Application of hsa-miR-16-5p in preparation of medicine for treating muscle injury

Abstract

The invention relates to the technical field of microRNA (Ribonucleic Acid) medicines, in particular to an application of hsa-miR-16-5p in preparation of a medicine for treating muscle injury. The invention discloses that the hsa-miR-16-5p has the function of inhibiting the expression of the Tgfbr3 gene, and the repair of muscle injury can be realized; the function of inhibiting fibroblast / adipogenic progenitor cell adipodifferentiation is also achieved, then muscle fat infiltration and muscle fat degeneration are inhibited, and the effect of further promoting muscle injury repair is achieved. As a microRNA drug, the microRNA drug can be used in muscle injury repair, muscle fat infiltration or muscle fatty degeneration can be effectively inhibited, and meanwhile, promotion of muscle injury repair is realized by regulating and controlling a signal channel in which Tgfbr3 participates. According to the technical scheme, the technical problem that RNA drugs capable of effectively treating muscle injuries are lacked in the prior art can be solved, and the RNA drugs have ideal application and popularization prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of microRNA drugs, and particularly relates to the application of hsa-miR-16-5p in the preparation of drugs for treating muscle injuries. Background Art

[0002] Strenuous exercise, trauma, toxic effects of chemical substances, etc. can all lead to muscle injuries, which may involve tearing, strain or contusion of muscle fibers, and the integrity of muscle fibers is damaged. There are various methods for treating muscle injuries, aiming to relieve symptoms, promote healing and restore function. Drug treatment mainly targets relieving pain and inflammation. In recent years, with the development of regenerative medicine, the application of stem cell therapy and growth factors has also begun to be explored as a potential treatment strategy, aiming to promote tissue repair and regeneration. However, these new technologies are still in the research stage, and their safety and effectiveness are being further evaluated.

[0003] Adipose infiltration refers to the appearance of adipocytes in the interstitial tissue of organs that normally do not contain adipocytes, mainly occurring in tissues such as the heart, pancreas, skeletal muscle, etc. Adipose infiltration in skeletal muscle (muscle steatosis) is an overlooked health risk factor, which can lead to fat deposition in skeletal muscle. Fat deposition includes the deposition of adipocytes between muscles and the deposition of adipocytes within muscles. The former refers to the deposition of adipocytes in the available space between skeletal muscles, and the latter includes all adipocytes inserted between viable skeletal muscle fibers in the skeletal muscle bed. It should be noted that adipose infiltration in skeletal muscle is an underappreciated health risk factor. It not only causes internal fat deposition in muscles, ultimately leading to muscle steatosis (a disease 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, etc., but is also closely related to the occurrence and development of various diseases.

[0004] The fatty infiltration of skeletal muscle is associated with the occurrence and development of various diseases. During the pathological development of muscle atrophy, with the decrease in the number and volume of muscle fibers, a large number of adipocyte infiltrations and fiber depositions will appear in the atrophied muscle, resulting in the obstruction of muscle regeneration and further promoting the progression of muscle atrophy. The accumulation of adipocytes is not only seen in myopathy, but also in severe neurogenic atrophy, type II diabetes, obesity or age-related muscle atrophy. The accumulation of muscle adipocytes is related to the destructive pathological changes of muscle fibers (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.). There are also literature reports that intramuscular fat deposition, including intermuscular and intramuscular adipocyte depositions, is closely related to obesity, diabetes and metabolic syndrome. Intramuscular fattening is an early muscle structure change that occurs before the appearance of strength and functional abnormalities, and before the metabolic changes associated with obesity and diabetes. The accumulation of fat in muscle, together with muscle fatty degeneration and muscle fiber degeneration, significantly reduces muscle mass and is related to 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] In the prior art, there is still a lack of effective therapeutic drugs for simultaneously treating muscle injury (promoting muscle regeneration and repair) and muscle fatty infiltration (muscle fatty degeneration), and there is an urgent need to further study and screen the corresponding drugs. Summary of the Invention

[0006] The present invention aims to provide an application of miRNA in the preparation of a drug for treating muscle injury, so as to solve the technical problem in the prior art that there is a lack of RNA drugs capable of effectively treating muscle injury.

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

[0008] An application of miRNA in the preparation of a drug for treating muscle injury, which is a double-stranded RNA with a sense strand as shown in SEQ ID NO.2 and an antisense strand as shown in SEQ ID NO.3; the double-stranded RNA is used to form hsa-miR-16-5p with a sequence as shown in SEQ ID NO.1.

[0009] Furthermore, the double-stranded RNA or hsa-miR-16-5p is used to promote regeneration and repair after muscle injury.

[0010] Furthermore, the double-stranded RNA or hsa-miR-16-5p is used to inhibit fat deposition after muscle injury.

[0011] Furthermore, the hsa-miR-16-5p is enriched in exosomes derived from platelet-rich plasma.

[0012] This technical solution also provides an application of miRNA in the preparation of a drug for inhibiting muscle fat infiltration, which is a double-stranded RNA with a sense strand as shown in SEQ ID NO.2 and an antisense strand as shown in SEQ ID NO.3.

[0013] Furthermore, the double-stranded RNA is used to form hsa-miR-16-5p with a sequence as shown in SEQ ID NO.1.

[0014] Furthermore, the double-stranded RNA or hsa-miR-16-5p is an inhibitor of adipogenic differentiation of fibroblast / adipocyte progenitor cells.

[0015] Furthermore, the double-stranded RNA or hsa-miR-16-5p is an inhibitor of Tgfbr3 gene expression.

[0016] Furthermore, muscle fat infiltration is caused by muscle injury.

[0017] The principle and beneficial effects of this technical solution are as follows:

[0018] By deeply studying the role of platelet-rich plasma (PRP) in inhibiting muscle tissue fat infiltration and muscle fatty degeneration, the inventors found that exosomes derived from PRP have significant effects. To further clarify its mechanism, the inventors conducted the following series of experiments:

[0019] Component analysis and function verification:

[0020] The inventors first removed DNA, RNA, or proteins from PRP exosomes separately and performed 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 of PRP-derived exosomes affecting the adipogenic differentiation of these progenitor cells.

[0021] miRNA profiling:

[0022] To identify the specific active components, the inventors comprehensively analyzed the miRNA composition in exosomes using high-throughput sequencing technology. The study found that hsa-miR-16-5p was highly enriched in exosomes, suggesting that this miRNA might be one of the key factors playing a role.

[0023] Synthesis and functional study of miRNA mimics:

[0024] Based on the above findings, the inventors synthesized double-stranded RNA mimics (mimics, used to form hsa-miR-16-5p in cells and tissues) matching the mature sequence of hsa-miR-16-5p and specifically studied its role in inhibiting the adipogenic differentiation of fibroblast / adipocyte progenitor cells. The results showed that hsa-miR-16-5p 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-miR-16-5p was the Tgfbr3 gene.

[0025] Verification in in vivo models:

[0026] In a muscle injury model established by glycerol treatment, the inventors further verified the function of hsa-miR-16-5p. The research results showed that hsa-miR-16-5p effectively prevented muscle tissue fat infiltration caused by muscle injury, thereby promoting the muscle injury repair process. Therefore, hsa-miR-16-5p 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 injuries with symptoms of muscle fat infiltration.

[0027] In summary, this study reveals the importance of hsa-miR-16-5p enriched in PRP-derived exosomes in inhibiting adipogenic differentiation of fibroblast / adipogenic progenitor cells and promoting muscle injury repair related to 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-miR-16-5p is promising for the prevention and treatment of muscle injury and its related fat infiltration problems. hsa-miR-16-5p helps maintain the normal structure and function of muscle tissue and prevent inappropriate fat infiltration. In a muscle injury and fat infiltration model established by glycerol treatment, hsa-miR-16-5p 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. Brief Description of the Drawings

[0028] Figure 1 This is the experimental research result of the in vitro inhibition of adipogenic differentiation of fibroblast / adipogenic progenitor cells by has-miR-16-5p in Example 1 of the present invention.

[0029] Figure 2 This is the experimental result of verifying the target gene of has-miR-16-5p by the dual-luciferase reporter system in Example 2 of the present invention.

[0030] Figure 3 This is the effect of hsa-miR-16-5p on cell adipogenic differentiation under the overexpression of Tgfbr3 in Example 2 of the present invention.

[0031] Figure 4 This is the experimental research result of the in vivo inhibition of fat infiltration after muscle injury by has-miR-16-5p in Example 3 of the present invention.

[0032] Figure 5 This is the experimental research result of the in vivo promotion of muscle regeneration after muscle injury by has-miR-16-5p in Example 3 of the present invention.

[0033] Figure 6 This is the experimental research result of the in vivo promotion of the expression of the early muscle regeneration marker eMyHC by has-miR-16-5p after muscle injury in Example 3 of the present invention.

[0034] Figure 7 This is the identification result of the morphology and markers of exosomes and the effect of PRP-derived exosomes on the adipogenic ability of adipogenic fibroblast / adipogenic progenitor cells detected by Oil Red staining in Example 4 of the present invention.

[0035] Figure 8This is the effect of Oil Red staining detection in Example 4 of the present invention on the adipogenic ability of fibroblast / adipogenic progenitor cells during adipogenic differentiation after removing DNA, RNA, or proteins in PRP exosomes respectively.

[0036] Figure 9 This is the miRNA high-throughput sequencing detection result of Example 4 of the present invention (showing the short fragment sequence reads and proportions of different miRNAs in PRP exosome samples of young and old people). Specific embodiments

[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-miR-16-5p

[0039] The mature sequence of hsa-miR-16-5p is: 5’-UAGCAGCACGUAAAUAUUGGCG-3’ (SEQ ID NO.1).

[0040] According to the mature sequence of hsa-miR-16-5p, double-stranded RNA hsa-miR-16-5p mimics were synthesized, which includes a sense strand and an antisense strand:

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

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

[0043] In the sequence listing, the U in the above SEQ ID NO.1-SEQ ID NO.3 is represented by T substitution. 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-mentioned hsa-miR-16-5p mimic and scRNA, the lyophilized powder of hsa-miR-16-5p mimic and scRNA was prepared into a stock solution for later use. The hsa-miR-16-5p 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 methods of the existing technology. The fibroblast / adipogenic progenitor cells were inoculated at a density of 3×10 5 cells / mL and cultured in DMEM medium for 72 h. Then, the complex formed by the above-mentioned amount of RNA and INVIDNA RNA Transfection Reagent was used to transfect the fibroblast / adipogenic progenitor cells according to the method described in the kit. After 24 h, the medium was replaced with a medium without RNA and INVIDNA RNA Transfection Reagent for cell culture until 96 h. The cells were then subjected to detection of relevant transcriptional expression, and the proportion of perilipin + cells was counted at the same time point to reflect the situation of cell adipogenic differentiation. Perilipin, as a marker, is mainly used to identify and study cell types containing lipid droplets, especially adipocytes and cells differentiating into adipocytes. As one of the main proteins on the surface of lipid droplets, perilipin plays an important role in regulating lipid metabolism.

[0046] After the above treatment, the samples were subjected to immunofluorescence detection to determine the adipogenic differentiation of fibroblast / adipogenic progenitor cells. The experimental results are shown in Figure 1 . The red fluorescence in the image indicates Perilipin, which is used to show the progenitor cells differentiating into adipocytes; the blue fluorescence is labeled with Hoechst 33342, which shows the cell nucleus. The cells labeled with Perilipin were counted, and the percentage of Perilipin + cells in the total cells was calculated. The statistical results are shown in the bar chart on the right in Figure 1 . Figure 1 The proportion of perilipin + cells was statistically analyzed (counting 80 - 150 cells in 3 random fields of view). It can be seen that transfection of fibroblast / adipogenic progenitor cells with hsa-miR-16-5p mimic can effectively inhibit perilipin +The number of cells undergoing adipogenic differentiation of cells, namely fibroblast / adipocyte progenitor cells, decreases. hsa-miR-16-5p mimic is a synthetic double-stranded RNA molecule designed to mimic the function of human miRNA hsa-miR-16-5p. This mimic can be transfected into cells to increase the expression level of the specific miRNA hsa-miR-16-5p, thereby studying its function and mechanism of action in cells. In this protocol, hsa-miR-16-5p mimic is transfected into fibroblast / adipocyte progenitor cells to form hsa-miR-16-5p miRNA with a sequence such as SEQ ID NO.1 in the cells, which plays a role in inhibiting adipogenic differentiation of cells.

[0047] Prior art literature reports that miRNA-16-5p is significantly upregulated during the differentiation of 3T3-L1 preadipocytes into mature adipocytes. Overexpression of miRNA-16-5p promotes the expression of mature adipocyte-specific genes and the accumulation of lipid droplets in vitro and in vivo. miRNA-16-5p promotes adipocyte differentiation by inhibiting EPT1. For details, see the literature: Jingjing Xu, microRNA-16–5p promotes 3T3-L1 adipocyte differentiation through regulating EPT1, Biochemical and Biophysical Research Communications, Volume 514, Issue 4, 5 July 2019, Pages 1251-1256. From the prior art reports, it can be seen that the efficacy of miRNA-16-5p is to promote adipocyte differentiation. After seeing such a technical revelation (the prior art gives a negative technical revelation and there is a certain technical bias), those skilled in the art tend to think that miRNA-16-5p cannot be used to inhibit muscle fatty degeneration (that is, it cannot be used to inhibit adipogenic differentiation of fibroblast / adipocyte progenitor cells). However, through the research of this technical solution, it is found that hsa-miR-16-5p mimics can effectively inhibit adipogenic differentiation of fibroblast / adipocyte progenitor cells, creating a basis for further exploring new uses of hsa-miR-16-5p.

[0048] Example 2: hsa-miR-16-5p regulates the adipogenic differentiation ability of fibroblast / adipocyte progenitor cells by targeting Tgfbr3

[0049] (1) Detection by dual-luciferase reporter system that the Tgfbr3 gene is a target gene of hsa-miR-16-5p

[0050] The gene Tgfbr3 (Transforming Growth Factor Beta Receptor III) encodes the Transforming Growth Factor Beta 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. A plasmid with the Tgfbr3 3'UTR sequence was constructed using the dual-luciferase reporter system. 10 μl of DMEM was mixed well with 0.16 μg of the target plasmid (m-tgfbr3-3’UTR) with the Tgfbr3 3'UTR sequence and 5 pmol of hsa-miR-16-5p (the aforementioned double-stranded hsa-miR-16-5p mimics) / Negative.Control (NC, NC mimics, scRNA), and then placed at room temperature (Solution A). Then, 10 μl of DMEM was mixed well with 0.3 μl of the transfection reagent (Solution B), placed at room temperature for 5 min, and then Solution A and Solution B were mixed well. Before transfection, the cells were replaced with fresh medium, and then the transfection mixture was added and mixed well. The cells were cultured at 37°C and 5% CO2. After 6 h of transfection, the fresh medium was replaced, and the cells were collected for detection after 48 h of transfection. 20 μl of cell lysate was taken, 100 μl of Luciferase Reaction Reagent equilibrated to room temperature was added, and after mixing, the fLuc signal was detected using a microplate reader; then 100 μl of Luciferase Reaction Reagent II equilibrated to room temperature was added, and after mixing, the rLuc signal was detected using a microplate reader.

[0051] WT is the wild-type m-tgfbr3-3’UTR sequence, Mut is the mutant m-tgfbr3-3’UTR, NC mimics is the control miRNA, and the ratio of the fLuc signal to the rLuc signal was calculated. The experimental results are shown in Figure 2 , and the dual-luciferase reporter system detected that the Tgfbr3 gene is a target gene of hsa-miR-16-5p.

[0052] Compared with the NC group, hsa-miR-16-5p significantly downregulated the luciferase expression of m-tgfbr3-3UTR-WT (P < 0.001), indicating that there is a binding interaction between hsa-miR-16-5p and m-tgfbr3-3’UTR-WT in this experiment. See the comparison between the WT+NC mimics group and the WT+hsa-miR-16-5p group for details.

[0053] After mutation, compared with the NC group, hsa-miR-16-5p failed to down-regulate the luciferase expression of m-tgfbr3-3’UTR-MUT (P>0.05), indicating successful mutation. See the comparison between the Mut+NC mimics group and the Mut+hsa-miR-16-5p group for details. hsa-miR-16-5p realizes its regulatory function through the 3’UTR of the tgfbr3 gene, and the tgfbr3 gene is the target of hsa-miR-16-5p.

[0054] More specifically, the Tgfbr3 (Transforming Growth Factor Beta Receptor III) gene encodes the Transforming Growth Factor Beta Receptor III (TGF-β Receptor III), also known as beta-glycan. This receptor is expressed on the cell surface and is an important part of the TGF-β signaling pathway. The Tgfbr3 gene is closely related to muscle injury repair and adipogenic differentiation of fibroblast / adipogenic progenitor cells. Especially regarding its role in muscle injury repair, the Tgfbr3 gene participates in the TGF-β signaling pathway, and during muscle injury repair, the TGF-β signaling pathway is involved in regulating various cell 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 experiments on a muscle injury model induced by glycerol demonstrated that treating muscle injury with hsa-miR-16-5p mimics can effectively and comprehensively promote muscle injury repair. The inventor analyzed that the reason is that hsa-miR-16-5p can play a comprehensive positive regulatory role in aspects such as neonatal muscle fiber formation, extracellular matrix synthesis and deposition, inflammatory response, and fat deposition during muscle injury repair by acting on the target gene Tgfbr3 gene.

[0055] (2) hsa-miR-16-5p can inhibit the adipogenic differentiation of fibroblast / adipogenic progenitor cells overexpressing the Tgfbr3 gene

[0056] In vitro, transfect the Tgfbr3 overexpression plasmid (TGFBR OE) or empty vector (vector) and hsa-miR-16-5p (the aforementioned double-stranded hsa-miR-16-5p mimics) or scRNA, and then induce the adipogenic differentiation of fibroblast / adipogenic progenitor cells with an adipogenic differentiation induction medium, and detect the adipocyte formation ability by oil red staining. That is, in fibroblast / adipogenic progenitor cells transfected with the Tgfbr3 overexpression plasmid or transfected with the empty vector, transfect hsa-miR-16-5p or scRNA according to the aforementioned method, and detect the adipocyte formation ability by oil red staining. See the experimental results in Figure 3(n = 3, the percentage of the Oil Red area in the total field of view was statistically analyzed), showing the regulatory effect of Oil Red staining in detecting the overexpression of Tgfbr3 on the inhibition of adipogenic differentiation of fibroblast / adipocyte progenitor cells by hsa-miR-16-5p. In fibroblast / adipocyte progenitor cells with overexpression of the Tgfbr3 gene, the overexpression of the Tgfbr3 gene leads to a decrease in the adipogenic differentiation of fibroblast / adipocyte progenitor cells. At the same time, increasing the amount of hsa-miR-16-5p in these cells will reduce the area of adipocytes, that is, reduce the adipogenic differentiation of fibroblast / adipocyte progenitor cells ( Figure 3 the blue statistical column in

[0057] Example 3: In Vivo Experimental Study on the Promotion of Muscle Injury Repair by hsa-miR-16-5p

[0058] 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 reactions when locally injected into muscle at a high concentration, thus simulating muscle injury. One hour after glycerol injection, 4 μg of hsa-miR-16-5p (the aforementioned double-stranded hsa-miR-16-5p mimics) or NC (scRNA) 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. This model can be used to study the mechanisms of skeletal muscle regeneration, repair, fat infiltration, and test potential treatment strategies. By locally injecting a high concentration of glycerol to induce necrotic injury of muscle tissue, after the injury, fat deposition will occur in skeletal muscle, affecting muscle regeneration and repair.

[0059] 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 4 (n = 4, detecting the percentage of the positive area of perilipin + in the area of the section specimen). Figure 4 The green part in Figure 4 is labeled with Perilipin to show adipocyte infiltration, the red part is labeled with Phalloidine to show the cytoskeleton of tissue cells; the blue part is labeled with Hoechst 33342 to show the cell nucleus. + The statistical chart on the right is the statistics of the area of the region labeled with Perilipin.Figure 4 The experimental results show that treatment with hsa-miR-16-5p can reduce fat infiltration in muscle tissue and inhibit muscle adipogenesis (muscle fatty degeneration).

[0060] In addition, the hsa-miR-16-5p treatment in this protocol can not only inhibit the adipogenesis of fibroblast / adipogenic progenitor cells, but also promote the regeneration and repair process after skeletal muscle injury. Phalloidin is a toxin commonly used to label and visualize actin fibers (F-actin). It has a high affinity for F-actin, and the actin structure in the cytoskeleton can be observed through fluorescently labeled phalloidin. When studying the degree of recovery from muscle injury, the use of phalloidin staining can help evaluate the integrity of the muscle fiber structure and its repair effect. Figure 4 It can be seen that the hsa-miR-16-5p group has a more regular and compact arrangement of the cytoskeleton of the tissue labeled by Phalloidine compared to the negative control, indicating that after the injection of double-stranded hsa-miR-16-5p mimics, the recovery of muscle damage is more ideal. It can be seen that hsa-miR-16-5p not only has the effect of inhibiting the adipogenic differentiation of fibroblasts / adipogenic progenitor cells, but also has the effect of promoting the recovery of muscle damage (specifically, promoting the regeneration and repair of damaged muscle fibers). A drug can be used in the treatment of muscle damage, not only to effectively inhibit muscle fatty degeneration, but also to promote the regeneration and repair of skeletal muscle fibers, improve the regularity and tightness of muscle fiber arrangement, and restore the integrity of muscle fiber structure.

[0061] In addition to the above studies, the inventors also conducted the following studies: According to the above method, after constructing a glycerol injury model, scRNA (control group) or hsa-miR-16-5p (the above double-stranded hsa-miR-16-5p mimics) was injected into the muscle, and the muscles were collected for frozen sections on the 14th day after injury. The cross-sectional area of ​​myotubes was detected by immunofluorescence histochemistry using phalloidin. The experimental results are detailed in Figure 5 , showing that injection of hsa-miR-16-5p promoted muscle regeneration and the muscle fiber area was effectively increased (scale bar = 100 μm).

[0062] According to the above method, after the glycerol injury model was constructed, scRNA (control group) or hsa-miR-16-5p (the above double-stranded hsa-miR-16-5p mimics) was injected into the muscle, and the muscle was collected for frozen sections on the third day after injury. Figure 6, The expression area of the early muscle regeneration marker eMyHC was detected by immunohistochemistry, and laminin was used to show the shape of myotubes (scale bar = 50 μm). The experimental results showed that hsa-miR-16-5p had the effect of promoting the regeneration of damaged muscle fibers.

[0063] This further demonstrated that hsa-miR-16-5p could be applied to the treatment of muscle injury because it could not only effectively inhibit muscle fatty degeneration, but also promote the regeneration and repair of skeletal muscle fibers. Some studies reported that hsa-miR-16-5p promoted myoblast apoptosis and inhibited myoblast differentiation through SESN1. However, in this study, it was found that hsa-miR-16-5p could effectively promote muscle repair and regeneration in the scenario of muscle injury, promoting the repair of skeletal muscle injury, which was different from the existing research results. The inventor analyzed that the reason was that muscle injury involved complex processes such as inflammatory response, synthesis and deposition of extracellular matrix, and neonatal muscle fibers, which was different from the ordinary muscle cell growth and differentiation process and environment. hsa-miR-16-5p showed its significant ability to repair muscle injury in the scenario of muscle injury.

[0064] Example 4: Discovery of hsa-miR-16-5p

[0065] It was found that platelet-rich plasma (PRP) had 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 could 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 was the key component mediating the inhibition of adipogenesis by exosomes derived from PRP. Through miRNA high-throughput sequencing, hsa-miR-16-5p was screened out as a key molecule that might inhibit adipogenesis by exosomes derived from PRP.

[0066] The exosomes in PRP were extracted by the conventional ultracentrifugation method in the prior art for identification. The experimental results are shown in Figure 7 . Transmission electron microscopy was used to detect the morphology of exosomes ( Figure 7 upper left), and Western blotting was used to detect the expression of typical markers of exosomes ( Figure 7 upper right). Oil red staining was used to detect the effect of exosomes derived from PRP on the adipogenic ability of fibroblast / adipogenic progenitor cells during adipogenic differentiation ( Figure 7 lower, n = 4, detecting the percentage of the positive area of perilipin + occupying the area of the section specimen), indicating that the use of exosomes derived from PRP could effectively inhibit the adipogenic differentiation of fibroblast / adipogenic progenitor cells.

[0067] The DNA, RNA, or proteins in PRP exosomes were removed separately, 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 RNA removal, suggesting that RNA is a key component of PRP-derived exosomes affecting the adipogenic differentiation of fibroblast / adipogenic progenitor cells. Through Oil Red staining detection, the experimental results are shown in Figure 8 (n = 5, detecting perilipin + percentage of the positive area accounting for the area of the section specimen).

[0068] High-throughput sequencing of miRNAs was used to detect the miRNA components enriched in PRP-derived exosomes, and it was found that hsa-miR-16-5p was highly enriched in such exosomes ( Figure 9 ). In PRP-derived exosomes, the number and proportion of short fragment sequences (reads) of hsa-miR-16-5p were found to be relatively high through sequencing. Therefore, in this technical solution, double-stranded mimics were designed based on the mature sequence of hsa-miR-16-5p, and then its effect on inhibiting muscle fattening was studied, and a potential RNA drug for the treatment of related diseases was discovered.

[0069] The above are only embodiments of the present invention, and common general technical solutions and / or characteristics in the solutions are not described in detail here. It should be noted that for those skilled in the art, without departing from the technical solution of the present invention, several modifications 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 should be subject to the content of its claims, and the specific implementation manners and the like recorded in the specification can be used to interpret the content of the claims.

Claims

1. Use of a miRNA in the preparation of a medicament for treating muscle injury, characterized in that: It is a double-stranded RNA with a sense strand as shown in SEQ ID NO.2 and an antisense strand as shown in SEQ ID NO.3; the double-stranded RNA is used to form hsa-miR-16-5p with a sequence as shown in SEQ ID NO.

1.

2. Use of an miRNA according to claim 1 in the preparation of a medicament for treating muscle injury, characterized in that: The double-stranded RNA or hsa-miR-16-5p is used to promote regeneration and repair after muscle injury.

3. Use of a miRNA according to claim 2 in the preparation of a medicament for treating muscle injury, characterized in that: The double-stranded RNA or hsa-miR-16-5p is used to inhibit fat deposition after muscle injury.

4. Use of a miRNA according to claim 3 in the preparation of a medicament for treating muscle injury, characterized in that: The hsa-miR-16-5p is enriched in exosomes derived from platelet-rich plasma.

5. Use of a miRNA in the preparation of a drug for inhibiting muscle fat infiltration, characterized in that: It is a double-stranded RNA with a sense strand as shown in SEQ ID NO.2 and an antisense strand as shown in SEQ ID NO.

3.

6. Use of a miRNA according to claim 5 in the preparation of a drug for inhibiting muscle fat infiltration, characterized in that: The double-stranded RNA is used to form hsa-miR-16-5p with a sequence as shown in SEQ ID NO.

1.

7. Use of a miRNA according to claim 5 or 6 in the preparation of a drug for inhibiting muscle fat infiltration, characterized in that: The double-stranded RNA or hsa-miR-16-5p is an inhibitor of adipogenic differentiation of fibroblast / adipocyte progenitor cells.

8. Use of a miRNA according to claim 5 or 6 in the preparation of a drug for inhibiting muscle fat infiltration, characterized in that: The double-stranded RNA or hsa-miR-16-5p is an inhibitor of Tgfbr3 gene expression.

9. Use of a miRNA according to claim 5 or 6 in the preparation of a drug for inhibiting muscle fat infiltration, characterized in that: Muscle fat infiltration is caused by muscle injury.

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