CN115944750A - A codable circRNA related to buffalo skeletal muscle development and its application - Google Patents

Abstract

The invention relates to encoding circRNA related to the development of buffalo skeletal muscle and application thereof, wherein the encoding circRNA is circMYBPCC, the sequence of the encoding circMYBPCC is shown as SEQ ID NO:1, the overexpression of the circMYBPCC promotes the proliferation and differentiation of myoblasts, the expression of the circMYBPCC is interfered to inhibit the proliferation and differentiation of the myoblasts, and a circMYBPCC nucleic acid compound formed by the circMYBPCC and liposome is used as a component of a medicine for treating muscle injury and is locally injected to a muscle injury part. The invention verifies the annular structure of the buffalo source circMYBPCC and the potential of the protein which can be coded by the annular structure, finds that after the administration of the circMYBPCC, the regeneration and repair of injured muscles can be remarkably promoted, and provides a new selection target point for molecular regulation and control of the development of buffalo muscles.

Description

A codable circRNA related to buffalo skeletal muscle development and its application

technical field

The invention relates to the field of biomedical technology, in particular to an encoding circRNA related to the development of buffalo skeletal muscle and its application.

Background technique

Skeletal muscle plays a vital role in locomotion, metabolism and homeostasis. Skeletal muscle development is inseparable from the proliferation and differentiation of myoblasts, which is not only regulated by genes, signaling pathways, transcription factors and miRNA, but also complexly regulated by circRNA. CircRNAs are a special class of non-coding RNAs that are produced by a non-canonical splicing event called back-splicing, lacking a 5' end cap and a 3' end poly(A) tail, and are closed by covalent bonds. Circular, not easily degraded by exonucleases, and more stable in structure than linear RNA. Studies have found that circRNAs are involved in biological processes such as body growth, development, and disease occurrence and development, and play important functions in cell proliferation and differentiation, cell cycle, cell metabolism, cell apoptosis, and maintenance of pluripotency. With the deepening of research, circRNA has been proved to be able to encode polypeptides, and the encoded functional peptides are involved in the regulation of skeletal muscle development. CircZNF609 is one of the first endogenous circRNAs reported to bind to polysomes for translation, and CircZNF609 has been shown to promote myoblast proliferation; circRNA CircFAM188B encodes a protein that regulates proliferation and differentiation of chicken myoblasts. These findings suggest that circRNAs can regulate myogenesis by encoding polypeptides.

Currently on the market, there are flurbiprofen Babu ointment, Yunnan Baiyao aerosol, swelling and pain aerosol spray, etc. for external use in the treatment of muscle damage. Oral Chinese patent medicines for promoting blood circulation and removing blood stasis, such as Huoxue Zhitong Capsules, Sanqi Powder or Sanqi Capsules, Hulisan Capsules, etc. If the local swelling is particularly severe, you can use detumescence and dehydration drugs, such as intravenous mannitol. If it causes damage to the nerves at the same time, it is necessary to take oral nerve-nourishing drugs, such as methylcobalamin or vitamin B1. After the injury will cause obvious pain, non-steroidal anti-inflammatory analgesic drugs should be selected according to the degree of pain. For mild pain, celecoxib capsules can be used for pain relief; for moderate pain, ibuprofen sustained-release capsules can be selected , If the injury is serious and severe pain occurs, choose tramadol sustained-release tablets. Mild muscle damage can be recovered in about two weeks, while serious muscle damage will take a long time to gradually recover. These drugs have different effects on muscle damage. Congestion and muscle tissue fragments after muscle damage will cause the body's inflammatory immune response. Anti-inflammatory and analgesic drugs can effectively reduce the degree of inflammatory immune response and reduce the pain of patients, but they will prolong the time for muscle repair. Nerve-nourishing drugs such as methylcobalamin can effectively help remodel nerve cells in muscles and promote muscle regeneration, but they cannot directly promote muscle regeneration.

It can be seen that the drugs currently used clinically do not directly promote the regeneration of muscle tissue. The therapeutic drugs are mainly represented by relieving the pain of patients, providing nutrition and other auxiliary functions, and mainly relying on the self-regeneration of human muscles to start the repair function. Nucleic acid drugs can directly promote the proliferation and differentiation of muscle cells, and promote the regeneration of muscle tissue, but there are no reports of nucleic acid drugs that directly treat damaged muscles.

In response to this problem, the present invention provides a buffalo-derived circRNA that can promote muscle repair after injury. By significantly promoting myoblast growth and related gene expression, it can help muscle fiber remodeling and promote muscle regeneration. Buffalo meat has low fat content, low cholesterol and low calories, making it suitable for all kinds of people. With the rapid development of molecular biology technology, the expression of related genes can be controlled at the molecular level. Therefore, using related genes to improve the growth performance and meat quality of buffalo can improve the growth rate and meat quality of buffalo faster and more accurately.

Contents of the invention

In order to solve the above problems, the present invention provides a codable circRNA related to the development of buffalo skeletal muscle and its application.

The present invention provides an application of a codable circRNA related to the development of buffalo skeletal muscle in the preparation of a drug for treating muscle damage. The codable circRNA is circMYBPCC, and its sequence is shown in SEQ ID NO: 1. Overexpression of circMYBPCC promotes growth Myocyte proliferation and differentiation, interference with circMYBPCC expression inhibits myoblast proliferation and differentiation.

Further, the mouse-derived sequence of the circMYBPCC is shown in SEQ ID NO:3.

Further, the human sequence of the circMYBPCC is shown in SEQ ID NO:4.

Further, the circMYBPCC and liposome form a circMYBPCC nucleic acid complex as a component of a drug for treating muscle damage.

Further, when the circMYBPCC nucleic acid complex is used as a component of a drug for treating muscle damage, the dosage per kilogram of damaged muscle is 0.5-2 mg.

Further, the drug for treating muscle damage is an injection, which is locally injected at the muscle damage site.

Compared with the prior art, the present invention has the following beneficial effects in application:

1. The present invention first verified the ring structure of buffalo-derived circMYBPCC and its potential to encode a protein, and prepared its polyclonal antibody. Further functional verification at the cellular level found that circMYBPCC promoted the proliferation and differentiation of buffalo myoblasts; at the same time, a mouse muscle injury model was constructed, and it was found that after administration of circMYBPCC, it could significantly promote the regeneration and repair of damaged muscles.

2. The present invention discovers for the first time that circMYBPCC can encode a polypeptide, and can promote the proliferation and differentiation of buffalo myoblasts, providing a new selection target for the molecular regulation of buffalo muscle development.

3. According to the buffalo-derived circMYBPCC sequence, the present invention provides a mouse-derived circMYBPCC sequence and a human-derived circMYBPCC sequence. It has been verified by mouse experiments that the circMYBPCC provided by the present invention has a therapeutic effect on muscle damage and can be used as an RNA drug, and It has the characteristics of rapid onset of action and high safety, and has great potential application value in the treatment of muscle damage through local intramuscular injection.

4. The present invention adopts the form of developing the circMYBPCC nucleic acid complex into an injection to inject and administer the local muscles that cause muscle damage. The circMYBPCC nucleic acid complex can be translated into a circMYBPCC polypeptide in the muscle, and then the circMYBPCC polypeptide can significantly promote muscle fiber repair. Compared with the current clinical muscle injury drugs (such as: anti-inflammatory and analgesic drugs can effectively reduce the degree of inflammatory immune response and reduce patient pain, but will prolong the time for muscle repair; drugs that nourish nerves such as methylcobalamin can effectively help nerve cells in muscles remodeling, but cannot directly promote muscle regeneration), circMYBPCC polypeptide nucleic acid complex can directly and effectively promote the repair of damaged muscles, filling the gap in the treatment of muscle damage.

Description of drawings

Figure 1, the identification of the circMYBPCC ring structure in the present invention;

Fig. 2, the expression of circMYBPCC encoded peptide in buffalo muscle in the present invention;

Figure 3. After overexpressing circMYBPCC in the present invention, the expression efficiency detection;

Fig. 4, after interfering with circMYBPCC in the present invention, the interference efficiency detection;

Fig. 5, CCK8 test detects cell proliferation after overexpressing circMYBPCC in the present invention;

Fig. 6. Flow cytometry detection of cell cycle after overexpression of circMYBPCC in the present invention;

Fig. 7, EdU test detects cell proliferation after interfering with circMYBPCC in the present invention;

Fig. 8, after overexpressing circMYBPCC in the present invention, detect the expression of myoblast proliferation marker genes at RNA and protein levels;

Figure 9, the detection of the expression of myoblast proliferation marker genes at the RNA and protein levels after interfering with circMYBPCC in the present invention;

Figure 10, the detection of the expression of myoblast differentiation marker genes at the RNA and protein levels after overexpressing circMYBPCC in the present invention;

Fig. 11 , detection of myoblast differentiation marker gene expression at RNA and protein levels after interfering with circMYBPCC in the present invention;

Fig. 12, the influence of overexpressing circMYBPCC on the number of buffalo myoblast differentiation myotubes detected by immunofluorescent staining of cells of the present invention;

Fig. 13, the slice analysis chart of mouse skeletal muscle injury model construction in the present invention;

Figure 14. The present invention detects the expression level of circMYBPCC in mouse muscles after administration of circMYBPCC;

Figure 15, the results of HE staining of muscle tissue after 3 days and 7 days of administration of circMYBPCC according to the present invention;

Fig. 16 , 7 days after administration of circMYBPCC in the present invention, the expression of myoblast proliferation, differentiation and apoptosis marker genes at RNA and protein levels was detected.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Experimental materials and their sources:

(1) pCD2.1-CIR was purchased from Wuhan Point Biotechnology Company; siRNA was designed and synthesized by Guangzhou Ruibo Biotechnology Company;

(2) 3-month-old fetal cows were collected from a slaughterhouse in Nanning, Guangxi; 4-5 month-old New Zealand white rabbits were from the animal experiment base of Guangxi University in Nanning, Guangxi; 4-week-old C57BL/6 strain SPF (specific pathogen-free) experimental mice Purchased from Hunan SJA Experimental Animal Co., Ltd.;

(3) The antibodies used in Western Blot and immunofluorescence staining are as follows: anti-β-actin (66009-1-Ig), CDK2 (60312-1-Ig), MyoD1 (18943-1-AP), MyoG (67082-1-Ig), Ig), MyHC (22281-1-AP), goat anti-mouse (SA00001-1), goat anti-rabbit (SA00001-2), (FITC)–Goat Anti-Rabbit (SA00003-2), (FITC)–Goat Anti-mouse (SA00003-1) (USA, proteintech); CyclinD1 (WL01435a), PCNA (WL03213) (Shenyang, Wanlei Biology);

(4) Primer synthesis: synthesized by Shanghai Sangon Biotechnology Service Co., Ltd.;

qPCR Master Mix(南京，诺唯赞)；Trizol Reagent试剂(美国，Invitrogent)；无水乙醇(上海，国药集团)；Ribo-Zero^TMGold Kits(美国，Epicentre；RNase R(广州，吉赛)；弗氏完全佐剂、弗氏不完全佐剂、青链霉素双抗、RIPA裂解液、PMSF蛋白酶抑制剂、4X蛋白上样缓冲液(含DTT)、ECL超敏发光液A、ECL超敏发光液B(北京，索莱宝)；E.coli DH5α感受态大肠杆菌(北京，全式金)；胶回收试剂盒(北京，天根)；去内毒素质粒DNA小量提取试剂盒(美国，OMEGA)；去内毒素质粒DNA小量提取试剂盒(北京，天漠)；BamHI、Kpn I限制性内切酶(日本，TaKara)；T4 DNA连接酶(美国，BioLabs)；EdU试剂盒(广东，锐博)；心脏毒素(Cardiotoxin，CTX)(上海，恪敏)；生理盐水、载玻片、苏木精染料、伊红染料、中性树脂、OTX、4％多聚甲醛、蔗糖(北京，索莱宝)；手术刀片、镊子、包埋盒、手术剪(江苏，世泰)。(5) Horse serum, fetal bovine serum, trypsin-EDTA (0.25%) (US, Gibco); 2X Taq PCR Mastermix (Beijing, Tiangen); DMEM (US, corning); HiScript 1st Strand cDNA Synthesis Kit, CCK-8Cell Counting Kit, HiScript III RT SuperMix for qPCR(+gDNA wiper), 2XCham QTM Universal

qPCR Master Mix (Nanjing, Novozyme); Trizol Reagent (US, Invitrogent); absolute ethanol (Shanghai, Sinopharm); Ribo-Zero ^TM Gold Kits (US, Epicentre); RNase R (Guangzhou, Jisai); Freund's complete adjuvant, Freund's incomplete adjuvant, penicillin-streptomycin double antibody, RIPA lysate, PMSF protease inhibitor, 4X protein loading buffer (including DTT), ECL ultra-sensitive luminescence solution A, ECL ultra-sensitive Luminescence solution B (Beijing, Suo Laibao); E.coli DH5α competent Escherichia coli (Beijing, Quanjin); Glue recovery kit (Beijing, Tiangen); Endotoxin-free plasmid DNA mini-extraction kit (USA , OMEGA); endotoxin-free plasmid DNA mini-extraction kit (Beijing, Tianmo); BamHI, Kpn I restriction endonuclease (Japan, TaKara); T4 DNA ligase (USA, BioLabs); EdU kit ( Guangdong, Ruibo); cardiotoxin (Cardiotoxin, CTX) (Shanghai, Kemin); normal saline, glass slide, hematoxylin dye, eosin dye, neutral resin, OTX, 4% paraformaldehyde, sucrose ( Beijing, Suo Laibao); surgical blades, tweezers, embedding cassettes, surgical scissors (Jiangsu, Shitai).

Example 1: Isolation and culture of buffalo primary myoblasts

The primary buffalo myoblasts were isolated by enzymatic digestion, and the aseptic operation was strictly controlled in this process. The specific steps are as follows:

1. The fetal membranes were peeled off, and the fetal cattle were washed 3 times with PBS containing 2% double antibody;

2. Cut the skin with sterile ophthalmic scissors, peel off the fascia and other tissues with tweezers, separate the longissimus dorsi muscle, wash it in 75% alcohol for 3 seconds, wash it with PBS containing 2× double antibody for 3 times, and wash the surface alcohol Afterwards, transfer to 37°C preheated PBS solution containing 2× double antibody and fully soak;

3. Transfer to a sterile ultra-clean bench, and use a new set of sterile surgical instruments to remove blood vessels and connective tissue between muscles;

4. Use sterile ophthalmic scissors to cut the muscle tissue into minced meat as small as possible, collect it into a 15mL centrifuge tube, add an appropriate amount of type I collagenase, and put it in a 37°C water bath for 1.5h;

5. Discard the supernatant by centrifugation, add an appropriate amount of trypsin to digest, and digest at 37°C for 30 minutes;

6. When the digestion is complete, add an equal volume of termination medium to terminate the digestion;

7. Centrifuge to obtain primary buffalo myoblasts, resuspend the myoblasts in warm DMEM (37°C), filter through 100-mesh and 70-mesh cell sieves, collect the filtrate, discard the supernatant by centrifugation, and keep the precipitated buffalo raw Generation of myoblasts;

8. Resuspend the buffalo primary myoblasts in DMEM containing 20% FBS and 1% penicillin-streptomycin, and culture them in an incubator at 37°C with 5% CO ₂ for 2 hours;

9. Aspirate the culture supernatant of buffalo primary myoblasts and inoculate them into a new cell culture plate for purification;

10. Change the medium with the primary culture medium every two days, and continue the culture to obtain primary buffalo myoblasts.

Embodiment 2: Utilize PCR method to amplify and determine circMYBPCC and its nucleotide sequence

1. Myoblast total RNA extraction and cDNA synthesis

(1) Harvest 1 to 5× ¹⁰ primary buffalo myoblasts of Example 1, discard the medium in the culture dish, wash once with pre-cooled PBS, discard the PBS, add 1mL Trizol, and pipette the myoblasts down, collected into a RNase-free 1.5ml centrifuge tube, and centrifuged at 4°C to discard the supernatant;

(2) Add 500 μL of pre-cooled chloroform to centrifuge tube 1, shake vigorously for 30 seconds, place on ice for 10 minutes, centrifuge at 12000 r/min at 4°C for 10 minutes;

(3) Aspirate the uppermost aqueous phase into a new 1.5mL centrifuge tube. Add 500 μL of pre-cooled isopropanol, shake vigorously for 30 s, place on ice for 5 min, centrifuge at 12000 r/min at 4 °C for 10 min;

(4) Discard the supernatant, wash with 1ml 75% ethanol, flick and mix well, centrifuge at 12000r/min at 4°C for 5min;

(5) Discard the supernatant, and dry the precipitated RNA in the ultra-clean Taichung wind;

(6) RNA-free ddH ₂ O dissolves RNA; use a spectrophotometer to detect the quality and concentration of total RNA.

2. Use HiScript 1st Strand cDNA Synthesis Kit to synthesize cDNA

(1) Remove genomic DNA: add the following liquids to an RNase-free PCR tube: template RNA (the amount of total RNA is 1pg ^-1 μg), 4×gDNA wiper Mix 4μL, RNase-free ddH ₂ O to 16μL, mix well ; PCR instrument 42 ℃, 2min;

(2) Prepare the reverse transcription reaction system: add 5×HisScriptqRT SuperMix 4μL directly into the centrifuge tube of the first step of reaction and mix well;

(3) Reverse transcription reaction: 37°C, 15min; 85°C, 5s. The product was stored in a -20°C refrigerator.

3. PCR amplification of the full length of circMYBPCC

The buffalo myoblast cDNA was used as a template for PCR amplification. Primers used: circMYBPCC-full-length-upstream primer: GGGGTACCAGCCAAGAGAACTATGCAGG; circMYBPCC-full-length-downstream primer: TTGATGATCTGCATCTCGAATGGATCCCG. Carry out PCR amplification operation according to 2X Taq PCR Master mix instructions, and use agarose gel to detect the obtained PCR product. The gel detection result is shown in Figure 1A. The PCR amplification product is a single band, and the size of the target gene is cut. The bands of the same size were recovered by gel, and the recovered products were sent to Shanghai Sangong for sanger sequencing. The obtained sequence circMYBPCC was 378bp in full length, and its nucleotide sequence was shown in SEQ ID NO:1.

Embodiment 3: Utilize qPCR and RNase R enzyme digestion method to prove the circular structure of circMYBPCC

1. Design primers: β-actin-buffalo-upstream primer: CTGGCATTGTCATGGACTCTG;

β-actin-buffalo-downstream primer: GCTCGGCTGTGGTGGTAAA; design a back primer across the circMYBPCC loop structure adapter: circMYBPCC-backward primer-upstream primer: TGCAGGAAACTACAGATGTGA; circMYBPCC-backward primer-downstream primer: GACTGTTGGCATTCTGCTTGT.

2. RNase R enzyme digests RNA

RNase R enzyme can digest linear RNA, but not circular RNA, so it can be used to identify circular RNA. Treat buffalo muscle RNA samples with RNase R, add 1 μL (20U/μL) RNaseR, 3 μL 10x RNase R Buffer, 6 μL ddH2O to 20 μL (total 20 μg) buffalo muscle RNA, mix well and incubate at 37°C for 15 minutes for treatment. After digestion, heat at 75°C for 10s to inactivate the enzyme. Use a reverse transcription kit to reverse synthesize RNA into cDNA for subsequent qPCR detection.

3. qPCR detection

The fluorescent quantitative kit ChamQ Universal SYBR qPCR Master Mix was used to detect cDNA samples reversely synthesized from RNase R-treated buffalo muscle RNA. The reaction was carried out on the real-time fluorescent quantitative PCR system-7500 system, with β-actin as the linear internal reference, using 2 ^-ΔΔCT method to calculate relative expression of genes (n=3) As shown in Figure 1B, after RNase R treatment, qPCR detected that the expression of Actin gene representing linear transcripts was significantly decreased, while the relative expression of circMYBPCC transcripts was significantly This is because RNase R digested the mRNA of the Actin gene and could not digest the circular structure of circMYBPCC. The qPCR results showed that circMYBPCC was resistant to the digestion of RNase R, indicating that circMYBPCC was a stable circular RNA.

Example 4: circMYBPCC coding ability identification

1. Peptide antigen design and antibody preparation

ORF Finder was used to predict the open reading frame (ORF) and internal ribosome entry point (IRES) of the full-length sequence of circMYBPCC. Predict the epitope of B cells recognizing the polypeptide encoded by circMYBPCC through the online website based on the neural network algorithm (https://webs.iiitd.edu.in/raghava/abcpred/), the parameters are: ABCpred, Threshold>0.8, length=16aa. Determine the amino acid sequence of the antigenic epitope located at the N-terminal with the highest scoring antigenic peptide: RCEVTYKDKFDSCSFD, which was synthesized by Shanghai Sangon Biological Company;

The synthesized peptide was dissolved in PBS to immunize rabbits; for the first immunization, 10 mg of the dissolved peptide was emulsified with Freund's complete adjuvant; for booster immunization, 5 mg of the peptide was emulsified with Freund's incomplete adjuvant. The method of multi-point subcutaneous injection on the back of the rabbit's neck was adopted, and a total of 4 immunizations were carried out, with an interval of 1 week between each immunization; 7 days after the last immunization, blood samples were drawn from the rabbit's heart, and the antiserum was separated by centrifugation.

2. Immunofluorescence

Harvest buffalo myoblasts in the proliferation and differentiation stages, rinse with PBS for 3 times, fix with 4% paraformaldehyde for 30 minutes, wash with PBS for 3 times; permeabilize with 0.1% Triton X-100 for 30 minutes, wash with PBS for 3 times; 2% goat Serum was blocked for 1 h, diluted circMYBPCC primary antibody was added dropwise to cover, and incubated overnight at 4°C. After discarding the primary antibody, wash with PBS, add diluted fluorescent secondary antibody, and incubate at room temperature in the dark for 1 hour; after washing with PBS, incubate with DAPI solution for 15 minutes at room temperature; finally wash three times with PBS, and observe with a fluorescence microscope. As shown in Figure 2A by immunofluorescence, myoblasts in the proliferating phase were spindle-shaped, and the circMYBPCC polypeptide (fluorescence signal) was mainly expressed in the cytoplasm of the myoblasts in the proliferating phase. The myoblasts at the differentiation stage in Fig. 2A are multinucleated and long strips, and the circMYBPCC polypeptide (fluorescent signal) is expressed in both the nucleus and the cytoplasm of the myoblasts at the differentiation stage.

3. Protein extraction and Western Blotting

Extract proteins from cell or tissue samples with RIPA Lysis Buffer containing 1% PMSF. Protein concentration was determined with BCA kit. The protein was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to the NC membrane; after incubation with the specific primary antibody and the corresponding secondary antibody, the resulting image was captured by the ChemiDoc XRS+ system;

The test results show that there are IRES and ORF on circMYBPCC, and it can encode a protein containing 439 amino acids. Its amino acid sequence is shown in SEQ ID NO: 2, and the expected size of the encoded protein is 50kDa. The detection results of the Western Blotting test are shown in Figure 2B. Using the antibody described in the first step of Example 4, a 50KDa circMYBPCC protein band was specifically detected in the buffalo muscle tissue protein lysate, which is consistent with the theoretical size of the protein. It indicated that the protein encoded by circMYBPCC was expressed in buffalo muscle tissue.

Example 5: Designing and constructing the overexpression vector and interfering RNA of circMYBPCC

1. Design primers for the full-length sequence of circMYBPCC, and add protective bases and enzyme cleavage sites to the upstream and downstream primers. The primer sequence is: (the underlined part is the restriction site)

PCD2.1-circMYBPCC-F:

GG GGTACC AGCCAAAGAGAACTATGCAGG;

PCD2.1-circMYBPCC-R:

TTGATGATCTGCATCTCGAAT GGATCC CG;

Using buffalo myoblast cDNA as a template, the full length of circMYBPCC was cloned. Ligate the amplified full-length sequence of circMYBPCC to the pCD2.1-CIR vector to obtain the overexpression vector pCD2.1-circMYBPCC;

As shown in Figure 3, in order to detect that the overexpression vector can express circMYBPCC in large quantities in myoblasts, pCD2.1-circMYBPCC was transfected into myoblasts, and the expression level of circMYBPCC was detected by qPCR after 18-24 hours of culture. The qPCR results showed that the expression level of circMYBPCC after transfection with pCD2.1-circMYBPCC increased by nearly 17 times compared with normal myoblasts, indicating that the overexpression vector pCD2.1-circMYBPCC can express circMYBPCC in large quantities, which can be used for subsequent functional verification.

2. According to the full-length sequence of circMYBPCC, its interference fragment was designed and synthesized by Guangzhou Ruibo Biotechnology Company: CTTGAAGTACTTGAATGGT;

As shown in Figure 4, the circMYBPCC interference fragment was transfected into myoblasts and cultured for 18-24 hours, and the expression of circMYBPCC in myoblasts was detected by qPCR. The results showed that the expression of circMYBPCC in the circMYBPCC interference group was significantly reduced by about 80% compared with the control group, indicating that the successful design of the circMYBPCC interference fragment can be used for subsequent functional verification.

Example 6: Detection of the effect of overexpressing or interfering with circMYBPCC on the proliferation ability of buffalo myoblasts

According to different test requirements, the buffalo primary myoblasts in Example 1 were inoculated into different types of cell culture dishes. When the myoblast density reached 60%, the pCD2.1-circMYBPCC plasmid and si-circMYBPCC (pCD2.1 Plasmid, si-NC as control) were transfected into buffalo myoblasts. After the transfection, the myoblasts were continued to be cultured until the density of the myoblasts reached about 80%, and the myoblasts were processed for subsequent experiments.

6.1. Detection of myoblast proliferation by CCK8

6.1.1 Inoculate the buffalo myoblasts in the logarithmic phase into a 96-well plate, and when the cell density reaches 60%, transfect the myoblasts with pCD2.1 and pCD2.1-circMYBPCC, and continue to culture;

6.1.2 When the myoblast density reaches about 80%, use the complete medium to prepare CCK8 dilution at a ratio of 10%; discard the complete medium in 96 wells, and add 100 μL of CCK8 dilution to each well; place in the incubator Continue culturing for 4h in medium;

6.1.3 Use a microplate reader to measure the OD value at a wavelength of 450nm (the entire operation process should be protected from light), and conduct statistical analysis;

6.1.4 The calculation formula is: myoblast proliferation percentage=[(A-C)/(B-C)]×100%, A: the absorbance value of the experimental group (containing medium, pCD2.1-circMYBPCC transfected myoblasts and CCK8 Absorbance value of Solution) B: Absorbance value of control group (absorbance value of culture medium, pCD2.1 transfected myoblasts, CCK8 Solution) C: blank group absorbance value (absorbance of culture medium, CCK8 Solution) .

As shown in Figure 5, the proliferation of buffalo myoblasts was detected by CCK8, and the cell proliferation ratio of the overexpressed circMYBPCC group was significantly higher than that of the control group. Taking the proliferation rate of myoblasts in the control group transfected with pCD2.1 as a reference, the proliferation rate of the overexpressed circMYBPCC group was Proliferation rate increased by approximately 20%. It shows that increasing the expression of circMYBPCC can promote the proliferation of myoblasts.

6.2. Cell cycle detection by flow cytometry

6.2.1 Cultured cells: Inoculate buffalo myoblasts in the logarithmic phase into 96-well plates. When the density of myoblasts reaches 60%, transfect myoblasts with pCD2.1 and pCD2.1-circMYBPCC respectively, and continue to culture When the density reaches 80%, proceed to the next test;

6.2.2 Collection and fixation of myoblasts: Discard the medium and wash once with PBS; add an appropriate amount of 0.25% trypsin to completely digest the myoblasts at 37°C, then add termination medium to terminate the digestion, and collect them into a 1.5ml centrifuge tube , centrifuge and discard the supernatant; wash with PBS 3 times, discard the supernatant; add 250 μL of pre-cooled PBS to resuspend the cells, add dropwise 750 μL of pre-cooled absolute ethanol, mix well, and fix at -20°C for 24 hours;

6.2.3 Myoblast staining: take out the myoblasts, centrifuge to discard the supernatant, and wash with PBS for 3 times; add FxCycleTM PI/RNAse Solution to the myoblast pellet and incubate at room temperature in the dark for 30 minutes, and wash once with PBS;

6.2.4 Detection: Myoblasts were resuspended in PBS, then detected by flow cytometry, and the results were analyzed using ModFit software.

As shown in Figure 6, Figure 6A is the result of cell cycle analysis using ModFit software, Figure 6A1 is the control group, Figure 6A2 is the overexpression circMYBPCC group, and the leftmost peaks in Figure 6A1 and Figure 6A2 represent cells in the G1/G0 phase The proportion of cells, the broad peak in the middle indicates the proportion of cells in S phase, and the rightmost peak indicates the proportion of cells in G2/M phase. Figure 6B shows the statistical results of G1/G0, S and G2/M periods. It can be seen from Figure 6B that compared with the control group, the proportion of cells in the G1/G0 phase of the overexpressed circMYBPCC group was significantly down-regulated, and the proportion of cells in the S phase was significantly increased. There was no significant difference in the proportion of cells in the M phase, which indicated that the myoblasts in the overexpression circMYBPCC group had a higher proportion of cells in the proliferation cycle, which was consistent with the result that circMYBPCC promoted cell proliferation.

6.3. EdU detection of myoblast proliferation

6.3.1 Inoculate buffalo myoblasts in the logarithmic phase into 96-well plates. When the cell density reaches 60%, transfect the cells with si-NC and si-circMYBPCC, and continue to culture for 18 hours. When the density reaches 80%, follow the EdU The kit is used for detection, and the detection steps are as follows;

6.3.2 Use the complete medium to dilute the EdU solution at a ratio of 1000:1 to prepare a 50 μmol/L EdU dilution. Add 100 μL of EdU dilution solution to each well, incubate in the incubator for 2 hours; discard the liquid, wash the cells twice with PBS, 5 minutes each time;

6.3.3 Add 50 μL of 4% paraformaldehyde to each well, fix at room temperature for 30 minutes, discard the liquid, add 50 μL of 2 mg/mL glycine to each well, and incubate for 5 minutes; after discarding glycine, wash once with PBS; then add 100 μL of 0.5 % TritonX-100 solution, permeabilize myoblasts for 10 minutes, wash myoblasts with PBS for 5 minutes;

6.3.4 Add 100 μL of Apollo staining reaction solution to each well, incubate at room temperature in the dark for 30 minutes; discard the liquid, add 100 μL of 0.5% TritonX-100 to each well in PBS to wash on a decolorizing shaker for 3 times, each time for 10 minutes; add 100 μL to each well Washed twice with methanol, 5min each time, washed once with PBS;

6.3.5 DNA staining: Prepare 1×Hoechst33342 reaction solution with ddH ₂ O at a ratio of 100:1, add 100 μL of 1×Hoechst33342 reaction solution to each well, incubate at room temperature in the dark for 30 minutes, discard the reaction solution; wash with PBS 3 times, each time 5min;

6.3.6 Imaging analysis of myoblasts using fluorescence microscopy.

As shown in Figure 7, the effect of circMYBPCC interfering with buffalo myoblasts on myoblast proliferation was analyzed by calculating the ratio of Hochest-stained and EdU-stained nuclei. In Figure 7A, Hochest can stain all nuclei, and EdU dye only stains proliferating nuclei. The ratio of EdU-stained nuclei to Hochest-stained nuclei in the interference circMYBPCC group and the control group was statistically calculated respectively, and the cell proliferation rates of the two groups were analyzed. As shown in Figure 7B, the nuclei of newly proliferated myoblasts in the interfering circMYBPCC group were significantly reduced compared with the control group, indicating that interfering with circMYBPCC significantly inhibited the proliferation of myoblasts.

6.4. Detect the expression of proliferation-related genes by qPCR and Western blotting test

The RNA was reverse-transcribed into cDNA by Nanjing Nuoweizan Reverse Transcription Kit, and the expression changes of myogenic proliferation-related genes PCNA, cyclinD1 and CDK2 were detected at the RNA level by quantitative PCR. The expression changes of circMYBPCC and myogenic proliferation-related genes PCNA, cyclinD1 and CDK2 at the protein level were detected by WB technology.

As shown in Figure 8, the qPCR test results showed that compared with the control group, overexpression of circMYBPCC significantly increased the transcriptional expression of PCNA, CDK2 and cyclinD1 genes (Figure 8A); Western blotting showed that myoblast-related proliferation genes (PCNA, cyclinD1 and CDK2 ) were significantly up-regulated, and the expression of circMYBPCC-encoded peptide was also significantly up-regulated after overexpression of circMYBPCC (Fig. 8B).

Correspondingly, as shown in Figure 9, after interfering with buffalo myoblast circMYBPCC, the transcriptional expression levels of CDK2, PCNA and cyclinD1 genes were detected by qPCR, and the gene expression of the interfering circMYBPCC group was significantly lower than that of the control group (Figure 9A); Western blotting The detection also found that the protein expression levels of related myoblast proliferation genes (PCNA, cyclinD1 and CDK2) in the interference circMYBPCC group were significantly lower than those in the control group (Figure 9B), and the expression level of circMYBPCC-encoded peptides was also significantly decreased after interference with circMYBPCC (Figure 9B). 9B). These results indicate that the proliferation of buffalo myoblasts lacking circMYBPCC is inhibited.

Example 7: Detection of the effect of overexpressing or interfering with circMYBPCC on the differentiation ability of buffalo myoblasts

The buffalo myoblasts isolated in Example 1 were inoculated into a 6-well plate until the cell density reached more than 60%. ) were transfected into buffalo myoblasts. After the transfection, the myoblasts were continued to be cultured until the density of the myoblasts reached about 80%, and they were replaced with DMEM containing 2% horse serum to induce differentiation. After the buffalo myoblasts were induced to differentiate for 5-6 days, the total RNA of the cells was extracted, and the total protein of the myoblasts was extracted at the same time. Nanjing Novizan reverse transcription kit was used for reverse transcription, RNA was reverse transcribed into cDNA, and the expression changes of myogenic differentiation-related genes MyoD1, MyoG and MyHC were detected at the RNA level by qPCR technology. Western blotting was used to detect the expression changes of circMYBPCC and myogenic differentiation-related genes MyoD1, MyoG and MyHC at the protein level. Myoblasts were seeded into 24-well plates, transfected and induced to differentiate in the same steps as the above method, and myotubes and their number were observed by immunofluorescence technology after 5 days of differentiation.

As shown in Figure 10A, differentiation was induced for 5 days after transfection, and the changes in transcriptional expression of myoblast differentiation-related genes were detected. It was found that after overexpression of circMYBPCC, the expressions of MyoD, MyHC and MyoG were significantly higher than those of the control group. As shown in Figure 10B, Western blotting detected changes in protein levels, and MyoD, MyHC, and MyoG protein expressions were also significantly upregulated. After interfering with circMYBPCC, MyoD, MyHC and MyoG transcriptional expression (Fig. 11A) and protein expression (Fig. 11B) were significantly decreased. These results suggest that circMYBPCC promotes buffalo myoblast differentiation.

Differentiated myotubes and myocyte nuclei were then stained by immunofluorescence staining technique (Fig. 12A). The number of myotubes after overexpression of circMYBPCC was observed and counted. The results showed that after overexpression of circMYBPCC, the number of myoblast myotubes in the circMYBPCC overexpression group was significantly increased compared with the control group ( FIG. 12B ).

Example 8: Effect of circMYBPCC Administration on Muscle Damage in Mice

According to the buffalo-derived circMYBPCC sequence in the above embodiments, the mouse-derived circMYBPCC sequence can be determined. We downloaded the mouse MYBPCC gene from NCBI, and found a sequence highly similar to the buffalo-derived circMYBPCC sequence through homology comparison, with a homology greater than 87%, and the predicted polypeptide encoded by the mouse-derived circMYBPCC sequence was also predicted to be similar to the buffalo-derived circMYBPCC sequence Has homology (55%). Therefore, it can be determined that the sequence is a mouse-derived circMYBPCC sequence, and its sequence is shown in SEQ ID NO:3.

Inject 50 mL of CTX solution (10 mM) into the tibialis anterior muscle of 5-week-old mice to induce skeletal muscle damage. After 24 h of CTX treatment, the tibialis anterior muscle tissues of the left hind leg (injected with 50 mL of normal saline) and right hind leg (injected with 50 mL of CTX) were collected respectively. % and 30% sucrose solutions were dehydrated, each concentration was dehydrated for 24 hours; after embedding and making frozen sections, H&E staining was used to analyze the damage. As shown in Figure 13, the H&E staining analysis of the injury after sectioning, compared with the control group, the HE staining of the tissue sections of the muscle injury model group showed that the muscle fiber gap in the injury group was obviously widened, the shape of the muscle fiber changed, and vacuolation appeared, accompanied by a large amount of inflammation cell infiltration.

Inject 50 μL of the nucleic acid complex consisting of 20-25 μg pCD2.1-circMYBPCC plasmid and Entransterin vivo transfection reagent into the tibialis anterior muscle of the left hind leg of 8 mice in the experimental group. The control group was injected with 50 μL of a nucleic acid complex consisting of 20-25 μg pCD2.1 plasmid and Entransterin vivo transfection reagent, and the expression of circMYBPCC in the two groups of samples at 7 days after muscle injury was detected by qPCR, as shown in Figure 14. The expression level of circMYBPCC in the circMYBPCC group was significantly higher than that in the control group. And as shown in Figure 15, compared with the control group, the number of broken muscle fibers in the group transfected with circMYBPCC on day 3 of muscle injury was significantly reduced; in the control group on day 7 of muscle injury, there were only a small number of new muscle fibers, while most of the group transfected with circMYBPCC Damaged muscle fibers are replaced by new muscle fibers.

Then, in order to explore the expression changes of muscle regeneration genes, the expression of relevant genes was detected by qPCR. As shown in Figure 16A, compared with the control group, the mRNA levels of proliferation and differentiation-related genes (MyOG, MyoD, MyHC, and PCNA) in the muscle of mice overexpressing circMYBPCC group were significantly increased. Further, western blotting technology was used to detect the expression of related gene protein levels. As shown in Figure 16B, the protein expression levels of circMYBPCC protein and proliferation and differentiation genes (PCNA, MyoD and MyoG) were significantly up-regulated. In summary, circMYBPCC promotes the repair of muscle damage in mice, and promotes the transcription and protein expression of related genes, which can be used as a nucleic acid drug to treat muscle damage.

Example 9: Determining the sequence of human circMYBPCC

The human-derived circMYBPCC sequence can be determined by the buffalo-derived circMYBPCC sequence. We downloaded the human MYBPCC gene from NCBI. Through homology comparison, we found a sequence highly similar to the buffalo-derived circMYBPCC sequence. The homology was greater than 90%, and the human The polypeptide encoded by the circMYBPCC sequence is also predicted to have homology (93%) with the buffalo-derived circMYBPCC. Therefore, it can be determined that the sequence is a human-derived circMYBPCC sequence. The human source circMYBPCC sequence is shown in SEQ ID NO:4. According to the experimental results of Examples 1-8, it can be deduced that the human circMYBPCC sequence shown in SEQ ID NO: 4 can be used to develop drugs for the treatment of human muscle damage.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

Claims (6)

1. An application of encoding circRNA related to the development of buffalo skeletal muscle in preparing a medicament for treating muscle injury is characterized in that the encoding circRNA is circMYBPCC, the sequence of which is shown in SEQ ID NO:1, the over-expression of the circMYBPCC promotes the proliferation and differentiation of myoblasts, and the expression of the circMYBPCC is interfered to inhibit the proliferation and differentiation of the myoblasts.

2. The use of the codable circRNA associated with skeletal muscle development in buffalo according to claim 1 in the preparation of a medicament for the treatment of muscle injury, wherein the mouse-derived sequence of circMYBPCC is set forth in SEQ ID No. 3.

3. The use of the codable circRNA related to buffalo skeletal muscle development according to claim 1, wherein the human sequence of circMYBPCC is shown in SEQ ID No. 4 for the preparation of a medicament for the treatment of muscle injury.

4. Use of a circMYBPCC encoding nucleic acid associated with skeletal muscle development in buffalos as claimed in any one of claims 1 to 3 in the manufacture of a medicament for the treatment of muscle damage, wherein the circMYBPCC forms a circMYBPCC nucleic acid complex with liposomes as a component of a medicament for the treatment of muscle damage.

5. Use of the encodable circRNA for the preparation of a medicament for the treatment of muscle damage as claimed in any one of claims 1 to 3, wherein said circMYBPCC forms a circMYBPCC nucleic acid complex with liposomes as a component of a medicament for the treatment of muscle damage in an amount of 0.5-2mg per kg of damaged muscle.

6. Use of the encodable circRNA for the preparation of a medicament for the treatment of muscle damage associated with the development of skeletal muscle of buffalo according to any one of claims 1 to 3, wherein said medicament for the treatment of muscle damage is an injectable formulation.

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