Introduction
The
domesticated pigeon (Columba livia)
has been bred for hundreds of years for the production of meat and ornamentals (Sales 2003) and for use as an experimental
animal in some research fields such as toxicology (Zeid et al.
2019) and behavior (Wilkie et al. 1981). Pigeon meat is
gaining popularity among consumers in Europe, China and the United States
(Pomianowski et al. 2009) which has greatly increased the number of farms
breeding pigeons for the production of meat and meat products. In China, pigeon
has become the fourth-largest domestic poultry by breeding scale, following
chicken, duck and goose. Unlike other poultry, pigeons are
altricial birds with an extraordinary growth rate during early development.
Specifically, reported that the weight of breast meat increased 173.3-fold
between 1-day-old and 35-day-old birds (Gao et al. 2016). The mechanism
underlying this rapid growth in pigeon pectoral muscles has not been fully
elucidated. Skeletal muscle growth is a complicated process involving cell
proliferation, apoptosis, differentiation and the transformation of muscle fiber
types (Gan et
al. 2018). In post-hatch birds,
the development of muscle cells occurs exclusively through an increase in
myofiber size (hypertrophy) (Remignon et al. 1995). This process is
mediated by activated muscle satellite cells (MSCs) (Harding et al. 2016), which
proliferate and fuse with muscle fibers, ultimately cause an increase in the
DNA content and protein synthetic capacity of the developing muscles (Yin et al.
2014). MSCs are a
multipotential mesenchymal stem cell population (Harding
et al. 2016). When skeletal muscle suffers an injury, SMSCs become active and begin to fuse to form a
new myotube (Relaix and Zammit 2012). In broilers,
MSCs are highly proliferative and actively differentiating one-week post-hatch,
after which, the MSC population decreases dramatically (Halevy et al. 2000, 2001).
Moreover, temporarily reducing SMSC activity via irradiation of the turkey Pectoralis
causes a decrease in mature muscle size (Mozdziak
et al. 1997). Therefore, the
mitotic activity of SMSCs in early life governs the ability of muscle to meet
its full potential genetic size (Simone and Vieira 2004).
Myogenesis is controlled by myogenic regulatory
factors including Myf5, MyoD and MRF4 (Pownall et al. 2002). As
post-transcriptional regulators of myogenic gene expression, miRNAs also have a
vital effect on myocyte differentiation (Zhang et al.
2017), microRNAs (miRNAs)
can influence cellular processes such as proliferation, apoptosis and
differentiation (Siengdee et al. 2015). As skeletal muscle precursors, SMSCs are also
modulated by miRNAs (Zhang et al. 2015). For example, miR-192
significantly attenuated SMSCs differentiation by targeting the 3′
untranslated region (UTR) of sheep retinoblastoma 1(RB1) (Zhao et al. 2016), and miR-143 modulates the differentiation and
proliferation of SMSCs by targeting IGFBP5
(Zhang et al. 2017). To date, miRNA
expression profiles in SMSCs of some domestic species (e.g., bovine)
have been surveyed using high-throughput sequencing (Zhang et al. 2016).
However, there have been no studies regarding miRNA identification in pigeon
SMSCs. Here, we have identified the miRNAs in pigeon SMSCs and differentiated myotubes. Our study will be instrumental in promoting a
better understanding of the roles of miRNAs during the differentiation of
pigeon MSCs.
Materials and Methods
Cell culture
Three 16-day-old pigeon
embryo eggs were purchased from the FengMao pigeon breeding farm (Mianyang,
China). Isolation of pigeon MSCs based on our previously established method (Lin et al.
2019). Briefly, 16-day-old pigeon embryos were dissected, followed by removing bilateral pectoral muscles and soaking in PBS (Hyclone, Utah, U.S.A.).
The pectoral muscles were finely minced and dissociated in 0.1% collagenase
type IV (Sigma, U.S.A.) for about 45 min. Subsequently, the cell suspension was
filtered using a 40-μm nylon mesh
(BD, Falcon™). The cells were collected after centrifugation at 1500 r·min–1
for 3 min and resuspended in DMEM (Hyclone, Utah, U.S.A.) containing 20% FBS
(Natocor, Argentina) with antibiotics (Solarbio, China). Subsequently, cells
were seeded in 96-well or 24-well plate and cultured in CO2
incubator (Thermo, U.S.A.) at 37°C and 5% CO2 with saturating
humidity. The culture medium was refreshed every 48 h until the fifth day. Based on
our previous finding that pigeon SMSCs can be automatically differentiated into
myotubes in common growth medium without addition of horse serum,
induction of differentiation was merely to have the SMSCs incubated in DMEM
medium containing 20% FBS for 5 days (Lin et al. 2019).
Immunofluorescence staining
Pigeon SMSCs and myotubes were
fixed with 4% paraformaldehyde for 15 min. After washing with PBS, cells
were permeabilized with 0.5% Triton X-100 for 15 min. Next, cells were blocked
by goat serum (Solarbio, China) for 30 min.
Subsequently, cells were incubated with primary antibody against mouse Anti-MyHC (1:500, Abcam, U.S.A.) overnight at 4℃. Followed
by washing twice with PBS,
cells were incubated with the secondary antibody (FITC labeled goat anti-mouse
IgG, 1:500, Abcam, U.S.A.) for 1 h and the cell nuclei were
counterstained for 10 min with DAPI. Then, digital photomicrographs were taken.
RNA
extraction and high-throughput sequencing
Total RNA was respectively extracted from pigeon
SMSCs and myotubes with TRIzol reagent
(Invitrogen, U.S.A.). Each differentiation stage had three replicates that came
from the three pigeon embryos. The quantity of total RNA
was assessed using an Agilent 2100 Bioanalyzer. For samples of high-throughput
sequencing, the RNAs from three replicates at each differentiation stage were
pooled as one RNA sample. Small RNA ranging from 10–45 nt in length was purified by polyacrylamide gel electrophoresis
and ligated using adaptors. The ligated RNA was reverse-transcribed to cDNA and
amplified by PCR. Finally, the libraries were sequenced on a BGIseq-500
sequencing platform.
Identification
and differential expression analysis of miRNAs
Raw reads were filtered to remove the low
quality-reads, repeated sequences and the adaptors, and the remaining reads was
called clean data. Subsequently, filtered sequences were mapped to the pigeon
reference genome (ColLiv2, GenBank assembly accession:
GCA_001887795.1) with stringent criteria (0 mismatch for full length) using
Bowtie software. Next, mappable reads were extended in the reference genome as
predicted miRNA precursors. Only candidate precursors that perfectly matched to
known pigeon (Columba livia) mature
miRNAs annotated by miRBase (Release 22.0) were identified as known pigeon
miRNAs (Kozomara et al. 2019).
Subsequently, to identify the conserved miRNAs, we performed alignments between
remaining candidate precursors and seed sequences of mature miRNAs from
chicken, zebra finch and other mammals, allowing no mismatch. Novel miRNAs were
further predicted using miRDeep2 (FriedläNder et al. 2008). EdgeR was used for
differential expression analysis between SMSCs and
myotubes in the OmicShare tools (Robinson et al. 2010). The miRNAs with |log2(fold change) |>1 and false discovery rate
(FDR) <0.001 were identified differentially expressed miRNAs.
Prediction and functional annotation of target genes
The TargetScan (Garcia
et al. 2011) and RNAhybrid (Jan and Marc 2006) were used to predict
the target genes of differentially expressed miRNA. The R package
ClusterProfiler was used for GO enrichment and KEGG pathway analysis (Yu et al.
2012).
Dual-luciferase
reporter assay
To validate
the miRNA-target interactions between representative DE miRNA and its target
gene. Fagments
(50 bp each) of 3′-UTR of Mef2a
(XM_005514073.2) containing the wild-type or mutant binding sites for
cli-miR-181a-5p were synthesized (Tsingke, China). The sequences were cloned
into the pmirGLO plasmid (Promega, USA). 100 ng recombinant pmirGLO vector were
co-transfected with 50 nM of the cli-miR-181a mimics or NC miRNA (GenePharma,
China) into HeLa cells by Lipofectamine 3000 (Invitrogen, U.S.A.). After 48h of
cell culture, dual-luciferase activity was measured using the Dual-Luciferase
Reporter Assay System kit (Promega, U.S.A.) according to the manufacturer’s
protocols.
qRT-PCR
miRNAs were
reverse-transcribed using Mir-X miRNA First-Strand Synthesis Kit, and qPCR
assays were carried out using SYBR® Premix Ex Taq™ II on the CFX96 Real-Time
PCR System (Bio-Rad, USA). Relative miRNA levels were normalized against U6
snRNA, and calculated using the 2-ΔΔCt method. Primer
sequences are listed in Table S1.
Fig. 1: Morphology and immunofluorescence identification of pigeon SMSCs. A. Morphological changes during differentiation of pigeon SMSCs (40×). B.
Immunofluorescence staining of MHC in SMSCs and myotubes
(100×)
Fig. 2: Length distributions of the pigeon SMSCs and myotubes
miRNA libraries. Blue columns represent length distributions of reads in the SMSCs
library; red columns represent length distributions of reads in the myotubes library
Statistical analysis
Statistical analyses
were performed by using the SPSS 19.0 software. Student’s t-test was applied to
compare the means between two groups. P <
0.05 was regarded as statistically significant.
Results
Evaluation of pigeon SMSCs differentiation
Terminal
differentiation and myotube formation were evaluated by morphology and Myosin
heavy chain (MHC) immunofluorescence staining. SMSCs were successfully isolated
from pectoral muscle tissue of 16-day pigeon embryos. After
incubation in growth medium (without horse serum) for 5 days, SMSCs were
automatically differentiated into myotubes (Fig. 1A). Furthermore, immunofluorescence
staining showed MHC protein was normally expressed in myotubes, but almost not
in undifferentiated SMSCs (Fig. 1B).
Overview of miRNA sequencing data
To explore miRNA
profiles in pigeon SMSCs and differentiated myotube, two pooled total RNA
samples (from three biological replicates, respectively) at each
differentiation stage were used to construct sequencing libraries. As a result,
we totally obtained 23.98 million raw reads. The adaptor sequences, contamination
and low-quality reads were then trimmed and the remaining reads were regarded
as high-quality clean reads. The proportion of high-quality clean reads account
for 93.12 and 87.30% in the SMSCs and myotubes libraries, respectively. As
shown in Fig. 2, the majority (≥ 83.42%) of the small RNAs have a length
within the range between 21 and 24 nt, which is a common length for miRNAs.
65.97 and 79.30% of the high-quality clean reads in the two libraries were
mapped to the pigeon genome, respectively (Table 1). The mappable reads were
used for further miRNA identification.
Table 1: Mapping the clean reads to the reference pigeon genome
Mapping statistics |
SMSCs |
Myotubes |
Raw Reads |
11,932,387 |
12,049,737 |
High-quality clean Reads |
11,111,427 |
10,519,865 |
Mapped Reads |
7,330,625 |
8,341,898 |
Unmapped Reads |
3,780,802 |
2,177,967 |
Mapped Ratio (%) |
65.97 |
79.30 |
Unmapped Ratio (%) |
34.03 |
20.70 |
Table 2: Pigeon miRNAs identified in two sRNA libraries
Group (number of pre-miRNA/miRNA) |
SMSCs |
Myotubes |
Total |
Pigeon known miRNAs |
162/290 |
155/272 |
166/297 |
Pigeon conserved miRNAs |
156/185 |
159/201 |
219/261 |
Pigeon putative novel miRNAs |
80/99 |
75/104 |
99/131 |
Fig. 3: miRNAs expression profiles in pigeon SMSCs and myotubes. A.
The number of expressed miRNAs at the two differentiation stages. B. Top 10 unique miRNAs with the
highest expression during SMSC differentiation. Plot with different color
represents the percentage of each miRNA expression in each library. The 6
overlapped miRNAs in the top 10 miRNAs in the two libraries are connected by
lines
Identification of miRNAs and expression profiles
in pigeon SMSCs
We totally identified 689
mature miRNAs in two differentiation stages of pigeon SMSCs (Table 2). These
miRNA candidates were classified into three types: known miRNAs,
conserved miRNAs and putative novel miRNAs. There are
297 pigeon known miRNAs corresponded to 166 pigeon known pre-miRNAs (Table S2).
261 pigeon conserved miRNAs corresponded to 219 other species pre-miRNAs (Table
S3). 131 putative novel miRNAs corresponded to 99 candidate pre-miRNAs (Table
S4). Besides, 462 miRNAs were shared by the two libraries, while 112 miRNAs
only expressed in SMSCs, and 115 miRNAs merely in myotubes (Fig. 3A).
To unveil the potential
roles of miRNAs in pigeon SMSCs and myotubes, identified miRNAs were ranked by
expression abundance (Fig. 3B). The miRNAs expression profile was different
between SMSCs and myotubes. Of note, the expression abundance of top 10 unique miRNAs accounts for 65.7 and 72.7% of the total counts in
these two libraries, respectively. Also, the top 10 unique miRNAs across two
SMSCs differentiation stages involves 14 kinds of unique miRNAs. Among these
miRNAs, six miRNAs (miR-21-5p, miR-26-5p, miR-92-3p, let-7a-5p, miR-27b-3p, miR-143-3p) overlapped.
Differentially expressed (DE) miRNAs analysis and
qPCR validation
To screen the
differential miRNAs between pigeon SMSCs and myotubes libraries, differential
expression analysis by taking |log2(fold
change) |>1 and FDR <0.001 as criteria were performed after removing
miRNAs of less than 11 count reads. We totally identified 193 miRNAs that were
differentially expressed during SMSCs differentiation (Table S5). Among these
DE miRNAs, 74 miRNAs were up-regulated, while another 119 miRNAs were
down-regulated (Fig. 4).
To confirm the small RNA-seq results, we
selected 8 miRNAs to conduct a qPCR assay. As shown in Fig. 4C, 6 miRNAs
(miR-181-5p, miR-429-3p, miR-119-5p, miR-126-3p, miR-200a-3p, miR-214-3p) were down-regulated in myotubes, while the other
2 miRNAs (miR-184a-3p and miR-133a-3p) were up-regulated when compared with
SMSCs. These results of qPCR assay were highly consistent with small RNA-seq.
Functional enrichment analysis
Fig. 4: Differential miRNAs
analysis and qPCR validation. A. Volcano plot of miRNA in SMSCs versus mytubes. B.
The upregulated and downregulated
number of the DE miRNAs. C. qRT-PCR validation of 8 DE miRNAs.
Red bars represent the miRNA relative expression
abundance determined by the miRNA sequencing reads
(normalized). Blue bars represent the miRNA relative
expression abundance determined by qRT-PCR (mean ± SE).
* above the error bars for each miRNA
show significant differences at P <
0.05
Differentially
expressed miRNAs with reading numbers higher than 1000 were selected to predict
the target genes. There is a total of 5284 genes having target sites for these
miRNAs. GO categorization of these target genes were
enriched in 237 terms including regulation of the biosynthetic process, cell
differentiation, cell development, anatomical structure morphogenesis, embryo
development, tube development (Fig. 5A, Table S6). KEGG pathways analysis
indicated these target genes were principally enriched in 25 pathways, such as
Endocytosis, Regulation of actin cytoskeleton, MAPK, Wnt, mTOR and TGF-beta
signal pathway (Fig. 5B, Table S6). Above results were closely linked to the
differentiation of skeleton muscle satellite cells.
Target verification of miR-181a-5p
Based on in silico
analysis, 3’-UTR region of Mef2a has
potential binding sites for cli-miR-181a-5p (Fig. 6A–B), we speculated that
cli-miR-181a-5p can directly target 3’UTR region of Mef2a and a dual-luciferase reporter assay was carried out. As
shown in Fig. 6C, cli-miR-181a-5p conspicuously decreased luciferase activity
of wild-type reporter of Mef2a
3′-UTR, whereas luciferase activity with Mef2a 3′-UTR mutant construct exhibited no statistically
significant difference between the cells transfected with cli-miR-181-5p mimics
and NC. These findings indicated that cli-miR-181a-5p can directly target Mef2a.
Discussion
The establishment of a
cellular research model through the isolation of muscle satellite cells from
different animal species is critical approach for understanding the mechanisms
of myogenic differentiation. Based on our previously established method, we
here isolated from the eggs of 16-day-old pigeon embryo. Interestingly, after
being cultivated
in growth medium without horse serum for 5 days, we observed numerous fused myotubes.
Combined with our results from MHC immunofluorescence staining, this confirmed
that the pigeon SMSCs had successfully differentiated. It is notable that horse
serum was not essential for pigeon SMSC differentiation, as it is close to
indispensable for the differentiation of SMSCs isolated from some other species
(e.g., bovine, mouse) into myotubes. Reported that
“spontaneous” differentiation of skeletal myoblast (e.g., Sol8 cells) is
associated with autocrine secretion of IGF -II (Florini
et al. 1991). We
speculate that this factor may also be associated with the “spontaneous”
differentiation of pigeon SMSCs; this will need to be confirmed by further
studies.
Fig. 5: Go functional enrichment and KEGG analysis of target genes of high
expressed DE miRNAs in SMSCs and myotubes.
A. Gene ontology enrichment (top20) analyzed by clusterProfiler. GeneRatio: the ratio of the number of target genes in the
GO category to that of the annotated genes in the GO database. B. Kyoto Encyclopedia of
Genes and Genomes (KEGG) pathway enrichment analysis (top15). GeneRatio: the ratio
of the number of target genes in the KEGG category to that of the annotated
genes in the KEGG database
Fig. 6: Cli-miR-181a-5p bioinformatics analyses and
dual-luciferase reporter assay. A.
Prediction of cli-miR-181a-5p binding
sites in 3’UTR of Mef2a.Luciferase
reporter plasmids contain WT or MUT putative
miR-181a-5p target sites. B. Schematic alignment of the free energy scores (RNAhybrid) for miRNA-181a-5p–Mef2a hybridization. C.
A dual-luciferase reporter assay was performed by co-transfecting luciferase
reporter containing the 3’UTR of Mef2a
(wild-type or mutant) with miR-181a-5p mimics or miR-control
into HeLa cells. Luciferase activity was determined
48 h after transfection. Three independent experiments were performed in
triplicate and all data were expressed as mean ± SE. * P < 0.05
MiRNAs play crucial roles in
myogenic differentiation by participating in an orchestrated process of gene
regulation (Horak et al. 2016). Certain miRNAs exclusively
expressed in the striated muscle are called myomiRs (McCarthy 2008). While miRNAs expressed in SMSCs and muscle tissues of
some species (e.g., bovine (Zhang et al.
2016), chicken (Li et al. 2011), duck (Gu et al.
2014)) have been identified, none have been reported in
pigeons. Here, we totally identified 689 miRNAs from two differentiation stages
of pigeon SMSCs, which is more than that previously reported (617) in bovine (Zhang et al.
2016), Among the top 10 unique miRNAs with the highest expression during pigeon
SMSC differentiation, three miRNAs (miR-21-5p, miR-199-3p and let-7a-5p) are
also ranked within the top ten in bovine SMSCs (Zhang
et al. 2016).
Some of identified
pigeon miRNAs have been previously reported to participate in skeletal
myogenesis and muscle development, for example, miR-21, miR-133,
miR-1, miR-27b, miR-499, miR-26 and miR-181 (Luo
et al. 2013; Bai et al. 2015). Among these, miR-21 exhibited the highest
abundance in both two libraries and its expression level was higher in myotubes
than in SMSCs. Reported that the miRNA-21 facilitates myogenesis by targeting
TGFβI (Bai et al. 2015). These results suggest that miRNA-21 also plays
a crucial role during skeletal myogenesis in pigeons. In addition, miR-133,
miR-1 and miR-499 are muscle-specific miRNAs. Of these, miR-133a suppresses
myoblast proliferation and promotes myoblast differentiation (Horak et al. 2016);
miR-1 can directly target HDAC4 to promote myogenesis (Chen et al. 2006);
overexpression of miR-499 reduces Mstn 3′UTR activity (Bell et al.
2010). Our results indicated that expression levels of miR-133a-3p,
miR-1 and miR-499 are conspicuously up-regulated during myogenic
differentiation in pigeons, which confirms the functional conservation of these
myomiRNAs between pigeons and other species. Additional miRNAs with a high
abundance in SMSCs and myotubes of pigeons have also been implicated in a
variety of physiological processes. For example, miR-184 regulated cell
proliferation by targeting SOX7 (Wu et al. 2014) and AKT2 (Foley et al. 2010), let-7a down-regulates
MYC and reverses MYC-induced cell growth (Sampson
et al. 2007), and miR199a-5p
inhibits insulin sensitivity via the suppression of ATG14-mediated autophagy (Li et al.
2018). Notably, a previous report documented that miR-181a paticipated
in muscle regeneration (Naguibneva et al. 2006); however, there is also evidence that miR-181 negatively regulates
myotube size (Soriano-Arroquia et al. 2016). In our study, the
expression level of miR-181a-5p was reduced during pigeon SMSCs
differentiation, which is consistent with research on bovine SMSCs (Zhang et al. 2016).
Furthermore, we found that cli-miR-181a-5p may directly target Mef2a which interacts with MRF family
members to promote myogenic differentiation (Luo
et al. 2013). Hence, the
down-regulation of cli-miR-181a-5p in myotubes may reflect the establishment of
a differentiated phenotype via the enhancement of of Mef2a expression.
The target genes of
highly abundant DE miRNAs were enriched in the GO categories ‘cellular
developmental process’, ‘cell differentiation’, ‘anatomical structure
morphogenesis’ and ‘tube development’. These results are in line with our
morphological observations and immunofluorescence analysis of pigeon SMSC
differentiation. Moreover, a KEGG pathway analysis revealed an enrichment of
these target genes mainly in the MAPK, Wnt, mTOR and TGF-beta signaling
pathways. Studies in other species have also demonstrated that these pathways play crucial
roles in myogenic differentiation (Liu et al.
2004; Keren et al. 2006; Tanaka et al. 2011). These findings
imply that pigeon miRNAs regulate SMSC differentiation also via similar signaling pathways as other
species.
Conclusion
In this
study, we identified 297 known miRNAs, 261 conserved miRNAs and 131 novel
miRNAs in pigeon SMSCs and myotubes using small RNA sequencing and proved that Mef2a is a direct target of
cli-miR-181a-5p. We infer that these identified miRNAs could play vital roles
during the myogenic differentiation of pigeon SMSCs, and these findings improve
our understanding of muscle differentiation and development in pigeons.
Acknowledgements
Differentially
expressed miRNAs were determined using the OmicShare tools, a free online
platform for data analysis (http://www.omicshare.com/tools).
Author Contributions
Xun Wang,
Xuewei Li and Mingzhou Li designed the experiments. Zhenhao Lin, Lei Liu, Peiqi Yan and Anan Jiang
performed experiments. Yi Luo, Siyuan
Feng, Qianzi Tang and Keren Long conducted bioinformatics analysis. Xun
Wang, Ling Zhao, Haifeng Liu, Long Jin and Jideng Ma statistically analyzed the
data and made illustrations.
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