circMAPKBP1 functions as a miR-143-y sponge to regulate quail myoblast proliferation and differentiation

Abstract

The quail (Coturnix japonica) is an economically important poultry species and a useful avian model for muscle biology. Although circular RNA (circRNA)–miRNA competitive endogenous RNA (ceRNA) networks have been described in chicken and duck skeletal muscle, their roles in quail remain poorly defined. We identified circMAPKBP1, generated from the mitogen-activated protein kinase binding protein 1 (MAPKBP1), as highly expressed in quail skeletal muscle and myoblasts. Gain- and loss-of-function experiments in quail myoblast 7 (QM7) cells showed that circMAPKBP1 promotes proliferation while inhibiting differentiation. Dual-luciferase reporter assays demonstrated direct binding between circMAPKBP1 and miR-143-y. Overexpression of miR-143-y promoted both proliferation and differentiation, and its expression pattern was reciprocal to that of circMAPKBP1 during differentiation. RNA-seq of miR-143-y-overexpressing cells revealed significant enrichment of downregulated genes in the mitogen-activated protein kinase (MAPK) signaling pathway. These results establish, for the first time, that the circMAPKBP1/miR-143-y axis modulates quail myogenesis, with the sponge mechanism most clearly explaining the opposing effects on differentiation. The findings provide molecular targets and a mechanistic framework for genetic improvement of muscle traits in poultry.

Introduction

The quail (Coturnix japonica) has emerged as an ideal model organism for avian genetics and developmental biology owing to its small body size, short generation interval, robust disease resistance, and distinctive genetic features (Serralbo et al., 2020; Zhang et al., 2024a). As the primary tissue determining meat production performance in poultry, skeletal muscle undergoes a tightly orchestrated developmental program encompassing myoblast proliferation, differentiation, and myofiber fusion and maturation (Buckingham et al., 2003; Zhang et al., 2024c). Although it is well established that skeletal muscle development is governed by an intricate network of transcription factors and signaling pathways (Mohammadabadi et al., 2021), the regulatory mechanisms specific to quail skeletal muscle—particularly the ncRNA-mediated layers of control—remain incompletely defined.

In recent years, ncRNAs have become a central focus of poultry functional genomics (Li et al., 2019; Wang et al., 2025). CircRNAs, a class of ncRNAs distinguished by their covalently closed loop structure, can sequester miRNAs through complementary base pairing, thereby alleviating miRNA-mediated post-transcriptional repression of target mRNAs—a mechanism known as the ceRNA hypothesis (Tay et al., 2014; Liu et al., 2025). CircRNA-miRNA-mRNA regulatory axes have been reported in several studies on the skeletal muscle development of chickens and ducks (Cai et al., 2022; Liu et al., 2025). However, compared with these species, the expression landscape and functional significance of ncRNAs during quail skeletal muscle development remain poorly characterized, and systematic functional validation is lacking. This knowledge gap has hindered efforts to improve quail meat production through molecular genetic strategies. circMAPKBP1 was identified from our previous whole-transcriptome sequencing of quail skeletal muscle (Zhang et al., 2024a) and satellite cell differentiation (Liu et al., 2026), where it exhibited high expression. Consistent with this, circRNAs derived from the MAPKBP1 locus have also been reported as abundantly expressed in embryonic duck breast muscle (Liu et al., 2023a). Here, we sought to systematically characterize the binding relationship between circMAPKBP1 and miR-143-y, define their regulatory roles in myoblast proliferation and differentiation, and identify downstream target genes. To achieve this, we employed overexpression and knockdown strategies, dual-luciferase reporter assays, RNA-seq, and functional phenotypic analyses. This study reveals, for the first time, the mechanism of action of the circMAPKBP1/miR-143-y regulatory axis in quail myogenesis, extends our understanding of ncRNA-mediated regulation in quail skeletal muscle, and provides a theoretical foundation and molecular targets for future improvements in quail meat yield and quality through marker-assisted selection.

Materials and methods

Ethics statement

All procedures involving quail embryos were approved by the Animal Ethics Committee of Jiangxi Agricultural University (Approval No.: [2018]30).

Experimental animals and cell culture

Korean quail hatching eggs were obtained from breeding stock maintained at the Poultry Research Institute of Jiangxi Agricultural University (Nanchang, China). Egg incubation and sample collection were performed as previously described (Liu et al., 2023b). Hindlimb muscle tissues (encompassing thigh and gastrocnemius muscles) were collected from quails at embryonic day 7 (E7) through post-hatch day 1 (P1), snap-frozen in liquid nitrogen, and stored at −80°C. The QM7 myoblast cell line (CRL-1962, ATCC, Manassas, VA) was cultured in M199 medium (Gibco, Waltham, MA) supplemented with 15% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco). Myogenic differentiation was induced by switching to M199 medium containing 1% FBS, with differentiation medium replaced every 24 h.

RNA extraction, reverse transcription, and quantitative Real-time PCR (qRT-PCR)

Total RNA was extracted from tissues or cells using TRIzol reagent (Takara, Japan). RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher). Genomic DNA was removed, and reverse transcription was conducted using the PrimeScript^RT reagent Kit (Takara). qRT-PCR was performed in 20 μL reactions containing ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), forward and reverse primers, and template cDNA. All reactions were run on a Bio-Rad CFX Duet system (Bio-Rad, Hercules, CA). The expression level of gene was calculated using the 2^−ΔΔCt method (Schmittgen and Livak, 2008). The amplification protocol consisted of an initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 small nuclear RNA (U6) served as internal reference genes. All primer sequences and related information are provided in Supplementary Table 1.

Validation of circMAPKBP1 circular structure and subcellular localization

To verify the circular structure of circMAPKBP1, convergent and divergent primers were designed according to the backsplice junction sequence and the quail MAPKBP1 reference sequence (XM_015864358.1) (Supplementary Table 1). PCR was performed using both genomic DNA and cDNA templates derived from QM7 cells. PCR products were analyzed by agarose gel electrophoresis. For subcellular localization analysis, nuclear and cytoplasmic RNA fractions were isolated from QM7 cells using the Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek, Thorold, Canada). The extracted RNAs were reverse-transcribed into cDNA, and the relative distribution of circMAPKBP1 in the nuclear and cytoplasmic fractions was determined by qRT-PCR.

Vector construction, oligo synthesis, and cell transfection

For circMAPKBP1 overexpression, the circMAPKBP1 sequence was synthesized with flanking restriction sites by Tsingke Biotechnology (Changsha, China) and ligated into the pCD25-cir circular RNA overexpression vector (Geneseed Biotech, Guangzhou, China) following the manufacturer's instructions. For dual-luciferase reporter assays, wild-type (WT) and binding site mutant (MT) fragments of circMAPKBP1 containing the miR-143-y binding sites were cloned into the pmirGLO vector (Promega, Madison, WI). siRNA targeting circMAPKBP1 (si-circMAPKBP1) and its negative control, as well as miR-143-y mimic, inhibitor, and corresponding negative controls (NC), were designed and synthesized by Tsingke Biotechnology (Supplementary Table 1). Transfections were performed using Lipofectamine 8000 (Beyotime, Shanghai, China) at 70%–80% cell confluence, and samples were harvested 24–48 h post-transfection.

Cell proliferation and differentiation assays

CCK-8 assay: QM7 cells were seeded in 96-well plates. Following transfection, 10 μL of CCK-8 reagent (MeilunBio, Dalian, China) was added at 12, 24, 36, and 48 h time points. After 2 h incubation, absorbance at 450 nm was measured using a multi-mode microplate reader (Bio-Rad).

EdU assay: QM7 cells were seeded in 24-well plates. At 36 h post-transfection, EdU working solution (MeilunBio) was added and incubated for 2 h. Cells were then fixed, permeabilized, and stained, and nuclei were counterstained with DAPI (Servicebio, Wuhan, China). Five random fields per well were imaged using an inverted fluorescence microscope (TS2R-FL, Nikon, Tokyo, Japan) for cell counting and statistical analysis.

Western blotting: Total protein was isolated with RIPA lysis buffer containing PMSF and protease inhibitors (Solarbio, Beijing, China). Proteins were resolved by SDS-PAGE (Beyotime) and transferred to PVDF membranes (0.45 μm; Monad, Suzhou, China). Membranes were incubated overnight at 4°C with primary antibodies against myosin heavy chain (MYHC, MF20, Developmental Studies Hybridoma Bank, Iowa, IA; 0.3 μg/mL) and GAPDH (Proteintech, Wuhan, China; 1:20,000). After incubation with secondary antibody (L3032, Signalway Antibody, Greenbelt, MD; 1:30,000), signals were developed with ECL solution (Beyotime) and detected using an Amersham Imager 680 (General Electric, Fairfield, CT).

MYHC immunofluorescence: After 4 days of induced differentiation, QM7 cells were fixed, permeabilized, and blocked. Cells were incubated with MYHC primary antibody (MF20, 3 μg/mL) overnight at 4°C, followed by Cy3-conjugated secondary antibody (A0521, Beyotime; 1:500) for 1 h. Nuclei were counterstained with DAPI (Servicebio). Images were captured using an inverted fluorescence microscope (TS2R-FL), and myotube area was quantified using ImageJ v1.8.0.

Target prediction and dual-luciferase reporter assay

RNAhybrid (v2.1) (Krüger and Rehmsmeier, 2006) and miRanda (v3.3) (John et al., 2004) were used to predict miR-143-y binding sites within circMAPKBP1. For dual-luciferase assays, WT or MT reporter vectors were co-transfected with miR-143-y mimic or NC into QM7 cells in 48-well plates. After 48 h, firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay System kit (DL101-01, Vazyme) on a Fluorescence/Multi-Detection Microplate Reader (Biotek, Winooski, VT). Results are expressed as the firefly/Renilla ratio. Each treatment group included six technical replicates. miR-143-y sequence conservation was assessed using miRBase (https://www.mirbase.org/) (Kozomara et al., 2019) and microRNAviewer (https://people.csail.mit.edu/akiezun/microRNAviewer/index.html) (Kiezun et al., 2012).

RNA-seq library preparation, sequencing, and bioinformatics analysis

Total RNA was extracted from QM7 cells transfected with miR-143-y mimic or NC (three biological replicates per group). RNA concentration and purity were evaluated using a NanoDrop 2000, integrity was assessed by agarose gel electrophoresis, and RQN values were determined using an Agilent 5300. mRNA was enriched from total RNA using Oligo(dT) magnetic beads. Following fragmentation, reverse transcription, adaptor ligation, and dUTP degradation, libraries were PCR-amplified and purified. Paired-end sequencing (PE150) was carried out on the DNBSEQ-T7 platform (BGI, Shenzhen, China).

Raw reads were filtered using Fastp (https://github.com/OpenGene/fastp) (Chen et al., 2018). High-quality reads were aligned to the quail reference genome GCA_001577835.2 (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_001577835.2/) using HISAT2 (http://ccb.jhu.edu/software/hisat2/index.shtml) (Kim et al., 2015). Gene and transcript expression levels were quantified using RSEM (http://deweylab.github.io/RSEM/) (Li and Dewey, 2011).

Differential expression analysis was performed with DESeq2 (http://bioconductor.org/packages/stats/bioc/DESeq2/), applying thresholds of |log2(FC)| > 1 and P-adjust < 0.05. Enrichment analyses of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed on the differentially expressed genes (DEGs) using Goatools (https://github.com/tanghaibao/GOatools) (Klopfenstein et al., 2018) and the scipy package in Python (https://scipy.org/install/) (Virtanen et al., 2020), respectively. After identifying the significantly downregulated genes, potential targets of miR-143-y were predicted using miRanda. Targets with a score > 100 and a minimum free energy (MFE) < −15 kcal/mol were considered as candidates. In addition to miRanda, target genes of miR-143-y were independently predicted using RNAhybrid with a MFE threshold of < −20 kcal/mol.

Statistical analysis

Data were processed using Excel 2024 and GraphPad Prism 9.0. Pairwise comparisons were performed using independent-samples Student's t-test; multiple-group comparisons were conducted using one-way ANOVA followed by Tukey's post-hoc test. Data are presented as mean ± SEM, and P < 0.05 was considered statistically significant.

Results

Identification and expression characteristics of circMAPKBP1

Analysis of our previously generated whole-transcriptome and circRNA sequencing data from quail satellite cells at different developmental stages revealed that the MAPKBP1 gene produces three circular RNAs via back-splicing (Fig. 1A). Quantifying expression abundance identified a circRNA composed of exons 26, 27, and 28—designated circMAPKBP1—that was abundantly expressed in both quail skeletal muscle and satellite cells (Fig. 1B, C). By contrast, circRNAs formed from exons 7–27 and exons 6–21 exhibited negligible expression and were excluded from further analysis. To examine the presence of MAPKBP1-derived circRNAs in other poultry species, we analyzed circRNA sequencing data from duck embryonic skeletal muscle and various chicken breeds. Although the primary sequence of circMAPKBP1 is not highly conserved, short circRNAs with high expression levels were detected in the skeletal muscle of both ducks and chickens (Fig. 1D, E).

Fig. 1.

Identification and expression characteristics of circMAPKBP1 in quail. (A) Schematic representation of the MAPKBP1 structure and the formation of three candidate circRNAs. (B) Expression levels of circMAPKBP1 in quail skeletal muscle at embryonic day 9 (E9) and E14. (C) Expression profiles of circMAPKBP1 in quail satellite cells (DA: satellite cells after differential adhesion; GM: proliferating satellite cells; DM4: differentiated myotubes after 4 d). (D-E) Expression of circMAPKBP1 in duck skeletal muscle (D) and chicken skeletal muscle (E) from different breeds (WRR: Recessive White Rock chicken; XH: Xinghua chicken). (F) PCR validation of circMAPKBP1 using convergent and divergent primers in cDNA and gDNA. (G) Subcellular localization of circMAPKBP1 in QM7 cells, with GAPDH and U6 serving as cytoplasmic and nuclear controls, respectively. (H) Relative expression of circMAPKBP1 during the differentiation of QM7 cells (GM, DM1–DM5). (I) Spatiotemporal expression profile of circMAPKBP1 in quail embryos from E7 to P1. (J) Validation of the circMAPKBP1 overexpression vector (ov-circMAPKBP1) efficiency in QM7 cells. (K) Knockdown efficiency of si-circMAPKBP1 in QM7 cells. *P < 0.05, **P < 0.01, ***P < 0.001; different letters indicate significant differences (P < 0.05).

To confirm the circular configuration of circMAPKBP1, we performed PCR with divergent primers designed to amplify across the backsplice junction. A specific product was amplified from cDNA but not from genomic DNA, verifying its circular structure (Fig. 1F). Nucleocytoplasmic fractionation assays indicated that circMAPKBP1 is predominantly cytoplasmic (Fig. 1G), consistent with a post-transcriptional regulatory function. Spatiotemporal expression profiling revealed dynamic changes in circMAPKBP1 levels during QM7 cell differentiation (Fig. 1H) and across quail embryonic development (Fig. 1I). Finally, we constructed a circMAPKBP1 overexpression vector and siRNA. qRT-PCR confirmed that the overexpression construct markedly elevated circMAPKBP1 levels, whereas the siRNA effectively depleted circMAPKBP1 in QM7 cells, establishing the experimental tools necessary for functional dissection (Fig. 1J, K).

circMAPKBP1 promotes quail myoblast proliferation

To investigate the biological function of circMAPKBP1 in myoblast proliferation, we transfected QM7 cells with the circMAPKBP1 overexpression vector (ov-circMAPKBP1) or siRNA (si-circMAPKBP1). Overexpression of circMAPKBP1 significantly upregulated the cell cycle-related genes CCNC, CCND1, and CCND2 (Fig. 2A), whereas circMAPKBP1 knockdown markedly reduced their expression (Fig. 2B), suggesting that circMAPKBP1 influences proliferation through cell cycle regulation. CCK-8 assays demonstrated that circMAPKBP1 overexpression enhanced the proliferative capacity of QM7 cells (Fig. 2C), while circMAPKBP1 depletion suppressed proliferation (Fig. 2D). EdU incorporation assays corroborated these findings: the proportion of EdU-positive cells was significantly increased upon circMAPKBP1 overexpression (Fig. 2E) and significantly decreased upon knockdown (Fig. 2F). Collectively, these data establish that circMAPKBP1 promotes the proliferation of QM7 myoblasts.

Fig. 2.

circMAPKBP1 promotes the proliferation of quail myoblasts. (A-B) Relative mRNA expression levels of cell cycle-related genes (CCNC, CCND1, CCND2, and CCNE2) in QM7 cells following overexpression (A) or knockdown (B) of circMAPKBP1. (C-D) CCK-8 cell viability assay of QM7 cells transfected with ov-circMAPKBP1/pCD25-cir (C) or si-circMAPKBP1/si-NC (D). (E-F) EdU assay in QM7 cells after overexpression (E) or knockdown (F) of circMAPKBP1. Scale bars = 100 μm (E) or 50 μm (F). *P < 0.05, **P < 0.01, ***P < 0.001; ns = not significant.

circMAPKBP1 inhibits quail myoblast differentiation

To assess the role of circMAPKBP1 in myogenic differentiation, we examined the expression of differentiation markers following circMAPKBP1 gain- and loss-of-function. Overexpression of circMAPKBP1 significantly suppressed the expression of key myogenic genes, including myocyte enhancer factor 2C (MEF2C), MYC proto-oncogene (MYC) and myogenin (MYOG) (Fig. 3A), whereas circMAPKBP1 knockdown significantly upregulated MEF2C and MYOG (Fig. 3B). Western blot analysis further showed that circMAPKBP1 overexpression reduced MYHC protein levels (Fig. 3C, D), while circMAPKBP1 depletion increased MYHC abundance (Fig. 3E, F). Immunofluorescence staining for MYHC revealed that circMAPKBP1 overexpression significantly decreased myotube area (Fig. 3G), whereas circMAPKBP1 knockdown significantly increased myotube formation (Fig. 3H). Together, these results demonstrate that circMAPKBP1 acts as a negative regulator of quail myoblast differentiation.

Fig. 3.

circMAPKBP1 inhibits myogenic differentiation of quail myoblasts. (A-B) Relative mRNA expression levels of myogenic differentiation-related genes (MEF2C, MYC, MYHC, and MYOG) in QM7 cells after overexpression (A) or knockdown (B) of circMAPKBP1. (C-D) Western blot analysis (C) and quantification (D) of MYHC protein expression in QM7 cells transfected with ov-circMAPKBP1 compared to the pCD25-cir. (E-F) Western blot analysis (E) and quantification (F) of MYHC protein expression in QM7 cells transfected with si-circMAPKBP1 compared to the si-NC. (G-H) Representative immunofluorescence images of MYHC (red) and DAPI (blue) in QM7 cells, with the corresponding quantification of myotube area after overexpression (G) or knockdown (H) of circMAPKBP1. Scale bars = 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns = not significant.

circMAPKBP1 is a direct target of miR-143-y

Bioinformatics analysis using RNAhybrid predicted miR-143-y binding sites within the circMAPKBP1 sequence with low MFE (Fig. 4A), suggesting a potential targeting relationship. Transfection of miR-143-y mimic and inhibitor into QM7 cells achieved significant overexpression and knockdown, respectively (Fig. 4B, C). Notably, miR-143-y overexpression significantly downregulated circMAPKBP1 levels (Fig. 4D), indicating that miR-143-y negatively regulates circMAPKBP1. Dual-luciferase reporter assays confirmed this interaction: the miR-143-y mimic significantly reduced luciferase activity from the WT circMAPKBP1 reporter but not the MT reporter (Fig. 4E), establishing a direct targeting relationship. Spatiotemporal expression analysis showed that miR-143-y levels peak at embryonic stage E16 (Fig. 4F) and at the DM2 stage of QM7 differentiation, followed by a progressive decline (Fig. 4G)—a pattern opposite to that of circMAPKBP1. Sequence alignment revealed that miR-143-y is highly conserved across quail and other species (Fig. 4H), suggesting an evolutionarily conserved function in skeletal muscle development. These findings support a model in which circMAPKBP1 sponges miR-143-y to modulate quail skeletal muscle development.

Fig. 4.

Verification of the targeting relationship between circMAPKBP1 and miR-143-y. (A) Bioinformatics prediction of the binding site between circMAPKBP1 and miR-143-y. (B-C) Transfection efficiency of miR-143-y mimic (B) and inhibitor (C) in QM7 cells. (D) Relative expression of circMAPKBP1 in QM7 cells following overexpression of miR-143-y. (E) Dual-luciferase reporter assay in QM7 cells co-transfected with miR-143-y mimic and luciferase reporter vectors containing wild-type (WT) or mutant (MT) circMAPKBP1 sequences. (F) Spatiotemporal expression profile of miR-143-y in quail embryos from embryonic day 8 (E8) to post-hatch day 1 (P1). (G) Temporal expression profile of miR-143-y during QM7 cell differentiation (GM, DM1–DM5). (H) Sequence conservation analysis of miR-143-y across various species. *P < 0.05, **P < 0.01, ***P < 0.001; ns = not significant; different letters indicate significant differences (P < 0.05).

miR-143-y promotes quail myoblast proliferation

To define the biological role of miR-143-y in myoblast proliferation, we transfected QM7 cells with miR-143-y mimic or inhibitor. miR-143-y overexpression significantly upregulated the cell cycle gene CCNE2 (Fig. 5A), whereas miR-143-y inhibition significantly downregulated CCND2 (Fig. 5B). CCK-8 assays demonstrated that miR-143-y overexpression enhanced QM7 cell proliferation (Fig. 5C), while miR-143-y knockdown exerted an inhibitory effect (Fig. 5D). EdU assays confirmed that the percentage of EdU-positive cells was significantly higher in the miR-143-y overexpression group (Fig. 5E) and significantly lower in the knockdown group (Fig. 5F). These results establish that miR-143-y positively regulates QM7 myoblast proliferation.

Fig. 5.

miR-143-y promotes the proliferation of quail myoblasts. (A-B) Relative mRNA expression levels of cell cycle-related genes (CCNC, CCND1, CCNE2, CCND2, and CDK2) in QM7 cells after overexpression (A) or knockdown (B) of miR-143-y. (C-D) CCK-8 cell viability assay of QM7 cells transfected with miR-143-y mimic (C) or miR-143-y inhibitor (D). (E-F) EdU assay in QM7 cells after overexpression (E) or knockdown (F) of miR-143-y. Scale bars = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001; ns = not significant.

miR-143-y promotes quail myoblast differentiation

We next examined the effect of miR-143-y on myogenic differentiation. miR-143-y overexpression significantly upregulated MEF2C and MYOG expression (Fig. 6A), whereas miR-143-y knockdown significantly downregulated both genes (Fig. 6B). Western blot analysis confirmed that miR-143-y overexpression increased MYHC protein levels (Fig. 6C, D), while miR-143-y depletion reduced MYHC abundance (Fig. 6E, F). Immunofluorescence staining further demonstrated that miR-143-y overexpression significantly increased myotube area (Fig. 6G), whereas miR-143-y knockdown impaired myotube formation (Fig. 6H). Thus, miR-143-y promotes QM7 myoblast differentiation. To further validate the ceRNA relationship at the functional level, a rescue experiment was performed by co-transfecting circMAPKBP1 overexpression vector with miR-143-y mimic in differentiating QM7 cells. Overexpression of circMAPKBP1 alone suppressed the expression of myogenic markers MEF2C and MYOG. Co-transfection of miR-143-y mimic partially restored the expression of both MEF2C and MYOG compared with the circMAPKBP1 overexpression group (Figure S1). These results indicate that miR-143-y can counteract the inhibitory effect of circMAPKBP1 on myoblast differentiation, supporting the proposed sponge mechanism.

Fig. 6.

miR-143-y promotes myogenic differentiation of quail myoblasts. (A-B) Relative mRNA expression levels of myogenic differentiation-related genes (MEF2C, MYOD1, and MYOG) in QM7 cells after overexpression (A) or knockdown (B) of miR-143-y. (C-D) Western blot analysis (C) and quantification (D) of MYHC protein expression in QM7 cells transfected with miR-143-y mimic compared to the mimic NC. (E-F) Western blot analysis (E) and quantification (F) of MYHC protein expression in QM7 cells transfected with miR-143-y inhibitor compared to the inhibitor NC. (G-H) Representative immunofluorescence images of MYHC (red) and DAPI (blue) in QM7 cells, with the corresponding quantification of myotube area after overexpression (G) or knockdown (H) of miR-143-y. Scale bars = 100 μm. *P < 0.05, **P < 0.01, ****P < 0.0001; ns = not significant.

Transcriptomic profiling of miR-143-y overexpression in QM7 cells

To gain mechanistic insight into how miR-143-y regulates QM7 cell proliferation and differentiation, we performed RNA-seq on miR-143-y mimic- and NC-transfected cells. Venn diagram analysis revealed the overlap of expressed genes between the two groups (Fig. 7A, Supplementary Table 2). Principal component analysis (PCA) showed clear separation between the two groups, confirming the reliability of the experimental design (Fig. 7B). Differential expression analysis identified 101 significant DEGs (22 upregulated, 79 downregulated) (Fig. 7C, Supplementary Table 3). KEGG pathway enrichment analysis of the downregulated genes revealed significant enrichment in the MAPK signaling pathway, ribosome, and protein processing in the endoplasmic reticulum (Fig. 7D, E, Supplementary Table 4). GO analysis indicated that downregulated genes were primarily associated with cellular processes, cellular anatomical entities, and binding activities (Fig. 7F, Supplementary Table 5). Using stringent criteria (MFE < −15 kcal/mol and alignment score > 100), we constructed an interaction network between miR-143-y and its predicted downregulated targets (Fig. 7G, Supplementary Table 6). An independent RNAhybrid analysis (MFE < −20 kcal/mol) was also performed, and the predicted targets are provided in Supplementary Table 7. These results suggest that miR-143-y acts through multiple targets, particularly by negatively regulating components of the MAPK signaling pathway, to influence QM7 cell proliferation and differentiation.

Fig. 7.

Transcriptome analysis of miR-143-y overexpression in QM7 cells. (A) Venn diagram showing the overlap of expressed genes between the miR-143-y mimic and NC groups. (B) Principal component analysis (PCA) of the transcriptomic data. (C) Volcano plot of differentially expressed genes (DEGs) between the mimic and NC groups. (D) KEGG enrichment analysis of significantly downregulated genes. (E) Chord diagram of KEGG pathway enrichment. (F) GO enrichment analysis of significantly downregulated genes. (G) Interaction network visualization between miR-143-y and its predicted downregulated target genes (thresholds: MFE < −15 kcal/mol and binding score > 100).

Discussion

Building on our previous whole-transcriptome and circRNA profiling of quail skeletal muscle (Zhang et al., 2024a), this study identifies circMAPKBP1 as a highly expressed circRNA in quail skeletal muscle and myoblasts. Functional experiments establish that circMAPKBP1 promotes QM7 myoblast proliferation while inhibiting differentiation. Mechanistically, we demonstrate that circMAPKBP1 exerts its effects by directly binding and sequestering miR-143-y. RNA-seq analysis further reveals that miR-143-y-regulated DEGs are significantly enriched in the MAPK pathway. To our knowledge, this represents the first systematic characterization and functional validation of the circMAPKBP1/miR-143-y regulatory axis in quail skeletal muscle, providing a theoretical framework and candidate molecular targets for the genetic improvement of poultry muscle development and meat quality.

MAPKBP1 encodes a scaffold protein for the JNK MAPK pathway and participates in cellular stress responses, NOD2 signaling, and inflammation regulation, with expression documented in skeletal muscle and other tissues (Lecat et al., 2012; Cohen-Katsenelson et al., 2013). The MAPK family (p38, ERK, JNK) plays central roles in myoblast proliferation, differentiation, and fusion: p38 MAPK typically promotes myotube formation and differentiation, whereas the JNK pathway can exert inhibitory or adaptive regulation depending on developmental or stress context (Zetser et al., 1999; Segalés et al., 2016). In recent years, circRNA profiling in poultry embryonic muscle has revealed numerous differentially expressed circRNAs that participate in myogenic regulatory networks, many through ceRNA mechanisms (Cai et al., 2022; Liu et al., 2023b; Zhang et al., 2024b). Hundreds of differentially expressed circRNAs have been catalogued in chicken leg muscle across embryonic stages (Wu et al., 2022). In chicken skeletal muscle satellite cells, PDLIM5 promotes proliferation and differentiation through p38-MAPK activation (He et al., 2021). Although the high expression and functional phenotype of circMAPKBP1 (pro-proliferation, anti-differentiation) may partially reflect its host gene's involvement in MAPK regulation, our data indicate that its primary mode of action is through the ceRNA mechanism. Consistent with this, other circRNAs derived from signaling scaffold or kinase-associated genes frequently reshape downstream pathway activity by sponging miRNAs (Zhang et al., 2024c). The MAPKBP1 locus in quail appears to be under post-transcriptional regulation that may modulate MAPK-related signaling output during myogenesis. miR-143-y in quail is highly homologous to mammalian miR-143-3p, with strong sequence conservation suggestive of retained core functions in vertebrate myogenesis. In mouse C2C12 myoblasts, miR-143-3p is upregulated during differentiation, and its overexpression inhibits MYOD, MYOG, MYF5, and MYHC expression while reducing myotube formation, possibly through Wnt pathway modulation (Du et al., 2016). In bovine skeletal muscle satellite cells, miR-143 similarly increases during differentiation and suppresses proliferation and differentiation by targeting IGFBP5 (Zhang et al., 2017). Recent work further demonstrates that miR-143-3p negatively regulates myogenic marker expression and myoblast fusion (Choe et al., 2025). In contrast, our data in quail QM7 cells show that miR-143-y overexpression significantly promotes both proliferation and differentiation, and its dynamic expression peak during QM7 differentiation is opposite to that of circMAPKBP1. This functional divergence across species may reflect non-conserved evolution of target gene repertoires, or avian-specific post-transcriptional regulatory networks. Although circRNAs are generally resistant to exonuclease-mediated degradation owing to their covalently closed structure, elevated miRNA levels can still promote circRNA turnover through an Ago2-dependent mechanism. For example, miR-671 has been shown to direct Ago2-mediated cleavage of the circular RNA CDR1as (Hansen et al., 2011), and similar miRNA-dependent degradation has been reported for other circRNAs. Consistent with these findings, we previously observed that overexpression of miR-22-5p reduced circFBLN2 abundance in duck myoblasts (Liu et al., 2025). The decrease in circMAPKBP1 following miR-143-y overexpression in the present study may therefore reflect an analogous process under conditions of high miRNA abundance, rather than contradicting the proposed sponge relationship. The high sequence conservation of miR-143-y and its positive regulatory role in QM7 cells provide valuable data for cross-species comparisons of myogenic regulatory mechanisms.

Prior studies have identified multiple miR-143-3p targets, including IGFBP5, Wnt pathway components, and the recently reported CHRNA1 (Zhang et al., 2017; Choe et al., 2025). Our RNA-seq analysis identified 101 significant DEGs (22 upregulated, 79 downregulated) following miR-143-y overexpression, with downregulated genes enriched in the MAPK signaling pathway, ribosome, and endoplasmic reticulum protein processing. Target prediction analysis indicated that UGGT1, ABI2, and BICD2 are potential target genes of miR-143-y among the significantly downregulated genes. Among these, ABI2 participates in actin cytoskeleton remodeling and Abl signaling, critical for myoblast migration and fusion (Grove et al., 2004); UGGT1 mediates glycoprotein quality control in the endoplasmic reticulum, particularly important under the high secretory demand of differentiating myoblasts (D’Alessio et al., 2010; Ferris et al., 2013); and BICD2 regulates intracellular trafficking (Matanis et al., 2002). The MAPK pathway is a central regulator of myogenesis: p38-MAPK directly mediates proliferation and differentiation in chicken skeletal muscle satellite cells (He et al., 2021; Ryu et al., 2022). By downregulating MAPK pathway components, miR-143-y may relieve specific inhibitory signals, thereby simultaneously supporting both proliferation and differentiation. Dual-luciferase reporter assay demonstrated that circMAPKBP1 could sponge miR-143-y, suggesting that it may regulate these target genes and the MAPK signaling pathway through a ceRNA mechanism.

Several limitations of this study should be acknowledged. First, the scarcity of quail-specific commercial antibodies and the small embryo size render primary cell isolation low-yield and technically challenging. Second, the similar regulatory effects of circMAPKBP1 and miR-143-y on QM7 proliferation raise the possibility that circMAPKBP1 may regulate proliferation through miR-143-y-independent mechanisms, or may involve additional parallel regulatory pathways, warranting further investigation. Future studies should focus on elucidating how MAPKBP1 and its derived circRNA regulate quail skeletal muscle development through MAPK-dependent and -independent pathways, and should employ in vivo models to validate the physiological relevance of the circMAPKBP1/miR-143-y axis.

Conclusion

This study identifies the circMAPKBP1/miR-143-y axis as a regulator of quail myoblast proliferation and differentiation. circMAPKBP1 promotes proliferation while inhibiting differentiation, whereas miR-143-y promotes both processes; the two molecules therefore exhibit opposing effects specifically during differentiation. Direct binding assays and reciprocal expression patterns support the conclusion that circMAPKBP1 functions as a molecular sponge for miR-143-y. RNA-seq further implicates the MAPK signaling pathway in miR-143-y-mediated regulation. These findings provide the first functional characterization of this ceRNA axis in quail skeletal muscle and supply candidate molecular targets for marker-assisted improvement of muscle yield and meat quality in poultry. Additional mechanisms may contribute to the proliferative effects of circMAPKBP1 and warrant further investigation.

Data availability

All data are available from the corresponding author upon reasonable request. The raw RNA-seq data have been deposited in the Genome Sequence Archive (GSA) under accession number CRA050571 (https://ngdc.cncb.ac.cn/gsa).

Author contributions

Biao Chen and Qingjie Zeng conceived and designed the study, supervised the research, critically reviewed the manuscript for intellectual content, approved the final version, and accept full accountability for all aspects of the work. Jing Liu and Hongxia Jiang conducted the majority of the experiments and data analysis, drafted the original manuscript, critically reviewed the manuscript, approved the final version, and accept full accountability for all aspects of the work. Xuwen Huang, Yuxiang Wang, and Guiyan Wang contributed to sample collection and experimental procedures, participated in data interpretation, critically reviewed the manuscript, approved the final version, and accept full accountability for all aspects of the work. Xiaolong Hu, Huirong Mao, and Sanfeng Liu contributed to data analysis and interpretation, critically revised the manuscript for intellectual content, approved the final version, and accept full accountability for all aspects of the work. All authors have read and approved the final manuscript and agree to be accountable for the accuracy and integrity of the work.

Disclosures

All authors declare no conflicts of interest. The authors disclose the use of ChatGPT for grammar revision and language polishing of the manuscript. After revision, all authors have read, agreed to, and take responsibility for the final version.

Supplementary files

Supplementary Table 1. Primer and oligonucleotide information.

Supplementary Table 2. Genes detected by RNA-seq after overexpression of miR-143-y.

Supplementary Table 3. Differentially expressed genes in the mimic vs. NC comparison.

Supplementary Table 4. KEGG enrichment analysis of significantly downregulated genes.

Supplementary Table 5. GO enrichment analysis of significantly downregulated genes.

Supplementary Table 6. Target prediction results for significantly downregulated genes and miR-143-y using miRanda.

Supplementary Table 7. Target prediction results for significantly downregulated genes and miR-143-y using RNAhybrid.