CircACLY regulates proliferation, differentiation, and apoptosis of Jingyuan chicken myoblasts by sponging gga-miR-6660-3P

Abstract

Circular RNAs (circRNAs) are key gene regulators that are involved in many fundamental biological processes. Skeletal muscle cannot develop normally without the involvement of circRNA. While circRNA plays a pivotal role in skeletal muscle development, its functions and mechanisms within the muscle development of local chicken breeds remain to be thoroughly elucidated. This study, using the Jingyuan chicken as a model, investigated the regulatory role of circACLY in skeletal muscle development and inosine monophosphate (IMP) synthesis. Experiments verified that circACLY is a circRNA that is mainly localized to the cytoplasm. Bioinformatics analysis and experimental validation demonstrated that circACLY is a target of gga-miR-6660-3p, which in turn regulates the expression of Ectonucleotide Triphosphate Diphosphohydrolase 7 (ENTPD7). Functional assays demonstrated that gga-miR-6660-3p inhibits myoblast proliferation and differentiation capacity while suppressing the de novo IMP synthesis pathway. However, circACLY reverses the inhibitory effects of gga-miR-6660-3p on these processes. This study systematically elucidates the regulatory mechanism of the circACLY/gga-miR-6660-3p/ENTPD7 axis in the development of quail myoblasts and IMP synthesis, providing a theoretical basis for deepening the understanding of the molecular basis of skeletal muscle development in local chicken breeds and the genetic improvement of meat quality traits.

A circular RNA, circACLY, promotes healthy muscle development by trapping an antagonistic RNA molecule that blocks growth, offering new pathways to enhance muscle health and combat muscle-related disorders.

Introduction

The Jingyuan chicken, also known as the Jingning chicken, was originally named the Guyuan chicken. As an indigenous breed of the Loess Plateau, it exhibits exceptional adaptability to high-altitude, cold, and arid climates, along with multiple advantages such as strong disease resistance, tolerance for coarse feed, and suitability for grazing (Huang et al 2022). As one of China’s protected livestock and poultry breeds, the conservation and research of Jingyuan Chicken germplasm resources are crucial for maintaining avian genetic diversity in the country (Yu et al 2023). Additionally, the Jingyuan chicken is renowned for its outstanding meat production performance, delicious meat quality, low cholesterol content, and rich polyunsaturated fatty acids with special nutritional value for humans (Wang et al 2022), making it an excellent dual-purpose breed for both eggs and meat. With the rapid advancement of science and technology, omics sequencing techniques are increasingly applied in genetic breeding research for cattle and poultry (Beauclercq et al 2022). Differentially methylated genes (DMGs) in breast and leg muscles of 180-d-old Jingyuan chickens were identified using MeRIP-seq and RNA-seq, combined with weighted gene co-expression network analysis (WGCNA) to identify key DMGs that regulate muscle development (Zhang et al 2024). Furthermore, integrating transcriptomic and metabolomic analyses with WGCNA explored key regulatory genes and metabolites governing dynamic changes in meat quality during the growth process of Jingyuan chickens at 42, 126, and 180 d of age (Zhao et al 2025). Transcriptomics and non-targeted metabolomics technologies were employed to identify differentially expressed genes (DEGs) and metabolites (DEMs) in 180-d-old Jingyuan chickens meat quality influenced by adding different doses of fresh corn extract (FCE) (CON, 0.3% FCE, 0.6% FCE, and 0.9% FCE) (Tian et al 2025). These omics studies hold significant practical value for systematically elucidating the growth and development mechanisms of Jingyuan chicken muscle, as well as for the breeding of high-quality broilers and the conservation and utilization of germplasm resources.

Skeletal muscle supports the normal life activities of livestock and poultry and is important source of human nutrition. Poultry is an essential short-cycle, inexpensive source of high-quality protein (Godfray et al 2010; Toomer et al 2019). The development of skeletal muscle in poultry is regulated by various factors, including genetics, nutrition, disease, and environment, and genetic selection, and breeding is an effective long-term solution to skeletal muscle growth and development in poultry (Flisar et al 2014). The genetic factors controlling skeletal muscle development include genes and non-coding (nc) RNAs.

Circular RNAs (circRNAs) do not have 3′ poly(A) and 5′ caps like linear RNAs; they are abundantly and stably expressed in eukaryotic tissues or cells and are resistant to RNA exonucleases such as RNase R (Jeck et al 2013; Ashwal-Fluss et al 2014). Circular endogenous ncRNAs are highly conserved (Jeck et al 2013; Ashwal-Fluss et al 2014). They do not have free 3′ and 5′ ends and cannot be detected by molecular techniques that rely on poly(A) (Ashwal-Fluss et al 2014). Cyclized exons can be back-spliced to form a covalent loop, which is different from classical linear splicing (Ashwal-Fluss et al 2014). Early mapping algorithms for transcriptome analysis could not directly assemble sequenced fragments to the genome; thus circRNAs were considered as byproducts of aberrantly spliced linear transcripts (Ashwal-Fluss et al 2014). Later findings showed that aberrant circRNA splicing is strictly regulated (Ashwal-Fluss et al 2014). Along with the application of high-throughput sequencing technology, numerous circRNAs have been identified that are involved in the development and differentiation of skeletal muscle in pigs (Wang et al 2019), cattle (Liu et al 2020), sheep (Cao et al 2018), and chickens (Shen et al 2019).

Myoblast proliferation, differentiation and apoptosis are fundamental to poultry growth and development, but these processes are influenced by a complex set of factors that depend on the precise expression of genes, cytokines, and ncRNAs in time and space (Ouyang et al 2018; Shen et al 2019). Ectonucleoside triphosphate diphosphohydrolase 7 (ENTPD7) hydrolyzes nucleotides and is involved in important regulatory processes in the purine metabolic pathway; it plays key roles in oxidative stress, DNA damage and cellular senescence (Seo et al 2014; Tordella et al 2016; Tani et al 2021). One of the most abundant classes of metabolites in animal cells comprises purines, which are the most basic intracellular survival substances. In addition to synthesizing genetic DNA and RNA, purine nucleotides are also a source of cellular energy, transducers of intracellular signals, cofactors for nicotinamide adenine dinucleotide (NADH) and coenzyme A, and participate in cell proliferation, differentiation and apoptosis (Chan et al 2015; Pedley and Benkovic 2017; Yin et al 2018). The roles of ncRNAs in poultry myocytogenesis are prominent during the development of complex organisms. A study of skeletal muscle development in chick embryos of different ages has shown that circRBFOX2S interacts with miR-206 to promote cell proliferation (Ouyang et al 2018). Circular FGFR2 produced by exons 3 to 6 of fibroblast growth factor receptor 2 (FGFR2), is differentially expressed in chicken embryonic skeletal muscle development and regulates myoblast proliferation and differentiation by sponging miR-133a-5p and miR-29b-1-5p (Chen et al 2019). Knockdown of circTMTC1 from the transmembrane O-mannosyltransferase targeting cadherins 1 (TMTC1) gene accelerates the proliferation and differentiation of chicken skeletal muscle satellite cells (Shen et al 2019). MicroRNA-128-3p targets the myostatin (MSTN) gene, inhibits its expression, and promotes the differentiation of chicken bone marrow mesenchymal stem cells, whereas circTMTC1 inhibits the differentiation of chicken embryonic stem cells by adsorbing miR-128-3p (Shen et al 2019).

Our previous high-throughput sequencing findings revealed that exons 2 and 3 of the chicken ATP citrate lyase (ACLY) gene in Jingyuan chickens expresses a differentially expressed circular RNA that we termed circACLY. We predicted that gga-miR-6660-3p has potential binding sites for circACLY and ENTPD7. The present study aimed to determine the role of gga-miR-6660-3p with respect to circACLY and ENTPD7, as well as the regulatory mechanisms of gga-miR-6660-3p and circACLY in skeletal muscle development.

Materials and methods

Ethics statement

This study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). This study proceeded according to the principles of the Basel Declaration and recommendations of the Statute on the Administration of Laboratory Animals, and the Ningxia University Institutional Animal Care and Use Committee approved the protocol (1 November 2020; approval ID: NXUC3127859).

Primers

All primers were designed using Premier Primer 5.0 software (Premier Biosoft International, Palo Alto, CA, USA) and commercially synthesized (Zhongke Yutong Biotech. Co., Ltd., Xian, China). Tables S1 and S2 show details of the primers.

Animal samples collection

The 180-d-old Jingyuan chickens used in this study were procured from the Jingyuan Chicken National Breeding Farm, Pengyang County, Guyuan City, Ningxia Hui Autonomous Region, China. After 12 h of fasting, the chickens were slaughtered, and leg muscle tissues were promptly collected, and the samples were quickly packed into freezing tubes and placed in liquid nitrogen for rapid freezing at 80 °C until RNA extraction.

RNA exaction, cDNA synthesis, polymerase chain reaction (PCR), and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from leg muscle and myoblasts of Jingyuan chickens by the Trizol method (Takara, Japan), and total RNA integrity was detected by 1% agarose gel electrophoresis, then optical density (OD) was measured at a ratio of 260/280 using a NanoDrop One/OneC Ultra-micro UV-Visible Spectrophotometer (Thermo-Fisher Scientific Inc., Waltham, MA, USA). Reverse transcription was performed using the HiScript II 1st Strand cDNA Synthesis Kit (Novozymes, USA). Specifically, mRNA was reverse transcribed using the kit’s Oligo(dT)23VN primer, while circRNA was reverse transcribed using the random hexamer primers provided in the same kit. For miRNA, reverse transcription was carried out using specific primers for gga-miR-6660-3p and neck-loop primers (Zhongke Yutong Biotech. Co., Ltd.), instead of the Oligo(dT)23VN. The reaction system was 20 μL, Total RNA 2 μL, 5×gDNA wiper Mix 2 μL, RNase-free ddH2O 6 μL, mixed by gently pounding with a pipette and placed in a water bath at 42 °C for 2 min, followed by the addition of 10×RT Mix 2 μL, Hiscript Ш Enzyme Mix 2 μL, Oligo(dT)20VN 1 μL, Random hexamers 1 μL, RNase-free ddH2O 4 μL, mixed well and then put in PCR (polymerase chain reaction) amplifier (Bio-rad, USA) at 37 °C for 15 min and 85 °C for 15 s to obtain cDNA products. Based on the mRNA sequences of the gene and GAPDH provided by the NCBI database (https://pubmed.ncbi.nlm.nih.gov/), the gene primers were designed using Primer Premier 5.0 software (Premier Biosoft International, USA) and synthesized by Zhongke Yutong Technology Service Company Limited. The primer sequences are shown in Tables S1 and S2. In this study, a Bio-Rad CFX96 Real-Time Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used to perform RT-qPCR experiments with a total amplification system of 20 μL. Among them, Bester SYBR Green qPCR Master Mix (Accurate, China) 10.0 μL, PCR Forward Primer (10.0 μmol/liter) 1.0 μL, PCR Reverse Primer (10.0 μmol/liter) 1.0 μL, cDNA template 1.0 μL, and RNase Free H2O 7.0 μL. Amplification procedure: 95 °C for 30 s, 95 °C for 10 s, setting annealing and extension temperatures based on the Tm value of the primer (Annealing and extension temperatures are listed in Table S1), 40 cycles, each sample was repeated three times and the relative amount of mRNA in each sample was determined using 2^−ΔΔCt.

Validation and subcellular localization of circACLY

Convergent and divergent primers were designed to validate circACLY and confirm its 3’ and 5’ junction sites based on the NCBI reference sequences of ACLY (NCBI Reference Sequence: NM_001030540.2). We used cDNA from Jingyuan chicken myoblasts for PCR using 2×Phanta Flash Master Mix (Dye Plus) (Vazyme, Nanjing, China). All sequenced PCR products (Zhongke Yutong Biotech. Co., Ltd.) were analyzed using DNASTAR 7.1 (DNASTAR, Madison, WI, USA). Total RNA and control RNA were incubated for 30 min at 37 °C with 2.5 μg of RNase R (Geneseed, Guangzhou, China), then their components were resolved by 1% agarose gel electrophoresis and used to synthesize cDNA. We investigated the subcellular localization of circACLY We used Cytoplasmic & Nuclear RNA Purification Kits (Norgen Biotek Corp., Thorold, ON, Canada) to isolate myoblast nuclear and cytoplasmic RNAs. These were resolved by 1% agarose gel electrophoresis and used to synthesize cDNA.

Cell culture and transfection

Chicken primary myoblasts were isolated from leg muscles of E11 Jingyuan chicken embryos (Animal Genetic Breeding and Reproduction Laboratory, Ningxia, China) as follows. Briefly, the skin and bone were removed from legs, then muscle tissues were rinsed in PBS, minced into ∼1-mm sections, then digested with collagenase I (Gibco, Grand Island, NY, USA) at 37 °C for 25 min, with shaking every 2 min. Digestion was terminated by adding equal volumes of DMEM/F-12 complete medium (Sartorius AG., Göttingen, Germany) containing 20% FBS and 0.2% penicillin/streptomycin (Gibco, Grand Island, NY, USA). Cell detach solution was filtered through a nylon mesh with 70 μm and 40 μm pores (Greiner GmbH., Pleidelsheim, Germany). The filtered cells were centrifuged at 1,500 rpm for 5 min and maintained in DMEM/F-12 complete medium at 37 °C under a humidified 5% CO₂ atmosphere. We cultured 293 T cells (Fenghui Bio, Hunan, China) in high-glucose DMEM medium (Sartorius AG.) supplemented with 10% fetal bovine serum (FBS) and 0.2% penicillin/streptomycin (Gibco, Grand Island, NY, USA). The growth medium was replaced with differentiation medium (2% horse serum and 0.2% penicillin/streptomycin) when the myoblasts reached 90% confluence. Plasmids harboring DNA, miRNA mimic, mimic negative control (mimic NC), miRNA inhibitors, inhibitor negative control (inhibitor NC) were transiently transfected into cells using the Zeta Life Advanced DNA RNA transfection reagent (Zeta Life, Menlo Park, CA, USA).

Vector construction and synthesis of RNA oligonucleotides

Exons 2 and 3 of the ACLY gene were amplified using EcoRI and BamHI enzymatic primers to construct a circACLY overexpression vector, then target fragments were cloned into the pCD25-ciR cyclization vector (Geneseed Biotech, Guangzhou, China). The gga-miR-6660-3p mimic, mimic NC, gga-miR-6660-3p inhibitor and inhibitor NC were synthesized (RiboBio Co., Ltd., Guangzhou, China). Table S2 shows the sequence information. We constructed the psiCHECK-2 Dual-Luciferase reporter vector by amplifying wild-type sequences in the 3’UTR of ENTPD7 and part of the region in circACLY that includes the predicted binding sites for gga-miR-6660-3p and inserted them into NotI and XhoI restriction sites in psiCHECK-2 vectors (Promega, Madison, WI, USA), as described by the manufacturer. Synthesized mutant sequences (Zhongke Yutong Co. Ltd.) were also inserted into psiCHECK-2 vector.

5-Ethynyl-2′-deoxyuridine (EdU) assays

We assayed EdU using BeyoClick EdU Cell Proliferation Kits with Alexa Fluor 594, Immunol Staining Fix Solution, QuickBlock Blocking Buffer for Immunol Staining and Enhanced Immunostaining Permeabilization Solution (all from Beyotime Biotechnology, Jiangsu, China) as described by the manufacturer. After transfection for 48 h, myoblasts and controls were incubated with 10 µM EdU for 2 h at 37 °C in 6-well plates. The cells were washed twice and stained with click reaction solution. Myoblasts that stained positive for EdU were counted using an IXplore Standard fluorescent microscope (100×magnification) (Olympus Optical Co., Ltd., Tokyo, Japan). The myoblast proliferation rate ratio was determined as the ratio of EDU- to Hoechst 33342-stained cells.

Cell Counting Kit 8 (CCK-8) assays

Myoblasts seeded in 96-well plates were incubated in DMEM/F-12 complete medium, and transfected. Cell proliferation was determined after 48 h using Enhanced Cell Counting Kit-8 (Beyotime, Jiangsu, China) as described by the manufacturer. Myoblasts were incubated with CCK-8 solution (10 μL) in darkness for 4 h at 37 ◦C under a 5% CO₂ atmosphere, then absorbance was measured using a Fluorescence/Multi-Detection Microplate Reader (Bio-Rad Laboratories Inc.) at a wavelength of 450 nm.

Targeted relationship prediction and dual-luciferase reporter assay

We predicted relationships between target genes and miR-6660-3p using miRDB (http://mirdb.org/miRDB/), TargetScan (http://www.targetscan.org/vert_72/), and RNAhybrid (http://bibiserv2.cebitec.uni-bielefeld.de/rnahybrid). We seeded 293 T cells in 48-well plates then transfected them 24 h later with wild-type psiCHECK-2-ENTPD7 plasmids and mimic, mutated psiCHECK-2-ENTPD7 plasmids and mimic, and psiCHECK-2 plasmids and mimic NC. A target relationship between circACLY and gga-miR-6660-3p was determined after 48 h using Dual-GLO Luciferase Assay System kits (Promega, Madison, WI, USA) as described by the manufacturer. Firefly and Renilla Luciferase signals were identified using a Fluorescence/Multi-Detection Microplate Reader (BioTek Instruments, Winooski, VT, USA). Firefly luciferase activities were normalized to Renilla luminescence in each well.

Enzyme-linked immunosorbent assays (ELISA)

Myoblasts were cultured in 6-well plates and transfected for 72 h. Protein suspensions were prepared using Whole Cell Lysis Assay kits. Protein levels were measured using chicken ELISA kits (R&D Systems Inc., Minneapolis, MN, USA) as described by the manufacturer.

Statistical analysis

All assays were conducted in triplicate. Data are presented as the means ± SEM of three independent experiments. The significance of differences in means between two groups was analyzed using two-tailed t-tests. Multiple comparison data were assessed by one-way ANOVA followed by least significant difference (LSD) and Duncan tests using SPSS 23.0 (IBM Corp., Armonk, NY, USA). Values with P < 0.05 were considered significantly different.

Results

Validation and subcellular localization of circACLY in myoblasts

Previous sequencing data revealed that the ACLY gene generates circRNAs (data not shown). The genomic structure of ACLY shows the regions formed by circACLY (Figure 1A). CircularACLY is a circRNA that spans two exons. We investigated the expression pattern of circACLY across different developmental stages. The relative expression of circACLY was significantly higher in E11 embryos compared to muscle tissue from 180-d-old adult chickens (Figure 1B). We investigated the full-length sequence and the cyclization junction site of circACLY, using PCR, with convergent and divergent primers (Figure 1C). The full-length sequences and cyclization junction sites of circACLY were amplified (Figure 1D and E), and sequencing findings showed that circACLY was generated from exons 2 and 3 of the ACLY gene. We applied RNase R digestion to further verify the existence of circACLY. The RNA bands disappeared compared with the control (Figure 1F). Quantitative RT-PCR of circACLY, GAPDH and β-actin also showed that linear RNA was digested, whereas circACLY expression did not significantly change (Figure 1G and H). We investigated the subcellular location of circACLY function by PCR amplification of nuclear and cytoplasmic cDNAs using U6 and circACLY convergent primers. The results showed that U6 was absent in cytoplasmic RNA and that cytoplasmic and nuclear RNAs were clearly separated. The brightness of nuclear and cytoplasmic bands indicated that circACLY was more predominant in the cytoplasm (Figure 1I–K). These results validated the existence, cellular localization, and differential expression of circACLY during skeletal muscle development in chickens.

Figure 1.

Differential expression, validation, and subcellular localization of circACLY. A) Circular RNA (circACLY) derived from ACLY. B) Detection of circACLY expression in E11 embryonic leg muscle tissues and at 180 D by qRT-PCR. C) Principles of circACLY divergent primer design. D) Full-length circACLY sequence amplified using primer pair. E) Divergent primer amplified circACLY cyclization junction site. F) Ribonuclease R digestion of total RNA. G) Digested linear RNA detected by qRT-PCR. H) Quantitative RT-PCR shows that circACLY resisted RNase R digestion. I) Cytoplasmic and nuclear RNA extraction electrophoresis. J) Cytoplasmic and nuclear RNA isolation detected by PCR amplification of U6. K) circACLY PCR amplification gel electrophoresis. All values are shown as means ± SEM of three independent experiments. ***P < 0.001.

Validation of targeting relationship between circACLY-miR-6660-3p-ENTPD7

Many Circular RNAs function as sponges for miRNAs to derepress miRNAs on target genes. Here, we predicted using RNAhybrid that circACLY would be a target of miR-6660-3P (Figure 2A–C). The seed sequence of miR-6660-3p matched one site in circACLY. Both miRDB and TargetScan predicted that miR-6660-3p would bind the target gene ENTPD7 (Figure 2D). To determine a targeting relationship between circACLY and each of miR-6660-3p, and ENTPD7, we amplified sequences containing miR-6660-3p with circACLY and ENTPD7 3’UTR binding sites ligated to psiCHECK-2 vector and transfected them into 293 T cells (Figure 2E–G). The transfection efficiency of the vector and miRNA in 293 T cells was assessed by qRT-PCR and as fluorescence emission. More miR-6660-3p was expressed after transfection with the mimic than mimic NC (Figure 2H). Both transfected circACLY and ENTPD7 3’UTR wild-type and mutant vectors expressed the target sequence more efficiently than the psiCHECK-2 vector (Figure 2I and J). Quantitative RT-PCR showed that ENTPD7 3’UTR expression in 293 T cells did not change in groups B (psiCHECK-2), C (psi-circ-wt), and D (mimic). However, more ENTPD7 3’UTR was expressed in groups A (mimic with psi-ENTPD7 3’UTR), E (mimic, psi-ENTPD7 3’UTR and psi-circ-wt) and F (psi-ENTPD7 3’UTR) ENTPD7 3’UTR than in groups B, C, and D, and was significantly higher in groups E and F, than A (Figure 2K). We then co-transfected 293 T cells with circACLY wild vector and miR-6660-3p mimic and a mutant vector with miR-6660-3p mimic. The psiCHECK-2 and mimic NC were the negative control transfected cells. The luminescence activities were significantly higher in the cells transfected with circACLY mutant vector and miR-6660-3p mimic and in the negative control than in cells transfected with the circACLY wild vector and miR-6660-3p mimic (Figure 2L). The results of the same groups and experiments using ENTPD7 3’UTR wild vector, ENTPD7 3’UTR mutant vector with miR-6660-3p mimic and with mimic NC (Figure 2M) revealed significantly more luminescence activity of the ENTPD7 3’UTR mutant vector with the miR-6660-3p mimic and NC than the ENTPD7 3’UTR wild vector with the miR-6660-3p mimic. Overall, these results indicated that miR-6660-3p acts on the target gene ENTPD7, whereas circACLY inhibited the effects of miR-6660-3p on the target gene.

Figure 2.

Validation of targeting relationship between circACLY-miR-6660-3p-ENTPD7. A) Putative binding sites for miR-6660-3p on circACLY. B) RNAhybrid predicts potential binding of miRNAs by circACLY. C) Potential binding site sequence of miR-6660-3p on circACLY and ENTPD7 3'UTR. D) Differentially expressed genes (DEGs) determined by RNA-seq miRDB and TargetScan screening for miR-6660-3p target genes. E) PCR gel electrophoresis of circACLY wild-type and mutant vector cultures. F) PCR gel electrophoresis of ENTPD7 wild-type and mutant vector cultures. G) circACLY and ENTPD7 3'UTR target fragment insertion sites. H) Quantitation and fluorescence staining following transfection with miR-6660-3p mimics and mimic NC transfected into 293T cells. I) Quantitation and fluorescence staining of circACLY wild-type and mutant vectors and negative control (psiCHECK-2) transfected into 293T cells. J) Quantitation analysis and fluorescence staining of 293T cells transfected with ENTPD7 3'UTR wild-type and mutant vectors and control (psiCHECK-2). K) Expression of ENTPD7 3'UTR in 293T cells transfected with A (mimic with psi-ENTPD7 3'UTR), B (psiCHECK-2), C (psi-circ-wt), D (mimic), E (mimic, psi-ENTPD7 3'UTR and psi-circ-wt), and F (psi-ENTPD7 3'UTR). L) Validation of circACLY and miR-6660-3p binding sites. M) Validation of ENTPD7 and miR-6660-3p binding sites. All values are shown as means ± SEM of three independent experiments. **P < 0.01; ***P < 0.001.

Myoblast proliferation is inhibited by miR-6660-3p

We transfected myoblasts with miR-6660-3p mimic (50 nM) or inhibitor (100 nM) to determine the effect of miR-6660-3p on their proliferation. Myoblasts transfected with miR-6660-3p mimic/mimic NC and miR-6660-3p inhibitor/inhibitor NC were analyzed by qRT-PCR. Compared to their respective NC, the expression of miR-6660-3p was significantly increased in myoblasts transfected with the mimic, and significantly decreased in those transfected with the inhibitor (Figure 3A and B). RT-qPCR results showed that knock-down of miR-6660-3p significantly promoted ENTPD7 expression, while the ectopic expression significantly suppressed its expression (Figure 3C and D). The ectopic expression of miR-6660-3p significantly inhibited the expression of genes that are hallmarks of cell proliferation, whereas knock-down of miR-6660-3p significantly promoted the expression of such genes (Figure 3E and F). We assessed the proliferation and viability of myogenic using CCK-8 assays. The results showed that cells were less viable when transfected with miR-6660-3p mimic than mimic NC and more viable in those transfected with miR-6660-3p inhibitor than inhibitor NC (Figure 3G and H). After transfection with miR-6660-3p mimic/mimic NC and miR-6660-3p inhibitor/inhibitor NC, we examined protein levels of proliferation signature genes using an ELISA. The results showed that the ectopic expression and knock-down of miR-6660-3p significantly suppressed and promoted the protein levels of genes that are markers of cell proliferation, respectively (Figure 3I–L). Assays of EdU revealed a significantly lower rate of dividing cells under ectopic expression than the mimic NC group (Figure 4A). Conversely, miR-6660-3p knock-down significantly increased the abundance of EdU stained cells compared with inhibitor NC (Figure 4B). Overall, these results indicated that miR-6660-3p suppressed myoblast proliferation.

Figure 3.

MicroRNA-6660-3p inhibits myoblast proliferation. A) Detection of miR-6660-3p mRNA expression following miR-6660-3p knock-down in myoblasts. B) Detection of miR-6660-3p mRNA expression following miR-6660-3p overexpression in myoblasts. C) Detection of mRNA expression of target gene ENTPD7 following miR-6660-3p knock-down in myoblasts. D) Detection of mRNA expression of target gene ENTPD7 following miR-6660-3p overexpression in myoblasts. E) Following miR-6660-3p overexpression in myoblasts, the expression levels of proliferation marker genes in myoblasts were detected by qRT-PCR. F) Following miR-6660-3p knock-down in myoblasts, the expression levels of proliferation marker genes in myoblasts were detected by qRT-PCR. G) CCK8 assay was used to detect the viability of myoblasts after treatment with miR-6660-3p overexpression. H) CCK8 assay was used to detect the viability of myoblasts after treatment with miR-6660-3p knock-down. I–J) Following miR-6660-3p overexpression in myoblasts, the levels of CDK1 and PCNA proteins in the myoblasts were detected using ELISA. K–L) Following miR-6660-3p knock-down in myoblasts, the levels of CDK1 and PCNA proteins in the myoblasts were detected using ELISA. All values are shown as means ± SEM of three independent experiments. *P < 0.05; ^**P < 0.01; ^***P < 0.001.

Figure 4.

Myoblast proliferation is inhibited by miR-6660-3p. A) Using EDU labeling to assess proliferation in myoblasts following miR-6660-3p overexpression treatment. B) Using EDU labeling to assess proliferation in myoblasts following miR-6660-3p knockout treatment. All values are shown as means ± SEM of three independent experiments. *P < 0.05; ^**P < 0.01.

Myoblast differentiation is repressed by miR-6660-3p

We examined the expression of miR-6660-3p and myogenic differentiation signature genes by inducing myogenic differentiation to determine whether miR-6660-3p is involved. The expression of miR-6660-3p continuously decreased, whereas myoblast differentiation gene expression continuously increased during culture for 5 d in differentiation (DM), compared with growth (GM) medium (day 0) (Figure 5A, B–E). We evaluated expression of the myoblast differentiation marker genes Myosin Heavy Chain (MyHC), Myoblast Determination Protein 1 (MyOD1), Myogenic factor 5 (Myf5) and Myogenin (MyOG) by qPCR in myoblasts transfected with miR-6660-3p mimic (or mimic NC) and inhibitor (or inhibitor NC). Overexpressed miR-6660-3p notably inhibited MyHC, MyOD1, Myf5, and MyOG expression compared with cells transfected with mimic NC (Figure 5F). In contrast, miR-30a-3p knock-down promoted MyHC, MyOD1, Myf5 and MyOG expression compared with inhibitor NC (Figure 5G). The results of ELISAs showed that ectopic miR-6660-3p expression and knock-down respectively inhibited (Figure 5H–I) and promoted (Figure 5J and K) protein levels of MyHC and MyOG. These results revealed that miR-6660-3p could repress myoblast differentiation.

Figure 5.

Myoblast differentiation is repressed by miR-6660-3p. A) Expression profile of miR-6660-3p in myoblasts during induced differentiation. B–E) Expression profiles of Myf5, MyHC, MyOD1, and MyOG in myoblasts during induced differentiation. F) Following miR-6660-3p overexpression in myoblasts, the expression levels of differentiation marker genes in myoblasts were detected by qRT-PCR. G) Following miR-6660-3p knock-down in myoblasts, the expression levels of differentiation marker genes in myoblasts were detected by qRT-PCR. H–I) Following miR-6660-3p overexpression in myoblasts, the levels of MYOG and MYHC proteins in the myoblasts were detected using ELISA. J–K) Following miR-6660-3p knock-down in myoblasts, the levels of MYOG and MYHC proteins in the myoblasts were detected using ELISA. All values are shown as means ± SEM from three independent experiments. *P < 0.05; ^**P < 0.01; ^***P < 0.001.

Myoblast apoptosis is promoted by miR-6660-3p

We transfected myoblasts with miR-6660-3p mimic/mimic NC and inhibitor/inhibitor NC to determine how miR-6660-3p affects myoblast apoptosis. The results of qRT-PCR showed that ectopic expression and knockdown of miR-6660-3p significantly promoted and repressed the expression of genes that are hallmarks of cell apoptosis, respectively (Figure 6A and B). The ELISA results showed that ectopic expression and knockdown of miR-6660-3p significantly promoted and suppressed the protein levels of genes that are markers of cell apoptosis, respectively (Figure 6C–F). Furthermore, significantly more cells were undergoing apoptosis with ectopic miR-6660-3p expression than mimic NC (Figure 6G). Conversely, miR-6660-3p knockdown decreased the numbers of apoptotic and necrotic cells compared with inhibitor NC (Figure 6G). These results suggested that miR-6660-3p promotes apoptosis and necrosis in myoblasts.

Figure 6.

Myoblast apoptosis is promoted by miR-6660-3p. A) Following miR-6660-3p overexpression in myoblasts, the expression levels of apoptosis marker genes BAK1, FAS, BID, and Caspase 9 were detected by qRT-PCR. B) Following miR-6660-3p knock-down in myoblasts, the expression levels of apoptosis marker genes BAK1, FAS, BID, and Caspase 9 were detected by qRT-PCR. C–D) Following miR-6660-3p overexpression in myoblasts, the levels of BID and FAS proteins in the myoblasts were detected using ELISA. E–F) Following miR-6660-3p knock-down in myoblasts, the levels of BID and FAS proteins in the myoblasts were detected using ELISA. G) Assays of apoptosis in myoblasts with over-expressed and inhibited miR-6660-3p. All values are shown as means ± SEM of three independent experiments. *P < 0.05; ^**P < 0.01; ^***P < 0.001.

Circular ACLY promotes myoblast proliferation and eliminates inhibitory effect of miR-6660-3p on myoblast proliferation

We transfected chicken myoblasts with the circACLY overexpression vector pCD-25-circACLY to determine the role of circACLY in skeletal muscle cell proliferation. The relative expression of circACLY was detected by qRT-PCR at 48 h post transfection. The effects of circACLY overexpression were significant in myoblasts (Figure 7A and B). Considering the interaction between circACLY and miR-6660-3p, we co-transfected circACLY with miR-6660-3p mimics to assess whether the inhibition of proliferation by miRNA could be blocked by overexpressed circACLY. Therefore, we assessed proliferation using CCK-8 assays, qRT-PCR to detect proliferation marker genes, ELISA to analyze protein levels and EdU incorporation in chicken myoblasts transfected with pCD25-circACLY/pCD25-circ or co-transfected with circACLY and miR-6660-3p mimics. The qRT-PCR and ELISA results showed that circACLY overexpression or co-transfected circACLY and miR-6660-3p mimic significantly promoted the mRNA expression and protein levels of myoblast proliferation marker genes (Figure 7C–H). The CCK-8 assay results showed that cells transfected with vectors overexpressing circACLY or co-transfected with circACLY and miR-6660-3p mimic, were more viable than negative control cells (Figure 7I and J). The EdU assay indicated that the rate of cell division was significantly higher in myoblasts overexpressing circACLY or co-transfected with circACLY and miR-6660-3p mimics than in negative controls (Figure 8A and B). Thus, the qRT-PCR, ELISA and EdU findings confirmed that circACLY promotes myoblast proliferation and eliminates the inhibition of chicken myoblast proliferation by overexpressed miR-6660-3p.

Figure 7.

Circular ACLY promotes myoblast proliferation and eliminates inhibitory effects of miR-6660-3p on myoblast proliferation. A–B) Detection of circACLY mRNA expression following circACLY overexpression in myoblasts. C) Following circACLY overexpression in myoblasts, the expression levels of proliferation marker genes in myoblasts were detected by qRT-PCR. D) Following co-transfection circACLY overexpression and miR-6660-3p mimic into myoblasts, the expression levels of proliferation marker genes in myoblasts were detected via qRT-PCR. E–F) Following circACLY overexpression in myoblasts, the levels of CDK1 and PCNA proteins in myoblasts were detected using ELISA. G–H) Following co-transfection circACLY overexpression and miR-6660-3p mimic into myoblasts, the levels of CDK1 and PCNA proteins in myoblasts were detected using ELISA. I) CCK8 assay was used to detect the viability of myoblasts after treatment with circACLY overexpression. J) CCK8 assay was used to detect the viability of myoblasts co-transfected with circACLY overexpression and miR-6660-3p mimic. All values are shown as means ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 8.

Circular ACLY promotes myoblast proliferation and eliminates inhibitory effects of miR-6660-3p on myoblast proliferation. A) Using EDU labeling to assess proliferation in myoblasts following circACLY overexpression treatment. B) The effect of co-transfection circACLY overexpression and miR-6660-3p mimic on proliferation in myoblasts following EDU detection. All values are shown as means ± SEM of three independent experiments. **P < 0.01.

Circular ACLY promotes myoblast differentiation and eliminates miR-6660-3p inhibition of myoblast differentiation

We analyzed expression of the differentiation marker genes, MyHC, MyOD1, Myf5 and MyOG in myoblasts transfected with pCD-25-circACLY/pCD-25, or pCD-25-circACLY with miR-6660-3p mimics/control using qRT-PCR to determine the potential role of circACLY in myoblast differentiation. Overexpressed circACLY and circACLY co-transfected with miR-6660-3p mimics significantly promoted the expression of these genes, indicating that circACLY promotes myoblast differentiation. Considering the relationship between circACLY and miR-6660-3p, circACLY abolished the inhibition of myoblast differentiation by miR-6660-3p (Figure 9A and B). We quantified protein levels of myoblast differentiation genes in myoblasts overexpressing circACLY or co-transfected with circACLY and miR-6660-3p using an ELISA. Both conditions significantly promoted the protein levels of these genes (Figure 9C–F). These results showed that circACLY promotes myocyte differentiation and eliminates the miR-6660-3p-induced inhibition of myocyte differentiation.

Figure 9.

Circular ACLY promotes myoblast differentiation and eliminates its inhibition by miR-6660-3p. A) Following circACLY overexpression in myoblasts, the expression levels of differentiation marker genes in myoblasts were detected by qRT-PCR. B) Following co-transfection circACLY overexpression and miR-6660-3p mimic into myoblasts, the expression levels of differentiation marker genes in the myoblasts were detected by qRT-PCR. C–D) Following circACLY overexpression in myoblasts, the levels of MYHC and MYOG proteins in myoblasts were detected using ELISA. E–F) Following co-transfection circACLY overexpression and miR-6660-3p mimic into myoblasts, the levels of MYHC and MYOG proteins in myoblasts were detected using ELISA. All values are shown as means ± SEM from three independent experiments. *P < 0.05; **P < 0.01.

circACLY inhibits myoblast apoptosis and eliminates the apoptosis-promoting effect of miR-6660-3p on myoblast

We assessed the effects of circACLY on apoptosis by transfecting myoblasts for 48 h with the pCD-25-circACLY/pCD-25 vector overexpressing circACLY, or pCD-25-circACLY cotransfected with miR-6660-3p mimics. Expression of the apoptosis marker genes, BCL2 Antagonist/Killer 1 (BAK1), BH3 Interacting Domain Death Agonist (BID), FS-7-associated surface antigen-cell surface death receptor (FAS), and Caspase9, was analyzed by qRT-PCR. Both conditions significantly inhibited the expression of apoptosis marker genes, indicating that circACLY inhibits myocyte apoptosis and abolishes the ability of miR-6660-3p to promote it (Figure 10A and B). The results of an ELISA showed that both conditions significantly inhibited the protein levels of apoptosis marker genes (Figure 10C–F). The rate of apoptotic cells was significantly lower under both conditions compared with the controls (Figure 10G).

Figure 10.

CircACLY inhibits myoblast apoptosis and abolishes the apoptosis-promoting effect of miR-6660-3p on myoblast. A) Following circACLY overexpression in myoblasts, the expression levels of apoptosis marker genes BAK1, FAS, BID, and Caspase 9 were detected by qRT-PCR. B) Following co-transfection circACLY overexpression and miR-6660-3p mimic into myoblasts, the expression levels of apoptosis marker genes BAK1, FAS, BID, and Caspase 9 were detected by qRT-PCR. C–D) Following circACLY overexpression in myoblasts, the levels of FAS and BID proteins in myoblasts were detected using ELISA. E–F) Following co-transfection circACLY overexpression and miR-6660-3p mimic into myoblasts, the levels of FAS and BID proteins in myoblasts were detected using ELISA. G) Assays of apoptosis in myoblasts with overexpressing circACLY and miR-6660-3p mimics. All values are shown as means ± SEM of three independent experiments. *P < 0.05; **P < 0.01.

MicroRNA-6660-3p inhibits inosine monophosphate synthesis de novo in purine metabolic pathway

Ectonucleoside triphosphate diphosphohydrolase 7 is downstream of inosine monophosphate (IMP) synthesis de novo in the purine metabolic pathway (Figure 11A), which is inextricably linked to cell proliferation and apoptosis as an intermediate product of RNA and DNA synthesis (Figure 11B). Given the interaction between miR-6660-3p and ENTP7, we postulated that miR-6660-3p is involved in regulating the pathway of de novo IMP synthesis in the purine metabolic pathway. We transfected miR-6660-3p mimic/mimic NC and miR-6660-3p inhibitor/inhibitor NC into myoblasts and assessed the effects of their overexpression and inhibition on the de novo synthesis of IMP using qRT-PCR. The ectopic expression and knock-down of miR-6660-3p significantly inhibited and promoted the expression of genes that are markers of this pathway, respectively (Figure 12A and B), and likewise suppressed and promoted the levels of their proteins (Figure 12C–G). Thus, miR-6660-3p inhibits the de novo synthesis of IMP in the purine metabolic pathway.

Figure 11.

Synthesis de novo and metabolism of inosine monophosphate in purine metabolic pathway. A) Purine metabolic pathway. B) Inosine monophosphate metabolism.

Figure 12.

MicroRNA-6660-3p inhibits the de novo synthesis of inosine monophosphate in the purine metabolic pathway. A) Following miR-6660-3p overexpression in myoblasts, qRT-PCR was used to detect expression levels of genes marking the de novo synthesis pathway of IMP. B) Following miR-6660-3p knock-down in myoblasts, qRT-PCR was used to detect expression levels of genes marking the de novo synthesis pathway of IMP. C–D) Following miR-6660-3p overexpression in myoblasts, the levels of ADSL and GART proteins in myoblasts were detected using ELISA. E–F) Following miR-6660-3p knock-down in myoblasts, the levels of ADSL and GART proteins in myoblasts were detected using ELISA. All values are shown as means ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. ADSL, adenylosuccinate lyase; AIRS, aminoimidazole ribonucleotide synthetase; GARS, glycinamide ribonucleotide synthetase; GART, glycinamide ribonucleotide formyltransferase; GPAT, glycerol-3-phosphate acyltransferase, chloroplastic; PAICS, phosphoribosylamino-imidazole carboxylase and phosphoribosylaminoimidazolesuccino-carboxamide synthase; PPAT, phosphoribosyl pyrophosphate amidotransferase.

CircularACLY eliminates the inhibitory effect of miR-6660-3p on the de novo synthesis pathway of inosine monophosphate in the purine metabolic pathway

Circular ACLY sponges miR-6660-3p and deregulates the miR-6660-3p inhibition of ENTPD7. We investigated the effects of circACLY on the de novo synthesis pathway of IMP in the purine metabolic pathway. The expression of marker genes of the IMP de novo synthesis pathway in myoblasts transfected with pCD-25-circACLY/pCD-25 or pCD-25-circACLY with miR-6660-3p mimic/control for 48 h was analyzed by qRT-PCR. Both overexpressed circACLY and circACLY co-transfected with miR-6660-3p mimics significantly promoted the expression of IMP synthesis pathway marker genes ab initio (Figure 13A and B) and the levels of their proteins (Figure 13C–F). These findings suggested that circACLY promotes the synthesis of IMP de novo and eliminates inhibition of its pathway by miR-6660-3p.

Figure 13.

Circular ACLY eliminates inhibitory effect of miR-6660-3p on de novo synthesis pathway of IMP in purine metabolic pathway. A) Following circACLY overexpression in myoblasts, qRT-PCR was used to detect expression levels of genes marking the de novo synthesis pathway of IMP. B) Following co-transfection circACLY overexpression and miR-6660-3p mimic into myoblasts, qRT-PCR was used to detect expression levels of genes marking the de novo synthesis pathway of IMP. C–D) Following circACLY overexpression in myoblasts, the levels of ADSL and GART proteins in myoblasts were detected using ELISA. E–F) Following co-transfection circACLY overexpression and miR-6660-3p mimic into myoblasts, the levels of ADSL and GART proteins in myoblasts were detected using ELISA. All values are shown as means ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. ADSL, adenylosuccinate lyase; AIRS, aminoimidazole ribonucleotide synthetase; GARS, glycinamide ribonucleotide synthetase; GART, glycinamide ribonucleotide formyltransferase; GPAT, glycerol-3-phosphate acyltransferase, chloroplastic; IMP, inosine monophosphate; PAICS, phosphoribosylamino-imidazole carboxylase and phosphoribosylaminoimidazolesuccino-carboxamide synthase; PPAT, phosphoribosyl pyrophosphate amidotransferase.

Discussion

Circular RNAs have been identified in various cell types in many species and they play regulatory roles in several biological processes (Cao et al 2018; Shen et al 2019; Liu et al 2020). Circular RNAs have spatial and temporal specificity in tissue cells, indicating that their functions vary among tissues at different stages (Cao et al 2018; Shen et al 2019; Liu et al 2020). Circular RNAs are differentially expressed during the developmental stages of chicken skeletal muscle and play key regulatory roles in skeletal muscle development (Chen et al 2018, 2019). Circular FGFR2 and circHIPK3 regulate the proliferation and differentiation of chicken myoblasts by sponging adsorbed miRNAs (Chen et al 2018, 2019). The present study found that a circular RNA produced by the ACLY gene, circACLY, is expressed at significantly different levels at various stages of skeletal muscle development in chickens. This indicated that circACLY functions in regulating skeletal muscle development.

Circular RNAs containing microRNA response elements (MREs) function as molecular sponges that effectively inhibit miRNA function according to bioinformatics-based findings of competing endogenous RNA (ceRNA) networks (Salmena et al 2011). Here, we predicted a binding site for circACLY to miR-6660-3p using RNAhybrid. We also found a possible targeting relationship between miR-6660-3p and ENTPD7 using miRDB and TargetScan. Dual luciferase reporter assays are useful for verifying binding relationships among circRNA, miRNA and mRNA (Clément et al 2015). Using these assays, we confirmed that circACLY and miR-6660-3p bind to the predicted sites of miR-6660-3p and ENTPD7, respectively. We designed a co-transfection assay in which psi-circ-WT containing the miR-6660-3p binding site derepressed the inhibitory effect of miR-6660-3P on ENTPD7. We confirmed that circACLY, miR-6660-3p, and ENTPD7 interact. Therefore, we speculated that circACLY, miR-6660-3p and ENTPD7 play important regulatory roles in skeletal muscle development. This is consistent with the conjecture of (Zhou et al 2022) suggesting that gga-miR-6660-3p may play important roles in the muscle development of Bian chickens.

MicroRNAs are ubiquitous in various cell types or tissues and play irreplaceable regulatory roles in plant and animal life. MicroRNAs are involved in the growth and development of skeletal muscle (Chen et al 2018; Ouyang et al 2018). Research has found that miR-206 is expressed in satellite cells and participates in muscle development and regeneration (Ma et al 2015), while miR-34c and miR-34a-5p are associated with skeletal muscle growth and development in Bian Chickens (Zhou et al 2022). The role of miR-6660-3p in poultry skeletal muscle has remained unclear but we found that it regulated the proliferation, differentiation and apoptosis of chicken skeletal muscle. Cycline, a key component of the core cell cycle machinery, activates cyclin-dependent kinase 2 (CDK2), which drives cell cycle progression (Clément et al 2015; Geng et al 2018; Chu et al 2021). Cyclin D1 is a key protein that regulates the G1 phase of the cell cycle by driving the cell cycle from the G1 to the S phase through activating G1-specific cell cycle protein-dependent kinase 4 (CDK4) (Coqueret 2002). Proliferating cell nuclear antigen (PCNA) functions as a sliding clamp during DNA synthesis, a polymerase switch factor and a recruitment factor, thus participating in DNA synthesis and the cell cycle, and the absence of these functions, or the intracellular absence or minimal levels of PCNA, leads to apoptosis (Paunesku et al 2001; Strzalka and Ziemienowicz 2011). Cyclin-dependent kinase 1 (CDK1) plays an important role in mitosis initiation and forms a complex with cyclin-b1 and cyclin-b2 to regulate the G2/M phase of the cell cycle (Malumbres and Barbacid 2005). Here, overexpressed and knocked down miR-6660-3p suppressed and promoted the expression of proliferation marker genes. In addition, miR-6660-3p reduced cell proliferation rates. The findings of circACLY functioning as a molecular sponge of miR-6660-3p were notable. Overexpressed circACLY promoted the expression of proliferation marker genes, and increased cell proliferation rates. The results of rescue experiments showed that circACLY eliminated the inhibition of myoblast proliferation by miR-6660-3p. This finding is consistent with the results reported by Ouyang et al (2018), who demonstrated that circSVIL promotes myoblast proliferation, antagonizes miR-203 functions, and promotes the embryonic skeletal muscle development in Xinghua chicken embryos. Furthermore, Zhao et al (2022) also found that circCCDC91 directly binds to the miR-15 family by activating the IGF1-PI3K/AKT signaling pathway, thereby promoting myoblasts proliferation in Tianfu broiler chicken and alleviating skeletal muscle atrophy.

Skeletal myogenesis occurs after cell cycle termination, which is coordinated by various regulatory transcription factors such as MyOD1, MyOG, Myf5, and MyHC (Tajsharghi and Oldfors 2013; Zammit 2017). The myogenic regulatory factor family consisting of MYOD1, MYf5, and MYOG links the genetic control of developmental and regenerative myogenesis, thus facilitating myoblast differentiation into myotubes (Zammit 2017). Myosin heavy chain is the molecular motor of muscle and forms the backbone of sarcomere thick filaments (Tajsharghi and Oldfors 2013). Assays of differentiation induction revealed that miR-6660-3p plays an important role in myoblast differentiation. MicroRNA-6660-3p inhibited, whereas circACLY promoted the expression of myoblast differentiation marker genes. Further rescue experiments showed that circACLY deregulated the repression of myogenic cell differentiation by miR-6660-3p. As Wang et al. (2023) discovered, circIGF2BP3 enhances the expression levels of MyHC, MyoD1, MyoG, and Myomaker, thereby promoting myoblast differentiation in Yuexi frizzled feather chickens. Shen et al. (2021) also observed that when miR-218-5p and circITSN2 were co-overexpressed, circITSN2 eliminated the inhibition effect of miR-218-5p on proliferation and differentiation in chicken primary myoblast (CPM).

Coordinated regulation of myoblast proliferation, differentiation and apoptosis is necessary for the formation of skeletal muscle during development. Downregulation of MyoD mediates the expression of miR-1 and miR-206, activates transcription of the anti-apoptotic proteins B-cell lymphoma 2 (BCL-2) and B-cell lymphoma-extra large (BCL-xL), and enhances myoblast survival (Hirai et al 2010). Apoptosis is an active process of programmed death that involves the activation, expression and regulation of a series of genes, including BAK1, BID, FAS, and Caspase9 (Brentnall et al 2013; Edlich 2018; Lan et al 2021). The apoptosis-promoting BAK1 and BID proteins can commit a cell to programmed death by permeabilizing the outer mitochondrial membrane (OMM) and subsequently initiating the caspase cascade (Harada and Grant 2003; Edlich 2018). The FAS gene is ubiquitously expressed in cells, and the Fas death receptor induces apoptosis by interacting with its ligand FasL (Zhang et al 2012). MicroRNA-6660-3p promoted, whereas circACLY inhibited the expression of apoptosis signature genes and suppressed apoptosis. The results of rescue experiments suggested that circACLY regulates apoptosis in myocytes by blocking miR-6660-3p.

We investigated the potential mechanism of action of circACLY adsorbed to miR-6660-3p in regulating myoblast proliferation, differentiation and apoptosis. We confirmed that the ENTPD7/purine metabolic pathway is a target of miR-6660-3p. The ectonucleoside triphosphate diphosphohydrolase (ENTPDase) family of eight members regulates extracellular adenosine triphosphate (ATP) levels and participates in cellular activities (Robson et al 2006). Ectonucleotide triphosphate diphosphohydrolase 7 regulates the proliferation and apoptosis of lung cancer cells, and down-regulating ENTPD7 inhibits lung cancer cell proliferation and promotes apoptosis via inhibiting the Ras/Raf/MEK/ERK pathway. In addition, ENTPD7 regulates senescence. Ectonucleotide triphosphate diphosphohydrolase 7 affects oxidative stress, DNA damage and senescence, and its expression of ENTPD7 or the inhibition of nucleotide ­synthesis in AT-Rich Interaction Domain 1B (ARID1B) gene-deficient hepatocellular carcinoma cells leads to the re-establishment of senescence (Tordella et al 2016). Most free-living organisms require the synthesis or acquisition of purines and pyrimidines that form the basis of nucleotides for survival and reproduction (Goncheva et al 2022). Purine nucleotides are synthesized de novo in organisms except for a few tissue sites and the products participate in many cell functions, such as serving as building blocks for DNA and RNA; purine metabolites also provide cells with energy (ATP and GTP), signaling molecules (ATP, Camp, and cGMP) and cofactors (FAD, NAD+, NADP+, and coenzyme A) that promote cell survival and proliferation (Ben-Sahra et al 2016; Pedley and Benkovic 2017; Goncheva et al 2022). Inosine monophosphate is a key intermediate in purine metabolism that is enzymatically converted to AMP or GMP, whereas the synthesis of inosine-5'-monophosphate de novo is catalyzed by a enzymes that sequentially assemble a purine base to phosphoryl pyrophosphate (PRPP) in ten steps (Pareek et al 2021). We investigated whether circACLY affects myoblast proliferation, differentiation and apoptosis through miR-6660-3p regulation of the ENTPD7/purine metabolic pathway. Our findings showed that miR-6660-3p negatively regulated the pathway of IMP synthesis de novo in the ENTPD7/purine metabolic pathway. CircACLY blocked the inhibitory effects of miR-6660-3p on the synthesis of IMP de novo in the ENTPD7/purine metabolic pathway.

Conclusions

Circular ACLY acts on the ENTPD7/purine metabolic pathway by functioning as a miR-6660-3p sponge to regulate myoblast proliferation, differentiation and apoptosis.

Abbreviations: 

ACLY

ATP citrate lyase

ADSL

adenylosuccinate lyase

AIRS

aminoimidazole ribonucleotide synthetase

ARID1B

AT-rich interaction domain 1B

ATP

adenosine triphosphate

BCL-2

B-cell lymphoma 2

BCL-xL

B-cell lymphoma-extra large

CCK-8

Cell Counting Kit 8

CDK1

cyclin-dependent kinase 1

CDK2

cyclin-dependent kinase 2

CDK4

cycle protein-dependent kinase 4

ceRNA

competing endogenous RNA

circRNAs

circular RNAs

CPM

chicken primary myoblast

DEGs

differentially expressed genes

DEMs

differentially expressed metabolites

DMGs

differentially methylated genes

EdU

5-ethynyl-2’-deoxyuridine

ELISA

enzyme-linked immunosorbent assays

ENTPD7

ectonucleotide triphosphate diphosphohydrolase 7

ENTPDase

ectonucleoside triphosphate diphosphohydrolase

FBS

fetal bovine serum

FCE

fresh corn extract

FGFR2

fibroblast growth factor receptor 2

GARS

glycinamide ribonucleotide synthetase

GART

glycinamide ribonucleotide formyltransferase; GPAT, glycerol-3-phosphate acyltransferase, chloroplastic

IMP

inosine monophosphate

LSD

least significant difference

mimic NC

mimic negative control

MREs

microRNA response elements

MSTN

myostatin

Myf5

myogenic factor 5

MyHC

myosin heavy chain

MyOD1

myoblast determination protein 1

MyOG

myogenin

NADH

nicotinamide adenine dinucleotide

NC

negative control

Nc

non-coding

OD

optical density

OMM

outer mitochondrial membrane

PAICS

phosphoribosylamino-imidazole carboxylase and phosphoribosylaminoimidazolesuccino-carboxamide synthase

PCNA

proliferating cell nuclear antigen

PCR

polymerase chain reaction

PPAT

phosphoribosyl pyrophosphate amidotransferase

PRPP

phosphoryl pyrophosphate

qRT-PCR

quantitative real-time PCR

RNase R

ribonuclease R

TMTC1

transmembrane O-mannosyltransferase targeting cadherins 1

WGCNA

weighted gene co-expression network analysis

Author contributions

Ruimin Ma (Software [true], Writing—original draft [true], Writing—review & editing [true]), Ying Zhou (Conceptualization [true], Methodology [true], Resources [true]), Weizhen Wang (Conceptualization [true], Methodology [true]), Ling Zhu (Conceptualization [true], Methodology [true], Resources [true]), Tong Zhang (Conceptualization [true], Methodology [true], Resources [true]), Jinli Tian (Conceptualization [true], Methodology [true], Resources [true]), Lijuan Yang (Conceptualization [true], Methodology [true], Resources [true]), Hua Wang (Methodology [true], Supervision [true]), Lin Xue (Methodology [true], Supervision [true]), Siyu Chen (Methodology [true], Supervision [true]), Xiaohua Tian (Resources [true], Software [true]), Xiaoyun Ji (Resources [true], Software [true]), Zhengyun Cai (Funding acquisition [true], Project administration [true], Resources [true]), Yaling Gu (Funding acquisition [true], Project administration [true], Resources [true]), and Juan Zhang (Funding acquisition [true], Project administration [true], Resources [true])

Supplementary data

Supplementary data are available at Journal of Animal Science online.

Conflict of interest statement. The authors declare no real or perceived conflicts of interest.

Data Availability

The datasets generated and analyzed during the current study are available in the National Center for Biotechnology Information (NCBI) repository (https://www.ncbi.nlm.nih.gov/sra/PRJNA903370).

Institutional review board

This study proceeded according to the principles of the Basel Declaration and recommendations of the Statute on the Administration of Laboratory Animals, and the Ningxia University Institutional Animal Care and Use Committee approved the protocol (1 November 2020; approval ID: NXUC3127859).