An engineered circular RNA circmiR-29b and its application in preparing a drug for treating muscular atrophy

Abstract

The present invention provides the use of engineered circular RNA circmiR‑29b in the preparation of a drug for treating muscular atrophy, and belongs to the field of biomedical technology. The present invention provides a circular RNA circmiR‑29b, comprising an active sequence and a random sequence; the active sequence is connected by 6 to 13 repetitions in series, and the random sequence is inserted between the active sequences; the nucleotide sequence of the active sequence is shown in SEQ ID NO: 1. The present invention delivers circmiR‑29b to skeletal muscle through AAV8, and can stably express circmiR‑29b in skeletal muscle, which can effectively inhibit various types of muscular atrophy. Therefore, the present invention also provides a gene therapy based on the AAV8 viral vector to deliver engineered circular RNA circmiR‑29b to achieve the purpose of treating muscular atrophy.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to an application of an engineered circular RNA circmiR-29b in preparing a drug for treating muscular atrophy. Background Art

[0002] Gene therapy is the use of molecular biology methods to deliver nucleic acid fragments to specific organs, edit genes to interfere with target gene expression, or directly express corresponding products to achieve the purpose of treating diseases. The key to gene therapy is to find the right target gene and choose the right delivery method. Gene therapy is mainly used for gene mutation diseases such as hemophilia, sickle cell anemia and muscular dystrophy.

[0003] Skeletal muscle is controlled by human consciousness. It can cause skeletal muscle fiber contraction through somatic nerve stimulation, realize the basic activities of the human body including flexion and extension, and is an important component of the human motor system. Muscular atrophy is a group of skeletal muscle cell degenerative diseases. The main manifestations of muscular atrophy are muscle fiber loss and muscle cell protein loss. Systemic diseases such as sepsis, AIDS, heart failure, and renal failure, as well as acute physical injuries such as severe burns and major trauma, and diseases such as cancer can cause muscular atrophy.

[0004] Currently, there is gene therapy for spinal muscular atrophy (SMA). Since SMA is an autosomal negative genetic disease, usually caused by a mutation in the survival motor neuron 1 (SMN1) gene, gene therapy for SMA treats SMA by delivering gene fragments that can express normal SMN1 protein to motor neurons to stably express SMN1 protein for a long time. However, there are many mechanisms that cause muscle atrophy, and gene therapy for SMA is not applicable to muscle atrophy caused by other factors. Therefore, it is necessary to find a universal muscle atrophy regulatory gene and intervene in it in order to achieve widespread muscle atrophy treatment.

[0005] In the occurrence and development of muscular atrophy, non-coding RNA plays an important role. Studies have shown that a variety of small RNAs such as miR-1, miR-29b, miR-133, miR-23a, miR-21, miR-27, miR-628, miR-431 and miR-206 play a regulatory role in the occurrence of muscular atrophy. For example, the invention patent of publication number CN108690846A discloses that the purpose of treating muscular atrophy is achieved by inhibiting the expression of miR-29b gene, and discloses that the inhibitor achieves the purpose of treating muscular atrophy by inhibiting the expression of miR-29b in cells and tissues, or destroying the stability of miR-29b in cells and tissues, or reducing the activity of miR-29b in cells and tissues, or reducing the effective action time of miR-29b in cells or tissues. Small interfering RNA (siRNA) specifically binds to miR-29b, thereby affecting the regulatory effect of miR-29b, thereby achieving the purpose of treating muscular atrophy. However, currently, linear siRNA is unstable and easily degraded in cells, and linear siRNA has limited ability to reduce the expression of miR-29b in the body and cannot reach the inhibitory level of miR-29b required for the treatment of muscular atrophy. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a circular RNA circmiR-29b, which serves as a molecular sponge of miR-29b to reduce the expression level of miR-29b in cells.

[0007] The present invention also aims to provide a use of circular RNA circmiR-29b in the preparation of a method for preventing and / or treating muscular atrophy.

[0008] The present invention provides a circular RNA circmiR-29b, comprising an effective sequence and a random sequence; the effective sequence is connected in a manner of 6 to 13 times of serial repetition, and the random sequence is inserted between the effective sequences;

[0009] The nucleotide sequence of the effective sequence is shown in SEQ ID NO: 1.

[0010] Preferably, the active sequence is connected in series by 10 to 12 repetitions.

[0011] Preferably, the length of the random sequence is 12 to 24 nt.

[0012] Preferably, the nucleotide sequence of the random sequence is as shown in SEQ ID NO:2.

[0013] Preferably, the nucleotide sequence of the circular RNA circmiR-29b is as shown in SEQ ID NO:3.

[0014] The present invention provides an adeno-associated virus recombinant vector, on the basis of which the circular RNA circmiR-29b is inserted.

[0015] Preferably, the adeno-associated virus vector comprises an AAV8 vector.

[0016] Preferably, the adeno-associated virus vector further comprises a 5' guide looping sequence and a 3' guide looping sequence;

[0017] The nucleotide sequence of the 5' guide looping sequence is shown in SEQ ID NO: 4;

[0018] The nucleotide sequence of the 3' guide looping sequence is shown in SEQ ID NO: 5;

[0019] The insertion position of the circular RNA circmiR-29b is between the 5' guide circularization sequence and the 3' guide circularization sequence.

[0020] The present invention provides a delivery system of circular RNA circmiR-29b. The delivery system is an adeno-associated virus containing the circular RNA circmiR-29b or is obtained by packaging the adeno-associated virus recombinant vector.

[0021] The present invention provides the use of the RNA circmiR-29b, the adeno-associated virus recombinant vector or the delivery system in preparing a drug for preventing and / or treating muscle atrophy.

[0022] The circular RNA circmiR-29b provided by the present invention comprises an effective sequence and a random sequence; the effective sequence is connected by 6 to 13 repetitions in series, and the random sequence is inserted between the effective sequences; the nucleotide sequence of the effective sequence is shown in SEQ ID NO: 1. The present invention designs an effective sequence that can be strictly complementary to the nucleotide sequence of miR-29b based on the nucleotide sequence of miR-29b, and prepares an engineered circular RNA circmiR-29b. Experiments show that compared with the inhibitory efficiency (44.8%) of the linear effective sequence on miR-29b, the inhibitory efficiency of the circular RNA circmiR-29b provided by the present invention reaches more than 90%, which can greatly reduce the expression of miR-29b, thereby achieving the purpose of treating muscle atrophy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Schematic diagram of AAV-dMCK-circmiR-29b constructed in an embodiment of the present invention, wherein dMCK is a muscle-specific promoter, 5'intron sequence is a 5' guide looping sequence, 3'intron sequence is a 3' guide looping sequence, and the insertion position of the artificially synthesized circular RNA is the yellow fragment position;

[0024] FIG2 is a result of the circular circmiR-29b specifically reducing the expression of miR-29b in an embodiment of the present invention, wherein Figure 2A The relative expression results of miR-29b in the AAV8-circmiR-29b group and the control group. Figure 2B The results of the luciferase experiment of the AAV8-circmiR-29b group and the control group in the embodiment of the present invention;

[0025] FIG3 is an example of circmiR-29b protecting muscle atrophy at the myotube level in the present invention, wherein Figure 3A The results of immunofluorescence staining in the AAV8-circmiR-29b group and the control group; Figure 3B Statistical graph of immunofluorescence staining results in the AAV8-circmiR-29b group and the control group; ***, p<0.001;

[0026] FIG4 shows that circmiR-29b delivered by AAV protects muscle atrophy induced by the Den model in an embodiment of the present invention, wherein Figure 4A This is the anatomical morphology of the gastrocnemius muscle of mice in the experiment of AAV8-circmiR-29b preventing muscle atrophy caused by Den model. Figure 4B The weight changes of gastrocnemius muscle samples of mice in each group; Figure 4C The results of miR-29b expression in gastrocnemius muscle of mice in each group are shown in Figure 2. Figure 4D The WGA staining and muscle fiber diameter statistics of gastrocnemius muscles of each group of mice are shown in the figure. Figure 4E HE staining of gastrocnemius muscle and statistical results of muscle fiber diameter in each group of mice; *, p<0.05, **, p<0.01, ***, p<0.001;

[0027] FIG5 shows that circmiR-29b delivered by AAV protects against muscle atrophy induced by the immobilization (IMO) model, where Figure 5A This is the anatomical morphology of the gastrocnemius muscle of mice in the experiment of AAV8-circmiR-29b preventing muscle atrophy caused by IMO model. Figure 5B The weight changes of gastrocnemius muscle samples of mice in each group are shown in Table 1. Figure 5C The results of miR-29b expression in gastrocnemius muscle of mice in each group are shown in Figure 2. Figure 5D The WGA staining and muscle fiber diameter statistics of gastrocnemius muscles of each group of mice are shown in the figure. Figure 5E HE staining of gastrocnemius muscle and statistical results of muscle fiber diameter in each group of mice; *, p<0.05, **, p<0.01, ***, p<0.001;

[0028] FIG6 shows that the delivered circmiR-29b protects against muscle atrophy induced by the AngII sustained-release pump (AngII) model, wherein Fig. 6A This is the anatomical morphology of the gastrocnemius muscle of mice in the experiment of AAV8-circmiR-29b preventing muscle atrophy caused by AngII model. Figure 6B The results of the grasping force test at the end of each group of mouse models. Figure 6C The weight changes of gastrocnemius muscle samples of mice in each group are shown in Table 1. Fig.6D The results of miR-29b expression in gastrocnemius muscle of mice in each group are shown in Figure 2. Fig. 6E The WGA staining and muscle fiber diameter statistics of gastrocnemius muscles of each group of mice are shown in the figure. Fig. 6F HE staining of gastrocnemius muscle and statistical results of muscle fiber diameter of mice in each group; *, p<0.05, **, p<0.01, ***, p<0.001;

[0029] FIG7 shows the delivered circmiR-29b for the treatment of muscle atrophy induced by SWI model, wherein Fig. 7A The gastrocnemius atrophy of mice at different days of SWI. Figure 7B This is the anatomical morphology of the gastrocnemius muscle of mice in the AAV8-circmiR-29b treatment of SWI model-induced muscle atrophy experiment. Figure 7C The weight changes of gastrocnemius muscle samples of mice in each group are shown in Table 1. Fig.7D The results of miR-29b expression in gastrocnemius muscle of mice in each group are shown in Figure 2. Fig. 7E The WGA staining and muscle fiber diameter statistics of gastrocnemius muscles of each group of mice are shown in the figure. Figure 7F HE staining of gastrocnemius muscle and statistical results of muscle fiber diameter of mice in each group; *, p<0.05, **, p<0.01, ***, p<0.001;

[0030] Figure 8 It is a standard curve diagram for detecting virus titer in the embodiment of the present invention. DETAILED DESCRIPTION

[0031] The present invention provides a circular RNA circmiR-29b, comprising an effective sequence and a random sequence; the effective sequence is connected in a manner of 6 to 13 repeated series, and the random sequence is inserted between the effective sequences; the nucleotide sequence of the effective sequence is shown in SEQ ID NO:1.

[0032] In the present invention, the active sequence is a sequence that is strictly complementary to miR-29b. The active sequence is connected in series by 6 to 13 repetitions to increase the probability of binding to miR-29b. The number of repetitions of the active sequence of the present invention is preferably 10 to 12 times. The present invention has been experimentally proven that by increasing the number of active sequences, the expression of miR-29b can be effectively reduced, thereby improving the inhibition efficiency and making the treatment effect of muscle atrophy better, and the more repetitions, the better the prevention and treatment effect.

[0033] In the present invention, the length of the random sequence preferably includes 12 to 24 nt. Experiments have shown that the random sequence of 12 to 24 nt has the best effect. As the spacer sequence is further extended (greater than 24 nt), the inhibitory effect is significantly weakened. The present invention has no special requirements for the specific nucleotide sequence of the random sequence, and a random sequence well known in the art can be used. In an embodiment of the present invention, the nucleotide sequence of the random sequence is preferably as shown in SEQ ID NO: 2. In an embodiment of the present invention, the effect of the circular RNA is illustrated by taking an effective sequence repeated 12 times as an example. The nucleotide sequence of the circular RNA circmiR-29b is preferably as shown in SEQ ID NO: 3.

[0034] The present invention provides an adeno-associated virus recombinant vector, on the basis of which the circular RNA circmiR-29b is inserted.

[0035] In the present invention, the adeno-associated virus vector preferably includes an AAV8 vector. In view of the fact that AAV8 can accurately target muscle cells and deliver the circular RNA circmiR-29b gene fragment into muscle cells, the AAV8 vector is selected to construct an adeno-associated virus recombinant vector. The adeno-associated virus vector preferably also includes a 5' guide circulogenesis sequence and a 3' guide circulogenesis sequence; the 5' guide circulogenesis sequence and the 3' guide circulogenesis sequence are from exon 2 of the Slc8a1 gene in the mouse genome. The nucleotide sequence of the 5' guide circulogenesis sequence is shown in SEQ ID NO:4; the nucleotide sequence of the 3' guide circulogenesis sequence is shown in SEQ ID NO:5. The insertion position of the circular RNA circmiR-29b is preferably between the 5' guide circulogenesis sequence and the 3' guide circulogenesis sequence. In an embodiment of the present invention, the method of delivering circular RNA circmiR-29b by a delivery system is illustrated by taking the recombinant AAV8 recombinant vector as an example. The spectrum of the recombinant AAV8 recombinant vector is shown in Figure 1 , which contains a dMCK promoter, and the dMCK promoter can achieve efficient expression of circular RNA circmiR-29b.

[0036] In the present invention, the method for constructing a recombinant vector containing the circular RNA circmiR-29b preferably comprises the following steps:

[0037] 1) Based on AAV-dMCK-EGFP, a fragment (SEQ ID NO: 10) was inserted between the EcoRI restriction sites, and the vector was linearized with XbaI. Two fragments, a 3' guide loop sequence (186 nt) and a reverse 5' guide loop sequence (870 nt), were inserted by homologous recombination to obtain a recombinant plasmid containing a guide loop sequence; the recombinant plasmid containing a guide loop sequence was then linearized using EcoRI single restriction enzyme digestion;

[0038] 2) Artificially synthesizing RNA circmiR-29b and cloning it into T plasmid to obtain recombinant T plasmid containing RNA circmiR-29b;

[0039] 3) The 5' guide circularization sequence (1060 nt) was amplified by PCR. After the PCR product was digested with EcoRI, the linearized T vector and the PCR product were connected using T4 ligase at room temperature for 30 min to obtain a recombinant T plasmid. Finally, the linearized recombinant T plasmid was double-digested with EcoRI and NdeI, and the circmiR-29b fragment was connected to the linearized recombinant plasmid containing the guide circularization sequence under the action of T4 ligase to obtain a recombinant vector containing circular RNA circmiR-29b (AAV-dMCK-circmiR-29b plasmid).

[0040] In the present invention, the AAV-dMCK-EGFP plasmid is reported in the prior art, see Prevention of Muscle Wasting by CRISPR / Cas9-mediated Disruption of Myostatin In Vivo; doi: 10.1038 / mt.2016.192.'; 'CRISPR / Cas9-Mediated miR-29b Editing as a Treatment of Different Types of Muscle Atrophy in Mice; doi: 10.1016 / j.ymthe.2020.03.005.

[0041] In the present invention, the inserted fragment 5'-ATGTCGACAAACGTGCTAAAGCTTGAATTCAAACGTGCTACATATGTCTAGACTCGAGAT-3' (SEQ ID NO: 11) can increase the multiple cloning site, and the restriction endonuclease cleavage sites added are: SalI, AccI, HincII, HindIII, EcoRI, NdeI, XbaI, and XholI.

[0042] The primers for amplifying the 3' guide loop sequence (186 nt) are as follows:

[0043] Infusion-3p-F: AACGTGCTACATATGTCTAGGAGGTGGAGGGGAAGACT (SEQ ID NO: 12);

[0044] Infusion-3p-R: GATTCTTAGGTGGTTTCTTGAATTGCACTTGT (SEQ ID NO: 13).

[0045] The primers for amplifying the reverse 5' guide looping sequence (870 nt) are as follows:

[0046] Infusion-5p-Inverted-F: CAAGAAACCACCTAAGAATCCATCAGTGACTAAAC (SEQ ID NO: 14);

[0047] Infusion-5p-Inverted-R: ATGAATTATCTCGAGTCTAGTAGAATCGCCACTCCTGCATC (SEQ ID NO: 15).

[0048] The primers for PCR amplification of the 5' guide loop sequence (1060 nt) are as follows:

[0049] Insert-5p-F:GTCGACTAGAATCGCCACTCCTGCAT(SEQ ID NO:16)

[0050] Insert-5p-R: AAGCTTTTGGGTGGGAGACTTAATCG (SEQ ID NO: 17).

[0051] In the present invention, the above PCR amplification system is a 50 μl reaction system, and the specific components are as follows

[0052] The PCR amplification program is shown in Table 1 below:

[0053]

[0054] The present invention provides a delivery system of circular RNA circmiR-29b. The delivery system is an adeno-associated virus containing the circular RNA circmiR-29b or is obtained by packaging the adeno-associated virus recombinant vector.

[0055] The present invention has no particular limitation on the method for packaging the adeno-associated virus containing circular RNA circmiR-29b, and any adeno-associated virus packaging scheme known in the art may be used.

[0056] The present invention provides the use of the RNA circmiR-29b, the adeno-associated virus recombinant vector or the delivery system in preparing a drug for preventing and / or treating muscle atrophy.

[0057] In the present invention, the dosage form of the drug preferably includes an injection. The injection contains the delivery system. The concentration of the delivery system is preferably 1×10 11 VG / up to 1×10 12 VG / piece, more preferably 1×10 12 VG / piece. The dosage of the injection is 1×10 12 VG / mouse, once injected, effective in 3 weeks.

[0058] In the present invention, the types of muscle atrophy include muscle atrophy caused by denervation (Den model), muscle atrophy caused by disuse (IMO model, SWI model), and muscle atrophy caused by heart failure (AngII model).

[0059] In the present invention, the circmiR-29b is transcribed and cyclized by eukaryotic cells themselves, the sequence guiding its correct cyclization comes from the natural circular RNA Slc8a1 circRNA, and the DNA fragment is delivered to muscle tissue by the AAV8 vector. Therefore, the present invention also provides the use of circmiR-29b in preventing and / or treating muscle atrophy.

[0060] The engineered circular RNA circmiR-29b provided by the present invention and its application in the preparation of a drug for treating muscular atrophy are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0061] Example 1

[0062] Method for constructing adeno-associated virus recombinant vector

[0063] 1. Design of circular RNA circmiR-29b fragment

[0064] According to the nucleotide sequence of miR-29b (UAGCACCAUUUGAAAUCAGUGUU, SEQ ID NO: 6), the activation sequence of miR-29b (AACACTGATTTTCCTGGTGCTA, SEQ ID NO: 1) was designed. The sequence was repeated 12 times in a row, and 11 random sequences (AAACGTGCTACG, SEQ ID NO: 2) were inserted between each repetition to obtain the sequence of the engineered circular RNA (AACACTGATTTTCCTGGTGCTAAAACGTGCTACGAACACTGATTTTCCTGGTGCTAAAACGTGCTACGAACACTGATTTTCCTGGTGCTAAAACGTGCTACGAACACTGATTTTCCTGGTGCTAAAACGTGCTACGAACACTGATTTTCCTGGTGCTAAAACGTGCTACGAACACTGATTTTCCTGGTGCTAAAACGTGCTACGAACACTGATTTTCCTGGTGCTAAAACGTGCTACGAACACTGATTTTCCTGGTGCTAAAACGTGCTACGAACACTGATTTTCCTGGTGCTAAAACGTGCTACGAACACTGATTTTCCTGGTGCTAAAACGTGCTACGAACACTGATTTTCCTGGTGCTAAAACGTGCTACGAACACTGATTTTCCTGGTGCTA, SEQ ID NO: 3).

[0065] 2. Construction of recombinant vector

[0066] The engineered circular sequence circmiR-29b sequence was artificially synthesized by BGI with EcoRI and NdeI restriction sites added at both ends, and cloned into a T vector to obtain a T vector containing the circmiR-29b sequence.

[0067] The AAV-dMCK-circmiR-29b plasmid construction process is as follows:

[0068] The plasmid construction skeleton was AAV-dMCK-EGFP (this plasmid was donated by Ding Qiurong's research group at the Institute of Nutrition, Chinese Academy of Sciences). First, the 5'-atgtcgacaaacgtgctaaagcttgaattcaaacgtgctacatatgtctagactcgagat-3' sequence (SEQ ID NO: 11) was inserted between the EcoRI restriction site to increase the multiple cloning site. The restriction endonuclease sites added were: SalI, AccI, HincII, HindIII, EcoRI, NdeI, XbaI, and XholI.

[0069] The vector was then linearized using XbaI, and primers were designed for PCR amplification of the 3'intron sequence (186nt) and the 5'inverted intron sequence (870nt), and the two fragments were cloned into the vector using homologous recombination. The PCR primers were: Infusion-3p-F: AACGTGCTACATATGTCTAGGAGGTGGAGGGGAAGACT (SEQ ID NO: 12); Infusion-3p-R: GATTCTTAGGTGGTTTCTTGAATTGCACTTGT (SEQ ID NO: 13); Infusion-5p-Inverted-F: CAAGAAACCACCTAAGAATCCATCAGTGACTAAAC (SEQ ID NO: 14); Infusion-5p-Inverted-R: ATGAATTATCTCGAGTCTAGTAGAATCGCCACTCCTGCATC (SEQ ID NO: 15).

[0070] The plasmid was then linearized using EcoRI single restriction enzyme digestion, and primers were designed to amplify the 5' intron sequence (1060 nt). The primers for amplifying the 5' guide loop sequence (1060 nt) are as follows:

[0071] Insert-5p-F:GTCGACTAGAATCGCCACTCCTGCAT(SEQ ID NO:16)

[0072] Insert-5p-R: AAGCTTTTGGGTGGGAGACTTAATCG (SEQ ID NO: 17).

[0073] The above PCR amplification systems are all 50 μl reaction systems, and the specific components are as follows:

[0074]

[0075] The PCR amplification program is shown in Table 1:

[0076]

[0077] After digesting the PCR product with EcoRI, T4 ligase was used to react at room temperature for 30 min to connect the linearized plasmid and the PCR product. Finally, the plasmid was linearized using EcoRI and NdeI double digestion, and the circmiR-29b fragment synthesized by BGI was cut from the T vector using EcoRI and NdeI, and the circmiR-29b fragment was separated and purified by gel electrophoresis, and then the circmiR-29b fragment and the linearized plasmid were connected using T4 ligase at room temperature for 30 min.

[0078] Example 2

[0079] AAV8-dMCK-circmiR-29b virus packaging method, the specific steps are as follows:

[0080] 293T cells were inoculated in DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin solution for culture. 293T cells were inoculated in 10 cm cell culture dishes at a density of 4 million cells per dish. After 24 hours, 1 ml of serum-free DMEM medium containing 10 μg AAV8, 10 μg pAAV-dMCK-circmiR-29b, 10 μg Helper, and 90 μg PEI MAX was added to each dish for transfection. After 12 hours of transfection, fresh DMEM complete medium was replaced, and the virus in the cells and culture was collected after 48 hours. To collect the virus in the culture medium: add 25 ml of 40% PEG-8000 per 100 ml of culture medium, stir overnight at 4 ° C, and centrifuge at 2800g, 15 ° C for 15 minutes. Add 1 mL of cell lysis buffer to the virus pellet and resuspend. Collection of viruses in cells: Resuspend the cell pellet in 5 ml of cell lysis buffer and thaw the cells three times in a -80°C refrigerator and a 37°C water bath. Mix the virus suspension in the culture medium with the frozen-thawed cell suspension, add 1M MgCl2 to a final concentration of 1mM, add Benzonase to a final concentration of 250U / mL, and incubate at 37°C for 45 minutes. Centrifuge at 4000rpm for 4 minutes at 4°C, and take the supernatant. Purify the virus using iodixanol gradient density centrifugation. See the schematic diagram of the purified AAV8 virus carrying the target gene for details. Figure 1 .

[0081] Virus titer detection method: dilute the viral vector plasmid pAAV-dMCK-circmiR-29b to 1ng / μl, dilute the plasmid 13 times to obtain standard concentrations 1 to 14, and use the 2-fold diluted Standard DNA dilution to prepare the standard curve. Take 5μl of the purified virus AAV8-dMCK-circmiR-29b prepared above, use the kit according to the instructions: extract viral gDNA, dilute to 50μl viral gDNA, then dilute the viral gDNA 100 times and use qPCR to detect the viral titer.

[0082] The qPCR reaction system is as follows:

[0083]

[0084] The sequences of the upstream primer F and downstream primer R used in the qPCR reaction are as follows:

[0085] Upstream primer F: GAGGTGGAGGGGAAGACTTT (SEQ ID NO: 7);

[0086] Downstream primer R: TAGAATCGCCACTCCTGCAT (SEQ ID NO: 8).

[0087] The qPCR reaction procedure was as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec, and 40 cycles.

[0088] Since the cycle number is linearly related to the logarithm of the Standard DNA dilution concentration (see Figure 8 ), and the virus titer was obtained by linear fitting. The virus titer was 3.057×10 13 VG / ml.

[0089] After titer determination, the AAV8-circmiR-29 virus can be directly used in animal experiments or frozen at -80°C.

[0090] Example 3

[0091] 1. A method for culturing C2C12 cells for differentiating myotubes, the specific steps are as follows: C2C12 cells are inoculated in a DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin solution. The medium for inducing C2C12 cell differentiation is a DMEM medium containing 2% horse serum and 1% penicillin-streptomycin solution. The medium is changed every 24 hours during differentiation, and differentiation is generally completed in 3 to 5 days. The cell culture environment is a 37°C constant temperature incubator with saturated humidity and 5% CO2.

[0092] 2. Targeting and functional verification of circmiR-29b delivered by AAV8-circmiR-29 virus: The target gene fragment capable of expressing circmiR-29b was delivered to muscle cells via AAV8-circmiR-29 virus, and then the expression of miR-29b was detected using fluorescent quantitative PCR.

[0093] The specific method is as follows: first, use TRIzol Reagent to extract total RNA in cells, take 400ng RNA and add specific RT primers (purchased from Ruibo Company), and use Bio-rad Company's reverse transcription kit to perform reverse transcription experiments: The reverse transcription reaction system is as follows:

[0094] 400ng RNA 5.5μl

[0095] miR-29b RT primer (5nM) 1μl

[0096] 5s RT primer (5nM) 1μl.

[0097] Among them, miR-29b RTprimer was purchased from Ruibo Company.

[0098] The volume of the mixture was 7.5 μl, and then the RT primer neck loop structure was fully opened at 70°C for 10 min, and annealed at 4°C for 5 min to allow the miRNA to fully combine with the specific RT primer.

[0099] Then add: 2 μl of 5×iScript reaction mixture, 0.5 μl of iScript reverse transcriptase, and then proceed as follows:

[0100] Priming 25℃ 5min

[0101] Reverse Transcription 46℃ 20min

[0102] RT inactivation 95℃ 1min

[0103] After obtaining the RT product, it was diluted 400 times and the expression of miR-29b was detected by qPCR. The specific method is as follows.

[0104] Prepare qPCR reaction system, the reaction system is 10μl:

[0105] SYBR Green 5μl

[0106] Primer mixture (F+R, 5nM) 1μl

[0107] cDNA (Diluted) 4μl

[0108] Among them, the upstream and downstream primers of miR-29b and internal reference 5s were purchased from Ruibo Company.

[0109] The qPCR reaction procedure is as follows:

[0110]

[0111]

[0112] 3. Detection of the specificity of circmiR-29b for miR-29b by luciferase binding assay

[0113] The luciferase reporter gene experiment method is as follows: 293T cells were inoculated into 12 wells at a concentration of 100,000 cells / ml. When the cell density reached about 80%, the constructed PGL-circmiR-29b plasmid and Renilla plasmid were transfected into 293T cells at a dose of 1 μg per well. After 12 hours, the medium was changed and the cells were transfected with miR-29a, miR-29b, and miR-29c mimics at a final concentration of 50 nM. The medium was changed 6 to 8 hours after transfection. After 24 hours, the cells were collected and used Promega's The dual luciferase reporter gene assay kit was used to detect whether there was specific binding between the miR-29 family and circmiR-29b.

[0114] The PGL-circmiR-29b plasmid was constructed as follows: the circmiR-29b fragment was cloned into PGL3-basic. The PGL3-basic plasmid was first linearized using the XbaI restriction endonuclease, and then the circmiR-29b fragment was amplified using PCR and cloned into the linearized vector using homologous recombination.

[0115] The PCR primers were: PGL3-R-circmiR-29b-F: CGATCTAAGTAAGCTAGTGACTCCGTCTCTCATATGATCG (SEQ ID NO: 9);

[0116] PGL3-R-circmiR-29b-R: CCGGAATGCCAAGCTAGTGTCTTGCGTCTCTGAATTC (SEQ ID NO: 10).

[0117] The PCR amplification system is a 50 μl reaction system, and the specific components are as follows:

[0118]

[0119] The PCR amplification program is shown in Table 1 below:

[0120]

[0121] The results are shown in Figure 2. The results in Figure 2 show that circmiR can specifically reduce the expression of miR-29b and does not affect other members of the miR-29 family. AAV-delivered circmiR-29b has an inhibitory effect on miR-29b. AAV virus was selected with an MOI of 10 6 C2C12 differentiated myotubes were infected, and the expression of miR-29b was detected 48 h after virus infection. circmiR-29b significantly reduced the expression of miR-29b ( Figure 2A ). The results of luciferase experiments showed that circmiR-29b specifically adsorbed miR-29b and had no effect on miR-29a and miR-29c of the same family ( Figure 2B ).

[0122] Example 4

[0123] 1. AAV8-circmiR-29 virus was used to transfect C2C12 differentiated myotubes, and the gene fragment capable of synthesizing circmiR-29b was integrated into the AAV8 vector, and packaged into AAV8 virus with the assistance of AAV8 capsid plasmid and Helper plasmid. After separation and purification, the AAV virus titer was determined, and 1×10 7 The virus was added to the cell culture medium at a concentration of 50 μM VG / ml, dexamethasone (Dex, 50 μM) was added 48 h later, and the diameter of myotubes was detected by immunofluorescence staining 24 h later.

[0124] 2. Establishment of Dex (dexamethasone)-induced muscular atrophy model at the cellular level

[0125] Myotubes differentiated from C2C12 cells were grouped as follows: Group 1 was the control virus + PBS group; Group 2 was the control virus + Dex group; Group 3 was the AAV8-circmiR-29b virus + PBS group; Group 4 was the AAV8-circmiR-29b virus + Dex group. AAV8 virus containing circmiR-29b (or control virus, i.e., virus without circmiR-29b) was added to the cell culture medium at an MOI of 10. 6 After infecting myotubes, fresh differentiation medium was replaced after 12 hours. After culturing in an incubator for 36 hours, dexamethasone (Dex, 50 μM) (or PBS) was added to the myotubes. After 24 hours, the medium was removed and the cells were fixed with paraformaldehyde. Immunofluorescence staining with MF-20 antibody was then performed to detect the diameter of the myotubes.

[0126] The results are shown in Figure 3. Immunofluorescence staining detected that circmiR-29b protected against muscle atrophy and reversed the decrease in myotube diameter caused by dexamethasone (Dex, 50 μM) ( Figure 3A and Figure 3B ). AAV-delivered circmiR-29b protects against muscle atrophy at the myotube level.

[0127] Example 5

[0128] 1. AAV8-circmiR-29 virus was injected into the gastrocnemius muscle of mice, and the gene fragment capable of synthesizing circmiR-29b was integrated into the AAV8 vector and packaged into AAV8 virus with the assistance of AAV8 capsid plasmid and Helper plasmid. After separation and purification, the AAV virus titer was determined, with a titer of 1×10 12 The virus was injected orthotopically into the gastrocnemius muscle at a dose of 100 VG / mouse, and the muscle atrophy model was established 3 weeks later.

[0129] 2. Establishment of Den (nerve cutting) induced muscle atrophy model.

[0130] Wild-type male mice aged 8-10 weeks were taken and anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 10 μl / g. After anesthesia, the mice were fixed on a constant temperature blanket with their abdomen facing down using medical tape, and hair removal cream was used to remove the hair at the base of the right hind thigh of the mice. The depilatory part was disinfected with 75% ethanol, and then the epidermis was cut open with microscissors to expose the muscle tissue. Then, the deep muscle was separated by blunt separation to expose the sciatic nerve. After finding it, the sciatic nerve was lifted with curved forceps and 3-5 mm nerve was cut with microscissors to ensure that there was no possibility of kissing when the nerve was cut. Then, the muscle and epidermis were sutured, and the mice were kept warm on a constant temperature blanket and waited for recovery. After that, they were transferred to a mouse cage and raised for 7 days before being killed. The mice were weighed, the tibia length, and the gastrocnemius muscle was weighed and kept for subsequent testing.

[0131] The specific method is as follows:

[0132] The experimental groups were divided into 4 groups, namely control virus + sham operation group, control virus + Den group, AAV8-circmiR-29b virus + sham operation group and AAV8-circmiR-29b virus + Den group.

[0133] Three weeks before the establishment of the Den model, mice were treated by intramuscular injection using a disposable 1 ml sterile syringe. The specific method is as follows:

[0134] Group 1: control virus + sham operation group, the control virus was injected into the gastrocnemius muscle of mice at 50 μl / mouse, and sham operation was performed three weeks later;

[0135] Group 2, control virus + Den group, was injected with control virus into the gastrocnemius muscle of mice at 50 μl / mouse, and Den surgery was performed three weeks later;

[0136] Group 3: AAV8-circmiR-29b virus + sham operation group, mice were injected with AAV8-circmiR-29b virus into the gastrocnemius muscle, and then underwent sham operation three weeks later;

[0137] In the fourth group, AAV8-circmiR-29b virus + Den group, AAV8-circmiR-29b virus was injected into the gastrocnemius muscle of mice, and Den surgery was performed three weeks later.

[0138] The experiment ended after 1 week, and the mice were killed. The gastrocnemius muscles of the mice were dissected and weighed using an analytical balance. The muscle samples were then stained with WGA and HE to count the changes in the cross-sectional area of ​​the muscle fibers. Tissue samples were taken to extract total RNA from the gastrocnemius muscle tissue, and fluorescent quantitative PCR technology was used to detect changes in miR-29b.

[0139] The results are shown in Figure 4. Mouse gastrocnemius muscle samples significantly atrophied in the Den model, and circmiR-29b was able to significantly protect against the loss of muscle weight ( Figure 4A and Figure 4B ). Fluorescence quantitative PCR showed that after the Den model mice were injected with AAV8-circmiR-29b, the expression of miR-29b was significantly reduced ( Figure 4C WGA and HE staining showed that the muscle fiber diameter was significantly protected after the injection of AAV8-circmiR-29b in Den model mice ( Figure 4D and Figure 4E ). It can be seen that AAV-delivered circmiR-29b protects muscle atrophy induced by the Den model.

[0140] Example 6

[0141] 1. AAV8-circmiR-29 virus was injected into the gastrocnemius muscle of mice, and the gene fragment capable of synthesizing circmiR-29b was integrated into the AAV8 vector, and packaged into sufficient AAV8 virus with the assistance of AAV8 capsid plasmid and Helper plasmid. After separation and purification, the AAV virus titer was determined, with a titer of 1×10 12 The virus was injected orthotopically into the gastrocnemius muscle at a dose of 100 VG / mouse, and the muscle atrophy model was established 3 weeks later.

[0142] 2. Establishment of IMO (fixation) induced muscle atrophy model.

[0143] Wild-type male mice aged 8 to 10 weeks were taken, and 1% sodium pentobarbital was injected intraperitoneally at a dose of 10 μl / g to anesthetize the mice. After anesthesia, the mice were fixed on a constant temperature blanket with their abdomen facing down using medical tape, and the hair at the base of the thigh of the right hind limb of the mouse was removed using depilatory cream. The depilated part was disinfected with 75% ethanol, and then an orthopedic fixation needle was inserted from the Achilles tendon of the right hind limb of the mouse and penetrated upward into the tibia of the mouse, so that the right hind limb of the mouse could not be flexed and extended. The excess orthopedic fixation needle was cut off, and the inserted needle part was disinfected. The mice were kept warm on a constant temperature blanket and waited for resuscitation. After that, they were transferred to a mouse cage and raised for 7 days. The mice were killed, and the weight and tibia length of the mice were weighed. The gastrocnemius muscle was weighed and samples were kept for subsequent testing. One week after the model was established, the mice were dissected and the gastrocnemius muscle of the mice was weighed. The weight of the gastrocnemius muscle of the mice with successful models was significantly reduced.

[0144] The specific method is as follows:

[0145] Three weeks before the IMO model was established, mice were treated by intramuscular injection using a disposable 1 ml sterile syringe. The experimental groups were divided into 4 groups. The specific method is as follows:

[0146] Group 1: control virus + sham operation group, the control virus was injected into the gastrocnemius muscle of mice at 50 μl / mouse, and sham operation was performed three weeks later;

[0147] Group 2, control virus + IMO group, was injected with control virus into the gastrocnemius muscle of mice at 50 μl / mouse, and IMO surgery was performed three weeks later;

[0148] Group 3: AAV8-circmiR-29b virus + sham operation group, mice were injected with AAV8-circmiR-29b virus into the gastrocnemius muscle, and then underwent sham operation three weeks later;

[0149] In the fourth group, AAV8-circmiR-29b virus + IMO group, AAV8-circmiR-29b virus was injected into the gastrocnemius muscle of mice, and IMO surgery was performed three weeks later.

[0150] The experiment ended after 1 week, and the mice were killed. The gastrocnemius muscles of the mice were dissected and weighed using an analytical balance. The muscle samples were then stained with WGA and HE to count the changes in the cross-sectional area of ​​the muscle fibers. Tissue samples were taken to extract total RNA from the gastrocnemius muscle tissue, and the changes in miR-29b were detected using fluorescent quantitative PCR technology.

[0151] The results are shown in Figure 5. Mouse gastrocnemius muscle samples significantly atrophied in the IMO model, and circmiR-29b was able to significantly protect against the loss of muscle weight ( Figure 5A and Figure 5B). Fluorescence quantitative PCR showed that the expression of miR-29b was significantly reduced after AAV8-circmiR-29b was injected into IMO model mice ( Figure 5C WGA and HE staining showed that the muscle fiber diameter was significantly protected after AAV8-circmiR-29b injection in IMO model mice ( Figure 5D and Figure 5E ). As can be seen, AAV-delivered circmiR-29b protected against muscle atrophy induced by the immobilization (IMO) model.

[0152] Example 7

[0153] 1. Establishment of AngII (AngII sustained-release pump)-induced muscle atrophy model.

[0154] Wild-type male mice aged 8 to 10 weeks were taken and anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 10 μl / g. After anesthesia, the mice were fixed on a constant temperature blanket with their abdomen facing downward using medical tape, and the hair on the back of the neck of the mice was removed using depilatory cream. The depilated part was disinfected with 75% ethanol, and then a 3 mm long incision was made on the back of the mouse using microscissors. Then, an Alzet sustained-release pump filled with 200 μl 2 mg / ml AngII solution was inserted subcutaneously from the incision on the back of the mouse. The sustained-release pump model was Model 2001 (ALZET), and the AngII release efficiency was 1.46 mg / kg / day. The sustained-release pump of the control group was filled with PBS. Then, the epidermis was sutured, and the mice were placed on a constant temperature blanket to keep warm and wait for recovery. After that, they were transferred to a mouse cage and raised for 7 days before being killed. The mice were weighed, the tibia length, and the gastrocnemius muscle was weighed and sampled for subsequent testing. One week after the model was established, the mice were dissected and their gastrocnemius muscles were weighed. The weight of the gastrocnemius muscles of mice with successful model was significantly reduced.

[0155] 2. AAV8-circmiR-29 virus was injected into the gastrocnemius muscle of mice, and the gene fragment capable of synthesizing circmiR-29b was integrated into the AAV8 vector, and packaged into sufficient AAV8 virus with the assistance of AAV8 capsid plasmid and Helper plasmid. After separation and purification, the AAV virus titer was measured, with a titer of 1×10 12 The virus was injected orthotopically into the gastrocnemius muscle at a dose of 100 VG / mouse, and the muscle atrophy model was established 3 weeks later.

[0156] The specific method is as follows:

[0157] Three weeks before the establishment of the AngII model, mice were treated by intramuscular injection using a disposable 1 ml sterile syringe. The experimental groups were divided into 4 groups. The specific method is as follows:

[0158] Group 1: Control virus + PBS group, the control virus was injected into the gastrocnemius muscle of mice at 50 μl / mouse, and the pump was implanted three weeks later;

[0159] Group 2, control virus + AngII group, was injected into the gastrocnemius muscle of mice at 50 μl / mouse, and the pump was implanted three weeks later;

[0160] Group 3: AAV8-circmiR-29b virus + PBS group, AAV8-circmiR-29b virus was injected into the gastrocnemius muscle of mice, and the pump was implanted three weeks later;

[0161] In the fourth group, AAV8-circmiR-29b virus + AngII group, AAV8-circmiR-29b virus was injected into the gastrocnemius muscle of mice, and the pump was implanted three weeks later.

[0162] The experiment ended after 1 week, and the mice were killed. The gastrocnemius muscles of the mice were dissected and weighed using an analytical balance. The muscle samples were then stained with WGA and HE to count the changes in the cross-sectional area of ​​the muscle fibers. Tissue samples were taken to extract total RNA from the gastrocnemius muscle tissue, and the changes in miR-29b were detected using fluorescent quantitative PCR technology.

[0163] The results are shown in Figure 6. In the AngII model, circmiR-29b can significantly restore the grip strength of mice and protect the loss of muscle weight ( Figure 6A to Figure 6C ). After the injection of AAV8-circmiR-29b into AngII model mice, the expression of miR-29b was significantly reduced ( Fig.6D WGA and HE staining showed that the muscle fiber diameter was significantly protected after AAV8-circmiR-29b injection in IMO model mice ( Fig. 6E and Fig. 6F ). It can be seen that the delivered circmiR-29b protects muscle atrophy induced by AngII sustained-release pump (AngII) model.

[0164] Example 8

[0165] 1. Establishment of SWI (spiral wire winding fixation) induced muscle atrophy model.

[0166] Wild-type male mice aged 8 to 10 weeks were taken, and 10 μl / g of 1% sodium pentobarbital was injected intraperitoneally to anesthetize the mice. After anesthesia, the mice were fixed on a constant temperature blanket with their abdomen facing down using medical tape. The hind limbs of the mice were first wrapped with medical tape. Be careful not to wrap it too tightly to affect the normal blood flow of the mice. Subsequently, the hind limbs of the mice were spirally wrapped and fixed using a 2 mm thick wire, while the mice in the control group were free to move. The mice were killed after being fixed for 3, 5, and 7 days, and the gastrocnemius muscles were dissected and their weights were measured. The results showed that the gastrocnemius muscles of mice underwent obvious atrophy at 3 days of fixation, and the muscle atrophy gradually worsened with the extension of fixation time ( Fig. 7A ). This indicates that the muscle atrophy model mouse was successfully constructed.

[0167] 2. On the 4th day after the establishment of the SWI model, when muscle atrophy had already occurred, the AAV8-circmiR-29 virus was injected into the gastrocnemius muscle of mice, and the gene fragment capable of synthesizing circmiR-29b was integrated into the AAV8 vector and packaged into sufficient AAV8 virus with the assistance of AAV8 capsid plasmid and Helper plasmid. After separation and purification, the AAV virus titer was determined, with a value of 1×10 12 The virus was injected in situ into the gastrocnemius muscle at a dose of 1.5 VG / mouse. The mice were killed 4 weeks later, and their body weight and tibia length were measured. The gastrocnemius muscle was weighed and samples were kept for subsequent testing.

[0168] The specific method is as follows:

[0169] On the 4th day of SWI model establishment, mice were treated by intramuscular injection using a disposable 1 ml sterile syringe. The experimental groups were divided into 4 groups. The specific method is as follows:

[0170] Group 1 control virus + control group, both were injected with control virus into the gastrocnemius muscle of mice at 50 μl / mouse;

[0171] Group 2, control virus + SWI group, the control virus was injected into the gastrocnemius muscle of mice at 50 μl / mouse;

[0172] Group 3 AAV8-circmiR-29b virus + control group, AAV8-circmiR-29b virus was injected into the gastrocnemius muscle of mice;

[0173] In the fourth group, AAV8-circmiR-29b virus + SWI group, AAV8-circmiR-29b virus was injected into the gastrocnemius muscle of mice.

[0174] The experiment ended after 4 weeks, and the mice were killed. The gastrocnemius muscles were dissected and weighed using an analytical balance. The muscle samples were then stained with WGA and HE to count the changes in the cross-sectional area of ​​the muscle fibers. Tissue samples were taken to extract total RNA from the gastrocnemius muscle tissue, and the changes in miR-29b were detected using fluorescent quantitative PCR.

[0175] The results are shown in Figure 7. In the SWI model, circmiR-29b can significantly protect against the loss of muscle weight ( Figure 7B to Figure 7C ). After the SWI model mice were injected with AAV8-circmiR-29b, the expression of miR-29b was significantly reduced ( Fig.7D WGA and HE staining showed that the muscle fiber diameter was significantly protected after AAV8-circmiR-29b injection in IMO model mice ( Fig. 7E and Figure 7F ). It can be seen that the delivered circmiR-29b protects muscle atrophy induced by the SWI model.

[0176] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. Sequence Listing <110> Shanghai University <120> An engineered circular RNA circmiR-29b and its application in preparing a drug for treating muscular atrophy <160> 17 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty two <212> DNA <213> Artificial Sequence <400> 1 aacactgatt ttcctggtgc ta 22 <210> 2 <211> 12 <212> DNA <213> Artificial Sequence <400> 2 aaacgtgcta cg 12 <210> 3 <211> 396 <212> DNA <213> Artificial Sequence <400> 3 aacactgatt ttcctggtgc taaaacgtgc tacgaacact gattttcctg gtgctaaaac 60 gtgctacgaa cactgatttt cctggtgcta aaacgtgcta cgaacactga ttttcctggt 120 gctaaaacgt gctacgaaca ctgattttcc tggtgctaaa acgtgctacg aacactgatt 180 ttcctggtgc taaaacgtgc tacgaacact gattttcctg gtgctaaaac gtgctacgaa 240 cactgatttt cctggtgcta aaacgtgcta cgaacactga ttttcctggt gctaaaacgt 300 gctacgaaca ctgattttcc tggtgctaaa acgtgctacg aacactgatt ttcctggtgc 360 taaaacgtgc tacgaacact gattttcctg gtgcta 396 <210> 4 <211> 1060 <212> DNA <213> Artificial Sequence <400> 4 tagaatcgcc actcctgcat catttggtct cttgagccca ctatagactt aatatgtgtt 60 ttggaataat catggaaaga atgacattgg cactgagaag gaacatttta tggctgactc 120 tgcagccttc tagctcttc catacagctc agcaaaggcc tggttatctc ttcttcctgt 180 tgcacactgc ctgcaacttc gaaagcaggc tgtgtataca gatgtgaaga actgtgcttg 240 aagacccatg tgaccagctg atgatctatc agaagtccca acttgttgaa atggggggag 300 gggaaggact gtgtcttaat gaaaacaggc tttgaaagaa ctgcacctaa aatatgagaa 360 tactgcatct tcagtgaaat aatttccttc acaaggacaa atactttga gctttattgc 420 caatgagctt gaagatttct ctgttcccaa ggaaccttgg agcagtagga tttttataaa 480 acacaataaa gatgtactcc agatttaag ataatcagca tgcattgtat tgggattatt 540 tcagagtctg agtttcaaga tgtggctctt aataaggtga ttttgtcaat gaaattttt 600 ccaaagatat catactaagt caaatggaaa aaaaaattca cttgtgcaac aaggaagaat 660 ttacttaatg cttcaagagg tttgctttct ggctgttagt atgtaccagc atgtgctcaa 720 tagaactgtg cggtaatggg acttttcatt ttcagggagt catatgtgtg tcttgctcca 780 tgaacgggac tacatgtaga aaatacacaa acagagagat tgaattgtaa atattacaaa 840 agattgttta gtcactgatg gattcttagg ataatgattc cacagagatt ttatgatata 900 gaaagttttg gtataagtga ttcaaataag ttattaatga tgctttgact ctagtgatgt 960 gatttctctt cttcgtcgtc ttttttttt tttttaactg caggttggag cagttggaag 1020 tcttattgta caacatgctt cgattaagtc tcccacccaa 1060 <210> 5 <211> 1056 <212> DNA <213> Artificial Sequence <400> 5 gaggtggagg ggaagacttt gaggacacct gtggagagct cgaattccag aacgatgaaa 60 tagtgtaagt tacctggtta cctgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgcctgtg 120 tgtgtgtgtg tgtatctttt tgcatgtgag tatgtactct tttaacaagt gcaattcaag 180 aaaccaccta agaatccatc agtgactaaa caatcttttg taatatttac aattcaatct 240 ctctgtttgt gtattttcta catgtagtcc cgttcatgga gcaagacaca catatgactc 300 cctgaaaatg aaaagtccca ttaccgcaca gttctattga gcacatgctg gtacatacta 360 acagccagaa agcaaacctc ttgaagcatt aagtaaaattc ttccttgttg cacaagtgaa 420 tttttttttc catttgactt agtatgatat ctttggaaaa aatttcattg acaaaatcac 480 cttattaaga gccacatctt gaaactcaga ctctgaaata atcccaatac aatgcatgct 540 gattatctta aatcctggag tacatcttta ttgtgtttta taaaaatcct actgctccaa 600 ggttccttgg gaacagagaa atcttcaagc tcattggcaa taaagctcaa aagtatttgt 660 ccttgtgaag gaaattattt cactgaagat gcagtattct catattttag gtgcagttct 720 ttcaaagcct gttttcatta agacacagtc cttcccctcc ccccatttca acaagttggg 780 acttctgata gatcatcagc tggtcacatg ggtcttcaag cacagttctt cacatctgta 840 tacacagcct gctttcgaag ttgcaggcag tgtgcaacag gaagaagaga taaccaggcc 900 tttgctgagc tgtatggaag agcctagaag gctgcagagt cagccataaa atgttccttc 960 tcagtgccaa tgtcattctt tccatgatta ttccaaaaca catattaagt ctatagtggg 1020 ctcaagagac caaatgatgc aggagtggcg attcta 1056 <210> 6 <211> 23 <212> RNA <213> Artificial Sequence <400> 6 uagcaccauu ugaaaucagu guu 23 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 gaggtggagg ggaagacttt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 tagaatcgcc actcctgcat 20 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <400> 9 cgatctaagt aagctagtga ctccgtctct catatgatcg 40 <210> 10 <211> 37 <212> DNA <213> Artificial Sequence <400> 10 ccggaatgcc aagctagtgt cttgcgtctc tgaattc 37 <210> 11 <211> 60 <212> DNA <213> Artificial Sequence <400> 11 atgtcgacaa acgtgctaaa gcttgaattc aaacgtgcta catatgtcta gactcgagat 60 <210> 12 <211> 38 <212> DNA <213> Artificial Sequence <400> 12 aacgtgctac atatgtctag gaggtggagg ggaagact 38 <210> 13 <211> 32 <212> DNA <213> Artificial Sequence <400> 13 gattcttagg tggtttcttg aattgcactt gt 32 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <400> 14 caagaaacca cctaagaatc catcagtgac taaac 35 <210> 15 <211> 41 <212> DNA <213> Artificial Sequence <400> 15 atgaattatc tcgagtctag tagaatcgcc actcctgcat c 41 <210> 16 <211> 26 <212> DNA <213> Artificial Sequence <400> 16 gtcgactaga atcgccactc ctgcat 26 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <400> 17 aagcttttgg gtgggagact taatcg 26

Claims

1. Use of a circular RNA circmiR-29b in the preparation of a drug for preventing and / or treating muscle atrophy, wherein the circular RNA circmiR-29b comprises an active sequence and a random sequence; the active sequences are connected in series by 6 to 13 repetitions, and the random sequence is inserted between the active sequences; the nucleotide sequence of the active sequence is shown in SEQ ID NO: 1; The nucleotide sequence of the circular RNA circmiR-29b is shown in SEQ ID NO:

3.

2. Use of an adeno-associated virus recombinant vector in the preparation of a drug for preventing and / or treating muscular atrophy, wherein the adeno-associated virus recombinant vector is an adeno-associated virus vector into which the circular RNA circmiR-29b described in claim 1 is inserted.

3. The application according to claim 2, characterized in that: The adeno-associated virus vector includes an AAV8 vector.

4. The use according to claim 2 or 3, characterized in that: The adeno-associated virus vector further comprises a 5' guide looping sequence and a 3' guide looping sequence; The nucleotide sequence of the 5' guide looping sequence is shown in SEQ ID NO: 4; The nucleotide sequence of the 3' guide looping sequence is shown in SEQ ID NO: 5; The insertion position of the circular RNA circmiR-29b is between the 5' guide circularization sequence and the 3' guide circularization sequence.

5. Use of a circular RNA circmiR-29b delivery system in the preparation of a drug for preventing and / or treating muscular atrophy, wherein the delivery system is an adeno-associated virus containing the circular RNA circmiR-29b described in claim 1 or is obtained by packaging the adeno-associated virus recombinant vector described in any one of claims 2 to 4.

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