as osteoporosis and bone formation disorder-related diseases. In addition to having multi-directional differentiation potential, BMSCs also have the characteristics of low immunogenicity and easy separation and culture, so they have also become important candidate cells for promoting bone regeneration and treating bone metabolism-related diseases and bone formation disorder-related diseases, etc.

[0003] Osteoporosis is a metabolic bone disease characterized by reduced bone density and damaged bone microstructure. Its main characteristics are insufficient bone formation and excessive bone resorption. BMSCs can improve the symptoms of osteoporosis by promoting OB generation and function and increasing bone density. Bone formation disorder-related diseases are a class of diseases caused by genetic or acquired factors, characterized by abnormal bone development or bone metabolism disorders, mainly manifested as reduced bone density, increased bone fragility, skeletal deformities or growth retardation, including osteogenesis imperfecta (brittle bone disease), osteomalacia, rickets, fibrous dysplasia of bone, etc.; Kannan Sridharan et al. found that transplanting BMSCs can significantly increase the bone density and bone strength of animals with osteogenesis imperfecta models and reduce the fracture incidence. Osteopetrosis is a rare genetic bone disease, mainly characterized by increased bone density but fragile bone structure and easy fracture. BMSCs can improve the symptoms of osteopetrosis and reduce the fracture incidence 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. Finding targets related to the osteogenic differentiation of BMSCs and developing new treatment strategies based on them have important clinical significance.

[0004] Long non-coding RNAs (LncRNAs) are a special class of non-coding RNAs, usually composed of more than 200 nucleotides, and do not have protein-coding functions. Although lncRNAs do not participate in protein synthesis, they play important roles in gene expression regulation, cell fate determination, and tissue function regulation. Some studies have found that LncRNA-ORLNC1 acts as a competing endogenous RNA (ceRNA) for miR-296, and miR-296 is significantly upregulated 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 the differentiation of human MSCs by inhibiting the expression of transforming growth factor β1 (TGF-β1); while lncRNA AK016739 inhibits OB differentiation, and interfering with lncRNA AK016739 can promote bone formation in ovariectomized osteoporosis model mice. The above studies provide a solid theoretical basis for the importance of lncRNAs in regulating osteogenic differentiation. However, most of the existing studies on lncRNAs are carried out in vitro, and the in vivo studies on lncRNA-regulated bone formation are relatively limited, which may not fully simulate the actual physiological functions and pathological changes of BMSCs in vivo and is not conducive to the development of bone regeneration drugs and the like. Summary of the Invention

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

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

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

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

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

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

[0011] The sixth object of the present invention is to provide the use of the RNA expression promoter in the preparation of a drug for preventing and / or treating osteoporosis.

[0012] The seventh object of the present invention is to provide the use of the RNA expression promoter in the preparation of a drug for treating bone formation disorder-related diseases.

[0013] The eighth object of the present invention is to provide the use of the RNA expression promoter in the preparation of a drug for improving the symptoms of osteopetrosis.

[0014] The ninth object of the present invention is to provide the use of the RNA expression promoter in constructing bone organoids or in the preparation of a preparation for constructing bone organoids.

[0015] The above objects of the present invention are achieved by the following technical solutions:

[0016] When studying the effect of Estrogen Receptor-α (ERα) on bone metabolism, the present invention found that trabecular bone rarefaction occurred in ERα gene knockout (ERα - / - ) mice, presenting an osteoporosis state. By comparing and screening the transcriptomic sequencing (RNA-seq) results of wild-type (WT) mice and ERα - / - mice and conducting experiments related to the osteogenic differentiation of BMSCs, a lncRNA capable of regulating the osteogenic differentiation of BMSCs was discovered, named lncRNAGm20628, and its nucleotide sequence is shown in SEQ ID NO.1. On this basis, by constructing a mouse cranial defect model, the present invention found that a stem cell scaffold constructed with mBMSCs overexpressing the lncRNA Gm20628 can promote the repair of mouse cranial defects. That is, the present invention confirmed the key role of lncRNAGm20628 in the bone formation process, and also confirmed that lncRNA Gm20628 has great potential in promoting the osteogenic differentiation of BMSCs and enhancing bone regeneration, and can become a new prevention and treatment target for bone metabolism-related diseases such as osteoporosis. Therefore, the present invention claims protection for lncRNA Gm20628 and its application in regulating the osteogenic differentiation of BMSCs.

[0017] The present invention provides a long non-coding RNA, named lncRNA Gm20628, and its nucleotide sequence is shown in SEQ ID NO.1.

[0018] In view of the fact that regulating the expression level of the lncRNA Gm20628 can regulate the osteogenic differentiation of mBMSCs. Therefore, the present invention claims protection for the use of a substance capable of regulating the expression level of the lncRNA Gm20628 in regulating the osteogenic differentiation of mBMSCs.

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

[0020] Specifically, when the expression level of the lncRNA Gm20628 increases, it promotes the osteogenic differentiation of mBMSCs; when the expression level of the lncRNA Gm20628 decreases, it inhibits the osteogenic differentiation of mBMSCs.

[0021] Optionally, the substance capable of increasing the expression level of the lncRNA Gm20628 is an overexpression plasmid of the lncRNA Gm20628; the substance capable of decreasing the expression level of the lncRNA Gm20628 is a microRNA targeting the lncRNA Gm20628 designed based on RNA interference technology, etc.

[0022] In a specific embodiment of the present invention, the substance capable of decreasing the expression level of the lncRNA Gm20628 is an antisense oligonucleotide targeting the lncRNA Gm20628.

[0023] Optionally, the antisense oligonucleotide is C*T*A*C*AGTATCTGGTGA*C*T*C*G, where * in the sequence represents phosphorothioate modification.

[0024] Optionally, the antisense oligonucleotide is G*G*A*C*TTCAAGACGGAC*C*T*G*C, where * in the sequence represents phosphorothioate modification.

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

[0026] The present invention also claims the use of an expression promoter of the lncRNA Gm20628 in the preparation of a preparation for bone defect repair.

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

[0028] The present invention also claims the use of an 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 the inhibition of the osteogenic differentiation ability of BMSCs, resulting in insufficient generation of osteoblasts. Since the expression promoter of lncRNA Gm20628 can promote the osteogenic differentiation of BMSCs, it can be used to treat related diseases.

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

[0031] The present invention also claims the application of the expression promoter of lncRNA Gm20628 in the preparation of a drug for improving the symptoms of osteopetrosis.

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

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

[0034] Specifically, the expression promoter of 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 of lncRNA Gm20628 and / or increase its expression level.

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

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

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

[0039] The present invention compares WT mice with ERα - / -Transcriptomic sequencing results of mice revealed a lncRNA named lncRNA Gm20628, whose nucleotide sequence is shown in SEQ ID NO.1. On this basis, the present invention found that overexpressing lncRNA Gm20628 in mouse bone marrow mesenchymal stem cells (mBMSCs) can effectively promote the osteogenic differentiation of mBMSCs, and further promote the bone defect repair in mice with skull injuries. Therefore, the present invention 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 invention is not only beneficial to the treatment of diseases related to bone marrow mesenchymal stem cells, but also beneficial to the construction of bone organoids. Description of the Drawings

[0040] Figure 1 Volcano plot of 105 differentially expressed lncRNAs discovered by RNA sequencing and Real-time PCR detection results of 5 lncRNAs selected from the differentially expressed lncRNAs; A in the figure is the 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 Results of nuclear / cytoplasmic localization experiment of lncRNA Gm20628, determination of alkaline phosphatase (ALP) activity in lncRNA Gm20628 overexpressing and knockdown cells, qRT-PCR detection of lncRNA Gm20628 after mBMSCs were cultured in osteogenic induction medium for different times, and methyl thiazolyl tetrazolium detection results of lncRNA Gm20628 overexpressing cells; A in the figure is the result of nuclear / cytoplasmic localization experiment of lncRNA Gm20628; B in the figure is the result of determination of ALP activity in lncRNA Gm20628 overexpressing and knockdown cells; C in the figure is the qRT-PCR detection result of lncRNA Gm20628 after mBMSCs were induced and cultured in osteogenic induction medium for different times; D in the figure is the methyl thiazolyl tetrazolium detection result of lncRNA Gm20628 overexpressing cells; ***P<0.001; ****P<0.0001 in the figure.

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

[0043] Figure 4 The results of the effects of overexpressing lncRNA Gm20628 on bone defect repair in vivo; A in the figure is the three-dimensional reconstruction images of the skulls of mice in the group overexpressing lncRNA Gm20628 and its corresponding control group; B in the figure is the trabecular bone number and structural model index of mice in the group overexpressing lncRNA Gm20628 and its corresponding control group; C in the figure is the scanning imaging results of hematoxylin and eosin (H&E) staining of mice in the group overexpressing lncRNA Gm20628 and its corresponding control group; the left figure in the figure is the control group, and the right figure is the group overexpressing lncRNA Gm20628; D in the figure is the scanning imaging results of Masson trichrome staining of mice in the group overexpressing lncRNA Gm20628 and its corresponding control group; the left figure in the figure is the control group, and the right figure is the group overexpressing lncRNA Gm20628. Detailed implementation manners

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

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

[0046] All animal experiments in the present invention have been approved by the Animal Protection Committee of Jinan University and are carried out in accordance with the institutional guidelines of the National Institutes of Health of the United States.

[0047] The nucleotide sequence of lncRNA Gm20628 in the embodiments of the present invention is shown in SEQ ID NO.1. Example 1 ERα - / - Detection of the expression of differential lncRNAs in mice

[0048] 1. Experimental method

[0049] (1) Experimental animals

[0050] The experimental animals used in the present invention are C57BL / 6J (WT) and ERα - / - mice; ERα - / - breeding pairs of mice (heterozygous females, heterozygous males, Cat#026176) are from JACKSON Laboratory and are based on C57BL / 6J.

[0051] (2) Detection of differential LncRNAs by transcriptome sequencing (RNA-seq)

[0052] After anesthetizing the experimental mice (three-month-old female C57BL / 6J WT and ERα - / - ), separate the two thighs, carefully strip the muscle and other tissues, retain the femur, and gently wipe the residual tissue on the bone surface with a gauze dipped in saline; to prevent degradation, grind the bone tissue with liquid nitrogen and then use Trizol reagent (Invitrogen, Carlsbad, CA, USA) for RNA extraction, and extract the total RNA of the femurs of ERα - / - and WT mice according to the method provided by the reagent manufacturer; use Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA) to evaluate the concentration and purity of the extracted RNA; the qualified RNA is used to construct a complementary deoxyribonucleic acid library from the total RNA using the Small RNA Sample Prep Kit (Illumina, San Diego, CA, USA); use an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) to detect the integrity and size of the cDNA; finally, sequence the qualified cDNA on a single-end HiSeq Xten platform (Illumina, San Diego, CA, USA).

[0053] 2. Experimental results

[0054] By RNA sequencing, the present invention is in ERα - / -105 differentially expressed lncRNAs were found in mice. Among them, 62 lncRNAs were upregulated and 43 lncRNAs were downregulated. The volcano plot of the 105 differentially expressed lncRNAs discovered by RNA sequencing is shown as Figure 1 A in Figure 1 . To verify the lncRNA sequencing results, the present invention randomly selected 5 lncRNAs including lncRNA Gm20628 for Real-time PCR detection, and the results are shown as Figure 1 B in

[0055] . Through Real-time PCR detection, it was confirmed that the RNA sequencing results were reliable and the expression of lncRNA Gm20628 in the femurs of ERα - / - mice was indeed significantly lower than that in WT mice, and further experiments on lncRNA Gm20628 were carried out subsequently.

[0055] Example 2 Effects of LncRNA Gm20628 on the proliferation and differentiation of BMSCs

[0056] 1. Experimental methods

[0057] The present invention overexpressed lncRNAGm20628 by constructing an overexpression plasmid, and inhibited its expression by designing antisense oligonucleotides (ASO) targeting lncRNA Gm20628 to explore the effects of LncRNA Gm20628 on the proliferation and differentiation of BMSCs. The experiment was divided into 5 groups, namely the overexpression control group (sh-NC), the LncRNA Gm20628 overexpression group (sh-LncRNA Gm20628), the knockdown control group (ASO-NC), the knockdown group 1 (ASO1-LncRNA Gm20628), and the knockdown group 2 (ASO2-LncRNA Gm20628).

[0058] (1) Cell culture

[0059] The experimental cells used in the present invention were mBMSCs, purchased from Procell, and cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) medium supplemented with 15% fetal bovine serum (CellMax) in an environment of 37°C and 5% CO2; when the cell confluence reached 80-90%, mBMSCs were passaged or cryopreserved with trypsin (Solarbio, Beijing, China) without adding EDTA; only the 3rd to 8th generation cells were used for the experiment.

[0060] (2) Cell transfection

[0061] Entrusted Shanghai Quanyang Biotechnology Company to complete the construction of the overexpression plasmid of lncRNA Gm20628 and the packaging of lentivirus. The obtained recombinant lentivirus containing the overexpression plasmid of lncRNA Gm20628 (CMV-LncRNAGm20628-EF1-copGFP-T2A-Puromycin) was placed in complete medium and added to mBMSCs at a fusion rate of 20-30% for culture (10 9 TU / mL, MOI = 10), and the overexpression group of lncRNA Gm20628 was constructed; in the same method as above, the recombinant lentivirus containing the control empty plasmid (CMV-EF1-copGFP-T2A-Puromycin) was used to infect mBMSCs, and the overexpression control group was constructed; after culturing the two groups of cells for 16 h, the supernatant was replaced with fresh complete medium for continued culture. After 72 h, the cells that were not successfully transfected were screened with 20 μg / mL puromycin (BioTianmei), and the cells were collected after 48 h for RNA and protein extraction.

[0062] The ASO (ASO-Gm20628) targeting lncRNA Gm20628 and the corresponding negative control were synthesized by RiboBio; among them, ASO-Gm20628 has two sequences as follows (the * in the sequence represents phosphorothioate modification);

[0063] ASO1: C*T*A*C*AGTATCTGGTGA*C*T*C*G;

[0064] ASO2: G*G*A*C*TTCAAGACGGAC*C*T*G*C;

[0065] Dissolve ASO-Gm20628 and the corresponding negative control in RNase-free water respectively to make their final concentration 20 μM; take 2 μL of ASO-Gm20628 solution and mix it with 50 μL of Opti-MEM (Invitrogen, USA). At the same time, dilute 4 μL of Lipofectamine 3000 reagent (Invitrogen, USA) in 50 μL of Opti-MEM; after incubating for 5 min, mix the two solutions and continue to incubate for 30 min; then, inoculate mBMSCs into each well of a 6-well plate at a density of 1.5×10 6 cells / well and culture them in DMEM basal medium; add the ASO-Gm20628 / Lipofectamine mixture to each well, and collect the cells after 48 h for RNA and protein extraction.

[0066] (3) Localization of LncRNA

[0067] To localize lncRNA Gm20628, a nuclear and cytoplasmic separation kit (BestBio) was used to separate and extract the nuclear and cytoplasmic RNAs of mBMSCs, 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 for qRT-PCR detection are shown in Table 1, and the primers were designed and synthesized by Shanghai Sangon Biotech Co., Ltd.

[0068] Table 1 Primers for qRT-PCR detection

[0069]

[0070]

[0071] (4) Alkaline phosphatase assay (ALP)

[0072] mBMSCs overexpressing lncRNA Gm20628 (sh-LncRNAGm20628) and the corresponding control group (sh-NC), as well as mBMSCs with knockdown of lncRNA Gm20628 (ASO1-LncRNA Gm20628 and ASO2-LncRNA Gm20628) and the corresponding control group (ASO-NC) were seeded into 6-well plates at a density of 2×10 3 cells / well. According to the kit instructions, an ALP kit (A059-2-2, Nanjing Jianjian Bioengineering Research Institute) was used to complete the ALP activity detection of each group of cells, and a multi-well spectrophotometer (BioTek, Synergy H4) was used to measure the absorbance (OD) at a wavelength of 520 nm.

[0073] (5) 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

[0074] mBMSCs overexpressing lncRNA Gm20628 and the corresponding control group mBMSCs were seeded into 96-well plates at a density of 3000 cells / well and cultured overnight at 37 °C in serum-free medium, in triplicate; 20 μL of MTT solution (5 mg / mL) (Beingmate) was added to each well, and after 4 h of culture, the supernatant was carefully discarded; a microplate reader (Tecan Sunrise, Hombrechtikon, Switzerland) was used to measure the OD of each well at a wavelength of 490 nm.

[0075] (6) RNA extraction and qRT-PCR

[0076] The mBMSCs were cultured in osteogenic induction medium (Guangzhou Peiyu Biological Products Co., Ltd., product number AAPR99-F500). Cells were collected at 0, 3, 7, 10, and 14 days of culture respectively. RNA was extracted using Trizol at 4°C. After reverse transcribing the extracted RNA into cDNA, qRT-PCR analysis was performed using ABI7500 in triplicate. The expression of lncRNA Gm20628 was detected by qRT-PCR, and GAPDH was used as an internal reference.

[0077] In addition, RNA was extracted from lncRNA Gm20628 overexpressing cells and knockdown cells cultured for 72 h respectively. After reverse transcribing the extracted RNA into cDNA, qRT-PCR analysis was performed using an ABI7500 fluorescence quantitative PCR instrument (the primers used are shown in Table 1) in triplicate. The expression of osteogenic differentiation-related factors was detected by qRT-PCR, and GAPDH was used as an internal reference. The reaction system was as follows: qPCR 5 μL of Green Master Mix, 0.2 μL of PCR Forward Primer, 0.2 μL of PCR Reverse Primer, 0.2 μL of template, and 4.4 μL of DEPC water; The cycling conditions were: 95°C for 2 min, then 40 cycles of 95°C for 15 s and 60°C for 30 s.

[0078] (7) Western Blotting analysis

[0079] LncRNA Gm20628 overexpressing cells and knockdown cells cultured for 48 h were lysed with RIPA lysis buffer containing 10% PMSF (Shanghai Baiyoutian Biotechnology Co., Ltd.) for 15 min, and centrifuged at 12,500 rpm for 15 min at 4°C; after removing the supernatant, the protein concentration of each sample was determined using a BCA protein assay kit (Invitrogen, Carlsbad, CA, USA); an equal amount of protein (10 - 20 μg) from each sample was loaded and separated on 10% glycine SDS-PAGE, and the separated proteins were electrotransferred to a 0.2 μm polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA) at a constant current of 300 mA; after blocking with 5% (w / v) non-fat milk in tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 60 min, the membrane was incubated overnight at 4°C with the following antibodies diluted 1:1000; antibodies: GAPDH (5174S, CST), ALP (PA5-106391, Thermo Fishe), BMP-2 (70583S, CST), RUNX2 (12556S, CST), COL1A1 (91144S, CST); the incubation was carried out in Quick Block TM primary antibody dilution buffer for Western Blot (Shanghai Baiyoutian Biotechnology Co., Ltd.), and then the membrane was incubated with a secondary antibody appropriately conjugated to horseradish peroxidase; the signals on the membrane were detected using a Bio-Rad Gel Doc XR system (Hercules, California, USA), and the reaction bands were quantified and analyzed using Image J software (version 1.52a). The protein expression levels were normalized to the GAPDH control.

[0080] 2. Experimental Results

[0081] The results of the nuclear / cytoplasmic localization experiment of LncRNA Gm20628 are shown in Figure 2 A in the figure. It can be seen from this figure that lncRNA Gm20628 is mainly expressed in the nuclei of mBMSCs.

[0082] The results of the ALP activity assay in LncRNA Gm20628 overexpressing and knockdown cells are shown in Figure 2 B in the figure. It can be seen from this figure that overexpression of lncRNA Gm20628 can significantly promote the osteogenic differentiation of mBMSCs, while knockdown of lncRNA Gm20628 inhibits this process.

[0083] After mBMSCs were cultured in osteogenic induction medium for different times, the qRT-PCR detection results of lncRNA Gm20628 are shown in Figure 2As shown in C of the figure. It can be seen from this figure that as the induction culture time increases, the expression level of lncRNA Gm20628 rises, but starting from the 10th day, the expression level of lncRNA Gm20628 gradually decreases, indicating that it plays an important role in bone formation.

[0084] The MTT detection results of lncRNA Gm20628 overexpressing cells are as Figure 2 shown in D of the figure. It can be seen from this figure that the OD value of the lncRNA Gm20628 overexpressing cell group increased significantly, indicating that the overexpression of lncRNA Gm20628 promoted the proliferation of mBMSCs. The above results indicate that overexpressing lncRNA Gm20628 can promote the proliferation of BMSCs cells and their osteogenic differentiation.

[0085] The results of the effects of overexpressing or knocking down lncRNA Gm20628 on the expression of osteogenesis-related factors ALP, BMP-2, RUNX2, and COL1A1 in mBMSCs are as Figure 3 shown. From Figure 3 it can be seen that after overexpressing lncRNA Gm20628, the mRNA and protein expression levels of osteogenesis-related factors ALP, BMP-2, RUNX2, and COL1A1 increased significantly ( Figure 3 in A, B, and F of the figure). The results of knocking down lncRNA Gm20628 showed that the expression of osteogenesis-related factors decreased significantly ( Figure 3 in C - E of the figure).

[0086] Example 3 Effects of overexpressing lncRNA Gm20628 on bone defect repair in vivo

[0087] 1. Skull defect experiment

[0088] The experimental animals used in this example were 10 female Sprague Dawley rats (weight 180 - 220 g) (Bio-science CO, Beijing, China). They were divided into two groups for experiments, namely the lncRNA Gm20628 overexpressing group (sh-Gm20628) and its corresponding control group (sh-NC).

[0089] After the rats were completely anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3 mL / 100 g), they were lying on the operating table in the prone position; after shaving and skin disinfection, a median incision of about 2 cm was made; after separating the soft tissues 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 with a high-speed skull drill, while paying attention to keeping the dura mater intact. The mBMSCs of the lncRNA Gm20628 overexpressing group and its corresponding control group were respectively at 1.0×10 7Resuspended at a concentration of cells / mL, and inoculated into gelatin (diameter 3.5 mm) at a volume of 200 μL per scaffold; subsequently, these scaffolds were implanted into the rat skull defect area for transplantation, implanted at the skull defect, and then the skin was sutured with 4-0 sutures. To prevent wound infection, 200,000 units of penicillin were intramuscularly injected within 3 days after surgery, and then the rats were fed under conventional conditions. Six weeks after the surgery, all rats were sacrificed by intraperitoneal injection of a lethal dose of chloral hydrate; the skull specimens were collected, fixed with 4% paraformaldehyde (Solarbio, China) at room temperature for 48 h, and then stored in PBS for Micro-CT scanning (Bruker SkyScan 1276, Belgium) and histological analysis.

[0090] 2. Experimental results

[0091] Three-dimensional reconstruction maps of the skulls of mice in the group overexpressing lncRNA Gm20628 and its corresponding control group are shown in Figure 4 Figure A. As can be seen from this figure, the bone regeneration of the control group mice was restricted, mainly manifested at the defect edge, and no bone bridge was formed in the entire defect area. Compared with the control, the bone generation and bone integration in the group overexpressing lncRNA Gm20628 were much better, almost filling the entire effect area.

[0092] The trabecular bone number (Tb.N) and structure model index (SMI) of mice in the group overexpressing lncRNA Gm20628 and its corresponding control group are shown in Figure 4 Figure B. As can be seen from this figure, compared with the control group, the Tb.N in the group overexpressing lncRNA Gm20628 was 1.4 times that of the control group (P = 0.039). In addition, the SMI in the group overexpressing lncRNA Gm20628 was significantly lower than that of the control group (P = 0.021) (2.01 ± 0.16 vs 2.59 ± 0.22), indicating that the group of lncRNA Gm20628 formed more trabeculae with a lamellar structure.

[0093] To master the specific recovery situation, the present invention also carried out histological analyses such as H&E and Masson's trichrome staining, and the results were scanned and imaged.

[0094] The scanned imaging results of H&E staining of mice in the group overexpressing lncRNA Gm20628 and its corresponding control group are respectively shown in Figure 4C in the figure; the left figure in the figure is the H&E staining scanning imaging result of the control group mice, and the right figure in the figure is the H&E staining scanning imaging result of the mice in the group overexpressing lncRNA Gm20628. In the control group, fibrous connective soft tissue (ST) was formed at the defect site, but very limited regenerated bone tissue (RT) could be observed at its edge. On the contrary, in the group overexpressing lncRNA Gm20628, denser RT was present at both the edge and the center of the defect.

[0095] The Masson's trichrome staining scanning imaging results of the mice in the group overexpressing lncRNA Gm20628 and its corresponding control group are as Figure 4 shown in D in the figure; the left figure in the figure is the Masson's trichrome staining scanning imaging result of the control group mice, and the right figure in the figure is the Masson's trichrome staining scanning imaging result of the mice in the group overexpressing lncRNA Gm20628. Consistent with the H&E scanning results, the Masson's trichrome staining results showed that in the control group, immature bone formation (marked by triangles) was mainly observed at the center of the defect, while very little mature bone formation (marked by stars) was present at the edge of the defect; different from the control group, the calcium defect in the group overexpressing lncRNA Gm20628 was almost filled with mature bone formation.

[0096] The above results indicate that overexpression of lncRNA Gm20628 can significantly promote the osteogenic differentiation and bone formation of BMSCs, thereby promoting bone defect repair, and can provide new targets and treatment regimens for the treatment of related diseases such as osteoporosis and bone injuries.

[0097] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A long non-coding RNA, characterized in that, The nucleotide sequence of the RNA is as shown in SEQ ID NO.

1.

2. Use of a substance capable of regulating the expression level of the RNA recited in claim 1 in regulating osteogenic differentiation of bone marrow mesenchymal stem cells.

3. Use of a substance capable of regulating the expression level of the RNA recited in claim 1 in the preparation of a preparation for regulating osteogenic differentiation of bone marrow mesenchymal stem cells.

4. The application according to claim 2 or 3, characterized in that When the expression level of the RNA increases, it promotes osteogenic differentiation of bone marrow mesenchymal stem cells; when the expression level of the RNA decreases, it inhibits osteogenic differentiation of bone marrow mesenchymal stem cells.

5. Use of an expression promoter of the RNA recited in claim 1 in the preparation of a preparation for enhancing bone regeneration.

6. Use of an expression promoter of the RNA recited in claim 1 in the preparation of a preparation for bone defect repair.

7. Use of an expression promoter of the RNA recited in claim 1 in the preparation of a drug for preventing and / or treating osteoporosis.

8. Use of an expression promoter of the RNA recited in claim 1 in the preparation of a drug for treating bone formation disorder-related diseases.

9. Use of an expression promoter of the RNA recited in claim 1 in the preparation of a drug for improving the symptoms of osteopetrosis.

10. Use of an expression promoter of the RNA recited in claim 1 in constructing bone organoids or in the preparation of a preparation for constructing bone organoids.

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