A biological identification method and application of bovine skeletal muscle development-related circular RNA circMYH8

Abstract

This invention discloses a biological identification method and application of bovine skeletal muscle development-related circular RNA circMYH8. The circular RNA circMYH8 is derived from the sequencing results of circular RNA in the longissimus dorsi muscle of Qinchuan cattle at different developmental stages, and is identified as circular RNA through biological identification. The interfering fragment circMYH8-si of circMYH8 is synthesized and transfected into primary cultured bovine skeletal muscle cells, which significantly improves the proliferation efficiency of primary bovine muscle cells. It can serve as an important candidate molecule for transgenic breeding of beef cattle, providing a theoretical basis for improving local cattle breeds in my country using molecular breeding technology, thereby accelerating the breeding process of high-yield Qinchuan cattle.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a biological identification method and application of bovine skeletal muscle development-related circular RNA circMYH8. Background Technology

[0002] Qinchuan cattle are a local yellow cattle breed in my country, bred for draft purposes over a long period. They are characterized by good meat quality and tolerance to roughage, but suffer from low meat yield and slow growth. In recent years, the continuously developing molecular breeding technology has the advantages of shortening the breeding cycle and improving breeding efficiency. However, the molecular research foundation on bovine muscle development is still weak, greatly limiting the application and development of bovine molecular breeding technology. Therefore, in-depth research on the molecular mechanisms related to muscle development in Qinchuan cattle is a prerequisite for improving the Qinchuan cattle breed using molecular breeding technology.

[0003] Skeletal muscle is the largest organ in mammals, accounting for approximately 40% of total body weight, and maintains basic functions such as metabolism, respiration, and movement. In vertebrates, skeletal muscle primarily originates from the myodermal sarcomere of the mesoderm. Myogenic progenitor cells proliferate and differentiate into spindle-shaped mononuclear myoblasts, which then migrate to the muscle-forming site. Through a series of proliferation, differentiation, and fusion processes, they eventually form mature muscle fibers. A portion of myoblasts do not fuse to form satellite cells for muscle regeneration in case of injury. The number of vertebrate skeletal muscle fibers remains constant during the fetal stage; after birth, muscle mass increases mainly through hypertrophy of muscle fibers. Primary bovine skeletal muscle cells are isolated from the longissimus dorsi muscle tissue of prenatal cattle and serve as an in vitro model cell for studying the early proliferation process of bovine skeletal muscle.

[0004] Skeletal muscle growth and development is an extremely complex process, and the study of its transcriptional regulatory mechanisms has always been a hot topic in the field of molecular genetics. With the rapid development of transcriptome sequencing technology, significant progress has been made in the functional identification of genes related to skeletal muscle growth and development and in the study of transcriptional regulatory mechanisms. In recent years, research on non-coding RNAs has gradually emerged, and their regulation of skeletal muscle development has also become a research hotspot. Circular RNAs are a class of non-coding RNAs that lack a 5' cap and a 3' poly(A) tail, forming a closed circular structure through covalent bonds. Compared with linear mRNAs, the covalently closed structure of circular RNAs makes them less susceptible to degradation by the exonuclease RNase R, exhibiting higher stability. Based on their origin, circular RNAs are mainly divided into four categories: exon circular RNAs, exon-intron circular RNAs, intron circular RNAs, and intergenic circular RNAs. Circular RNAs are products of backsplicing, exhibiting spatiotemporal specificity and tissue / cell specificity. Numerous studies have confirmed that circular RNAs are widely involved in regulating the growth and development of animal organisms. Among them, the most extensive involvement is in the regulation of ceRNAs, which act as molecular sponges of miRNAs. In addition, circular RNAs can bind to functional proteins and participate in splicing regulation, transcriptional regulation, epigenetic modification, and protein translation regulation.

[0005] In the field of skeletal muscle development research, studies on the regulation of skeletal muscle cell proliferation by circular RNA are gradually increasing. For example, bovine circRILPL1 promotes bovine myoblast proliferation by adsorbing miR-138; bovine circCPE can adsorb miR-133a to promote bovine myoblast proliferation; chicken circPTPN4 competitively binds to miR-499-3p to promote chicken myoblast proliferation; and bovine circSVIL promotes bovine myoblast proliferation by inhibiting STAT1 phosphorylation. circMYH8, located on chromosome 19 of Qinchuan cattle, is formed by the circularization of exons 29-33 of the MYH8 gene. Sequencing results of circular RNA from the longissimus dorsi muscle of Qinchuan cattle at different developmental stages show that circMYH8 is highly expressed in the longissimus dorsi muscle of fetal cattle, and may be involved in early bovine skeletal muscle development. Summary of the Invention

[0006] The purpose of this invention is to provide a biological identification method and application of bovine skeletal muscle development-related circular RNA circMYH8, in order to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for biological identification of bovine skeletal muscle development-related circular RNA circMYH8, comprising the following steps:

[0008] Two sets of primers, divergent primer and convergent primer, for circMYH8 were designed and synthesized. PCR amplification was performed using bovine longissimus dorsi muscle cDNA, RNase R-treated bovine longissimus dorsi muscle cDNA, and bovine longissimus dorsi muscle gDNA as templates, respectively. Identification was performed by sequencing and agarose gel electrophoresis.

[0009] Preferably, the circMYH8 divergent primer is:

[0010] Upstream primer F1: 5′-TAGAGGAAGCAGAGGTGAAT-3′,

[0011] Downstream primer R1: 5′-CTGACGTTTCAGTTCCTCAATC-3′;

[0012] The convergent primer for circMYH8 is:

[0013] Upstream primer F1: 5′-GCACTGGCCTTGGATGAAAC-3′,

[0014] Downstream primer R1: 5′-CCAGTTCCTTCCTTTTGGCCT-3′.

[0015] Preferably, the RNase R treatment process is as follows: treat total RNA from bovine longissimus dorsi muscle at 37°C with 20 U / μL RNase R for 4 h.

[0016] Preferably, the PCR amplification system (20 μL) consists of: 1 μL of 50 ng / μL template DNA, 1 μL each of upstream and downstream primers corresponding to the 10 pmol / L primer divergent primer or convergent primer, 10 μL of PCR Mix, and 7 μL of deionized water.

[0017] Preferably, the PCR reaction procedure includes the following steps: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 65℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 15 cycles; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 20 cycles; 72℃ extension for 10 min.

[0018] Preferably, the expected agarose gel electrophoresis results for circMYH8 are as follows: using bovine longissimus dorsi cDNA or RNaseR-treated bovine longissimus dorsi cDNA as a template, the circMYH8 divergent primer can successfully amplify the product, but when using bovine longissimus dorsi gDNA as a template, the circMYH8 divergent primer cannot amplify the product; using bovine longissimus dorsi cDNA or bovine longissimus dorsi gDNA as a template, the circMYH8 convergent primer can successfully amplify the product, but when using RNaseR-treated bovine longissimus dorsi cDNA as a template, the circMYH8 convergent primer cannot amplify the product.

[0019] A method for promoting the proliferation of bovine skeletal muscle primary cells includes: reducing the expression level of the bovine circMYH8.

[0020] Furthermore, the expression level of bovine circMYH8 was reduced by inhibiting circMYH8-si.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention successfully identified bovine circular RNA circMYH8 from exons 29-33 of the MYH8 gene and conducted functional studies on it. The results showed that bovine circular RNA circMYH8 can regulate the proliferation of bovine skeletal muscle cells. Interference with circMYH8 can significantly improve the proliferation efficiency of primary bovine skeletal muscle cells. It can be used as an important candidate molecule for transgenic breeding of beef cattle, providing a theoretical basis for improving local cattle breeds in my country using molecular breeding technology, thereby accelerating the breeding process of high-yield Qinchuan cattle. Attached Figure Description

[0022] Figure 1 This is the genome mapping and Sanger sequencing data of bovine circMYH8 in this invention.

[0023] Figure 2 The results of agarose gel electrophoresis identification of bovine circMYH8 in this invention are shown.

[0024] Figure 3 The figure shows the EdU results for interfering with circMYH8 in this invention; * indicates significant differences.

[0025] Figure 4 The figures show the flow cytometry results of interfering with circMYH8 in this invention; * indicates significant differences. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1: Biological identification of circMYH8

[0028] 1.1 Source of laboratory animals and collection of samples

[0029] The experimental animals used in this invention are Qinchuan cattle, the top breed of yellow cattle among my country's five major breeds. Samples of the longissimus dorsi muscle from healthy fetal cattle were collected from Qinbao Livestock Co., Ltd. in Baoji, Shaanxi, China. To prevent or reduce RNA degradation, the collected tissue samples were placed in enzyme-free centrifuge tubes and stored at -80°C.

[0030] 1.2 Extraction of total RNA

[0031] (1) Take 50-100 mg of tissue sample, put it into an RNase-free mortar, add liquid nitrogen and grind it into powder. When the liquid nitrogen has basically evaporated, add 1 mL of Trizol reagent and grind it thoroughly. After the sample melts, transfer it into an RNase-free 1.5 mL centrifuge tube and let it stand at room temperature for 15 min.

[0032] (2) Centrifuge at 4°C, 12000 r / min for 10 min in an ultracentrifuge.

[0033] (3) Transfer the supernatant to a new RNase-free 1.5 mL centrifuge tube.

[0034] (4) Add 0.2 mL of chloroform (1 / 5 of the volume of Trizol), shake vigorously for 15 s, and let stand on ice for 5 min.

[0035] (5) Centrifuge at 4℃, 12000r / min for 15min, and carefully remove the centrifuge tube.

[0036] (6) Carefully transfer the supernatant to a new 1.5 mL centrifuge tube (be careful not to aspirate the middle protein layer), and add an equal volume of isopropanol to the supernatant.

[0037] (7) Gently invert and mix well, then let stand on ice for 10 minutes.

[0038] (8) Centrifuge at 4℃, 12000r / min for 10min.

[0039] (9) Discard the supernatant, add 1 mL of 75% ethanol (prepared with RNase-free water), and wash.

[0040] (10) Centrifuge at 4℃, 12000r / min for 10min. Repeat steps (9) and (10) once.

[0041] (11) Discard the supernatant and dry the precipitate at room temperature for 5-10 minutes (excessive drying will greatly reduce the solubility of RNA).

[0042] (12) Dissolve in 30 μL of DEPC water for subsequent experiments or storage at -80℃. After extraction, the concentration and OD value of total RNA were detected using NanoDrop2000. The OD260 / 280 ratio of RNA should be between 1.8 and 2.0 to ensure that there is no protein or phenol contamination and no degradation.

[0043] 1.3 Isolation, extraction, and purification of gDNA

[0044] Refer to the method of Sambrock et al (2002).

[0045] 1.4 circMYH8 primer design

[0046] Primers for circRNA were designed using the NCBI Primer Design Tool. Unlike mRNA, circRNA does not have 5' and 3' ends; instead, it is a closed circular structure formed by reverse splicing during RNA cleavage. Therefore, primer design differs from that for mRNA. When designing primers for circRNA, the circularization site should be included; that is, divergent primers should be designed. When designing primers: the Tm value should not be lower than 40℃; the GC content should ideally be between 40% and 60%; the Tm values ​​and GC contents of the upstream and downstream primers should be similar; the primer length should ideally be between 18 and 25 nt; and the length of the circRNA amplification product should ideally be between 100 bp and 250 bp, and should not be too long.

[0047] circMYH8's Divergent Primer:

[0048] Upstream primer F1: 5′-TAGAGGAAGCAGAGGTGAAT-3′,

[0049] Downstream primer R1: 5′-CTGACGTTTCAGTTCCTCAATC-3′.

[0050] To verify that the selected RNA is circular RNA, a control group needs to be set up. Therefore, primers for its mRNA, namely convergent primers, were designed.

[0051] circMYH8's convergent primer:

[0052] Upstream primer F1: 5′-GCACTGGCCTTGGATGAAAC-3′,

[0053] Downstream primer R1: 5′-CCAGTTCCTTCCTTTTGGCCT-3′.

[0054] 1.5 RNase R treatment

[0055] RNase R, or Ribonuclease R, is a ribonuclease derived from *E. coli* that can cleave and degrade RNA. It can digest almost all linear RNA molecules, but it is less effective at digesting circular RNA and lasso structures; therefore, it can be used for the identification of circRNAs. Treating total RNA from the longissimus dorsi muscle of cattle with 20 U / μL RNase R for 4 hours, followed by reverse transcription, allows for PCR detection of the stability of circMYH8.

[0056] 1.6 The extracted total RNA was reverse transcribed.

[0057] RNA concentration and OD260 / 280 values ​​were determined using a Nano Drop 2000. If there was no contamination and no degradation, reverse transcription was performed using a Takara kit.

[0058] (1) Perform the genomic DNA removal reaction. Add 1 μL of gDNA Clean Reagent, 2.0 μL of 5×gDNA Clean Buffer, 1 μg of Total RNA to an RNase-free PCR tube, and finally add RNase-free ddH2O to 10 μL. Incubate at 42°C for 2 min in a PCR instrument.

[0059] (2) Reverse transcription reaction: Add 1.0 μL of Evo M-ML V Rtase Enzyme Mix, 1.0 μL of RT Primer Mix, 4.0 μL of 5×RTase Reaction Buffer Mix I to the PCR tube from the previous step, and finally add RNase-free ddH2O to 20 μL. Incubate at 37℃ for 15 min and 85℃ for 5 s in a PCR instrument to complete the reverse transcription process of total RNA.

[0060] 1.7 PCR amplification and product sequencing

[0061] 1.7.1 PCR Amplification

[0062] Using circMYH8 primers, the above-mentioned reverse transcription sample was used as a template for PCR amplification. The PCR amplification system (20 μL) consisted of: 1 μL of 50 ng / μL template DNA, 1 μL each of the forward and reverse primers corresponding to the 10 pmol / L divergent primer or convergent primer, 10 μL of PCR Mix, and 7 μL of deionized water. The PCR reaction program used included the following steps: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 65℃ annealing for 30 s, and 72℃ extension for 1 min, for a total of 15 cycles; 95℃ denaturation for 30 s, 50℃ annealing for 30 s, and 72℃ extension for 1 min, for a total of 20 cycles; and 72℃ extension for 10 min.

[0063] 1.7.2 Agarose gel electrophoresis

[0064] We used 2% agarose gel electrophoresis to check if the length of the amplified product matched the length of our target sequence. The steps for 2% agarose gel electrophoresis are as follows:

[0065] (1) Prepare a 2% agarose gel containing nucleic acid dye, spot the sample, and electrophore at 120V for 30 minutes;

[0066] (2) When the fragments are clearly separated, image them using the BIO-RAD Gel Doc 2000 gel imaging system;

[0067] (3) The sequence length of the amplified product was analyzed by comparing the positions of markers and fragment bands in agarose gel electrophoresis imaging.

[0068] 1.8 Sanger sequencing

[0069] If agarose gel electrophoresis analysis reveals that the sequence length of the amplified product is the same as the target fragment sequence length, Sanger sequencing is performed to check the sequencing results for the amplification of circularization sites. If circularization sites are amplified, it confirms that it is circular RNA (see [link to article]). Figure 1 ).

[0070] 1.9 Amplification of circMYH8 in different PCR templates

[0071] PCR amplification was performed using bovine skeletal muscle cDNA, RNase R-treated cDNA, and gDNA as templates with divergent primer and convergent primer, respectively. The procedure used was the same as in 1.6.1. The PCR amplification products were analyzed by 2% agarose gel electrophoresis to determine the amplification status of circMYH8. Positive results for circMYH8 identification were as follows: When using bovine longissimus dorsi muscle cDNA or RNase R-treated bovine longissimus dorsi muscle cDNA as a template, the circMYH8 divergent primer successfully amplified the product; however, when using bovine longissimus dorsi muscle gDNA as a template, the circMYH8 divergent primer failed to amplify the product. Similarly, when using bovine longissimus dorsi muscle cDNA or bovine longissimus dorsi muscle gDNA as a template, the circMYH8 convergent primer successfully amplified the product; however, when using RNase R-treated bovine longissimus dorsi muscle cDNA as a template, the circMYH8 convergent primer failed to amplify the product (see [link to relevant documentation]). Figure 2 ).

[0072] Example 2: circMYH8 Function Verification

[0073] 2.1 Isolation and Culture of Primary Skeletal Muscle Cells

[0074] (1) First, place the whole fetal calf in a large petri dish, wipe the skin with an alcohol swab, then peel off the skin and fascia with a scalpel, take an appropriate amount of muscle tissue and rinse it three times with sterile PBS.

[0075] (2) Place the muscle tissue in a culture dish, add a small amount of PBS, cut the muscle tissue into small pieces with sterile scissors, add it to a 10mL centrifuge tube and let it stand for minutes. After removing the supernatant, add 0.2% type II collagenase at 4 times the volume of the tissue and digest it in a 37℃ water bath shaker for 3 hours.

[0076] (3) Filter the digested cells through a 400-mesh filter and wash the filtered cells three times with sterile PBS (1500 r / min).

[0077] (4) The cells were suspended in a complete culture medium containing 20% ​​fetal bovine serum and cultured for a longer period.

[0078] 2.2 Design and Synthesis of circMYH8 Interference Fragments

[0079] siRNA design: (1) Fragment length: 19-21 nt; (2) GC content: 40%-60%; (3) Avoid more than 4 consecutive A / T sequences; (4) Avoid repetition at the first position as much as possible; (5) The sequence position should be evenly distributed on both sides of the circularization site; (6) Generally, a TT overhang is added to the 3' end of the interfering fragment. The sequence of circMYH8-si is: GGAAGCAGAGGTGAATATT. After designing, it is sent to the company for synthesis.

[0080] 2.3 Effects of circMYH8 on the growth and development of bovine primary muscle cells

[0081] Synthetic circMYH8-si was transfected into bovine muscle primary cells to detect the effect of circMYH8 on bovine muscle cell proliferation. The detection methods included EdU and flow cytometry.

[0082] 2.4 Cell transfection

[0083] (1) Culture the cells and seed them into 12-well plates. Before transfection, replace the culture medium in the wells with OPTI-MEM medium.

[0084] (2) Taking a 12-well plate as an example: depending on the concentration of the vector or RNA oligonucleotide, dissolve 50 pmol of RNA oligonucleotide in 100 μL of OPTI-MEM medium (A), and at the same time dissolve 1.5 μL of R0531 in 100 μL of OPTI-MEM medium and let it stand at room temperature for 5 min (B).

[0085] (4) Slowly add (A) to (B), mix gently, and incubate at room temperature for 15 minutes.

[0086] (5) Add the mixture of (A) and (B) to the corresponding well plate, shake the culture medium well using the "cross method", and place it in an incubator to continue culturing.

[0087] 2.5 EdU detection of cell proliferation

[0088] Following the instructions of the Ribobio Cell-Light EdU Apollo567 In Vitro Kit, cells were treated in 96-well plates, and EdU images were acquired using a fluorescence microscope (DM5000B; Germany). The results showed that interference with circMYH8 significantly promoted the proliferation of bovine myocardial primary cells (see [link to kit]). Figure 3 ).

[0089] 2.6 Flow cytometry for cell cycle detection

[0090] (1) Inoculate bovine myocardial primary cells into medium-sized dishes at a seeding density of 15% to 20%. When the cell density reaches about 50%, transfect the cells. When the cell confluence is about 80%, digest the cells with trypsin and collect them into 2 mL centrifuge tubes. Wash the cells twice with PBS.

[0091] (2) After washing the cells with PBS, centrifuge and discard the supernatant. Add pre-cooled 70% ethanol and fix overnight at 4°C.

[0092] (3) Cell staining: Centrifuge to collect cells, wash cells twice with 1 mL PBS, add 500 μL LPI staining solution (PBS containing 50 μg / mL propidium iodide (PI), 100 μg / mL RNase A, 0.2% Triton X-100), and incubate at 4°C in the dark for 30 minutes.

[0093] (4) Flow cytometry was used to detect and count 20,000-30,000 cells, and the data were analyzed using cell cycle simulation software. The results showed that interfering with circMYH8 increased the proportion of primary bovine muscle cells in S phase (see [link to relevant documentation]). Figure 4 ).

[0094] The above results successfully identified circMYH8 as a bovine circular RNA. Interference with circMYH8-si in primary bovine muscle cells increased EdU cell proliferation activity and the proportion of cells in S phase. These results indicate that interfering with circMYH8 can promote the proliferation of primary bovine muscle cells, and circMYH8 can serve as an important candidate molecule for transgenic beef cattle breeding.

[0095] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of bovine circular RNA circMYH8 in promoting the proliferation of bovine skeletal muscle primary cells, characterized in that, The expression level of circMYH8 was suppressed by interfering with circMYH8-si, the sequence of which is GGAAGCAGAGGTGAATATT. circMYH8 is derived from exons 29-33 of the MYH8 gene, located on bovine chromosome 19.

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