Novel circRNA, application and kit

Abstract

The invention discloses a novel circRNA (Ribonucleic Acid), application and a kit, and relates to the field of animal genetic breeding, and the nucleotide sequence of the circ-06958 is shown as SEQ ID NO.1 in a sequence table. The invention discloses application of Circ-06958 in preparation of a porcine skeletal muscle satellite cell proliferation reagent. According to the invention, a new circRNA sequence-CircRNA is successfully cloned and identified, the circ-06958 is determined to be the porcine skeletal muscle satellite cell proliferation marker through a molecular biological method, and the porcine skeletal muscle satellite cell proliferation can be promoted by increasing the expression quantity of the circ-06958.

Description

Technical Field

[0001] The present invention belongs to the field of animal genetic breeding, and specifically relates to a marker Circ-06958 associated with the proliferation of pig skeletal muscle satellite cells and an application thereof. Background Art

[0002] Muscle growth and development and its intrinsic characteristics determine the quantity and quality of meat products, and are directly linked to the economic benefits of pig production. They have long been the focus of pig producers and pig breeding experts. Skeletal muscle is one of the important components of muscle tissue, mainly composed of muscle fibers and surrounding connective tissue, and plays an important regulatory role in muscle movement, endocrine regulation, energy consumption, metabolic hemostasis, etc. The number of muscle fibers is closely related to the growth rate and meat production potential of livestock and poultry. After the birth of an animal, the number of muscle fibers remains basically unchanged, and the change in their diameter is the fundamental reason for the increase in muscle mass. Skeletal muscle satellite cells are myogenic stem cells in skeletal muscle, which have the potential for proliferation and differentiation and can self-renew. Their proliferation ability largely determines the number of muscle fibers in fetal skeletal muscle. After birth, skeletal muscle satellite cells are usually in a quiescent state. During the process of muscle loss repair and regeneration, they will be reactivated to enter the proliferation and differentiation process to help damaged tissues return to normal. Skeletal muscle lacking satellite cells cannot regenerate after acute injury. Therefore, skeletal muscle satellite cells play an important role in muscle development. Revealing the proliferation and differentiation mechanism of skeletal muscle satellite cells will help improve meat production and quality and promote the process of pig molecular breeding.

[0003] The proliferation and differentiation of skeletal muscle satellite cells are jointly regulated by transcription factors, functional genes, non-coding RNA and epigenetic modifications. Circular RNA (circRNA) is a single-stranded, closed, circular non-coding RNA recently discovered. CircRNA is formed by reverse splicing of mRNA precursors and lacks a 5' phosphate cap structure and a 3' polyadenylic acid tail. Therefore, it is insensitive to nuclease degradation and has stronger stability. In addition, the circRNA sequence is highly conserved. The high degree of conservation and stability indicates the functional importance of circRNA. CircRNA can absorb miRNA as a molecular sponge and play an important role in biological processes such as gene expression regulation and cell signal transduction. It can also directly participate in the physiological and pathological processes of cells by encoding polypeptide chains or affecting the function and localization of proteins. Studies have shown that circRNA plays an important regulatory role in the growth and development of animal skeletal muscle. For example, CircZfp609 regulates mouse myoblast differentiation by competitively binding to miR-194-5p, and CircHIPK3 promotes chicken skeletal muscle development. However, there are relatively few studies on circRNA. The identification of circRNA related to skeletal muscle development is of great significance for in-depth revelation of the regulatory mechanism of muscle generation. Summary of the invention

[0004] The purpose of the present invention is to provide Circ-06958, uses and kits.

[0005] The present invention uses high-throughput sequencing technology to analyze the mRNA, miRNA and lncRNA omics in pig muscle tissue, performs bioinformatics analysis on the sequencing results, predicts new circRNAs, and clones and verifies circRNAs using molecular biological experimental techniques. A new circRNA, Circ-06958, was identified in pig muscle tissue. It is formed by reverse splicing of exons 11-13 of the long-chain acylCoA synthetase 1 (ACSL1) gene, and experiments such as overexpression and cell proliferation marker gene detection have determined that Circ-06958 promotes the proliferation of pig skeletal muscle satellite cells.

[0006] The present invention successfully cloned and identified a new circRNA sequence, Circ-06958. Through molecular biological methods, it was determined that Circ-06958 is a marker for the proliferation of pig skeletal muscle satellite cells. Increasing the expression of Circ-06958 can promote the proliferation of pig skeletal muscle satellite cells. A detection kit containing a Circ-06958 specific primer pair was designed, and the expression level of Circ-06958 was analyzed using the kit. If the expression of Circ-06958 is increased compared with the control group, it indicates that the proliferation efficiency of pig skeletal muscle satellite cells is high.

[0007] A novel circRNA of the present invention is Circ-06958, and the nucleotide sequence of Circ-06958 is shown in the sequence table SEQ ID NO.1.

[0008] The application of the circRNA of the present invention, the application of Circ-06958 for preparing a pig skeletal muscle satellite cell proliferation reagent.

[0009] Furthermore, the Circ-06958 is used to prepare a reagent for detecting or enhancing the proliferation of porcine skeletal muscle satellite cells.

[0010] Furthermore, the Circ-06958 expression level is positively correlated with the proliferation of pig skeletal muscle satellite cells.

[0011] Furthermore, Circ-06958 detection of porcine skeletal muscle satellite cell proliferation is achieved using Circ-06958-RT-F and Circ-06958-RT-R primers, and the nucleotide sequences of Circ-06958-RT-F and Circ-06958-RT-R are as follows:

[0012] Circ-06958-RT-F:GGACAAGCAAATACCACGC;

[0013] Circ-06958-RT-R:ATCGGAGGAAGGACTGAATG.

[0014] The present invention provides a kit for detecting the proliferation of porcine skeletal muscle satellite cells, wherein the kit comprises a kit for detecting the expression level of Circ-06958, and the Circ-06958 nucleotide sequence is shown in the sequence table SEQ ID NO.1.

[0015] Furthermore, the kit detects the expression level of Circ-06958 using Circ-06958-RT-F and Circ-06958-RT-R primers, and the nucleotide sequences of Circ-06958-RT-F and Circ-06958-RT-R are as follows:

[0016] Circ-06958-RT-F:GGACAAGCAAATACCACGC;

[0017] Circ-06958-RT-R:ATCGGAGGAAGGACTGAATG.

[0018] The present invention has the following beneficial effects:

[0019] (1) A new circRNA, Circ-06958, was cloned and identified in the muscle tissue of pigs.

[0020] (2) Circ-06958 promotes the proliferation of porcine skeletal muscle satellite cells.

[0021] (3) Circ-06958 can be used as a marker for the proliferation capacity of porcine skeletal muscle satellite cells.

[0022] (4) The expression level of Circ-06958 can identify the proliferation ability of pig skeletal muscle satellite cells.

[0023] (4) A kit for detecting the proliferation ability of pig skeletal muscle satellite cells is provided.

[0024] (5) Provided is a method for promoting the proliferation of porcine skeletal muscle satellite cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the circular structure of Circ-06958; Circ-06958 is formed by the circular connection of exons 11-13 of the ACSL1 gene;

[0026] Figure 2 Circ-06958 circularization identification results; lanes 4 and 5 are DNA molecular weight standards, lanes 1 and 6 are divergent primer amplification results, lanes 2 and 7 are convergent primer amplification results, and lanes 3 and 8 are internal reference β-actin amplification results;

[0027] Figure 3 The sequencing results of Circ-06958; arrows indicate reverse splicing sites;

[0028] Figure 4 Reverse transcription-PCR analysis of Circ-06958's RNase R resistance; Lane 1 is a DNA molecular weight standard; Lanes 2 and 4 are divergent primer amplification results, and Lanes 3 and 5 are convergent primer amplification results;

[0029] Figure 5 Real-time PCR method to identify Circ-06958 RNase R enzyme resistance diagram; **, p < 0.01;

[0030] Figure 6 Circ-06958 overexpression efficiency detection diagram; **, p<0.01;

[0031] Figure 7 CCK-8 analysis of the effect of Circ-06958 on the proliferation of skeletal muscle satellite cells; *, p<0.05; **, p<0.01;

[0032] Figure 8 EdU analysis of the effect of Circ-06958 on the proliferation of skeletal muscle satellite cells; EV indicates empty vector; OE indicates overexpression vector. **, p<0.01;

[0033] Fig. 9 Effect of Circ-06958 on the expression of PCNA protein, a marker gene for cell proliferation; **, p<0.01. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed by the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify them according to the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0035] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.

[0036] Embodiment 1

[0037] Circ-06958 circularization identification and sequence analysis

[0038] 1. Total RNA extraction: Total RNA was extracted using the Trizol method.

[0039] 2. Genomic DNA extraction: Genomic DNA was extracted using the conventional phenol-chloroform method.

[0040] 3. Reverse transcription and cDNA synthesis: using PrimeScript from Takara TM RT reagent Kit with gDNAEraser kit was used for reverse transcription to synthesize cDNA, and the reverse transcription primers were random primers.

[0041] 4. Circ-06958 prediction: Bioinformatics analysis revealed that there is a circular transcript of the long-chain acylCoA synthetase 1 (ACSL1) gene, named Circ-06958. The circular structure characteristics are shown in Figure 1 .

[0042] 5. Primer design: According to the predicted sequence of Circ-06958 obtained by high-throughput sequencing, Circ-06958-full-F / R primers (see Table 4) were designed to amplify the full length of Circ-06958; divergent primers (Circ-06958-DiV-F, Circ-06958-DiV-R) across the splice site and reverse convergent primers (Circ-06958-CoN-F, Circ-06958-CoN-R) were designed for circularization identification of Circ-06958; at the same time, amplification primers (β-actin-F, β-actin-R) were designed according to the porcine β-actin sequence published in GenBank. The primer sequences are shown in Table 1.

[0043] Table 1 Circ-06958 cloning and identification primers

[0044]

[0045] 6. PCR amplification and sequence analysis: PCR amplification was performed using cDNA and gDNA of the longissimus dorsi muscle of pig as templates, β-actin as an internal reference, and the amplification primers were the divergent primer Circ-06958-DiV-F / R and the convergent primer Circ-06958-CoN-F / R. The PCR reaction system is shown in Table 2; reaction conditions: 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 57℃ annealing for 30s, 72℃ extension at 1000bp / min, 32 cycles, 72℃ final extension for 7min, and storage at 16℃.

[0046] The results showed that when cDNA was used as template, both the convergent primer and the divergent primer could amplify a single target band; when genomic DNA was used as template, only the convergent primer could amplify a band, and the divergent primer had no amplified band. This proved that Circ-06958 is a circular sequence and is truly expressed in muscle tissue. The PCR circularization identification results are shown in Figure 2 .

[0047] The PCR products were purified using the gel recovery kit from Novozymes ( After purification using the Gel DNA Extraction MiniKit, the clones were connected to the pMD-18T vector and transformed into E. coli DH5α. After plasmid extraction and identification, the positive clones were sent to Beijing BGI Technology Co., Ltd. for sequencing. The sequencing results are shown in Figure 3 .

[0048] Table 2 PCR amplification system

[0049]

[0050] Embodiment 2

[0051] Stability Analysis of Circ-06958

[0052] The stability of Circ-06958 was analyzed by RNase R digestion experiment. The total RNA of the longissimus dorsi muscle was treated with RNase R, reverse transcribed to synthesize cDNA, and conventional PCR amplification was performed using Circ-06958-DiV-F / R and Circ-06958-CoN-F / R primers, respectively. The results showed that both pairs of primers could amplify bands in the cDNA samples not treated with RNase R, while the linear transcripts of the parental gene basically disappeared in the cDNA treated with RNase R. The PCR amplification results are shown in Figure 4 .

[0053] At the same time, the real-time PCR method was used to analyze the changes in the expression of the linear transcripts (ACSL1) of Circ-06958 and the parental gene before and after RNase R digestion. The results showed that RNase R treatment did not lead to a decrease in the relative expression level of CircRNA, while the relative expression level of the linear transcript was significantly reduced. The results of real-time PCR analysis are shown in Figure 5 .

[0054] The total RNA extraction, reverse transcription and PCR amplification methods are shown in Example 1. The real-time PCR reaction was performed using Takara's TB Premix Ex Taq TM II (Tli RNaseH Plus) kit was used in strict accordance with the instructions. Circ-06958-RT-F and Circ-06958-RT-R were used as primers, and β-actin was used as the internal reference (see Table 3). Three independent experiments were performed, with three replicates for each sample, and the relative expression of the target gene was calculated using the 2-ΔΔCt method.

[0055] Table 3 Real-time PCR primers

[0056]

[0057] Embodiment 3

[0058] Effect of Circ-06958 on the proliferation of skeletal muscle satellite cells

[0059] 1. Skeletal muscle satellite cells: isolated and preserved by the laboratory in the early stage. Cells were revived and cultured. The specific operations are as follows:

[0060] (1) Cell recovery: The frozen cells were removed from liquid nitrogen and quickly placed in 37°C water. The cells were shaken to promote thawing to obtain a cell suspension. The suspension was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the suspension was resuspended in complete medium, and the suspension was transferred to a 6 cm culture dish. The cells were cultured in a 37°C, 5% CO2 cell culture incubator.

[0061] (2) Passaging: On the second day, observe the cell density and contamination. If there is no contamination, continue culturing with complete medium and pass the cells when the cell density reaches 90%.

[0062] 2. Overexpression vector construction

[0063] The full-length sequence of Circ-06958 was amplified using primers Circ-06958-Full-F / R (see Table 4), and the full-length sequence and the empty vector pCD2.1-CIR were double-digested with KpnI and BamHI, respectively. After the digestion products were recovered and purified, they were ligated with T4 DNA enzyme. The digestion and ligation system is as follows:

[0064] Enzyme digestion system: total system 20 μL, including 1 μL each of KpnI and BamHI, 2 μL of 10× Buffer, 1 μg of DNA, and ddH 2 O to 20 μL. Reaction conditions: incubate in a 37°C water bath for 30 min.

[0065] Ligation system: 1.0 μL each of T4 DNA ligase and Buffer, linearized vector and full-length DNA sequence were added to the reaction mixture at a mass ratio of 1:3-1:10, and dd H 2 O to 10 μL. Reaction conditions: 16°C overnight.

[0066] The ligation products were transformed into Escherichia coli DH5α, and after plasmid extraction and identification, the positive clones were sent to Beijing BGI Technology Co., Ltd. for sequencing identification.

[0067] Table 4 Circ-06958 full-length amplification primers

[0068]

[0069] Note: The underline indicates the KpnI and BamHI recognition sites

[0070] 3. Cell transfection: Cells were inoculated into culture plates. When the confluence reached 60%, the overexpression vector and the empty vector were transfected into porcine skeletal muscle satellite cells respectively. The transfection reagent was Lipofectamine 2000. The operation was performed strictly according to the instructions. The blank group, control group, and experimental group were set up with 3 replicates each.

[0071] 4. Overexpression efficiency detection

[0072] 48 hours after transfection, cells were collected, total RNA was extracted, reverse transcribed into cDNA, and real-time PCR amplification was performed. The blank group, control group, and experimental group were set up with 3 replicates each. It was determined that the constructed overexpression vector can achieve Circ-06958 overexpression in skeletal muscle satellite cells. The detailed methods of RNA extraction, reverse transcription, and real-time PCR amplification are shown in Example 1. The amplification results are shown in Figure 6 .

[0073] 5.CCK-8 assay

[0074] 10 μL of enhanced CCK-8 solution was added to the cells at 0, 24, 48, 72, 96, and 120 hours after transfection, and the cells were incubated for 2 hours. The absorbance was measured at 450 nm, and statistical analysis was performed based on the obtained values ​​to draw a cell growth activity curve. The results showed that after overexpression of Circ-06958, the activity of skeletal muscle satellite cells was significantly increased (p<0.01). Figure 7 .

[0075] 5. EdU Detection

[0076] Cells were collected 24 hours after transfection and the cell proliferation was detected according to the BeyoClick EdU-555 cell proliferation detection kit of Beyotime. It was found that the number of skeletal muscle satellite cells increased significantly after overexpression of Circ-06958 (p<0.05). Figure 8 .

[0077] 6. Detection of proliferation marker gene protein expression

[0078] Western blotting was used to analyze the effect of Circ-06958 on the expression of proliferating cell nuclear antigen (PCNA), a cell proliferation marker gene. Cells were collected 48 hours after transfection, and total cell protein was extracted using RIPA buffer from Bio-Tech. The concentration was determined using a BCA protein concentration assay kit. 25 to 30 μg of total protein was loaded onto an SDS-PAGE gel and electrophoresed for 2 hours to separate total protein. The electrophoresis results were transferred to a PVDF membrane, blocked in 5% skim milk for 1 hour, and a 5000-fold diluted primary antibody (Proteintech) was added. The membrane was incubated overnight at 4°C. After the PVDF membrane was washed and recovered, a fluorescent secondary antibody was added and incubated for 1 hour. β-tubulin was used as an internal reference. The hybridization results were detected using a UVP ChemStudioTM PLUS touch instrument from Analytik Jena. Results are shown in Fig. 9 .

Claims

1. A novel circRNA, characterized in that The circRNA is Circ-06958, and the nucleotide sequence of Circ-06958 is shown in the sequence table SEQ ID NO.

1.

2. The use of circRNA as claimed in claim 1, characterized in that The Circ-06958 is used to prepare a pig skeletal muscle satellite cell proliferation reagent.

3. The use according to claim 2, characterized in that The Circ-06958 is used to prepare a reagent for detecting or increasing the proliferation of pig skeletal muscle satellite cells.

4. The use according to claim 3, characterized in that The Circ-06958 expression level is positively correlated with the proliferation of pig skeletal muscle satellite cells.

5. The use according to claim 2 or 3, characterized in that Circ-06958 detection of porcine skeletal muscle satellite cell proliferation is achieved using Circ-06958-RT-F and Circ-06958-RT-R primers, and the nucleotide sequences of Circ-06958-RT-F and Circ-06958-RT-R are as follows: Circ-06958-RT-F:GGACAAGCAAATACCACGC; Circ-06958-RT-R:ATCGGAGGAAGGACTGAATG.

6. A kit for detecting the proliferation of porcine skeletal muscle satellite cells, characterized in that The kit contains a kit for detecting the expression level of Circ-06958, and the Circ-06958 nucleotide sequence is shown in the sequence table SEQ ID NO.

1.

7. The kit according to claim 6, characterized in that The kit detects the expression level of Circ-06958 using Circ-06958-RT-F and Circ-06958-RT-R primers. The nucleotide sequences of Circ-06958-RT-F and Circ-06958-RT-R are as follows: Circ-06958-RT-F:GGACAAGCAAATACCACGC; Circ-06958-RT-R:ATCGGAGGAAGGACTGAATG.

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