Skeletal C2C12 myoblasts (CRL-1772; ATCC) were removed from the liquid nitrogen tank and allowed to resuscitate. The cells were then cultured in a carbon dioxide incubator at 37°C with 5% CO₂. For experiments, only those cells that had been passaged less than 30 times were used. The cells were exposed to a high-sugar medium containing 2% horse serum for induction of myogenic differentiation. The cell lines present in this study were obtained from American Type Culture Collection (ATCC).

miR-182-5p mimic and miR-182-5p inhibitor were purchased from RiboBio (Guangzhou, China). The C2C12 cells were cultured in 6-well culture plates and grown to approximately 50–60% confluence, after which they were transfected with miR-182-5p mimic or miR-182-5p inhibitor using the riboFECTTMCP reagent (Ribobio, GuangZhou), following the manufacturer’s instructions.

Cells were transfected for 24 h and then the transfection mixture was replaced with a myogenic differentiation medium to induce myogenic differentiation on days 2, 4, 6, and 8. The degree of myogenic differentiation of Giemsa-stained C2C12 cells was observed under a microscope according to the manufacturer’s instructions. As described below, the cells were washed with phosphate-buffered saline (PBS), treated with 500 μL of anhydrous methanol for 10 min, and then stained with 400 μL of Giemsa dye for 1 h. Subsequently, the stained cells were washed with distilled water to remove the excess dye, observed under a microscope, and photographed.

Cells were seeded into 4-well plates and washed with PBS. The assay was performed according to the manufacturer’s instructions. The cells were fixed with 200 μL of cell tissue fixative for 15 min, permeabilized with 200 μL of 0.5% TritonX-100 for 10 min, blocked with 200 μL of 1% bovine serum albumin for 30 min, and then incubated overnight with 100 μL of primary antibody at 4°C. Thereafter, these cells were incubated with 100 μL of secondary antibody for 1 h and stained with DAPI. Before incubation with each new reagent, the cells were washed three times with 200 μL PBS for 5 min each time. The stained cells were with antifluorescence quenching sealer and observed under a confocal microscope.

Total RNA, including miRNAs, was extracted from C2C12 cells using the PrimeScript RT reagent Kit with gDNA Eraser kit, following the manufacturer’s instructions. The concentration of total RNA was measured using a microspectrophotometer, and 100 ng RNA was reverse-transcribed to cDNA using the Thermal Sciences First Strand cDNA Synthesis Kit. The cells were induced in 6-well cell culture plates, and the myogenic differentiation medium was discarded. The cells were collected in trypsin-free tubes via trypsin digestion. RNA was extracted on ice in an enzyme-free environment. The PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) was used for miRNA reverse transcription. MicroRNA reverse transcription primers were synthesized by Rebo. Real-time PCR of the marker gene and miR-182-5p was performed using TB Green Premix Ex Taq II (Tli RNase H Plus). The primers used in the qRT-PCR experiments were synthesized by BGI.

Proteins were extracted using a Mammalian Protein Extraction Kit. The Mammalian Protein Extraction Reagent was cooled in advance of protein extraction. The bicinchoninic acid assay was used to determine the protein concentration for sample loading. Protein samples separated via SDS-PAGE were transferred onto nitrocellulose membranes using the wet transfer method. The membranes were then blocked by incubating them in 5% skim milk powder at 37°C for 1 h on a shaker. The following primary antibodies were used: Anti-GAPDH (Ab9485, Abcam), Anti-MYOD (PA5-23078, Thermo Fisher), and Anti-α-Tublin (11224-1-AP, Proteintech). Heavy chain (Ab11083, Abcam), primary antibodies were specifically conjugated to the target protein and incubated overnight at 4°C. The membranes were then rinsed three times with TBST and incubated with horseradish peroxidase-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L) (SA00001-2, Proteintech) for 1 h at room temperature. The blots were developed using Pierce™ ECL Western Blotting Substrate and visualized with TANon-5200. The ImageJ software (NIH, USA) was used for densitometric analysis to determine the protein expression.

A dual-luciferase reporter vector (E1960; Promega) containing target gene-binding site (s) was constructed. This vector was subsequently transfected into 293 T cells using Lipofectamine^® 2000 Reagent via liposome-mediated transfection. The luciferase activity was measured using a Dual-Luciferase Reporter Assay System. Firefly/Renilla ratios were normalized to the MUT + NC group (set as 100%).

Statistical analyses were performed using GraphPad Prism 9.5 (GraphPad Software). Between two groups, differences were assessed by unpaired two-tailed Student’s t-test. For multi-group comparisons, one-way ANOVA with Tukey’s post hoc test was applied. Data represent mean ± SEM of three biologically independent experiments. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Prior to transfection experiments, we systematically determined the optimal concentration of the miR-182-5p mimic via dose–response screening. Quantitative analysis revealed that transfection with 50 nM miR-182-5p mimic resulted in significantly higher relative expression levels of miR-182-5p than achieved with other concentrations (p < 0.001), establishing this concentration as optimal for subsequent C2C12 cell experiments (Figure 1A). To investigate the role of miR-182-5p in myogenic differentiation, we performed a time-course analysis using Giemsa staining. During days 2–6 of differentiation, the mimics-transfected cells exhibited greater length and larger diameter myotubes than the controls (Figures 1B–F). Molecular analysis revealed a significant upregulation of myogenic differentiation markers at both the transcriptional and translational levels. qRT-PCR showed elevated mRNA expression of Myh4, Myod, and Myog in transfected cells throughout the differentiation timeline (days 2–8; Figures 1G–I). Western blot analysis confirmed the corresponding increase in MYH4 and MYOD protein levels, with statistically significant differences maintained across all time points (Figures 1J–L). Immunofluorescence analysis revealed that miR-182-5p-transfected cells consistently developed longer and thicker myotubes compared with controls from days 2 to 8 of differentiation (Figure 2). These results established that miR-182-5p overexpression enhanced the myogenic differentiation of C2C12 cells.

We further identified 100 nM as the optimal miR-182-5p inhibitor concentration that was most effective in suppressing the expression of miR-182-5p in C2C12 cells (Figure 3A). While no significant morphological changes were observed on day 2 post-transfection, the inhibitor-transfected cells progressed to thinner myotubes than the controls by day 4 and 6 (Figures 3B–D). Quantitative morphometric analysis using the ImageJ software further revealed length and diameter were significantly reduced in miR-182-5p-suppressed cells from days 2 to 6 of differentiation (Figures 3E,F). Molecular characterization revealed the coordinated downregulation of myogenic markers across multiple layers of regulation. mRNA levels of Myh4, Myod, and Myog in inhibitor-transfected cells were significantly reduced throughout the differentiation period (days 2–8; Figures 3G–I). Correspondingly, western blot analysis confirmed marked reduction in MYH4 and MYOD protein expression, consistent with the observed alteration in the morphology of cells (Figures 3J–L). This phenotypic impairment was corroborated by immunofluorescence analysis, which revealed reduced myotube length and diminished diameter in the inhibitor-treated groups (Figure 2). Collectively, these findings demonstrated that miR-182-5p suppression attenuates the myogenic differentiation of C2C12 cells.

The candidate target genes predicted through multiple databases (ENCORI, miRDB, miRWalk, RNAhybrid, and TargetScan7.2) include: Arf4, Rere, Zbtb7a, Aebp2, Qk, Nuak1, Dab2ip, Casp2, Paip2, Tns3, Jmjd1c, 4930402H24Rik. Based on the binding probability scores of miR-182-5p target genes predicted by RNAhybrid, we selected Zbtb7a, Qk, Nuak1, Dab2ip, Casp2, and Tns3 for dual-luciferase assays. The results confirmed that Zbtb7a is a direct target of miR-182-5p. Cotransfection of cells with pmir-GLO-Zbtb7a-WT and miR-182-5p mimic resulted in a significant reduction in the Firefly/Renilla ratio, confirming the binding specificity (Figures 4A–D). To determine whether miR-182-5p regulates Zbtb7a expression during myogenesis, we evaluated Zbtb7a expression during the differentiation of C2C12 cells transfected with miR-182-5p mimic. Consistent with the identification of Zbtb7a as the direct target of miR-182-5p, qRT-PCR showed that the relative mRNA expression of Zbtb7a was significantly lower in cells transfected with miR-182-5p mimic than in the miR-182-5p mimic NC group (Figure 4E). These changes in Zbtb7a expression were reflected at the protein level, as determined using western blotting (Figures 4F,G), confirming that miR-182-5p negatively regulates Zbtb7a expression during the myogenic differentiation of C2C12.

To further identify Zbtb7a as a target gene for miRNA-182-mediated regulation of myogenic differentiation, its expression was knocked down using Zbtb7a-siRNA. Zbtb7a-siRNA-transfected C2C12 cells exhibited significantly increased induction of C2C12 myogenic differentiation compared with Zbtb7a-siRNA NC-transfected C2C12 cells (Figure 5).