miRNA-223 upregulated by MYOD inhibits myoblast proliferation by repressing IGF2 and facilitates myoblast differentiation by inhibiting ZEB1
Citation: Cell Death and Disease
miRNA-223 upregulated by MYOD inhibits myoblast proliferation by repressing IGF2 and facilitates myoblast differentiation by inhibiting ZEB1
Guihuan Li 0 1 2
Wen Luo 0 1
Bahareldin A Abdalla 0 1
Hongjia Ouyang 0 1
Jiao Yu 0 1
Fan Hu 0 1
Qinghua Nie 0 1
Xiquan Zhang 0 1
0 Department of Animal Genetics , Breeding and Reproduction , College of Animal Science, South China Agricultural University , Guangzhou 510642, Guangdong Province , China
1 Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, and Key Lab of Chicken Genetics , Breeding and Reproduction , Ministry of Agriculture, South China Agricultural University , Guangzhou 510642, Guangdong Province , China
2 Department of Cell Biology, School of Basic Medical Sciences, Southern Medical University , Guangzhou 510515 , China
Skeletal muscle differentiation can be regulated by various transcription factors and non-coding RNAs. In our previous work, miR-223 is differentially expressed in the skeletal muscle of chicken with different growth rates, but its role, expression and action mechanism in muscle development still remains unknown. Here, we found that MYOD transcription factor can upregulate miR-223 expression by binding to an E-box region of the gga-miR-223 gene promoter during avian myoblast differentiation. IGF2 and ZEB1 are two target genes of miR-223. The target inhibition of miR-223 on IGF2 and ZEB1 are dynamic from proliferation to differentiation of myoblast. miR-223 inhibits IGF2 expression only in the proliferating myoblast, whereas it inhibits ZEB1 mainly in the differentiating myoblast. The inhibition of IGF2 by miR-223 resulted in the repression of myoblast proliferation. During myoblast differentiation, miR-223 would be upregulated owing to the promoting effect of MYOD, and the upregulation of miR-223 would inhibit ZEB1 to promote myoblast differentiation. These results not only demonstrated that the well-known muscle determination factor MYOD can promote myoblast differentiation by upregulate miR-223 transcription, but also identified that miR-223 can influence myoblast proliferation and differentiation by a dynamic manner regulates the expression of its target genes. Cell Death and Disease (2017) 8, e3094; doi:10.1038/cddis.2017.479; published online 5 October 2017
MicroRNAs (miRNAs) are small non-coding RNA of 20?25
nucleotides that mainly transcript from introns or intergenic
regions, having critical roles in the regulation of gene
expression at posttranscriptional levels.1 It have been reported
that miRNAs widely participate in chicken embryo growth,
maturation, muscle cell proliferation and differentiation, cell
migration, dosage compensation effect of Z chromosome and
a series of life activities.2?7 In our preliminary study, we have
used high-throughput RNA sequencing to study breast muscle
transcriptome in Recessive White Rock (fast-growing chicken)
and Xinghua chicken (slow-growing chicken), and pointed out
that miR-223 can be used as a candidate gene associating
with broiler growth.8
The miR-223 is highly conserved among vertebrates. As the
first miRNA identified in human hemopoietic system,9 miR-223
was confirmed to participate in the regulation of hematopoietic
cell proliferation and differentiation,10 and become a research
hotspot. There are some competitive binding sites for NFI-A
and C/EBP alpha in the upstream of miR-223. C/EBP alpha
enhances miR-223 transcription activity, whereas NFI-A
inhibits the transcription activity of miR-223.10 The
upregulation of miR-223 expression will promote granulocyte and
megakaryocyte generation, and the decline of miR-223
expression will cause hematopoietic stem cells to differentiate
into erythrocyte.11,12 In addition, miR-223 was confirmed to
regulate cell proliferation, differentiation, migration and signal
transduction in mammals.13 However, the roles of miR-223 in
muscle development still remain unclear.
Insulin-like growth factor-2 (IGF2) is a member of insulin-like
growth factor family that can play a role in all insulin-activated
target cells.14 It is well-known that IGF2 can be acted as the
intermediate messenger of growth hormone, which can
transfer the growth hormone to target organs, and regulate
the development of organisms.15 IGF2 is conserved during
evolution, the gene structure and exons location in chicken are
similar to that in mammal, although the non-coding sequences
were very different.16 In mammals, IGF2 is a kind of
multifunctional cell regulation factor that has an important role
in the muscle cell proliferation and differentiation.17 In chicken,
IGF2 is significantly associated with broilers growth traits and
carcass traits, such as birth weight, breast muscle weight,
abdominal fat weight and glandular stomach weight.18?20
Zinc finger E-box binding homeobox 1 (ZEB1) is a crucial
nuclear transcription factor, it can directly bind to the E2-box
[5-CATTC(G)] sequence in different gene promoter region,
repressing the activity of gene transcription.21 In mammal,
ZEB1 has three inhibiting zones.22 Zone-I mainly regulates
T-lymphocyte differentiation. No inhibitory activity was found in
zone-II. Zone-III is associated with muscle differentiation. In
chicken, ZEB1 is also called DeltaEF1 (delta-crystallin/E2-box
factor 1), which is highly homologous with mice and can also
recognize the E2-box sequence (CATTCG).23 In the process
of chicken embryo development, ZEB1 was expressed in
somite, notochord, urogenital system, nervous system and
other parts of the chicken embryo, suggesting that it may
involve in cell development process.24 In addition, ZEB1 can
inhibit the activity of viral gene transcription by targeting the
E2-box sequence exists in the promoter region of chicken
infectious anemia virus, result in the decrease of the virus
MYOD and MYOG are critical transcription factors in skeletal
muscle differentiation and can regulate the transcription of
most myogenesis-related genes.26?28 During myoblast
differentiation, both of these two factors are upregulated their
expression.29?31 MYOD and MYOG regulates their target
genes transcription by binding to E-box promoter elements
containing the site CANNTG.28 Recently, many myogenic
miRNAs were found to be regulated by MYOD and MYOG,32
demonstrating an indirect role of these transcription factors in
regulating muscle differentiation.
In this study, we investigated the function and regulation of
miR-223 in avian skeletal muscle development. We found that
miR-223 is regulated by MYOD transcription factor, which can
bind to the promoter region of the gga-miR-223 gene. IGF2
and ZEB1 are two target genes of miR-223, and the
upregulation of miR-223 can inhibit myoblast proliferation
and promote myoblast differentiation by its repression on
these two target genes. In addition, miR-223 represses IGF2
expression only in proliferating myoblast, and it has a more
significant inhibiting effect on ZEB1 in differentiating myoblast
than in proliferating myoblast. These results identified a role for
miR-223 in avian myoblast proliferation and differentiation
through dynamic regulates its target genes expression, and
found another regulatory pathway of MYOD to shape myoblast
miR-223 expression is related to skeletal muscle
development. In our previous miRNA sequencing data, we found
that miR-223 exhibited differentially expression between the
skeletal muscles of chickens with different growth rate.8 To
further understand the relationship between miR-223 and
skeletal muscle development, we detected its expression in
breast muscle during chicken embryonic development.
miR-223 was gradually upregulated its expression from
embryo day 10 (E10) to E13. However, its expression was
continually downregulated after E13 (Figure 1a). In addition,
in situ hybridization results also showed that the miR-223
expression was upregulated from E10 to E13 and
downregulated from E14 to E19 (Figure 1b). Haematoxylin?eosin
staining of the breast muscle showed that the muscle fibers
are blurry, irregular and may in the stage of cell differentiation
and fusion between E10 and E13 (Figure 1c). After E13, the
cross sections of muscle fibers were relatively regular and
becoming more clearly defined (Figure 1c). Together with the
expression data of miR-223, these results suggesting that
miR-223 upregulated its expression during the active stage of
muscle cell differentiation and fusion, whereas it
downregulated its expression during muscle fiber maturation.
miR-223 inhibits myoblast proliferation. To further
determine the roles of miR-223 in chicken skeletal muscle
development, we transfected miR-223 mimics and inhibitors
into chicken primary myoblast (CPM), respectively. CCK-8
assay indicated that miR-223 overexpression inhibited
myoblast proliferation (Figure 2a), whereas miR-223
loss-offunction promoted myoblast proliferation (Figure 2b). In
addition, by analyzing cell cycle of the transfected myoblasts,
we found that miR-223 overexpression can decrease cell
population in the S phase and increase cell population in the
G1/0 phase (Figure 2c). miR-223 overexpression can also
inhibit the expression of cell cycle-promoting genes and
enhance cell cycle-inhibiting genes (Figure 2d). On the
contrary, inhibition of miR-223 significantly increased cell
population in the S phase and decreased cell population in
the G1/0 phase (Figure 2e). miR-223 inhibition also
enhanced the expression of cell cycle-promoting genes and
repressed cell cycle-inhibiting genes (Figure 2f). EdU staining
showed that the proliferation rate of miR-223-transfected cells
was significantly reduced compared with that of the control
cells (Figure 2g and h), whereas miR-223 loss-of-function
promoted cell proliferation rate (Figure 2g and h).
Furthermore, these roles of miR-223 were also existed in the qm-7
cells (Figure 2i-p), indicating that miR-223 can inhibit avian
The inhibitory effect of miR-223 on myoblast proliferation
was achieved by its target gene IGF2. IGF2 is an important
growth factor that can functioning as growth promoting
hormone during cell development. Here, we found that the
3?-untranslated regions (3?-UTR) of IGF2 mRNA has a
potential binding site of miR-223 (Figure 3a). To validate
whether IGF2 is a target gene of miR-223, we constructed
two dual-luciferase reporters with the wide-type and mutant
3?-UTR of IGF2, respectively. Results shown that miR-223
significantly repressed the luciferase activity of the wide-type
reporter, whereas it has no effect on the luciferase activity of
the mutant reporter (Figure 3b). In addition, miR-223
significantly inhibited the mRNA and protein expression of
IGF2 gene in CPM (Figure 3c), and the inhibition of miR-223
released IGF2 mRNA and protein expression (Figure 3d).
The above results indicated that IGF2 is a direct target gene
Although IGF2 is a well-known growth promoting-gene, its
roles in chicken myoblast proliferation have not been
examined. In this study, we transfected pSDS-IGF2 and three
IGF2 siRNAs in poultry myoblast to overexpressing or
inhibiting IGF2 expression, respectively. pSDS-IGF2
transfection significantly increased IGF2 mRNA and protein
expression, whereas the si-170, which is specifically designed for
chicken IGF2 mRNA, significantly repressed IGF2 mRNA and
protein expression (Figure 3e-h). IGF2 overexpression
promotes myoblast proliferation through CCK-8 assay (Figure 3i
and j) and cell cycle analysis (Figure 3k and l), whereas IGF2
inhibition repressed myoblast proliferation (Figure 3m-p).
Furthermore, the rescue assay shown that miR-223
overexpression can inhibit the myoblast proliferation, and the
transfection of IGF2 overexpression vector could counteract
the inhibitory effect of miR-223 on myoblast proliferation
(Figure 3q). Together, these results argue that the inhibitory
effect of miR-223 on myoblast proliferation was achieved by its
target gene IGF2.
miR-223 promotes myoblast differentiation. miR-223 was
upregulated its expression during the active stage of chicken
skeletal muscle differentiation and fusion. Next, we examined
the expression and function of miR-223 during myoblast
differentiation and fusion. miR-223 expression was
significantly upregulated from the proliferation to the differentiation
of both CPM and qm-7 cells (Figure 4a and b), suggesting its
involvement in these processes. Therefore, we transfected
miR-223 mimic and inhibitor into the myoblasts. After
transfection, the cells were induced to differentiation and
fusion. Overexpression of miR-223 significantly increased the
expression of muscle differentiation marker genes (Figure 4c
and d), whereas the inhibition of miR-223 decreased the
expression of these marker genes (Figure 4e and f). In
addition, miR-223 promotes the formation of myotubes
(Figure 4g), and the size of the myotube area was
significantly increased after transfection of miR-223
(Figure 4h). Therefore, these results demonstrated that
miR-223 can promote myoblast differentiation.
ZEB1 is another miR-223 target gene, functioning as an
inhibitor of myoblast differentiation. By using the
TargetScan online software to predict the target genes of
miR-223, we found that the 3?-UTR of ZEB1 mRNA has a
potential binding site of miR-223 (Figure 5a). Notably, this
binding site is conserved among vertebrates (Figure 5b).
The dual-luciferase report assay suggested that miR-223
directly binds to the predicted target site of ZEB1-3?-UTR
(Figure 5c). miR-223 overexpression inhibited the mRNA
and protein expression level of ZEB1 (Figure 5d and e),
and the inhibition of miR-223 promoted the mRNA and
protein expression level of ZEB1 (Figure 5f and g).
Therefore, the above results indicated that ZEB1 is another
target gene of miR-223.
During myoblast proliferation and differentiation, ZEB1 protein
was gradually downregulated its expression (Figure 5h). To test
the function of ZEB1 in myoblast differentiation, we synthesized
siRNAs specifically target for chicken ZEB1 mRNA. Transfection
of these siRNAs significantly inhibited the mRNA and protein
expression level of ZEB1 in CPM and qm-7 (Figure 5i and j). In
addition, ZEB1 inhibition significantly increased the expression of
muscle differentiation marker genes in both of these two cells
(Figure 5k and l), suggesting ZEB1 is a repressor of myoblast
differentiation. Rescue assay shown that ZEB1 overexpression
is able to counteract the promotion effect of miR-223 on myoblast
differentiation (Figure 5m). Therefore, ZEB1 is another miR-223
target gene that can function as an inhibitor of myoblast
The inhibition of miR-223 on IGF2 and ZEB1 is different
between myoblast proliferation and differentiation. The
above results shown that both IGF2 and ZEB1 are target
genes of miR-223. However, IGF2 is able to promote
myoblast differentiation in mammal myoblast.33 Its expression
was increased during CPM and qm-7 differentiation (Figure
6a and b). This expression trend is similar to miR-223, which
can directly inhibit IGF2 expression. In addition, specifically
inhibition of IGF2 by siRNA significantly downregulated
muscle differentiation marker genes (Figure 6c and d),
indicating its promoting effect in myoblast differentiation.
These results suggested that miR-223 cannot restrict IGF2
expression and function during myoblast differentiation.
On the other hand, ZEB1, another miR-223 target gene that
can inhibit myoblast differentiation, is unable to regulate cell
cycle in proliferating myoblast (Figure 6e and f). Therefore, we
were interest in how the miR-223 regulates the ZEB1 and
IGF2 expression in the proliferating and differentiating
myoblast, respectively. For ZEB1, miR-223 inhibits its
expression in both proliferation and differentiation stages of myoblast
(Figure 6g), and the inhibitory effect of miR-223 on ZEB1 has
no significant difference between these two stages. For IGF2,
miR-223 inhibits its expression only in the proliferation stage of
myoblast (Figure 6g), and the miR-223 expression shown no
significant difference between the proliferation and
differentiation stages (Figure 6h). To further validate these results, we
transfected dual-luciferase reporter inserted with the 3?-UTR
of ZEB1 or IGF2 into the proliferating and differentiating
myoblasts, respectively. The reporter activity inserted with
ZEB1-3?-UTR was significant lower in differentiating
myoblasts compared with that in proliferating myoblasts (Figure 6i).
However, the reporter activity inserted with IGF2-3?-UTR was
significant higher in differentiating myoblasts compared with
that in proliferating myoblasts (Figure 6i). Together, these
results suggested that the inhibition of miR-223 on IGF2 and
ZEB1 is different between myoblast proliferation and
MYOD regulates miR-223 transcription by binding to the
E-box 1 region. To further understand the structure of the
gga-miR-223 gene, we isolated its full-length pri-miR-223 by
using 5? and 3? rapid amplification of cDNA ends (RACE).
The obtained gga-miR-223 gene was 2228 bp in length
(Figure 7a). Next, we analyzed the upstream region of the
gga-miR-223 gene to find the core promotor region. Four
fragments, including 668-bp, 1020-bp, 1508-bp and 1932-bp
upstream regions of the gga-miR-223 transcription start site
(TSS) were amplified and cloned into the pGL3-basic vector.
Forty-eight hours after transfected these reporter vectors
into the myoblasts, the relative luciferase activity was
measured to analyze the promoter activity of these four
reporters. Results shown that the four reporters have similar
relative luciferase activity (Figure 7b), suggesting that the
shortest one or no reporter have the core promoter region.
In addition, we found that there are two E-boxes located in
the 1932-bp upstream regions of the gga-miR-223 TSS
(Figure 7a). It is well-known that the MYOG and MYOD
transcription factors can bind to the E-box region and
promote the transcription activity of myogenic genes.34?36
Considering that MYOD, MYOG and miR-223 are all
upregulated their expression during myoblast differentiation,
we next examined whether MYOD and MYOG can bind to
these two E-box regions and promote gga-miR-223
transcription. By mutated these two regions, respectively, we
found that the relative luciferase activity was significantly
reduced when we mutated E-box 1, whereas there is no
change of the luciferase activity when we mutated E-box 2
(Figure 7b). This result suggesting that the E-box 1 is
important for the transcription activity of the promoter region.
Next, chromatin immunoprecipitation assay (ChIP) results
indicated that the MYOD can bind to the E-box 1 region
(Figure 7c). To avoid the effect of endogenous MYOD, we
used DF-1 cell to test the regulation of MYOD on the
ggamiR-223 gene promoter and miR-223 transcription. MYOD
overexpression promoted the relative luciferase activity of
the pGL3-1932 reporter, but this overexpression had no
effect on the luciferase activity of the mutated pGL3-1932
reporter (Figure 7d). In addition, MYOD overexpression
promoted miR-223 expression (Figure 7e). For primary
myoblast, MYOD loss-of-function not only inhibited the
expression of miR-223 and muscle differentiation marker
genes, but also promoted the expression of miR-223 target
genes IGF2 and ZEB1 (Figure 7f). Furthermore, myoblast
proliferation was also being inhibited when the cell was
transfected with si-MYOD (Figure 7g). Together, these
results demonstrated that the MYOD regulates
ggamiR-223 transcription by binding to the E-box 1 region.
In this study, we identified miR-223 as another miRNA that has
a role in avian skeletal muscle development. miR-223
regulates myoblast proliferation and differentiation by
balancing the expression of its target genes IGF2 and ZEB1. In
addition, MYOD promotes miR-223 transcription during
myoblast differentiation by binding to the gga-miR-223 gene
promoter region (Figure 8). These findings not only revealed a
new miRNA-mediated pathway functions on skeletal muscle
differentiation, but also found an interest regulatory mode of
miRNA in its target genes.
MYOD belongs to the myogenic basic-helix-loop-helix
transcription factors, and is an important regulator during
skeletal muscle differentiation. MYOD has the ability to initiate
a lot of muscle-related genes transcription by recruitment of
transcription factors and histone acetyltransferases.28,34,35
Many muscle development related genes have been found to
be regulated by MYOD during myoblast differentiation.28,34 In
this study, we found that MYOD can bind to the E-box, which is
located in the promoter region of miR-223 gene, and promote
miR-223 expression in chicken myoblast. In addition, the
expression of miR-223 and MYOD are upregulated during
chicken myoblast differentiation,29 demonstrating a positive
association between these two regulators. Besides, some of
the muscle-specific miRNAs, such as miR-1, miR-133 and
miR-206, can also be regulated by MYOD during myoblast
differentiation.37,38 Therefore, these findings suggested that
MYOD is able to regulate myoblast differentiation through
activate various myogenic miRNAs expression.
ZEB1 is a transcriptional repressor. During muscle
differentiation, ZEB1 is able to repressed muscle gene
transcription, and the inhibition of ZEB1 can promote
myoblast differentiation and fusion.39 Although the
repression effect of ZEB1 in muscle differentiation has been well
documented, however, its expression pattern during muscle
differentiation still remains unknown. In this study, we have
shown that ZEB1 protein was gradually downregulated
during chicken myoblast differentiation, and the decrease of
ZEB1 is, at least in part, owing to the inhibition of miR-223.
In addition, ZEB1 can also repress myoblast differentiation
in avian, but it cannot regulate myoblast proliferation, even
though it has been shown to have positive role in many
cancer cell proliferation.40?42 MYOD can occupy
G/Ccentered E-boxes in the promoters of many
musclerelated genes and initiate the transcription of these genes
during myoblast differentiation.43 Interestingly, ZEB1 can
also bind to the G/C-centered E-boxes in myoblast, but its
binding results in the repression of muscle-related gene
expression.39 Therefore, our results suggesting that MYOD
inhibits ZEB1 expression by promoting miR-223 expression
during myoblast differentiation.
IGF2 has long been established to have important roles in
muscle development,44 and it is also a critical growth factor
for chicken.19 However, the function of IGF2 in avian
myoblast has not been verified before. Here, we reported
that IGF2 can both promote avian myoblast proliferation and
differentiation, and we also found that miR-223 has the
ability to inhibit IGF2 expression. Notably, miR-223 inhibits
IGF2 expression only in the proliferating myoblast. Its
inhibition effect on IGF2 expression was significantly
reduced from proliferating myoblast to differentiating
myoblast. In addition, the inhibitory effect of miR-223 on ZEB1 in
the proliferating myoblast was more significant than that in
the differentiating myoblast. It is generally understood that a
miRNA can bind to multiple target genes, and the inhibiting
effect of miRNA on these target genes would be different
because of their target sites with different functional
properties.45 However, it is rare to see that a miRNA has
different binding efficiency on one target gene during cell
development. Previous study has shown that synaptic
stimulation can relieve the repression of miRNA to its target
genes.46,47 A miRNA-mediated inhibition of target gene
translation can also be relieved when the cells subjected to
different stress conditions, and this relief is depend on the
binding of HuR, which is an AU-rich element RNA-binding
protein (RBP), to the 3?UTR of the target gene.48 Dead end 1
(Dnd1) is another RBP evolutionary conserved among
vertebrates, and also has the ability to counteracts the
inhibition roles of several miRNAs by binding to the 3?UTR of
target genes.49 However, the precise mechanism of how
miR-223 dynamic regulates IGF2 and ZEB1 during
myoblast proliferation and differentiation still need to be
explored. Collectively, these and our findings suggest that
the binding of miRNA to its target genes is dynamic in
different cellular environment, and this binding can be
rapidly responded to the specific cellular needs.48
In summary, this work has shown that the avian myoblast
proliferation and differentiation are regulated by
MYODmiR-223-IGF2/ZEB1 pathway. In the proliferating avian
myoblast, miR-223, which is upregulated by MYOD, inhibits
IGF2 expression to represses myoblast proliferation. In the
differentiating avian myoblast, the upregulation of MYOD
promotes miR-223 expression, and miR-223 inhibits ZEB1
rather than IGF2 to facilitate myoblast differentiation and
Materials and Methods
Animals. The hatching eggs of Xinghua chickens were bought from the Chicken
Breeding Farm of South China Agricultural University, Guangdong, China. More
than ten chick embryos for each group were selected for pectorals and leg muscle
separation. All the tissue samples were frozen in liquid nitrogen and stored at ? 80 ?
C for the subsequent DNA and RNA extraction. With the sex-specific primers, the
sex of each embryo was determined by PCR amplification.
RNA extraction, cDNA synthesis and quantitative real-time
PCR. The total RNA was extracted from muscle tissues or cells using RNAiso
reagent (Takara, Otsu, Japan). The reverse transcription reaction for mRNA was
performed with PrimeScript RT reagent Kit (Perfect Real-Time) (Takara) according
to manufacturer?s manual. The reverse transcription reaction for miRNA was using
ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). The specific Bulge-loop miRNA
qRT-PCR Primer for miR-223 and U6 (one RT primer and a couple of qPCR primers
for each gene) were designed by RiboBio (RiboBio, Guangzhou, China). With KAPA
SYBR FAST qPCR Kit (KAPA Biosystems, Wobrun, MA, USA), qPCR program was
carried out in Bio-rad CFX96 Real-Time Detection system (Bio-rad, Hercules, CA,
USA), and the method was as described.50 All reactions were run in triplicate.
Cell culture. QM-7 cell culture: QM-7 cells were cultured in M199 (Gibco,
Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (Hyclone,
Logan, UT, USA), 10% tryptose phosphate broth solution (Sigma Life Science, St.
Louis, MO, USA) and 0.2% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA).
The differentiation of QM-7 was induced by M199 supplemented with 10% tryptose
phosphate broth solution (Sigma) and 0.2% penicillin/streptomycin.
CPM isolation and culture: The leg muscle of E11 chickens were used to isolate
CPM. When the skin and bones removed, the leg muscles were rapidly chopped into
small pieces in petri dish containing DMEM (Gibco) media supplemented with 20%
fetal bovine serum (Hyclone) and 0.2% penicillin/streptomycin. The muscle
suspending liquid was shaken by vortexing 1 min and filtered to obtain single cell.
Repeat the vortexing and filtering steps for 4?6 times to release enough cells. The
cells were then collected by centrifugation at 1000 ? g for 5 min at room temperature.
Decant medium and discard. Add DMEM supplemented with 20% FBS and 0.2%
penicillin/streptomycin to resuspend cells completely. Serial plating was needed to
remove fibroblasts and enrich myoblasts. The differentiation of myoblasts was
induced by DMEM supplemented with 0.2% penicillin/streptomycin.
Immunofluorescence. The immunofluorescence was performed in CPM
cultured in 24-well plates. Cells were fixed in 4% paraformaldehyde for 20 mins at
room temperature and then washed three times with PBS (5 min for every turn).
Subsequently, cells were treated with 0.1% Triton X-100 for 20 minutes and were
blocked with goat serum for 30 min. Then, the cells were incubated with anti-MyHC
(DSHB, Iowa City, IA, USA) at 4 ?C overnight. After washed with PBS, the cells were
treated with FITC-conjugated anti-rabbit IgG antibody and incubated in dark for 1 h.
The cell nuclei were stained with DAPI (Beyotime, Jiangsu, China). The total
myotube area was calculated and measured as previously described.50
The 5?- and 3?-RACE. The SMARTer RACE cDNA Amplification Kit (Takara)
was used to perform both 5?- and 3?-RACE according to the user manual. The 5?
and 3? RACE-Ready first-strand cDNA was synthesized using total RNA extracted
from chicken skeletal muscle. Two rounds of PCR reaction were preformed. First
round was carried out with Universal Primer Mix (UPM) provided by the supplier
inside the kit and miR-223 specific outer primer, and second round with Nested
Universal Primer (NUP) also provided by the supplier inside the kit and miR-223
specific inner primer. Both 5?- and 3?-RACE PCR products were cloned and
sequenced. All the primers used in RACE PCR are summarized in Supplementary
pmirGLO dual-luciferase reporters: The 3?UTR fragment of IGF2 (NCBI
Reference Sequence: NM_001030342.1) and ZEB1 (NM_205131.1) containing the
binding sites were amplified by PCR from chicken embryonic leg muscle cDNA and
then cloned into pmirGLO vector. The mutant vectors were constructed by PCR
mutagenesis. Five seed sequences were successful mutated from ACTGA to
CTGAG for IGF2-3?UTR vector, and from CTGAC to ACTGT for ZEB1-3?UTR
Gene overexpression vector: The IGF2 overexpression vector was
constructed according to the user manual of Easy Ligation Kit (Sidansai, Shanghai,
China). IGF2 coding sequence (NCBI Reference Sequence: NM_001030342.1) was
amplified from chicken embryonic leg muscle cDNA by PCR. The PCR product was
cloned into the pSDS-20117 vector (Sidansai). The successful IGF2 overexpression
vector was confirmed by DNA sequencing. The MYOD overexpression vector was
constructed as previously described.29
miR-223 promoter reporter plasmid: A 1932-bp fragment of the miR-223
promoter was isolated by PCR using the primers listed in Supplementary file. After
the PCR product was digested with KpnI and SmaI restriction sites, the insertion
was ligated into the pGL3-basic vector (Promega, Madison, WI, USA) to create the
expression vector pGL3-1932. After pGL3-1932 was sequenced, this construct was
used as a template, and pGL3-1508, pGL3-1020 or pGL3-668 was isolated by PCR.
Site-directed mutagenesis of E-box 1 and E-box 2 was carried out by PCR
amplification and DpnI digestion to remove the parental DNA.
Transfections. Transfections were performed with Lipofectamine 3000 reagent
(Invitrogen) according to the manufacturer?s direction. Nucleic acids were diluted in
OPTI-MEM Medium (Gibco). All experiments were carried out at least three times
RNA oligonucleotides. The miR-223 mimics, negative control (NC), miR-223
inhibitors, miRNA inhibitor NC, siRNA against chicken IGF2, ZEB1 and MYOD were
all purchased from GenePharma (GenePharma, Shanghai, China).
Dual-luciferase reporter assay. The miRNA target verification assay was
performed in QM-7 cells. Wild-type or mutant IGF2-3? UTR dual-luciferase reporter
(200 ng) and miR-223 mimic or NC duplexes (50 nM) were co-transfected into
QM-7 cells using the Lipofectamine 3000 reagent (Invitrogen) in 48-well plates. For
the promoter assays, qm-7 cells and DF-1 cells were co-transfected with reporter
plasmid and MYOD overexpression vector or control vector, and the TK-Renilla
reporter was also co-transfected to each sample as an internal control. After 48 h
transfection, cells were washed by PBS twice and the activities of Firefly and Renilla
luciferase were measured according to the manual of Luc-pair Duo-Luciferase
Assay Kit 2.0 (GeneCopoeia, Rockville, MD, USA). All the data were acquired by
averaging the results from four independent repeats.
Cell cycle analysis. After 36 h transfection, primary myoblast or QM-7 cells
cultured in 12-well plates were fixed in 75% ethanol overnight at ? 20 ?C. With the
Cell Cycle Analysis Kit (Thermo Fisher Scientific, Waltham, MA, USA), the cells
were analyzed by a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA,
USA). All the data were acquired by averaging the results from three independent
CCK-8 assay. Primary myoblast or QM-7 cells were cultured in 96-well plates.
A total of 10 ?l of Cell counting kit-8 reagent was added into each well and
incubated for 1 h. The assay was repeated at different time points of 12, 24, 36,
48 h or every 24 h after transfection. The absorbance was measured at 450 nm by
a Model 680 Microplate Reader (Bio-Rad). All the data were acquired by averaging
the results from six independent experiments.
Western blot. Western blot was performed as previously described.50 The
following antibodies were used: anti-IGF2 (Santa Cruz Biotechnology, CA, USA),
anti-ZEB1 (abcam, Cambridge, MA, USA) and anti-GAPDH (Bioworld, St Louis
Park, MN, USA).
ChIP assays. ChIP assay was performed as previously described.29 Samples
were done in triplicate. The primer sequences for ChIP-qPCR analysis are shown in
miRNA in situ hybridization assay. gga-miR-223 miRCURY LNA probe
and a scrambled probe were synthesized (Exiqon, Copenhagen, Denmark). 10 ?m
thick cryosections from breast muscle tissues of Xinhua chicken were fixed in 4%
PFA for 15 min at room temperature, and then treated for 10 min with Proteinase K.
miRNA in situ hybridization was performed using the FISH kit (Exonbio Lab,
Guangzhou, China). DNA was counterstained with DAPI (1 mg/ml). Images of
miRNA signals in slides were captured by a Leica DMi8 fluorescent microscope.
EdU assay. EdU assay was performed as previously described with the
following modifications.29 Twelve hours after transfection, primary myoblasts were
exposed to 10 ?M 5-ethynyl-2?-deoxyuridine (EdU; RiboBio) for 24 h at 37 ?C, and
the QM-7 cells were exposed to 50 ?M EdU for 2 h at 37 ?C. In addition, for primary
myoblasts, 1 ? Apollo reaction cocktail (RiboBio) was added to the cells and
incubated for 30 min. Whereas, in QM-7 cells, 1 ? Apollo reaction cocktail was
added to the cells and incubated for 20 min. The EdU-stained cells were visualized
under a Leica DMi8 fluorescent microscope. The proliferation rate was calculated by
the number of EDU-stained cells normalized to the number of Hoechst
Statistical analysis. Each experiment was repeated three times, and all
results are represented as the mean ? S.E.M. Independent sample t-test was used
to perform the statistical significant difference between groups.
Ethics standards. All experimental protocols were approved by the South
China Agricultural University Institutional Animal Care and Use Committee (approval
ID: SCAU#0014). And the methods were carried out in accordance with the
regulations and guidelines established by this committee.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements. This work was supported by Natural Scientific
Foundation of China (31472090), China High-Tech Programs (2013AA102501), the China
Agriculture Research System (CARS-41-G03), and the Program for New Century
Excellent Talents in University (NCET-13-0803), and the Foundation for High-level
Talents in Higher Education of Guangdong, China.
Springer Nature remains neutral with regard to jurisdictional claims in published maps
and institutional affiliations.
Cell Death and Disease is an open-access journal
published by Nature Publishing Group. This work is
licensed under a Creative Commons Attribution 4.0 International
License. The images or other third party material in this article are
included in the article?s Creative Commons license, unless indicated
otherwise in the credit line; if the material is not included under the
Creative Commons license, users will need to obtain permission from
the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/
Supplementary Information accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis)
1. Bartel DP . MicroRNAs: genomics, biogenesis, mechanism, and function . Cell 2004 ; 116 : 281 - 297 .
2. Rengaraj D , Lee BR , Lee SI , Seo HW , Han JY . Expression patterns and miRNA regulation of DNA methyltransferases in chicken primordial germ cells . PLoS ONE 2011 ; 6 : e19524 .
3. Gschwend AR , Weingartner LA , Moore RC , Ming R. The sex-specific region of sex chromosomes in animals and plants . Chromosome Res 2012 ; 20 : 57 - 69 .
4. Kang L , Cui X , Zhang Y , Yang C , Jiang Y . Identification of miRNAs associated with sexual maturity in chicken ovary by Illumina small RNA deep sequencing . BMC Genomics 2013 ; 14 : 352 .
5. Huang HY , Liu RR , Zhao GP , Li QH , Zheng MQ , Zhang JJ et al. Integrated analysis of microRNA and mRNA expression profiles in abdominal adipose tissues in chickens . Sci Rep 2015 ; 5 : 16132 .
6. Han B , Lian L , Li X , Zhao C , Qu L , Liu C et al. Chicken gga-miR-103-3p targets CCNE1 and TFDP2 and inhibits MDCC-MSB1 cell migration . G3 (Bethesda) 2016 ; 6 : 1277 - 1285 .
7. Li Y , Wang X , Yu J , Shao F , Zhang Y , Lu X et al. MiR -122 targets the vanin 1 gene to regulate its expression in chickens . Poult Sci 2016 ; 95 : 1145 - 1150 .
8. Ouyang H , He X , Li G , Xu H , Jia X , Nie Q et al. Deep sequencing analysis of miRNA expression in breast muscle of fast-growing and slow-growing broilers . Int J Mol Sci 2015 ; 16 : 16242 - 16262 .
9. Chen CZ , Li L , Lodish HF , Bartel DP . MicroRNAs modulate hematopoietic lineage differentiation . Science 2004 ; 303 : 83 - 86 .
10. Fazi F , Rosa A , Fatica A , Gelmetti V , De Marchis ML , Nervi C et al. A minicircuitry comprised of microRNA-223 and transcription factors NFI-A and C/EBPalpha regulates human granulopoiesis . Cell 2005 ; 123 : 819 - 831 .
11. Masaki S , Ohtsuka R , Abe Y , Muta K , Umemura T . Expression patterns of microRNAs 155 and 451 during normal human erythropoiesis . Biochem Biophys Res Commun 2007 ; 364 : 509 - 514 .
12. Yuan JY , Wang F , Yu J , Yang GH , Liu XL , Zhang JW . MicroRNA-223 reversibly regulates erythroid and megakaryocytic differentiation of K562 cells . J Cell Mol Med 2009 ; 13 : 4551 - 4559 .
13. Yang W , Lan X , Li D , Li T , Lu S. MiR-223 targeting MAFB suppresses proliferation and migration of nasopharyngeal carcinoma cells . BMC Cancer 2015 ; 15 : 461 .
14. Rinderknecht E , Humbel RE . Primary structure of human insulin-like growth factor II . FEBS Lett 1978 ; 89 : 283 - 286 .
15. Vasilatos-Younken R , Scanes CG . Growth hormone and insulin-like growth factors in poultry growth: required, optimal , or ineffective? Poult Sci 1991 ; 70 : 1764 - 1780 .
16. Darling DC , Brickell PM . Nucleotide sequence and genomic structure of the chicken insulin-like growth factor-II (IGF-II) coding region . Gen Comp Endocrinol 1996 ; 102 : 283 - 287 .
17. Gerrard DE , Okamura CS , Ranalletta MA , Grant AL . Developmental expression and location of IGF-I and IGF-II mRNA and protein in skeletal muscle . J Anim Sci 1998 ; 76 : 1004 - 1011 .
18. Amills M , Jimenez N , Villalba D , Tor M , Molina E , Cubilo D et al. Identification of three single nucleotide polymorphisms in the chicken insulin-like growth factor 1 and 2 genes and their associations with growth and feeding traits . Poult Sci 2003 ; 82 : 1485 - 1493 .
19. Tang S , Sun D , Ou J , Zhang Y , Xu G , Zhang Y . Evaluation of the IGFs (IGF1 and IGF2) genes as candidates for growth, body measurement, carcass, and reproduction traits in Beijing You and Silkie chickens . Anim Biotechnol 2010 ; 21 : 104 - 113 .
20. Gholami M , Erbe M , Garke C , Preisinger R , Weigend A , Weigend S et al. Population genomic analyses based on 1 million SNPs in commercial egg layers . PLoS ONE 2014 ; 9 : e94509 .
21. van Grunsven LA , Taelman V , Michiels C , Opdecamp K , Huylebroeck D , Bellefroid EJ . deltaEF1 and SIP1 are differentially expressed and have overlapping activities during Xenopus embryogenesis . Dev Dyn 2006 ; 235 : 1491 - 1500 .
22. Postigo AA , Dean DC . Differential expression and function of members of the zfh-1 family of zinc finger/homeodomain repressors . Proc Natl Acad Sci USA 2000 ; 97 : 6391 - 6396 .
23. Sekido R , Murai K , Funahashi J , Kamachi Y , Fujisawa-Sehara A , Nabeshima Y et al. The delta-crystallin enhancer-binding protein delta EF1 is a repressor of E2-box-mediated gene activation . Mol Cell Biol 1994 ; 14 : 5692 - 5700 .
24. Funahashi J , Sekido R , Murai K , Kamachi Y , Kondoh H . Delta-crystallin enhancer binding protein delta EF1 is a zinc finger-homeodomain protein implicated in postgastrulation embryogenesis . Development 1993 ; 119 : 433 - 446 .
25. Miller MM , Jarosinski KW , Schat KA . Negative modulation of the chicken infectious anemia virus promoter by COUP-TF1 and an E box-like element at the transcription start site binding deltaEF1 . J Gen Virol 2008 ; 89 : 2998 - 3003 .
26. Berkes CA , Tapscott SJ . MyoD and the transcriptional control of myogenesis . Semin Cell Dev Biol 2005 ; 16 : 585 - 595 .
27. Braun T , Gautel M. Transcriptional mechanisms regulating skeletal muscle differentiation, growth and homeostasis . Nat Rev Mol Cell Biol 2011 ; 12 : 349 - 361 .
28. Cao Y , Kumar RM , Penn BH , Berkes CA , Kooperberg C , Boyer LA et al. Global and genespecific analyses show distinct roles for Myod and Myog at a common set of promoters . EMBO J 2006 ; 25 : 502 - 511 .
29. Luo W , Li E , Nie Q , Zhang X . Myomaker, regulated by MYOD, MYOG and miR- 140 -3p, promotes chicken myoblast fusion . Int J Mol Sci 2015 ; 16 : 26186 - 26201 .
30. Davis RL , Weintraub H , Lassar AB . Expression of a single transfected cDNA converts fibroblasts to myoblasts . Cell 1987 ; 51 : 987 - 1000 .
31. Hasty P , Bradley A , Morris JH , Edmondson DG , Venuti JM , Olson EN et al. Muscle deficiency and neonatal death in mice with a targeted mutation in the myogenin gene . Nature 1993 ; 364 : 501 - 506 .
32. Luo W , Nie Q , Zhang X. MicroRNAs involved in skeletal muscle differentiation . J Genet Genomics 2013 ; 40 : 107 - 116 .
33. Ge Y , Sun Y , Chen J . IGF-II is regulated by microRNA-125b in skeletal myogenesis . J Cell Biol 2011 ; 192 : 69 - 81 .
34. Blum R , Vethantham V , Bowman C , Rudnicki M , Dynlacht BD . Genome-wide identification of enhancers in skeletal muscle: the role of MyoD1 . Genes Dev 2012 ; 26 : 2763 - 2779 .
35. Cao Y , Yao Z , Sarkar D , Lawrence M , Sanchez GJ , Parker MH et al. Genome-wide MyoD binding in skeletal muscle cells: a potential for broad cellular reprogramming . Dev Cell 2010 ; 18 : 662 - 674 .
36. Aziz A , Liu QC , Dilworth FJ . Regulating a master regulator: establishing tissue-specific gene expression in skeletal muscle . Epigenetics 2010 ; 5 : 691 - 695 .
37. Rosenberg MI , Georges SA , Asawachaicharn A , Analau E , Tapscott SJ . MyoD inhibits Fstl1 and Utrn expression by inducing transcription of miR-206 . J Cell Biol 2006 ; 175 : 77 - 85 .
38. Koutsoulidou A , Mastroyiannopoulos NP , Furling D , Uney JB , Phylactou LA . Expression of miR-1 , miR- 133a , miR -133b and miR-206 increases during development of human skeletal muscle . BMC Dev Biol 2011 ; 11 : 34 .
39. Siles L , Sanchez-Tillo E , Lim JW , Darling DS , Kroll KL , Postigo A . ZEB1 imposes a temporary stage-dependent inhibition of muscle gene expression and differentiation via CtBP-mediated transcriptional repression . Mol Cell Biol 2013 ; 33 : 1368 - 1382 .
40. Hou LK , Yu Y , Xie YG , Wang J , Mao JF , Zhang B et al. miR -340 and ZEB1 negative feedback loop regulates TGF-beta- mediated breast cancer progression . Oncotarget 2016 ; 7 : 26016 - 26026 .
41. Zhang Y , Liu G , Wu S , Jiang F , Xie J , Wang Y . Zinc finger E-box-binding homeobox 1: its clinical significance and functional role in human thyroid cancer . Onco Targets Ther 2016 ; 9 : 1303 - 1310 .
42. Gu Y , Zhao Y , Zhou Y , Xie Y , Ju P , Long Y et al. Zeb1 is potwntial regulator of Six2 in the proliferation, apoptosis and migration of metanephric mesenchyme cells . Int J Mol Sci 2016 ; 17 : pii: E1283 .
43. Soleimani VD , Yin H , Jahani-Asl A , Ming H , Kockx CE , van Ijcken WF et al. Snail regulates MyoD binding-site occupancy to direct enhancer switching and differentiation-specific transcription in myogenesis . Mol Cell 2012 ; 47 : 457 - 468 .
44. Florini JR , Magri KA , Ewton DZ , James PL , Grindstaff K , Rotwein PS . "Spontaneous" differentiation of skeletal myoblasts is dependent upon autocrine secretion of insulin-like growth factor-II . J Biol Chem 1991 ; 266 : 15917 - 15923 .
45. Brennecke J , Stark A , Russell RB , Cohen SM . Principles of microRNA-target recognition . PLoS Biol 2005 ; 3 : e85 .
46. Ashraf SI , McLoon AL , Sclarsic SM , Kunes S . Synaptic protein synthesis associated with memory is regulated by the RISC pathway in Drosophila . Cell 2006 ; 124 : 191 - 205 .
47. Schratt GM , Tuebing F , Nigh EA , Kane CG , Sabatini ME , Kiebler M et al. A brain-specific microRNA regulates dendritic spine development . Nature 2006 ; 439 : 283 - 289 .
48. Bhattacharyya SN , Habermacher R , Martine U , Closs EI , Filipowicz W. Relief of microRNAmediated translational repression in human cells subjected to stress . Cell 2006 ; 125 : 1111 - 1124 .
49. Kedde M , Strasser MJ , Boldajipour B , Oude VJ , Slanchev K , le Sage C et al. RNA-binding protein Dnd1 inhibits microRNA access to target mRNA . Cell 2007 ; 131 : 1273 - 1286 .
50. Luo W , Li G , Yi Z , Nie Q , Zhang X. E2F1-miR- 20a -5p/20b-5p auto-regulatory feedback loop involved in myoblast proliferation and differentiation . Sci Rep 2016 ; 6 : 27904 .
r The Author(s) 2017