RESEARCH ARTICLE
MicroRNA-497 Inhibits Cardiac Hypertrophy
by Targeting Sirt4
Yimin Xiao
1,2
, Xiaofei Zhang
1,2
, Shihao Fan
1,2
, Guanghao Cui
1
, Zhenya Shen
1
*
1 Department of Cardiovascular Surgery of the First Affiliated Hospital & Institute for Cardiovascular Science,
Soochow University, Suzhou, China, 2 Department of Cardiovascular Surgery, Shanghai Yodak
Cardiothracic Hospital, Shanghai, China
*
uuzyshen@aliyun.com
Abstract
Cardiac hypertrophy is an adaptive enlargement of the myocardium in response to overload
pressure of heart. From abundant studies, a conclusion is drawn that many microRNAs
(miRNAs) are associated with cardiac hypertrophy and heart failure. To investigate the role
of microRNA-497 (miR-497) in myocardial hypertrophy, two models were established in this
study from cell level to integral level. Cardiac hypertrophy was induced by using angiotensin
II (Ang II) in vitro and was created by transverse abdominal aortic constriction (TAC) in vivo.
There was a significant decrease expression of miR-497 in cardiac hypertrophy models.
Moreover, overexpression of miR-497 inhibited myocardial hypertrophy both in vitro and in
vivo without heart function variation. In addition, luciferase reporter assays demonstrated
that Sirt4 was a direct target gene of miR-497. Taking together, our study indicates that
miR-497 modulates cardiac hypertrophy by targeting Sirt4 and may serve as a potential
therapeutic substance in the course.
Introduction
World Health Organization announced that a total number of 17,500,000 people die from
heart failure each year, which account for 30% of the death case [
1]. Cardiac hypertrophy is a
major determinant for the development of heart failure and is associated with a higher risk of
sudden cardiac death (SCD) [
2]. Though cardiac hypertrophy is not a disease in essence, it’s a
symptom of a serious condition which may place human’s life in great danger by evolving into
heart failure under sustainable high pressure.
MiRNAs are endogenous, single-stranded, short non-coding RNAs that act as regulators of
gene expression by promoting the degradation or inhibiting the translation of target mRNAs
[
3]. It is reported that miRNAs are involved in varieties of normal function of eukaryotic cells,
including proliferation, apoptosis, development and so on [4]. Relationships between miRNAs
dysregulation and human disease are investigated from variable disease. MiR-497, being one
of miR-15 family members, is mainly identified as a tumor suppressor in various cancers, such
as hepatocellular carcinoma [
5], osteosarcoma [6–7] and breast cancer [8]. Recently, miR-15
family members were identified as novel regulators in cardiac hypertrophy and fibrosis by
inhibition of the TGFβ pathway [
9]. However the exact function of miR-497 in cardiomyocytes
PLOS ONE | DOI:10.1371/journal.pone.0168078 December 16, 2016 1 / 10
a11111
OPEN ACCESS
Citation: Xiao Y, Zhang X, Fan S, Cui G, Shen Z
(2016) MicroRNA-497 Inhibits Cardiac
Hypertrophy by Targeting Sirt4. PLoS ONE 11(12):
e0168078. doi:10.1371/journal.pone.016807 8
Editor: Guo-Chang Fan, University of Cincinnati
College of Medicine, UNITED STATES
Received: October 8, 2016
Accepted: November 28, 2016
Published: December 16, 2016
Copyright: © 2016 Xiao et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the manuscript and supporting information
files.
Funding: The authors received no specific funding
for this work.
Competing Interests: The authors have declared
that no competing interests exist.
or heart remains uncertain. Our study is focused on the exact effect of miR-497 in cardiac
hypertrophy, which may offer a new outlook for human being on it.
In this study, our experimental data demonstrated a direct myocardial role for miR-497 as a
critical regulator in the development of cardiac hypertrophy and provided a useful model to
elucidate its mechanisms in the pathogenesis of cardiac hypertrophy. We offered further proof
for the first time that the overexpression of miR-497 significantly suppressed cardiac hypertro-
phy by inhibiting the expression of Sirt4. These findings disclosed a new connection between
Sirt4 and miR-497 in the development of cardiac hypertrophy and failure.
Materials and Methods
Primary cardiomyocyte isolation and culture
Neonatal mouse cardiomyocytes were separately isolated from the hearts of 1–2-day-old new-
born C57BL6 mice as described previously [
10]. Isolated cardiomyocytes were seeded in pre-
coated 24-well plates at 1.5×10
4
cells per well in B/D medium (Life Technologies, Foster City,
CA) containing 50% Dulbecco’s minimum essential medium (DMEM, Life Technologies)
together with 10% fetal calve serum (FCS, Hyclone, Logan, UT, USA). The cells were cultivated
in an incubator at 37˚C in a humid atmosphere consisting of 5% CO2 and the cultures were kept
in a semi-confluent condition to prevent cell from cellular differentiation. Furthermore, Bromo-
deoxyuridine of 0.1 mM was added to the culture media for the first 72 hours to further minimize
contamination from fibroblasts. The elimination of fibroblasts and other proliferations was per-
formed through preplating for 90 minutes at 37˚C to achieve the great purity of cardiomyocytes.
Animal model
All mice were purchased from Laboratory Animal Centre, Soochow University and were fed
on autoclaved food in the pathogen-free animal room with access to the reasonable and con-
trollable temperature, light and humidity. All experiments and animal care procedures were
approved by Ethics Review Board of Soochow University and done following institutional
guidelines. Firstly body weight was measured for individual mice. After mice were anesthe-
tized with ketamine (100 mg/kg), TAC was carried out to induce the cardiac hypertrophy of
mice. To be brief, a 4–0 suture was employed to tie two circles around the abdominal aorta by
21-gauge needle to decrease the outer aortic by 0.3 mm approximately, after which the needle
was eliminated, while the sham group was subjected to the same cutting and stitching course
as the experimental group in order to control variables. 8 weeks later, the mice were anesthe-
tized with ketamine, and the cardiac dimensions and function were analyzed with 7.5-MHz
pulse-wave Doppler echocardiography (Philips IE33). Then, the mice were killed by decapita-
tion, and hearts were gathered for further evaluation.
Histological analysis
First, the myocardial tissue samples were fixed by immersion in 4% paraformaldehyde for 24
to 48 h. Then, myocardial tissue was embedded in paraffin and stained with haematoxylin and
eosin (H&E). The cardiomyocyte and paraffin-embedded tissue sections were incubated with
a mouse monoclonal (α-actin) antibody (1:100, Abcam, LA, USA) for at 4˚C overnight. An
Alexa Fluor 594 goat anti-mouse antibody (1:200, Abcam, LA, USA) was used as a secondary
antibody. DNA in the nucleus of cardiomyocytes was stained with 0.025 μg/ml DAPI at room
temperature without any exposure to light. Finally, the slides were mounted with Vectashield
and 4’,6-diamidino-2-phenylindole mounting medium without being exposed to light at 4˚C
overnight, and fluorescently labelled cells were closely scrutinized using a fluoview 1000
MicroRNA-497 Suppresses Cardiac Hypertrophy
PLOS ONE | DOI:10.1371/journal.pone.0168078 December 16, 2016 2 / 10
confocal microscope (Olympus, Osaka, Japan). A random collection of 10 cardiomyocytes
images was created for the calculation of cardiomyocyte area using Image J software. Quantifi-
cation of fluorescence intensities was carried out using MetaMorph software (Boyce Scientific,
St. Louis, MO).
Recombinant lentivirus construction and infection
Both the primary miR-497 and negative control sequence were synthesized by Genepharma
(Shanghai, China) inserted into the Pglv3/h1/GFP plasmid vector (Genepharma, Shanghai,
China) to construct specific miRNA-overexpressing lentivirus. The negative control sequence
is a random sequence that has been extensively tested in mouse cell lines and tissues and vali-
dated to not produce identifiable effects on known miRNA function. The pseudoviral particles
were produced using a lentivector packaging system (Genepharma) according to the manufac-
turer’s instructions. Cells were infected with the miR-497 lentivirus (Leti-miR-497) or negative
control lentivirus (Leti-NC) at an MOI of 50. TAC mice were subject to chest reopening with
injection of indicated lentivirus (3.5×10
7
viral particles per mice). The infection efficiency was
evaluated by observing GFP expression under a fluorescence microscope after 48 h.
Real-time quantitative PCR (qRT-PCR) analysis
Total RNA from cardiomyocytes and myocardial tissue was isolated and obtained using Trizol
(Invitrogen, Carlsbad, CA). Briefly, 2 μg of extracted RNA was reverse transcribed to cDNA
with the application of reverse transcriptase (Takara, Tokyo, Japan). The expression levels
were quantitatively analysed using a standard SYBR green PCR kit (Promega, Madison, WI,
USA). GAPDH was used as an internal control. The primer sequences were as follows: MHC,
forward: 5’-CCTCGCAATATCAAG GGAAA-3’, reverse: 5’-TACAGGTGCATCAGCTCCA
G-3’; ANP, forward: 5-GGGGGTAGGATTG ACAGGAT -3’, reverse: 5’-CTCCAGGAGGGT
ATTCACCA-3’; GAPDH, forward: 5’-AAGAAGGTG GTGAAGCAGGC-3’, reverse: 5’-TCC
ACCACCCAGTTGCTGTA-3’.
MiRNAs were quantified by using TaqMan MicroRNA Assays (Applied Biosystems, Foster
City, CA). The first strand cDNA synthesis of each miRNA was performed through reverse
transcription with use of TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems).
U6 was applied as the internal control for target genes. The performance of analysis was per-
formed by 2
−ΔΔCT
method.
Tritium-leucine incorporation assay
Neonatal cardiomyocytes were cultured on 24-well plates at a density of 4×10
5
cells/ml. After
the incubation with Ang II, the cardiomyocytes were treated with 37 kBq tritium-leucine
(China Institute of Atomic Energy, Beijing, China) with a final concentration of 3.7×10
4
Bq/ml
for 48 h. The incubation was finished by putting 100 μl of 50% TCA into the plates, followed
by measurement of the incorporated tritium-leucine with the application of Packard Tri-Carb
2100TR Liquid Scintillation Analyzer (GMI, Inc, Ramsey, MN, USA).
Western blot
Cardiomyocytes were collected and lysed after which equivalent quantity of protein was loaded
and separated by 10% SDS-PAGE. Then all the separated protein was electrotransferred onto
polyvinylidene fluoride (PVDF) membranes. The membrane was blocked by 5% skim milk for
1h, incubated with primary antibodies (Sirt4, 1:1000, Santa Cruz Biotechnology, Inc., Dallas,
Texas, USA; GAPDH, 1:2000, Cell Signaling Technology, Boston, USA) at 4˚C overnight.
MicroRNA-497 Suppresses Cardiac Hypertrophy
PLOS ONE | DOI:10.1371/journal.pone.0168078 December 16, 2016 3 / 10
Afterwards horseradish peroxidase-conjugated secondary antibody (1:10,000, Abcam, LA,
USA) was also incubated on the membrane at 37˚C for 1 h. Proteins were scanned and
detected by enhanced chemiluminescence (Bio-Rad Laboratories, Hercules, CA, USA) using a
ChemiDoc MP system (Bio-Rad Laboratories). Image J software was used to analyze densito-
metric results of band.
Plasmid construction
To construct a luciferase reporter vector, 3’-untranslated region (3’UTR) of Sirt4 was synthe-
sized by PCR with the involvement of restriction enzyme cutting site. For sequence point
mutation, site-directed mutagenesis of potential target site in the Sirt4 3’UTR was performed
using a QuikChange Site-Directed Mutagenesis kit (Promega, Madison, WI, USA). The wild-
type and mutant 3’UTR of Sirt4 were cloned into downstream of the luciferase open reading
frame in the pMIR-report vector (Ambion, Carlsbad, CA, USA).
Luciferase reporter assays
For luciferase assays, primary cardiomyocytes were infected recombinant lentivirus
(MOI = 50) for 48 h, then transfected with 2 μg of the 3’UTR-luciferase reporter vector using
Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) in 12-well plates.
A quantity of 2 μg of pMIR vector plasmid was put into the well at the same time as a standard
control. Cells were gathered after the 72 h-long incubation for the measurement of luciferase
activities with the application of the Dual-Luciferase Reporter Assay System (Beyotime Bio-
technology, Beijing, China) according to the manufacturer’s protocol.
Statistical analysis
All experiment data were analyzed statistically by SPSS 13.0 software (SPSS, Inc, Chicago, IL,
USA) and were expressed as mean ± standard deviation. Comparison of parameters between
two groups was performed by Student’s t test (where distributions were normal) or Mann-
Whitney U test (where distributions were significantly skewed). P<0.05 indicated that differ-
ences were statistically allowable.
Results
Down-regulation of miR-497 in cardiac hypertrophy
To identify the expression of miR-497 in cardiac hypertrophy, we established Ang II-induced
primary cardiomyocytes hypertrophy and TAC mice model. The levels of ANP and β-MHC
were used to assess the extent of myocardial hypertrophy. The relative cell area increased sig-
nificantly in response to the treatment with Ang II (
Fig 1A and 1B), and the expression levels
of ANP and β-MHC were up-regulated obviously compared with control group (
Fig 1C). Simi-
larly, cardiac hypertrophy was further identified by the elevated ratio of heart weight to tibial
length (HW/TL), the cell area and ANP and β-MHC levels in the TAC group (
Fig 1E–1H). In
addition, transthoracic echocardiography revealed that the interventricular septal end-diastolic
thickness (IVSd), interventricular septal end-systolic thickness (IVSs), left ventricular posterior
wall end-diastolic (LVPWd) and end-systolic thickness (LVPWs) markedly elevated in the
TAC group compared with the sham group, while left ventricular end-diastolic dimension
(LVIDd) and leftventricular end-systolic dimension (LVIDs) was significant reduced in
TAC group. The fractional shortening (FS) and ejection fraction (EF) revealed no difference
between the two groups (
Table 1). The expression of miR-497 was significantly reduced in
Ang II-induced cardiomyocytes and TAC mice (Fig 1D and 1I).
MicroRNA-497 Suppresses Cardiac Hypertrophy
PLOS ONE | DOI:10.1371/journal.pone.0168078 December 16, 2016 4 / 10
miR-497 suppresses cardiac hypertrophy in vitro and in vivo
To explore the role of miR-497 in cardiac hypertrophy, we generated a miR-497-overexpres-
sing recombinant lentivirus (Leti-miR-497). 48 h after infection, the level of miR-497 was sig-
nificantly enhanced in cardiomyocytes (
Fig 2A and 2B). Overexpression of miR-497 obviously
inhibited Ang II-induced cardiomyocytes hypertrophy (cell area, ANP and β-MHC levels) in
Leti-miR-497 group compared with control group (
Fig 2C–2E). Cardiomyocyte protein syn-
thesis was determined by tritium-leucine incorporation. The tritium-leucine incorporation
assay showed that overexpression of miR-497 reversed Ang II-induced cardiomyocytes protein
synthesis (Fig 2F). Similarly, in order to identify the role of miR-497 in cardiac hypertrophy
in vivo, we infected Leti-miR-497 and control adenovirus in TAC mice. 48 h after infection,
the level of miR-497 was significantly enhanced in cardiomyocytes of TAC mice (
Fig 3A and
3B
). Our result showed that cardiac hypertrophy (ratio of HW/TL, and cell area, ANP and
β-MHC levels) was observably reversed after being infected with Leti-miR-497 (
Fig 3C–3F).
Transthoracic echocardiography demonstrated that overexpression of miR-497 decreased
Fig 1. miR-497 is down-regulated in hypertrophic cardiomyocytes. Establishment of hypertrophic cardiomyocytes treated with Ang II for
48h. a: Immunofluorescence assay of α-actin was performed to identify cells. b: Relative cell area of cardiomyocytes detected by randomly
measuring 10 cells from each section. c: ANP and β-MHC mRNA expression measured by qRT-PCR. d: miR-497 determined by qRT-PCR. e:
Establishment of a mouse model of TAC-induced hypertrophy and histological analysis of hearts from different groups using H&E staining. f: The
ratio of heart weight to tibial length. g: Relative cell area of cardiomyocytes detected by randomly measuring 10 cells from each section. h: ANP and
β-MHC mRNA expression measured by qRT-PCR assay. i: miR-497 determined by qRT-PCR. Data are presented as the mean ± SD; *, P < 0.05,
n = 5.
doi:10.1371/journal.pone.0168078.g001
MicroRNA-497 Suppresses Cardiac Hypertrophy
PLOS ONE | DOI:10.1371/journal.pone.0168078 December 16, 2016 5 / 10