circRNAs Related to the Proliferation of Porcine Skeletal Muscle Satellite Cells and Their Applications

Abstract

The present invention provides a circRNA related to the proliferation of porcine skeletal muscle satellite cells and its application. Based on whole transcriptome sequencing, the present invention first discovers that circular RNA circLMO7 is highly expressed in the muscle tissue of Jinfen white pigs, and its expression level significantly decreases at 1 day, 90 days, and 180 days. The objective existence of circLMO7 is confirmed by PCR, Sanger sequencing, and RNase R digestion experiments; further, real-time fluorescence quantitative PCR is used to confirm that the expression level of circLMO7 continuously decreases at 1 day, 90 days, and 180 days in Jinfen white pigs; the EdU, CCK-8, and real-time fluorescence quantitative PCR experiments are used to further verify that circLMO7 plays a significant upregulating role in the proliferation process of porcine skeletal muscle satellite cells. The present invention proposes that circLMO7 can be used as a biomarker to identify the proliferation of porcine skeletal muscle satellite cells.

Description

Technical Field

[0001] The present invention relates to the field of molecular genetics technology. Specifically, it relates to a circRNA related to the proliferation of porcine skeletal muscle satellite cells and its application. Background Art

[0002] Pork is one of the most important meat sources in China, accounting for nearly 40% of the total meat consumption. As a tissue with a relatively large proportion in the animal body, skeletal muscle directly determines the meat production performance of livestock. Skeletal muscle development is a highly coordinated process, mainly including the proliferation and differentiation of mesenchymal stem cells located in the mesoderm into myoblasts, the fusion of myoblasts into multinucleated myofibers, and the maturation of myofibers. These processes are strictly and complexly controlled by the expression of myogenic genes such as myogenic regulatory factors (MRFs) and paired box (PAX) genes. At the same time, non-coding RNAs (ncRNAs), such as microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA), their regulatory roles in skeletal muscle growth and development have been gradually revealed.

[0003] CircRNA is a covalently closed circular RNA molecule formed by back-splicing. Due to the development of high-throughput RNA sequencing (RNA-seq), thousands of circRNAs have been identified in eukaryotes (including humans, mice, cattle, and chickens). The structures of most circRNAs are very stable, with high abundance and spatio-temporal expression specificity, indicating that circRNAs may play an important role in the regulation of gene expression in biological organisms. In recent years, the research on circRNAs as diagnostic and therapeutic targets for related diseases in medicine has attracted extensive attention, while there are few reports on circRNAs related to porcine skeletal muscle satellite cells. Exploring new circRNAs that play important roles in porcine skeletal muscle growth and development can not only reveal new mechanisms of the functions of skeletal muscle satellite cells but also provide new diagnostic and therapeutic targets for muscle diseases. Summary of the Invention

[0004] The purpose of the present invention is to provide a circRNA related to the proliferation of porcine skeletal muscle satellite cells and its application.

[0005] To achieve the purpose of the present invention, in the first aspect, the present invention provides a circRNA related to the proliferation of porcine skeletal muscle satellite cells. The circRNA is circLMO7, and its corresponding cDNA sequence is shown in SEQ ID NO:1.

[0006] In the second aspect, the present invention provides the application of the circRNA as a marker for the proliferation of porcine skeletal muscle satellite cells in the preparation of detection reagents or kits for identifying the proliferation of porcine skeletal muscle satellite cells.

[0007] In a third aspect, the present invention provides a pair of primers for detecting the circRNA, including an upstream primer shown in SEQ ID NO:2 and a downstream primer shown in SEQ ID NO:3.

[0008] In a fourth aspect, the present invention provides a kit for identifying the proliferation of porcine skeletal muscle satellite cells, and the kit includes reagents for detecting the expression level of circLMO7.

[0009] Furthermore, the kit includes the primers shown in SEQ ID NO:2-3.

[0010] In a fifth aspect, the present invention provides an inhibitor of circLMO7, and the inhibitor is siRNA shown in SEQ ID NO:8-9, SEQ ID NO:10-11 or SEQ ID NO:12-13, or a combination of the above three pairs of siRNA.

[0011] In a sixth aspect, the present invention provides a method for identifying the proliferation efficiency of porcine skeletal muscle satellite cells, and the method includes: detecting the relative expression level of circLMO7 in porcine skeletal muscle satellite cells, and if the expression level of circLMO7 is significantly reduced, it indicates that the proliferation efficiency of porcine skeletal muscle satellite cells is low.

[0012] Furthermore, the expression level of circLMO7 is detected using the primers shown in SEQ ID NO:2-3.

[0013] By means of the above technical solutions, the present invention has at least the following advantages and beneficial effects:

[0014] The present invention first discovers a novel circRNA, circLMO7, in pigs. Through gain-of-function and loss-of-function of circLMO7, it is found that circLMO7 can affect the proliferation rate of porcine skeletal muscle satellite cells, thereby providing a circRNA marker related to the proliferation of porcine skeletal muscle satellite cells. Using this method, the proliferation of porcine skeletal muscle satellite cells can be identified, and new ideas and methods are provided for clinical research on muscle growth and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the PCR product sequencing result of circLMO7 in the longissimus dorsi muscle tissue of 1-day-old Jinfen white pigs in a preferred embodiment of the present invention.

[0016] Figure 2 It is the result of the circLMO7 RNase R enzyme digestion test in a preferred embodiment of the present invention. * indicates significant difference, and ** indicates extremely significant difference. NC represents the blank control group without adding Rnase R enzyme.

[0017] Figure 3 Expression changes of circLMO7 in the longissimus dorsi muscle tissues of Jinfen white pigs at 1 day old, 90 days old and 180 days old in the preferred embodiment of the present invention. Different capital letters indicate extremely significant differences.

[0018] Figure 4 Overexpression efficiency of circLMO7 in porcine skeletal muscle satellite cells in the preferred embodiment of the present invention. NC represents the control group, and OE represents the circLMO7 overexpression group. * indicates significant difference, and ** indicates extremely significant difference.

[0019] Figure 5 Interference efficiency of circLMO7 in porcine skeletal muscle satellite cells in the preferred embodiment of the present invention. NC represents the control group, and Smart Silence represents the circLMO7 interference group. * indicates significant difference, and ** indicates extremely significant difference.

[0020] si-NC represents the blank control group; si-1, si-2, and si-3 respectively represent three pairs of small interfering RNAs.

[0021] Figure 6 and Figure 7 In the preferred embodiment of the present invention, the effect of circLMO7 on the proliferation ability of porcine skeletal muscle satellite cells was detected by EdU staining and Image J statistical analysis. * indicates significant difference, and ** indicates extremely significant difference. NC represents the blank control group.

[0022] Figure 8 and Figure 9 In the preferred embodiment of the present invention, the effect of circLMO7 on the proliferation ability of porcine skeletal muscle satellite cells was detected by the CCK-8 method. * indicates significant difference, and ** indicates extremely significant difference. NC represents the blank control group.

[0023] Figure 10 and Figure 11 In the preferred embodiment of the present invention, the expression changes of proliferation-related genes were detected by qPCR 48 hours after transfection with Smart Silence. * indicates significant difference, and ** indicates extremely significant difference. NC represents the blank control group. Detailed implementation manners

[0024] The present invention provides a circular RNA circLMO7 and its applications. First, through high-throughput sequencing and cloning sequencing, the present invention discovers and identifies a new circular RNA circLMO7. Subsequently, siRNA and overexpression vectors of circLMO7 are designed and synthesized, and transfected into porcine skeletal muscle satellite cells by liposome method to interfere with and overexpress the expression level of circLMO7 in porcine skeletal muscle satellite cells. By changing the expression of circLMO7 in porcine skeletal muscle satellite cells, the proliferation rate of porcine skeletal muscle satellite cells is changed.

[0025] The present invention also provides a detection method for a circRNA marker related to the proliferation of porcine skeletal muscle satellite cells. Using this method, the proliferation of porcine skeletal muscle satellite cells can be identified, and new ideas and methods can be provided for clinical research on muscle growth and development.

[0026] The present invention adopts the following technical solutions:

[0027] The present invention provides a circRNA marker related to the proliferation of porcine skeletal muscle satellite cells. The circRNA marker is circLMO7, and its sequence is shown as SEQ ID NO:1.

[0028] The present invention also provides detection primers for a circRNA marker related to the proliferation of porcine skeletal muscle satellite cells, namely circLMO7. The detection primers are divergent primers for specifically amplifying circLMO7.

[0029] The present invention also provides a product for identifying the proliferation of porcine skeletal muscle satellite cells. The product detects the expression level of circLMO7 in a sample through bioinformatics prediction technology and nucleic acid amplification technology. The nucleic acid amplification technology is selected from polymerase chain reaction, reverse transcription polymerase chain reaction, transcription-mediated amplification, ligase chain reaction or nucleic acid sequence-based amplification.

[0030] The present invention also provides small interfering RNA of the above circular RNA circLMO7. The small interfering RNA is a siRNA composition. The inhibitor is siRNA shown as SEQ ID NO:8-9, SEQ ID NO:10-11 or SEQ ID NO:12-13, or a combination of the above three pairs of siRNA.

[0031] The present invention also provides an overexpression vector of the above circular RNA circLMO7. The overexpression vector is pCD2.1-circLMO7.

[0032] The present invention also provides a method for identifying the proliferation efficiency of porcine skeletal muscle satellite cells. The system of treating circLMO7 with small interfering candidate substances can significantly inhibit the proliferation of porcine skeletal muscle satellite cells; the system of treating circLMO7 with an overexpression vector using candidate substances can significantly promote the proliferation of porcine skeletal muscle satellite cells. The results confirm that the circLMO7 sequence has the effect of promoting the proliferation of porcine skeletal muscle satellite cells.

[0033] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Unless otherwise specified, the examples are carried out under conventional experimental conditions, such as the Molecular Cloning Experimental Manual by Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions. Example 1 Verification of circLMO7

[0034] 1. Materials

[0035] Sample collection: After slaughtering 1-day-old Jinfen white pigs, the longissimus dorsi muscle tissue was quickly collected, frozen in liquid nitrogen and stored at -80°C.

[0036] Reagents: Trizol, PrimeScript RT reagent Kit, DNA marker, 2×SYBR Premix ExTaqⅡ were purchased from Takara (Japan); 2×Es Taq Master Mix (Dye) was purchased from Kangwei Century Biotechnology Co., Ltd. (Jiangsu); 50×TAE buffer, nucleic acid dye, agarose were purchased from Solarbio Science & Technology Co., Ltd. (Beijing); RNase R and primers were purchased from Sangon Biotech Co., Ltd. (Shanghai).

[0037] 2. Methods

[0038] 2.1 Extraction of total RNA

[0039] Take out the longissimus dorsi muscle tissue of 1-day-old Jinfen white pigs from the -80°C refrigerator, add 700 μL of Trizol reagent, and completely grind it with a tissue homogenizer and then let it stand for 10 min for sufficient lysis. Centrifuge at 12000 r·min -1 After centrifugation for 10 min, take the supernatant into a new sterile RNase-free EP tube, add 250 μL of chloroform, shake vigorously for 15 s and then let it stand for 10 min for stratification. Carefully aspirate the supernatant into a new sterile RNase-free EP tube, add an equal volume of isopropanol and gently invert it up and down, and let it stand on ice for 10 min. Centrifuge at 12000 r·min -1After centrifugation for 15 min, discard the supernatant and keep the precipitate. Add an appropriate amount of 75% ethanol to wash the precipitate three times, then invert it on filter paper to volatilize the residual ethanol. Add an appropriate amount of DEPC water, mix well, and let it stand on ice for 20 min. Use a nucleic acid and protein analyzer to detect the purity and concentration of RNA. Qualified RNA (OD 260 / OD 280 within the range of 1.9 - 2.1) is stored at -80 °C for subsequent experiments.

[0040] 2.2 RNase R digestion assay

[0041] Add 1 μL of RNase R and DEPC water to the experimental group and control group of 5 μg of porcine longissimus dorsi muscle RNA respectively, incubate in a 37 °C water bath for 15 min, and store at -80 °C for later use.

[0042] 2.3 Reverse transcription reaction

[0043] Remove genomic DNA: Add RNA and genomic DNA removal reagent to 10 μL (reaction program: 42 °C, 2 min); Reverse transcription: Mix the above reaction liquid with buffer and reverse transcriptase, etc., and make the final volume to 20 μL (reaction program: 37 °C, 15 min; 85 °C, 5 s), and store in a -20 °C refrigerator.

[0044] 2.4 Primer design

[0045] According to the sequence of circLMO7 (SEQ ID NO:1), design divergent primers for circLMO7, primers for LMO7 and 18s through NCBI, as follows:

[0046] Upstream primer for circLMO7: 5′-ACACAAAACGGTGGGAGGAA-3′ (SEQ ID NO:2)

[0047] Downstream primer: 5′-TGATTGGGCTTAGCTTTCGC-3′ (SEQ ID NO:3)

[0048] Upstream primer for 18S rRNA: 5′-ATGCCAGAGTCTCGTTCGTTAT-3′ (SEQ ID NO:4)

[0049] Downstream primer: 5′-CGGACAGGATTGACAGATTGAT-3′ (SEQ ID NO:5)

[0050] Upstream primer for LMO7: 5′-CTGTCCACACCAATAGCAGGT-3′ (SEQ ID NO:6)

[0051] Downstream primer: 5′-TGTGCTTTTCTTCCCAGCCA-3′ (SEQ ID NO:7)

[0052] 2.5 PCR amplification

[0053] The reaction system was as follows: 5 μL of 2×Es Taq Master Mix, 0.5 μL each of the divergent upstream and downstream primers of circLMO7 (10 pmol / μl), 1 μL of cDNA, and made up to 10 μL with ddH2O.

[0054] The reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, for 33 cycles; continued extension at 72°C for 5 min. After PCR amplification, agarose gel electrophoresis was performed to analyze the size of the obtained fragments.

[0055] 2.6 Real-time fluorescence quantitative PCR

[0056] The total reaction volume was 10 μL (0.5 μL each of the upstream and downstream primers, 1 μL of cDNA, 5 μL of 2×SYBR Premix Ex TaqⅡ, and made up with RNase Free ddH2O). The reaction program was as follows: 30 s at 95°C; 5 s at 95°C, 30 s at 60°C, 30 s at 72°C, for 45 cycles; 15 s at 95°C, 30 s at 65°C, 30 s at 95°C to generate a melting curve. Each sample was technically replicated 3 times, and the 2 -△△CT method was used to analyze the detection results, and the results were expressed as mean ± standard error.

[0057] 3. Results

[0058] circLMO7 is a new circRNA obtained by screening through a large number of bioinformatics analyses such as differential expression analysis, GO and KEGG enrichment analysis, and downstream miRNA prediction in the whole transcriptome sequencing of the longissimus dorsi muscle tissues of Jinfen white pigs at 1, 90, and 180 days of age. Its corresponding sequence is shown in SEQ ID NO:1. After PCR amplification with the specific divergent primers of circLMO7, the target band was detected by agarose gel electrophoresis. The size of the obtained band was 239 bp, which was consistent with the expected fragment size. The results of further sequencing were as Figure 1 shown, verifying that the amplified fragment was the junction sequence of circLMO7 and confirming the real existence of this circRNA. The results of the RNase R digestion experiment were as Figure 2 shown. The stability of circLMO7 was significantly higher than that of its parental gene LMO7 (XM_021065678.1) under RNase R digestion, proving the stability characteristic of circRNA.

[0059] Example 2 Temporal expression characteristics of circLMO7

[0060] 1. Materials

[0061] Samples: The longissimus dorsi muscle tissues of Jinfen white pigs at 1-day-old, 90-day-old, and 180-day-old were collected and placed in cryotubes. After being quickly immersed in liquid nitrogen for freezing, they were stored at -80 °C.

[0062] Reagents: The same as in Example 1.

[0063] 2. Methods

[0064] 2.1 Extraction and reverse transcription of total RNA

[0065] The specific method was the same as in Example 1.

[0066] 2.2 Real-time fluorescence quantitative PCR

[0067] The specific method was the same as in Example 1.

[0068] 3. Results

[0069] The experimental results were as Figure 3 shown. The expression of circLMO7 was the highest in the longissimus dorsi muscle tissues of 1-day-old Jinfen white pigs, showing a trend of continuous decrease in its expression with the increase of the age of Jinfen white pigs, suggesting that circLMO7 may be involved in regulating the process of pig muscle growth and development.

[0070] Example 3 Role of circLMO7 in the proliferation process of porcine skeletal muscle satellite cells

[0071] 1. Materials

[0072] Cells: Porcine skeletal muscle satellite cells, isolated and cultured in the laboratory in the early stage.

[0073] Reagents: Fetal bovine serum (FBS), high-glucose DMEM, and 0.25% trypsin were purchased from Gibco (USA); penicillin-streptomycin mixture, PBS powder, and 4% paraformaldehyde were purchased from Solaibio (Beijing); CCK-8 kit, EdU proliferation detection kit, and LipoFiter TM transfection reagent were purchased from Hanheng Biotechnology Co., Ltd. (Shanghai), and transfection reagent riboFECTTM CP was purchased from Ribobio Co., Ltd. (Guangzhou).

[0074] 2. Methods

[0075] The present invention is directed to the circLMO7 sequence, and siRNA sequences (Smart Silence) that inhibit its expression are synthesized. SmartSilence is the siRNA shown in SEQ ID NO: 8-9 (si-1), SEQ ID NO: 10-11 (si-2), or SEQ ID NO: 12-13 (si-3), or a combination of the above three pairs of siRNAs. The specific sequences are as follows:

[0076] 5′-GGAAAGUCUGUAACGAGAGTT-3′(SEQ ID NO:8)

[0077] 5′-CUCUCGUUACAGACUUUCCTT-3′(SEQ ID NO:9)

[0078] 5′-AGUCUGUAACGAGAGGAGATT-3′(SEQ ID NO:10)

[0079] 5′-UCUCCUCUCGUUACAGACUTT-3′(SEQ ID NO:11)

[0080] 5′-AUGGAAAGUCUGUAACGAGTT-3′(SEQ ID NO:12)

[0081] 5′-CUCGUUACAGACUUUCCAUTT-3′(SEQ ID NO:13)

[0082] The sequence of the NC group is:

[0083] 5′-UUCUCCGAACGUGUCACGUTT-3′(SEQ ID NO:14)

[0084] 5′-ACGUGACACGUUCGGAGAATT-3′(SEQ ID NO:15)

[0085] The circLMO7 interference sequence Smart Silence and the control sequence Smart Silence NC used in the experiment were both synthesized by Shanghai GenePharma Co., Ltd. The overexpression vectors pCD2.1 and pCD2.1-circLMO7 were purchased from Wuhan KingRiver Biotechnology Co., Ltd.

[0086] 2.1 Detection of circLMO7 interference and overexpression efficiency

[0087] Porcine skeletal muscle satellite cells were seeded in 12-well plates and transfected when the cell density reached about 60%. 4 μL of LipoFiter was diluted with 100 μL of DMEM solution respectivelyTM And process the substances (4 μL si-NC, 4 μL si-circLMO7, 4 μg OE-NC, 4 μg OE-circLMO7), gently mix and let stand for 5 min; then gently pipette and mix well, incubate at room temperature for 15 min; make up the mixed solution to 1 mL with DMEM solution respectively, gently mix and add to the cells, and change to medium without antibiotics after 6 h. Collect the cells after culturing in the incubator for 48 h, and detect the overexpression efficiency by real-time fluorescence quantitative PCR method.

[0088] si-NC represents the interference blank control group, si-circLMO7 represents the interference group, OE-NC represents the overexpression blank control group, and OE-circLMO7 represents the overexpression group.

[0089] 2.2 EdU assay for cell proliferation

[0090] Seed the cells in 24-well plates. When the cell density reaches about 60%, transfect with OE-NC, OE-circLMO7, si-NC, and si-circLMO7 respectively, with three replicates for each treatment. At 48 h after transfection, add 50 μL of 1000:1 diluted EdU solution to each well and incubate in the incubator for 2 h. After washing twice with PBS, add 200 μL of 4% paraformaldehyde and fix at room temperature for 30 min, then add 200 μL of 2 mg·mL -1 glycine, incubate on a shaker for 5 min. After washing 3 times with PBS, add 200 μL of 0.5% Triton X-100 and incubate on a shaker at room temperature for 30 min. After washing once with PBS, add 200 μL of Apollo staining solution and incubate in the dark at room temperature for 30 min, then wash 3 times with 0.5% Triton X-100 on a shaker; add DAPI staining solution and incubate in the dark at room temperature for 30 min. After washing 3 times with PBS, perform fluorescence microscopy imaging analysis.

[0091] 2.3 CCK-8 assay for cell proliferation

[0092] Seed the cells in 96-well plates. When the cell density reaches about 60%, transfect with OE-NC, OE-circLMO7, si-NC, and si-circLMO7 respectively, with three replicates for each treatment. The transfection method is the same as that shown in 2.1 of Example 3. Add 10 μL of CCK-8 solution at 0 h, 24 h, 36 h, and 48 h after transfection respectively, mix well and incubate in the incubator for 3 h, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD value at 450 nm and analyze.

[0093] 2.4 Primer design

[0094] Using the mRNA sequences of porcine proliferation-related marker genes PCNA (NM_001291925.1), Ki67 (NM_001101827.1), CDK1 (NM_001159304.2), and CDK4 (NM_001123097.1) published in the NCBI database, primers were designed as follows:

[0095] Forward primer for PCNA: 5′-GTGATTCCACCACCATGTTC-3′ (SEQ ID NO:16)

[0096] Reverse primer: 5′-TGAGACGAGTCCATGCTCG-3′ (SEQ ID NO:17)

[0097] Forward primer for Ki67: 5′-ACCAGGCACAATGGATGGAAA-3′ (SEQ ID NO:18)

[0098] Reverse primer: 5′-TTCTTGCGGATTCAGGGACA-3′ (SEQ ID NO:19)

[0099] Forward primer for CDK1: 5′-GAGCGACGCTGACGTGGTA-3′ (SEQ ID NO:20)

[0100] Reverse primer: 5′-TGGATGTGGTAGATCCCAGCTT-3′ (SEQ ID NO:21)

[0101] Forward primer for CDK4: 5′-GGCCCTCAAGAGCGTAAGAG-3′ (SEQ ID NO:22)

[0102] Reverse primer: 5′-GACATCCATCAGCCGGACAA-3′ (SEQ ID NO:23)

[0103] 2.5 Real-time fluorescence quantitative PCR for detecting cell proliferation

[0104] Cells were seeded in 12-well plates and transfected when the cell density reached about 60%. The transfection method was the same as that shown in 2.1 of Example 3. After 48 h of transfection, cells in different treatment groups were collected, RNA was extracted and reverse-transcribed into cDNA, and the changes in key proliferation genes such as PCNA, Ki67, CDK1, and CDK4 were detected by real-time fluorescence quantitative PCR. The specific operation method was the same as that in 2.6 of Example 1.

[0105] 3. Results

[0106] 3.1 Overexpression efficiency of circLMO7

[0107] By constructing an overexpression vector of circLMO7, the results of real-time fluorescence quantitative PCR are as follows Figure 4 shown. Compared with the control group, the overexpression vector can significantly increase the expression level of circLMO7 in porcine skeletal muscle satellite cells, indicating that the designed and synthesized overexpression vector can effectively promote the expression of circLMO7.

[0108] 3.2 Interference efficiency of Smart Silence on circLMO7

[0109] To study the function of circLMO7, the present invention inhibits the expression of circLMO7 through siRNA-mediated interference experiments. The results of real-time fluorescence quantitative PCR are as follows Figure 5 shown. Compared with the control group, the second pair (si-2) of the three pairs of Smart Silence synthesized can significantly reduce the expression level of circLMO7 in cells, indicating that the designed and synthesized Smart Silence can effectively inhibit the expression of circLMO7.

[0110] 3.3 Effect of circLMO7 on the proliferation of porcine skeletal muscle satellite cells

[0111] The present invention detects the effect of interfering with and overexpressing circLMO7 on the proliferation of porcine skeletal muscle satellite cells through EdU, CCK-8 and real-time fluorescence quantitative PCR assays. The results are as follows Figures 6 - 11 shown. The EdU, CCK-8 and real-time fluorescence quantitative PCR assays all show that interfering with circLMO7 can significantly reduce the proliferation rate of porcine skeletal muscle satellite cells, and overexpressing circLMO7 can promote the proliferation of porcine skeletal muscle satellite cells.

[0112] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention. Sequence Listing <110> Shanxi Agricultural University, Modern Agricultural Industry Development Center, Taigu District, Jinzhong City <120> circRNA related to the proliferation of porcine skeletal muscle satellite cells and its application <130> KHP221114632.2 <160> 23 <170> SIPOSequenceListing 1.0 <210> 1 <211> 554 <212> DNA <213> Pig (Sus scrofa) <400> 1 taacgagagg agaacttggg gcaccaatgt ggagcattgg ccaagagcac aagaaacctc 60 agactcctct tgttacttgg aagaggaaga agaaaagaca agcataccca acacggtaaa 120 ggatgatctt tacgtgcgaa agctaagccc aatcatgcca agcccaggga atgcttttga 180 tcagtttctt cccaaatgct ggatcccagg agatgtgaac tggaaacgaa taaaaaggga 240 aacttacaaa ccgtggtata aagaatttca gggattcaga tcagttcagt gatatgagaa 300 tcagcataaa ccagacgcct gggaacagtc ttgactttgg gtttaccgta aaatgggctt 360 tttccgggat cttcgtagca tcagttgaag caggagccca gcagaatttt ctcagctaca 420 ggtagatgat gaaattattg ctgtcaacaa caccaagttt tcctataagg acacaaaacg 480 gtgggaggaa gccatggcta atgctcagga aaccggaaac ctggtgatgg atatcaggcg 540 ctatggaaag tctg 554 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 acacaaaacg gtgggaggaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 tgattgggct tagctttcgc 20 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <400> 4 atgccagagt ctcgttcgtt at 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <400> 5 cggacaggat tgacagattg at 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <400> 6 ctgtccacac caatagcagg t 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 tgtgcttttc ttcccagcca 20 <210> 8 <211> 21 <212> DNA / RNA <213> Artificial Sequence <400> 8 ggaaagucug uaacgagagt t 21 <210> 9 <211> 21 <212> DNA / RNA <213> Artificial Sequence <400> 9 cucucguuac agacuuucct t 21 <210> 10 <211> 21 <212> DNA / RNA <213> Artificial Sequence <400> 10 agucuguaac gagaggagat t 21 <210> 11 <211> 21 <212> DNA / RNA <213> Artificial Sequence <400> 11 ucuccucucg uuacagacut t 21 <210> 12 <211> 21 <212> DNA / RNA <213> Artificial Sequence <400> 12 auggaaaguc uguaacgagt t 21 <210> 13 <211> 21 <212> DNA / RNA <213> Artificial Sequence <400> 13 cucguuacag acuuuccaut t 21 <210> 14 <211> 21 <212> DNA / RNA <213> Artificial Sequence <400> 14 uucuccgaac gugucacgut t 21 <210> 15 <211> 21 <212> DNA / RNA <213> Artificial Sequence <400> 15 acgugacacg uucggagaat t 21 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 gtgattccac caccatgttc 20 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <400> 17 tgagacgagt ccatgctcg 19 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <400> 18 accaggcaca atggatggaa a 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <400> 19 ttcttgcgga ttcagggaca 20 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <400> 10 gagcgacgct gacgtggta 19 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <400> 21 tggatgtggt agatcccagc tt 22 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <400> 22 ggccctcaag agcgtaagag 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <400> 23 gacatccatc agccggacaa 20

Claims

1. circRNA related to the proliferation of porcine skeletal muscle satellite cells, characterized in that, The circRNA is circLMO7, and its corresponding cDNA sequence is shown in SEQ ID NO:

1. Use of a reagent for detecting the expression level of the circRNA according to claim 1 in the preparation of a detection reagent or kit for identifying the proliferation of porcine skeletal muscle satellite cells.

3. Primers for detecting the circRNA recited in claim 1, characterized in that, It includes an upstream primer shown in SEQ ID NO:2 and a downstream primer shown in SEQ ID NO:

3.

4. A kit for identifying the proliferation of porcine skeletal muscle satellite cells, characterized in that, The kit includes a reagent for detecting the expression level of circLMO7; Among them, the cDNA sequence corresponding to circLMO7 is shown in SEQ ID NO:

1.

5. The kit according to claim 4, wherein The kit includes the primers shown in SEQ ID NO:2-3.

6. The inhibitor of the circRNA according to claim 1, characterized in that, The inhibitor is the siRNA shown in SEQ ID NO:10-11.

Images