Application of circular RNA circDdb1 as target spot in preparation of medicine for preventing and treating muscle atrophy

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of circular RNAcircDdb1 as a target spot in preparation of a medicine for preventing and treating muscular atrophy. The circular RNAcircDdb1 disclosed by the invention can be used as a target spot for preventing and treating muscle atrophy, and the condition of muscle atrophy of an organism can be effectively improved by inhibiting the expression of the circular RNAcircDdb1 in the organism. Experiments prove that the inhibition of the circular RNA circDdb1 can improve angiotensin II, denervation and fixation induced muscle atrophy, improve the muscle strength of a mouse injected with the angiotensin II, improve the gastrocnemius muscle weight of a muscle atrophy model mouse induced by various factors, and improve the muscle strength of the mouse. The muscle fiber cross section area of a muscle atrophy model mouse induced by various factors is increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of circular RNA circDdb1 as a target in the preparation of drugs for preventing and treating muscle atrophy. Background Art

[0002] Skeletal muscle is an organ that maintains the body's autonomous movement and posture, and can serve as the largest protein reserve in the body. Skeletal muscle changes the rate of protein synthesis and degradation through signaling pathways that affect protein turnover, and constantly adjusts its quality and function to adapt to physiological and pathological stimuli. However, under some pathological stimuli, the rate of protein degradation in skeletal muscle is greater than the rate of protein synthesis, which in turn leads to muscle atrophy. The main pathological characteristics of muscle atrophy are skeletal muscle dystrophy, and the cross-sectional area of ​​muscle fibers is significantly reduced or disappears. In addition, muscle atrophy can cause patients to have decreased muscle strength or disability, and in severe cases, death, which is very unfavorable for the patient's prognosis. At the same time, a variety of causes, including chronic systemic diseases, heart failure, disuse, aging, denervation, and intrinsic muscle diseases, are often accompanied by the occurrence of muscle atrophy. Therefore, improving muscle strength and maintaining the size of muscle fiber cross-sectional area are key links in preventing muscle atrophy. Although muscle atrophy has considerable consequences for the quality of life of patients worldwide, there are almost no effective therapeutic drugs except rehabilitation and nutritional therapy. Therefore, finding effective targets for the treatment of muscle atrophy has important medical value for the treatment of muscle atrophy.

[0003] In eukaryotes, about 2% of the RNA transcribed from the genome can translate proteins, and more than 70% is non-coding RNA. As the name implies, non-coding RNA is RNA that does not translate proteins, which mainly includes microRNA, long non-coding RNA and circular RNA. Non-coding RNA regulates gene expression at the transcription, RNA processing, translation and epigenetic levels. Therefore, non-coding RNA participates in the regulation of a variety of biological functions. It can modify chromatin by interacting with protein complexes, thereby regulating the growth and development of organisms, and can also participate in the regulation of oxidative stress-mediated genes and the proliferation and migration of vascular smooth muscle cells. In addition, in patients with diseases such as cancer, diabetes, mitochondrial damage and chronic kidney disease, non-coding RNA has been found to be abnormally expressed. Therefore, non-coding RNA plays an important role in a variety of human diseases.

[0004] Circular RNA is a type of closed circular non-coding RNA that does not contain a 5' cap structure and a 3' terminal poly(A) tail, and is therefore not affected by RNA exonucleases and is not easily degraded. They are produced by reverse splicing events. In recent years, thousands of circular RNAs have been identified in eukaryotic organisms including fungi, protists, plants, worms, fish, insects, and mammals using high-throughput RNA sequencing (RNA-seq) and circular RNA-specific bioinformatics algorithms, and have been found to have tissue-specific expression patterns. Circular RNAs have been shown to play an important regulatory role in the study of diabetes, cardiovascular disease, chronic inflammatory diseases, and neurological diseases.

[0005] However, current research on the function of circular RNA in the field of skeletal muscle mainly focuses on skeletal muscle proliferation and differentiation, and no relevant reports have been found in skeletal muscle atrophy. Summary of the invention

[0006] The purpose of the present invention is to use the circular RNA circDdb1 as a target in the preparation of a drug for preventing and treating muscle atrophy. The circular RNA circDdb1 can be used as a target for preventing and treating muscle atrophy, and by inhibiting the expression of the circular RNA circDdb1 in the body, the muscle atrophy of the body can be effectively improved.

[0007] The present invention provides the use of circular RNA circDdb1 as a target in the preparation of a drug for preventing and treating muscle atrophy.

[0008] The present invention also provides the use of an agent for inhibiting the expression of circular RNA circDdb1 in the preparation of a drug for preventing and treating muscle atrophy.

[0009] Preferably, the reagent comprises a biological material for knocking out or knocking down the circular RNA circDdb1.

[0010] Preferably, the reagent comprises one or more of a nucleic acid, a nucleic acid construct and a viral particle that interferes with the expression of the circular RNA circDdb1.

[0011] Preferably, the nucleic acid comprises shRNA or siRNA, and the nucleotide sequence of the shRNA is shown in SEQ ID NO.1;

[0012] The siRNA includes si-circDdb1-1 or si-circDdb1-2; the si-circDdb1-1 is composed of the sense chain shown in SEQ ID NO.5 and the antisense chain shown in SEQ ID NO.6; the si-circDdb1-2 is composed of the sense chain shown in SEQ ID NO.7 and the antisense chain shown in SEQ ID NO.8.

[0013] Preferably, the nucleic acid construct comprises an interfering viral vector, and the interfering viral vector contains the shRNA shown in SEQ ID NO.1.

[0014] Preferably, the initial vector in the interfering viral vector includes an adenoviral vector, a retroviral vector or a lentiviral vector.

[0015] Preferably, the interfering viral vector comprises pENN-sh-circDdb1, and the nucleotide sequence of pENN-sh-circDdb1 is shown in SEQ ID NO.2.

[0016] The present invention also provides a drug for preventing and treating muscle atrophy, which comprises an agent for inhibiting the expression of circular RNA circDdb1 and a pharmaceutically acceptable excipient.

[0017] Preferably, the reagent comprises one or more of a nucleic acid, a nucleic acid construct and a viral particle that interferes with the expression of the circular RNAcircDdb1;

[0018] The nucleic acid comprises shRNA or siRNA, and the nucleotide sequence of the shRNA is shown in SEQ ID NO.1;

[0019] The siRNA includes si-circDdb1-1 or si-circDdb1-2; the si-circDdb1-1 is composed of the sense strand shown in SEQ ID NO.5 and the antisense strand shown in SEQ ID NO.6; the si-circDdb1-2 is composed of the sense strand shown in SEQ ID NO.7 and the antisense strand shown in SEQ ID NO.8;

[0020] The nucleic acid construct comprises an interfering viral vector, wherein the interfering viral vector contains the shRNA shown in SEQ ID NO.1.

[0021] Beneficial effects:

[0022] The present invention provides the use of circular RNA circDdb1 as a target in the preparation of a drug for preventing and treating muscle atrophy. Specifically, the present invention also provides the use of an agent for inhibiting the expression of circular RNA circDdb1 in the preparation of a drug for preventing and treating muscle atrophy. The circular RNA circDdb1 of the present invention can be used as a target for preventing and treating muscle atrophy, and by inhibiting the expression of the circular RNA circDdb1 in the body, the muscle atrophy of the body can be effectively improved. Experiments have shown that the inhibition of the circular RNA circDdb1 can improve angiotensin II, denervation and fixation-induced muscle atrophy at the experimental animal level, improve the muscle strength of angiotensin II-injected mice, increase the gastrocnemius weight of mice with muscle atrophy induced by multiple factors, and increase the muscle fiber cross-sectional area of ​​mice with muscle atrophy induced by multiple factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0024] Figure 1 This is a schematic diagram of the experimental process of the virus injection and angiotensin II-induced mouse muscle atrophy model in Example 2;

[0025] Figure 2 The effect of intramuscular injection of AAV8-pENN-sh-circDdb1 virus on the muscle strength of mice with angiotensin II-induced muscular atrophy in the mouse grasping strength test in Example 2;

[0026] Figure 3 The improvement effect of intramuscular injection of AAV8-pENN-sh-circDdb1 virus on angiotensin II-induced muscle atrophy in mice was detected by gastrocnemius muscle morphology and weight in Example 2;

[0027] Figure 4 The WGA staining in Example 2 was used to detect the effect of intramuscular injection of AAV8-pENN-sh-circDdb1 virus on the cross-sectional area of ​​gastrocnemius muscle fibers in mice with angiotensin II-induced muscular atrophy;

[0028] Figure 5 HE staining was used to detect the effect of intramuscular injection of AAV8-pENN-sh-circDdb1 virus on the cross-sectional area of ​​gastrocnemius muscle fibers in mice with angiotensin II-induced muscular atrophy in Example 2;

[0029] Figure 6 This is a schematic diagram of the experimental process of the virus injection and denervation-induced mouse muscle atrophy model in Example 3;

[0030] Figure 7The improvement effect of intramuscular injection of AAV8-pENN-sh-circDdb1 virus on denervation-induced muscle atrophy in mice was detected by gastrocnemius muscle morphology and weight in Example 3;

[0031] Figure 8 The WGA staining in Example 3 was used to detect the effect of intramuscular injection of AAV8-pENN-sh-circDdb1 virus on the cross-sectional area of ​​gastrocnemius muscle fibers in mice with denervation-induced muscular atrophy;

[0032] Fig. 9 HE staining was used to detect the effect of intramuscular injection of AAV8-pENN-sh-circDdb1 virus on the cross-sectional area of ​​gastrocnemius muscle fibers in mice with denervation-induced muscular atrophy in Example 3;

[0033] Fig.10 This is a schematic diagram of the experimental process of virus injection and fixation to induce mouse muscle atrophy model in Example 4;

[0034] Fig.11 The improvement effect of intramuscular injection of AAV8-pENN-sh-circDdb1 virus on muscle atrophy induced by immobilization in mice was detected by gastrocnemius muscle morphology and weight in Example 4;

[0035] Fig.12 The WGA staining in Example 4 was used to detect the effect of intramuscular injection of AAV8-pENN-sh-circDdb1 virus on the cross-sectional area of ​​gastrocnemius muscle fibers in mice with immobilization-induced muscular atrophy;

[0036] Fig.13 HE staining was used to detect the effect of intramuscular injection of AAV8-pENN-sh-circDdb1 virus on the cross-sectional area of ​​gastrocnemius muscle fibers in mice with immobilization-induced muscular atrophy in Example 4;

[0037] Fig.14 is the sequence map of the pENN plasmid in Example 1;

[0038] Fig.15 The results of silencing circDdb1 in C2C12 myotube cells and myotube atrophy models by si-circDdb1-1 and si-circDdb1-2 in Example 5;

[0039] Fig.16 The expression of MuRF-1 and Atrogin-1 in the myotube atrophy model induced by dexamethasone, tumor necrosis factor-α, and angiotensin II in which circDdb1 expression was knocked down in Example 5. DETAILED DESCRIPTION

[0040] The present invention provides the use of circular RNA circDdb1 as a target in the preparation of a drug for preventing and treating muscle atrophy. The accession number of the RNA circDdb1 in the present invention in circBase is mmu_circ_0007604.

[0041] Specifically, the present invention also provides the use of an agent for inhibiting the expression of circular RNAcircDdb1 in the preparation of a drug for preventing and treating muscle atrophy. The agent of the present invention preferably includes a biological material for knocking out or knocking down the circular RNAcircDdb1. The agent of the present invention preferably includes one or more of a nucleic acid, a nucleic acid construct and a viral particle that interferes with the expression of the circular RNAcircDdb1, and more preferably a nucleic acid, a nucleic acid construct or a viral particle that interferes with the expression of the circular RNAcircDdb1.

[0042]

[0043] The virus particles of the present invention are preferably obtained by packaging the nucleic acid construct with a virus. The method of packaging the virus is not particularly limited and can be performed by conventional virus packaging methods in the art or by entrusting a third party.

[0044] The siRNA of the present invention preferably includes si-circDdb1-1 or si-circDdb1-2; the si-circDdb1-1 preferably consists of the sense strand shown in SEQ ID NO.5 and the antisense strand shown in SEQ ID NO.6; the si-circDdb1-2 preferably consists of the sense strand shown in SEQ ID NO.7 and the antisense strand shown in SEQ ID NO.8. The present invention preferably achieves the inhibition of the expression of circular RNA circDdb1 by transfecting the siRNA through cells.

[0045] The circular RNA circDdb1 of the present invention can be used as a target for preventing and treating muscle atrophy. By inhibiting the expression of the circular RNA circ Ddb1 in the body, the muscle atrophy of the body can be effectively improved.

[0046] The present invention also provides a drug for preventing and treating muscular atrophy, the drug comprising an agent for inhibiting the expression of circular RNAcircDdb1 and a pharmaceutically acceptable excipient. The agent of the present invention preferably comprises one or more of nucleic acids, nucleic acid constructs and viral particles that interfere with the expression of the circular RNAcircDdb1, more preferably nucleic acids, nucleic acid constructs or viral particles that interfere with the expression of the circular RNAcircDdb1; the relevant features of the nucleic acids, nucleic acid constructs and viral particles of the present invention have been defined and preferred in the above technical scheme, and will not be repeated. The excipients of the present invention preferably include but are not limited to pharmaceutically acceptable carriers, diluents or excipients. The present invention does not specifically limit the dosage of the agent in the drug, and it can be adjusted routinely according to the preparation requirements.

[0047] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0048] Example 1

[0049] The construction method of circular RNA-circDdb1 interference virus is as follows:

[0050] 1. Construction of pENN-sh-circDdb1 plasmid:

[0051] 1) Primer design

[0052] The mmu_circ_0007604 sequence was searched, and primers were designed across the reverse splicing site, as shown in Table 1. The knockdown plasmid used pENN as the vector (prepared by the laboratory, published in doi:10.1002 / advs.202206732.), with BamHI and EcoR I as restriction sites. The plasmid map of the pENN vector is shown in Fig.14 shown.

[0053] Table 1 Primer sequences

[0054]

[0055] 2) Plasmid construction

[0056] Knockdown plasmid construction

[0057] a) Annealing the primers according to the system described in Table 2:

[0058] Table 2 Annealing system (50 μL)

[0059]

[0060] Reaction conditions: 95°C, 4 min; naturally cooled to room temperature;

[0061] b) Annealing the carrier according to the system described in Table 3:

[0062] Table 3 Enzyme digestion system (30 μL)

[0063]

[0064] Reaction conditions: 37°C, 30 min;

[0065] c) The annealed product is connected to the carrier according to the connection system in Table 4:

[0066] Table 4 Connection system (10 μL)

[0067]

[0068] Reaction conditions: 16°C, overnight ligation;

[0069] d) Transformation of ligation products:

[0070] d1. Mix 5 μL of ligation product with 50 μL of competent cells Stabl3 and place on ice for 20 min.

[0071] d2, heat shock at 42°C for 45 seconds, and place on ice for another 3 minutes;

[0072] d3, add 500 μL of LB liquid medium without ampicillin, and incubate at 37°C, 200 rpm constant temperature shaker for 45 min;

[0073] d4, centrifuge at 3000 rpm for 5 min, discard the supernatant, mix the remaining 200 μL, apply to LB solid medium containing ampicillin, and culture in a 37°C incubator for 14 h;

[0074] d5. Select a single clone, inoculate it into 6 mL of LB liquid medium containing ampicillin, culture it at 37°C in a shaking incubator for 16 h, extract the plasmid, and send it to the company for sequencing;

[0075] d6. The recombinant plasmid with the correct sequencing result was stored at -20°C for future use, thus obtaining the pENN-sh-circDdb1 plasmid.

[0076] 2. Adeno-associated virus packaging, separation and purification:

[0077] a. Plate 293T cells at a density of 4 million per dish in a 10 cm cell culture dish, about 40 dishes per batch;

[0078] b. When the cell density reaches about 90%, transfection is performed. 10 μg AAV Construct (pENN-sh-circDdb1 prepared in step 1), 10 μg helper plasmid, 10 μg AAV serotype (adeno-associated virus / AAV serotype plasmid, AAV8 plasmid was used in this experiment), 90 μL PEI (total volume of three plasmids: PEI volume = 1:3-1:4) and 1 mL serum-free DMEM are added dropwise to each dish after mixing and standing for 15 minutes;

[0079] c. 10h-12h after transfection, replace with fresh complete medium. After 48h, collect the upper layer of cell medium and cells into a 50mL centrifuge tube;

[0080] d. Centrifuge the virus collected in a 50 mL centrifuge tube at 4000 rpm, 4°C, for 30 min;

[0081] e. Collect the supernatant in a culture medium bottle, add 1 / 4 volume of PEG 8000, stir for 1 hour at 4°C, and let stand overnight. Resuspend the cell pellets after centrifugation one by one in the last 50mL centrifuge tube with 2mL cell lysis buffer (Lysis buffer: 150mM NaCl, 20mM Tris pH=8.0), add 2mL cell lysis buffer and repeat the above steps, and collect together with the previous 2mL cell lysis buffer, and freeze in -80°C anhydrous ethanol overnight;

[0082] f. The virus supernatant that has been left to stand overnight was packaged, and the supernatant was discarded at 4000 rpm for 30 min;

[0083] g. Add 2 mL of cell lysis buffer (Lysis buffer: 150 mM NaCl, 20 mM Tris pH = 8.0) to the remaining virus pellet after centrifugation in f, resuspend each one in the last 50 mL centrifuge tube, then add 2 mL of cell lysis buffer and repeat the above steps, and collect together with the previous 2 mL of cell lysis buffer;

[0084] h. Freeze and thaw the cell pellet collected in e for three times (-80°C anhydrous ethanol bath, 37°C water bath), mix it with the virus pellet resuspension in g into a tube, add cell lysis buffer and make up to 20 mL;

[0085] i. Add the corresponding amount of 1M MgCl2 to a final concentration of 1mM, add the corresponding amount of Merck nuclease to a final concentration of 250U / mL, shake and mix, and incubate at 37℃ for 1h;

[0086] j. Centrifuge at 4000 rpm, 4°C for 30 min and collect the supernatant;

[0087] k. Slowly add 4 mL of 17% by volume, 4 mL of 25% by volume, 4 mL of 40% by volume, and 3 mL of 60% by volume iodixanol to the centrifuge tube from bottom to top;

[0088] 1. Slowly add the concentrated supernatant and cell lysate after centrifugation to the top layer of the centrifuge tube, and fill the centrifuge tube with cell lysis buffer;

[0089] m.60000rpm, 4℃, centrifugation for 2h, rotor 70Ti, acceleration 6, deceleration 9 (BECKMAN COULTER, OptimaXPN-100Ultracentrifuge);

[0090] n. Use a syringe to draw 40% concentration of iodixanol and transfer it to the concentrator;

[0091] o. Fill the concentrator tube with 1X PBS, invert to mix, centrifuge at 4000 rpm, 4°C for 1 h, repeat 3 times;

[0092] p. The liquid volume is finally concentrated to 500-1000 μL, and the liquid sample is the AAV virus we prepare;

[0093] q. Take 5 μL AAV virus, use tissue DNA purification kit to extract AAV virus DNA, dilute the viral DNA 100 times and use it for virus titer detection;

[0094] r. Use gradient dilutions of AAV for detection, and use the virus titer determination method to make a standard curve using AAV standard plasmid to absolutely quantify viral DNA.

[0095] The virus titers of AAV8-pENN-sh-control prepared multiple times (the preparation steps are the same as AAV8-pENN-sh-circDdb1, the only difference is that the target gene expression plasmid is pENN empty plasmid) are: 3.4×10 12 vg / mL, 4.4×10 12 vg / mL and 4.9×10 12 vg / mL;

[0096] The titers of AAV8-pENN-sh-circDdb1 prepared multiple times were: 3.7×10 12 vg / mL, 3.4×10 12 vg / mL and 4.5×10 12 vg / mL;

[0097] The AAV virus after titer determination, i.e., AAV8-pENN-sh-circDdb1 virus, can be directly used for animal experiments or frozen at -80°C.

[0098] Example 2

[0099] 1. The AAV8-pENN-sh-circDdb1 virus was injected into the muscle (gastrocnemius) of the right hind limb of C57BL / 6 male mice through an insulin needle. The virus dose injected per mouse was 1×10 11 vg;

[0100] The injection method and injection dose of the control virus AAV8-pENN-sh-control were the same as those of the AAV8-pENN-sh-circDdb1 virus. Three weeks later, the muscle atrophy model was established.

[0101] 2. Establishment of angiotensin II (Ang II)-induced muscle atrophy model, the process is as follows Figure 1 As shown, the dose of AAV8 virus injected intramuscularly into each mouse was 1×10 11 vg, 3 weeks after virus injection, mice were treated with angiotensin II pump for 1 week, and then sacrificed and evaluated.

[0102] Inject angiotensin II (Ang II) buffer (2 μg / μL) into the minipump in advance and wrap it with tin foil to avoid light. The buffer is 0.9% (mass fraction) saline containing 0.006% (volume fraction) acetic acid. First, 4% chloral hydrate was injected into the abdominal cavity of the mouse at a dose of 10 μL / g for anesthesia. After anesthesia, the mouse was fixed in a prone position. Depilatory cream was used to remove hair on the right side of the back of the mouse. 75% ethanol was used to disinfect the skin on the right side of the back. Then, microscissors were used to cut the skin horizontally in the area near the neck on the right side of the back, and the skin was separated from the muscle tissue with a blunt instrument. Then, the minipump was implanted into the skin of the mouse with the pump head facing forward, and the skin was sutured with medical sutures. Finally, it was placed on a 37°C electric blanket to wake it up and transferred to a breeding cage for breeding;

[0103] In the control group, PBS solution was injected into the minipump, and the rest of the steps were the same as the experimental group. After seven days, the mice were killed and samples were taken for detection and analysis.

[0104] 3. According to the steps in 1 and 2, the experimental groups were set up and divided into 4 groups, namely AAV8-pENN-sh-control virus control group + PBS group, AAV8-pENN-sh-control virus control group + Ang II group, AAV8-pENN-sh-circDdb1 virus intervention group + PBS group, AAV8-pENN-sh-circDdb1 virus intervention group + Ang II group. Three weeks after the virus was injected into the mice by intramuscular injection, Ang II was used to induce muscle atrophy.

[0105] The specific method is as follows:

[0106] The first group, AAV8-pENN-sh-control virus control group + PBS group, was 10 11 The dose of vg virus / mouse was 1:1. AAV8-pENN-sh-control virus was injected intramuscularly into the gastrocnemius muscle of mice. Three weeks later, a minipump containing PBS was implanted into the mice. One week later, the mice were killed and samples were taken for testing.

[0107] The second group, AAV8-pENN-sh-control virus control group + Ang II group, was 10 11 The dose of vg virus / mouse was 1:1. AAV8-pENN-sh-control virus was injected intramuscularly into the gastrocnemius muscle of mice. After three weeks, Ang II was used to induce muscle atrophy in mice. After another week, the mice were killed and samples were taken for testing.

[0108] The third group, AAV8-pENN-sh-circDdb1 virus intervention group + PBS group, was treated with 10 11 The AAV8-pENN-sh-circDdb1 virus was injected intramuscularly into the gastrocnemius muscle of mice at a dose of vg virus / mouse. Three weeks later, a minipump containing PBS was implanted into the mice. One week later, the mice were killed and samples were taken for testing.

[0109] The fourth group, AAV8-pENN-sh-circDdb1 virus intervention group + Ang II group, was 10 11 The AAV8-pENN-sh-circDdb1 virus was injected intramuscularly into the gastrocnemius muscle of mice at a dose of vg virus / mouse. Three weeks later, Ang II was used to induce muscle atrophy in the mice. After another week, the mice were killed and samples were taken for testing.

[0110] After four weeks, the mice were tested for grip strength to check changes in their muscle strength. The method for testing the grip strength of small animals is as follows: C57BL / 6 male mice were placed on a small animal grip strength meter to adapt for 5 minutes, then the tails of the mice were grabbed and pulled horizontally at a constant speed, and the maximum readings of the four legs were recorded. This was repeated three times to obtain the average value. The right hind legs were then fixed, and the grip strength of the three legs was continued to be tested. The test was repeated three times to obtain the average value. Finally, the grip strength value of the right hind legs was the difference between the average grip strength of the four legs and the average grip strength of the three legs. The test results are as follows: Figure 2 As shown in the figure, it was found that intramuscular injection of AAV8-pENN-sh-circDdb1 virus could improve the muscle strength of mice with AngII-induced muscle atrophy, while the control virus injection group had no improvement effect. The mice were then killed and the gastrocnemius muscles were isolated.

[0111] The weight of the gastrocnemius muscle of mice was measured by an electronic analytical balance. Figure 3 As shown, A is the morphological diagram of the gastrocnemius muscle of mice in the angiotensin II-induced muscular atrophy model; B is the statistical diagram of the gastrocnemius muscle weight of mice with angiotensin II-induced muscular atrophy. It was found that intramuscular injection of AAV8-pENN-sh-circDdb1 virus can increase the gastrocnemius muscle weight of mice with Ang II-induced muscular atrophy, while the control virus injection group had no improvement effect.

[0112] The OCT samples of the mouse gastrocnemius muscle were frozen and sectioned, and WGA staining was used to detect the size of the cross-section of the mouse gastrocnemius muscle fibers. The method for WGA staining of the mouse gastrocnemius muscle tissue is as follows:

[0113] The OCT samples of the mouse gastrocnemius muscle were frozen and sectioned at a thickness of 10 μm, and the cut tissues were adsorbed onto adhesive slides and stored at -80°C for later use. The specific method of WGA staining is shown in Table 5:

[0114] Table 5. Relevant parameters of WGA staining steps

[0115]

[0116] After washing with PBS buffer, 50% glycerol was added for sealing (protected from light), and photographed under a fluorescence microscope (CarlZeiss Microscopy GmbH), and then the size of the cross-sectional area of ​​the mouse gastrocnemius muscle fibers was analyzed using ImageJ software.

[0117] The results are as follows Figure 4 As shown, A is the WGA staining image of angiotensin II-injected mice after intramuscular injection of AAV8-pENN-sh-circDdb1 virus; B is the WGA statistical result of the cross-sectional area of ​​the gastrocnemius muscle fibers of angiotensin II-injected mice after intramuscular injection of AAV8-pENN-sh-circDdb1 virus. It was found that intramuscular injection of AAV8-pENN-sh-circDdb1 virus can increase the cross-sectional area of ​​the gastrocnemius muscle fibers of mice with Ang II-induced muscular atrophy, while the control virus injection group had no improvement effect.

[0118] At the same time, paraffin sections were performed on the paraffin samples of the mouse gastrocnemius muscle, and the size of the cross-section of the mouse gastrocnemius muscle fibers was further detected by HE staining. The method of HE staining of the mouse gastrocnemius muscle was as follows:

[0119] The paraffin sections of the mouse gastrocnemius muscle paraffin samples were sectioned at a thickness of 5 μm, and the cut paraffin samples were adsorbed on adhesive slides and placed in an oven at 65°C to remove some of the paraffin. Subsequently, dewaxing treatment was performed, and the specific method is shown in Table 6:

[0120] Table 6 Steps of dewaxing in HE staining of mouse gastrocnemius muscle

[0121]

[0122] Then, the sections were rinsed with tap water for 20 minutes to remove excess reagents, and then soaked with distilled water to be stained. Then, HE staining kit (Keygen Biotechnology) was used for tissue staining. The specific method is shown in Table 7:

[0123] Table 7 Specific steps for tissue staining using HE staining kit (Keygen Biotechnology)

[0124]

[0125] After staining, the slides were dried overnight at room temperature and then sealed with neutral gum (Sinopharm Group). They were photographed and observed under an upright biological microscope (Leica). ImageJ software was then used to analyze the cross-sectional area of ​​the gastrocnemius muscle fibers of mice.

[0126] The results are as follows Figure 5 As shown, A is the HE staining image of angiotensin II injected mice after intramuscular injection of AAV8-pENN-sh-circDdb1 virus; B is the HE statistical result of the cross-sectional area of ​​the gastrocnemius muscle fibers of angiotensin II injected mice after intramuscular injection of AAV8-pENN-sh-circDdb1 virus. Intramuscular injection of AAV8-pENN-sh-circDdb1 virus can increase the cross-sectional area of ​​the gastrocnemius muscle fibers of mice with Ang II-induced muscular atrophy, while the control virus injection group has no improvement effect.

[0127] Example 3

[0128] 1. AAV8-pENN-sh-circDdb1 virus was injected into the muscle of the right hind limb of C57BL / 6 male mice through an insulin needle. The virus dose injected per mouse was 1×10 11 vg, the injection method and injection dose of the control virus AAV8-pENN-sh-control were the same as those of the AAV8-pENN-sh-circDdb1 virus. Three weeks later, the muscle atrophy model was established.

[0129] 2. Establishment of the denervation (Den)-induced muscle atrophy model, the process is as follows Figure 6 As shown, the dose of AAV8 virus injected intramuscularly into each mouse was 1×10 11 vg, Three weeks after virus injection, mice were denervated for 1 week, and then sacrificed and evaluated.

[0130] First, the mice were anesthetized with 4% chloral hydrate at a dose of 10 μL / g. Then, the right hind limb of the mouse was depilated with a depilatory cream. The mouse was fixed on the experimental table in a prone position, and a small incision was made horizontally in the skin of the right hind limb near the outer thigh with microscissors, and then the muscle tissue was torn apart with micro-tipped forceps to expose a white sciatic nerve. The sciatic nerve was picked out and a small section was cut off with microscissors to prevent it from being joined. After that, the wound was sutured with medical sutures, placed on a 37°C electric blanket to wake it up and transferred to a breeding cage for breeding. The control group did not need to cut the sciatic nerve, and the rest of the steps were the same as the experimental group. The mice were killed seven days later and samples were taken for detection and analysis.

[0131] 3. Set up the experimental groups according to the steps in 1 and 2, and divide them into 4 groups, namely AAV8-pENN-sh-control virus control group + sham group, AAV8-pENN-sh-control virus control group + Den group, AAV8-pENN-sh-circDdb1 virus intervention group + sham group, AAV8-pENN-sh-circDdb1 virus intervention group + Den group. Three weeks after the virus was injected into mice by intramuscular injection, Den induced muscle atrophy. The specific method is as follows:

[0132] The first group, AAV8-pENN-sh-control virus control group + sham group, was 10 11 The dose of vg virus / mouse was 1:1. AAV8-pENN-sh-control virus was injected intramuscularly into the gastrocnemius muscle of mice. Three weeks later, sham surgery was performed to induce muscle atrophy. One week later, the mice were killed and samples were taken for testing.

[0133] The second group, AAV8-pENN-sh-control virus control group + Den group, was 10 11 The dose of vg virus / mouse was 1:1. AAV8-pENN-sh-control virus was injected intramuscularly into the gastrocnemius muscle of mice. Three weeks later, Den was used to induce muscle atrophy in mice. One week later, the mice were killed and samples were taken for testing.

[0134] The third group, AAV8-pENN-sh-circDdb1 virus intervention group + sham group, was 10 11 The AAV8-pENN-sh-circDdb1 virus was injected intramuscularly into the gastrocnemius muscle of mice at a dose of vg. Three weeks later, a sham operation was performed to induce muscle atrophy. One week later, the mice were killed and samples were taken for testing.

[0135] The fourth group, AAV8-pENN-sh-circDdb1 virus intervention group + Den group, was 10 11 The AAV8-pENN-sh-circDdb1 virus was injected intramuscularly into the gastrocnemius muscle of mice at a dose of vg virus / mouse. Three weeks later, Den was used to induce muscle atrophy in the mice. After another week, the mice were killed and samples were taken for testing.

[0136] Four weeks later, the mice were killed and the gastrocnemius muscles were isolated. The weight of the gastrocnemius muscles was measured using an electronic analytical balance. Figure 7As shown, A is the morphological diagram of the gastrocnemius muscle of mice in the denervation-induced muscle atrophy model; B is the statistical diagram of the gastrocnemius muscle weight of mice with denervation-induced muscle atrophy. It was found that intramuscular injection of AAV8-pENN-sh-circDdb1 virus can increase the weight of the gastrocnemius muscle of mice with Den-induced muscle atrophy, while the control virus injection group had no improvement effect.

[0137] The OCT samples of mouse gastrocnemius were frozen and sectioned, and WGA staining was used to detect the size of the cross-sectional area of ​​the mouse gastrocnemius muscle fibers. Figure 8 As shown, A is the WGA staining image of denervated mice after intramuscular injection of AAV8-pENN-sh-circDdb1 virus; B is the WGA statistical result of the cross-sectional area of ​​the gastrocnemius muscle fibers of denervated mice after intramuscular injection of AAV8-pENN-sh-circDdb1 virus. It was found that intramuscular injection of AAV8-pENN-sh-circDdb1 virus can increase the cross-sectional area of ​​the gastrocnemius muscle fibers of mice with Den-induced muscular atrophy, while the control virus injection group had no improvement effect.

[0138] Paraffin sections of the mouse gastrocnemius muscle were made, and the size of the cross-section of the mouse gastrocnemius muscle fibers was further detected by HE staining. Fig. 9 As shown, A is the HE staining image of denervated mice after intramuscular injection of AAV8-pENN-sh-circDdb1 virus; B is the HE statistical result of the cross-sectional area of ​​the gastrocnemius muscle fibers of denervated mice after intramuscular injection of AAV8-pENN-sh-circDdb1 virus. It was found that intramuscular injection of AAV8-pENN-sh-circDdb1 virus can increase the cross-sectional area of ​​the gastrocnemius muscle fibers of mice with Den-induced muscular atrophy, while the control virus injection group had no improvement effect.

[0139] Example 4

[0140] 1. AAV8-pENN-sh-circDdb1 virus was injected into the muscle of the right hind limb of C57BL / 6 male mice through an insulin needle. The virus dose injected per mouse was 1×10 11 vg, the injection method and injection dose of the control virus AAV8-pENN-sh-control were the same as those of the AAV8-pENN-sh-circDdb1 virus. Three weeks later, the muscle atrophy model was established.

[0141] 2. Establishment of the immobilization (Imo) induced muscle atrophy model, the process is as follows Fig.10 As shown, the dose of AAV8 virus injected intramuscularly into each mouse was 1×10 11 vg, 3 weeks after virus injection, mice were fixed for 1 week and then sacrificed and evaluated.

[0142] First, the mice were anesthetized with 4% chloral hydrate at a dose of 10 μL / g. The mice were then fixed on the experimental table in a prone position, with the soles of the feet fixed at an angle of 180° to the tibia. A disposable needle with a diameter of 0.45 mm was used to pass through the mouse radix and talus from the soles of the feet and insert into the backbone. The needle that was not inserted into the sole of the foot was removed. Finally, it was placed on a 37°C electric blanket to wake it up, and then transferred to a breeding cage for breeding. The needle was removed from the control group, and the rest of the steps were the same as the experimental group. The mice were killed seven days later and samples were taken for detection and analysis.

[0143] 3. Set up the experimental groups according to the steps in 1 and 2. The experiment was divided into 4 groups, namely AAV8-pENN-sh-control virus control group + sham group, AAV8-pENN-sh-control virus control group + Imo group, AAV8-pENN-sh-circDdb1 virus intervention group + sham group, AAV8-pENN-sh-circDdb1 virus intervention group + Imo group. Three weeks after the virus was injected into mice by intramuscular injection, Imo induced muscle atrophy. The specific method is as follows:

[0144] The first group, AAV8-pENN-sh-control virus control group + sham group, was 10 11 The dose of vg virus / mouse was 1:1. AAV8-pENN-sh-control virus was injected intramuscularly into the gastrocnemius muscle of mice. Three weeks later, sham surgery was performed to induce muscle atrophy. One week later, the mice were killed and samples were taken for testing.

[0145] The second group, AAV8-pENN-sh-control virus control group + Imo group, was 10 11 The dose of vg virus / mouse was 1:1. AAV8-pENN-sh-control virus was injected intramuscularly into the gastrocnemius muscle of mice. Three weeks later, Imo was performed to induce muscle atrophy in mice. One week later, the mice were killed and samples were taken for testing.

[0146] The third group, AAV8-pENN-sh-circDdb1 virus intervention group + sham group, was 10 11 The AAV8-pENN-sh-circDdb1 virus was injected intramuscularly into the gastrocnemius muscle of mice at a dose of vg. Three weeks later, a sham operation was performed to induce muscle atrophy. One week later, the mice were killed and samples were taken for testing.

[0147] The fourth group, AAV8-pENN-sh-circDdb1 virus intervention group + Imo group, was 10 11The AAV8-pENN-sh-circDdb1 virus was injected intramuscularly into the gastrocnemius muscle of mice at a dose of vg virus / mouse. Three weeks later, Imo was performed to induce muscle atrophy in mice. After another week, the mice were killed and samples were taken for testing.

[0148] Four weeks later, the mice were killed and the gastrocnemius muscles were isolated. The weight of the gastrocnemius muscles was measured using an electronic analytical balance. Fig.11 As shown, A is the morphological diagram of the gastrocnemius muscle of mice in the immobilization-induced muscle atrophy model; B is the statistical diagram of the gastrocnemius muscle weight of mice with immobilization-induced muscle atrophy. It was found that intramuscular injection of AAV8-pENN-sh-circDdb1 virus could increase the weight of the gastrocnemius muscle of mice with Imo-induced muscle atrophy, while the control virus injection group had no improvement effect.

[0149] The OCT samples of mouse gastrocnemius were frozen and sectioned, and WGA staining was used to detect the size of the cross-sectional area of ​​the mouse gastrocnemius muscle fibers. Fig.12 As shown, A is the WGA staining image of fixed mice after intramuscular injection of AAV8-pENN-sh-circDdb1 virus; B is the WGA statistical result of the cross-sectional area of ​​the gastrocnemius muscle fibers of fixed mice after intramuscular injection of AAV8-pENN-sh-circDdb1 virus. It was found that intramuscular injection of AAV8-pENN-sh-circDdb1 virus can increase the cross-sectional area of ​​the gastrocnemius muscle fibers of mice with Imo-induced muscular atrophy, while the control virus injection group had no improvement effect.

[0150] At the same time, the paraffin samples of the mouse gastrocnemius muscle were paraffin sectioned, and the size of the cross-section of the mouse gastrocnemius muscle fibers was further detected by HE staining. The results are as follows Fig.13 As shown, A is the HE staining image of fixed mice after intramuscular injection of AAV8-pENN-sh-circDdb1 virus; B is the HE statistical result of the cross-sectional area of ​​the gastrocnemius muscle fibers of fixed mice after intramuscular injection of AAV8-pENN-sh-circDdb1 virus. It was found that intramuscular injection of AAV8-pENN-sh-circDdb1 virus can increase the cross-sectional area of ​​the gastrocnemius muscle fibers of mice with immobilization-induced muscular atrophy, while the control virus injection group had no improvement effect.

[0151] The WGA staining and statistical results of the gastrocnemius muscle tissue of mice in the above examples show that intramuscular injection of AAV8-pENN-sh-circDdb1 virus can increase the cross-sectional area of ​​the gastrocnemius muscle fibers in mice with muscular atrophy induced by angiotensin II, denervation and fixation, while the control virus injection group has no improvement effect.

[0152] The HE staining and statistical results in the above examples show that intramuscular injection of AAV8-pENN-sh-circDdb1 virus can increase the cross-sectional area of ​​gastrocnemius muscle fibers in mice with muscular atrophy induced by angiotensin II, denervation and fixation, while the control virus injection group has no improvement effect.

[0153] Example 5

[0154] The C2C12 cell transfection steps are as follows:

[0155] siRNA transfection

[0156] a. Dissolve the siRNA powder in ddH2O to a 20 μM stock solution (the final concentration when used is 100 nM);

[0157] b. For transfection, take a 24-well plate as an example, dissolve 2.5 μL siRNA in 50 μL Opti-MEM medium (mix gently and let stand for 5 min), 1.5 μL 2000 was dissolved in 50 μL Opti-MEM medium (mixed gently and allowed to stand for 5 min), 400 μL Opti-MEM medium was mixed by pipetting gently (total volume was about 50+50+400=500 μL), and allowed to stand for 20 min;

[0158] c. Add 500 μL of the mixture in step b to each well of C2C12 mature myotube cells, and replace the medium with 10% FBS complete medium after 4-6 hours;

[0159] d. Depending on the cell status, collect cells for testing after 24 to 72 hours. If functional verification experiments are to be performed later, test them again after drug treatment.

[0160] Table 8 siRNA sequences

[0161]

[0162] Note: The "dTdT" in the siRNA sequences in Table 8 refers to the overhanging DNA at both ends.

[0163] siRNA was transfected into C2C12 differentiated mature myotube cells, and the knockdown efficiency was verified by real-time fluorescence quantitative PCR; the primer sequences used for fluorescence quantitative PCR were as follows: mmu-circDdb1-F: 5'-CAGTGCTTTGGGGTCCTTTC-3' (SEQ ID NO.9); mmu-circDdb1-R: 5'-CCTGAGGGTGGATCTGAAGG-3' (SEQ ID NO.10).

[0164] The results showed that si-circDdb1-1 and si-circDdb1-2, which specifically targeted the reverse splicing site, could effectively silence circDdb1 in C2C12 myotube cells. Fig.15 A), and had no effect on the expression of Ddb1 mRNA ( Fig.15 (B).

[0165] The inventors constructed three cellular-level muscle atrophy models, namely, myotube atrophy models induced by dexamethasone, tumor necrosis factor-α, and angiotensin II. The construction methods are as follows:

[0166] 1) Dexamethasone (Dex) model: After C2C12 cells differentiated into mature myotubes, dexamethasone with a concentration of 25mM was diluted with differentiation medium to a final concentration of 50μM as the treatment group culture medium, and the control group was prepared with 998μL differentiation medium + 2μL anhydrous ethanol per ML culture medium; taking a 12-well plate as an example, the corresponding culture medium was added to each well of cells in the control group and the treatment group at 1ml / well, and the degree of myotube cell atrophy was detected after 24h. The differentiation medium is: DMEM containing 2% horse serum (HS) by volume, 1% penicillin by volume, and 1% streptomycin by volume.

[0167] Dexamethasone (Dex): The powder is prepared into a 25 mM stock solution with anhydrous ethanol and stored at 4°C.

[0168] 2) Tumor necrosis factor-α (TNF-α) model: After C2C12 cells differentiated into mature myotubes, TNF-α powder was diluted to 100 ng / mL with differentiation medium and added to mature myotubes. The degree of myotube atrophy was detected at 24 h and 48 h.

[0169] TNF-α: The powder was prepared into a 100 μg / mL stock solution with sterile double distilled water and stored at -20°C.

[0170] 3) Angiotensin II (Ang II) model: After C2C12 cells differentiated into mature myotubes, Ang II powder was diluted to 500 nM with differentiation medium and added to mature myotube cells. The culture plate was then wrapped with tin foil with one end slightly open to maintain normal cell respiration and placed in an incubator away from light. The degree of myotube cell atrophy was detected after 48 hours.

[0171] Ang II: The powder was prepared into 5 mM with Ang II buffer in a centrifuge tube, wrapped with tin foil, and stored at -20°C.

[0172] Two siRNAs (si-circDdb1-1 and si-circDdb1-2) with better transfection inhibition effects were used for functional loss experiments (siRNA transfection experiments).

[0173] Immunofluorescence staining results showed that the diameter of myotubes induced by dexamethasone, tumor necrosis factor-α and angiotensin II decreased, and knocking down the expression of circDdb1 could improve the diameter of myotubes to a certain extent ( Fig.15 (CE).

[0174] At the same time, real-time fluorescence quantitative PCR was used to detect the expression of muscle atrophy marker genes MuRF-1 and Atrogin-1; the primer sequences for detecting MuRF-1 and Atrogin-1 were as follows: mmu-MuRF-1-F: 5'-GTGTGAGGTGCCTACTTGCTC-3' (SEQ ID NO.11), mmu-MuRF-1-R: 5'-GCTCAGTCTTCTGTCCTTGGA-3' (SEQ ID NO.12); mmu-Atrogin-1-F: 5'-CAGCTTCGTGAGCGACCTC-3' (SEQ ID NO.13); mmu-Atrogin-1-R: 5'-GGCAGTCGAGAAGTCCAGTC-3' (SEQ ID NO.14).

[0175] exist Figure 15-16 In the figure, the NC group represents the negative control. The nucleotide sequence of the positive chain of the negative control siRNA transfected in the cells is 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO.15), and the nucleotide sequence of the antisense chain is 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO.16), which were synthesized by Tuoran Biotechnology Co., Ltd.

[0176] The results showed that the expression of circDdb1 was significantly upregulated in the atrophy of myotube cells induced by dexamethasone, tumor necrosis factor-α, and angiotensin II. After knocking down the expression of circDdb1, the expression of the two marker genes decreased ( Fig.16 These results suggest that reducing the expression of circDdb1 can alleviate muscle atrophy at the cellular level.

[0177] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of circular RNA circDdb1 as a target in the preparation of drugs for the prevention and treatment of muscular atrophy.

2. Application of agents that inhibit the expression of circular RNA circDdb1 in the preparation of drugs for preventing and treating muscular atrophy.

3. The use according to claim 2, characterized in that: The reagent includes biological materials for knocking out or knocking down the circular RNA circDdb1.

4. The use according to claim 2 or 3, characterized in that: The reagent includes one or more of a nucleic acid, a nucleic acid construct and a virus particle that interferes with the expression of the circular RNA circDdb1.

5. The use according to claim 4, characterized in that: The nucleic acid comprises shRNA or siRNA, and the nucleotide sequence of the shRNA is shown in SEQ ID NO.1; The siRNA includes si-circDdb1-1 or si-circDdb1-2; the si-circDdb1-1 is composed of the sense chain shown in SEQ ID NO.5 and the antisense chain shown in SEQ ID NO.6; the si-circDdb1-2 is composed of the sense chain shown in SEQ ID NO.7 and the antisense chain shown in SEQ ID NO.

8.

6. The use according to claim 4, characterized in that: The nucleic acid construct comprises an interfering viral vector, wherein the interfering viral vector contains the shRNA shown in SEQ ID NO.

1.

7. The use according to claim 6, characterized in that: The initial vector in the interfering viral vector includes an adenoviral vector, a retroviral vector or a lentiviral vector.

8. The use according to claim 7, characterized in that: The interfering viral vector includes pENN-sh-circDdb1, and the nucleotide sequence of pENN-sh-circDdb1 is shown in SEQ ID NO.

2.

9. A drug for preventing and treating muscle atrophy, characterized in that: The drug comprises an agent for inhibiting the expression of circular RNA circDdb1 and a pharmaceutically acceptable excipient.

10. The drug according to claim 9, characterized in that The reagent includes one or more of a nucleic acid, a nucleic acid construct and a virus particle that interferes with the expression of the circular RNA circDdb1; The nucleic acid comprises shRNA or siRNA, and the nucleotide sequence of the shRNA is shown in SEQ ID NO.1; The siRNA includes si-circDdb1-1 or si-circDdb1-2; the si-circDdb1-1 is composed of the sense strand shown in SEQ ID NO.5 and the antisense strand shown in SEQ ID NO.6; the si-circDdb1-2 is composed of the sense strand shown in SEQ ID NO.7 and the antisense strand shown in SEQ ID NO.8; The nucleic acid construct comprises an interfering viral vector, wherein the interfering viral vector contains the shRNA shown in SEQ ID NO.1.

Images