Samples of the LDM and IMF were collected from three male LW and three BM at 7 days of age. Porcine skeletal muscle satellite cells (PSCs) were isolated from porcine leg muscle tissues using the method described by Li et al [28]. All animal experiments received approval from the Experimental Animal Center of Jilin University and were conducted in accordance with the ethical guidelines outlined in the Guide for Ethical Use of Animals provided by the Animal Welfare and Research Ethics Committee of Jilin University (approval no. KT202003090).

The 3T3-L1, C2C12, and 293T cell lines used in the experiments were kindly provided by Prof. Hongsheng Ouyang from the College of Animal Science at Jilin University. These cell lines were cultured in a CO₂ incubator at 37°C using Dulbecco’s Modified Essential Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with penicillin, streptomycin (Gibco), and 10% fetal bovine serum (FBS; Gibco), referred to as M1 medium. PSCs were maintained in DMEM/F-12 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 20% FBS, 0.5% penicillin/streptomycin, and 0.5% chick embryo extract in an incubator with 5% CO₂ at 37°C.

For adipogenic differentiation, 3T3-L1 cells were cultured until 90% confluence (day 0), after which the initial differentiation medium (stage I) was replaced with M1 medium containing 10 μg/mL insulin (Yuanye Bio-Technology, Shanghai, China), 1 μM dexamethasone (Sigma-Aldrich), 200 μM indomethacin (Yuanye Bio-Technology), and 0.5 mM IBMX (Sigma-Aldrich). On day 2, the medium was changed to DMEM with 10% FBS and 10 μg/mL insulin. From day 9 onwards, insulin was excluded, and cells were maintained in M1 medium with medium replacement every 2 days.

For myogenic differentiation, C2C12 cells were cultured in DMEM supplemented with 2% horse serum (Solarbio, Beijing, China) once they reached 90% confluence. The culture medium was replaced every 48 h. After 6 days of differentiation, visible myotubes were formed. These cells were then treated with DMEM supplemented with 0.5% bovine serum albumin (Beyotime Biotechnology, Shanghai, China) and 500 μM oleic acid for 24 h to induce lipid accumulation within the myotubes.

For the psiCHECK-2 dual-luciferase reporter vector, the 3′UTR fragment of FBXO11, which contains the miR-21-5p binding site, was cloned into the psiCHECK-2 vector. These fragments, designated FBXO11-WT and FBXO11-Mut, were provided by Sangon Biotech (Shanghai, China). RNA oligonucleotides used in this study, including miR-21-5p mimic, mimic-negative control (NC), miR-21-5p inhibitor, inhibitor-NC, si-FBXO11, and si-NC, were sourced from RiboBio Co., Ltd. (Guangzhou, China). Cells were seeded in six-well plates and cultured for 12 h prior to transfection with the mimics and their respective controls. Transfection of oligonucleotides and plasmids was conducted using LipoPlus Reagent (SageCreation, Beijing, China) following the manufacturer’s protocol, with all experiments performed in at least three independent replicates.

For luciferase reporter analysis, 293T cells were seeded into 96-well plates and cultured until reaching 80% confluence. Cells were then co-transfected with either FBXO11-WT or FBXO11-Mut constructs alongside ssc-miR-21-5p mimics or controls. After 48 h of transfection, firefly, and Renilla luciferase activities were quantified using a Fluorescence/Multi-Detection Microplate Reader (BioTek, Winooski, VT, USA) in conjunction with a Dual-Luciferase Reporter Gene Assay kit II (Beyotime Biotechnology). The luciferase activity was normalized to the corresponding firefly luciferase activity to ensure consistency across samples.

C2C12 cells were cultured in 24-well plates lined with cell slides, then transfected with ssc-miR-21-5p mimic, mimic-NC, ssc-miR-21-5p inhibitor, inhibitor-NC, si-FBXO11, or siRNA-NC. These cells were subsequently induced for myogenic differentiation over 6 days. After the differentiation period, the medium was removed, and cells were rinsed three times with PBS before being fixed with 4% paraformaldehyde for 20 min at room temperature. Following fixation, cells were washed thrice with PBS and permeabilized using 0.1% Triton X-100 for 30 min. The cells were then blocked with 10% FBS for 30 min and incubated overnight with anti-Myosin Heavy Chain (1:200; R&D Systems, Minneapolis, MN, USA). After three 5-min PBS washes, the cells were incubated with secondary antibodies (FITC-labeled Goat Anti-Mouse IgG (H+L), 1:100; Bioworld, St. Louis Park, MN, USA) for 2 h at room temperature, followed by additional PBS washes. Nuclei were stained with DAPI (Beyotime Biotechnology) for 5 min. Images were captured using an inverted fluorescence microscope (Nikon, Tokyo, Japan), and the data were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

C2C12 cells were seeded into 96-well plates at a density of 2000 cells per well and transfected with miR-21-5p mimic, mimic-NC, miR-21-5p inhibitor, inhibitor-NC, si-FBXO11, or siRNA-NC. Cell proliferation was evaluated at 0, 24, 48, 72, and 96 h using a Cell Counting Kit-8 (CCK-8) (SAINT-BIO, Shanghai, China) according to the manufacturer’s instructions.

Lipid droplet formation in cultured cells was assessed using Oil Red O staining. Differentiated C2C12 myotubes were treated with oleic acid for 24 h and subsequently stained. Cells were first rinsed with PBS and fixed with 4% paraformaldehyde (Beyotime Biotechnology) for 20 min. Following fixation, the cells were briefly washed in 60% isopropanol and stained with Oil Red O working solution (Solarbio) for 20 min. Stained cells were washed with 60% isopropanol for 30 s and rinsed 3 to 5 times with PBS. To quantify lipid content, Oil Red O was extracted using isopropanol, and the optical density was measured at 510 nm. After washing with Oil Red O buffer for 1 min, cells were covered with PBS and observed under a microscope (Nikon).

Total RNA was isolated from tissues or cells using TRIzol reagent (Takara, Kusatsu, Japan). For mRNA, cDNA synthesis was performed using the TransScript Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen Biotech, Beijing, China). miRNA reverse transcription was conducted using the miRNA First Strand cDNA Synthesis Tailing Reaction Kit (Sangon Biotech). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the TransStart Green qPCR SuperMix (TransGen Biotech) on an ABI PRISM 7900HT thermocycler (Applied Biosystems, Foster City, CA, USA). The comparative Ct (2^−ΔΔCt) method was employed for data analysis. All reactions were performed in triplicate. For normalization, GAPDH served as the internal reference for mRNA analysis, whereas U6 was used as the internal control for miRNA quantification. Primers used in the qRT-PCR experiments are detailed in Supplement 1.

The procedure for Western Blot analysis was adapted slightly from previously established protocols. Primary antibodies utilized in this study included FBXO11 (1:500; Santa Cruz Biotechnology, CA, USA), MyoD (1:500; Santa Cruz Biotechnology), and GAPDH (Bioworld, 1:10,000). The secondary antibody used was an horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Bioworld, 1:10,000).

All experiments were performed in three independent replicates, and the results are presented as mean±standard error of the mean. Statistical analysis was conducted using GraphPad Prism Software (Version 8; La Jolla, CA, USA). Comparisons between two groups were made using Student’s t-test to determine statistical significance. A p-value of <0.05 was considered to be statistically significant (* p<0.05, ** p<0.01, *** p< 0.001).

The pig BodyMap transcriptome dataset (GEO: GSE162147) [29] demonstrated that miR-21-5p was abundantly and differentially expressed across various porcine tissues, including skeletal muscle and adipose tissues at distinct anatomical sites (Figures 1A, 1B). Additionally, analysis of miR-21-5p expression during the differentiation of porcine intramuscular adipocytes (GEO: GSE193608) [19] revealed significant downregulation (Figure 1C). The expression of miR-21-5p in LDM and IMF and PSCs of two pig breeds: LW and BM, was compared. The results showed that miR-21-5p was differentially expression in these tissues and cells between the breeds (Figures 1D, 1E). The mature sequence of miR-21-5p is highly conserved among mammals, with notable sequence similarity across human, mouse, and pig species (Figure 1F). To further examine its expression patterns during differentiation, C2C12 cells and 3T3-L1 cells were used as models. In C2C12 cells, miR-21-5p expression progressively increased during myogenic differentiation. Conversely, in 3T3-L1 cells, miR-21-5p exhibited a three-phase expression change with an overall decreasing trend, mirroring the pattern observed in porcine intramuscular adipocytes (Figures 1G, 1H). These results highlighted that miR-21-5p shows contrasting expression trends during myogenic and adipogenic differentiation, suggesting distinct roles in regulating skeletal muscle development and IMF deposition.

Mouse C2C12 cells were utilized as a model system because primary porcine cells exhibit low transfection efficiency, and no porcine cell lines are currently available. Transfected C2C12 cells with synthetic mimics or inhibitors of miR-21-5p were analyzed for miR-21-5p expression and several cell cycle-related genes using RT-qPCR. Additionally, CCK-8 assays were conducted to evaluate the proliferation of C2C12 cells at various time points post-transfection. The results demonstrated that transfection with miR-21-5p mimics or inhibitors significantly altered miR-21-5p expression levels (Figures 2A, 2D). Overexpression of miR-21-5p markedly increased the mRNA levels of PCNA, CCND1, and CDK6, thereby promoting C2C12 cell proliferation (Figures 2B, 2C). Conversely, knockdown of miR-21-5p reduced the mRNA expression levels of PCNA, CDK4, and CDK6, resulting in inhibited proliferation of C2C12 cells (Figures 2E, 2F). These findings indicate that miR-21-5p acts as a positive regulator of myoblast proliferation in vitro.

To investigate the role of miR-21-5p in myoblastic differentiation, C2C12 cells were transfected with miR-21-5p mimics or inhibitors and induced to differentiate in a low-serum medium. After 6 days, myotube formation was evaluated using immunofluorescence staining for myosin heavy chain (MyHC), while miR-21-5p levels and the expression of myogenesis-related genes were analyzed by RT-qPCR and western blotting. Overexpression or inhibition of miR-21-5p resulted in significant upregulation or downregulation of miR-21-5p expression, respectively (Figure 3A). Compared to NC, miR-21-5p overexpression enhanced myotube formation, whereas inhibition impaired it (Figures 3F, 3G). Additionally, miR-21-5p overexpression elevated the mRNA levels of MyoD, MyoG, and MyHC (Figure 3B), as well as the protein expression level of MyoD tended to increase (Figure 3D), while its inhibition reduced these markers (Figures 3C, 3E). These findings suggest that miR-21-5p positively regulates myogenic differentiation in C2C12 cells by modulating the expression of myogenesis-related genes.

Oleic acid has been shown to influence the lipid deposition in C2C12-derived myotubes. The effect of ssc-miR-21-5p on oleic acid-induced lipid accumulation in differentiated C2C12 cells was examined. At day 6 of differentiation, C2C12 cells were transfected with miR-21-5p mimics or inhibitors and exposed to 500 μmol/L oleic acid. Lipid accumulation was assessed using oil red O staining, and the expression of lipogenic factors was measured by RT-qPCR. Transfection with miR-21-5p mimics or inhibitors altered the expression of miR-21-5p when compared with the NC (Figure 4A). The results of oil red O staining showed that overexpression of miR-21-5p inhibited intracellular lipid deposition in myotubes and significantly decreased intracellular triglyceride concentrations (Figures 4D, 4E). However, the amount of lipid and the intracellular triglyceride concentration was increased after knockdown of miR-21-5p (Figures 4F, 4G). RT-qPCR analysis revealed that miR-21-5p overexpression significantly downregulated the mRNA levels of FABP4, FAS, CEBPα, and CEBPβ (Figure 4B), while its inhibition upregulated FABP4, PPARγ, CEBPα, and CEBPβ (Figure 4C). These findings demonstrate that miR-21-5p inhibits oleic acid-induced lipid deposition in C2C12 myotubes.

MiR-21-5p plays a critical role in the proliferation and differentiation of C2C12 cells as well as lipid deposition; however, its underlying regulatory mechanism remains unclear. To investigate this mechanism, potential target genes of miR-21-5p were identified using four miRNA target gene prediction tools: TargetScan, StarBase, miRDB, and miRWalk. The intersection of these prediction results was analyzed (Figure 5A). Homology analyses of porcine and mouse sequences were conducted for regions before and after 100 bp of their 3′UTR binding sites, resulting in the selection of three candidate target genes with high homology and binding sites within 300 bp: PBRM1, PDCD4, and FBXO11. To evaluate the targeting potential of these genes, miR-21-5p mimics and inhibitors were transfected into PSCs (Figure 5B). Among the candidate genes, FBXO11 exhibited the most significant downregulation or upregulation compared to NC (Figure 5C). A dual luciferase reporter gene assay was conducted to confirm whether FBXO11 is a direct target gene of miR-21-5p (Figure 5D). Additionally, the expression of FBXO11 was assessed by overexpressing or knocking down miR-21-5p in C2C12 cells (Figures 2A, 2D). Western blot analysis revealed that miR-21-5p overexpression significantly suppressed FBXO11 expression, while knockdown of miR-21-5p enhanced FBXO11 expression (Figure 5E). These findings collectively confirm the targeting relationship between miR-21-5p and FBXO11.

Previous studies have suggested that FBXO11 plays a pivotal role in the development of skeletal muscle [24,25]. To investigate the effect of FBXO11 on C2C12 cell proliferation, three distinct si-FBXO11 constructs were transfected into C2C12 cells. Among these, si-FBXO11-3 resulted in the most pronounced reduction in FBXO11 mRNA and protein expression (Figures 6A, 6B). Following transfection with si-FBXO11-3, a CCK-8 assay was performed to evaluate cell proliferation at different time points, while RT-qPCR was used to analyze the expression of cell cycle-related genes. The results indicated that FBXO11 silencing significantly upregulated the mRNA expression of PCNA, CDK4, and CDK6, thereby enhancing C2C12 cell proliferation (Figures 6C, 6D). To explore the role of FBXO11 in myogenic differentiation, C2C12 cells transfected with si-FBXO11 or siRNA-NC were induced to differentiate in a low-serum medium for six days. Transfection efficiency and myotube formation were assessed using MyHC immunofluorescence staining, while RT-qPCR and western blotting were used to evaluate the expression of myogenesis-related genes. FBXO11 silencing significantly reduced its expression levels (Figure 6E) and enhanced myotube formation relative to the control (Figure 6H). Moreover, FBXO11 inhibition markedly increased the mRNA levels of MyoG, MyoD, and MyHC (Figure 6F), as well as the protein level of MyoD (Figure 6G). These results demonstrate that FBXO11 suppression promotes C2C12 proliferation and myogenic differentiation in vitro. Interestingly, these effects contrast with the role of miR-21-5p in regulating C2C12 cell proliferation and differentiation, suggesting that miR-21-5p exerts its influence on these processes by targeting FBXO11.

To investigate whether miR-21-5p inhibits lipid deposition in myocytes by targeting FBXO11 and to validate the role of FBXO11 in lipid deposition in C2C12 myocytes induced by oleic acid, si-FBXO11, and siRNA-NC were transfected into C2C12 cells undergoing myogenic differentiation for six days, followed by oleic acid induction. Transfection efficiency was assessed, and Oil Red O staining was employed to evaluate lipid deposition, alongside the measurement of mRNA expression levels of adipogenesis-related factors. si-FBXO11 effectively downregulated FBXO11 mRNA expression compared to NC (Figure 7A). Oil Red O staining revealed that FBXO11 inhibition reduced intracellular lipid accumulation in myotubes and significantly lowered intracellular triglyceride levels (Figures 7C, 7D). Moreover, RT-qPCR analysis demonstrated that suppressing FBXO11 expression markedly decreased the mRNA levels of adipogenesis-related genes, including FABP4, CEBPα, and CEBPβ (Figure 7B). These results indicate that FBXO11 silencing effectively inhibits lipid accumulation induced by oleic acid in myotubes derived from C2C12 myoblasts.