Animals and cells
The muscle, heart, liver, spleen, kindey and cartilage tissues were collected from the female Bama Xiang pig (BM) and the Landrace pig (LP) at 7-days age, respectively. The porcine skeletal muscle satellite cells (PSCs) were isolated from porcine leg muscle tissues at 7 days of age, following the method [22]. Briefly, the collected leg muscle tissues were washed with PBS supplement with 0.5% penicillin/streptomycin, then the sterile muscle tissues were minced into small pieces and were 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, then we resuspended precipitation by PBS, and filtered by 40 µm again, next centrifuged at 3000 rpm for 15 min. After removing the supernatant, we resuspended precipitation by complete culture medium, next the cells was transferred to the culture dish. To separate and purify satellite cells, after 2 h, the suspension was transferred to new culture dish. All animal experiments were performed in accordance with the rules and regulations of the Animal Care and Experimentation Committee of Jilin University (Changchun, China).
Cell Culture
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.
Immunofluorescence Staining
The PSCs cultured in 96-well plates were first treated with 4% formaldehyde for 15 min and then permeabilized by 0.1% Triton X-100. Next, after being blocked in 10% FBS for 30 min, the cells were sequentially incubated overnight with anti-Pax7 (Abcom, US; 1:100). Thereafter, the cells were treated with an 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. Then the images were captured with an inverted luorescence microscope (Nikon, Japan) and the data were analyzed in Image J software (National Institutes of Health, Bethesda, MD, USA).
Plasmid Construction and Cell Transfection
The 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 fragment named IGF-1R-WT and IGF-1R-MT were synthesized by Genewiz (Beijing, China). The sequences are listed in Table 1.
The IGF-1R overexpression vector: The full coding sequence (CDS) 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 were shown in Table 1. The oligonucleotides and plasmids were transfected using Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s protocol with at least three replications.
Table 1 Oligonucleotide sequences used in this study
Fragment Name
|
Sequence (5’ to 3’)
|
Y-56 mimics
|
AGAGGAGGAAGAGGAGGAGGA
CUCCUCCUCUUCCUCCUCUUU
|
mimics-NC
|
UUCUCCGAACGUGUCACGUTT
ACGUGACACGUUCGGAGAATT
|
Y-56 inhibitor
|
UCCUCCUCCUCUUCCUCCUCU
|
inhibitor-NC
|
CAGUACUUUUGUGUAGUACAA
|
si-IGF-1R
|
CCUCGAGCUAGAGAACUUTT
AAGUUCUCUAGCUCCGAGGTT
|
Dual-Luciferase Reporter Assay
The psiCHECK-2 dual-luciferase reporter vector (200 ng) containing a wild-type or mutant IGF-1R 3’UTR 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, the 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).
Quantitative real-time PCR
Total RNA was isolated from the collected tissues and the PSCs after transfection using RNAiso Plus (Takara, Ostu, Japan). cDNA synthesis for mRNA was carried out using the PrimeScript RT reagent Kit (Takara, Japan). The reverse transcription reaction for miRNA was performed with the PrimeScript RT reagent Kit (Takara, Japan) using specific Bulge-loop miRNA qRT-PCR Primer for Y-56 and U6 that were synthesized by Genewiz (Beijing, China). qRT-PCR reactions were carried out with 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 for qRT-PCR data analysis. All amplifications were performed in triplicate, and the mRNA level of the target genes was normalized to the GAPDH gene as an internal reference. The primers used for qRT-PCR are listed in Table 2.
Table 2. Primers used in this study
Primer Name
|
Primer Sequences (5’to 3’)
|
Size (bp)
|
IGF-1R
|
F: TGCCCTTCAGGCTTCATC
R: TCTTCTCTTCCTCACAGACTTTG
|
91
|
CDK4
|
F: TTCGAGCATCCCAATGTTGTC
R: GTCTCGATGAACGATGCAGTTG
|
225
|
PCNA
|
CAATTTGGCCATGGGCGTGA
GGTGTCTGCATTATCTTCTGCC
|
103
|
Cyclin D1
|
F: TGCATCTACACCGACAACTCCA
R: GTTGGAAATGAACTTCACGTCTGT
|
222
|
GAPDH
|
F: TCGGAGTGAACGGATTTGGC
R: TGACAAGCTTCCCGTTCTCC
|
189
|
U6
|
F: CTCGCTTCGGCAGCACA
R: AACGCTTCACGAATTTGCGT
|
|
Cell counting kit-8 assay
The cell proliferation rate of the PSCs was assessed at 0 h, 24 h, 48 h, 72 h and 96 h after transfection using a Cell Counting Kit-8 (CCK-8) (Beyotime, China). Briefly, the PSCs were seeded in a 96-well format at a density of 5000 cells/well and were then transfected with different miRNAs, vectors or siRNAs as described above. 10 μL of CCK-8 solution was then added to each well containing 100 μL of the culture medium and then the plate was incubated for 30 min at 37℃, and the absorbance was read at a wavelength of 450 nm using a microplate reader (TECAN, Switzerland).
EdU staining
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-56inhibitor, 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, then were fixed with 4% paraformaldehyde for 15 min and were permeabilized with 0.5% Trixon-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 luorescence microscope (Nikon, Japan) and the data were analyzed in Image J (National Institutes of Health, Bethesda, MD, USA).
Flow cytometry
The PSCs were cultured in a 6-well culture plate at a density of 4 × 105 per well and were treated with Y-56 mimics, Y-56inhibitor, pcDNA3.1+IGF1R and si-IGF-1R for 48 h. Then, the cells were digested with 0.25% trypsin and terminated with DMEMF-12 containing 20% FBS, next the cells were collected and fixed in cold 70% ethanol overnight at 4 °C. The cells were then washed twice and stained with 50mg/mL propidium iodide (PI) for 30 min. Finally, the cell cycles were analyzed by flow cytometry (Becton Dickinson, USA).
Western blotting analysis
The PSM cells 48 h after transfection were harvested to extract the proteins using the lysis buffer (KeyGen Biotech, China) complement with protease and protein phosphatase inhibitor on ice. Protein concentration was determined using BCA Protein Assay Kit (Beyotime, China) and 30 μg protein per sample was loaded and separated using a 5% stacking gel and a 12% separating gel. Separated proteins were transferred to PVDF membrane, which was then blocked in 5% BSA for 2 h at room temperature and incubated with primary antibodies at 4 °C overnight. After washing thrice (10 min once) in TBST, membranes were incubated with HRP conjugated goat anti-rabbit IgG or HRP conjugated goat anti-mouse IgG (Bioworld Technology, USA, 1:2000) 2 h at room temperature.
The Western blot was visualized by Enhanced Chemiluminescence (ECL, Bioworld, USA) performed on 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 (proliferating cell nuclear antigen) rabbit polyclonal antibody, CDK4 (cyclin-dependent kinase 4) rabbit polyclonal antibody, Cyclin D1 rabbit polyclonal antibody (Cell Signaling Technology, USA, 1:2000) and β-actin polyclonal antibody (BBI, China, 1:2000).
Statistical Analysis
All experimental results are presented as the mean ± SEM, with at least three independent replications. The statistically significant difference between groups was tested by one-way ANOVA using GraphPad Prism version 6.01. We considered P < 0.05 to be statistically significant. * P < 0.05; ** P < 0.01; *** P < 0.001.