CircCOPS8 inhibits the proliferation of buffalo myoblasts by binding to IGF2BP3 and promoting tumor suppressor gene expression

doi:10.21203/rs.3.rs-3236591/v1

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Research Article

CircCOPS8 inhibits the proliferation of buffalo myoblasts by binding to IGF2BP3 and promoting tumor suppressor gene expression

https://doi.org/10.21203/rs.3.rs-3236591/v1

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Background

Buffalo is a characteristic species in Guangxi, whose meat is nutrient-dense and good for the human body because of its polyunsaturated fatty acids and conjugated linoleic acid. CircRNA is a type of non-coding RNA contributing to the growth of skeletal muscle via molecular sponges, protein scaffolds, and coded short peptides.

Results

Our prior RNA-seq results presented that the expression of circCOPS8 in the longissimus muscle of buffalo and cattle was considerably different. According to the results of western blotting and qPCR (P < 0.05), circCOPS8 improved the differentiation and suppressed the proliferation of buffalo myoblasts. However, the outcomes of the mouse muscle injury model showed that circCOPS8 hindered mice from repairing their damaged muscles. Further study on the mechanism of RIP-qPCR proved that circCOPS8 could bind to IGF2BP3 (P < 0.05). Transcriptome sequencing along with the outcomes of qPCR further demonstrated that circCOPS8 may also prevent cell growth by activating tumor suppressor genes (P < 0.05).

Conclusion

CircCOPS8 inhibits cell proliferation by binding to IGF2BP3 and promoting ATR gene expression. This study described the function and mechanism of circCOPS8, setting the theoretical groundwork for research of circRNAs regulating buffalo muscle growth.

Muscles

Noncoding RNA

Proliferation

Protein

Skeletal muscle makes up roughly 30–40% of the adult animal body weight and is an important component of animal body [1]. In the embryonic stage, mesodermal mesenchymal cells give rise to satellite cells of the skeletal muscle, which can multiply [2, 3]. Skeletal muscles lose their ability to proliferate when they fuse with muscle fibers or myotubes [2, 4, 5]. When skeletal muscles unite with muscle fibers or myotubes, they loss of the ability to proliferate. Skeletal muscle cell growth during the embryonic stage is activated by the Pax3 gene [4]. Muscle cells in mammals that express Pax7 stop going through the cell cycle and have less Pax3 gene expression. By stimulating the expression of the MyoD and Myf5 genes, the Pax7 gene subsequently causes myoblast development [6]. After differentiation, the cell membranes of myocytes dissolve and fuse with each other to form multinucleated myotubes [1, 7]. In order to create mature muscle fibers that would eventually mature into contractile muscles, myotubes produced contractile proteins [8].

CircRNAs are a non-coding RNA molecule with a closed loop structure, without a 5'cap and a 3'poly (A) structure, which made them more stable and not easily degraded [9, 10]. According to research, circRNAs can control the development of muscles by adsorbing miRNAs to sponges, serving as protein scaffolds, and competitively interacting with proteins [11, 12]. For instance, circNfix acted as a sponge for miR-204 to enhance MEF2C expression and promote myotube formation [13]. Chicken skeletal muscle satellite cells were stimulated to proliferate but were prevented from differentiating by the protein circFAM188B-103AA, which is encoded by circFAM188B [14]. CircHipk2 binds to the ribosomal protein Rpl7 and inhibits myoblastic proliferation while promoting differentiation [15].

Buffalo meat is nutrient-dense, with polyunsaturated fatty acids and conjugated linoleic acid [16]. Regular consumption of buffalo meat can lower blood lipid levels and reduce the risk of cardiovascular disease [16]. The marketability of buffalo meat is poor due to their older slaughter age [17, 18]. Our earlier RNA-seq findings (Data Number SAMN21421329) shown that the expression of circCOPS8 in the longissimus dorsi muscle of buffalo was substantially higher than that of cattle. Functionally, the findings demonstrated that circCOPS8 could prevent buffalo myoblasts from proliferating while encouraging their differentiation and death. We have demonstrated that circCOPS8 can work with IGF2BP3 to play a part in the mechanism. Buffalo myoblasts overexpressing circCOPS8 were also subjected to transcriptome sequencing and qPCR verification, which suggested that circCOPS8 may activate tumor suppressor genes and hence reduce the expression of cell proliferation marker genes. Our findings thereby advance the theoretical underpinnings of non-coding RNA regulation of muscle development and provides new insight into the mechanism by which circCOPS8 influences the development of buffalo myoblasts.

Authenticity verification and carrier construction of circCOPS8

CircCOPS8, which derives from buffalo's COPS8 (also known as COP9) on chromosome 18, has a size of 253 kb. For the production of circCOPS8, the COP9 exons 2, 3, and 4's transcripts are spliced together and cycled (Fig. 1A). On the basis of RNA-seq investigations conducted early, the expression of circCOPS8 in buffalo was much higher than that in cattle (Fig. 1B). We carried out RNase R digestion and qPCR in order to further confirm the validity of circCOPS8, and the outcomes showed that circCOPS8 really did exist (Fig. 1C). Further confirming its validity is the fact that circCOPS8 can be amplified from the buffalo longissimus dorsi cDNA processed with RNase R (Fig. 1D). The amplified circCOPS8 was compatible with the sequencing sequence, based on the findings of the bacterial liquid sequencing (Fig. 1E). Then, we constructed the circCOPS8 vector and transferred it to buffalo myoblasts. And qPCR outcomes proved that buffalo myoblasts successfully overexpressed circCOPS8(Fig. 1F-H).

CircCOPS8 inhibits myoblast proliferation

WB and qPCR results showed that the expression of CyclinD1, CDK2 and PCNA decreased significantly (Fig. 2A and B). Based on the results of cell cycle detection by flow cytometry, overexpression of circCOPS8 reduced the number of cells in the G2 and S phases and suppressed cell proliferation (Fig. 3A and B). Buffalo myoblast proliferation decreased substantially as seen by EdU and CCK-8 (Fig. 2C and D). In summary, circCOPS8 significantly lowered the growth of buffalo myoblasts.

CircCOPS8 promotes the differentiation of myoblasts

After buffalo myoblasts effectively overexpressed circCOPS8, differentiation of the myoblasts was initiated (Fig. 3B and F). The expression of cell differentiation marker genes such MyoD and MyoG increased, according to the results of qPCR and western blotting (Fig. 3D and E). In accordance with the results of cell immunofluorescence, there were more myotubes in the experimental group than in the control group (Fig. 3C). Therefore, circCOPS8 promotes bovine myoblast differentiation.

CircCOPS8 inhibits the repair of mouse muscle cells

By NCBI, the mRNA sequences of circCOPS8 and mouse COPS8 were compared, and the results showed that 86% of them were identical (Fig. 4A). In order to further explore the effect of circCOPS8 on the repair of muscle injury in mice, we constructed a muscle injury model in mice. And qPCR showed that circCOPS8 was successfully overexpressed in tibialis anterior mice (Fig. 4C). The results of qPCR and western blotting showed that the expression levels of CDK2, CyclinD1 and PCNA decreased, while the expression levels of MyoD and MyoG increased (Fig. 4D and E). This suggests that circCOSP8 can enhance the expression of a proliferation marker gene while suppressing the expression of a differentiation marker gene in a mouse muscle injury model. When mouse muscle tissue was stained with H&E on the third and seventh days, we noticed that the experimental group had more inflammatory cells than the control group, and the effectiveness of muscle fiber regeneration was poor (Fig. 4B). In conclusion, circCOPS8 prevents mice from repairing muscle damage.

Our current discoveries shown that circCOPS8 dramatically reduced myoblast proliferation in both mice and bovine tissue, as well as in mice muscle recovery. As a result, we investigated circCOPS8's impact on apoptosis in more detail. After overexpressing circCOPS8, we found that the expression of apoptotic marker genes such p53, Caspase-3, and Pdcd5 dramatically increased in buffalo myoblasts and muscle tissues of mice (Fig. 4F). This indicates that in buffalo and mouse myocytes, circCOPS8 induces apoptosis.

Quality control analysis of sequencing data

To ensure the reliability of data analysis, the reads with sequencing linker containing N bases and low sequencing quality were filtered (Fig. 5A). The result showed Q30 ≥ 93.53%, which meant good data quality (Fig. 5B). Finally identified in pCD2.1 group 44648410–47532024 clean reads; 44885006–47747422 cleans reads were identified in the circCOPS8 group (Fig. 5C). According to the comparison results, the proportions of reads in the exon region, intron region and intergenic region of the genome were counted. The results showed that the largest proportion of data was matched to exon region (86.06%), while the proportions in intron region and intergenic region were 4.25% and 13.36% (Fig. 5D).

GO and KEGG analyses

The samples exhibited a good correlation, and a total of 2293 differential genes with annotation data were found, of which 1298 were down-regulated and 995 were up-regulated (Fig. 6A-E). The differentially expressed genes were mapped to 113 GO terms, mainly including ribonucleoprotein complex assembly (GO:0022618), immune response process (GO:0002252), and antigen processing and presentation (GO:0019882) (Fig. 6F and G). To further reveal the biological functions of these differentially expressed genes, we performed KEGG analysis. The results showed that the differentially expressed genes were mainly enriched in the ribosome (bbub03010) and oxidative phosphorylation (bbub00190) pathways (Fig. 6H and I). Among them, the pathways related to cell proliferation and differentiation included cell senescence (bbub04218), cell cycle (bbub04110), DNA replication (bbub03030), longevity regulation (bbub04211) and apoptosis (bbub04215).

Mechanisms of circCOPS8

CircCOPS8 may function as a molecular sponge, a protein scaffold, and a coded short peptide because RNA-FISH studies indicate that it is localized in the cytoplasm. First of all, we compared the miRNA database, and the results showed that circCOPS8 had almost no bound miRNA. By using OFR Finder, we were able to anticipate its ORF region, and the results showed that it did[19]. In order to further forecast the IRES locus of circCOPS8, the IRESite website was utilized[20]. It was discovered that the confidence in the finding was not high, meaning that circCOPS8 likely had no IRES locus (Supplement file, Table S10). As a result, we went one step further and predicted the circCOPS8 m6A modification site using the SRAMP website (Supplement file, Table S9)[21]. The results showed that circCOPS8 contained two potent m6A binding sites (Fig. 7A and B). Comparing the amino acid sequence encoded by circCOPS8 with the protein database-NMDC[22], it was found that the amino acid sequence of circCOPS8 was highly similar to that of 6R6H-HY and other proteins, which were all components of the COP9 signal complex (Fig. 7C). Then circCOPS8 may encode a small peptide through m6A modification site, and its function is similar to that of protein encoded by its source gene COP9.

Two websites, RBPsuite and catRAPID (Supplement file, Table S7 and S8), were used to predict the proteins that circCOPS8 might bind to, and the intersection was taken and the histogram was drawn (Fig. 7D)[20, 23]. Since IGF2BP3 is related to cell proliferation and m6A, it is examined whether it binds to circCOPS8. RIP-qPCR results showed that circCOPS8 combined with IGF2BP3 (Fig. 7E). The upstream pathway genes of proliferation marker genes were found through KEGG website and compared with the differential genes of transcriptome[24], and the gene action pathway map was drawn. By qPCR verification of the genes in the mechanism map, the differential genes consistent with the transcriptome sequencing results were drawn into the final gene pathway map (Fig. 7F and G). The results show that circCOPS8 can activate tumor suppressor genes to inhibit the expression of cell proliferation-related genes, such as ATR can simultaneously inhibit the expression of CCND1 and CDK2/4, and PTEN can inhibit the expression of PCNA and CCND1.

COP9 signalsome subunit 8 (COPS8) is one of the eight subunits of the COP9 signalsome (CSN), a key multifunctional evolutionarily conserved protein complex in cells and essential in plants and animals. Thus, circCOPS8 derived from this gene may play an important role in the cell cycle. In buffalo muscle cells and mouse muscle injury models, we discovered from WB and other experiments that circCOPS8 improved the expression of differentiation marker genes while inhibiting the expression of proliferation markers. CircCOPS8 prevented mice from healing muscle damage, according to the results of H&E section staining. Therefore, we further explored the effect of circCOPS8 on apoptosis, and the results showed that circCOPS8 significantly promoted the expression of apoptosis marker genes. The function of circRNAs varies depending on where it is located in the cell. CircRNAs mainly function in the nucleus by controlling gene transcription. By adsorbing proteins, miRNA, or small peptides that code for genes, circRNAs may operate in the cytoplasm. Due to the fact that circCOPS8 is found in the cytoplasm, we have predicted its potential mode of operation through a number of sources. The findings suggested that via adsorbing protein or m6A binding sites, circCOPS8 may encode a small peptide. CircCOPS8 binding proteins can be divided into three categories, such as DGCR8, AGO and other proteins involved in the synthesis and splicing of miRNAs [25, 26]. Proteins such as FIF4A3 and TIAIL1 are linked to the death of cancer cells, while proteins such as FUS are involved in gene repair [27, 28]. IGF2BP3 has an impact on cell proliferation and has a role in muscle development. Results from RIP-qPCR shown that circCOPS8 may bind to IGF2BP3 and prevent cell growth. Second, the small peptide that circCOPS8 may encode may play a part in the creation of the COPS9 signal complex. We identified a route of circCOPS8 suppressing cell growth using transcriptome data together with qPCR confirmation. The findings demonstrated that circCOPS8 suppressed the expression of cell proliferation markers by activating tumor suppressor genes. For instance, PTEN suppresses CDK4 and CCND1 expression while ATR inhibits CDK2/4 and CCND1 expression. The ATR gene is a key gene that stops the cell cycle, while the PTEN gene regulates the synthesis of a specific protein[29–33]. The protein acts as a tumour suppressor, regulating the cell division cycle by preventing cells from growing too quickly or dividing uncontrollably.

Buffalo has better disease resistance than cattle, which have virtually no circCOPS8 expression in the longissimus dorsi muscle. Our results demonstrate that co-expression of circCOPS8 and IGF2BP3 inhibits cell proliferation. By promoting the expression of tumor suppressor genes, circCOPS8 simultaneously prevents the production of genes relevant to the cell cycle. Hence, in order for buffalo to have a consistent and balanced resistance to illness, does circCOPS8 play a significant role in the regulation of disease resistance? This may require further exploration. In conclusion, we explored the function and mechanism of circCOPS8, which provided a theoretical basis for the research on the related function and mechanism of circRNAs regulating buffalo muscle development.

Synthesis of cDNA and real-time qPCR

TRIzol reagent to tissue cells was added to extract total RNA (Life, California, USA). 1µg of total RNA was added with 1–3µL of 20U/µL RNase R (Geneseed, Guangzhou, China), incubated at 37°C for 60 min, and RNA was converted into cDNA by using HiScript III reverse transcription kit (Vazyme, Nanjing, China). Real-time PCR (Vazyme, Nanjing, China) was performed using AceQ qPCR SYBR Master Mix on an ABI 7500 machine (Thermo, MA, USA). The primers are shown in Table S1.

Construction vector

The vector of plasmid pCD2.1-circCOPS8 was constructed. The cloned circCOPS8 sequence was ligated into pCD2.1-ciR vector (Geneseed, Guangzhou, China) through Kpn I and Bam HI restriction sites. The primers are shown in Table S2.

Cell isolation and culture

The longissimus dorsi muscle (Native buffalo in Guangxi, about 90 days old) of buffalo fetuses were collected and the primary muscle cells were isolated. The skin on the calf's back was cut to remove fascia, intramuscular fat and connective tissue. Muscles were cut off and put into a sterile Petri dish filled with PBS. The tissues were washed with ultrapure water, PBS (2% penicillin/streptomycin, Gibco, MA, USA), 75% alcohol, PBS (1% penicillin/streptomycin, Gibco, MA, USA) in turn, and then put into a sterile culture dish. A proper amount of PBS (1% penicillin/streptomycin, Gibco, MA, USA) was added into the culture dish and the tissues were cut into soybean size with sterile ophthalmic scissors. The tissues were transferred into a 15mL centrifuge tube and stood at constant temperature for 3min. Collagenase I (Sigma, Missouri, USA) was added after the supernatant was discarded and double the volume had been added. The tissues were equally blown, then placed on a shaking table to digest for 1h (37°C, 2000rpm). The tissue was centrifuged for 5min at a speed of 1500rpm. The supernatant was discarded, and the mixture was then properly digested in a shaking table with 0.25% trypsin (37°C, 20min). Complete medium was added to stop digestion. The cell fluid was transferred to a new 50 mL centrifuge tube in an ultra-clean station, and filtered twice (1500r/rpm, 6min) with a 100 µm cell filter (Gibco, MA, USA). The supernatant was discarded and DMEM containing 20% fetal bovine serum (Gibco, MA, USA) and 1% penicillin/streptomycin (Gibco, MA, USA) was added. After culturing in a cell incubator for 2h (37°C, 5% CO₂), the liquid was transferred from the cell culture dish to a new culture dish. This step was repeated twice. When the amount of myoblast fusion reached 80%, DMEM (Gibco, MA, USA) containing 2% horse serum was used instead of the previous culture medium to induce cell differentiation.

Fluorescence in situ hybridization.

Fluorescence in situ hybridization experiments were performed with in situ hybridization reagents (Servicebio, Wuhan, China) according to the manufacturer's instructions. Cell-climbing slices of buffalo myoblasts were fabricated and the cells were cultured at room temperature for 30min with FISH fixative. The cells were penetrated with 0.2% Triton X-100 PBS (room temperature, 5min), and then cultured with pre-hybridization solution (37°C, 1h). The fixed cells were added to the fluorescently labeled hybridization solution of the circCOPS8 probe and incubated in the dark at 4℃ overnight. The next day, the cells were washed 3–5 times with PBS, DAPI solution was added, and the cells were left at room temperature for 5min (dark and moist). Finally, the cells were observed by the fluorescence microscope (Nikon, Tokyo, Japan) and fluorescence photos were taken.

CCK-8, EdU and cell cycle assay

CCK-8, EdU and cell cycle testing were all carried out using kits. The cells were treated according to the instructions of CCK-8 kit (Vazyme, Nanjing, China), and the absorbance was detected at 450nm by microplate reader. According to the instructions of Apollo 567 (Ribobio, Guangzhou, China) of EdU, the proliferation of bovine skeletal muscle cells was detected, and the cells were observed by the fluorescence microscope (Nikon, Tokyo, Japan) and fluorescence photos were taken. All the above experiments were performed according to the manufacturer's instructions.

Western blotting

1% PMSF RIPA lysate buffer (Solarbio, Beijing, China) was added into cell tissue to extract protein. The BCA kit (Solarbio, Beijing, China) was used to determine the protein concentration. 10% SDS-PAGE (Bio-RAD, California, USA) electrophoresis was used to isolate proteins. The protein was transferred to a 0.22 µm nitrocellulose membrane (PALL, New York, USA) by a membrane transfer apparatus. The binding protein bound to primary antibodies (ABclonal, Wuhan, China) specific for CyclinD1, CDK2, PCNA, MyoG, MyoD and β-actin. The membrane was washed with TBS and TBST, and the second antibody (ABclonal, Wuhan, China) was incubated for 1h at room temperature. ECL Plus chromogenic solution (Solarbio, Beijing, China) was dripped on the film, and the image was captured by ChemiDoc XRS + system (Bio-Rad, California, USA).

Establishment of skeletal muscle injury model

The experimental mice were SPF grade Kunming mice (n = 12, 4–5 weeks old, purchased from the Experimental Center of Guangxi Medical University). The anterior tibial muscle of mice was injected with 50µL myocardial toxin solution (CTX) 48h later, the tissues were taken for frozen section and H&E staining was used to detect the muscle injury. After the creation of effective muscle damage models, mice were injected with the pCD2.1-circCOPS8 plasmid and utilized as the experimental group for in vivo transfection reagent complex (Engreen Bio-system Co., Ltd.) in the left tibialis anterior muscle. The in vivo transfection reagent combination (Engreen Bio-system Co., Ltd.) and pCD2.1 plasmid were injected into the mouse right anterior tibial muscle, which served as the control group. In total, there were three injections, 12, 48 and 96h after the CTX induced muscle injury according to the three injections. After 144h of CTX treatment, RNA was extracted from the tibialis anterior and reverse transcribed. The research protocol and animal care procedures were approved by the Animal Care Committee of the College of Animal Science and Technology of Guangxi University (GXU-2022-309).

H&E staining and tissue immunofluorescence

The collected tibialis anterior samples were subjected to H&E staining and tissue immunofluorescence. The tissue was fixed with a 4% neutral formaldehyde fixative for 24h and subjected to gradient dehydration. The tissues were trimmed into 0.5cm³ cubes and immersed in 1:1 mixture of OCT embedding medium (SAKURA, Japan) and 30% sucrose at room temperature for 2h. The tissues were then transferred to new OCT embedding medium and immersed at room temperature for 6h. The tissues, together with the OCT embedding medium, were frozen and sectioned with a microtome to a thickness of 510µm. The sections were incubated with 0.5% Triton X at room temperature for 20 min and blocked with 10% BSA at room temperature for 1h. The sections were incubated overnight at 4℃ with the addition of primary antibodies, CyclinD1, PCNA, MyoD and MyHc (ABclonal, Wuhan, China). The tissue sections were washed with PBS three times and the secondary antibodies were incubated at room temperature for 60 min. The sections were incubated for 15 min in a 0.1% DAPI solution, followed by 3 washings with PBS. Finally, the fluorescence signals were observed under a fluorescence microscope (Leica, Wetzlar, Germany). The average fluorescence intensity of the protein (n = 7) was analyzed and counted using Image J software. The data in the figures are expressed as mean ± SEM.

RIP

Buffalo myoblasts that have been pCD2.1-circCOPS8 and pCD2.1-ciR plasmid transfected. The experiment was completed in accordance with the kit's instructions (Geneseed, Guangzhou, China). The eluted and purified RNA was analyzed by qPCR.

Statistical analysis

The difference between the two groups was statistically significant using the multi-t test (FDR < 0.01) of plate prism v6.01 software. P < 0.05 was considered significant and P < 0.01 was extremely significant.

Transcriptome sequencing

Preparation of sequencing samples

Purification of total RNA: Two groups of buffalo myoblast samples, including the experimental group transfected with the pCD2.1-circCOPS8 plasmid and the blank group transfected with the pCD2.1-ciR plasmid, with three biological replicates in each group. RNA is extracted from tissues or cells. The completeness and total amount of extracted RNA are measured by the Agilent 2100 bioanalyzer.

Library construction and quality inspection

The database was constructed using total RNA as the initial RNA. After mRNA with poly A tail was enriched with Oligo(dT) magnetic beads, the database was constructed according to the general database construction method of NEB or the chain-specific database construction method. After the completion of library construction, the library was diluted to 1.5ng/µl using Qubit 2.0 Fluorometer for preliminary quantification, and then the insert size of the library was detected by Agilent 2100 bioanalyzer. After the insert size met the expectation, the effective concentration of the library was accurately quantified by qRT-PCR (the effective concentration of the library was higher than 2nM) to ensure the quality of the library.

Computer sequencing

After library qualification, different libraries were sequenced by Illumina according to the requirements of effective concentration and target out-of-machine data volume. Four fluorescently labeled dNTP, DNA polymerase and adapter primers were added to the sequencing flow cell for amplification. When each sequencing cluster extends the complementary strand, the corresponding fluorescence can be released by adding a fluorescently labeled dNTP. The sequence information of the segment to be measured can be obtained by the sequencer by capturing the fluorescence signal and converting the optical signal to the sequenced peak via computer software.

Data quality control

The sequenced fragments were converted into sequence data (reads) by base recognition of CASAVA from the image data obtained by high-throughput sequencing instrument. The raw data obtained by sequencing contains a small number of reads with sequencing linkers or low sequencing quality. To ensure the quality and reliability of the data analysis, the raw data needs to be filtered. It mainly includes the removal of reads with adapter, the removal of reads containing N (N indicates that the base information cannot be determined), and the removal of low-quality reads (reads in which the number of bases of Qphred ≤ 20 accounts for more than 50% of the entire read length). The Q20, Q30, and GC contents of the clean data are calculated. All subsequent analyses were based on high-quality analysis performed by clean data.

Sequence assembly and expression evaluation of transcripts

The reference genome and gene model annotations are downloaded. The reference genome was indexed using HISAT2 v2.0.5 and paired terminal clean reads were compared to the reference genome using HISAT2 v2.0.5. The FPKM is calculated for each gene using feature counts.

Identification of differential genes and functional annotations

The difference expression between the two comparative combinations was analyzed using DESeq2 software (1.20.0). The resulting p-values were adjusted using the method of Benjamini and Hochberg to control the false detection rate. Genes with P values < 0.05 were marked as significantly different by DESeq2. The p-values were adjusted using the Benjamini & Hochberg method. The corrected p-value and |log2foldchange| are used as thresholds for the saliency difference expression.

Differential gene enrichment analysis

GO and KEGG enrichment analysis of differentially expressed genes were implemented by the clusterProfiler R package (3.4.4).

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

CircRNA

Circular RNA

MiRNA

MicroRNA

RNA-seq

RNA Sequencing

qPCR

Quantitative Polymerase Chain Reaction

MRFs

Myogenic Factors 5

Myf6

Myogenic Factors 6

MyoD

Myoblast Determination Protein

MyoG

Myogenin

Pax3

Paired Box 3

Pax7

Paired Box 7

SiRNA

Short Interfering RNA

CeRNA

Competing Endogenous RNA

RNA-FISH

RNA-fluorescence in Situ Hybridization

BSA

Albumin from Bovine Serum

Western Blot

IRES

Internal Ribosome Entry Site

IGFs

Insulin-like Growth Factor

RBP

RNA Binding Protein

PCNA

Proliferating Cell Nuclear Antigen

CDK2

Cyclin Dependent Kinase 2

ATM

Ataxia Telangiectasia-mutated

Map3k20

Protein Kinase Kinase Kinase 20

Supporting Information

The online version contains supplementary material available at https://orcid.org/my-orcid?orcid=0000-0002-5604-0592

Acknowledgements

Not applicable.

Authors’ contributions

P.S, Y.D., M.S. and W.Q. did major experiments. M.C. and P.S. were responsible for writing the manuscript. X.Q., P.S., and M.J. were in charge of various experiments and data analysis. H.L., D.S., Q.L. and J.W. built the main experimental framework. M.C., L.Z., and Y.H. were in charge of refining and polishing the study.

Fundings

This work was supported by grants from the National Natural Science Foundation of China (32102514, 31972996 and U20A2051), the Guangxi Natural Science Foundation (2020GXNSFBA297148), the Guangxi Science and Technology Major Project (GuiKe AA22068099), the Bama County Program for Talents in Science and Technology, Guangxi, China (202101265 and 202100174), and the State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources (SKLCUSA-a202203). We are also grateful to the supported of Guangxi Bagui Scholar Program.

Availability of data and materials

All data are available upon request from the corresponding author.

Ethics approval and consent to participate

All of the procedures described in this study, including animal experiments, were approved by the animal experimental ethics committee of Guangxi University (No: GXU-2022-309). Our experiment is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

Consent for publication

Not applicable.

Competing interests

All authors declare that they have no competing interests.

Authors’ details

¹State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi Key Laboratory of Animal Breeding and Disease Control, College of Animal Science and Technology, Guangxi University, Nanning, China.²Guangdong Provincial Key Laboratory of Animal Molecular Design and Precise Breeding, School of Life Science and Engineering, Foshan University, Foshan, China.

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No competing interests reported.

Fig.S1CircCOPS8sequenceinthebovinegenome.jpg
Figure S1. CircCOPS8 sequence in the bovine genome
Fig.S2RNAFISHprobeprimer.jpg
Figure S2. RNA-FISH probe primer.
TableS1ProbesequencesofqPCR.xlsx
Table S1. Probe sequences of qPCR.
TableS2Primersforvectorconstruction.xlsx
Table S2. Primers for vector construction.
TableS3Transcriptomesequencingqualitycontroldata.xlsx
Table S3. Transcriptome sequencing quality control data.
TableS4Upregulatedanddownregulatedgenes.xlsx
Table S4. Up-regulated and down-regulated genes.
TableS5GOenrichmentanalysis.xlsx
Table S5. GO enrichment analysis.
TableS6KEGGenrichmentanalysis.xlsx
Table S6. KEGG enrichment analysis.
TableS7BindingProteinpredictionCatRAPID.xlsx
Table S7. Binding Protein prediction-CatRAPID.
TableS8BindingProteinpredictionRBPsuite.xlsx
Table S8. Binding Protein prediction-RBPsuite.
TableS9Predictionofm6Abindingsites.xlsx
Table S9. Prediction of m6A binding sites.
TableS10PredictionofIRESlocus.xlsx
Table S10. Prediction of IRES locus.
SupplementarydocumentofWesternBlottinggelelectrophoresismap.pdf
Table S11. Supplementary document of Western Blotting gel electrophoresis map.

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CircCOPS8 inhibits the proliferation of buffalo myoblasts by binding to IGF2BP3 and promoting tumor suppressor gene expression

Status:

Version 1

Abstract

Background

Results

Conclusion

Figures

Introduction

Results

Authenticity verification and carrier construction of circCOPS8

CircCOPS8 inhibits myoblast proliferation

CircCOPS8 promotes the differentiation of myoblasts

CircCOPS8 inhibits the repair of mouse muscle cells

Quality control analysis of sequencing data

GO and KEGG analyses

Mechanisms of circCOPS8

Discussion

Materials and methods

Synthesis of cDNA and real-time qPCR

Construction vector

Cell isolation and culture

CCK-8, EdU and cell cycle assay

Western blotting

Establishment of skeletal muscle injury model

H&E staining and tissue immunofluorescence

RIP

Statistical analysis

Transcriptome sequencing

Preparation of sequencing samples

Library construction and quality inspection

Computer sequencing

Data quality control

Sequence assembly and expression evaluation of transcripts

Identification of differential genes and functional annotations

Differential gene enrichment analysis

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1