Tissue samples collection
In this study, the animal samples have been approved by the Northwest Agriculture and Forestry University's Animal Care and Use Committee. At a local slaughterhouse in (Xi'an; China), all specimens from Qinchuan bovine embryonic phase (90 days) were collected, including Skeletal muscle, stomach, intestine, spleen, heart, liver, lung, and kidney, and washed by (DEPC) water, and stored at − 80 °C until RNA extraction.
RNA extraction and Real-time PCR
Total cell or tissue RNAs were extracted using the manufacturer's instructions for TRIzol Reagent (Takara, Dalian, China). Next, using the PrimerScript RT reagent kit with gDNA Eraser (Takara, Dalian, China), RNA was reversed to complementary DNA (cDNA). And a nucleoplasmic separation kit (PARIS kit, Life Technologies, Carlsbad, CA, USA) was used to separate the nuclear and cytoplasmic fractions. For treatment with RNase R, 1 µg of total RNA was incubated at 37 °C for 20 mints with two units µg− 1 of RNase R and then purified using an RNeasy MinElute cleaning kit (Qiagen, Hilden, Germany). SYBR Green PCR master mix reagent kit (Takara, Dalian, China) was used to conduct qRT-PCR. The GAPDH and U6 (for miRNA) were used as an internal control for data normalization with three biological replicates. The relative expression level of mRNA was calculated using the 2−∆∆Ct technique. All primers used in this study are listed in Table 1.
To explore the possible functions of circMYL1 during bovine skeletal muscle development, we downloaded the sequencing data of circRNAs of bovine muscle tissue from NCBI: accession ID,GSE87908;databankURL,https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=atglausktpsjlel&acc=GSE87908 (Text S1). For the construction of circMYL1 overexpression plasmids, using PCR to amplify the linear sequences of circMYL1, and the cDNA template was produced from the bovine primary myoblasts RNA by RT-PCR. The obtained linear sequences were then cloned into the KpnI and BamHI -restriction sites of the pCD2.1 vector (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction, to produce the overexpression vector PCD2.1- circMYL1. The MYOG 3'UTR-WT fragment, including the miR-2400 binding site, was amplified and inserted into the psiCHECK-2 vector (MYOG-WT) (Promega, Madison, WI, USA) at the 3 ' end of the Renilla gene using the XhoI and NotI restriction enzymes (Takara, Dalian, China) and T4 DNA ligase. Mutant type psiCHECK-2- MYOG 3'UTR-MUT (MYOG-MUT) was created using overlapping PCR to mutate complementary to the miR-2400 seed region. MYOG -WT and MYOG -MUT are eventually incorporated in the same way into the psiCHECK-2 vector. A miR-2400 sensor (with a perfect miR-2400 binding site) was created by inserting two sequences that complement the mature miR-2400 sequence after the Rluc psiCHECK-2 vector. Consequently, the fluorescence activity alteration will react to miR-2400 reflecting. The PCK-circMYL1 vector was also obtained using the same procedure. Primer sequences are shown in Table 2. All constructs were verified by sequencing (Sangon Biotech, Shanghai, China).
The bovine primary myoblasts, as reported previously , were isolated and cultured from bovine longissimus muscle. The HEK293T cells (ATCC, USA) and bovine primary myoblasts were grown in growth medium containing high-glucose Dulbecco's modified Eagle's medium (DMEM; Hyclone, USA) and supplemented by 10% or 20% fetal bovine serum (FBS) (Hyclone, USA) and 1% penicillin-streptomycin (Invitrogen, USA) and incubated at 37 °C with 5% CO2. The differentiation of bovine primary myoblast was induced by replacing growth medium with the differentiation medium (DMEM containing 2% horse serum) when bovine primary myoblasts confluence reached approximately 70%~80%.
The bovine primary myoblasts were cultured in six-well plates. When the confluence of the cells reached to 40% for proliferation or 70%~80% for differentiation the cells were transfected with PCD2.1-circMYL1 (2 µg/ml), 100 nM si-circMYL1 (RiboBio, Guangzhou, China), 50 nM miR-2400 mimics (RiboBio, Guangzhou, China) using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's protocol. Transcription was inhibited by adding 1 mM of Actinomycin D solution to the cell culture medium (Leagene, Beijing, China).
RNA Fluorescent In Situ Hybridization (RNA-FISH)
Specific probes to circMYL1 sequence were used to perform in situ hybridization in bovine primary myoblasts following instructions from the probe manufacturer (RiboBio, Guangzhou, China). The probe sequence is 5'- GGCATTCATCTCTTCATAGTTGATGCA − 3'. The cells were cultured in the 6-well plates in cover glass and grown to a confluence of 70% ~80%, and then fixed. After treatment with 0.1% of Triton X-100 transmembrane, cells were incubated overnight at 37 ° C with 20 mg/ml of circMYL1 probe. Then using DAPI to counterstain the nuclei. Photos have been obtained using a confocal laser microscope FV1200 (Olympus).
5-Ethynyl 2′-Deoxyuridine Assay (EdU)
Cell-Light EdU Apollo ® (RiboBio; China) was used to detect the proliferation of bovine primary myoblasts. The experiment was carried out following the protocols of the manufacturer. The bovine primary myoblast cells were grown in a 96-well plate and then transfected by treatments with three replicates in each group. Concisely, before immunostaining, the cells were incubated at 37 °Cwith 50 µM EdU for 2 hours after 24 hours of transfection. A fluorescent microscope (AMG EVOS, Seattle, WA) was used to capture all images.
Cell Counting Kit-8 (CCK8) assay
The cell counting reagent Kit-8(CCK8) (Multiscience, Hangzhou, China) was used to assess the proliferation of cells. The bovine primary myoblasts were grown in 96 well plates with eight independent replicates for each treatment. After 24 hours of cultured at 37 ° C, we added 10 µL of CCK-8 reagent to each well for 4 hours. Then an automatic microplate reader (Molecular Devices, USA) was used to measure the absorbance value of all samples at 450 nm.
Cell Cycle Assay by Flow Cytometry
Bovine primary myoblasts were seeded in 60 mm cell culture plates (2 × 106 cells/well). After 24 hours of transfected, we collected cells and washed with PBS buffer, and resuspended with 1 ml of DNA staining solution and 10 µL of permeabilization solution (Multisciences, Hangzhou, China). The suspension was then incubated for 30 minutes in the dark at room temperature. Flow Cytometry (FACS Canto TM II, BD Biosciences, USA) measured the cell cycle and, for each treatment group, there were three independent replicates.
Luciferase Activity Assay
For investigated the binding sites of circMYL1 with miR-2400, HEK293T cells were seeded in 96-well plates. Then, the miR-2400 mimics and PCK-circMYL1 or miR-2400 sensors and PCD2.1-circMYL1 are cotransfected with Lipofectamine 2000 into HEK293 T cells. The miR-2400 mimics and MYOG-WT or MYOG-MUT have also been cotransfected into cells. After 24 hours, the cells were then washed by Phosphate Buffered Saline (PBS) and collected using 100 µL Passive Lysis Buffer (PLB). Luciferase activity assay was performed using MPPC luminescence analyzer (HAMAMATSU, Beijing, China), and Dual-Luciferase® Reporter (DLR) Assay Kit (Promega, Madison, WI, USA). And the activities of Firefly luciferase were normalized in each well to Renilla luminescence.
Western Blotting Analysis
Total bovine primary myoblasts proteins were extracted from different treatment groups using RIPA lysis buffer containing 1 mM PMSF (Solarbio; Beijing, China). The protein extract was then boiled for 10 minutes in a loading buffer of 5 × SDS-PAGE. The protein was then separated by SDS-PAGE and transferred to a 0.2 µm polyvinylidene fluoride membranes (PVDF). Then incubated for 2 hours at room temperature with 5% skim milk in Tris Buffered Saline, with Tween (TBST) to block the PVDF membrane. Subsequently, in 4 °C overnight incubation with the primary anti-MYOD specific antibodies (Dilution 1:1000; ab16148; Abcam, England), anti-MYOG (1;1000; ab124800; Abcam, Cambridge, England), anti-MYH2 (Dilution 1:1000; WL02785; Wanleibio, China), anti- CDK2 (Dilution 1:1000; WL02028; Wanleibio, China), anti-PCNA (Dilution 1:500; WL03069; Wanleibio, China), anti-CyclinD1 (Dilution 1:1000; WL01435a; Wanleibio, China), anti-β-actin (1:1000; KM9001T, SungeneBiotech, China). After that, the PVDF membranes were washed with TBST buffer and then incubated at room temperature for 2 hours with the secondary antibody. The secondary antibodies were: goat anti-mouse IgG HRP (M21001S, Abmart, China), goat anti-rabbit IgG (H + L)-HRP (BA1054, Boster, China). Finally, ECL luminous liquid (DiNing, China) was used to detect the protein bands.
Primary bovine myoblasts seeded in 24-well plates and differentiation was induced to form the myotubes for four days. The differentiated cells were fixed for 30 minutes with 4% paraformaldehyde, then washed with PBS for 5 minutes three times. The cells were subsequently permeabilized by adding (Triton X-100) 0.5% for 15 minutes. The cells were then incubated overnight with the MyHC-antibody (1:250; GTX20015, GeneTex, USA), which was diluted in 1% Bovine Serum Albumin. The cells were washed with 1XPBS and incubated with IgG H&L secondary anti-mouse (Alexa Fluor 594) (1:500; RS3608, Immunoway, USA) and protected for 2 hours from light at room temperature. The nuclei of cells were stained for 15 minutes with DAPI (4′,6-diamidino-2-phenylindole) (Solarbio; Beijing, China). A fluorescence microscope was used to capture the Immunofluorescence images (DM5000B, Leica, Germany).
The data of the experiment are presented as mean ± SEM. SPSS 20.0 software was used for statistical analysis, and the student t-test was used to determine the variation between the groups. Variations in * P < 0.05and * *P < 0.01 were considered statistically significant.