CircINSR regulates fetal bovine muscle and fat development

Abstract

Background: The level of muscle development directly affects the production efficiency of livestock, and the content of intramuscular fat (IMF) is an important factor affecting meat quality. Nevertheless, the molecular mechanism of embryonic circular RNA in muscle and IMF development remains largely unknown.
Methods: In this study, we isolated myoblasts and intramuscular preadipocytes from fetal bovine skeletal muscle. Oil Red O and BODIPY staining were used to identify lipid droplets of preadipocytes, and anti-MyHC immunofluorescence was used to identify myotubes differentiated from myoblasts. Bioinformatics, dual fluorescence reporter system, and RNA immunoprecipitation were used to determine the interactions between circINSR and miR-15/16 family. Molecular and biochemical assays were used to confirm the role of circINSR in myoblasts and intramuscular preadipocytes.
Results: We found that the isolated myoblasts and preadipocytes can differentiate normally. Besides, circINSR severed as a sponge of miR-15/16 family, which targeting CyclinD1 and Bcl-2. CircINSR overexpression significantly promoted myoblasts and preadipocytes proliferation, and inhibited cell apoptosis. In addition, circINSR inhibited preadipocytes adipogenesis by alleviating the inhibition of miR-15/16 on target genes FOXO1 and EPT1.
Conclusions: Taken together, our study demonstrated circINSR as a regulator of embryonic muscle and IMF development.

Full text

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CircINSR regulates fetal bovine muscle and fat
development
Xuemei Shen
Northwest Agriculture and Forestry University https://orcid.org/0000-0003-3451-0204
Jia Tang
Northwest Agriculture and Forestry University
Wenxiu Ru
Northwest Agriculture and Forestry University
Xiaoyan Zhang
Northwest Agriculture and Forestry University
Yongzhen Huang
Northwest Agriculture and Forestry University
Xianyong Lan
Northwest Agriculture and Forestry University
Chuzhao Lei
Northwest Agriculture and Forestry University
Hui Cao
Shaanxi Province
Hong Chen (  chenhong1212@263.net )
Northwest Agriculture and Forestry University
Methodology article
Keywords: Bovine, Muscle development, Circular RNAs, Cell proliferation, Preadipocyte
DOI: https://doi.org/10.21203/rs.3.rs-79997/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
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Abstract
Background: The level of muscle development directly affects the production eciency of livestock, and
the content of intramuscular fat (IMF) is an important factor affecting meat quality. Nevertheless, the
molecular mechanism of embryonic circular RNA in muscle and IMF development remains largely
unknown.
Methods: In this study, we isolated myoblasts and intramuscular preadipocytes from fetal bovine skeletal
muscle. Oil Red O and BODIPY staining were used to identify lipid droplets of preadipocytes, and anti-
MyHC immunouorescence was used to identify myotubes differentiated from myoblasts.
Bioinformatics, dual uorescence reporter system, and RNA immunoprecipitation were used to determine
the interactions between circINSR and miR-15/16 family. Molecular and biochemical assays were used to
conrm the role of circINSR in myoblasts and intramuscular preadipocytes.
Results: We found that the isolated myoblasts and preadipocytes can differentiate normally. Besides,
circINSR severed as a sponge of miR-15/16 family, which targeting
CyclinD1
and
Bcl-2
. CircINSR
overexpression signicantly promoted myoblasts and preadipocytes proliferation, and inhibited cell
apoptosis. In addition, circINSR inhibited preadipocytes adipogenesis by alleviating the inhibition of miR-
15/16 on target genes
FOXO1
and
EPT1
.
Conclusions: Taken together, our study demonstrated circINSR as a regulator of embryonic muscle and
IMF development.
1. Introduction
In livestock production, the development of muscle and intramuscular fat (IMF) is an important factor in
ensuring meat quality. Bovine muscle development begins in the early embryonic stage, including the
proliferation of myoblast progenitor cells, as well as the proliferation and fusion of mononuclear
myoblasts form multinucleated myotubes. After birth, the number of muscle bers will not change, but
the bers become thicker(1, 2). Therefore, the level of muscle development during pregnancy directly
affects the meat production. Fat starts to develop in the second trimester(3). The content of the IMF is
related to the tenderness and juiciness of beef, which has always been a research hotspot. Increasing the
number of preadipocytes in the fetal muscle helps to deposit fat and marbling after birth(4). However, the
mechanism of muscle and IMF development is still unclear. In addition, premature adipogenesis and
maturation of intramuscular preadipocytes in the fetus may cause muscle tissue dysfunction(5).
Therefore, it is of great signicance to explore the molecular mechanism of muscle and IMF
development.
The development of biotechnology has greatly promoted the screening and research of key genes for
muscle and IMF development. Several critical genes have been demonstrated to mediate muscle
development and adipogenesis. For instance, peroxisome proliferative activated receptor (
PPARγ
) and
CCAAT/enhancer binding protein (
C/EBPα
) have been characterized as the regulators of adipogenesis(6,

Page 3/19
7). The myogenic regulatory factors (
MRFs
), myocyte enhancer factor (
MEF
2),
PAX3/PAX7
, and
myostatin (
MSTN
) have been reported to be effective in inducing myoblasts proliferation and
differentiation(8-12).
In addition to coding genes, a large number of non-coding RNAs have also been proven to regulate
muscle and fat development. For example, a large number of miRNAs and circRNAs have been reported
to participate in the physiological regulation of muscle and fat. CircRNAs have a covalent closed loop
structure, neither 5'-3' polarity nor polyadenylated tail(13). They can participate in physiological regulation
by sponging miRNAs or binding regulatory proteins(14). In existing reports, circFUT10 and circFGFR4
regulate the muscle development related genes through sponge miR-133a and miR-107(15, 16). CircINSR
adsorb miR-34a to promote the proliferation of bovine myoblasts(17). CircTshz2-1 and circArhgap5-2
have been shown to be indispensable regulators of fat formation(18). Despite these ndings, the role of
circRNAs in muscle and IMF development still requires further research.
In this study, we isolated fetal bovine myoblasts and intramuscular preadipocytes. The targeting
relationship between circINSR and miR-15/16 family and the effect of circINSR on the proliferation and
apoptosis of myoblasts and preadipocytes were analyzed in vitro. Importantly, we proved that circINSR
can target miR-15/16 to inhibit the premature differentiation of intramuscular preadipocytes, thereby
ensuring the normal of muscle function.
2. Materials And Methods
2.1 Progenitor cells isolation and cell lines.
Bovine fetuses for 120~180 days were collected from the slaughterhouse and transported to the
laboratory immediately. Using the enzyme digestion combined with the differential adhesion method, the
primary myoblasts and intramuscular preadipocytes were isolated from the longissimus dorsi as
previously described (19). The longissimus dorsi was isolated from the fetus, washed with phosphate
buffered saline (PBS), and minced into small fragments. It was then digested in Dulbecco’s modied
Eagle’s medium (DMEM) containing type IV collagenase (w/v, 0.2%; C5138, Sigma, USA) at 37°C with
continuous shaking for 2 hours. The cell plasma was ltered through the 200 μm lter, collected by
centrifugation and resuspended in DMEM. The cells were seeded in complete medium and incubated at
37 °C with 5% CO2. The adherent cells within two hours of inoculation were collected for adipogenesis
induction. After 24 hours of culture, the non-adherent cell suspension was re-inoculated. After repeated
operations for 2 days, adherent cells were collected for myogenesis induction. HEK-293T cells were
purchased from the American Type Culture Collection (ATCC) and were tested negative for mycoplasma
contamination.
2.2 Differentiation of myoblasts and preadipocytes.
Myoblasts were cultured in high-glucose DMEM supplemented with 20% fetal bovine serum (FBS, Gibco).
2 days after cells reached conuence, DMEM containing 2% horse serum was used for myogenesis

Page 4/19
induction. Myotube identication was performed on the 4th day of induction. Intramuscular
preadipocytes were cultured in F12/DMEM supplemented with 10% FBS. Adipocyte differentiation was
induced by M1 medium (containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1.5
μg/mL insulin). 2 days later, the M1 medium was replaced with M2 medium (containing 1.5 μg/mL
insulin). Then differentiation was induced for 8 days, during which the medium was changed once every
2 days. The M2 medium was changed every two days, and Oil Red O staining was performed on the 8th
day of adipogenic differentiation.
2.3 RNA extraction and real-time qPCR
Total RNA was isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized
with PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Tokyo, Japan). Real-time qPCR for RNA
analyses were performed using SYBR Green PCR Master Mix (Takara, Tokyo, Japan). MiRNAs specic
stem-loop primers were used for reverse transcription. The level of GAPDH was used to normalize the
expression of circRNA and mRNA, and the level of small nuclear U6 was used to normalize the expression
levels of miRNAs.
2.4 Vector construction and cell transfection
The second exon sequence of
INSR
gene was constructed into pCD2.1 vector and psi-CHECK2 vector.
Small interfering RNA (siRNA) oligonucleotides were designed to combine with the back-splice region of
circINSR (RiboBio, Guangzhou, China). The mimics of bta-miR-15a, bta-miR-15b, bta-miR-16a, and bta-
miR-16b were purchased from RiboBio (Guangzhou, China). The 3'-UTRs of
CCND
1 and
Bcl
-2 genes
containing the miR-15/16 binding sites were amplied by PCR enzyme mix (Platinum II Taq Hot-Start
DNA Polymerase, Invitrogen). The wild-type and mutant 3'-UTR gene sequences were cloned into the psi-
CHECK2 vector. The mimics (50 nM) or vectors (2 μg/mL) were transfected into cells with transfection
reagent (R0531, Thermo Fisher Scientic, USA). For overexpression of miR-15/16 family, a quarter of miR-
15a, miR-15b, miR-16a, and miR-16b mimics were selected and mixed in equal amounts for transfection.
2.5 Oil Red O and BODIPY staining
After 8 days of differentiation, the intramuscular preadipocytes were stained with Oil red O (O0625,
sigma, USA) and 4,4-diuoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503)
(D3922, Thermo Fisher Scientic). Oil Red O dyeing was performed according to the instructions. To
quantify staining in fat droplets, the 100% isopropanol was used to dissolve the lipid droplets, and
measure the absorbance at 510 nm. For BODIPY staining, the cells were washed twice with PBS to
remove residual 4% paraformaldehyde. Hank’s Balanced Salt Solution with 10 μM BODIPY 493/503 was
added to the cells and then incubated at 37°C for 30 minutes in the dark. The samples were washed 3
times with PBS and photographed immediately.
2.6 Immunouorescence analysis

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After 4 days of myogenic differentiation, the myoblasts were xed with 4% paraformaldehyde. After
washing with PBS, MyHC antibody (1:250, Heavy chain cardiac Myosin antibody, GTX20015, GeneTex,
USA) was added to incubate overnight at 4°C, and then the secondary antibody was added to incubate
for 2 hours. The nucleus was stained with DAPI. The myotube coverage area was analyzed by Image pro
plus software.
2.7 Dual Luciferase Reporter Assay
HEK-293T cells were co-transfected with miR-15/16 mimics and plasmid. After 24 hours of transfection,
the luciferase activity was detected with the Dual-Luciferase Reporter Assay Kit (E2920, Promega,
Fitchburg, WI, USA). The optical density of the resulting solution was assessed using the automatic
microplate reader (Molecular Devices, Sunnyvale, USA).
2.8 RNA-binding protein immunoprecipitation (RIP)
RNA-binding protein immunoprecipitation assay was performed using EZ-Magna RIP kit (17-701,
Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. The Argonaute2 (Ago2)
antibody (Abcam, UK) and IgG antibody were used for immunoprecipitation. The RNA was extracted from
the immunoprecipitation products of myoblasts and preadipocytes, and the abundance of circINSR and
miR-15/16 was detected by real-time qPCR.
2.9 5-Ethynyl-2`-deoxyuridine (EdU) and Cell Counting Kit-8 (CCK-8) assay
When the density of myoblasts and preadipocytes reached 40%-50%, the transfection was performed with
overexpression plasmid, siRNA or miRNA mimics. After 24 hours of transfection, the cell proliferation was
tested by EdU assay kit (RiboBio, Guangzhou, China). The nucleus was stained with Hoechst 33342. Use
a uorescence microscope to take pictures immediately after staining (AMG EVOS, Seattle, WA, USA).
Similarly, we also used the CCK-8 (Multisciences, Hangzhou, China) to detect the level of cell proliferation
after transfection. The optical density of CCK-8 at 450 nm was measured using an automatic microplate
reader (Synergy4, BioTek, Winooski, USA).
2.10 Cell cycle and apoptosis assay
We used ow cytometry and the Cell Cycle Testing Kit (Multisciences, Hangzhou, China) to analyze the
cell cycle. The myoblasts and preadipocytes were transfected when the cell growth density reached 50%.
After transfection for 24 hours the cells were collected and washed with PBS. Subsequently, follow the kit
instructions for staining. Flow cytometry analysis was performed on a BD Accuri C6 flow cytometer (BD
Biosciences, USA). Cell apoptosis assays were performed with Annexin V-PE/7-AAD Apoptosis Detection
Kit (RiboBio, Guangzhou, China) according to the manufacturer’s recommendations. Afterward, the
apoptosis rate was analyzed using ow cytometry (FACS Canto™ II, BD BioSciences, USA).
2.11 Western Blot analysis

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Proteins from cultured myoblasts and preadipocytes were lysed with RIPA buffer (Solarbio, Beijing,
China). Proteins were loaded onto 12% SDS-PAGE gels and transferred to polyvinylidene diuoride
(PVDF) membranes (Thermo Fisher Scientic). The membranes were incubated overnight with primary
antibodies specic for anti-GAPDH (#ab9485), anti-CyclinD1 (#ab226977), (Abcam, Cambridge, UK), anti-
Bcl-2 (#bs-0032R), anti-caspase-9 (#bs-0049R), anti-Bax (#bs-0127M), anti-FABP4 (#bsm-51247M)
(Bioss, Beijing, China), anti-PCNA (#WL01804), anti-CDK2 (#WL01543), anti-PPARγ (#WL01800) and anti-
C/EBPα (#WL01899) (Wanlei Bio, Shenyang, China) at 4℃. After incubation with secondary antibodies,
the membranes were quantied with the chemiluminescence system (Bio Rad, Hercules, CA, USA).
2.12 Statistical analyses
Data are expressed as the mean ± standard error (SEM) of at least 3 independent experiments. Statistical
analyses were performed using SPSS 19.0 statistical software (SPSS, Chicago, IL, USA). Statistically
signicant differences were calculated using Student`s
t
-test. A probability of 0.05 or less was considered
statistically significant.
3. Results
3.1 Myoblasts and intramuscular preadipocytes were isolated from the bovine fetus.
Skeletal muscle and intramuscular adipose tissue differentiated from mesoderm mesenchymal stem
cells (MSCs). Muscle begins to develop in the early embryonic period, and adipose tissue begins to occur
in the second trimester(20) (Fig. 1A). Enzymatic digestion combined with differential adhesion method
could roughly separate myoblasts and intramuscular preadipocytes. In this study, these isolated
preadipocytes were spindle-shaped and possess the common characteristics of broblasts. After 8 days
of adipogenic induction, small lipid droplets accumulated in some cells. The Oil Red O staining results
also intuitively indicated that the isolated cells had undergone adipogenic differentiation (Fig. 1B). The
results of BODIPY staining showed that 8 days of adipogenic induction caused lipid accumulation in
intramuscular preadipocytes (Fig. 1C). In addition, the real-time qPCR results showed that the
adipogenesis marker genes
PPARγ
and
C/EBPα
were signicantly increased (Fig. 1E). At the same time,
the results of MyHC immunouorescence showed that the isolated myoblasts were also induced into
some myotubes (Fig. 1D). And myogenic differentiation marker genes were also signicantly
overexpressed (Fig. 1F).
3.2 CircINSR serves as a sponge of miR-15/16.
Previous studies have shown that circINSR could adsorb miR-34a. In this study, after overexpression of
circINSR in myoblasts and preadipocytes, real-time qPCR results showed that the expression of miR-15a,
miR-15b, miR-16a, and miR-16b was signicantly reduced (Fig. 2A, B). The binding sites for miR-15/16
family on circINSR were predicted using Target Scan 7.0 and miRanda (Fig. 2C). The results of the dual
uorescence reporter system showed that the overexpression of miR-15/16 family could signicantly
inhibit the activity of Renilla luciferase in the psi-CHECK2-circINSR vector (Fig. 2D). In order to verify this

Page 7/19
adsorption, we performed Ago2-RIP assay in myoblasts and preadipocytes to detect the expression of
endogenous circINSR and miR-15/16 bound to Ago2 protein. The results showed that the miR-15/16
family can be enriched by Ago2 protein in both myoblasts and preadipocytes (Fig. 2F, G). In Ago2 protein
immunoprecipitation, the expression of circINSR was signicantly higher than that of IgG group (Fig. 2E).
3.3 CircINSR promotes myoblasts proliferation and inhibits apoptosis.
Existing studies have reported that
CyclinD
1 and
Bcl-
2 are the target genes of miR-15/16 family. We
connected the wild type and mutant type of the 3'-UTR sequence containing the binding site into the psi-
CHECK2 vector for verication of the dual uorescent reporter gene system (Fig. 3A). The results showed
that the overexpression of miR-15/16 family could signicantly inhibit the activity of Renilla luciferase in
the wild-type vector (Fig. 3B).
CyclinD1
gene is a key regulator of cell proliferation. Real-time qPCR results
showed that the overexpression of miR-15/16 mixed mimics could signicantly reduce the expression of
CyclinD
1 and other cell proliferation marker genes. However, co-transfection of circINSR and miR-15/16
could alleviate this inhibition (Fig. 3C). Further EdU results showed that the transfection of miR-15/16
mixed mimics could signicantly reduce the number of proliferating cells, and co-transfection with
circINSR could rescue this anti-proliferation effect (Fig. 3D, E). The CCK-8 cell proliferation analysis also
obtained the same result (Fig. 3F). The cell cycle results showed that miR-15/16 blocked the cell cycle
and reduced the number of cells entering the S phase, while co-transfection with circINSR alleviated this
inhibition (Fig. 3G, H).
In order to verify the targeting relationship between miR-15/16 and
Bcl-
2, we constructed wild-type and
mutant psi-CHECK2 vectors (Fig. 4A). The results of the uorescence report analysis indicate that
Bcl-
2
was a potential target gene of miR-15/16 (Fig. 4B). Real-time qPCR results showed that miR-15/16
signicantly inhibited the expression of
Bcl-
2 and promoted the expression of apoptosis marker genes
Bax
and
Caspase
9 (Fig. 4C). The subsequent ow cytometry analysis results showed that overexpression
of miR-15/16 promoted myoblasts apoptosis, and co-transfection of circINSR reduced the number of
apoptotic cells (Fig. 4D, E).
3.4 CircINSR promotes preadipocytes proliferation by sponging miR-15/16.
To investigate the role of circINSR in adipogenesis, preadipocytes were transfected with circINSR
overexpression vector and siRNA. The results of real-time qPCR showed that overexpression of circINSR
signicantly promoted the expression of cell proliferation marker genes (Fig. 5A), while interference with
circINSR inhibited the expression of these genes (Fig. 5B). The Western Blots showed the same results
(Fig. 5C). In addition, transfection of mixed miR-15/16 mixed mimics in preadipocytes could signicantly
inhibit the expression of cell proliferation-related genes, while co-transfection with circINSR could restore
this inhibition (Fig. 5D). The results of EdU (Fig. 5E, F) and CCK-8 (Fig. 5G) also showed that miR-15/16
inhibited cell proliferation, and co-transfection of miR-15/16 and circINSR alleviated this inhibition. Cell
cycle assay showed that miR-15/16 inhibited preadipocytes entering S phase, and co-transfection with
circINSR promoted cell proliferation (Fig. 5H, I).

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3.5 CircINSR inhibits preadipocytes apoptosis by sponging miR-15/16.
To further explore the function of circINSR in preadipocytes apoptosis, real-time qPCR was used to detect
the expression of apoptosis-related genes after overexpression and interference with circINSR. The results
showed that circINSR promoted the expression of anti-apoptotic gene
Bcl-2
and inhibited the expression
of pro-apoptotic genes
BAX
and
Caspase
9 (Fig. 6A, B). At the same time, Western Blots got the same
trend (Fig. 6C). In contrast, real-time qPCR results showed that overexpression of miR-15/16 inhibited
Bcl-
2 gene expression and promoted
BAX
and
caspase
9 expression. The co-transfection of circINSR inhibited
the apoptosis of preadipocytes (Fig. 6D). To further verify that circINSR can inhibit cell apoptosis by
adsorbing miR-15/16 family, we used Annexin V-PE/7-AAD staining combined with ow cytometry to
analyze the effects of co-transfection of miR-15/16 and circINSR on cell apoptosis. The results showed
that miR-15/16 promoted cell apoptosis, and co-transfection with circINSR rescued this anti-apoptotic
effect (Fig. 6E, F).
3.6 CircINSR inhibits preadipocytes differentiation.
According to the above results, circINSR could promote the proliferation of myoblasts and preadipocytes,
and inhibit cell apoptosis. We further studied the function of circINSR on the differentiation of
preadipocytes. The results showed that overexpression of circINSR could inhibit the expression of
adipogenic related genes
PPARγ
,
C/EBPα
, and
FABP4
(Fig. 7A, B). In contrast, interference with circINSR
promoted the expression of these genes (Fig. 7C, D). BODIPY staining can directly observe the formation
of lipid droplets in adipocytes through green uorescence. Given that the circINSR overexpression vector
carries GFP uorescence, we only analyzed the BODIPY staining after interference with circINSR. 8 days
after induction, the cells were conducted by BODIPY staining. The results showed that si-circINSR
signicantly increased the intensity of green uorescence in preadipocytes (Fig. 7E). In addition, the
staining results of Oil Red O also showed that overexpression of circINSR inhibited the lipogenesis of
precursor fat, and the accumulation of lipid droplets increased after interference with circINSR (Fig. 7F).
In order to further analyze whether the effect of circINSR on adipogenesis is related to miR-15/16, we
found two reported target genes of miR-15/16,
FOXO1
and
EPT1
(
SELENOI
) (Fig. 8A). Real-time qPCR
results showed that the expressions of
FOXO1
and
EPT1
were signicantly reduced after miR-15/16
overexpression, and co-transfection with circINSR rescued this inhibition (Fig. 8B). In addition, miR-15/16
promoted the expression of adipogenic genes, while circINSR inhibited the expression of adipogenic
genes (Fig. 8C). The results of Oil Red O staining showed that miR-15/16 promoted lipid accumulation in
preadipocytes, while co-transfection with circINSR inhibited adipogenesis (Fig. 8D, E).
4. Discussion
Myoblasts and intramuscular adipocytes are derived from mesenchymal stem cells (MSCs). Under
complex signal regulation, some MSCs differentiate into myogenic and non-myogenic cell lines.
Myogenic cells enter the process of muscle development, while non-myogenic cells enter the process of
adipogenesis or broblast development(20, 21). At about 180 days of gestation in bovine fetuses, the IMF

Page 9/19
begins to appear. But before that, the adipogenic progenitor cells have already begun to differentiate into
preadipocytes(22). Therefore, we can separate myoblasts and intramuscular preadipocytes by using
enzyme digestion combined with differential adhesion screening.
In this study, the isolated myoblasts were of high purity, and thick myotubes were labeled with anti-MyHC
uorescent antibody after 4 days of differentiation. However, the isolated preadipocytes contained MSCs,
broblasts and adipogenic progenitor cells, which affected the cell purity. Combining cell surface marker
proteins (such as CD140a) and ow cytometry sorting might be able to obtain higher purity
preadipocytes(23). Nevertheless, the preadipocytes were also successfully induced to differentiate, and
lipid droplets were identied using Oil Red O and BODIPY staining.
The development of muscle and fat is regulated by a complex signal network involving coding genes and
non-coding RNAs. In this study, the expression of
PPARγ
and
C/EBPa
in differentiated preadipocytes
increased signicantly. And after 4 days of myogenic differentiation,
MyOD
and
MyHC
expression
increased signicantly. These coding genes played an important role in the differentiation of muscle and
fat. In addition, more and more reports proved that non-coding RNAs are also involved in the regulation of
muscle and fat development.
The circINSR is highly homologous to human has_circ_0048966, formed by the head-to-tail splicing of
INSR
second exon (552 bp), and is mainly expressed in the cytoplasm. In previous research, circINSR
regulates cells proliferation and apoptosis through miR-34a-modulated
Bcl-
2 and
CyclinE
2
expression(17). In this study, we found that circINSR could also sponge the miR-15/16 family. The real-
time qPCR results showed that overexpression of circINSR in myoblasts and preadipocytes could
signicantly inhibit the expression of miR-15/16 family. Dual uorescence analysis and Ago2-RIP results
also illustrated this adsorption relationship. Therefore, according to the molecular mechanism of
sponging miRNAs, circRNAs should have the same targeting ability in view of the same seed sequence of
miRNAs family.
In animals, single-stranded miRNAs binds specic mRNAs through sequences that are imperfectly
complementary to the target mRNAs, mainly to the 3'-UTR(24). Existing studies have reported the
regulatory mechanism of
Bcl-
2 and
CyclinD
1 in cancer cells consisting of posttranscriptional down-
regulation by miR-15 and miR-16(25-27). In this study, we veried the interaction of miR-15/16 with
Bcl-
2
and
CyclinD
1. Overexpression of miR-15/16 in myoblasts and preadipocytes promoted cell proliferation
and inhibited apoptosis. Much more, the effect of miR-15/16 was counteracted when circINSR was co-
transfected. The results indicated that during the embryonic stage, circINSR could promote muscle
development and increase the number of intramuscular preadipocytes.
The number of intramuscular preadipocytes is the guarantee for the marbling during fattening. However,
the premature maturation of the IMF may lead to fetal muscle insuciency and wasted of nutrition
during pregnancy. For example, the early muscle tissue of Duchenne muscular dystrophy is manifested
as muscle ber regeneration and mild lipid droplets, and the late muscle bers are gradually replaced by
fat and connective tissue, which seriously affects muscle function. Studies have pointed out that miR-

Page 10/19
15/16 promotes adipogenesis by targeting
FOXO
1 and
EPT
1 genes(28, 29). In our results, the
overexpression of circINSR in preadipocytes inhibited the expression of key genes for adipogenic
differentiation and reduced lipid droplet formation. And the function of inhibiting adipogenesis is
achieved by sponging miR-15/16. Therefore, the function of circINSR to inhibit adipogenesis in the fetal
period can maintain the normal muscle function while ensuring the number of intramuscular
preadipocytes.
5. Conclusions
In conclusion, our results reveal that circINSR negatively regulates miR-15/16 family expression. CircINSR
promotes the proliferation of myoblasts and preadipocytes and inhibits apoptosis. And inhibit the
differentiation of intramuscular preadipocytes to ensure the normal development of embryonic muscle.
These results provide potential molecular targets for improving beef production and molecular breeding.
6. Abbreviations
IMF: intramuscular fat; AGO2: Argonaute2 protein;
PPARγ
: peroxisome proliferative activated receptor;
C/EBPα
: CCAAT/enhancer binding protein;
MRFs
: myogenic regulatory factors;
MEF
2: myocyte enhancer
factor; MSCs: mesoderm mesenchymal stem cells; INSR: insulin receptor; EdU: 5-ethynyl-20-deoxyuridine;
CCK-8: cell counting kit-8; circRNAs: circular RNAs; ceRNA: competitive endogenous RNA; MyHC: myosin
heavy chain isoforms; NC: negative control; 7-AAD: 7-aminoactinomycin; BODIPY 493/503: 4,4-diuoro-
1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene; RIP: RNA binding protein immunoprecipitation;
7. Declarations
Funding
This work was supported by the National Natural Science Foundation of China [Grant No. 31772574],
Major project of collaborative innovation of industry, university, research and application in Yangling
Demonstration Zone [2018CXY-05]. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Availability of data and material
The datasets used and analyzed during the current study are available from the corresponding author on
reasonable request.
Authors' contributions
H. Chen and X. M. Shen designed research; X. M. Shen, X. Y. Zhang, W. X. Ru and Y. Z. Huang performed
experiments and analyzed data. X. M. Shen wrote the paper. C. Z. Lei and X. Y. Lan contributed new

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analytic tools. H. Chen and J. Tang helped modify the language of this manuscript. The authors declare
they have no competing nancial interest and no conicts of interest.
Ethics approval and consent to participate
All animal experiments and study protocols were approved by the Animal Care Commission of the
College of Veterinary Medicine, Northwest A&F University.
Consent for publication
Not applicable.
Competing interests
The authors declare they have no competing nancial interest and no conicts of interest.
Acknowledgements
Not applicable.
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Figures
Figure 1

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Identication of isolated myoblasts and intramuscular preadipocytes. (A) The growth pattern of muscle
and fat during embryonic development of cattle. (B) The growth morphology of the isolated
preadipocytes on day 2 and day 4, and the Oil Red O staining intracellular lipid droplets at 8 days of
adipogenic differentiation. (C) BODIPY staining of preadipocytes at 8 days of adipogenic differentiation.
(D) Anti-MyHC immunouorescence staining of myoblasts at 4 days after myogenic differentiation. (E)
The expression of adipogenic marker genes PPARγ and C/EBPα after differentiation of preadipocytes. (F)
The expression of muscle differentiation marker genes MyHC and MyoD after differentiation of
myoblasts. Data are presented as means ± SEM. *P < 0.05, **P < 0.01.
Figure 2
CircINSR could sponging miR-15/16 family in myoblasts and preadipocytes. (A) The changes of miR-
15/16 family in myoblasts after overexpression of circINSR. (B) The changes of miR-15/16 family in
preadipocytes after overexpression of circINSR. (C, D) Luciferase reporter activity of circINSR-WT in HEK-
293T cells co-transfected with miR-15/16 mimics or mimics NC. (E) Ago2-RIP assay for the amount of
circINSR in myoblasts and preadipocytes. (F) Ago2-RIP assay for the amount of miR-15/16 family in
myoblasts. (G) Ago2-RIP assay for the amount of miR-15/16 family in preadipocytes. Data are presented
as means ± SEM. *P < 0.05.

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Figure 3
CircINSR promotes myoblasts proliferation by sponging the miR-15/16 family. (A) TargetScan predicted
that CyclinD1 3'-UTRs had binding sites for miR-15/16. (B) The uorescence activity changes after co-
transfection with dual uorescent reporter vectors and miR-15/16. (C) The expression of marker genes
related to cell proliferation after co-transfection of circINSR and miR-15/16 in myoblasts. (D, E) EdU assay
for myoblasts transfected with miR-15/16 mimics alone or co-transfected with circINSR. Scale bars, 200
μm. (F) CCK-8 assay for myoblasts transfected with miR-15/16 mimics alone or co-transfected with
circINSR. n=6. (G, H) Cell cycle assay for myoblasts transfected with miR-15/16 mimics alone or co-
transfected with circINSR. Data are presented as means ± SEM. *P < 0.05.

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Figure 4
CircINSR inhibits the apoptosis of bovine primary myocytes by sponging the miR-15/16 family. (A)
TargetScan predicted that Bcl-2 3'-UTRs had binding sites for miR-15/16. (B) The uorescence activity
changes after co-transfection with dual uorescent reporter vectors and miR-15/16. (C) The expression of
marker genes related to cell apoptosis after co-transfection of circINSR and miR-15/16 in myoblasts. (D,
E) Cell apoptosis was determined by Annexin V/7-AAD dual staining followed by ow cytometry. n=3.
Data are presented as means ± SEM. *P < 0.05.

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Figure 5
CircINSR promotes preadipocytes proliferation by sponging the miR-15/16 family. (A, B) In preadipocytes,
the effect of overexpression and interference with circINSR on proliferation marker genes. (C) The
expression of CCND1, CDK2, and PCNA was detected by Western Blots. (D) The expression of marker
genes related to cell proliferation after co-transfection of circINSR and miR-15/16 in preadipocytes. (E, F)
EdU assay for preadipocytes transfected with miR-15/16 mimics alone or co-transfected with circINSR.
Scale bars, 200 μm. (G) CCK-8 assay for preadipocytes transfected with miR-15/16 mimics alone or co-
transfected with circINSR. n=6. (H, I) Cell cycle assay for preadipocytes transfected with miR-15/16
mimics alone or co-transfected with circINSR. Data are presented as means ± SEM. *P < 0.05.

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Figure 6
CircINSR inhibits preadipocytes apoptosis by sponging the miR-15/16 family. (A, B) The mRNA levels of
cell apoptosis markers, including Bcl-2, Bax, and Caspase9 were detected by real-time qPCR in
preadipocytes transfected with circINSR or siRNA. (C) The protein expression of Bcl-2, Bax, and Caspase9
was detected by Western Blots. (D) The expression of marker genes related to cell apoptosis after co-
transfection of circINSR and miR-15/16 in preadipocytes. (E, F) Cell apoptosis was determined by
Annexin V/7-AAD dual staining followed by ow cytometry. n=3. Data are presented as means ± SEM. *P
< 0.05.
Figure 7

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CircINSR inhibits adipogenic differentiation of preadipocytes. (A, B) The expression of adipogenic marker
genes after overexpression of circINSR in preadipocytes was detected by real-time qPCR and Western
Blots. (C, D) The expression of adipogenic marker genes after interference with circINSR in preadipocytes
was detected by real-time qPCR and Western Blots. (E) Interference with circINSR in pre-adipocytes,
BODIPY staining to analyze lipid droplet deposition. The uorescence signal was analyzed by image J
software. (F) Lipid droplets in preadipocytes were stained with Oil Red O. Lipid content measured by
spectrophotometric analysis after dissolving in isopropanol. Data are presented as means ± SEM. *P <
0.05.
Figure 8
Adipogenic differentiation was regulated by circINSR through miR-15/16. (A) The reported target genes of
the miR-15/16 family. (B) The effect of miR-15/16 or co-transfection with circINSR on target genes in
preadipocytes. (C) The expression of adipogenic marker genes in preadipocytes was detected by real-time
qPCR. (D, E) Lipid droplets in preadipocytes were stained with Oil Red O. Lipid content measured by
spectrophotometric analysis after dissolving in isopropanol. Data are presented as means ± SEM. *P <
0.05. (F) Schematic diagram of circINSR regulating the proliferation, apoptosis and differentiation of
myoblasts and preadipocytes.