A novel circRNA, its uses, and a reagent kit

Abstract

A novel circRNA, its uses, and a reagent kit are disclosed, relating to the field of animal genetics and breeding. The nucleotide sequence of Circ-06958 of this invention is shown in SEQ ID NO.1 of the sequence listing. Circ-06958 is used in the preparation of a reagent for the proliferation of porcine skeletal muscle satellite cells. This invention successfully cloned and identified a novel circRNA sequence—Circ-06958. Molecular biological methods confirmed that Circ-06958 is a marker for the proliferation of porcine skeletal muscle satellite cells, and increasing the expression level of Circ-06958 can promote the proliferation of porcine skeletal muscle satellite cells.

Description

Technical Field

[0001] This invention belongs to the field of animal genetics and breeding, specifically relating to a biomarker Circ-06958 related to the proliferation of porcine skeletal muscle satellite cells and its applications. Background Technology

[0002] Muscle growth and development, along with its intrinsic characteristics, determine the quantity and quality of meat products, directly impacting the economic benefits of pig farming and thus attracting significant attention from pig producers and breeding experts. Skeletal muscle, a crucial component of muscle tissue, primarily consists of muscle fibers and surrounding connective tissue, playing a vital regulatory role in muscle movement, endocrine regulation, energy consumption, and metabolic hemostasis. The number of muscle fibers is closely related to the growth rate and meat production potential of livestock and poultry. After birth, the number of muscle fibers remains relatively constant, with changes in their diameter being the fundamental reason for increased muscle mass. Skeletal muscle satellite cells are myogenic stem cells in skeletal muscle, possessing proliferative and differentiation potential and capable of self-renewal. Their proliferative capacity largely determines the number of muscle fibers in fetal skeletal muscle. After birth, skeletal muscle satellite cells are typically in a quiescent state. During muscle loss repair and regeneration, they are reactivated to enter the proliferative and differentiation process, helping damaged tissues recover. Skeletal muscle lacking satellite cells cannot regenerate after acute injury. Therefore, skeletal muscle satellite cells play a crucial role in muscle development. Elucidating the proliferation and differentiation mechanisms of skeletal muscle satellite cells is beneficial for improving meat yield and quality and advancing the molecular breeding process for pigs.

[0003] The proliferation and differentiation of skeletal muscle satellite cells are jointly regulated by transcription factors, functional genes, non-coding RNAs, and epigenetic modifications. Circular RNA (circRNA) is a recently discovered type of single-stranded, closed, circular non-coding RNA. Formed by backsplicing of mRNA precursors, circRNAs lack a 5' phosphate cap and a 3' polyadenylated tail, making them less sensitive to nuclease degradation and more stable. Furthermore, circRNA sequences are highly conserved. This high degree of conservation and stability indicates the functional importance of circRNAs. circRNAs can act as molecular sponges to absorb miRNAs, playing crucial roles in biological processes such as gene expression regulation and cell signaling. They can also directly participate in cellular physiological and pathological processes by encoding polypeptide chains or influencing protein function and localization. Previous studies have shown that circRNAs play important regulatory roles 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 is relatively little research on circRNAs. Identifying circRNAs related to skeletal muscle development is of great significance for revealing the regulatory mechanisms of muscle formation. Summary of the Invention

[0004] The purpose of this invention is to provide Circ-06958, its uses, and a reagent kit.

[0005] This invention utilizes high-throughput sequencing technology to analyze the mRNA, miRNA, and lncRNA genomes in porcine muscle tissue. Bioinformatics analysis of the sequencing results is performed to predict novel circRNAs, which are then cloned and validated using molecular biology techniques. A novel circRNA, Circ-06958, was identified in porcine muscle tissue. This circRNA is formed by backsplicing exons 11-13 of the long-chain acylCoA synthetase 1 (ACSL1) gene. Overexpression and detection of cell proliferation marker genes confirm that Circ-06958 promotes the proliferation of porcine skeletal muscle satellite cells.

[0006] This invention successfully cloned and identified a novel circRNA sequence—Circ-06958. Molecular biological methods confirmed that Circ-06958 is a biomarker for porcine skeletal muscle satellite cell proliferation, and increasing Circ-06958 expression promotes porcine skeletal muscle satellite cell proliferation. A detection kit containing Circ-06958-specific primer pairs was designed. Using this kit to analyze Circ-06958 expression levels, an increase in Circ-06958 expression compared to the control group indicates high proliferation efficiency of porcine skeletal muscle satellite cells.

[0007] The present invention discloses a novel circRNA, wherein the circRNA is Circ-06958, and the nucleotide sequence of Circ-06958 is shown in SEQ ID NO.1 of the sequence listing.

[0008] The application of the circRNA of the present invention, specifically the application of Circ-06958 in the preparation of a porcine skeletal muscle satellite cell proliferation reagent.

[0009] Furthermore, the Circ-06958 is used in the preparation of reagents for detecting or enhancing the proliferation of porcine skeletal muscle satellite cells.

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

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

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

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

[0014] The present invention discloses a kit for detecting the proliferation of porcine skeletal muscle satellite cells, the kit comprising the ability to detect the expression level of Circ-06958, the nucleotide sequence of which is shown in SEQ ID NO.1 of the sequence listing.

[0015] Furthermore, the kit is used to detect the expression level of Circ-06958 using Circ-06958-RT-F and Circ-06958-RT-R primers, the nucleotide sequences of which 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 of the proliferative capacity of porcine skeletal muscle satellite cells.

[0022] (4) The expression level of Circ-06958 can be used to identify the proliferative capacity of porcine skeletal muscle satellite cells.

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

[0024] (5) A method for promoting the proliferation of porcine skeletal muscle satellite cells is provided. Attached Figure Description

[0025] Figure 1 A 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 The results of circulation identification of Circ-06958 are shown in the figure; lanes 4 and 5 are DNA molecular weight standards, lanes 1 and 6 are the results of amplification with divergent primers, lanes 2 and 7 are the results of amplification with convergent primers, and lanes 3 and 8 are the results of amplification with the internal control β-actin.

[0027] Figure 3 This is the sequencing identification result for Circ-06958; the arrow indicates the backsplicing site.

[0028] Figure 4 RNase R resistance of Circ-06958 was analyzed by reverse transcription PCR; lane 1 is the DNA molecular weight standard; lanes 2 and 4 are the results of amplification with divergent primers, and lanes 3 and 5 are the results of amplification with convergent primers.

[0029] Figure 5 Real-time PCR was used to identify the RNase R resistance of Circ-06958; **, p<0.01;

[0030] Figure 6 Overexpression efficiency detection plot of Circ-06958; **, p<0.01;

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

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

[0033] Figure 9 Effect of Circ-06958 on the expression of PCNA protein, a cell proliferation marker gene; **, p<0.01. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

[0035] The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0036] Example 1

[0037] Circulation identification and sequence analysis of Circ-06958

[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 to synthesize cDNA: using Takara's PrimeScript. TM The RT reagent kit with gDNAEraser was used for reverse transcription to synthesize cDNA. Random primers were used for reverse transcription.

[0041] 4. Circ-06958 Prediction: Bioinformatics analysis revealed a circular transcript in the long-chain acylCoA synthetase 1 (ACSL1) gene, named Circ-06958. Its circular structure characteristics are described below. Figure 1 .

[0042] 5. Primer Design: Based on the predicted sequence of Circ-06958 obtained from 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) and convergent primers (Circ-06958-CoN-F, Circ-06958-CoN-R) across splice sites were designed for circumspheric identification of Circ-06958. Simultaneously, amplification primers (β-actin-F, β-actin-R) were designed based on the porcine β-actin sequence published in GenBank. Primer sequences are shown in Table 1.

[0043] Table 1 Primers for Circ-06958 cloning and identification

[0044]

[0045] 6. PCR Amplification and Sequence Analysis: PCR amplification was performed using porcine longissimus dorsi muscle cDNA and gDNA as templates, with β-actin as an internal control. 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; the reaction conditions were: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension at 1000 bp / min for 32 cycles, final extension at 72℃ for 7 min, and storage at 16℃.

[0046] The results showed that when cDNA was used as a template, both convergent and divergent primers amplified a single target band; however, when genomic DNA was used as a template, only the convergent primer amplified a band, while the divergent primer did not amplify any band. This proves that Circ-06958 is a circular sequence and is truly expressed in muscle tissue. PCR circularization identification results are shown below. Figure 2 .

[0047] PCR products were extracted using a gel extraction kit from Novizan (Novazia). After purification using the Gel DNA Extraction Mini Kit, the DNA was ligated into the pMD-18T vector, transformed into E. coli DH5α, and after plasmid extraction and identification, positive clones were sent to Beijing BGI Genomics Co., Ltd. for sequencing. Sequencing results are shown below. Figure 3 .

[0048] Table 2 PCR amplification system

[0049]

[0050] Example 2

[0051] Stability analysis of Circ-06958

[0052] The stability of Circ-06958 was analyzed using an RNase R digestion assay. Total RNA from the longissimus dorsi muscle was treated with RNase R, and cDNA was synthesized via reverse transcription. Conventional PCR amplification was performed using primers Circ-06958-DiV-F / R and Circ-06958-CoN-F / R, respectively. The results showed that both primer pairs amplified bands in the untreated cDNA samples, while the linear transcripts of the parental gene were almost completely lost in the RNase R-treated cDNA. PCR amplification results are shown below. Figure 4 .

[0053] Meanwhile, real-time PCR was used to analyze the changes in the expression levels of linear transcripts (ACSL1) of Circ-06958 and its parental genes 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 real-time PCR analysis results are shown below. Figure 5 .

[0054] Total RNA extraction, reverse transcription, and PCR amplification methods are described in Example 1. Real-time PCR was performed using Takara's TB... Premix Ex Taq TM The II (Tli RNaseH Plus) kit was used, and the procedure was strictly followed according to the instructions. Three independent experiments were conducted using Circ-06958-RT-F and Circ-06958-RT-R as primers and β-actin as an internal control (see Table 3). Each sample was tested in triplicate, and the relative expression level of the target gene was calculated using the 2-ΔΔCt method.

[0055] Table 3 Real-time PCR primers

[0056]

[0057] Example 3

[0058] Effects of Circ-06958 on skeletal muscle satellite cell proliferation

[0059] 1. Skeletal muscle satellite cells: isolated and preserved in the laboratory previously. Cell resuscitation and culture were performed as follows:

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

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

[0062] 2. Construction of overexpression vectors

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

[0064] Enzyme digestion system: Total volume 20 μL, containing 1 μL each of KpnI and BamHI, 2 μL of 10× Buffer, 1 μg of DNA, and adjusted to 20 μL with ddH2O. Reaction conditions: Incubate at 37℃ 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 H2O was added to adjust the volume to 10 μL. Reaction conditions: 16°C overnight.

[0066] The ligation product was transformed into E. coli DH5α, and after plasmid extraction and identification, positive clones were sent to Beijing BGI Genomics Co., Ltd. for sequencing identification.

[0067] Table 4. Primers for full-length amplification of Circ-06958

[0068]

[0069] Note: Underlined sites indicate KpnI and BamHI recognition sites.

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

[0071] 4. Overexpression efficiency detection

[0072] Cells were collected 48 hours after transfection, total RNA was extracted, reverse transcribed into cDNA, and amplified by real-time PCR. Three replicates were set for each of the blank control, control, and experimental groups. The constructed overexpression vector was confirmed to achieve Circ-06958 overexpression in skeletal muscle satellite cells. Detailed methods for RNA extraction, reverse transcription, and real-time PCR amplification are described in Example 1. Amplification results are shown below. Figure 6 .

[0073] 5. CCK-8 detection

[0074] Cells at 0, 24, 48, 72, 96, and 120 h post-transfection were incubated with 10 μL of enhanced CCK-8 solution for 2 h, and absorbance was measured at 450 nm. Statistical analysis was performed based on the results, and cell growth and viability curves were plotted. The results showed that overexpression of Circ-06958 significantly increased the viability of skeletal muscle satellite cells (p<0.01). See results below. Figure 7 .

[0075] 5. EdU detection

[0076] Cells were collected 24 hours after transfection, and cell proliferation was assessed using the BeyoClick EdU-555 cell proliferation assay kit from Beyotime Biotechnology. Overexpression of Circ-06958 significantly increased the number of skeletal muscle satellite cells (p<0.05). Results are shown below. Figure 8 .

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

[0078] The effect of Circ-06958 on the expression of the cell proliferation marker gene, Proliferating Cell Nuclear Antigen (PCNA), was analyzed using Western blotting. Cells were collected 48 h after transfection. Total protein was extracted using RIPA buffer from Beyotime Biotechnology Co., Ltd., and the concentration was determined using a BCA protein assay kit. 25–30 μg of total protein was loaded onto an SDS-PAGE gel and electrophoresed for 2 h to separate the total protein. The electrophoresis results were transferred to a PVDF membrane, blocked with 5% skim milk for 1 h, and incubated overnight at 4°C with a 5000-fold diluted primary antibody (Proteintech). After washing and recovery from the PVDF membrane, a fluorescent secondary antibody was added and incubated for 1 h. β-tubulin was used as an internal control. Hybridization results were detected using an Analytik Jena UVP ChemStudio™ PLUS touch instrument. Results are shown below. Figure 9 .

Claims

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

1. The Circ-06958 is used for preparing a porcine skeletal muscle satellite cell proliferation reagent.

2. The use of a circRNA according to claim 1, characterized in that ​

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