The miRNA expression profiles were compared in the anterior pituitary between BM and LP. The different expression miRNAs were used in this study. A volcano plot was drawn to show an overview of the differentially expressed miRNAs. The x-axis represents the fold-change of miRNA expression, and the y-axis represents the significance of miRNA expression differences between pools. Differential expression analysis revealed that 41 miRNAs (32 upregulated and 9 downregulated) were regulated significantly in BM after false-discovery rate correction (Figure 1A) (fold change ≥ 1.5, P < 0.001).

Among the 32 upregulated miRNAs, there were four novel miRNAs (Y-56, Y-84, Y-71 and Y-44) in the top 10 expression levels in BM (Supplementary Table S1). Furtherly, Y-56 was as a candidate miRNA according to Mean (BM) ≥ 6,000, Log2 (BM/LP) ≥ 1 and p ≤ 0.001 (Supplementary Table S2). The expression of Y-56 in other porcine tissues was examined. The results showed that Y-56 was highly expressed in muscle tissues (Figure 1B). Furthermore, the expression levels of Y-56 in leg muscle tissues between LP and BM were compared. The results showed that Y-56 expression in BM was significantly higher than that in LP (Figure 1C; P < 0.001).

PSCs were isolated according to the above description and then identified by pax7 (Supplementary Figure S1). Y-56 mimics were transfected into the PSCs to reveal the effect of Y-56 on PSC proliferation. The results showed that expression in the Y-56 mimics group highly increased than that in the mimics-NC group (Figure 2A). The 5-ethynyl-2-deoxyuridine (EdU) staining results showed that the number of EdU-labeled positive cells were reduced in the Y-56 mimics group compared with the mimics-NC group (Figures 2B,C; P < 0.01). Cell counting kit-8 (CCK-8) assay also showed that Y-56 overexpression hampered PSC proliferation (Figure 2D). In addition, flow cytometry assays showed that the number of PSCs were lower in the G1 phase (P < 0.01), whereas the proportion of the PSCs increased in the S phase (Figures 2E,F; P < 0.01). When Y-56 was overexpressed, the results of qRT-PCR and Western blot showed that the expression levels of cyclin-dependent kinase 4 (CDK4), proliferating cell nuclear antigen (PCNA), and cyclin D1 substantially decreased (Figures 2G–I).

PSCs were treated with Y-56 inhibitor and inhibitor-NC to further confirm that Y-56 inhibits PSC proliferation. The Y-56 expression in the Y-56 inhibitor group was dramatically reduced (Figure 3A; P < 0.001). The number of EdU-positive cells markedly increased in the Y-56 inhibitor group (Figures 3B,C; P < 0.001). The number of S-phase cells significantly increased after the suppression of Y-56 expression (Figures 3E,F). qRT-PCR and Western blot data showed that Y-56 inhibition increased the expression of CDK4, PCNA, and cyclin D1 (Figures 3G,I). Therefore, Y-56 overexpression suppressed PSC proliferation, whereas Y-56 knockdown promoted PSC proliferation.

The results in Figures 2, 3 demonstrated that Y-56 inhibited PSC proliferation. TargetScan 7.2 and miRTar Base were used to predict potential target genes to better understand its regulatory mechanisms, and IGF-1R was a candidate target gene. The seed sequence of Y-56 is presented in Figure 4A, and the secondary structures formed by Y-56 after binding to the 3′UTR of IGF-1R is shown in Figure 4B. The corresponding minimum free energy formed by Y-56 and IGF-1R was −27.4 kcal/mol. Then, wild-type IGF-1R 3′UTR (IGF-1R-WT) and mutant IGF-1R 3′UTR (IGF-1R-MT) dual-luciferase reporter vectors were constructed accordingly. The dual-luciferase activity of IGF-1R-WT and Y-56 mimics co-transfected into 293T cells was higher than that of IGF-1R-WT co-transfected with mimics-NC, and the dual-luciferase activity of IGF-1R-MT with Y-56 mimics and mimics-NC appeared to have no effect (Figure 4D; P < 0.01). Next, the mRNA expression levels of IGF-1R in different porcine tissues were detected. The results showed that IGF-1R mRNA expression level was less expressed in the muscle than in other tissues (Figure 4C). Y-56 mimics were transfected into PSCs to verify whether Y-56 regulated the expression of endogenous IGF-1R. The results of qRT-PCR and Western blot suggested that IGF-1R mRNA and protein levels were reduced in the Y-56 mimics group than in the mimics-NC group, (Figures 4E–G; P < 0.01).

A pcDNA3.1+IGF-1R expression vector was constructed to address the function of IGF-1R in the regulation of PSC proliferation. The mRNA expression of IGF-1R was higher in the pcDNA3.1+IGF-1R group than in the pcDNA3.1 group (Figure 5A; P < 0.001). The EdU staining and CCK-8 assays were carried out to evaluate PSC proliferation, and the results showed that IGF-1R overexpression significantly increased the number of cells at the proliferative stage (Figures 5B–D; P < 0.001). After IGF-1R overexpression, the number of cells in the G1 and G2 phases were significantly reduced (P < 0.01), whereas the number of S-phase cells were increased (Figures 5E,F; P < 0.01). Furthermore, the results of qRT-PCR and Western blot showed that IGF-1R overexpression also significantly increased CDK4, PCNA, and cyclin D1 expression at the mRNA and protein expression levels (Figures 5G–I; P < 0.01).

Next, siRNA technology was employed to confirm the role of IGF-1R in PSCs. Si-IGF-1R downregulated IGF-1R expression efficiently at the mRNA level (Figure 6A; P < 0.001). EdU staining (Figures 6B,C) and CCK-8 assay (Figure 6D) showed that IGF-1R inhibition hampered PSC proliferation. In addition, the number of G1-phase cells significantly increased after si-IGF-1R transfection, but the number of cells in the S and G2 phases noticeably decreased (Figures 6E,F; P < 0.01). Furthermore, when si-IGF-1R was transfected into the PSCs, the expression of CDK4, PCNA, and cyclin D1 were suppressed at the mRNA and protein expression levels (Figures 5G–I; P < 0.01). Collectively, the results showed an opposite trend in IGF-1R overexpression, and all proofs demonstrated that IGF-1R could promote PSC proliferation.

pcDNA3.1+IGF-1R and Y-56 mimics were co-transfected to verify whether Y-56 regulated PSC proliferation through IGF-1R. The EdU and CCK-8 assays proved that the decreased proliferation capabilities of PSCs caused by Y-56 overexpression were retarded by IGF-1R overexpression (Figures 7A–C; P < 0.01). Moreover, the cell cycle changes also showed that IGF-1R overexpression reversed the inhibition by Y-56 mimics (Figures 7D,E; P < 0.01), and the mRNA and protein expression levels of CDK4, PCNA, and cyclin D1 showed the same trend (Figures 7F–H; P < 0.001). Collectively, our data suggest that the suppressing role of Y-56 in maintaining PSC proliferation was dependent on IGF-1R.

Previous studies reported that IGF-1R activation could trigger the AKT and ERK pathways. Therefore, we examined the possibility that Y-56 restrains the AKT and EKR pathways by targeting IGF-1R. Protein assay showed that Y-56 overexpression remarkably reduced the phosphorylation levels of AKT and ERK without changing the total AKT and ERK expression in PSCs, whereas Y-56 inhibition promoted p-AKT and p-ERK expression (Figures 8A,B; P < 0.01). In addition, IGF-1R or si-IGF-1R was overexpressed in PSCs, and the opposite results were obtained (Figures 8C,D). The protein expression levels of p-AKT and p-ERK were recovered in Y-56 mimics-transfected PSCs after transfection with pcDNA3.1+IGF-1R (Figures 8E,F; P < 0.01). The results suggested that Y-56 exerts a negative regulatory role in PSCs by directly targeting IGF-1R and triggers the AKT and ERK signaling pathways.

Muscle, heart, liver, spleen, kindey, and cartilage tissues were collected from female BM and LP at 7 days of age, 6 pigs per breed. PSCs were isolated from porcine leg muscle tissues at 7 days of age. Following the method (38). Briefly, the collected leg muscle tissues were washed with PBS supplemented with 0.5% penicillin/streptomycin. Then, the sterile muscle tissues were minced into small pieces and digested with collagenase II (Gibco, USA) at 37°C for 3–6 h. The suspension was filtered through a 70 μm cell strainer and centrifuged at 3000 rpm for 15 min at room temperature. The precipitation was resuspended in PBS, filtered by a 40 μm filter, and centrifuged at 3,000 rpm for 15 min. The supernatant was removed, and the precipitation was resuspended in complete culture medium. The cells were transferred to the culture dish. After 2 h, the suspension was transferred to new culture dish to separate and purify the satellite cells. All animal experiments were performed in accordance with the rules and regulations of the Animal Care and Experimentation Committee of Jilin University (Changchun, China).

The 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Sigma, USA) supplemented with 10% FBS (BI, Israel) and 0.5% penicillin/streptomycin (Gibco, USA) in an incubator with 5% CO₂ at 37°C. The PSCs were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, Sigma, USA) with 20% FBS, 0.5% penicillin/streptomycin and 0.5% chick embryo extract (CEE) and were cultured in an incubator with 5% CO₂ at 37°C.

PSCs were cultured in 96-well plates, treated with 4% formaldehyde for 15 min, and permeabilized with 0.1% Triton X-100. Next, the cells were blocked in 10% fetal bovine serum (FBS) for 30 min and sequentially incubated overnight with anti-Pax7 (Abcom, US; 1:100). Thereafter, the cells were treated with Alexa Fluor 594-conjugated anti-mouse IgG (Bioworld Technology, USA; 1:200) according to the manufacturer's instructions. Finally, the nuclei were stained with DAPI (Beyotime, Shanghai, China) for 5 min. Cell images were captured with an inverted fluorescence microscope (Nikon, Japan), and the data were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

For psiCHECK-2 dual-luciferase reporter vector: The 3′UTR fragment of IGF-1R containing the Y-56 binding site was cloned into the psiCHECK-2 dual-luciferase reporter vector, and the fragments named IGF-1R-WT and IGF-1R-MT were synthesized by Genewiz (Beijing, China). The sequences are listed in Table 1.

For IGF-1R overexpression vector: The full coding sequence of IGF-1R was synthesized by Genewiz (Beijing, China) and cloned into the pcDNA3.1(+) expression vector.

The RNA oligonucleotides used in this study, including Y-56 mimics, mimics-NC, Y-56 inhibitor, inhibitor-NC, si-IGF-1R, and si-NC, were obtained from Gene Pharma (Shanghai, China) and are shown in Table 1. The oligonucleotides and plasmids were transfected using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's protocol with at least three replications.

The psiCHECK-2 dual-luciferase reporter vector (200 ng) containing IGF-1R-WT or IGF-1R-Mut fragment was co-transfected with Y-56 mimics or mimics-NC duplexes (100 nM) into 293T cells in a 96-well plate with six independent repeats. After 48 h of transfection, firefly and Renilla luciferase activities were measured in a Fluorescence/Multi-Detection Microplate Reader (BioTek, Winooski, VT, USA) using a Dual-GLO Luciferase Assay System Kit (Promega, Madison, WI, USA).

The RNA samples were obtained from the different tissues of 6 pigs each breed. Total RNA was isolated from the collected tissues and PSCs after transfection using RNAiso Plus (Takara, Ostu, Japan). cDNA synthesis for mRNA was carried out using the PrimeScript RT Reagent Kit (Takara, Japan). Reverse transcription reaction for miRNA was performed with the PrimeScript RT Reagent Kit (Takara, Japan) using the specific Bulge-loop miRNA qRT-PCR primers for Y-56 and U6 that were synthesized by Genewiz (Beijing, China). qRT-PCR reactions were carried out using the SYBR Green PCR Master Mix (Thermo Fisher Scientific, MA) according to the manufacturer's instructions on an ABI PRISM 7900HT thermocycler (Applied Biosystems, Foster City, CA, USA). The comparative Ct (2^−ΔCt) method was used in qRT-PCR data analysis. All amplifications were performed in triplicate, and the mRNA levels of the target genes were normalized using the GAPDH gene as the internal reference. The primers used for qRT-PCR are listed in Table 2.

The cell proliferation rate of PSCs was assessed at 0, 24, 48, 72, and 96 h after transfection by CCK-8 (Beyotime, China). Briefly, the PSCs were seeded in a 96-well format at a density of 5000 cells/well and then transfected with different miRNAs, vectors, or siRNAs as described above. CCK-8 solution (10 μL) was then added to each well containing 100 μL of the culture medium, and then the plate was incubated for 30 min at 37°C. The absorbance was read at a wavelength of 450 nm using a microplate reader (Tecan, Switzerland).

The PSCs were seeded in 96-well plates at a concentration of 2 ×10³ per well. Then, the PSCs were treated with Y-56 mimics, Y-56 inhibitor, pcDNA3.1+IGF1R, and si-IGF-1R for 48 h and incubated with 50 μM EdU (RiboBio, Guangzhou, China) for 3 h. The cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton-X 100 for 20 min. At the end of each step, the cells were washed twice with PBS for 5 min. According to the kit, the cells were incubated in a mixture of reagents for 30 min. The nuclei were stained with DAPI for 5 min. The stained cells were finally observed on an inverted fluorescence microscope (Nikon, Japan), and the data were analyzed in ImageJ (National Institutes of Health, Bethesda, MD, USA).

The PSCs were cultured in a 6-well culture plate at a density of 4 × 10⁵ per well and treated with Y-56 mimics, Y-56 inhibitor, pcDNA3.1+IGF1R, and si-IGF-1R for 48 h. Then, the cells were digested with 0.25% trypsin and terminated with Dulbecco's modified Eagle medium/Nutrient Mixture F-12 containing 20% FBS. The cells were collected and fixed in cold 70% ethanol overnight at 4°C. The cells were washed twice and stained with 50 mg/mL propidium iodide (PI) for 30 min. Finally, the cell cycle was analyzed by flow cytometry (Becton Dickinson, USA).

The PSCs were harvested after 48 h of transfection to extract proteins using a lysis buffer (KeyGen Biotech, China) complemented with protease and protein phosphatase inhibitor on ice. Protein concentration was determined using Bicinchoninic Acid Protein Assay Kit (Beyotime, China), and 30 μg protein per sample was loaded and separated using 5% stacking gel and 12% separating gel. The separated proteins were transferred to polyvinylidene difluoride membrane, which was then blocked in 5% bovine serum albumin for 2 h at room temperature and incubated with primary antibodies at 4°C overnight. The membranes were washed thrice (10 min each) in tris-buffered saline with Tween and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG (Bioworld Technology, USA, 1:2,000) for 2 h at room temperature.

The Western blots were visualized by Enhanced Chemiluminescence (Bioworld, USA) performed on a Bio-Rad Gel Doc XR instrument (Bio-Rad, Hercules, CA, United States). Thereafter, the gray value of the target strip was analyzed using Geno Sens Analysis software, and the expression of the target proteins was analyzed by the ratio of the gray value of the target strip to the gray value of β-actin.

The primary antibodies used in this study were as follows: IGF-1R rabbit polyclonal antibody (Abcom, US, 1:1000), PCNA rabbit polyclonal antibody, CDK4 rabbit polyclonal antibody, cyclin D1 rabbit polyclonal antibody (Cell Signaling Technology, USA, 1:2,000), p-AKT rabbit polyclonal antibody, AKT rabbit polyclonal antibody, p-ERK rabbit polyclonal antibody, ERK rabbit polyclonal antibody, and β-actin polyclonal antibody (BBI, China, 1:2,000).

All experimental results are presented as the mean ± standard error of the mean (SEM) of at least three independent replications. The statistically significant difference between groups was tested by one-way ANOVA using GraphPad Prism version 6.01. P < 0.05 was considered statistically significant (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001).