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RAPID REPORT
Musculoskeletal Biology and Bioengineering
MicroRNA control of the myogenic cell transcriptome and proteome: the role of
miR-16
Seongkyun Lim,
1
David E. Lee,
1
Francielly Morena da Silva,
1
Pieter J. Koopmans,
1,2
Ivan J. Vechetti Jr,
3
Ferdinand von Walden,
4
Nicholas P. Greene,
1,2
and Kevin A. Murach
1,2
1
Department of Health, Human Performance, and Recreation, Exercise Science Research Center, University of Arkansas,
Fayetteville, Arkansas, United States;
2
Cell and Molecular Biology Graduate Program, University of Arkansas, Fayetteville,
Arkansas, United States;
3
Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United
States; and
4
Neuropediatrics, Department of Women’s and Children’s Health, Karolinska Institutet, Stockholm, Sweden
Abstract
MicroRNAs (miRs) control stem cell biology and fate. Ubiquitously expressed and conserved miR-16 was the first miR implicated
in tumorigenesis. miR-16 is low in muscle during developmental hypertrophy and regeneration. It is enriched in proliferating myo-
genic progenitor cells but is repressed during differentiation. The induction of miR-16 blocks myoblast differentiation and myo-
tube formation, whereas knockdown enhances these processes. Despite a central role for miR-16 in myogenic cell biology, how
it mediates its potent effects is incompletely defined. In this investigation, global transcriptomic and proteomic analyses after
miR-16 knockdown in proliferating C2C12 myoblasts revealed how miR-16 influences myogenic cell fate. Eighteen hours after
miR-16 inhibition, ribosomal protein gene expression levels were higher relative to control myoblasts and p53 pathway-related
gene abundance was lower. At the protein level at this same time point, miR-16 knockdown globally upregulated tricarboxylic acid
(TCA) cycle proteins while downregulating RNA metabolism-related proteins. miR-16 inhibition induced specific proteins associated
with myogenic differentiation such as ACTA2, EEF1A2, and OPA1. We extend prior work in hypertrophic muscle tissue and show that
miR-16 is lower in mechanically overloaded muscle in vivo. Our data collectively point to how miR-16 is implicated in aspects of myo-
genic cell differentiation. A deeper understanding of the role of miR-16 in myogenic cells has consequences for muscle developmen-
tal growth, exercise-induced hypertrophy, and regenerative repair after injury, all of which involve myogenic progenitors.
lncRNA; proteomics; RNA-sequencing; satellite cells; skeletal muscle
INTRODUCTION
MicroRNAs (miRNAs) destabilize mRNAs and/or prevent
their translation and are key regulators of myogenic stem cell
(satellite cell) biology (1). These small noncoding RNAs control
nearly every aspect of myogenic cell function including main-
tenance of quiescence (2–5), activation (6,7), proliferation
(8,9), self-renewal (10), migration (11), and differentiation (8,
12,13). The depletion of miRNAs from myogenic cells results
in swift cell death (2,8,14). Dysregulation of miRNAs in satel-
lite cells in vivo may negatively affect muscle development,
adaptability to exercise, the muscle microenvironment with
aging, and regenerative capacity after injury; all these proc-
esses depend on satellite cells (15–24). Furthermore, miRNAs
are released from satellite cells in extracellular vesicles (EVs)
and taken up by recipient cells throughout muscle in vivo (14,
25,26). Satellite cell communication throughout muscle via
miRNAs in EVs contributes to muscle adaptation during
loading (27,28). Understanding the role of miRNAs in myo-
genic progenitors may accelerate the discovery of miRNA-
based therapeutics that affect muscle plasticity via fusion-de-
pendent and/or -independent mechanisms (27,29).
miR-16 is highly conserved and ubiquitously expressed (30–
32). It was the first miRNA implicated in tumorigenesis and is a
highly influential miRNA in numerous cellular processes, spe-
cifically as it relates to tumor growth (32,33). In skeletal muscle
tissue that contains both mature muscle fibers and myogenic
satellite cells, miR-16 content rises during avian embryonic de-
velopment but begins to wane at birth concomitant with sec-
ondary myogenesis (34). Low miR-16 levels during development
areassociatedwithmusclehypertrophy(35,36). We reported
that muscle miR-16 levels decline after resistance exercise fol-
lowing a week of acclimation to training and that knockdown in
myotubes increases muscle protein synthesis (37). Skeletal mus-
cle miR-16 is also lower in regenerating relative to uninjured
muscle (6). The repression of miR-16 in muscle is therefore
Correspondence: N. P. Greene (npgreene@uark.edu); K. A. Murach (kmurach@uark.edu).
Submitted 24 February 2023 / Revised 20 March 2023 / Accepted 20 March 2023
http://www.ajpcell.org 0363-6143/23 Copyright ©2023 the American Physiological Society. C1101
Am J Physiol Cell Physiol 324: C1101–C1109, 2023.
First published March 27, 2023; doi:10.1152/ajpcell.00071.2023
Downloaded from journals.physiology.org/journal/ajpcell at Univ of Arkansas Fayetteville (130.184.252.076) on May 18, 2023.
associated with postnatal myofiber growth and regeneration as
well as the exercise-induced hypertrophic response. In myo-
genic precursor cells, miR-16 is induced upon activation (2,6,
38) and enriched during proliferation (2,14)butgradually
declines in culture (2) and with differentiation (34,36,39). Some
evidence suggests that miR-16-5p is high in quiescent satellite
cells but still drops precipitously during differentiation (1). miR-
16 overexpression prevents myogenic cell differentiation and
myotube formation in vitro (34), whereas knockdown enhances
these processes (34,40). MyoD contributes to myogenic cell dif-
ferentiation (41–48). miR-16 knockdown in murine myofiber-
associated myogenic cell culture may increase the proportion of
MyoD þsatellite cells by 3 days but reduces it by 5 days, point-
ing to an effect on myogenic cell behavior and fate (6). The liter-
ature collectively points to miR-16 having a key function in
myogenic cells and in determining skeletal muscle mass.
Despite its importance, how miR-16 exerts its effects in myo-
genic cells is incompletely defined.
The purpose of this investigation was to provide detailed
information on how miR-16 influences myogenic cell biol-
ogy. First, we evaluated miR-16 levels in mechanically over-
loaded muscle tissue in vivo, which is a well-established
model of rapid growth associated with satellite cell differen-
tiation and fusion (49). We then inhibited miR-16-5p in pro-
liferating C2C12 myoblasts and performed RNA-sequencing
(RNA-seq) and discovery proteomics. C2C12s express the sat-
ellite cell marker Pax7 (50) and can model the behaviors of
primary myogenic progenitors in vitro (51–53). Our experi-
ments point to a multifaceted role for miR-16 repression in
myogenic cell differentiation. We thus shed light on poten-
tial mechanisms whereby miR-16 influences satellite cell-
mediated muscle growth and regenerative potential in vivo
(6,36). We also provide general information on miR-16 tar-
gets, which may be informative in nonmuscle cell types
where miR-16 is enriched (30).
MATERIALS AND METHODS
Murine Experiment
Female C57BL6/J mice (2–3 mo of age) were utilized for in
vivo overload experiments. These experiments were per-
formed for a prior publication from our laboratory (54), and
the mice were injected with 5-ethenyl uridine 5 h before
being euthanized. Experiments were approved by the
Institutional Animal Care and Use Committee (IACUC) of
the University of Kentucky. Mice were housed in a tempera-
ture- and humidity-controlled room maintained on a 14:10-h
light-dark cycle, and food and water were provided ad libi-
tum throughout experimentation. Briefly, synergist ablation
surgery to overload the plantaris muscle was performed as
described by Murach et al. (54). Under isoflurane anesthesia,
a portion of the gastrocnemius and soleus was removed,
leaving the plantaris to be mechanically overloaded during
reambulation (n= 4). Sham-operated mice (no removal of
muscle) were controls (n=3).Seventy-twohoursaftersur-
gery, animals were euthanized in the morning via a lethal
dosage of pentobarbital sodium injected intraperitoneally
followed by cervical dislocation. Plantaris muscle tissue
was harvested and frozen in liquid nitrogen and stored for
downstream analyses. miR-16 level in tissue was evaluated
with an unpaired directional ttest in GraphPad Prism
(Boston, MA).
Cell Culture Experiments
C2C12 murine myoblasts (CRL-1772, ATCC, Manassas, VA)
were plated in six-well plates (5 10
4
cells/well) with 2 mL of
Dulbecco’smodified Eagle medium (DMEM) combined with
20% fetal bovine serum (FBS) and 1% penicillin-streptomycin
(P/S). Cells were incubated at 37Cwith5%CO
2
,andmedium
was changed every 48 h. Upon 40–50% confluence, cells were
washed with PBS for transfection (37). For proteomics five
technical replicates were utilized, and for transcriptomics
three control and two knockdown wells were employed. miR-
16 levels in cells were evaluated in Prism with an unpaired
directional ttest with n= 5 technical replicates.
Transfection of C2C12 Myoblasts
Plasmids encoding either an empty vector control
(pCMVmiR;PCMVMIR,Origene,Rockville,MD)ormiR-16-1
(miR-16-5p) inhibitor were transferred into DH5-a Escherichia
coli as we have previously described (37). Plasmid DNA was
amplifiedandisolatedfrombacteriawiththePureLinkHiPure
Plasmid Filter Maxiprep kit (K211017, Life Technologies,
Carlsbad,CA).FormiR-16inhibition,weutilizedanti-miRin-
hibitor (AM17000, Ambion, Austin, TX). Plasmid DNA (1 μg)
was diluted in 50 μL of Opti-MEM reduced serum medium
(31985088, Life Technologies), combined with 4 μLof
Lipofectamine 2000 (11668019, Life Technologies) diluted in
50 μL of Opti-MEM, and incubated for 20 min to allow lipid/
DNA complexes to form. Medium was replaced with Opti-
MEM, and lipid/DNA complexes were added and incubated
for 5 h at 37Cwith5%CO
2
. After 5-h incubation, medium
was replaced with normal growth medium for 18 h. The effi-
ciency of the inhibition of miR-16 was tested and validated
previously (37).
RNA Isolation, Quality Check, and qPCR for miR-16
Myoblast and muscle tissue RNA were isolated with
TRIzol reagent, which was then homogenized with a
Polytron or bullet homogenizer. After homogenization, RNA
was isolated with the phase separation method by addition
of chloroform or bromochloropropane followed by centrifu-
gation. The aqueous phase was transferred to a new 1.5-mL
tube, and an equal amount of 70% of diethyl pyrocarbonate
(DEPC)-treated ethanol was added. RNA isolation was fur-
ther processed with the Invitrogen RNA isolation kit
(K145002, Invitrogen, Carlsbad, CA) or the Zymo Direct-zol
kit (Zymo Research, Irvine, CA). RNA concentration was
determined with a BioTek Take3 microplate with a BioTek
PowerWave XS microplate reader as previously described
(55) or NanoDrop (ThermoFisher Scientific). RNA samples
were only accepted if 260 nm-to-280 nm ratio was >2.0.
Samples were stored at 80Cuntilfurtheruse.
Reverse transcription (RT) of miR-16 was performed with
the TaqMan MicroRNA Reverse Transcription Kit (4366596,
Applied Biosystems, Waltham, MA). Briefly, 200 ng of total
miRNA was added to a master mix comprised of 1 lLofa
10TaqMan probe (PN4427975, Applied Biosystems) for U6
and miR-16 each (RT:001973 and RT:000391, respectively,
Applied Biosystems), 0.3 lLofdNTPs,3lLofMultiScribe
miR-16 IN MYOGENIC CELLS
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RTase, 1.5 lLof10RTase buffer, 0.19 lLofRNaseinhibitor,
and nuclease-free water added up to 15 lLoftotalvolume.RT
reaction occurred with a hold step of 30 min at 16C, followed
by 30 min at 42Candfinally 5 min at 85C. Samples were held
at 4C until further analysis. RT-qPCR was performed with the
QuantStudio 3 Real-Time PCR system (Applied Biosystems). A
20-lL reaction composed of an adequate amount of TaqMan
probes plus TaqMan Fast Advanced Master Mix (4444556,
Applied Biosystems) was used to amplify cDNA. Samples
followed a protocol consisting of incubation at 95Cfor4
min, followed by 45 cycles of denaturation, annealing, and
extension at 95Cand60
C. TaqMan probes were meas-
ured at the end of the extension step of each cycle.
Fluorescence-labeled 20TaqMan probes (PN4427975,
Applied Biosystems) included U6 and miR-16 (TM:001973 and
TM:000391, respectively, Applied Biosystems). Results were
analyzed with QuantStudio Software. Cycle threshold (C
T
)
was determined, and the DC
T
value was calculated as the dif-
ference between C
T
value and U6 C
T
value. U6 C
T
values were
not different between experimental conditions. Final quantifi-
cation of gene expression was calculated with the DDC
T
method. Relative quantification was calculated as 2DDCTand
expressed as arbitrary units.
RNA-Sequencing and Transcriptomic Analyses
RNA-sequencing was performed by NovoGene as previ-
ously described by us (54). Standard 150-bp paired-end
sequencing was performed, and read counts were >20 mil-
lion. Raw counts from RNA-sequencing were used as inputs
into Partek Flow. Alignment was performed with STAR with
mmu39. After low-expressed genes were filtered, DESeq2
(version 1.34.0) was used for normalization and differential
analyses to identify differentially expressed genes (DEGs)
with pairwise comparisons (56). DEGs were identified with a
false discovery rate (FDR, step-up procedure) adjusted P
value <0.05. DEGs with adjusted Pvalue <0.05 were used
for downstream functional analysis using ConsensusPath DB
(57). Pathway analysis was conducted using the mouse over-
representation feature, up- or downregulated DEGs, and the
Reactome database with default settings. For pathway analy-
sis, qvalues were used to determine significance based on
DEGs with adjusted P<0.05.
Proteomics: FASP bHPLC-Orbitrap Fusion
Proteomics was performed at the University of Arkansas
for Medical Sciences Proteomics Core. Protein samples were
reduced, alkylated, and digested by filter-aided sample prep-
aration (58) with sequencing-grade modified porcine trypsin
(Promega). Tryptic peptides were separated into 46 fractions
on a 100 1.0-mm Acquity BEH C18 column (Waters) with
an UltiMate 3000 UHPLC system (Thermo) with a 50-min
gradient from 99:1 to 60:40 buffer A-to-Bratio under basic
pH conditions and then consolidated into 12 superfractions;
buffer A = 0.1% formic acid, 0.5% acetonitrile, and buffer B =
0.1% formic acid, 99.9% acetonitrile. Each superfraction was
then further separated by reverse-phase XSelect CSH C18 2.5-
μm resin (Waters) on an in-line 150 0.075-mm column
with an UltiMate 3000 RSLCnano system (Thermo). Peptides
were eluted with a 45-min gradient from 98:2 to 65:35 buffer
A-to-Bratio. Eluted peptides were ionized by electrospray
(2.4 kV) followed by mass spectrometric analysis on an
Orbitrap Fusion Tribrid mass spectrometer (Thermo). MS
data were acquired with the FTMS analyzer in profile mode
at a resolution of 240,000 over a range of 375 to 1,500 m/z.
After HCD activation, MS/MS data were acquired with the
ion trap analyzer in centroid mode and normal mass range
with normalized collision energy of 28–31% depending on
charge state and precursor selection range. Proteins were
identified by database search using MaxQuant (Max Planck
Institute) label-free quantification with a parent ion toler-
ance of 2.5 ppm and a fragment ion tolerance of 0.5 Da.
Scaffold Q þS (Proteome Software) was used to verify MS/
MS-based peptide and protein identifications. Protein identi-
fications were accepted if they could be established with
<1.0% false discovery and contained at least two identified
peptides. Protein probabilities were assigned by the Protein
Prophet algorithm (59). Protein abundance is presented with
intensity-based absolute quantification (iBAQ) values. To
determine differential expression and pathway analysis
between conditions, Pvalues based on unpaired nondirec-
tional ttests were employed with a cutoff of P<0.05.
Benjamini–Hochberg-adjusted Pvalues were subsequently
calculated from these Pvalues. Untranslated region (UTR)
sequences for genes that resulted in proteins of interest were
downloaded from UCSC Genome Browser and used for pre-
diction of miR-16 binding sites. The potential binding site
was identified with RNAhybrid software (60,61), which com-
bines thermodynamic and seed sequence information.
RESULTS
miR-16 Is Lower after 72-h Synergist Ablation
Mechanical Overload of the Plantaris Muscle
Previous work suggests that muscle miR-16 levels are
inversely related to postnatal muscle growth in chickens
(34,36) and are lower during regeneration in mice (24). We
showed that miR-16 is lower in muscle tissue after a bout of
resistance exercise in rats (37). Previous work showed signifi-
cant changes in global muscle miRNA levels in the early
phase of mechanical overload-induced hypertrophy (62). To
determine whether miR-16 declines during the early phase
of a well-characterized model of rapid loading-induced hy-
pertrophyinmice(49,63,64), we compared miR-16-5p levels
in 72-h mechanically overloaded (MOV) plantaris muscle to
sham-operated muscles. miR-16 was 28% lower in muscle tis-
sue during MOV (P=0.06)(Fig. 1A). Overall, low miR-16
appears characteristic of growing muscle in several settings
and species (6,35–37).
miR-16 Influences Ribosomal-, p53-, and Pol II
Transcription-Related mRNA Abundance
The in vitro study design is found in Fig. 1B.Thetransfec-
tion of a miR-16 inhibitor was effective at knocking down
miR-16 (miR-16 KD) in proliferating C2C12 myoblasts (Fig. 1C).
Inhibition of miR-16 in myoblasts upregulated mRNA levels
of Rps12,Rps26,Rplp1,Rpl22l1,andRpl35 (adj. P<0.05),
which encode ribosomal proteins (Fig. 1D). Additional ribo-
somal protein genes tended to be higher after miR-16 knock-
down (Rpl27 and Rpl27a,adj.P<0.10) (Supplemental Table
S1). The major processes downregulated by miR-16 inhibition
miR-16 IN MYOGENIC CELLS
AJP-Cell Physiol doi:10.1152/ajpcell.00071.2023 www.ajpcell.org C1103
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were Regulation of TP53 Activity through Methylation
(Reactome, q= 0.0014) and RNA Polymerase II Transcription
(Reactome, q= 0.0026) (Supplemental Table S2). In the for-
mer pathway, Atm,Ep300,andMdm2 were lower (adj. P<
0.05). In addition to those three genes, Aff4,Crebbp,Kmt2a,
Kmt2c, Mga,Nr2c2,andSox9 were lower after miR-16 knock-
down in the latter pathway (adj. P<0.05) (Fig. 1E). The long
noncoding RNA (lncRNA) H19 was elevated with miR-16 inhi-
bition (adj. P= 0.0035), and the lncRNAs Malat1 (adj. P=
0.059) and Xist (adj. P= 0.009) were lower (Supplemental
Table S1).
miR-16 Knockdown Increases Metabolism-Related
Proteins and Specific Markers of Myogenic Cell
Differentiation
Proteomics was performed in five technical replicates per
condition. After miR-16 knockdown, five proteins were upregu-
lated (EEF1A2, HIST1H1B, NPC2, PRPH, and TSEN54) and
seven proteins were downregulated (GLRX2, GOLPH3, GRK6,
LAPTM4A, NDC1, THAP4, and ZC3H11A) with an adjusted
Pvalue <0.05, and 899 proteins achieved significance at P<
0.05 (Supplemental Table S3). Proteins involved in different
aspects of Metabolism (Reactome, q= 0.0178) were upregulated
(assessed using proteins with P<0.05) (Fig. 2A)(Supplemental
Tables S3 and S4). Within the broad Metabolism pathway,
various tricarboxylic acid (TCA) cycle proteins were selec-
tively enriched (Reactome, q= 0.00293). Proteins related
to Metabolism of RNA (Reactome, q= 0.000456) were
most downregulated by miR-16 inhibition (Supplemental
Table S4). Markers of myogenic cell maturation, including
myosin light chains (MYL6B, MYL9, MYL12A, P<0.05) as
well as smooth muscle actin (ACTA2, P=0.04,adj.P=
0.41) (Fig. 2B), were upregulated with miR-16 KD. Specific
proteins that have a defined role in satellite cell behavior
were also altered. In addition to muscle-enriched EEF1A2
(P= 0.0000164, adj. P=0.018)(Fig. 2C)(65–67), OPA1 (P<
0.05, adj. P=0.25and0.30)(Fig. 2D)(68,69)andPTEN
(P<0.05, adj. P=0.41)(Fig. 2E)(70–72)wereelevatedby
miR-16 inhibition; the latter two proteins are of interest
butdidnotachievesignificance according to adjusted
Aff4 Atm Crebbp Ep300 Kmt2a Kmt2c Mdm2 Mga Nr2c2 Sox9
0
1000
2000
3000
4000
5000
DESeq2 Gene Expression
Regulation of p53 and Pol II Transcription mRNA Levels
Control
miR-16 KD
0.0
0.5
1.0
1.5
miR-16
*
*
*
**
*
*
*
*
miR-16 : U6
Rps12 Rps26 Rplp1 Rpl22l1 Rpl35
0
5000
10000
15000
DESeq2 Gene Expression
Ribosomal Protein mRNA Levels
Control
miR-16 KD
Sham MOV
0.0
0.5
1.0
1.5 miR-16
p=0.06
A
D
E
miR-16 : U6
BC
*
*
*
**
*
*
Figure 1. miR-16 levels in overloaded mus-
cle in vivo and RNA-sequencing (RNA-seq)
analysis in proliferating C2C12 myoblasts 18
h after miR-16 knockdown (KD). A:miR-16
levels in plantaris skeletal muscle after 72 h
of synergist ablation mechanical overload
of the plantaris (MOV) relative to sham oper-
ation. B: in vitro study design. C:miR-16lev-
els in control and KD myoblasts. D:levelsof
ribosomal protein genes in control and KD
myoblasts. E:levelsofp53-andPolIItran-
scription-related genes in control and KD
myoblasts. For RNA-seq experiments, n=3
control and n= 2 KD technical replicates.
adj. P<0.05.
miR-16 IN MYOGENIC CELLS
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Pvalues. OPA1 was detected twice (canonical 111 kDa and
truncated 34 kDa), and the levels of both isoforms were
higher with knockdown (P<0.05) but not according to
adjusted Pvalues.
An overlap in gene and protein expression would not neces-
sarilybeexpectedatadiscretetimepointsinceproteintransla-
tion lags behind changes in mRNA expression. Nevertheless,
comparison of gene to protein levels revealed agreement for
Bmpr2,Cltc,Lama5,Lpp,Mga,Nptx1,andSema5a,allofwhich
were lowered by miR-16 knockdown (P<0.05). Rpl27a,which
was trending to increase at the gene level, was elevated at the
protein level (P=0.01,adj.P=0.28).Sinceweobservedupregu-
lation of proteins with miR-16 knockdown, and miRNAs can
prevent protein translation without altering transcript levels
Normalized iBAQ
PTEN
110 kDa 34 kDa
Normalized iBAQ
OPA1
*
*
0.0
5.0×109
1.0×1010
1.5×1010
0.0
5.0×106
1.0×107
1.5×107
2.0×107
Normalized iBAQ
ACTA2
Control
miR-16 KD
A
-3 -2 -1 0
Dual Incision in GG-NER
Chromatin modifying enzymes
Chromatin organization
Oocyte meiosis - Mus musculus (mouse)
Death Receptor Signalling
DNA Replication
COPI-dependent Golgi-to-ER retrograde traffic
Toll-like Receptor Cascades
Nucleotide Excision Repair
SUMOylation of DNA damage response and repair proteins
Transport of Mature mRNA Derived from an Intronless Transcript
Resolution of Abasic Sites (AP sites)
Resolution of AP sites via the multiple-nucleotide patch replacement pathway
Free fatty acids regulate insulin secretion
Interleukin-2 signaling
Synthesis of DNA
MAP kinase activation
Interleukin-17 signaling
S Phase
Toll Like Receptor 3 (TLR3) Cascade
TRAF6 mediated induction of NFkB and MAP kinases upon TLR7/8 or 9 activation
MyD88 cascade initiated on plasma membrane
Toll Like Receptor 10 (TLR10) Cascade
Toll Like Receptor 5 (TLR5) Cascade
Mitochondrial translation termination
MyD88 dependent cascade initiated on endosome
MyD88:MAL(TIRAP) cascade initiated on plasma membrane
Toll Like Receptor TLR1:TLR2 Cascade
Toll Like Receptor TLR6:TLR2 Cascade
Toll Like Receptor 2 (TLR2) Cascade
Mitochondrial translation
Toll Like Receptor 7/8 (TLR7/8) Cascade
Activation of the AP-1 family of transcription factors
Inactivation of APC/C via direct inhibition of the APC/C complex
Inhibition of the proteolytic activity of APC/C
Cell Cycle, Mitotic
Golgi-to-ER retrograde transport
Toll Like Receptor 9 (TLR9) Cascade
MAPK targets/ Nuclear events mediated by MAP kinases
Metabolism
Citric acid cycle (TCA cycle)
Pyruvate metabolism and Citric Acid (TCA) cycle
Log10 q value
UPregulated Proteins: Reactome
Normalized iBAQ
EEF1A2
0.0
1.0×109
2.0×109
3.0×109
4.0×109
5.0×109
0.0
1.0×106
2.0×106
3.0×106
4.0×106
5.0×106
p=0.04
adj.
p=0.41
p=0.005
adj.
p=0.25
p=0.013
adj.
p=0.30 p=0.04
adj.
p=0.41
p<0.001
adj.
p=0.018
*
BC
DE
**
Figure 2. Proteomic analysis in proliferating
C2C12 myoblasts 18 h after miR-16 knock-
down (KD). A: Reactome pathway analysis
using upregulated differentially expressed
proteins (P<0.05). B–E: thermodynamic and
seed sequence target prediction for miR-16
and protein levels of ACTA2 (B), EEF1A2 (C),
OPA1 (D), and PTEN (E) in control and KD
myoblasts. iBAQ, intensity-based absolute
quantification. For all experiments, n= 5 tech-
nical replicates were utilized. P<0.05.
Created with BioRender.com.
miR-16 IN MYOGENIC CELLS
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(14,73), we performed a 30UTR binding affinity analysis of
miR-16 for ACTA2, EEF1A2, OPA1, and PTEN (Fig. 2, B–E),
using RNAhybrid (60,61).Basedonfreeenergy,theresultscol-
lectively suggested that miR-16 could regulate the levels of
these proteins. The highest complementarity between the miR-
16 seed sequence and 30UTR sequence was for EEF1A2, OPA1,
and PTEN (Fig. 2. C–E).
DISCUSSION
Satellite cells differentiate and begin fusing appreciably to
muscle fibers between days 4 and 5 of mechanical overload in
adult mice (25)and5 days after transplantation into muscle
of mice (74). In our 72-h overload experiments, the contribu-
tions of miR-16 from satellite cells versus other cell types in
muscle tissue (e.g., muscle fibers or immune cells) (75) cannot
be discerned. The influence of hypertrophy per se versus a
degeneration/regeneration response that can occur with syner-
gist ablation (19,76) is also unclear. Nevertheless, our observa-
tion of lower miR-16 in murine muscle tissue after 72 h of MOV
corresponds with reduced muscle miR-16 levels during early
in vivo regeneration after injury (6) and during recovery from
a bout of resistance exercise (37). To model miR-16 regulation
of myogenic cell behavior, we repressed it in proliferating
myoblasts and performed transcriptomic and proteomic profil-
ing.ThesedataprovideaframeworkforunderstandingmiR-
16’s role in myogenic cell fate.
miR-16-5p can induce p53 signaling in myogenic cells (34).
Repression of p53-related gene expression with miR-16
knockdown dovetails with this observation. In C2C12s in
vitro, the expression of ribosomal proteins is upregulated
during early differentiation (77,78). Enrichment of ribo-
somal protein genes is associated with satellite cell fusion
into the myofiber syncytium during hypertrophy (79). The
induction of ribosomal protein genes, specifically Rps26,by
miR-16 inhibition may characterize myogenic cells that are
primed for fusion (79). The lncRNA H19 was elevated with
miR-16 knockdown, whereas Malat1 and Xist were lower.
lncRNAs are not typically targeted for decay by miRNAs
since translation is seemingly required for RNA destabiliza-
tion (80). H19 induction (81–84)andMalat1 reduction (85)
are strongly implicated in myogenic cell differentiation, but
how miR-16 could affect lncRNA levels in myogenic cells
deserves further investigation. In concert with evidence sug-
gesting that miR-16 can target Myomaker, the gatekeeper of
myogenic cell fusion that gradually increases during differ-
entiation (86,87), our RNA-seq data collectively point to
declining miR-16 levels facilitating lineage progression to-
ward differentiation.
At the protein level, repressing miR-16 in proliferating myo-
blasts leads to several alterations that are indicative of miR-
16’s roles in the regulation of differentiation. The enrichment
of myosin light chains, noted in our data, is a sign of myo-
genic cell maturation (88,89). Upregulation of smooth muscle
actin (Acta2) is also strongly associated with myoblast differ-
entiation (90–93). Satellite cell differentiation is controlled by
a metabolic shift and progression from glycolysis to the TCA
cycle (94). Elevated TCA cycle proteins with miR-16 repres-
sion therefore seems intuitive from a metabolic perspective.
Induction and phosphorylation of EEF1A2 is linked to myo-
genic cell differentiation (65–67). EEF1A2 is muscle enriched
(95) and highly regulated during muscle development (96),
protects myotubes from cell death (65), and controls Utrophin
levels in skeletal muscle (97,98). EEF1A2 is also a core compo-
nent of cardiomyocyte differentiation (99). The causal func-
tions of EEF1A2 control by miR-16 in myoblast differentiation
deserve further study. Higher PTEN and OPA1 with miR-16 in-
hibition is noteworthy since both are implicated in reinforc-
ing satellite cell quiescence (68,70,71). PTEN can repress the
satellite cell identity gene Pax7 (72,100)butmayalsofacili-
tate a return to quiescence (70,71) that could ensue if differen-
tiation and fusion does not progress. Whereas OPA1 supports
quiescence by maintaining mitochondrial integrity (68), OPA1
induction and mitophagy is an essential component of suc-
cessful C2C12 myoblast differentiation (69).
The inhibition of miR-16 in proliferating myoblasts, which
occurs naturally during myogenic differentiation, reveals its
contributionstothisprocess.Specifically, we uncover miR-
16’s regulation of ribosomal, p53-related, and lncRNA gene
expression as well as metabolic- and muscle maturation-
related protein abundance. A more detailed understanding of
miR-16 dynamics, specifically in satellite cells in vivo during
different stages of myogenesis, regeneration, and hypertrophy,
will inform how miR-16 controls muscle mass in varying cir-
cumstances. The present study is limited to a single time point
with an immortalized cell line. We also do not report on myo-
genic cell behavior after miR-16 inhibition, although this has
been documented in detail elsewhere (6,34,40). Limitations
aside, we provide insights from two -omic layers to expand our
understanding of miR-16 regulation in myogenic cells.
DATA AVAILABILITY
Processed data are provided in Supplemental Tables S1–S4
and were deposited in GEO (GSE229134).
SUPPLEMENTAL DATA
Supplemental Tables S1–S4: https://doi.org/10.6084/m9.figshare.
22151624.
ACKNOWLEDGMENTS
The authors thank Dr. John J. McCarthy of the University of
Kentucky and Dr. Vandre C. Figueiredo of Oakland University for
critical feedback on this manuscript. The authors also thank the
University of Arkansas for Medical Sciences Proteomics Core for
conducting the proteomics and analysis.
The Graphical Abstract was generated with BioRender.
GRANTS
This work was supported by NIH Grant R00 AG063994 to K.A.M.
Support for proteomics was provided by an Arkansas Biosciences
Institute grant to N.P.G.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by
the authors.
AUTHOR CONTRIBUTIONS
N.P.G. and K.A.M. conceived and designed research; S.L., D.L.,
F.M., P.J.K., and F.v. performed experiments; S.L., F.M., P.J.K., I.J.V.,
miR-16 IN MYOGENIC CELLS
C1106 AJP-Cell Physiol doi:10.1152/ajpcell.00071.2023 www.ajpcell.org
Downloaded from journals.physiology.org/journal/ajpcell at Univ of Arkansas Fayetteville (130.184.252.076) on May 18, 2023.
and K.A.M. analyzed data; S.L., F.M., I.J.V., and K.A.M. interpreted
results of experiments; S.L., I.J.V., and K.A.M. prepared figures; S.L.,
D.L., and K.A.M. drafted manuscript; S.L., D.L., F.M., P.J.K., I.J.V., F.v.,
N.P.G., and K.A.M. edited and revised manuscript; S.L., D.L., F.M.,
P.J.K., I.J.V., F.v., N.P.G., and K.A.M. approved final version of
manuscript.
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