Gene 676 (2018) 9–15

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Gene
journal homepage: www.elsevier.com/locate/gene

Research paper

Osteoglycin post-transcriptional regulation by miR-155 induces cellular T

architecture changes in H9c2 cardiomyoblasts
Grasieli de Oliveiraa, Paula Paccielli Freirea, Ana Carolina Mieko Omotoa, Sarah Santiloni Curya,
⁎
Cesar Seigi Fuziwarab, Edna Teruko Kimurab, Maeli Dal-Pai-Silvaa, Robson Francisco Carvalhoa,
a
Department of Morphology, Institute of Biosciences of Botucatu, São Paulo State University, Botucatu, São Paulo, Brazil
b
Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Several studies have demonstrated dysregulated cardiac microRNAs (miRNAs) following cardiac stress and
Mimecan development of cardiac hypertrophy and failure. miRNAs are also differentially expressed in the inflammation
Cardiac cell morphology that occurs in heart failure and, among these inflammatory-related miRNAs, the miR-155 has been implicated in
Cardiac pathological hypertrophy the regulation of cardiac hypertrophy. Despite these data showing the role of miRNA-155 in cardiomyocyte
hypertrophy under a hypertrophic stimulus, it is also important to understand the endogenous regulation of this
miRNA without a hypertrophic stimulus to fully appreciate its function in this cell type. The first aim of the
present study was to determine whether, without a hypertrophic stimulus, miR-155 overexpression induces
H9c2 cardiac cells hypertrophy in vitro. The second objective was to determine whether osteoglycin (Ogn), a key
regulator of heart mass in rats, mice, and humans, is post-transcriptionally regulated by miR-155 with a potential
role in inducing H9c2 cells hypertrophy. Here, we show that, without a hypertrophic stimulus, miR-155 sig-
nificantly repressed Ogn protein levels, but induce neither alteration in morphological phenotype nor in the
expression of the molecular markers that fully characterize pathological hypertrophy of H9c2 cells. However,
most importantly, Ogn silencing in H9c2 cells mimicked the effects of miR-155 overexpression in inducing
cellular architecture changes that were characterized by a transition of the cell shape from fusiform to rounded.
This is a new role of the post-transcriptional regulation of Ogn by miR-155 in the maintenance of the cardiac cell
morphology in physiological and pathological conditions.

1. Introduction nucleotides that regulate gene expression in higher eukaryotes by

Cardiac remodeling involves a repertoire of pathophysiological re-
sponses including molecular, cellular, and extracellular matrix changes
that, in turn, promote alterations in size, shape, and function of the
heart after injury or stress stimulation [reviewed in (Kehat and
Molkentin, 2010)]. The identification of microRNAs (miRNAs) during
cardiac remodeling has opened up new fields of investigation aiming to
better understand gene expression regulation in cardiovascular diseases
[reviewed in (Liu and Olson, 2010; Small and Olson, 2011)]. miRNAs
are an evolutionarily conserved class of small noncoding RNAs of ~22
binding to complementary sequences mainly within the 3′ untranslated
region (3′UTR) of protein-coding mRNAs [reviewed in (Bartel, 2004;
Carrington and Ambros, 2003)].
Several studies have demonstrated dysregulated cardiac miRNAs in
adaptive and maladaptive hypertrophy and heart failure (HF) in re-
sponse to cardiac stress (Bagnall et al., 2012; Sucharov et al., 2008; van
Rooij et al., 2008, 2006). miRNAs are also differentially expressed in
the inflammation that occurs in HF and, among these inflammatory-
related miRNAs, the miR-155 has been implicated in the regulation of
cardiac hypertrophy under a hypertrophic stimulus. Seok et al. (2014)

Abbreviations: miRNAs, microRNAs; Ogn, osteoglycin/mimecan; 3′UTR, 3′ untranslated region; HF, heart failure; Socs1, suppressor of cytokine signaling 1; FoxO3a,
Forkhead box O3a; ECM, extracellular matrix; GM, growth medium; RIN, RNA integrity number; PBS, Phosphate Buffered Saline; PMSF, phenylmethylsulfonyl
fluoride, protease inhibitor; RIPA, Radio-Immunoprecipitation Assay; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TBS, Tris buffered
saline; Gapdh, Glyceraldehyde 3-phosphate dehydrogenase; BSA, Bovine serum albumin; DAPI, (4′,6-diamidino-2-phenylindole); ANP, Atrial Natriuretic Peptide;
BNP, Brain Natriuretic Peptide; Myh6, Myosin, Heavy Chain 6, Cardiac Muscle, Alpha; Myh7, Myosin, Heavy Chain 7, Cardiac Muscle, Beta; MYH, Myosin, Heavy
Chains Isoforms; Acta1, Actin, Alpha 1, Skeletal Muscle; Ppib, Peptidylprolyl isomerase B
⁎
Corresponding author.
E-mail address: robson.carvalho@unesp.br (R.F. Carvalho).

https://doi.org/10.1016/j.gene.2018.07.020
Received 12 April 2018; Received in revised form 1 July 2018; Accepted 6 July 2018
Available online 07 July 2018
0378-1119/ © 2018 Elsevier B.V. All rights reserved.
G. de Oliveira et al.
uncover the miR-155 as an inducer of pathological cardiomyocyte hy-
pertrophy by targeting Jarid2/jumonji, which is also known to critically
regulate the cardiovascular development (Seok et al., 2014); (Mysliwiec
et al., 2011). The miR-155 also targets the suppressor of cytokine sig-
naling 1 (Socs1) transcript in cardiac macrophages and promotes the
inflammatory response to pressure overload, where genetic loss or
pharmacological inhibition of miR-155 protected against cardiac hy-
pertrophy and HF (Heymans et al., 2013). Furthermore, the polyphenol
resveratrol, which has been reported to exhibit cardioprotective effects,
produced protective effects against cardiac hypertrophy by reducing
miR-155 levels (Fan et al., 2016). These authors also showed that
Forkhead box O3a (FoxO3a) is a miR-155 target transcript involved in
the anti-hypertrophic effect of resveratrol in hypertrophied cardio-
myocytes.
Despite these data demonstrating the role of miRNA-155 in cardi-
omyocyte hypertrophy under a hypertrophic stimulus, it is also im-
portant to understand the endogenous regulation of this miRNA
without a hypertrophic stimulus to fully appreciate its function in this
cell type. Thus, the first aim of the present study was to determine
whether, without a hypertrophic stimulus, miR-155 overexpression
induces H9c2 cardiac cell hypertrophy in vitro. Moreover, an important
issue to address in the miRNA field is that a single miRNA can regulate
multiple mRNAs (Pasquinelli, 2012), challenging the identification of
new miR-155-target transcripts that may be critical for understanding
the molecular basis of cardiomyocyte biology. Recently, we demon-
strated that miR-155 inhibits osteoglycin/mimecan (Ogn) expression to
control myogenic differentiation of C2C12 myoblasts (Freire et al.,
2017). Interestingly, Ogn is a secreted extracellular matrix (ECM)
component that has been considered a key regulator of heart mass in
rats, mice, and humans, and a novel biomarker to predict chronic HF
outcomes and left ventricular remodeling (Motiwala et al., 2014;
Petretto et al., 2008; Van Aelst et al., 2015). The second objective of the
study was to determine whether the post-transcriptional regulation of
osteoglycin by miR-155 may induce H9c2 cell hypertrophy in vitro.
Considering that reactivation/upregulation of the fetal cardiomyocyte
gene program, including the natriuretic peptide A (ANP), natriuretic
peptide B (BNP), skeletal muscle alpha actin (Acta1), and myosin heavy
chain beta (β-MHC or Myh7), and the downregulation of genes asso-
ciated with contractile function, such as myosin heavy chain beta (α-
MHC or Myh6), are molecular hallmarks of the pathological cardiac
hypertrophy (Chien et al., 1991; Depre et al., 1998; Izumo et al., 1988;
Rajabi et al., 2007), we evaluated the expression of these genes as
markers of H9c2 cells hypertrophy. We found that, without a hyper-
trophic stimulus, miR-155 significantly repressed Ogn protein levels,
but induce neither alteration in morphological phenotype nor in the
expression of the molecular markers that fully characterize pathological
hypertrophy of H9c2 cells. However, most importantly, Ogn silencing
in H9c2 cells mimicked the effects of miR-155 overexpression in in-
ducing cellular architecture changes that were characterized by a
transition of the cell shape from fusiform to rounded. This is a new role
of the post-transcriptional regulation of Ogn by miR-155 in the main-
tenance of the cardiac cell morphology in physiological and patholo-
gical conditions.
2. Materials and methods
2.1. Cell culture
H9c2 (2-1) rat BDIX cardiomyoblasts (ATCC, The Global
Bioresource Center, USA) were cultured at 37 °C and 5% CO2 in
Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific,
USA) growth medium (GM) enriched with 1% penicillin-streptomycin
(Thermo Fisher Scientific, USA) and 10% fetal bovine serum (Thermo
Fisher Scientific, USA). All experiments were carried out using at least
three independent replicates per group.
10
Gene 676 (2018) 9–15
2.2. Oligonucleotides and transfection
The miR-155 mimic (mirVana™ miRNA Mimic, assay ID: MC13058;
Thermo Fisher, USA), and the small interfering RNA (Silencer® Select
siRNA, assays ID: s70945 and s70947; Thermo Fisher Scientific, USA),
against Ogn transcript, and the respective negative controls (miRNA
Mimic Negative Control and Silencer® Select Negative Control No. 1
siRNA; Thermo Fisher Scientific, USA), were complexed with Opti-MEM
reduced serum medium (Thermo Fisher Scientific, USA) before trans-
fection. After the H9c2 cardiomyoblasts cultures have reached ap-
proximately 80% confluence, the cells were transfected with RNAiMAX
lipofectamine (Thermo Fisher Scientific, USA) mixed with 30 nmol of
each oligonucleotide for 12 h. Oligonucleotide concentration and in-
cubation time were previously standardized in our laboratory in muscle
cells. After transfection, the medium was changed to GM, and the cells
were analyzed after 48 h (Supplementary Fig. 1).
2.3. RNA isolation
H9c2 cardiomyoblasts total RNA was extracted using the TRIzol
reagent (Thermo Fisher Scientific, USA), as suggested by the manu-
facture. NanoVue Plus Spectrophotometer (GE Healthcare, USA) was
used to quantify RNA concentration and quality; subsequently, the ri-
bosomal RNAs were analyzed using the 2100 Bioanalyzer system
(Agilent, USA) to obtain the RNA integrity number (RIN) and RNA
quality. RNA preparations with A260/280 ratio of 1.8–2.0, A260/230
ratio > 2.0, and RIN > 9 were used for further analysis.
2.4. RT-qPCR
Total RNA samples (1 μg) were reverse transcribed into cDNA with
the High Capacity RNA-to-cDNA Master Mix (Thermo Fisher Scientific,
USA). The qPCR reactions (15 μL) were performed with Power SYBR™
Green Master Mix (Thermo Fisher Scientific, USA), as described by the
manufacturer, using specific primers (listed in Supplementary Table 1).
The reactions were run on a QuantStudio™ 12 K Flex System (Thermo
Fisher Scientific, USA) using 95 °C for 10 min followed by 40 cycles of
95 °C for 15 s and 60 °C for 1 min as cycling conditions. Finally, the raw
data was retrieved and imported into the Expression Suite Software
v1.0.3 (Thermo Fisher Scientific, USA). Relative quantification of
mRNA expression was evaluated using the 2−ΔΔCTmethod (Livak and
Schmittgen, 2001), and Ppib was used as reference gene to normalize
the mRNA expression data. This reference gene was selected based on
geNorm calculations (Vandesompele et al., 2002), and the expression
stability was further confirmed by the comparisons of Ct values be-
tween control and treated samples (miR-155 mimic or siRNA-Ogn)
samples (p > 0.80, Student t-test). Cutoffs for significant changes were
set at fold-change > 1.5 and p-value ≤ 0.05.
2.5. Western blot analysis
After transfections, the H9c2 cardiomyoblasts were harvested in
PBS (Phosphate Buffered Saline), mechanically lysated, and the cell
pellets were re-suspended and incubated in ice-cold RIPA buffer (Radio-
Immunoprecipitation Assay, Sigma, USA) containing 1% PMSF (pro-
tease inhibitor), on ice for 30 min. The lysated cells were centrifuged in
8.000 RCF for 10 min at 4 °C to collect the supernatant, and protein
concentration was determined by Bradford protein assay kit according
to the manufacturer's instructions (Bradford3, Bio-Rad Laboratories,
USA). Subsequently, the samples were prepared using Lammeli buffer
(Sigma, USA) at 100 °C for 10 min, for SDS-PAGE protocol.
Electrophoresis were performed in 10% polyacrylamide gels, and after
protein separation, proteins were electrotransferred to nitrocellulose
membranes (Bio-Rad, USA). Membranes were blocked for 2 h at room
temperature, using 5% nonfat dry milk in TBS buffer containing 0.5%
Tween 20 (TBST), and then incubated with specific antibodies against
G. de Oliveira et al. Gene 676 (2018) 9–15

Osteoglycin (Ogn, Santa Cruz Biotechnology, USA; 1:1000 dilution),

Myosin Heavy Chain (MYH, Santa Cruz Biotechnology, USA; 1:500
dilution), and Glyceraldehyde 3-phosphate dehydrogenase (Gapdh,
ABR Affinity BioReagents, USA; 1:2500 dilution), overnight at 4–8 °C.
Finally, secondary antibodies (rabbit or mouse, depending on the pro-
tein) were incubated for 2 h at room temperature to promote the
binding with primary antibody, and secondary antibody peroxidase
conjugated was detected using Enhanced Chemiluminescence
(Amersham Biosciences, USA), and autoradiography in ImageQuant™
LAS 4000 (GE Healthcare, USA). Proteins quantification of blots were
performed by ImageJ software (Version 1.49o, Wayne Rasband, NIH).
Targeted bands were normalized to the expression of GAPDH.

2.6. Immunofluorescence analysis

Firstly, 6-well plates were used to seed the H9c2 cardiomyoblasts

for immunoassay. After transfections, these cells were fixed in 4%
paraformaldehyde for 15 min at room temperature, and washed with
PBS and 0.1% TritonX-100 (Sigma, USA). The block was performed
during 1 h at room temperature using 3% BSA, 1% glycine, 8% fetal
bovine serum in PBS and 0.1% TritonX-100. After the block, the cells
were incubated overnight at 4°Cwith Ogn primary antibody, and sub-
sequently, with secondary antibody for 1 h, at room temperature.
Nucleus staining was performed with DAPI (4′,6-diamidino-2-pheny-
lindole) counter staining. All the images were acquired at room tem-
perature with a confocal microscope TCS SP5 (Leica Microsystems, UK). Fig. 1. miR-155 post transcriptionally represses Ogn protein expression in H9c2
Cellular width and length were measured by ImageJ software (Version cardiomyoblasts. RT-qPCR analysis show that miR-155 does not change Ogn
1.49o, Wayne Rasband, NIH). transcript levels (A); however, miR-155 represses Ogn protein expression, as
revealed by Western blot analysis (B). Western blot and RT-qPCR analysis
2.7. Statistical analysis confirm the efficiency of siRNA-Ogn on Ogn transcript (C) and protein (D) le-
vels in H9c2 cardiomyoblasts, respectively. RT-qPCR data were normalized by
the expression level of the reference Ppib gene, and western blot data were
All experimental data are expressed as the mean ± standard de-
normalized by Gapdh content. Results are expressed as mean ± SD of three
viation (SD). Data were analyzed using Student's t-test to establish
independent biological replicates (n = 3). Statistical difference was analyzed by
significance between the groups. p < 0.05 were considered statisti- Student's t-test. *p < 0.05 vs. Control (CT).
cally significant.

3. Results polyclonal antibody (MYH), reported to detect all muscle myosin heavy
chain isoforms as a molecular marker of protein synthesis or degrada-
3.1. miR-155 post transcriptionally represses Ogn protein expression in tion. However, the miR-155 mimetic treatment did not change MYH
H9c2 cardiomyoblasts protein levels when compared to control group (Fig. 2B).

We had previously identified and validated Ogn transcript
(NM_008760.4) as a miR-155 target on its 3′UTR in fibroblasts and
myoblasts (Freire et al., 2017).To confirm the effect of miR-155 in
regulating the expression of Ogn in cardiomyoblasts, we first trans-
fected H9c2 cells with miR-155 mimic and evaluated Ogn mRNA and
protein expression levels. The miR-155 transfection did not affect Ogn
transcript levels (Fig. 1A); however, resulted in a reduction on protein
levels (Fig. 1B). Finally, to simulate a potential miR-155 post tran-
scriptional regulation of Ogn in H9c2 cardiomyoblasts, we also
knocked-down Ogn with siRNA oligonucleotides and evaluated Ogn
mRNA and protein expression levels. This Ogn knockdown induced a
significant reduction in Ogn mRNA and protein expression levels in
H9c2 cells when compared to control group (Fig. 1C and D).
3.2. miR-155 alters the expression of cardiac stress markers in H9c2
cardiomyoblasts
Deregulation of molecular hallmarks of the pathological cardiac
hypertrophy (upregulation of the fetal cardiomyocyte gene program
ANP, BNP, Acta1, and Myh7; and the downregulation of Myh6) was
measured in the H9c2 cardiomyoblasts transfected with miR-155 by
RT-qPCR (Fig. 2A). The miR-155 transfection did not alter the expres-
sion levels of the ANP and Myh6 transcripts (Fig. 2A); however, the
Myh7and BNP levels were decreased, and the Acta1 levels were in-
creased when compared to the control group (Fig. 2A). We also used a
3.3. Ogn alters the expression levels of ANP in H9c2 cardiomyoblasts
To verify whether Ogn knockdown induce a similar pattern in the
expression of cardiac stress markers as those found for the miR-155
mimic transfected cells, the transcript levels of ANP, BNP, Acta1, β-
MHC/Myh7, and α-MHC/Myh6 genes were measured in the H9c2
cardiomyoblasts by RT-qPCR (Fig. 3). Ogn knockdown only increased
ANP mRNA expression levels in H9c2 cardiomyoblasts when compared
to the control group (Fig. 3A). We also analyzed the MYH protein ex-
pression by western-blot in H9c2 cells treated with siRNAs for Ogn.
However, this treatment also did not alter the MYH protein levels
(Fig. 3B).
3.4. miR-155 and Ogn induces cellular architecture changes in H9c2
cardiomyoblasts
Although the changes in expression profile of the molecular markers
of pathological hypertrophy were different among H9c2 cardiomyo-
blasts treated with miR-155 mimic or siRNAs for Ogn, we clearly noted
that both treatments had a similar pattern of cardiomyoblasts pheno-
typic changes with the cells acquiring a rounded shape (Fig. 4A and B).
These phenotypic changes in the cellular architecture was further
confirmed by a morphometric analysis that showed a decreased cellular
length of the H9c2 cardiomyoblasts transfected with either miR-155 or
siRNA-Ogn when compared to the respectively control groups (Fig. 4C

11
G. de Oliveira et al.
Fig. 2. miR-155 alters the expression of cardiac stress markers in H9c2 cardi-
omyoblasts. Relative expression levels of mRNAs were measured by RT-qPCR
analysis in H9c2 cardiomyoblasts transfected with miR-155 or control (CT) (A).
MYH protein expression levels were measured by Western blot analysis (B). RT-
qPCR data were normalized by the expression level of the reference Ppib gene,
and western blot data were normalized by Gapdh content. Results are expressed
as mean ± SD of three independent biological replicates (n = 3). Statistical
difference was analyzed by Student's t-test. *p < 0.05 vs. Control (CT). ANP:
Atrial Natriuretic Peptide; BNP: Brain Natriuretic Peptide; Myh6: Myosin,
Heavy Chain 6, Cardiac Muscle, Alpha; Myh7: Myosin, Heavy Chain 7, Cardiac
Muscle, Beta; MYH: Myosin, Heavy Chains Isoforms; Acta1: Actin, Alpha 1,
Skeletal Muscle; Ppib: Peptidylprolyl isomerase B; Gapdh: glyceraldehyde-3-
phosphate dehydrogenase.
and D). Interestingly, treatment with siRNA for Ogn increases the width
of H9c2 cells, while treatment with miR-155 did not alter the width
(Fig. 4C and D).
4. Discussion
In the present study, we identified that, without a hypertrophic
stimulus, miR-155 significantly repressed Ogn protein levels, but in-
duce neither alteration in morphological phenotype nor in the expres-
sion of the molecular markers that fully characterize pathological hy-
pertrophy of H9c2 cardiac cells. However, most importantly, Ogn
silencing in cardiomyoblast mimicked the effects of miR-155 over-
expression in inducing cellular architecture changes that were char-
acterized by a transition of the cell shape from fusiform to rounded
(results are summarized in Supplementary Table 2).
It was unclear whether changes in miR-155 expression direct induce
cardiac hypertrophy or constitute a response to a diverse stimuli that
are related to the onset of cardiac hypertrophy (Fan et al., 2016;
Heymans et al., 2013; Seok et al., 2014). Therefore, we selected a
controlled cell culture system to transfect H9c2 cardiomyoblasts with
miR-155, without a hypertrophic stimulus, and analyzed the gene ex-
pression levels of the pathological cardiac hypertrophy markers. This
12
Gene 676 (2018) 9–15
Fig. 3. Ogn alters the expression levels of ANP in H9c2 cardiomyoblasts.
Relative expression levels of mRNAs were measured by RT-qPCR analysis in
H9c2 cardiomyoblasts transfected with miR-155 or control (CT) (A). MYH
protein expression levels were measured by Western blot analysis (B). RT-qPCR
data were normalized by the expression level of the reference Ppib gene, and
western blot data were normalized by Gapdh content. Results are expressed as
mean ± SD of three independent biological replicates (n = 3). Statistical dif-
ference was analyzed by Student's t-test. *p < 0.05 vs. Control (CT). ANP:
Atrial Natriuretic Peptide; BNP: Brain Natriuretic Peptide; Myh6: Myosin,
Heavy Chain 6, Cardiac Muscle, Alpha; Myh7: Myosin, Heavy Chain 7, Cardiac
Muscle, Beta; MYH: Myosin, Heavy Chain Isoforms; Acta1: Actin, Alpha 1,
Skeletal Muscle; Ppib: Peptidylprolyl isomerase B; Gapdh: glyceraldehyde-3-
phosphate dehydrogenase.
miR-155 overexpression decreased BNP and Myh7, increased skeletal
α-actin, and induced no changes in the expression levels of ANP, and
Myh6. Despite these changes in the cardiomyocyte molecular markers
expression induced by miR-155 do not characterize cardiac hyper-
trophy, we clearly noted a change in the cell morphology, characterized
by a transition of the cell shape from fusiform to rounded.
Although we had not found studies showing that, without a hy-
pertrophic stimulus, in vitro miR-155 overexpression direct induces
cardiomyocyte hypertrophy, the role of miR-155 in signaling pathways
and cellular responses triggered by hypertrophic stimuli is very de-
bated. Previous study found that the mRNA expression of ANP, BNP,
and Myh7 was markedly increased in cardiomyocytes from neonatal
mice hypertrophied with isoprenaline and transfected with miR-155
(Fan et al., 2016). Furthermore, cardiomyocyte hypertrophy induced by
pressure overload or by calcineurin transgene was reduced in miR-155-
knockout mice hearts (Seok et al., 2014). These authors also showed
that the hypertrophy growth induced in vitro by phenylephrine is sup-
pressed in cardiomyocytes isolated from neonatal miR-155-knockout
mice or in neonatal rat cardiomyocytes with endogenous inhibition of
miR-155. This diverges from (Heymans et al., 2013), who showed that
cardiomyocyte-specific miR-155 overexpression and knockdown do not
G. de Oliveira et al.
affect heart failure susceptibility. These authors further confirmed in
vitro that miR-155 knockdown in cardiomyocytes does not affect their
response to hypertrophic signaling. The disparities in the role of miR-
155 in cardiomyocytes hypertrophy are clearly associated with dif-
ferent “in vivo” and “in vitro” hypertrophic stimuli, which induce dis-
tinct phenotypes in cardiomyocytes (Schaub et al., 1997).
Moreover, each of these miR-155 studies described above validated
a new target transcript for this miRNA, which includes Jarid2/jumonji,
Socs1, and FoxO3a; all coding proteins that participate in the cardio-
myocyte hypertrophy process (Fan et al., 2016; Heymans et al., 2013;
Seok et al., 2014). These studies demonstrate the importance of the
miRNA miR-155 in this process and challenge the fully comprehension
of the effects of this miRNA in the hypertrophy phenotype at the
transcript level. Considering that we have recently demonstrated that
miR-155 inhibits Osteoglycin (Ogn) expression to regulate proliferation
and differentiation of C2C12 myoblast cells (Freire et al., 2017), we also
tested whether this new miR-155-Ogn has a role in cardiomyocyte
hypertrophy. Interestingly, Ogn is a secreted extracellular matrix (ECM)
component which belongs to a small leucine-rich proteoglycan family
and first described to be implicated in bone formation (Bentz et al.,
1990, 1989; Madisen et al., 1990). Ogn has also been involved in co-
chlear development (Williamson et al., 2008), pituitary tumor biology
(Hu et al., 2005), skin collagen fibrin abnormalities (Tasheva et al.,
2002), and corneal transparency (Scott and Haigh, 1988; Tasheva et al.,
2004). Most importantly, Ogn has been considered a key regulator of
heart mass in rats, mice, and humans, and a novel biomarker to predict
chronic heart failure outcomes and left ventricular remodeling
(Motiwala et al., 2014; Petretto et al., 2008; Van Aelst et al., 2015).
Noteworthy, Ogn silencing in cardiomyoblast mimicked the effects
of miR-155 overexpression in the cell morphology; in both conditions,
the cells presented similar cellular morphological changes, character-
ized by a transition of the cell shape from fusiform to rounded. We also
noted that cardiomyoblasts affected by RNAi-induced knockdown of
Ogn, except for the upregulation of ANP gene, did not change the ex-
pression of the molecular markers of pathological hypertrophy. These in
vitro results are in accordance with those observed in Ogn−/− knockout
mice, which had no difference in left ventricular mass without per-
missive physiological perturbations required to reveal cardiovascular
phenotypes (Petretto et al., 2008). These authors also observed that
Ogn+/− and Ogn−/− mice show a significant attenuation in left ven-
tricular mass as compared to Ogn+/+ controls after subcutaneous an-
giotensin II infusion as a pro-hypertrophic stimulus.
What caused the similar pattern of the cell shape transition from a
fusiform to rounded in cardiomyoblasts with silenced Ogn or
13
Gene 676 (2018) 9–15
Fig. 4. miR-155 and Ogn induces cellular phenotypic changes
in H9c2 cardiomyoblasts. Ogn immunostaining (green) in
H9c2 cells treated with miR-155 (A) or siRNA-Ogn (B) and
respective controls; Nuclei were stained in blue by DAPI.
Cellular architecture was evaluated by morphometric analysis
(cellular length and width) of the H9c2 cardiomyoblasts
treated with miR-155 (C) or siRNA-Ogn (D) and respective
controls. Results are expressed as mean ± SD of three in-
dependent biological replicates (n = 3). Statistical difference
was analyzed by Student's t-test. *p < 0.05 vs. Control (CT).
Scales bars 50 μm. Ogn: Osteoglycin; DAPI: 4′,6-Diamidino-2-
phenylindole dihydrochloride. (For interpretation of the re-
ferences to color in this figure legend, the reader is referred to
the web version of this article.)
overexpressing miR-155 remains to be determined; however, an Ogn/
miR-155 pathway regulating ECM organization may have been in-
volved. Beyond the involvement of miRNAs in cardiomyocytes re-
modeling, these small non-coding RNAs, including miR-155, control
extracellular matrix (ECM) production in the heart through direct ef-
fects on fibroblasts and indirectly by interfering with cardiomyocyte
paracrine biology (Duisters et al., 2009; He et al., 2015; Orenes-Piñero
et al., 2013; van Rooij and Olson, 2009). Interestingly, Ogn has been
causally linked to cardiac integrity and function by maintaining a well-
structured ECM; the absence of osteoglycin leads to an improper col-
lagen assembly with consequent increased infarct rupture and wall
thinning after myocardium infarction without affecting cardiac in-
flammation or cardiomyocyte hypertrophy (Van Aelst et al., 2015).
Moreover, in vitro cardiomyocytes respond to patterns in ECM compo-
nents by detecting and transducing phenotypic information stored
within the tertiary structure of the surrounding matrix (Simpson et al.,
1994). Previous in vitro analyses also showed that Ogn knockdown in-
duces cellular senescence and promotes migratory activity of cardiac
fibroblasts; these Ogn-mediated effects are mimicked by the miR-22, a
miRNA that also targets the Ogn transcript in cardiac fibroblast
(Jazbutyte et al., 2013). Thus, our data demonstrate the importance of
an Ogn/miR-155 pathway that may regulate a well-structured ECM to
maintain both the cellular architecture and functional integrity of the
cardiomyoblasts.
The study design described here has limitations that should be
considered. Specifically, the biological implications of one single
miRNA and its impact on signaling pathways cannot be determined
with confidence using only the tools described herein. Although the
functionality of one single miRNAs is measurable, the regulation of
targets by miRNAs is subject to different levels of control, and recent
advances have presented that targets can also reciprocally control the
level and function of miRNAs (Pasquinelli, 2012). Thus, whether this
miR-155-induced morphological changes reflects only a direct effect on
Ogn, or whether this is related to a direct or indirect modulation of
others transcriptional and posttranslational factors cannot be dis-
tinguished.
5. Conclusion
In conclusion, we found that in vitro miR-155 overexpression does
not direct induce H9c2 cardiomyoblasts hypertrophy but rather induces
cellular architecture changes by the repression of Ogn. These results
show that the new post-transcriptional regulation of Ogn by miR-155
has a potential role in the maintenance of the cardiac cell morphology
G. de Oliveira et al.
in physiological and pathological conditions.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.gene.2018.07.020.
Conflict of interest
The authors declare that there are no conflicts of interest.
Competing interests
The authors state that they have no conflicts of interest.
Author contributions
RFC, PPF and GO conceived and designed the project, with input
from MDPS, CSF, and ETK. GO conducted the cell culture experiments.
GO, PPF and SSC carried out immunofluorescence and morphometric
analyses. PPF, SSC, ACMO, GO, and RFC performed RT-qPCR experi-
ments and data analyses. GO, SSC and PPF carried western blots ana-
lysis. GO, PPF, and RFC performed statistical analyses. GO, PPF, SSC,
ACMO, and RFC performed study design, data analyses and data in-
terpretation. GO, RFC wrote the main manuscript, with contributions
from all authors. All authors critically discussed the results and im-
plications, reviewed and approved the final version of the manuscript.
Acknowledgements
This study was supported by São Paulo Research Foundation
(FAPESP, grant # 2012/13961-6, RFC). GO was funded by a MSc.
FAPESP fellowship # 2014/14340-0.
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