2.1 Progenitor cells isolation and cell lines.
Bovine fetuses for 120~180 days were collected from the slaughterhouse and transported to the laboratory immediately. Using the enzyme digestion combined with the differential adhesion method, the primary myoblasts and intramuscular preadipocytes were isolated from the longissimus dorsi as previously described (19). The longissimus dorsi was isolated from the fetus, washed with phosphate buffered saline (PBS), and minced into small fragments. It was then digested in Dulbecco’s modified Eagle’s medium (DMEM) containing type IV collagenase (w/v, 0.2%; C5138, Sigma, USA) at 37°C with continuous shaking for 2 hours. The cell plasma was filtered through the 200 μm filter, collected by centrifugation and resuspended in DMEM. The cells were seeded in complete medium and incubated at 37 °C with 5% CO2. The adherent cells within two hours of inoculation were collected for adipogenesis induction. After 24 hours of culture, the non-adherent cell suspension was re-inoculated. After repeated operations for 2 days, adherent cells were collected for myogenesis induction. HEK-293T cells were purchased from the American Type Culture Collection (ATCC) and were tested negative for mycoplasma contamination.
2.2 Differentiation of myoblasts and preadipocytes.
Myoblasts were cultured in high-glucose DMEM supplemented with 20% fetal bovine serum (FBS, Gibco). 2 days after cells reached confluence, DMEM containing 2% horse serum was used for myogenesis induction. Myotube identification was performed on the 4th day of induction. Intramuscular preadipocytes were cultured in F12/DMEM supplemented with 10% FBS. Adipocyte differentiation was induced by M1 medium (containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1.5 μg/mL insulin). 2 days later, the M1 medium was replaced with M2 medium (containing 1.5 μg/mL insulin). Then differentiation was induced for 8 days, during which the medium was changed once every 2 days. The M2 medium was changed every two days, and Oil Red O staining was performed on the 8th day of adipogenic differentiation.
2.3 RNA extraction and real-time qPCR
Total RNA was isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized with PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Tokyo, Japan). Real-time qPCR for RNA analyses were performed using SYBR Green PCR Master Mix (Takara, Tokyo, Japan). MiRNAs specific stem-loop primers were used for reverse transcription. The level of GAPDH was used to normalize the expression of circRNA and mRNA, and the level of small nuclear U6 was used to normalize the expression levels of miRNAs.
2.4 Vector construction and cell transfection
The second exon sequence of INSR gene was constructed into pCD2.1 vector and psi-CHECK2 vector. Small interfering RNA (siRNA) oligonucleotides were designed to combine with the back-splice region of circINSR (RiboBio, Guangzhou, China). The mimics of bta-miR-15a, bta-miR-15b, bta-miR-16a, and bta-miR-16b were purchased from RiboBio (Guangzhou, China). The 3'-UTRs of CCND1 and Bcl-2 genes containing the miR-15/16 binding sites were amplified by PCR enzyme mix (Platinum II Taq Hot-Start DNA Polymerase, Invitrogen). The wild-type and mutant 3'-UTR gene sequences were cloned into the psi- CHECK2 vector. The mimics (50 nM) or vectors (2 μg/mL) were transfected into cells with transfection reagent (R0531, Thermo Fisher Scientific, USA). For overexpression of miR-15/16 family, a quarter of miR-15a, miR-15b, miR-16a, and miR-16b mimics were selected and mixed in equal amounts for transfection.
2.5 Oil Red O and BODIPY staining
After 8 days of differentiation, the intramuscular preadipocytes were stained with Oil red O (O0625, sigma, USA) and 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) (D3922, Thermo Fisher Scientific). Oil Red O dyeing was performed according to the instructions. To quantify staining in fat droplets, the 100% isopropanol was used to dissolve the lipid droplets, and measure the absorbance at 510 nm. For BODIPY staining, the cells were washed twice with PBS to remove residual 4% paraformaldehyde. Hank’s Balanced Salt Solution with 10 μM BODIPY 493/503 was added to the cells and then incubated at 37°C for 30 minutes in the dark. The samples were washed 3 times with PBS and photographed immediately.
2.6 Immunofluorescence analysis
After 4 days of myogenic differentiation, the myoblasts were fixed with 4% paraformaldehyde. After washing with PBS, MyHC antibody (1:250, Heavy chain cardiac Myosin antibody, GTX20015, GeneTex, USA) was added to incubate overnight at 4°C, and then the secondary antibody was added to incubate for 2 hours. The nucleus was stained with DAPI. The myotube coverage area was analyzed by Image pro plus software.
2.7 Dual Luciferase Reporter Assay
HEK-293T cells were co-transfected with miR-15/16 mimics and plasmid. After 24 hours of transfection, the luciferase activity was detected with the Dual-Luciferase Reporter Assay Kit (E2920, Promega, Fitchburg, WI, USA). The optical density of the resulting solution was assessed using the automatic microplate reader (Molecular Devices, Sunnyvale, USA).
2.8 RNA-binding protein immunoprecipitation (RIP)
RNA-binding protein immunoprecipitation assay was performed using EZ-Magna RIP kit (17-701, Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. The Argonaute2 (Ago2) antibody (Abcam, UK) and IgG antibody were used for immunoprecipitation. The RNA was extracted from the immunoprecipitation products of myoblasts and preadipocytes, and the abundance of circINSR and miR-15/16 was detected by real-time qPCR.
2.9 5-Ethynyl-2`-deoxyuridine (EdU) and Cell Counting Kit-8 (CCK-8) assay
When the density of myoblasts and preadipocytes reached 40%-50%, the transfection was performed with overexpression plasmid, siRNA or miRNA mimics. After 24 hours of transfection, the cell proliferation was tested by EdU assay kit (RiboBio, Guangzhou, China). The nucleus was stained with Hoechst 33342. Use a fluorescence microscope to take pictures immediately after staining (AMG EVOS, Seattle, WA, USA). Similarly, we also used the CCK-8 (Multisciences, Hangzhou, China) to detect the level of cell proliferation after transfection. The optical density of CCK-8 at 450 nm was measured using an automatic microplate reader (Synergy4, BioTek, Winooski, USA).
2.10 Cell cycle and apoptosis assay
We used flow cytometry and the Cell Cycle Testing Kit (Multisciences, Hangzhou, China) to analyze the cell cycle. The myoblasts and preadipocytes were transfected when the cell growth density reached 50%. After transfection for 24 hours the cells were collected and washed with PBS. Subsequently, follow the kit instructions for staining. Flow cytometry analysis was performed on a BD Accuri C6 flow cytometer (BD Biosciences, USA). Cell apoptosis assays were performed with Annexin V-PE/7-AAD Apoptosis Detection Kit (RiboBio, Guangzhou, China) according to the manufacturer’s recommendations. Afterward, the apoptosis rate was analyzed using flow cytometry (FACS Canto™ II, BD BioSciences, USA).
2.11 Western Blot analysis
Proteins from cultured myoblasts and preadipocytes were lysed with RIPA buffer (Solarbio, Beijing, China). Proteins were loaded onto 12% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific). The membranes were incubated overnight with primary antibodies specific for anti-GAPDH (#ab9485), anti-CyclinD1 (#ab226977), (Abcam, Cambridge, UK), anti-Bcl-2 (#bs-0032R), anti-caspase-9 (#bs-0049R), anti-Bax (#bs-0127M), anti-FABP4 (#bsm-51247M) (Bioss, Beijing, China), anti-PCNA (#WL01804), anti-CDK2 (#WL01543), anti-PPARγ (#WL01800) and anti-C/EBPα (#WL01899) (Wanlei Bio, Shenyang, China) at 4℃. After incubation with secondary antibodies, the membranes were quantified with the chemiluminescence system (Bio Rad, Hercules, CA, USA).
2.12 Statistical analyses
Data are expressed as the mean ± standard error (SEM) of at least 3 independent experiments. Statistical analyses were performed using SPSS 19.0 statistical software (SPSS, Chicago, IL, USA). Statistically significant differences were calculated using Student`s t-test. A probability of 0.05 or less was considered statistically significant.