Animal care
The care and experimental use of chickens were approved by the Institutional Animal Care and Use Committee, Seoul National University (SNU-170417-18-2). All chickens were maintained according to a standard management program at the University Animal Farm, Pyeongchang campus, Seoul National University, South Korea. The procedures for animal management, reproduction, and embryo manipulation adhered to the standard operating protocols of our laboratory.
Primary chicken myoblast (pCM) cell culture and induction of myotube differentiation
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 induce myotube differentiation 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. 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.
Construction of a miR146b-5p overexpression vector
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).
Transfection and selection of the miR-146b-5p overexpression vector
To establish miR-146b-5p-expressing myoblast cells, we co-transfected 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 the manufacturer’s protocol. After pCM cells were washed with PBS and refreshed with 2 mL culture medium without antibiotic-antimycotic, a plasmid 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. One day after lipofection, 10 µg/mL puromycin was added to select cells stably transfected with the transgene. GFP-expressing cells were observed with a fluorescent microscope (Carl Zeiss Axio Observer A1, Oberkochen, Germany).
Quantitative RT-PCR analyses
Total RNA was extracted with Trizol reagent (Invitrogen; cat#10296010) according to the manufacturer’s instructions. Total RNA was quantified with a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and cDNA synthesis was performed with 2 µg RNA and random primers (Invitrogen; cat#18080-051) under standard conditions. Quantitative RT-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 1). 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 1). The reaction was terminated by a final incubation at 72°C for 10 min, and melting curve profiles were analyzed.
Western blotting
Total protein was extracted with 1× radioimmunoprecipitation lysis buffer and separated on a 10% polyacrylamide gel followed by transfer 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.
Cell growth curve and statistical analyses
To calculate the cell growth curve, 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.
Library preparation and sequencing
For total mRNAs from rpCM cells or pCM-146b OE cells, the library was constructed with the QuantSeq 3′ mRNA-Seq Library Prep Kit (Lexogen, Vienna, Austria; cat#015) according to the manufacturer’s instructions. In brief, 500 ng total RNA was prepared, and an oligo-dT primer containing an Illumina-compatible sequence at its 5′ end was hybridized to the RNA and reverse transcription was performed. After the RNA template degraded, second-strand synthesis was initiated by a random primer containing an Illumina-compatible linker sequence at its 5′ end. The double-stranded library was purified with magnetic beads to remove all reaction components. The library was amplified to add the complete adapter sequences required for cluster generation. The finished library was purified from PCR components. High-throughput sequencing was performed as single-end 75 sequencing with NextSeq 500 (Illumina, USA).
Data analyses
QuantSeq 3′ mRNA-Seq reads were aligned with Bowtie2 [25]. Bowtie2 indices were generated from either the genome assembly sequence or the representative transcript sequences for alignment with the genome and transcriptome. The alignment file was used to assemble transcripts, estimate their abundance, and detect differential expression of genes. Differentially expressed genes were determined based on counts from unique and multiple alignments using coverage in Bedtools [26]. The read count data were processed based on the quantile normalization method with EdgeR in R and Bioconductor [27]. Gene classification was based on searches of the DAVID (http://david.abcc.ncifcrf.gov/) and Medline (http://www.ncbi.nlm.nih.gov) databases. Using mRNA next-generation-sequencing data, 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
Statistical analyses were conducted with SAS version 9.4 (SAS Institute, Cary, NC, USA). The significance of differences was analyzed with a general linear model procedure, and differences between groups were deemed significant at p < 0.05.