Animal care
The procedures for animal management, reproduction, and manipulation adhered to the standard operating protocols of our laboratory at the University Animal Farm, Pyeongchang campus, Seoul National University, South Korea [24].
Primary chicken myoblast (pCM) cell culture and myotube differentiation assay
According to our previous report [24], pCM cells were isolated from the pectoralis major of 10-day-old male chick embryos and maintained in Medium 199 (Invitrogen, Carlsbad, CA, USA; cat#11150-059) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT, USA; cat#SH30084.03), 2% chicken serum (Sigma-Aldrich, St. Louis, MO, USA; cat#C5405), and 1× antibiotic-antimycotic (Invitrogen; cat#15240-062) [24]. These cells were cultured in an incubator at 37°C in an atmosphere of 5% CO2 and 60–70% relative humidity. To differentiate into myotube at 80% confluency of cells, the cells were washed once in phosphate-buffered saline (PBS), and the differentiation medium containing 0.5% FBS and 1× antibiotic-antimycotic was changed. The differentiation medium was replaced with fresh differentiation medium daily according to our previous report [24]. The myotube-differentiated area was measured and analyzed in each well of regular pCM (rpCM) or pCM cells overexpressing miRNA-146b-5p (pCM-146b OE cells) after 4 days of differentiation. All experiments were performed in triplicate with both rpCM or pCM-146b OE cells.
miR146b-5p overexpression vector construction
To overexpress chicken microRNA-146b-5p (miR-146b-5p), we inserted miR-146b-5p into the piggyBac transposon transgene expression system vector (System Biosciences, Palo Alto, CA, USA; cat#PB513B-1) after Asc I digestion and ligation (piggyBac cytomegalovirus [CMV]-GFP-miRNA-146b-5p). The CMV and elongation factor-1 promoters controlled the expression of GFP-miRNA-146b-5p and the puromycin resistance gene, respectively (Figure 1A). The miRNA-146b-5p was synthesized as 5′-gct ggt gac gtc ccc tat gga att gag ttc tcc gct gtg aca ctt caa act gag aac tga att cca tag gcg atg tgg tca gca-3′ (Bionics, Seoul, Korea).
Development of the miR-146b-5p overexpressing myoblast
For miR-146b-5p-expressing myoblast cells, we conducted co-transfection of the transgene expression vector, piggyBac CMV-GFP (control), or piggyBac CMV-GFP-miRNA-146b-5p with piggyBac transposase using Lipofectamine 3000 (Invitrogen; cat#L3000015) according to our previous report [22]. Transgene DNA-lipid complex consisting of 7.5 µL Lipofectamine 3000 reagent in 250 µL Opti-MEM (Invitrogen; cat#31985-062) and 10 µL P3000 reagent with 2.5 µg piggyBac transgene vector and piggyBac transposase plasmid in 250 µL Opti-MEM was added to each well according to our previous report [22]. One day after lipofection, 10 µg/mL puromycin was added to develop stably transfected cells with the transgene. GFP-expressing cells were observed with a fluorescent microscope (Carl Zeiss Axio Observer A1, Oberkochen, Germany).
Analysis of gene expression by quantitative RT-PCR
Total RNA was extracted with Trizol reagent (Invitrogen; cat#10296010) and cDNA was synthesized with 2 µg RNA and random primers (Invitrogen; cat#18080-051) under standard conditions. Quantitative RT-PCR (qRT-PCR) for miRNA was conducted with the high-specificity miRNA QPCR Core Reagent Kit (Agilent Technology, Santa Clara, CA, USA; cat#600545). Each 20 µL RT-PCR reaction mix contained 2 µL cDNA, 2.5 µL PCR buffer, 1 µL dNTP mixture (2.5 mM), 1 U Taq DNA polymerase, and 10 pmol forward and reverse primers (Table 2). Quantitative RT-PCR analyses were performed with the iCycler iQ Real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and EvaGreen (Biotium, Fremont, CA, USA; cat#31000). The PCR parameters were as follows: an initial incubation at 94°C for 5 min, followed by 40 cycles at each condition (Table 2). The reaction was terminated by a final incubation at 72°C for 10 min, and melting curve profiles were analyzed.
Western blotting assay
After extraction with 1× radioimmunoprecipitation lysis buffer, total protein from each treated cells was separated on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad). The primary antibodies used were mouse anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA; cat#SC-47778), anti-PAX7 (R&D Systems, Minneapolis, MN, USA; cat#MAB1675), anti-MYOD (Santa Cruz Biotechnology; cat#MAB1675), anti-Desmin (Novus Biologicals, Littleton, CO, USA; cat#NB110-1790). Horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Bio-Rad; cat#170-6516 and 170-6515) were used as secondary antibodies. The blots were treated with ECL substrate solutions and exposed in a ChemiDoc XRS System (Bio-Rad) to detect chemiluminescence according to our previous report [24].
Cell growth analyses
For analysis of the cell growth, we subcultured pCM-GFP or pCM-146b OE cells in 24-well culture plates (2 × 104 cells/well). The total number of cells in each well was counted and analyzed statistically during a 5-day in vitro culture. In addition, the proliferative capacities were compared with a 5-bromo-2′-deoxyuridine (BrdU) flow kit (Becton, Dickinson and Company, Franklin Lakes, NJ, USA; cat#559619). Briefly, flow cytometry analyses of cell cycles were compared between rpCM and pCM-146b OE cells after the incorporation of BrdU.
RNA-sequencing and Data analyses
To analyze RNA-sequence data in rpCM cells or pCM-146b OE cells, we generated an RNA-sequencing library for each sample and carried out using Illumina HiSeq2500 (Illumina, San Diego, CA). Subsequently, these sequences were aligned and mapped against the chicken reference genome using TopHat for paired-end sequences according to our previous report [7]. Based on searches of the DAVID (http://david.abcc.ncifcrf.gov) and Medline (http://www.ncbi.nlm.nih.gov) databases, we conducted gene classification analysis. We identified differentially expressed genes (DEGs) from rpCM cells and pCM-146b OE cells with a p cutoff of 0.001 and a fold change cutoff of 1.5. Protein–protein association was analyzed with STRING analyses to identify all functional interactions of DEGs (https://string-db.org).
Statistical analyses
We conducted statistical analyses with SAS version 9.4 (SAS Institute, Cary, NC, USA) and the significance of differences was analyzed with a general linear model procedure.