Primary chicken myoblast (pCM) cell culture and induction of myotube differentiation
Primary chicken myoblast (pCM) cells were isolated from pectoralis major of 10-day-old male chick embryos and maintained in Medium 199 (Invitrogen), supplemented with 10% fetal bovine serum (FBS; HyClone), 2% chicken serum (Sigma-Aldrich) and 1 × antibiotic-antimycotic (Invitrogen) (Lee et al. 2019). These cells were cultured in an incubator 37℃ in an atmosphere of 5% CO2 and 60–70% relative humidity. To induce myotube differentiation at 80% confluency of cells, after washed one time by using PBS, the differentiation medium containing 0.5% FBS and 1 × antibiotic-antimycotic was changed. The differentiation medium was replaced with fresh differentiation medium daily.
Construction of miR146b overexpression vector
To overexpress chicken microRNA-146b (miR-146b), miR-146b were inserted into the piggyBac transposon transgene expression system vector (System Biosciences, Palo Alto, CA, USA) after Asc I digestion and ligation (piggyBac CMV-GFP-miRNA-146b). The cytomegalovirus (CMV) and elongation factor-1 (EF-1) promoter controlled the expression of GFP-miRNA-146b and puromycin resistance gene, respectively (Fig. 1A). The miRNA-146b were 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 miR-146b overexpression vector
To establish miR-146b expressing myoblast cells, the transgene expression vector, piggyBac CMV-GFP (control) or piggyBac CMV-GFP-miRNA-146b, was co-transfected with piggyBac transposase using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. After pCM cells were washed with phosphate-buffered saline (PBS) and refreshed with 2 ml of the culture media without antibiotic-antimycotic, the plasmid DNA-lipid complex consisting of 7.5 µl Lipofectamine 3000 reagent in 250 µl Opti-MEM (Invitrogen) and 10 ul P3000 reagent with 2.5ug piggyBac transgene vector and piggyBac transposase plasmid in 250 ul Opti-MEM was added to each well. One day after lipofection, 10ug/ml puromycin was added to select the cells stably transfected with the transgene.
Quantitative RT-PCR analysis
Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Total RNA was quantified using a NanoDrop 2000 (Thermo Scientific), and 2ug RNA were used for cDNA synthesis using random primers (Invitrogen) under standard conditions. Quantitative RT-PCR for miRNA was conducted by using High-specificity miRNA QPCR Core Reagent Kit (Agilent Technology, Santa Clara, CA, USA). Each 20 ul RT-PCR reaction mix contained 2 ul cDNA, 2.5 ul PCR buffer, 1 ul dNTP mixture (2.5 mM), 1unit Taq DNA polymerase, and 10 pmol forward and reverse primer (Table 1). Quantitative RT-PCR analysis was performed using the iCycler iQ Real-time PCR detection system (Bio-Rad) and EvaGreen (Biotium, Fremont, CA, USA). The PCR parameters were as follows: an initial incubation at 94℃ for 5 min, followed by 40 cycles at each condition (Table 1). The reaction was terminated by a final incubation at 72℃ for 10 min, and melting curve profiles were analyzed for the amplicons.
Table 1
List of primer sets for PCR analysis.
Gene | Forward | Reverse | Annealing Temp. (°C) | PCR size(bp) |
Actin | GATGATATTGCTGCGCTCGT | GTGCTCCTCAGGGGCTACTC | 60℃ | 618 |
Pax7 | AGGTACCAAGAGACGGGCTC | CTCGGCAGTGAAAGTGGTCC | 60℃ | 411 |
MyoD | ACACGTCGGACATGCACTTC | TCTGACTCCCCGCTGTAGTG | 55℃ | 433 |
SMAD4 | GCCCACCACAACATACTCCT | GCACTTGAGATCGAAGGCGT | 60℃ | 315 |
NUMB | GCTGCCCCAACTACTACTGC | ACAGGGCACTAATGCTGTCC | 55℃ | 310 |
ADCK3 | CTGTGCAGCAAACATGTCCT | GGCATCTTCCATTTCCTTGA | 60℃ | 366 |
RRM2B | GGACCTTCCTCACTGGAACA | TCCACTTCAGAGCCCAGTCT | 55℃ | 308 |
SGCB | CACGAGTTTCATCTGCCAAA | TCACTTGCACCTTGAACAGC | 55℃ | 343 |
CCND3 | TTTCTGGATGCTGGAGGTGT | ATGCAGAGCTTCTCCACAGT | 60℃ | 195 |
IRF2 | AATGCAGAGGGACGACTTCA | ACTGGGTGATGTCTGACGTT | 60℃ | 301 |
WNT5A | GATACCGCTTTGCCAAGGAG | GCCTACCTTGCGGAAATCAG | 60℃ | 224 |
PDGFRB | AGAGCTAGAGGACAGTGGGA | CATTGGAAGCTCGGATGGTG | 60℃ | 359 |
CCNB2 | TGAAATGTTGGTGGTAGGGC | GGAACAAGTATGCAAGTAGC | 60℃ | 209 |
CDC20 | GAGTCCTGAACCTGACCATG | CTGTACAGTGTGTAAGCCCA | 60℃ | 221 |
KIF23 | CCTTTCTTGTCAGGCCCTCT | TCTGTGAGCACGTTACCCTT | 60℃ | 348 |
KPNA2 | ACACAGAGCAAGGGGTTACA | TCCAAATTCAGGGCAATGCT | 60℃ | 332 |
PLK1 | CTGATGCTGTGGTGATGGTG | TCTCAACCTGGGCACGTTAA | 60℃ | 316 |
TOP2A | TCAACAAAGGCAGCAAGGTC | GGCTCGATTCATCCTGGAGA | 60℃ | 348 |
ID1 | TGATCGACTACATCTGGGACC | TCTGAGAAGGTTACGAGCCG | 60℃ | 251 |
sno RNA : GGGATGTAAAAAAATACTTGCTATC miR-146b : UGAGAACUGAAUUCCAUAGGCG | 60℃ | |
Western blotting
Total protein was extracted with 1 × radioimmunoprecipitation (RIPA) lysis buffer and separated on a 10% polyacrylamide gel followed by transfer to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The primary antibodies used were mouse anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Pax7 (R&D Systems, Minneapoils, MN, USA), anti-MyoD (Santa Cruz Biotechnology), anti-Desmin (Novus Biologicals, Littleton, CO). HRP-conjugated anti-mouse IgG or anti-rabbit IgG (Bio-Rad) 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 analysis
To calculate cell growth curve, pCM-GFP or pCM-146b OE cells were subcultured in 24-well culture plates (2 × 104 cells/well). Total cell number of each well was counted during in vitro culture of 5 days.
Library preparation and sequencing
For total mRNAs from regular pCM cells or pCM-146b OE cells, the construction of library was performed using QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen, Inc., Austria) according to the manufacturer’s instructions. In brief, each 500 ng total RNA were 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 degradation of the RNA template, 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 by using magnetic beads to remove all reaction components. The library was amplified to add the complete adapter sequences required for cluster generation. The finished library is purified from PCR components. High-throughput sequencing was performed as single-end 75 sequencing using NextSeq 500 (Illumina, Inc., USA).
Data analysis
QuantSeq 3’ mRNA-Seq reads were aligned using Bowtie2 (Langmead and Salzberg 2012). Bowtie2 indices were either generated from genome assembly sequence or the representative transcript sequences for aligning to the genome and transcriptome. The alignment file was used for assembling transcripts, estimating their abundances and detecting differential expression of genes. Differentially expressed gene was determined based on counts from unique and multiple alignments using coverage in Bedtools (Gentleman et al. 2004). The RC (Read Count) data were processed based on quantile normalization method using EdgeR within R using Bioconductor (Quinlan et al. 2010). Gene classification was based on searches done by DAVID (http://david.abcc.ncifcrf.gov/) and Medline databases (http://www.ncbi.nlm.nih.gov). Using mRNA next-generation-sequencing (NGS) data, differentially expressed genes (DEGs) from regular pCM cells and pCM-146b OE cells were identified with a p-value cutoff at 0.001 and a fold change cutoff at 1.5. Protein-protein association was analyzed using STRING analysis to identify all functional interactions of DEGs (https://string-db.org).
Statistical analysis
Statistical analysis was conducted using the SAS version 9.4 software (SAS Institute, Cary, USA). The significance of differences was analyzed using a general linear model procedure and the differences among groups were deemed to be significant when p < 0.05.