Use of microRNA-32-5p in preparation of anti-osteoporosis drugs

Abstract

The application discloses application of microRNA-32-5p in preparation of a medicine for preventing and treating osteoporosis, and a medicine composition taking microRNA-32-5p as an active ingredient. It is proved through experiments that knocking out the microRNA-32-5p gene significantly aggravates osteoporosis induced by estrogen deficiency; the microRNA-32-5p plays a bone protection role by promoting osteogenic differentiation of bone marrow mesenchymal stem cells. Therefore, the microRNA-32-5p can be applied to prevention and treatment of osteoporosis as a microRNA medicine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of microRNA-32-5p in the preparation of anti-osteoporosis drugs. Background Technology

[0002] Osteoporosis (OP) is a systemic bone disease characterized by decreased bone mass, damage to bone microstructure, and increased bone fragility, leading to a higher risk of fractures. It is commonly seen in postmenopausal women and the elderly. With the increasing aging of the global population, the prevalence of OP is rising annually. OP frequently induces fragility fractures, placing a heavy burden on patients, families, and society. Therefore, the development of novel drugs for the prevention and treatment of OP and the elucidation of their mechanisms of action are both necessary and urgent.

[0003] MicroRNAs are a class of non-coding RNAs, approximately 21–25 nucleotides (nt) in length, present in all eukaryotic cells. They exert post-transcriptional regulation of target genes by binding to the 3' untranslated region (UTR), inducing mRNA degradation or inhibiting its translation efficiency. MicroRNAs participate extensively in the regulation of various life activities in the body through a complex network of regulation. Studies have shown that microRNAs can regulate bone metabolism through multiple pathways, including regulating osteogenic and adipogenic differentiation, osteoclast differentiation, chondrogenic differentiation, and H-type bone angiogenesis. Therefore, identifying specific microRNAs that regulate bone metabolism and applying them to the preparation of drugs for treating osteoporosis could provide a new strategy for the prevention and treatment of osteoporosis. Summary of the Invention

[0004] The purpose of this invention is to provide the use of microRNA-32-5p in the preparation of an anti-osteoporosis drug; another purpose of this invention is to provide a pharmaceutical composition for the prevention and / or treatment of osteoporosis.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Application of MicroRNA-32-5p in the preparation of drugs for the prevention and treatment of osteoporosis.

[0007] Preferably, the osteoporosis includes postmenopausal osteoporosis and / or senile osteoporosis.

[0008] Preferably, the microRNA-32-5p has at least one of the following functions:

[0009] 1) Increase the volume fraction of cancellous bone in the distal femur;

[0010] 2) Increase the number of trabeculae;

[0011] 3) Reduce trabecular separation;

[0012] Preferably, the microRNA-32-5p can promote osteogenic differentiation of bone marrow mesenchymal stem cells.

[0013] Preferably, the microRNA-32-5p can bind to the specific nucleic acid aptamer of bone marrow mesenchymal stem cells and target bone marrow mesenchymal stem cells to exert an osteoproliferative effect.

[0014] A pharmaceutical composition having microRNA-32-5p as the active ingredient, said pharmaceutical composition comprising any one of microRNA-32-5p, its analogues, its agonists, or substances expressing microRNA-32-5p.

[0015] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipients. The present invention does not have any particular limitation on the specific types of pharmaceutically acceptable carriers and excipients, and any types commonly used in the art are acceptable. The carrier may be nanoparticles, liposomes, cholesterol, chitosan, etc.

[0016] Preferably, the substance expressing microRNA-32-5p includes a promoter, enhancer, or expression vector capable of upregulating microRNA-32-5p gene expression. This invention does not impose any particular limitation on the backbone vector of the expression vector; any vector commonly used in the art is acceptable.

[0017] Preferably, the nucleotide sequence of microRNA-32-5p is as shown in SEQ ID NO.1 or is a nucleotide sequence obtained by substitution, deletion, or addition of one or more bases to the sequence shown in SEQ ID NO:1. Specifically, the nucleotide sequence of microRNA-32-5p is: uauugcacauuacuaaguugca. In the ST.26 standard sequence listing, "uracil" in the RNA sequence shown in SEQ ID NO.1 is represented by "t".

[0018] The present invention also provides a pharmaceutical composition with microRNA-32-5p as the active ingredient, comprising any one of microRNA-32-5p, its analogues, its agonists, or substances expressing microRNA-32-5p.

[0019] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient; the carrier includes nanoparticles, liposomes, cholesterol, or chitosan.

[0020] Preferably, the substance expressing microRNA-32-5p includes a promoter, enhancer, or expression vector capable of upregulating microRNA-32-5p gene expression.

[0021] Preferably, the nucleotide sequence of the microRNA-32-5p is as shown in SEQ ID NO:1 or is a nucleotide sequence obtained by substitution, deletion or addition of one or more bases to the sequence shown in SEQ ID NO:1.

[0022] This invention discloses the application of microRNA-32-5p in the preparation of anti-osteoporosis drugs. Experiments demonstrated that knockout of the microRNA-32-5p gene significantly aggravated osteoporosis induced by bilateral ovariectomy (OVX) in mice, and revealed that microRNA-32-5p exerts its osteoprotective effect by promoting osteogenic differentiation of bone marrow mesenchymal stem cells. Therefore, microRNA-32-5p can be used as a microRNA-based drug in the prevention and treatment of osteoporosis. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 : Comparison of bone volume fraction in microRNA-32-5p gene knockout mice 3 months after OVX surgery in Example 1 of this invention;

[0025] Figure 2 : Comparison of trabecular bone count in microRNA-32-5p gene knockout mice 3 months after OVX surgery in Example 1 of this invention;

[0026] Figure 3 Comparison of trabecular bone separation in microRNA-32-5p gene knockout mice 3 months after OVX surgery in Example 1 of this invention;

[0027] Figure 4 Three-dimensional image of the distal femur of microRNA-32-5p gene knockout mice three months after OVX surgery in Example 1 of this invention;

[0028] Figure 5 Example 2 of this invention: Osteogenic induction of primary bone marrow mesenchymal stem cells in wild-type mice and microRNA-32-5p gene knockout mice for 10 days, Alizarin Red staining comparison image.

[0029] Figure 6 : In Example 2 of the present invention, primary bone marrow mesenchymal stem cells of microRNA-32-5p gene knockout mice were transfected with microRNA-32-5p mimic and then induced to differentiate into osteoblasts for 10 days. The comparison diagram of alizarin red staining is shown. Detailed implementation manners

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0031] Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the protection scope of the present invention.

[0032] Unless otherwise specified, all kinds of reagents and raw materials used in the present invention are commercially available products or products that can be prepared by known methods.

[0033] Example 1: Effect of MicroRNA-32-5p gene knockout on bone mass in OVX mice

[0034] (1) All the animals selected in this experiment were 12-week-old healthy female C57BL / 6J wild-type mice or microRNA-32-5p gene knockout mice.

[0035] (2) The pre-microRNA sequence of the miR-32-5p gene in C57BL / 6J mice was edited and constructed by CRISPR / Cas9 technology to obtain microRNA-32-5p gene knockout mice, which were propagated and expanded in the Animal Department of Nanhua University. Wild-type mice with the same background were purchased from Hunan Slack Jingda, license number: SCXK(Xiang)2016-0002.

[0036] (3) Mouse osteoporosis model: Female mice of the same age were divided into wild-type sham operation group (WT-Sham), wild-type OVX operation group (WT-OVX), and microRNA-32-5p gene knockout OVX operation group (MicroRNA-32-5p - / --OVX). Bone tissue specimens were taken 3 months after the operation, and the bone volume fraction (BV / TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp) of the distal femoral cancellous bone were detected respectively. The results ( Figure 1-3The results showed that after OVX surgery, microRNA-32-5p gene knockout mice had significantly lower BV / TV and Tb.N compared to the wild-type OVX group, while Tb.Sp significantly increased. Figure 4 Three-dimensional images of the distal femur in mice after OVX surgery.

[0037] Example 2: Effect of MicroRNA-32-5p gene knockout on osteogenic differentiation capacity of mouse bone marrow mesenchymal stem cells

[0038] (1) Extraction of primary mouse bone marrow mesenchymal stem cells

[0039] Three 4-week-old wild-type mice and three microRNA-32-5p gene knockout mice were selected. After anesthesia and cervical dislocation, the femurs and tibias of the mice were rapidly separated by soaking them in 75% ethanol for 5-10 minutes. The separated femurs and tibias were placed in culture dishes, and PBS containing 1% penicillin-dextrin was added. The muscles attached to the bone surface were quickly peeled off, and the femurs and tibias were then transferred to new serum-free α-MEM. The epiphyses at both ends were removed with ophthalmic scissors. The bone marrow cavity contents were washed out with complete culture medium (α-MEM + 15% FBS + 1% penicillin-dextrin), then centrifuged at 1000 rpm for 5 minutes. The supernatant was removed, and the cells were resuspended in 4 ml of complete culture medium and seeded in T25 culture flasks. After 24 hours of routine culture, the culture medium was completely replaced to remove non-adherent cells. Adherent cells containing bone marrow mesenchymal stem cells were cultured for another 2-3 days with normal medium changes. When the cells reached a density of approximately 80-90%, they were passaged by digestion with 0.25% trypsin / EDTA. Second-generation bone marrow mesenchymal stem cells are used for osteogenic induction.

[0040] (2) Osteogenic induction of mouse primary bone marrow mesenchymal stem cells

[0041] Bone marrow mesenchymal stem cells were trypsinized and seeded into 48-well plates. After routine culture until the density reached approximately 80-90%, the culture medium was replaced with osteogenic induction medium. Mouse bone marrow mesenchymal stem cell osteogenic differentiation induction kit (MUXMX-90021), with the culture medium changed every 2 days. After 10 days of induction, Alizarin Red staining was performed to detect the expression level of calcium nodules. The results are as follows: Figure 5 As shown, the amount of calcium nodules in bone marrow mesenchymal stem cells of microRNA-32-5p gene knockout mice was significantly reduced compared to wild-type mice. Osteogenic induction was performed after transfecting microRNA-32-5p mimic into bone marrow mesenchymal stem cells of microRNA-32-5p gene knockout mice, and the results were as follows. Figure 6 As shown, the amount of calcium nodules increased significantly. These results indicate that the microRNA-32-5p gene can promote osteogenic differentiation of bone marrow mesenchymal stem cells.

[0042] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of microRNA-32-5p in preparation of a drug for preventing and treating osteoporosis; The nucleotide sequence of the microRNA-32-5p is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, The osteoporosis includes postmenopausal osteoporosis and / or senile osteoporosis.

3. Use according to claim 1, characterized in that, The microRNA-32-5p has at least one of the following functions: 1) increasing the bone volume fraction of the distal femur cancellous bone; 2) increasing the trabecular bone number; 3) reducing the trabecular separation degree.

4. Use according to claim 1, characterized in that, The microRNA-32-5p can promote the osteogenic differentiation of bone marrow mesenchymal stem cells.

5. Use according to claim 4, characterized in that, The microRNA-32-5p can bind to a bone marrow mesenchymal stem cell specific nucleic acid aptamer, target the bone marrow mesenchymal stem cells and play a role in promoting osteogenesis.

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