Primary Intramuscular Precursor Adipocytes Isolation and Culture and Induced Differentiation
The intramuscular adipocytes were from the pectoral muscle tissue of 14-day-old Gushi chicken under sterile conditions. Pectoral muscle tissue was separated using a scalpel and then was digested with collagenase type I (solaibao, Beijing, China) at 37°C for 60 min. Briefly, the digested cell fraction was filtered sequentially passed through cell strainers (Biologix, Jinan, China) with pore sizes of 70 µm and 45 µm. The cell precipitate was centrifugated at 1000 rpm for 10 min. Subsequently, these cells were maintained in DMEM/F12 medium (BI, Massachusetts, USA), supplemented with 10% fetal bovine serum (BI, Massachusetts, USA), and 1% penicillin/streptomycin (Solarbio)) in an incubator with a 5% CO2 atmosphere at 37°C. After 2 hours, the medium was changed. Once upon the confluence of cells reaching 90%, according to the hormone "cocktail method" is used to induce differentiation [24], and the complete medium would be replaced with the differentiation inducing medium (0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 uM dexamethasone (Sigma), and 10 g/l insulin (Sigma)).
Cell Transfection
The miRNA mimic/inhibitor and siRNA interference sequences and their corresponding negative controls were purchased from Genechem (Shanghai, China). Cells were transfected with miRNA mimics, inhibitors and FDPS-SI with Liposome 2000 reagent (Invitgen, USA) according to the manufacturer's instructions. Transfection was carried out when the cell confluence was 60–70%, and the medium was changed to complete medium after 6 h.
Collection of Sequencing Samples
The transfected miR-128-3p-mimics, miR-128-3p-inhibitor and miR-128-3p-NC were divided into overexpression group (M group), interference group (SI group), and blank treatment group (NC group), respectively. The total RNA was extracted using Trizol reagent (Vazyme, Nanjing, China). The expression of miR-128-3p was detected by fluorescent quantitative PCR. The sample with the highest overexpression efficiency (n = 3) and interference efficiency (n = 3) was collected, respectively, and sent to Nanjing Parsono Gene Technology Co., Ltd. for transcriptome sequencing.
Transcriptome Data Analysis
After the removal of raw reads containing no insertion sequence, over 0.2% of poly-N, and low-quality paired reads, we obtained clean reads. The high-quality data (clean data) were mapped to the reference genome (GRCg7a) using TopHat2's upgraded HISAT2 software [25]. Read count values were aligned to each gene using HTSeq statistics as the gene's original expression quantity [26]. Differential gene expression analysis was conducted using DESeq [27], and differentially expressed genes (DEGs) were defined based on the following criteria: |log2 fold change| > 1 and the P value < 0.05. The DEGs were subjected to functional annotation and pathway enrichment analysis using the KOBAS server [28].
Q-PCR
Total RNA was extracted from tissues and harvested cells using Trizol (Vazyme, Nanjing, China). Reverse transcription of mRNA was performed using a HiScript II Q Select RT SuperMix for qPCR kit (Vazyme, Nanjing, China) according to manufacturer instructions. We designed the Q-PCR primers using Primer Premier 5, and the primer sequences were shown in supplementary table 1. The ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China) was used to conduct quantitative real-time PCR (Q-PCR). The total reaction volume was 10 ul, and consisted of 1 µl cDNA, 0.5 µl reverse and forward primers (per gene), 5 ul SYBR, and 3 ul double-distilled water. The reaction procedure was performed under the following conditions:95°C for 30 s, followed by 40 cycles at 95°C for 15 s, and 60°C for 34 s. Finally, the melting curve is collected at 60–95°C. Relative expression level was quantified by the 2−△△Ct [29] approach. β-actin and U6 was using as the normalization references for mRNA and miRNA, respectively.
CCK-8 Assay
The intramuscular adipocytes proliferation was detected after 12 h, 24 h, 36 h and 48 h of transfection using a Trans Detect CCK-8 kit (Toyohito, Japan) according to the manufacturer's protocol. 10 µL of CCK-8 solution was added to the cells and incubated at 37 ℃ constant temperature incubator for 2 h. Then, the absorbance value was measured at 450 nm using the microplate reader.
EdU Assay
The intramuscular adipocytes was detected using a Cell-Light EdU Apollo567 In Vitro Kit (RiboBio, Guangzhou, China) according to the manufacturer’s protocol after transfection for 24 h. Briefly, 100 mL of 50 mm EdU reagent was added to each well and incubated for 2 h at 37 ℃. After the staining, the camera was photographed in the dark under the fluorescent microscope. Finally, the Imaje J software was used to count the number of new cells.
Cell Cycle Assay through Flow Cytometry
The intramuscular adipocytes were seeded in 6 well cell culture plates. The cells were transfected with miR-128-3p mimic, miR-128-3p inhibitor, and negative control. After 48 h, Cells were collected, washed with cold PBS, and fixed with 70% ethanol at 20°C for 6h. Then the cells were washed with PBS and added for a final mass concentration of 50 mg/mL to incubat for 30 min at 37°C. Subsequently, 400 µl of 50 µg/mL propidium iodide (PI) solution was added, and the cells were stained in the dark for 30 min at 25°C. Finally, the cell cycle was observed by flow cytometry (FACS Calibur, BD Biosciences).
Oil-Red O Staining
The intramuscular adipocytes samples were washed three times with PBS (Gibco, Carlsbad, CA, USA) and fixed with 4% paraformaldehyde for 30 min [30]. Subsequently, cells were stained with Oil-Red O working solution (Sigma) for 20 min after being washed with PBS. Then the intramuscular adipocytes were photographed by microscope. Lipid droplets were dissolved by Isopropyl alcohol and the absorbance value was calculated at 490 nm using microplate reader.
Triglyceride Assay
The intramuscular adipocytes samples were treated with 0.25% trypsin until separation and centrifuged for 3 min at 1000 rpm. After all, TG content in cell homogenate was determined using a triglyceride content detection kit (APPLYGEN, Beijing, China) according to the manufacturer’s instructions. The protein concentrations were measured with the BCA Protein Assay Kit (EpiZyme, Shanghai, China) to normalize the TG content. The absorbance value was calculated at 550 nm using microplate reader.
Dual Luciferase Reporter Assay
The laboratory psiCHECK2 vector was used to construct the psiCHECK2-FDPS-3'UTR-WT and psiCHECK2-FDPS-3'UTR-MuT plasmids. They were transfected with miRNA-128-3p-mimics and psiCHECK2 vector into DF1 cells. After 48h, the samples were collected, and the fluorescence activity was detected using the Dual-Glo Luciferase Assay Systemt (Promega, Madison, WI, USA).
Statistical Analysis
Data analysis was performed with SPSS 26.0, All data was presented as “mean ± standard error (SEM)”. Significant differences between groups were analyzed using one-way ANOVA. Asterisks signify different significance levels (*P < 0.05, **P < 0.01, and ***P < 0.001).