AABArchives Animal BreedingAABArch. Anim. Breed.2363-9822Copernicus PublicationsGöttingen, Germany10.5194/aab-66-93-2023The role of miR-369-3p in proliferation and differentiation of preadipocytes
in Aohan fine-wool sheepThe role of miR-369-3p in proliferation and differentiation of preadipocytesXueShijieLiuKaidongZhaoLeZhouLishengGaoXiaoxiaoLiuLirongLiuNannanliu@sina.comHeJianninghexingxing104@163.comCollege of Animal Science and Technology, Qingdao Agricultural
University, Qingdao 266109, ChinaQingdao animal husbandry and veterinary research Institute, Qingdao
266032, ChinaChina Animal Health and Epidemiology Center, Qingdao 266032, China
MicroRNAs (miRNAs) are a large class of non-coding RNAs
that play important roles in the proliferation and differentiation of
adipocytes. Our previous sequencing analysis revealed higher expression of
miR-369-3p in the longissimus muscle of 2-month-old Aohan fine-wool sheep
(AFWS) compared to 12-month-old sheep (P<0.05), suggesting that
miR-369-3p may regulate fat deposition in AFWS. To test this, miR-369-3p
mimics, inhibitors, and negative controls (NCs) were constructed and
transfected into AFWS preadipocytes. After transfection with miR-369-3p
mimics, we found a decrease (P<0.05) in the expression of genes and
proteins related to cell proliferation and differentiation, detected by
RT-qPCR (quantitative reverse transcription PCR) and western blot analyses. Moreover, EdU (5-ethynyl-2′-deoxyuridine) detection and Oil Red O
staining showed a decrease (P<0.05) in cell proliferation and lipid
accumulation, respectively. The opposite trends (P<0.05) were
obtained after transfection with miR-369-3p inhibitors. In conclusion, the
results showed that miR-369-3p can inhibit the proliferation and
differentiation of AFWS preadipocytes, providing a theoretical basis to
further explore the molecular mechanism of fat deposition in sheep and other
domestic animals.
Introduction
Mutton is becoming increasingly popular as living standards improve and
dietary patterns change because of its unique taste, high protein content,
and low cholesterol content (Jia et al., 2023). Intramuscular fat (IMF)
content is a key indicator of meat quality, affecting meat colour, flavour,
tenderness, and other traits, and increasing IMF relative to other indicators
can improve the quality of meat (Mortimer et al., 2017). Therefore,
determining how to improve IMF content is essential to improving the quality
of meat.
MicroRNAs (miRNAs) are a class of 20–25 nt non-coding RNAs that regulate
gene expression by targeting mRNA (Iorio et al., 2005; Yekta et al., 2004).
Many miRNAs have been reported as regulators of fat formation and
metabolism. For example, miR-145 downregulates ADAM17 to promote the
decomposition of human adipocytes (Lorente-Cebrián et al., 2014),
miR-182 and miR-203 act in the differentiation of brown adipocytes (Kim et
al., 2014), and miR-155 directly targets and inhibits PPARγ expression to
regulate adipocytes (Karkeni et al., 2016). Studies relating to sheep show
that miR-27a regulated the formation of lipid droplets in sheep adipocytes
by targeting the CPT1B gene (Li et al., 2021), and miR-340-5p inhibits sheep
adipocyte differentiation by targeting ATF7 (Liu et al., 2020). In a preliminary
study, we evaluated the IMF content of 2-, 4-, 6-, and 12-month-old Aohan fine-wool sheep (AFWS). The
results showed that the IMF content in the 2- and 12-month-old sheep was
significantly different (Han et al., 2021a). Then we found many
differentially expressed miRNAs in back longissimus muscle between 2-month-
and 12-month-old AFWS by RNA-seq (Han et al., 2021b). Of these miRNAs, the
expression level of miR-369-3p at 2 months old was significantly higher than
at 12 months old (Table S1 in the Supplement). Therefore, miR-369-3p may have a
regulatory effect on intramuscular fat content.
There have been few studies regarding the regulation of miR-369-3p on fat,
whereas the majority of studies focused on disease. Galleggiante et al. (2019) showed that miR-369-3p inhibited the expression of C/EBPβ.
C/EBPβ is the first set of transcription factors regulating the process of
fat formation. Mouse embryonic fibroblasts (3T3-L1) lacking these genes
cannot complete the differentiation of fat induced by hormones and
interfere with normal expression of other adipogenic genes. C/EBPβ also
promotes cell mitosis in the process of fat formation. When induced in
vitro, precursor adipocytes reenter the cell division cycle at the early
stage of differentiation, and C/EBPβ promotes cell mitosis in this
process, which is required for adipocyte differentiation (Azad et al.,
2014). The binding of miR-369-3p to the 3′UTR region of TSPAN13 mRNA inhibited
expression of TSPAN13, leading to a decrease in CDK4 expression (Li et al., 2019; Lou
et al., 2017). CDK4 indirectly inhibits oxidative metabolism by controlling
E2F1 transcription factors in muscle and brown adipose tissue (BAT) (Blanchet et
al., 2011) and also promotes insulin signalling pathways in mature adipocytes
(Lagarrigue et al., 2016). In sheep, miR-369-3p targets the key gene
PDPK-1 that activates autophagy, and its expression is negatively correlated (Lie
et al., 2016; Hu et al., 2021). The miR-369-3p can also regulate and inhibit
the autophagy-related gene ATG10 (Liu et al., 2019).
Therefore, a significant amount of data has shown that miR-369-3p is closely
related to genes related to adipocyte proliferation and differentiation. To
further investigate the role of miR-369-3p in AFWS, AFWS preadipocytes were
used to study the effect of miR-369-3p on preadipocyte proliferation and
differentiation.
Materials and methodsMaterials
Samples of preadipocytes, back longissimus muscle, biceps femoris, shoulder
skin, pituitary, heart, lung, testicle, liver, and small intestine were
collected from 4 d old Aohan fine-wool male lambs obtained from Aohan
Breeding Farm in the city of Chifeng, Inner Mongolia. We anesthetized the sheep by
intravenous injection of sodium pentobarbital at a dose of 25 mg kg-1
following published protocols (Raj et al., 2004; Hawkins et al., 2016) and
then placed it in a closed room that was filled with carbon dioxide at a
rate of 20 % min-1. When the gas concentration had reached 80 %,
the sheep died.
Differentially expressed miRNAs in sheep muscle tissue
In a preliminary study, we found many differentially expressed miRNAs in
back longissimus muscle between 2-month- and 12-month-old AFWS by RNA-seq
(Han et al., 2021b). Of these miRNAs, the expression level of miR-369-3p at
2 months old was significantly higher than at 12 months old (Table S1).
Culture and passage of AFWS preadipocytes
After the 4 d old AFWS was killed, the longissimus muscle tissue was taken
and washed five times with PBS (phosphate-buffered saline)) containing 0.1 % penicillin / streptomycin.
We also removed the visible blood vessels and connective tissue in sterile
culture dishes. Then we cut the longissimus muscle tissue into a tissue
block of about 1.0 mm, adding 0.2 % collagenase II (Solarbio, Beijing,
China) before putting the tissue into a 37 ∘C water bath for
digestion in 1.5 h. Shaking it every 5 min, we added a complete
medium (Dulbecco's Modified Eagle Medium (DMEM) + 10 % fetal bovine serum (FBS) + 0.1 % penicillin / streptomycin) of equal volume
after digestion. Digestive solution was filtered with 200 mesh and 400 mesh
cell sieves, after which the cells were resuspended in the complete medium
and placed in the CO2 incubator for 3–4 h. The adherent cells were
preadipocytes. When the confluence degree of primary cell culture reached
80 %, the cells were passaged at a ratio of 1 : 2 and placed in a 37 ∘C, 5 % CO2 incubator.
Induction of preadipocyte differentiation
After 2 d of fusion, the cells were induced in the first stage after
high cell fusion (DMEM, FBS, penicillin / streptomycin,
methylisobutylxanthine, dexamethasone, insulin). Before induction, the cells
were collected for RNA and protein extraction, which was recorded as day 0.
After the first stage, some cells became round. After 2 d, the second
stage of induction (DMEM, FBS, insulin, penicillin / streptomycin) was
conducted. The culture medium was changed every 2 d, with cell
collection.
Transfection
After the cells were digested using trypsin, they were evenly distributed in
a six-well plate with 2 mL of complete medium and cultured overnight. When
the density reached about 60 %, the mimics, inhibitors, and negative
controls (NCs) (Jima Gene, Shanghai, China) were transfected into the cells
with GP-transfect-Mate (Jima Gene, Shanghai, China) according to the
manufacturer's instructions (A: miR-369-3p mimic, B: miR-369-3p mimic NC, C:
miR-369-3p inhibitor, D: miR-369-3p NC). After 48 h, the cells were collected
to detect the expression of miR-369-3p.
RNA extraction and reverse transcription
RNA was extracted from back longissimus muscle, biceps femoris, shoulder
skin, pituitary, heart, lung, testis, spleen, and small intestine of
4 d old AFWS lambs. After grinding, total RNA was extracted using a
SPARKeasy total RNA rapid extraction kit (Shandong Sikejie Biotechnology
Co., Ltd.). The cDNA was prepared from the RNA using an Evo M-MLV reverse
transcription kit (Aikerui Biotechnology, Hunan, China), followed by an
miRNA cDNA first island chain synthesis kit (Aikerui Biotechnology, Hunan,
China).
RT-qPCR (quantitative reverse transcription PCR)
The expression levels of mRNAs and miRNA were quantified by the CFX96
real-time PCR detection system (Bio-Rad, Hercules, CA, United States).
Primers were designed using Primer 5 (Table S2) and
synthesized by Tsingke Biological Technology (Tsingke, Qingdao, China).
Reverse transcription was conducted using 1 µg total RNA per sample
and the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa, Dalian, China). The
levels of U6 and GAPDH were used to normalize the expression levels of mRNA and
miRNAs, respectively. The sequences of the primers used are listed in Table 1. The 20 µL PCR reaction system consisted of 10 µL
SYBR® Premix Ex Taq II (TaKaRa), 0.5 µL forward primer
(10 µM L-1), 0.5 µL reverse primer (10 µM L-1), 1 µL
cDNA, and 8 µL ddH2O. The following thermocycling programme was
used: one cycle of 95 ∘C for 10 min; 45 cycles of 95 ∘C for 10 s, 60 ∘C for 10 s, and 72 ∘C for 10 s; and one cycle of 72 ∘C for 6 min. Three independent replicates were conducted for each sample. The
relative expression levels were calculated using the 2-ΔΔCt
method (Livak and Schmittgen, 2001).
Total protein extraction and western blot analysis
Total proteins were extracted using RIPA Lysis Buffer (Beyotime, Shanghai,
China), supplemented with phenylmethylsulfonyl fluoride (Service-Bio, Wuhan,
China) at a ratio of 100 : 1 on ice. The extracted protein concentration was
then measured by a BCA Protein Assay Kit (Solarbio, Beijing, China) (Gao et
al., 2019). Proteins (30 µg) were separated by 10 % SDS-PAGE
polyacrylamide gel electrophoresis and then transferred onto PVDF (polyvinylidene difluoride) membranes
(Beyotime). The membranes were blocked with 5 % skimmed milk for 2 h
and then incubated overnight at 4 ∘C with rabbit anti-PPARγ,
rabbit anti-C/EBPα, rabbit anti-CDK4, or rabbit anti-CyclinB at a dilution of
1 : 2000. The membranes were then incubated with a horseradish peroxidase (HRP)-conjugated secondary
antibody (1 : 2000) for 1 h at room temperature, followed by washing. Rabbit
anti-GAPDH was used as an internal control. All antibodies were purchased from
Proteintech (Chicago, IL, United States). The protein signals were detected
in a darkroom using a Pierce ECL Western Blotting Substrate kit (Thermo Fisher
Scientific, Dallas, TX, United States) according to the manufacturer's
instructions.
Oil Red O staining
Differentiation was induced in preadipocytes for 8 d, and then the cells
were stained with Oil Red O (ORO; Solarbio, Beijing, China). The culture medium
was removed, and cells were washed with PBS (phosphate buffered saline) and fixed with ORO fixative for 30 min. The fixative was removed, cells were rinsed with distilled water
twice, and 60 % isopropanol was added and incubated for 5 min before
removal. The ORO stain was then added and incubated for 15 min before
removal. The stained cells were washed five times with distilled water.
After washing, Mayer's hematoxylin staining solution was added, and cells were
re-stained for 2 min. After discarding, the stained cells were washed
five times with distilled water. ORO buffer was added, incubated for 1 min, and then discarded. Finally, the cells were covered with distilled
water and observed under a microscope (Leica Microsystems, Wetzlar,
Germany).
EdU detection of cell proliferation
After passage to the six-well plate, preadipocytes in good condition and at
the growth density of 70 % were selected for EdU (5-ethynyl-2′-deoxyuridine) staining using an EdU
cell proliferation detection kit (Beyotime, Shanghai, China). Cells were
fixed with 4 % paraformaldehyde and labelled with EdU, and then the nuclei
were stained using Hoechst 33342.
Statistical analysis
IBM SPSS Statistics 26.0 software was used to calculate the relative
expression level of genes using the 2-ΔΔCt method (Livak and
Schmittgen, 2001). The relative expression of genes was analysed by
an independent sample t test, and the results were expressed as mean ± SE
(standard error). A value of P<0.05 was considered a significant
difference.
ResultsExpression of miR-369-3p in different AFWS tissues
To explore whether miR-369-3p mainly plays a role in sheep muscle, its
expression on different AFWS tissues was determined. As shown in Fig. 1,
RT-qPCR showed that miR-369-3p was widely expressed in various tissues, with
higher expression levels in the back longissimus muscle and biceps
femoris. As shown in Fig. S1 in the Supplement, comparison of the miR-369-3p
sequences of 14 species revealed high conservation, indicating these miRNAs
may play important roles.
Expression of miR-369-3p in AFWS tissue.
The expression of miR-369-3p in preadipocyte differentiation
To explore the role of miR-369-3p in AFWS preadipocyte differentiation, its
expression on consecutive days following induction of differentiation was
determined. The results of RT-qPCR are shown in Fig. 2. The expression
levels of marker genes for adipocyte differentiation, PPARγ and
C/EBPα, peaked at 6 d, and the differences in expression level were
significant (P<0.05). The expression levels decreased after 6 d,
indicating a critical period for the differentiation of preadipocytes at 6 d, but the expression level of miR-369-3p reached the lowest value on the
sixth day, with a significant difference.
Temporal expression of miR-369-3p, PPARγ, and C/EBPα in AFWS preadipocyte differentiation (*P<0.05).
Transfection effect of the miR-369-3p mimic and inhibitor
To further investigate the expression of miR-369-3p, we transfected cells
with a mimic or inhibitor, as well as negative controls (NCs), as shown in
Fig. S2. After 48 h of transfection, the expression of
miR-369-3p in the mimic group was higher compared to the mimic NC group, and
the difference was significant (P<0.05). The expression of
miR-369-3p in the inhibitor group was lower compared to the NC group, and
this difference was significant (P<0.05). The observed changes in
expression indicated successful transfection of the miR-369-3p mimic and
inhibitor, allowing for subsequent experiments.
The expression changes of preadipocyte proliferation and
differentiation marker genes after transfection
To further investigate the role of miR-369-3p, the mimic or inhibitor
constructs were used, and the effects on the expression levels of
preadipocyte proliferation and differentiation marker genes were determined.
As shown in Fig. 3, after transfection of the miR-369-3p mimic for 48 h, the
expression levels of CDK4, CyclinB, and PPARγ and of C/EBPα were significantly decreased
(P<0.05). However, the opposite results were obtained after
transfection of the miR-369-3p inhibitor (P<0.05).
The expression levels of preadipocyte proliferation and
differentiation marker genes after transfection. (a) Relative
expression of CDK4 after overexpression of miR-369-3p. (b) Relative
expression of CDK4 after inhibition of miR-369-3p. (c) Relative expression of
CyclinB after overexpression of miR-369-3p. (d) Relative expression of
CyclinB after inhibition of miR-369-3p. (e) Relative expression of
PPARγ after overexpression of miR-369-3p. (f) Relative expression of
PPARγ after inhibition of miR-369-3p. (g) Relative expression of
C/EBPα after overexpression of miR-369-3p. (h) Relative expression of
C/EBPα after inhibition of miR-369-3p (*P<0.05).
Changes of protein expression after transfection
To further explore the effect of miR-369-3p on the proliferation and
differentiation of adipocytes, the total protein was extracted from
preadipocytes after transfection for 48 h in each transfected group. The
protein expression levels of adipocyte marker genes, CDK4, CyclinB, and PPARγ, as well as
C/EBPα, were determined, using the level of GAPDH as a control, as shown in
Figs. 4 and 5. The CDK4, CyclinB, and PPARγ as well as C/EBPα protein expression was
inhibited by miR-369-3p mimics and promoted by miR-369-3p inhibitors. The
results showed changes in adipocyte marker genes consistent with the
observed trends in RNA expression detected by RT-qPCR.
Effect of overexpression of miR-369-3p on the expression level of
CDK4, CyclinB, PPARγ, and C/EBPα. (a) Western blot analysis of
adipogenic genes. (b) Relative protein level of CDK4. (c) Relative protein
level of CyclinB. (d) Relative protein level of PPARγ. (e) Relative
protein level of C/EBPα (*P<0.05).
Effect of inhibition of miR-369-3p on the expression level of CDK4,
CyclinB, PPARγ, and C/EBPα. (a) Western blot analysis of
adipogenic genes. (b) Relative protein level of CDK4. (c) Relative protein
level of CyclinB. (d) Relative protein level of PPARγ. (e) Relative
protein level of C/EBPα (*P<0.05).
EdU detection of cell proliferation
To assess cell proliferation, EdU detection was performed using the
transfected cells, and the results are shown in Fig. 6. Cell proliferation
was significantly greater in the miR-369-3p inhibition group compared to the
NC group, and the opposite result was obtained in the mimic group (P<0.05). Thus, miR-369-3p acts to inhibit the proliferation of
preadipocytes.
(a) EdU detection of cell proliferation after
transfection. Blue fluorescence represents the nucleus stained with
Hoechst, and green fluorescence represents proliferating cells. (b) Ratio of
EdU-positive cell of inhibitor and NCs. (c) Ratio of EdU-positive cell of
mimic and mimic NCs (*P<0.05).
Oil Red O staining
After being induced for 8 d, the cells were stained with Oil Red O to
investigate the effect of miR-369-3p on lipid deposition, as shown in Fig. 7. There was significantly greater staining in the miR-369-3p inhibitor
group compared to the NC group, with the opposite results in the mimic group
(P<0.05). These results further verified that miR-369-3p can
inhibit the differentiation of preadipocytes.
Oil Red O staining after transfection. (a) The red areas represent
stained lipid droplets. (b) Oil Red O staining area of inhibitor and NCs. (c) Oil Red O staining area of mimic and mimic NCs (*P<0.05).
Discussion
As non-coding RNAs, miRNAs play important roles in a variety of tissues or
organs by regulating gene expression. In adipose tissue, miRNAs can regulate
adipocyte proliferation, differentiation, metabolism, and endocrine function
(Arner and Kulyté, 2015). Our previous work revealed
significantly lower expression of miR-369-3p in the longissimus dorsi muscle
of 12-month-old AFWS compared to that of 2-month-old sheep (Han et al.,
2021b) (Table S1). We found that the expression of miR-369-3p
in muscle was higher compared to other tissues. This suggests that
miR-369-3p acts in the growth and development of muscles. So far, studies on
miR-369-3p have mainly focused on diseases, including cancer and inflammation (Chen
et al., 2021; Scalavino et al., 2020; Xu and Liu, 2020). For example, in a
study of chronic inflammation, Galleggiante et al. (2019) found that the
upregulation of miR-369-3p could inhibit the expression of C/EBPβ, a key
transcription factor in the differentiation of preadipocytes (Cao et al.,
2017). There was another study showing that during 3T3-L1 adipocyte
differentiation, C/EBPβ is induced early to transactivate the expression
of C/EBPα and PPARγ (Guo et al., 2015). Members of the C/EBP and PPAR families
of transcription factors are the most notable in the differentiation of
preadipocytes into adipocytes. Furthermore, C/EBPα and PPARγ are
critical regulators of adipogenesis, since the lack of either of these
proteins prevents the development of white adipose tissue in mice (Linhart
et al., 2001; Rosen et al., 1999; Barak et al., 1999). Therefore, miR-369-3p
may inhibit the expression of C/EBPα and PPARγ by inhibiting the
expression of C/EBPβ, thus inhibiting the differentiation of adipocytes.
Autophagy removes multiple cellular components during fat differentiation
(Mizushima and Levine, 2010). Xu et al. (2018) showed that the expression of
the autophagy pathway was negatively correlated with fat decomposition.
ATG10 plays an important role in the formation and extension of autophagosomes
(Kim and Lee, 2014). Liu et al. (2019) found that miR-369-3p regulates the
proliferation and migration of EEC (endometrioid adenocarcinoma) cells,
suggesting that miR-369-3p can also inhibit adipocyte differentiation by
inhibiting the expression of ATG10 in human body. Lie et al. (2016) showed that
miR-369-3p targeted PDPK-1 (3-phosphoinositide-dependent protein kinase-1) in
sheep, and expression levels were negatively correlated. PDPK-1 is the key gene to
initiate autophagy (Hu et al., 2021), and the expression of PDPK-1 and
PPARγ resulted in a synergistic effect (Yin et al., 2006). Hence,
miR-369-3p may inhibit the autophagy process and the expression of
PPARγ by inhibiting the expression of PDPK-1 in sheep to negatively affect the
differentiation of adipose precursor cells.
Lou et al. (2017) found that in CRC (colorectal cancer) cell lines,
knocking down TSPAN13 led to the decrease of CDK4 expression, and miR-369-3p
inhibited TSPAN13 by binding to the 3'UTR region of TSPAN13 mRNA (Li et al., 2019).
Therefore, miR-369-3p likely inhibits the expression of CDK4 by downregulating
TSPAN13. In previous studies, overexpression of CDK4 has been shown to promote cell
proliferation by driving cell cycle progression (Retzer-Lidl et al., 2007;
Rodriguez-Puebla et al., 2002; An et al., 1999). Further, the proper
regulation of CyclinB is essential for the entry into mitosis (Krek and Nigg,
1991), and reducing the time between cell birth and mitosis increases the
rate of cell multiplication (Moxnes et al., 2004).
In summary, miR-369-3p negatively regulates proliferation and
differentiation of AFWS preadipocytes.
Conclusion
In this study, we found that miR-369-3p inhibits the proliferation and
differentiation of AFWS preadipocytes. This study provides a theoretical
basis for further exploration of the molecular mechanism of fat deposition
in sheep and other livestock.
The supplement related to this article is available online at: https://doi.org/10.5194/aab-66-93-2023-supplement.
Author contributions
SX, KL, JH, and NL: conceptualization. SX,
KL, LeZ, LiZ, XG, LL, NL, and JH: methodology. SX: software.
SX and JH: validation. SX and NL: formal analysis. SX: investigation. SX, KL, LeZ, LiZ, XG, LL, NL, and JH: resources. SX, KL, LeZ, LiZ, XG,
LL, NL, and JH: data curation. SX and
KL: writing – original draft preparation. SX, KL, and JH: writing – review and editing. SX: visualization. LiZ, JH, and NL: supervision. KL: project
administration. NL, LiZ, and JH: funding acquisition. All
authors have read and agreed to the published version of the manuscript.
Competing interests
The contact author has declared that none of the authors has any competing interests.
Ethical statement
The animal study was reviewed and approved by the Animal Administration and
Ethics Committee of Qingdao Agricultural University, Animal Science and
Technology College (permit no. DKY2021021).
Disclaimer
Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We thank the AFWS breeding farm of Chifeng for providing samples.
Financial support
The work was supported by the Shandong Province Nataral Science Foundation
(grant no. ZR2020MC167), the Earmarked Fund for Modern China Wool & Cashmere
Technology Research System (grant no. CARS-39-05), the Project of Shandong Province
Agricultural Variety Program (grant no. 2019LZGC012), and the Central Guidance on Local
Science and Technology Development Fund (grant no. YDZX2021082).
Review statement
This paper was edited by Henry Reyer and reviewed by Thais Correia Costa and one anonymous referee.
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