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The MicroRNA-210/Casp8ap2 Pathway
Alleviates Hypoxia-Induced Injury in
Myocardial Cells by Regulating Apoptosis
Kunsheng Li1,2, Jun Pan1, Qiuchang Li3, Shiliang Li6, Kai Li1, Yongqing Cheng1, Lin Chai1, Chao Li4, Junling Li3, Zhikun Fu5, Dongjin Wang1* and Yang Bai7*
1Department of Cardiothoracic Surgery, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, 321 Zhongshan Road, Nanjing 210008, Jiangsu Province, P.R. China
2Department of Cardiac Surgery, University of Heidelberg, 69120 Heidelberg, Germany
3Puyang Medical College, Shangyang Road and Wenyan Street, Puyang 457000, Henan Province, P.R. China
4School of life sciences, Anhui Medical University, 81 Meishan Road, Hefei 230000, Anhui Province, P.R. China
5Department of Cardiac Surgery, The 7th people’s hospital of Zhengzhou, 17 Jingnan wu Road, Zhengzhou 450000, Henan Province, P.R. China
6Department of Cardiac Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, P.R. China
7Division of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, P.R. China
Submission: June 26, 2020; Published: July 21, 2020
*Corresponding author: Dr. Yang Bai, Division of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jiefang Road, No 1095, Wuhan 430030, Hubei Province, P.R. China
Dr. Dongjin Wang, Department of Cardiothoracic Surgery, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, 321 Zhongshan Road, Nanjing 210008, Jiangsu Province, P.R. China/p>
How to cite this article:Kunsheng L, Jun P, Qiuchang L, Shiliang L, Kai L, et al. The MicroRNA-210/Casp8ap2 Pathway Alleviates Hypoxia-Induced Injury in
Myocardial Cells by Regulating Apoptosis and Autophagy. J Cardiol & Cardiovasc Ther. 2020; 16(3): 555936. DOI: 10.19080/JOCCT.2020.16.555937
Aim: This study was conducted to investigate the role of the miR-210/Caspase8ap2 pathway in apoptosis and autophagy in hypoxic myocardial cells.
Methods: The miR-control, miR-210 mimic and miR-210 inhibitor were transfected into rat myocardial H9C2 cells. The transfection efficiency of exogenous miR-210 was determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). H9C2 cells were then treated with CoCl2 for 24, 48 and 72 h to generate a myocardial injury model. The apoptosis of H9C2 cells was assessed by flow cytometry. Additionally, a western blot assay was used to determine the expression of the autophagy-associated proteins light chain 3 (LC3), p62 and Beclin-1, and apoptosis-associated proteins Caspase8ap2, cleaved caspase 8 and cleaved caspase 3.
Results: We determined that a 48 h hypoxia treatment duration in H9C2 cardiomyocytes induced myocardial injury. Additionally, the overexpression of miR-210 significantly inhibited cell apoptosis. MiR-210 suppressed autophagy by upregulating p62 and downregulating LC3II/I in hypoxic H9C2 cells. Since Caspase8ap2 was a putative target of miR-210, miR-210 mediated apoptosis and autophagy of H9C2 cells via suppressing Caspase8ap2. Furthermore, the expression of caspase 8, caspase 3 and Beclin-1 were decreased in response to miR-210.
Conclusions:miR-210 exhibits anti-apoptosis and anti-autophagy effects, which alleviate myocardial injury in response to hypoxia.
Coronary heart disease (CHD) is characterized by myocardial ischemia, anoxia and necrosis attributed to coronary
atherosclerosis, which results in heart defects. CHD is a leading cause of death and disability, with a 10-year mortality rise of 60% in China [1,2]. This devastating rise is mainly due to adverse trends in risk factors, such as blood pressure, cholesterol and
diabetes . Identifying mechanisms to inhibit myocardial cell
apoptosis under a hypoxic-ischemic state is a recent topic of high
interest in CHD research.
Many studies have shown that microRNAs (miRNAs), an
abundant class of endogenic non-coding RNAs with approximately
22 nucleotides, are involved in a wide variety of biological
processes by modulating the expression of target genes and
pathways [4,5]. For CHD, an increasing number of miRNAs have
been identified that play considerable roles in CHD development,
such as miR-320b , miR-224 , miR-423, miR-208a and miR-1
Among various miRNAs, miR-210 plays a significant role
in the hypoxia response on the account of its high sensitivity to
hypoxia. Recent evidence has emerged that suggests miR-210,
as the main hypoxia-induced miRNA, has pivotal implications in
cell survival and angiogenesis . During hypoxia stress, miR-
210 was potently upregulated in myocardial cells in vitro .
In addition, targeted miR-210 could effectively protect the heart
from myocardial infarctions and promote functional recovery
. However, little is known about the mechanism of miR-210 in
ischemia-hypoxia in myocardial cells.
Caspase-8-associated protein-2 (Caspase8ap2) consists of
1926 amino acids and is an integral member of the apoptosis
signalling complex that activates caspase-8 and downstream
caspase 3 by combining with the death receptor of procaspase-8
. Thus, Caspase8ap2 is considered a pro-apoptosis protein.
Kim et al.  reported that the apoptosis in mesenchymal
stem cells (MSCs) caused by ischemic preconditioning (IP) was
suppressed by miR-210 by repressing Caspase8ap2 .
The present study was conducted to investigate the role of the
miR-210/Caspase8ap2 pathway in apoptosis and autophagy in
hypoxic myocardial cells. The findings of this study can potentially
provide novel avenues for the treatment of myocardial injury
induced by hypoxia.
The rat myocardial cell line H9C2 (American Type Culture
Collection, Manassas, USA) was cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM, Gibco-BRL, Grand Island, USA)
supplemented with 10% fetal bovine serum (FBS; HyClone,
Logan, USA). All cells were maintained in a humidified incubator
containing 95% air and 5% CO2 at 37°C. Cells were cultured to
70-80% confluence, which was determined using a phase contrast
microscopy (Olympus, Tokyo, Japan). After reaching 70%-80%
confluence, H9C2 cells were treated with 400 μM CoCl2 for 24, 48
and 72 h.
Hypoxic H9C2 cells were seeded at a density of 3 × 104 cells/
well in six-well plates and allowed to adhere to the dish overnight.
The next day, the miR-negative control (NC), miR-210 mimic
and miR-210 inhibitor (GenePharma Co, Shanghai, China) were
transfected into H9C2 cells using Lipofectamine 2000 reagent (Life
Technologies, Gaithersburg, USA) according to manufacturer’s
Total RNA was extracted from normal H9C2 cells, hypoxic H9C2
cells, NC, miR-210 mimic and miR-210 inhibitor group with TRIzol
reagent (Invitrogen, Carlsbad, USA) following the manufacturer’s
instructions. Briefly, single-stranded complementary DNA (cDNA)
was synthesized from 2 μg of RNA via the RevertAid First Strand
cDNA Synthesis Kit (Fermentas, Ontario, Canada). Subsequently,
the generated miR-210 cDNA was amplified via qRT-PCR based
on the TaqMan microRNA assay protocol (Roche LightCycler 480
II System). Briefly, 20 μL reactions were incubated in a 96-well
optical plate at 94°C for 2 min and then subjected to 40 cycles of
94°C for 20 s and 60°C for 34 s. The relative expression of miR-
210 was normalized to the internal control (U6) using the 2–ΔΔCt
method. The primers for miR-210 and U6 were synthesized with
the miScript Primer Assay kit (Qiagen, Dusseldorf, Germany).
Apoptotic cells in NC, miR-210 mimic and miR-210 inhibitor
groups were quantified using the Annexin V-FITC apoptosis
detection kit (Beyotime Institute of Biotechnology, Jiangsu, China).
Both the attached cells and cells in the supernatant were collected
from the above four groups and fixed with 2% paraformaldehyde.
They were washed twice with cold phosphate-buffered saline
(PBS) and resuspended in 100 μl of the annexin-binding buffer.
Then, H9C2 cells were incubated with 2.5 μl of Annexin V for 15
min and analysed using an EPICS XL flow cytometer (Beckman-
Coulter, Fullerton, USA). Apoptotic cells were defined as the cells
that belonged to the right quadrant (right top + right bottom) in
flow cytometry plots. The experiment was independently repeated
three times, and an average apoptotic rate was calculated.
Standard western blotting was conducted for protein
expression assays in hypoxic H9C2 cells transfected with miR-
210 mimic, inhibitor and NC. Two days following transfection,
proteins were isolated with RIPA lysis buffer containing 1 mg
protease inhibitors (Applygen Technologies Inc., Beijing, China).
The protein contents were quantified using the Bicinchoninic
Acid (BCA) Protein Assay Kit (CoWin Biotech Co., Ltd., Beijing,
China). Primary antibodies against light chain 3 (LC3; ab48394),
p62 (ab56416), Caspase8ap2 (ab4052), cleaved caspase 8
(ab25901), cleaved caspase 3 (ab13847), Beclin-1 (ab62557),
and the internal controls b-actin and GAPDH were from Abcam
(Cambridge, UK). Furthermore, secondary antibodies labelled
with horseradish peroxidase were incubated with membranes for
1 h at 37°C. Samples were then transferred to a polyvinylidene
fluoride (PVDF) microporous membrane (Millipore, Boston, USA). Blots were imaged by a FluorChem E imager (Cell Biosciences,
San Jose, USA). The grey value of each band was determined with
ImageJ version 1.51.
For transfections, host H9C2 cells were plated in triplicate
into 24-well plates containing Lipofectamine 2000 (Invitrogen,
Carlsbad, USA) and 0.8 μg pEZX-miR-210 (or pEZX-miR-Sc) per
well. The precursor miR-210 expression clone was established
via a feline immunodeficiency virus using the lentiviral vector
system (pEZX-miR-210). Subsequently, we designed the luciferase
reporter targeting the 3-UTR of Caspase8ap2 to surround the
binding sites of miR-210. The Dual-Luciferase Reporter Assay
System kit (Promega, Madison, USA) was used to measure
the amount of luciferase and Renilla luciferase activity with a
fluorescence spectrophotometer. The relative transcriptional
activity was normalized to its corresponding value.
All data were expressed as the mean ± standard deviation
(SD). Statistical differences were evaluated by SPSS 19.0 software.
Statistical analysis was performed using a one-way analysis of
variance and Student’s t-test, and a P-value < 0.05 was considered
as statistically significant.
To generate an optimized model of anoxic myocardial cells,
H9C2 cells were treated with CoCl2 for 24, 48 and 72 h. The number
of apoptotic cells and relative miR-210 expression are shown in
Figure 1. With increasing CoCl2 concentrations and treatment
duration, the apoptosis ratio of H9C2 cells was enhanced (Figure
1A). Additionally, the relative expression of miR-210 increased
in a time-dependent manner (Figure 1B). We found that a 24 h
and 48 h hypoxia treatment provoked similar apoptosis ratios in
H9C2 cells. Therefore, a 48 h hypoxia duration was used in H9C2
cardiomyocytes for all subsequent experiments.
We evaluated the effects of miR-210 overexpression on cell
apoptosis in hypoxic cardiomyocytes by generating NC, miR-210
mimic and miR-210 inhibitor groups under hypoxia culture for
48 h. The relative expression of miR-210 was potently enhanced
in the miR-210 mimic group but was suppressed in the miR-
210 inhibitor group, which demonstrated the high transfection
efficiency in H9C2 cells (Figure 2A). Moreover, there was no
significant difference in the apoptosis ratio between hypoxia and
NC groups (data not shown). Overexpressing miR-210 caused a
decline in apoptosis, whereas silencing miR-210 had the opposite
effect. It was uncovered that overexpressing miR-210 inhibited
cell apoptosis in hypoxic H9C2 cells (Figure 2B and C).
Next, the expression of autophagy-associated proteins LC3
and p62 were assessed by western blot to investigate the effects
of miR-210 overexpression on autophagy in hypoxic H9C2 cells.
As shown in Figure 3, LC3 II/I was upregulated and p62 was
downregulated in the hypoxia group compared with the normoxia
group, which demonstrated that there was a certain degree of
autophagy in H9C2 cells. Furthermore, the expression of LC3 II/I
was significantly lower, and the expression of p62 was higher in
the miR-210 mimic group compared with other groups, which
indicated that miR-210 overexpression inhibited autophagy in
H9C2 cells under hypoxic conditions.
To explore whether Caspase8ap2 was a target gene of miR-
210, we performed a dual-luciferase reporter gene assay in H9C2
cells. Our computational studies identified consensus putative
target sites of miR-210 with high complementarity relevant to
the 3’UTR region of Caspase8ap2 (Figure 4A). For the luciferase
reporter assay, a dual-luciferase construct was generated. Remarkably decreased luciferase activity was observed on
pEZX-miR-201 co-transfecting with the pEZX-Luc-Caspase8ap2
3’UTR (Figure 4B; P < 0.01). These results showed that miR-210
could inhibit the activity of luciferase on the Caspase8ap2 3’UTR
reporter gene cattier.
To further investigate the mechanism of miR-210 in
regulating Caspase8ap2 and its associated signalling pathway, we
determined the effects of exogenous miR-210 on the expression
of Caspase8ap2 and downstream associated proteins by western
blot (Figure 5A). Beclin-1 was upregulated in the hypoxia
group compared with the normoxia group. Protein levels of
Caspase8ap2, cleaved caspase 8 and cleaved caspase 3 were
decreased in response to overexpressing miR-210 (Figure 5B and
C). Beclin-1, which regulates both apoptosis and autophagy, was
also downregulated in the miR-210 mimic group (Figure 5D). On
the other hand, the expression of Caspase8ap2 and other proteins
in response to silencing miR-210 were increased. Therefore, miR-
210 likely mediates apoptosis and autophagy in H9C2 cells via
targeted-regulation of Caspase8ap2.
Myocardial hypoxia is a negative prognostic marker
associated with myocardial injury and a poor patient outcome.
It is also a primary component of CHD microenvironment .
Recent work supports the idea that miRNAs are involved in the
adaptive response to myocardial hypoxia in cardiovascular
diseases. Liu et al.  suggested that miR-130a participated in
hypoxia/reoxygenation (H/R) induced injuries by accelerating
autophagy and relieving apoptosis in cardiomyocytes .
Moreover, the silencing of miR-122 alleviated cardiomyocyte
H/R injury by promoting GATA-4 expression . Li et al. 
reported that miR-7a/b protected H9C2 cells from hypoxiainduced
injury and promoted cardiac remodelling by decreasing
fibrosis and apoptosis . In particular, for the hypoxic response,
all published literature agrees that miR-210 is a robust target of
hypoxia-inducible factors. It is well known that the upregulation
of miR-210 is a hypoxic signature in vivo . Hence, miR-210- mediated myocardial hypoxia may represent an attractive target
for therapeutic intervention in CHD. However, the mechanisms
involved in miR-210 repair of myocardial hypoxia remains to be
elucidated. Our study was performed to identify the role of miR-
210 in a hypoxic H9C2 model, which might contribute to the
development of potential diagnostic and therapeutic approaches
in CHD. First, we determined that a 48 h hypoxia treatment
duration in H9C2 cardiomyocytes induced myocardial injury. Also,
miR-210 significantly inhibited cell apoptosis and autophagy in
hypoxic H9C2 cells. Since Caspase8ap2 was a putative target of
miR-210, miR-210 likely mediated apoptosis and autophagy of
H9C2 cells via suppressing Caspase8ap2. Taken together, the miR-
210/Caspase8ap2 pathway alleviated the effects of CoCl2 induced
injury in H9C2 cells by regulating apoptosis and autophagy. There
are few published articles on the effects of miR-210 on the other
two apoptotic signaling pathways in cardiomyocytes. Besides
of death receptor/ Caspase8 pathway, mitochondria/Caspase9
and ER stress/Caspase12 pathways are also major signaling
pathways in apoptosis [18,19]. Further studies on the association
between miR-210 and other potential target apoptotic genes in
cardiomyocytes with hypoxia need to be conducted.
In the miRNA field, extensive evidence consistently points
to a dominant role for miR-210 in the hypoxic stress response
. The induction of apoptosis in cells subjected to hypoxia has
been of great importance to cell survival and tissue homeostasis.
Numerous previous reports have supported an anti-apoptotic
role of miR-210 during hypoxia. For example, miR-210 exhibited
cytoprotective effects from apoptosis [10,21], while the
downregulation of miR-210 induced apoptosis [22,23]. Similar
results were verified in this study in which the overexpression
of miR-210 inhibited apoptosis, whereas silencing miR-210
strikingly enhanced apoptosis in hypoxic H9C2 cells. Further,
Caspase8ap2 has been predicted as a downstream target of miR-
210 . Caspase8ap2 was initially identified as a member of
the death signalling complex joining Fas and tumour necrosis
factor-α (TNF-α) during apoptosis . Its interaction with
death effectors promotes the delivery of caspase-8 from the
apoptosis pathway to activate the downstream caspase cascade.
Because of its role in apoptosis, we validated Caspase8ap2 for
its functional involvement in H9C2 apoptosis under hypoxic
conditions using the dual-luciferase reporter assay. Western blot
analysis demonstrated that miR-210 overexpression reduced
the protein level of Caspase8ap2, cleaved caspase 8 and caspase
3 upon exposure to hypoxia, suggesting that miR-210 inhibited
cardiomyocyte apoptosis by modulating the levels of Caspase8ap2.
The double-edged sword biological function of autophagy in
myocardial hypoxia injury was demonstrated by recent studies.
Moderate autophagy contributes to ATP generation and cellular
homeostasis when cells are exposed to non-serious injury.
However, uncontrolled excessive autophagy could be induced by
serious or long-term injury. Excessive autophagy may contribute
to autophagic cardiomyocyte death [25,26]. In our study, CoCl2
treatment for 48 h significantly inhibited cell survival. Hence, under
this serious injury condition, we assumed that excessive autophagy
was induced, and it may have contributed to cell death. Autophagy
and apoptosis are considered to be highly interconnected through
mechanisms that involve Beclin-1 . Beclin-1 is necessary for
autophagy to occur, it can direct autophagy-related proteins to
phagocytes, and can regulate autophagy formation and maturation
with other proteins. Although it is widely accepted that autophagy
can promote survival in response to milder stress, excessive and/
or long-term autophagy can also cause cell death by promoting
the excessive self-digestion of essential organelles and proteins.
In the current study, both the upregulation of adapter protein p62
and downregulation of autophagy indicators LC3 II/I and Beclin-1
showed that miR-210 may protect H9C2 cells from excessive
autophagy in response to hypoxia.
In conclusion, miR-210 collectively exerts anti-apoptosis
and anti-autophagy effects, which alleviates myocardial injury
in response to hypoxia. This study suggests that the miR-210/
Caspase8ap2 pathway is essential to protect cells from hypoxia.
Future studies aimed at further characterizing the underlying
mechanism of miR-210 in other apoptosis-associated pathways
should be conducted.
We would like to give our gratitude sincere to the reviewers for
their helpful comments on this paper. This study was supported
by The National Key Clinical Speciality Construction Project of