Application of hsa-let-7f-5p in preparation of medicine for treating muscle fatty degeneration

Abstract

The invention relates to the technical field of microRNA medicines, in particular to application of hsa-let-7f-5p in preparation of a medicine for treating muscle fatty degeneration. The invention discloses that hsa-let-7f-5p can be used for inhibiting muscle fat infiltration by inhibiting adipose differentiation of fibroblast / adipogenesis progenitor cells. The hsa-let-7f-5p can be used as a microRNA drug, and is applied to the treatment of muscle fat infiltration and muscle fat degeneration diseases, and fat infiltration-related diseases, such as muscle atrophy, muscle injury, obesity, diabetes and the like. Related nucleic acid fragments are wrapped in the lipidosome, so that the drug stability and the drug delivery efficiency can be improved. According to the technical scheme, the technical problem that medicines capable of effectively inhibiting muscle fatty degeneration and treating muscle injury are lacked in the prior art can be solved, and the medicine has ideal application and popularization prospects in the fields of sports medicine, rehabilitation treatment and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of microRNA drugs, and particularly to the application of hsa-let-7f-5p in the preparation of drugs for treating muscle steatosis. Background Art

[0002] Adipose infiltration refers to the phenomenon of finding adipocytes in the stroma of tissues or organs that usually contain few or no adipocytes. This situation is particularly common in tissues such as the heart, pancreas, and skeletal muscle. It is worth noting that adipose infiltration in skeletal muscle is a health risk factor that has not been fully recognized. It not only leads to the deposition of fat inside the muscle, ultimately causing muscle steatosis (a disorder characterized by abnormal accumulation of fat in muscle tissue), which may be caused by genetic factors, metabolic disorders, malnutrition, long-term inactivity, or certain diseases, but is also closely related to the occurrence and development of various diseases.

[0003] Specifically, fat deposition can be divided into two forms: intermuscular and intramuscular. The former refers to the deposition of adipocytes in the available space between skeletal muscles; the latter covers all adipocytes embedded between active skeletal muscle fibers. Muscle steatosis is closely related to skeletal muscle lesions. Regarding muscle atrophy, as the course of muscle atrophy progresses, while the number and volume of muscle fibers decrease, a large number of adipocyte infiltrations and fibrosis phenomena occur, which not only hinder muscle regeneration but also accelerate the process of muscle atrophy. When skeletal muscle is damaged, muscle fibers are damaged or the number decreases, accompanied by an inflammatory response. During this repair process, the occurrence of a large number of adipocyte infiltrations and fibrosis will further block muscle regeneration, thus affecting the recovery effect. Therefore, inhibiting skeletal muscle steatosis is of great significance for promoting the recovery after muscle injury.

[0004] In addition, studies have shown that intramuscular fat deposition (whether intermuscular or intramuscular) is closely associated with conditions such as obesity, diabetes, and metabolic syndrome (Uezumi A, Fukada S, Yamamoto N, Takeda S, Tsuchida K. Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle. Nat Cell Biol. 2010;12:143-52.). As one of the early muscle structure changes, intramuscular fattening occurs before the appearance of abnormal strength and function, and it is a prelude to the metabolic changes associated with obesity and diabetes. The combined effect of fat accumulation in muscle and muscle fiber degeneration significantly reduces muscle mass and is positively correlated with morbidity and mortality (Zamboni, M., Gattazzo, S. & Rossi, A.P. Myosteatosis: a relevant, yet poorly explored element of sarcopenia. Eur Geriatr Med 10, 5–6 (2019); Hausman GJ, Basu U, Du M, Fernyhough-Culver M, Dodson MV. Intermuscular and intramuscular adipose tissues: Bad vs. good adipose tissues. Adipocyte. 2014 Dec 10;3(4):242-55.).

[0005] Despite the growing importance of skeletal muscle fat degeneration, there is currently a lack of effective medical treatment for this condition in clinical practice. Especially when dealing with muscle injury or atrophy, there is no specific drug that can promote the muscle repair process by inhibiting muscle fat deposition. This field still awaits more research and exploration in order to find more effective treatment methods. Summary of the Invention

[0006] The present invention aims to provide the application of hsa-let-7f-5p in the preparation of a drug for treating muscle fat degeneration, so as to solve the technical problem in the prior art that there is a lack of drugs that can effectively inhibit muscle fat degeneration and treat muscle injury.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The application of hsa-let-7f-5p in the preparation of a drug for treating muscle steatosis, and its sequence is as shown in SEQ ID NO.1. RNA.

[0009] Furthermore, hsa-let-7f-5p is produced from double-stranded RNA; the sense strand of the double-stranded RNA is as shown in SEQ ID NO.2, and the antisense strand is as shown in SEQ ID NO.3.

[0010] Furthermore, the double-stranded RNA or hsa-let-7f-5p is an inhibitor of adipogenic differentiation of fibroblast / adipocyte progenitor cells.

[0011] Furthermore, the double-stranded RNA or hsa-let-7f-5p is an inhibitor of Tgfbr3 gene expression.

[0012] Furthermore, muscle fat infiltration is caused by muscle injury.

[0013] This technical solution also provides an application of a double-stranded RNA in the preparation of a drug for treating muscle injury, the sense strand of which is as shown in SEQ ID NO.2, and the antisense strand is as shown in SEQ ID NO.3; the double-stranded RNA is used to form hsa-let-7f-5p with a sequence as shown in SEQ ID NO.1.

[0014] Furthermore, the double-stranded RNA or hsa-let-7f-5p is used to promote regeneration and repair after muscle injury, and to inhibit fat deposition after muscle injury.

[0015] Furthermore, the hsa-let-7f-5p is enriched in exosomes derived from platelet-rich plasma.

[0016] This technical solution also provides a liposome vesicle encapsulating double-stranded RNA.

[0017] Furthermore, the raw materials of the liposome vesicle include SPC, DOTAP, cholesterol and DSPE-PEG-2k;

[0018] The liposome vesicle encapsulating double-stranded RNA is prepared by the following method: dissolving SPC, DOTAP, cholesterol and DSPE-PEG-2k in absolute ethanol to form a lipid mixture; adding a citrate buffer solution containing dissolved double-stranded RNA and ethanol to the lipid mixture; and then obtaining the liposome vesicle encapsulating double-stranded RNA after ultrasonic treatment, extrusion treatment with a liposome extruder and dialysis treatment.

[0019] The principle and beneficial effects of this technical solution are as follows:

[0020] By deeply studying the role of platelet-rich plasma (PRP) in inhibiting muscle tissue fat infiltration and muscle fatification, the inventors found that exosomes derived from PRP have significant effects. The inventors first removed DNA, RNA, or proteins from PRP exosomes respectively and conducted in vitro interventions on fibroblast / adipocyte progenitor cells undergoing adipogenic differentiation. The experimental results showed that after removing RNA, the inhibitory effect of PRP-derived exosomes on the adipogenic differentiation ability of fibroblast / adipocyte progenitor cells was significantly weakened. This indicates that RNA is a key component for PRP-derived exosomes to affect the adipogenic differentiation of these progenitor cells. To determine the specific active ingredient, the inventors comprehensively analyzed the miRNA composition in exosomes using high-throughput sequencing technology. The study found that hsa-let-7f-5p was highly enriched in exosomes, suggesting that this miRNA may be one of the factors playing a key role.

[0021] Based on the above findings, the inventors synthesized double-stranded RNA mimics (mimics, used to form hsa-let-7f-5p in cells and tissues) that match the mature sequence of hsa-let-7f-5p and specifically studied its role in inhibiting the adipogenic differentiation of fibroblast / adipocyte progenitor cells. The results showed that hsa-let-7f-5p mimics significantly inhibited the adipogenic differentiation ability of fibroblast / adipocyte progenitor cells, demonstrating the important role of this miRNA in this process. In addition, the study found that the target of hsa-let-7f-5p is the Tgfbr3 gene.

[0022] In a muscle injury model established by glycerol treatment, the inventors further verified the function of hsa-let-7f-5p. The research results showed that hsa-let-7f-5p effectively prevented muscle tissue fat infiltration caused by muscle injury, thereby promoting the repair process of muscle injury. Therefore, hsa-let-7f-5p can not only be used alone to inhibit the adipogenic differentiation of fibroblast / adipocyte progenitor cells, but also achieve an ideal effect when used in the treatment of muscle injury with symptoms of muscle fat infiltration.

[0023] To improve the stability of RNA drugs, this technical solution also attempted to prepare liposome vesicles encapsulating hsa-let-7f-5p. The instability of RNA molecules themselves and the challenges they face during in vivo delivery (such as enzymatic degradation, immunogenicity, poor targeting specificity, etc.) limit their wide application. As a carrier for RNA drugs, liposome vesicles can effectively overcome these problems. Liposomes can form a physical barrier to protect RNA from degradation by nucleases in the in vivo and in vitro environments. By optimizing the liposome composition, the chemical stability of RNA drugs can be further improved. Liposomes have better compatibility with the receptor cell membrane, promoting the uptake of RNA drugs by cells.

[0024] In summary, this study reveals the importance of hsa-let-7f-5p, which is enriched in PRP-derived exosomes, in inhibiting adipogenic differentiation of fibroblast / adipocyte progenitor cells and promoting muscle injury repair associated with fat infiltration. These findings not only deepen our understanding of the mechanism of exosome-mediated muscle repair but also provide a theoretical basis for the development of new therapeutic strategies. In particular, hsa-let-7f-5p shows promise for the prevention and treatment of muscle injury and its associated fat infiltration problems. hsa-let-7f-5p helps maintain the normal structure and function of muscle tissue and prevents inappropriate fat infiltration. In a muscle injury and fat infiltration model established by glycerol treatment, hsa-let-7f-5p effectively blocked fat infiltration in muscle tissue, which is of great significance for the prevention and treatment of pathological changes caused by muscle injury, especially in the fields of sports medicine, rehabilitation therapy, and age-related muscle degeneration.

[0025] When delving deeper into the role of platelet-rich plasma (PRP) in inhibiting muscle fat infiltration and muscle fattening, researchers found that PRP-derived exosomes showed significant effects. To identify their active components, researchers first removed DNA, RNA, or proteins from PRP exosomes and conducted in vitro intervention experiments on fibroblast / adipocyte progenitor cells. The results showed that when RNA was removed, the inhibitory effect of PRP-derived exosomes on adipogenic differentiation ability was significantly weakened, indicating that RNA is a key component.

[0026] To further identify the specific active components, researchers analyzed the miRNA composition in exosomes using high-throughput sequencing technology and found that hsa-let-7f-5p was highly enriched in exosomes, suggesting that it might be one of the main factors at play. Based on this finding, researchers synthesized double-stranded RNA mimics that matched the mature sequence of hsa-let-7f-5p (mimics, used to form hsa-let-7f-5p in cells and tissues) and studied its effect on inhibiting adipogenic differentiation. The experimental results showed that hsa-let-7f-5p mimics significantly reduced the likelihood of adipogenic differentiation, demonstrating the important role played by this miRNA in this process. In addition, the study also determined that the Tgfbr3 gene is the target of hsa-let-7f-5p.

[0027] In a glycerol-induced muscle injury model, researchers verified the function of hsa-let-7f-5p and found that it effectively prevented fat infiltration caused by muscle injury and promoted the repair of muscle tissue. This indicates that hsa-let-7f-5p not only can be used alone to inhibit adipogenic differentiation but also shows potential in treating muscle injuries accompanied by fat infiltration.

[0028] Considering the problems encountered during the in vivo delivery of RNA drugs, such as enzymatic degradation, immunogenicity, and poor targeting specificity, researchers have attempted to encapsulate hsa-let-7f-5p in liposomal vesicles to improve its stability. As a carrier, liposomes can not only form a physical barrier to protect RNA from nuclease damage but also enhance chemical stability and promote cellular uptake by optimizing their composition.

[0029] In summary, this study reveals the importance of hsa-let-7f-5p in PRP-derived exosomes in inhibiting adipogenic differentiation and promoting muscle injury repair. These findings not only enhance our understanding of the muscle repair mechanism mediated by exosomes but also provide a theoretical basis for the development of new therapeutic methods. Especially in the fields of sports medicine, rehabilitation therapy, and age-related muscle degeneration, hsa-let-7f-5p has broad application prospects, contributing to the prevention and treatment of fat infiltration problems caused by muscle injury and maintaining the normal structure and function of muscle tissue. Brief Description of the Drawings

[0030] Figure 1 This is the experimental research result of the in vitro inhibition of adipogenic differentiation of fibroblast / adipogenic progenitor cells by hsa-let-7f-5p in Example 1 of the present invention.

[0031] Figure 2 This is the effect of hsa-let-7f-5p on cellular adipogenic differentiation under the overexpression of Tgfbr3 in Example 2 of the present invention.

[0032] Figure 3 This is the experimental research result of the in vivo inhibition of muscle fattening after muscle injury by hsa-let-7f-5p in Example 3 of the present invention.

[0033] Figure 4 This is the identification result of the morphology and markers of exosomes in Example 4 of the present invention and the effect of oil red staining to detect the adipogenic ability of fibroblast / adipogenic progenitor cells induced by PRP-derived exosomes on adipogenic differentiation.

[0034] Figure 5 This is the effect of oil red staining in Example 4 of the present invention to detect the adipogenic ability of fibroblast / adipogenic progenitor cells on adipogenic differentiation after removing DNA, RNA, or protein in PRP exosomes respectively.

[0035] Figure 6 This is the miRNA high-throughput sequencing detection result in Example 4 of the present invention (showing the short fragment sequence reads and proportions of different miRNAs in PRP exosome samples from young and old people).

[0036] Figure 7Microscopic images of liposome vesicles encapsulating hsa-let-7f-5p in Example 5 of the present invention and experimental results of its therapeutic effect. Detailed implementation manners

[0037] The present invention will be further described in detail below in conjunction with embodiments, but the implementation manners of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels.

[0038] Example 1: Experimental study on the in vitro inhibition of adipogenic differentiation of fibroblast / adipogenic progenitor cells by hsa-let-7f-5p

[0039] The mature sequence of hsa-let-7f-5p is: 5’-UGAGGUAGUAGAUUGUAUAGUU-3’ (SEQ ID NO.1).

[0040] According to the mature sequence of hsa-let-7f-5p, double-stranded RNA hsa-let-7f-5p mimics were synthesized, which include a sense strand and an antisense strand:

[0041] Sense strand: 5’-UGAGGUAGUAGAUUGUAUAGUU-3’ (SEQ ID NO.2);

[0042] Antisense strand: 5’-CUAUACAAUCUACUACCUCAUU-3’ (SEQ ID NO.3).

[0043] In the sequence listing, U in SEQ ID NO.1-SEQ ID NO.3 above is replaced by T. At the same time, a negative control scRNA was synthesized for use in subsequent experiments.

[0044] The experiment was carried out according to the following method:

[0045] Before transfection with the above hsa-let-7f-5p mimic and scRNA, the lyophilized powders of hsa-let-7f-5p mimic and scRNA were prepared into stock solutions for use. The hsa-let-7f-5p mimic and scRNA were transfected into cells at a concentration of 80 μM (final concentration) with the assistance of INVIDNA RNA Transfection Reagent (Invigentech, a conventional transfection reagent). Muscle primary fibroblast / adipogenic progenitor cells were sorted by fluorescence-activated cell sorting (FACS), and fibroblast / adipogenic progenitor cells were cultured in vitro by conventional means of the prior art. The fibroblast / adipogenic progenitor cells were seeded at 3×10 5Inoculate at a quantity of cells / mL, and after culturing in DMEM medium for 72 h, use the complex formed by the RNA at the above dosage and INVIDNA RNA Transfection Reagent to transfect fibroblast / adipogenic progenitor cells according to the method described in the kit. After 24 h, replace the medium with a medium without RNA and INVIDNA RNA Transfection Reagent and culture the cells until 96 h of culture, and then detect the relevant transcriptional expression of the cells. Perform the same conventional adipogenic differentiation induction on the transfected fibroblast / adipogenic progenitor cells of different experimental groups, and use the adipogenic differentiation induction medium to induce the adipogenic differentiation of fibroblast / adipogenic progenitor cells (i.e., prepare a model for the adipogenic differentiation of cells), and perform Oil Red O staining at the same time point to detect the ability of adipocyte formation.

[0046] See the detailed experimental results in Figure 1 (n = 3, and the percentage of the Oil Red O area in the total area of the visual field was statistically analyzed), showing the Oil Red O staining conditions of different experimental groups. Increasing the amount of hsa-let-7f-5p in cells (transfecting hsa-let-7f-5p mimics) will reduce the area of adipocytes, that is, reduce the adipogenic differentiation of fibroblast / adipogenic progenitor cells.

[0047] Example 2: hsa-let-7f-5p regulates the adipogenic differentiation ability of fibroblast / adipogenic progenitor cells by targeting Tgfbr3

[0048] The gene encoded by Tgfbr3 (Transforming Growth Factor Beta Receptor III) is the transforming growth factor β receptor III (TGF-β Receptor III), also known as beta-glycan. This receptor is expressed on the cell surface and is an important component of the TGF-β signaling pathway. Overexpress the Tgfbr3 gene in normal cells and further induce and culture with the adipogenic differentiation induction medium to obtain an enhanced model of cell adipogenic differentiation for studying the effect of drugs on cell adipogenic differentiation. A large amount of adipogenic differentiation of fibroblast / adipogenic progenitor cells in muscle forms muscle fat infiltration and muscle fat degeneration.

[0049] Experiments have proven that hsa-let-7f-5p can inhibit the adipogenic differentiation of fibroblast / adipocyte progenitor cells overexpressing the Tgfbr3 gene. In vitro, transfect the Tgfbr3 overexpression plasmid (TGFBR OE) or the empty vector (vector). In the cells transfected with the Tgfbr3 overexpression plasmid (TGFBR OE), transfect hsa-let-7f-5p (the aforementioned double-stranded hsa-let-7f-5p mimics) or scRNA, and then induce the adipogenic differentiation of fibroblast / adipocyte progenitor cells with an adipogenic differentiation induction medium, and detect the adipocyte formation ability by Oil Red staining. That is, in fibroblast / adipocyte progenitor cells transfected with the Tgfbr3 overexpression plasmid or the empty vector, transfect hsa-let-7f-5p or scRNA according to the aforementioned method, and then use Oil Red staining to detect the adipocyte formation ability. The experimental results are shown in Figure 2 (n = 3, the percentage of the Oil Red area in the total area of the visual field was statistically analyzed), which shows the regulatory effect of Oil Red staining on the inhibition of adipogenic differentiation of fibroblast / adipocyte progenitor cells by hsa-let-7f-5p with overexpression of Tgfbr3. In fibroblast / adipocyte progenitor cells with overexpression of the Tgfbr3 gene, the overexpression of the Tgfbr3 gene will lead to a decrease in the adipogenic differentiation of fibroblast / adipocyte progenitor cells. At the same time, increasing the amount of hsa-let-7f-5p in these cells will reduce the area of adipocytes, that is, reduce the adipogenic differentiation of fibroblast / adipocyte progenitor cells.

[0050] Although the prior art has reported that one of the action targets of hsa-let-7f-5p is the Tgfbr1 gene and it can be applied to the treatment of osteoporosis. However, there are very large differences between the Tgfbr1 gene and the Tgfbr3 gene. For example, their structural characteristics are different (the former has serine / threonine kinase activity, while the latter lacks intrinsic kinase activity). They also differ in ligand binding mode, signal transduction function, tissue distribution, and biological effects. Thus, it can be seen that the Tgfbr3 gene is a newly discovered action target of hsa-let-7f-5p in this scheme. Through this action target, the inhibition of adipogenic differentiation of fibroblast / adipocyte progenitor cells is achieved, thereby treating muscle steatosis and muscle damage related to muscle steatosis.

[0051] More specifically, Tgfbr3 is expressed on the cell surface and is an important component of the TGF-β signaling pathway. The Tgfbr3 gene is closely related to muscle injury repair and adipogenic differentiation of fibroblast / adipocyte progenitors. Particularly regarding its role in muscle injury repair, the Tgfbr3 gene is involved in the TGF-β signaling pathway. During muscle injury repair, the TGF-β signaling pathway is involved in regulating a variety of cellular activities. For example, this pathway is related to the synthesis and deposition of neonatal muscle fibers, extracellular matrix, and the regulation of inflammatory responses in muscle injury. Subsequent experimental studies on a muscle injury model induced by glycerol have demonstrated that treating muscle injury with hsa-let-7f-5p mimics can effectively and comprehensively promote muscle injury repair. The inventors analyzed the reason that hsa-let-7f-5p can play a comprehensive positive regulatory role in aspects such as neonatal muscle fiber formation, synthesis and deposition of extracellular matrix, inflammatory response, and fat deposition during muscle injury repair by acting on the target gene Tgfbr3 gene.

[0052] Example 3: Experimental study on the inhibition of fat infiltration after muscle injury by hsa-let-7f-5p hsa-let-7f-5p

[0053] Construction of the model animal: A 50% (v / v) glycerol solution (50 μL) was locally injected into the tibialis anterior muscle of mice to induce an acute injury model of muscle fat infiltration. Glycerol, as a solvent, can cause the dissolution of muscle fibers and inflammatory responses when locally injected into muscle at a high concentration, thus mimicking muscle injury. One hour after glycerol injection, hsa-let-7f-5p (the aforementioned double-stranded hsa-let-7f-5p mimics) or NC (scRNA) (4 μg) was locally injected. After the administration was completed, specimens were taken on the 14th day after modeling for subsequent detection. Treating muscle tissue with glycerol is a method for establishing a muscle injury model. After such muscle injury, there will be phenomena of muscle fat infiltration and fatification. Therefore, it is also a related model of muscle fat infiltration (muscle fatty degeneration). This model can be used to study the mechanisms of skeletal muscle regeneration, repair, fat infiltration (muscle fatification), and to test potential treatment strategies. Necrotic injury of muscle tissue was induced by locally injecting a high concentration of glycerol. After the injury, fat deposition would occur in skeletal muscle, affecting muscle regeneration and repair.

[0054] The tibialis anterior muscle specimens were fixed in 4% paraformaldehyde for 12 hours, and then dehydrated overnight with 30% sucrose solution; the dehydrated samples were prepared into frozen sections with a thickness of 8 - 10 μm using a cryostat. Fluorescence immunohistochemistry was performed on the samples to determine the situation of fat infiltration. The experimental results are shown in detail in Figure 3 (n = 4, detecting perilipin + Percentage of the positive area in the area of the section specimen).Figure 3 The green part in is the Perilipin label, used to show adipocyte infiltration. The red part is the Phalloidine label, which demonstrates the tissue cytoskeleton. The blue part is the Hoechst 33342 label, showing the cell nuclei. Figure 3 The statistical chart on the right is for Perilipin + the statistical analysis of the area of the labeled region. From Figure 3 the experimental results, it can be seen that treatment with hsa-let-7f-5p can reduce the fat infiltration in muscle tissue, playing a role in inhibiting muscle fatification.

[0055] In addition, the hsa-let-7f-5p treatment of this protocol can not only inhibit the fatification of fibroblast / adipogenic progenitor cells, but also promote the regeneration and repair process after skeletal muscle injury. Phalloidine is a toxin commonly used to label and visualize actin filaments (F-actin). It has a high affinity for F-actin and can observe the actin structure in the cytoskeleton through fluorescently labeled phalloidine. When studying the degree of muscle injury recovery, phalloidine staining can help evaluate the integrity of the myofiber structure and its repair effect. From Figure 3 it can be seen that compared with the negative control, the hsa-let-7f-5p group has a larger average cross-sectional area of phalloidine-labeled myotubes and a more regular arrangement, indicating that after injecting double-stranded hsa-let-7f-5p mimics, the recovery degree of muscle injury is more ideal.

[0056] Example 4: Discovery of hsa-let-7f-5p

[0057] It was found that platelet-rich plasma (PRP) has the function of inhibiting muscle fat infiltration. This technical solution further studied its mechanism of action. By extracting exosomes from PRP, it was found that exosomes can significantly inhibit muscle fat infiltration by inhibiting the adipogenic differentiation ability of fibroblast / adipogenic progenitor cells. By screening out the active ingredients, it was found that RNA is the key component mediating the inhibition of adipogenesis by PRP-derived exosomes. Through miRNA high-throughput sequencing, hsa-let-7f-5p was screened out as a key molecule that may inhibit adipogenesis by PRP-derived exosomes.

[0058] The exosomes in PRP were extracted and identified by the conventional ultracentrifugation method of the existing technology. The experimental results are shown in detail in Figure 4 . Electron microscopy was used to detect the morphology of exosomes ( Figure 4 upper left) and Western blotting was used to detect the expression of typical exosome markers ( Figure 4Upper right), Oil Red staining was used to detect the effect of PRP-derived exosomes on the adipogenic ability of fibroblast / adipogenic progenitor cells during adipogenic differentiation. Figure 4 Below, n = 4, perilipin was detected + (Percentage of positive area in the area of the section specimen), indicating that the use of PRP-derived exosomes can effectively inhibit the adipogenic differentiation of fibroblast / adipogenic progenitor cells.

[0059] The DNA, RNA, or protein in PRP exosomes was removed respectively, and then fibroblast / adipogenic progenitor cells undergoing adipogenic differentiation were intervened in vitro. It was found that the inhibitory effect of PRP-derived exosomes on the adipogenic differentiation ability of fibroblast / adipogenic progenitor cells was significantly weakened after removing RNA, suggesting that RNA is a key component for PRP-derived exosomes to affect the adipogenic differentiation of fibroblast / adipogenic progenitor cells. Through Oil Red staining detection, the experimental results are shown in Figure 5 (n = 5, perilipin was detected + (Percentage of positive area in the area of the section specimen).

[0060] High-throughput sequencing of miRNAs was used to detect the miRNA components enriched in PRP-derived exosomes, and it was found that hsa-let-7f-5p was highly enriched in such exosomes. Figure 6 ) In PRP-derived exosomes, the number and proportion of short fragment sequences (reads) of hsa-let-7f-5p were found to be relatively high by sequencing. Therefore, in this technical solution, according to the mature sequence of hsa-let-7f-5p, double-stranded mimics were designed, and then the research on its effect of inhibiting muscle fattening was carried out, and then this potential RNA drug that can be used for the treatment of related diseases was discovered.

[0061] Example 5: Liposome vesicles encapsulating hsa-let-7f-5p

[0062] Wrap let-7f into liposomes, then locally inject to intervene in the glycerol-induced anterior tibial muscle injury model. Collect muscle samples on the 21st day for frozen section, and detect the percentage of the expression area of Perilipin in the total area of the sample section by immunofluorescence staining. For the experimental method, see Example 3. The injection volume of liposomal vesicles (lipo-miRNAs) encapsulating hsa-let-7f-5p and liposomal vesicles (lipo-scRNA) encapsulating scRNA is both 60 μL. The liposomal vesicles (lipo-miRNAs and lipo-scRNA) encapsulating hsa-let-7f-5p (double-stranded hsa-let-7f-5p mimics) or scRNA are prepared by the conventional method for preparing liposomal vesicles in the prior art, that is, wrapping RNA into vesicles formed by lipids. The general operation process is as follows: Take SPC (phosphatidylcholine), DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), cholesterol, DSPE-PEG-2k (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]) and dissolve them in 2 mL of absolute ethanol, then transfer to an eggplant-shaped flask to form a lipid mixture. Among them, the dosage ratio of the above raw materials can be carried out in the conventional manner of the prior art. For example: SPC:DOTAP:cholesterol:DSPE-PEG-2k = 40-60 mg:2-3 mg:2-3 mg:2-3 mg:2 mL. Dissolve RNA (i.e., RNA drug, double-stranded hsa-let-7f-5p mimics or scRNA) in a citrate buffer solution (50 mM citrate, pH = 4) containing 25% ethanol, slowly add it to the aforementioned lipid mixture, mix well and incubate for 20 minutes, and then process it with ultrasonic and a liposome extruder (200 nm filter membrane). Then dialyze with a nano-dialysis device (polycarbonate membrane, pore size 50 nm) to remove the unloaded RNA. Finally, supplement sterile water to make up the volume to 10 mL.

[0063] Figure 7 A is the electron microscope image of liposomal vesicles encapsulating hsa-let-7f-5p, Figure 7 B is the fluorescence immunohistochemical detection of the sample to determine the situation of fat infiltration (n = 4, detecting the percentage of the positive area of perilipin + in the area of the section specimen). Figure 7 The green part in B is the Perilipin (perilipin) label, used to show the infiltration of adipocytes. The red part is the Phalloidine (phalloidin) label, showing the cytoskeleton of tissue cells; the blue part is the Hoechst 33342 label, showing the cell nucleus. Figure 7 The statistical chart in C is the statistics of the area of the Perilipin + labeled area. FromFigure 7 From the experimental results, it can be seen that treatment with liposomal vesicles encapsulating hsa-let-7f-5p can reduce the fat infiltration in muscle tissue and play a role in inhibiting muscle fattening. Moreover, compared with the negative control (lipo-scRNA group), the average cross-sectional area of the myotubes labeled with Phalloidine in the lipo-miRNAs group is larger, and the arrangement is more regular and compact, indicating that after injection of liposomal vesicles encapsulating hsa-let-7f-5p, the degree of muscle injury recovery is more ideal.

[0064] RNA drugs, including small interfering RNA (siRNA), microRNA (miRNA), messenger RNA (mRNA), etc., have shown great potential in the treatment of various diseases. However, the instability of RNA molecules themselves and the challenges they face during in vivo delivery (such as enzymatic degradation, immunogenicity, poor targeting specificity, etc.) limit their widespread application. Liposomal vesicles, as carriers of RNA drugs, can effectively overcome these problems, and this scheme attempts to encapsulate liposomal vesicles outside RNA drugs.

[0065] The above are only examples of the present invention. Specific technical solutions and / or common knowledge such as characteristics well known in the art are not described in detail herein. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several deformations and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application shall be subject to the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.

Claims

1. Use of hsa-let-7f-5p in the preparation of a drug for treating muscle steatosis, characterized in that: Its sequence is shown in SEQ ID NO.

1.

2. Use of hsa-let-7f-5p according to claim 1 in the preparation of a medicament for treating muscle steatosis, characterized in that: It is produced from double-stranded RNA; the sense strand of the double-stranded RNA is shown in SEQ ID NO.2, and the antisense strand is shown in SEQ ID NO.

3.

3. Use of hsa-let-7f-5p according to claim 1 or 2 in the preparation of a medicament for treating muscle steatosis, characterized in that: The double-stranded RNA or hsa-let-7f-5p is an inhibitor of adipogenic differentiation of fibroblast / adipocyte progenitor cells.

4. Use of hsa-let-7f-5p according to claim 1 or 2 in the preparation of a medicament for treating muscle steatosis, characterized in that: The double-stranded RNA or hsa-let-7f-5p is an inhibitor of the expression of the Tgfbr3 gene.

5. Use of hsa-let-7f-5p according to claim 1 or 2 in the preparation of a medicament for treating muscle steatosis, characterized in that: Muscle fat infiltration is caused by muscle injury.

6. Use of a double-stranded RNA in the preparation of a medicament for treating muscle injury, characterized in that: Its sense strand is shown in SEQ ID NO.2, and its antisense strand is shown in SEQ ID NO.3; the double-stranded RNA is used to form hsa-let-7f-5p with a sequence shown in SEQ ID NO.

1.

7. Use of a double-stranded RNA according to claim 6 in the preparation of a medicament for treating muscle injury, characterized in that: The double-stranded RNA or hsa-let-7f-5p is used to promote regeneration and repair after muscle injury and to inhibit fat deposition after muscle injury.

8. Use of a double-stranded RNA according to claim 7 in the preparation of a medicament for treating muscle injury, characterized in that: The hsa-let-7f-5p is enriched in exosomes derived from platelet-rich plasma.

9. A liposome vesicle encapsulating the double-stranded RNA according to any one of claims 6-8.

10. A liposomal vesicle encapsulating double-stranded RNA according to claim 9, characterized in that: The raw materials of the liposome vesicle include SPC, DOTAP, cholesterol, and DSPE-PEG-2k; The liposome vesicle encapsulating the double-stranded RNA is prepared by the following method: dissolving SPC, DOTAP, cholesterol, and DSPE-PEG-2k in absolute ethanol to form a lipid mixture; adding a citric acid buffer solution containing the double-stranded RNA and ethanol to the lipid mixture; and then obtaining the liposome vesicle encapsulating the double-stranded RNA after ultrasonic treatment, extrusion treatment with a liposome extruder, and dialysis treatment.

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