- Email: [email protected]
Akt activation by targeting PDCD4
Journal Pre-proof MiR-340-5p alleviates oxygen-glucose deprivation/reoxygenation-induced neuronal injury via PI3K/Akt activation by targeting PDCD4 Yake Zheng, Peng Zhao, Yajun Lian, Shuang Li, Yuan Chen, Lihao Li PII:
S0197-0186(19)30441-3
DOI:
https://doi.org/10.1016/j.neuint.2019.104650
Reference:
NCI 104650
To appear in:
Neurochemistry International
Received Date: 8 August 2019 Revised Date:
28 November 2019
Accepted Date: 19 December 2019
Please cite this article as: Zheng, Y., Zhao, P., Lian, Y., Li, S., Chen, Y., Li, L., MiR-340-5p alleviates oxygen-glucose deprivation/reoxygenation-induced neuronal injury via PI3K/Akt activation by targeting PDCD4, Neurochemistry International (2020), doi: https://doi.org/10.1016/j.neuint.2019.104650. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
MiR-340-5p
alleviates
oxygen-glucose
deprivation/reoxygenation-induced
2
neuronal injury via PI3K/Akt activation by targeting PDCD4
3
Yake Zheng1, Peng Zhao2, Yajun Lian1,*, Shuang Li1, Yuan Chen1, Lihao Li1
4
1
5
Zhengzhou, China, 450052;
6
2
7
China, 450004;
8
* Corresponding author: Yajun Lian
9
Jianshe East Road, The First Affiliated Hospital of Zhengzhou University, Zhengzhou,
Department of Neurology, The First Affiliated Hospital of Zhengzhou University,
Department of Neurology, The First People’s Hospital of Zhengzhou, Zhengzhou,
10
China, 450052;
11
Email: [email protected]
12
Tel: 13838367143
13
1
1
Abstract
2
MicroRNA-340-5p (miR-340-5p), a suppressor of certain target genes in brain,
3
reportedly is decreased in peripheral circulation of acute stroke patients. However,
4
little is known regarding its role in regulating cerebral ischemia/reperfusion injury.
5
This study explores the effect of miR-340-5p on ischemia/reperfusion insults by
6
exposing rat hippocampal neurons to oxygen-glucose deprivation/reoxygenation
7
(OGDR) in vitro. We found miR-340-5p to be poorly expressed in these neurons after
8
OGDR stimulation. OGDR stimulation decreased cell viability, increased lactate
9
dehydrogenase (LDH) activity and cell apoptosis, all of which were significantly
10
inhibited by miR-340-5p overexpression and enhanced by miR-340-5p inhibition.
11
Using bioinformatics analysis, we identified mRNA encoding the pro-apoptotic factor,
12
programmed cell death 4 (PDCD4) as a putative target of miR-340-5p. A
13
dual-luciferase reporter assay suggested that miR-340-5p targeted the 3’-UTR of
14
PDCD4. PDCD4 was upregulated in cells exposed to OGDR, and miR-340-5p
15
negatively modulated expression of PDCD4. PDCD4 overexpression partly reversed
16
the neuroprotective effect of miR-340-5p during OGDR-induced injury. MiR-340-5p
17
overexpression significantly promoted the activation of PI3K/Akt signaling pathway
18
(P<0.05) in OGDR-exposed cells, and PDCD4 overexpression attenuated this effect
19
(P<0.05). Collectively, our results indicate that miR-340-5p might exerted
20
neuroprotective effects during OGDR injury by targeting PDCD4 and then activating
21
the PI3K/Akt pathway. These results indicated a novel target for treating cerebral
22
ischemic injury.
23
Keywords: miR-340-5p, oxygen glucose deprivation/reoxygenation (OGDR),
24
neuroprotection, PDCD4, PI3K/Akt
25 26
Abbreviations
27
miRNAs, microRNAs; IR, ischemia/reperfusion; PDCD4, programmed cell death 4;
28
OGDR, oxygen-glucose deprivation/reoxygenation; FBS, fetal bovine serum;
29
HEK293, human embryonic kidney 293 cells; LDH, lactate dehydrogenase; PI,
30
propidium iodide; PVDF, polyvinylidene difluoride 2
1
Introduction
2
Ischemic stroke is a brain disease caused by temporary cessation or insufficient
3
blood flow to brain. It is associated with high disability and mortality rates(Stegner et
4
al., 2019). The subsequent reperfusion and reoxygenation of blood can further
5
aggravate tissue injury and cell death(Galkin, 2019). Due to its high disability and
6
mortality, ischemic stroke brings heavy burden to the patients and the society(Patel
7
and McMullen, 2017). Understanding the complex pathophysiological mechanism
8
underlying stroke-related neuron damage may help identify effective treatments for
9
ischemic stroke.
10
MicroRNAs (miRNAs) are small (18–28 nucleotides), noncoding RNAs that
11
negatively modulate expression of their target genes by binding to the 3’-UTRs of
12
target mRNA. MiRNAs help modulate pathophysiological processes in cerebral
13
ischemia/reperfusion (IR) injury. For example, miR-34b targets Keap1 to protect
14
against focal cerebral IR injury in rats with middle cerebral artery occlusion (Huang et
15
al., 2019). MiR-424 inhibits oxidative stress to ameliorate focal cerebral IR injury in
16
mice (Liu et al., 2015). MiR-99a reduces cerebral IR-induced neuronal damage by
17
regulating the cell cycle and apoptosis (Tao et al., 2015). MiR-200a silencing protects
18
neural stem cells during cerebral IR injury (Ma et al., 2017). Ou et al. reports that
19
miR-375 exhibits protective effects against cerebral IR injury by targeting Ctgf (Ou et
20
al., 2017). Additionally, miR-182-5p mitigates neuronal injury after cerebral IR injury
21
by targeting Toll-like receptor 4 (Wang et al., 2018). In rats with sciatic nerve crush
22
injury, miR-340 dysregulation occurs in the injured nerves, which affects the removal
23
of cell debris and axon regeneration (Li et al., 2017). Furthermore, miR-340-5p is
24
downregulated in the peripheral blood of patients with acute stroke (Yoo et al., 2018).
25
These findings suggest that miRNAs are potential targets for cerebral IR injury.
26
However, the specific function of miR-340-5p in cerebral IR-induced neural injury
27
remains unknown.
28
Apoptosis is a critical mechanism in cerebral IR injury, and the programmed cell
29
death 4 (PDCD4) protein mediates this process. PDCD4 knockdown attenuates mouse
30
myocardial injury by inhibiting apoptosis(Xu et al., 2017). It also inhibits protein 3
1
translation and accelerates apoptosis (Colburn et al., 2006). A study reveals that
2
miR-499-5p protects against cardiomyocyte ischemic injury by directly targeting
3
PDCD4 (Li et al., 2016). Other studies shows that PDCD4 participates in regulation
4
of acute myocardial infarction (Gu et al., 2018). PDCD4 also directly binds to miR-21
5
and helps mediate ischemic neuron death (Yan et al., 2017). Therefore, targeting
6
PDCD4 may be a novel therapeutic strategy for cerebral IR injury.
7
In
this
study,
we
used
an
in
vitro
model
of
oxygen-glucose
8
deprivation/reperfusion (OGDR) injury in primary rat hippocampal neurons to
9
simulate cerebral IR injury. We explored the expression, effect, and potential
10
mechanism of miR-340-5p in OGDR-injured hippocampal neurons.
11
Materials and methods
12
Cell culture
13
Primary hippocampal neurons were isolated, as previously described (Krohn et al.,
14
1998) (provided by the Committee of Animal Care and Use at Zhengzhou University).
15
Briefly, the hippocampal tissues were dissected from neonatal SD rats and dissociated
16
using trypsin-EDTA (0.25%). The isolated cells were seeded on neurobasal media
17
(Life Technologies, Carlsbad, CA, USA) using GlutaMAX, B27, and glucose (4.5
18
mg/ml) for 7 days. Cells then were cultured in neurobasal medium with 5% fetal
19
bovine serum (FBS) and glucose (4.5 mg/ml) for 14 days. Animal experiments were
20
approved by The First Affiliated Hospital of Zhengzhou University. Human
21
embryonic kidney 293 (HEK293) cells (ATCC, Manassas, VA, USA) were grown in
22
DMEM (Life Technologies, Carlsbad, CA, USA). Upon reaching 70% to 80%
23
confluency, the HEK293 cells were collected for experiments. All cells were cultured
24
in an incubator containing 5% CO2 at 37 °C.
25
Oxygen-glucose deprivation/reperfusion model
26
Hippocampal neurons were cultured in OGDR conditions to mimic cerebral IR in
27
vitro. The OGDR model was prepared as previously described (Yang et al., 2015).
28
Briefly, hippocampal neurons were subjected to glucose-free DMEM (Gibco, Grand
29
Island, NY, USA) and incubated at 37 °C in hypoxic conditions (1% O2, 94% N2, 5%
30
CO2) for 2 h. Then, the cells were cultured in normal DMEM containing glucose in a 4
1
normoxic atmosphere (95% air, 5% CO2) for 24 h.
2
Cell transfection
3
Hippocampal neurons were transfected with a miR-340-5p mimic, miR-340-5p
4
inhibitor, and their respective negative controls, mimic Ctrl and inhibitor Ctrl
5
(GenePharma, Shanghai, China), using Lipofectamine 2000 (Invitrogen, Carlsbad,
6
CA, USA). MiR-340-5p mimic sequences: 5’-UUA UAA AGC AAU GAG ACU GA
7
UU-3’; mimic control sequences are: 5’- UUC UCC GAA CGU GUC ACG UTT-3’;
8
miR-340-5p inhibitor sequences: 5’-AAU CAG UCU CAU UGC UUU AU AA-3’;
9
inhibitor control sequences: 5’-CAC UAC UUU UGU GU AGUA CAA-3’. The
10
full-length of PDCD4 was amplified and cloned into the pcDNA3.1 plasmid to
11
construct pcDNA3.1-PDCD4. The pcDNA3.1-PDCD4 or pcDNA3.1 plasmid was
12
transfected into hippocampal neurons using Lipofectamine 2000.
13
Cell viability
14
MiRNA-transfected hippocampal neurons were seeded in 96-well plates (5×104
15
cells/well).
After
OGDR
exposure,
cells
were
incubated
with
3-(4,
16
5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 5 mg/ml; Sigma St.
17
Louis, MO, USA) for 4 h. Dimethyl sulfoxide (Sigma) was used to dissolve the
18
formazan crystals. Absorbance at 490 nm was detected to determine cell viability.
19
Lactate dehydrogenase assay
20
Neuronal cells in each group were cultured into 96-well plates, and the collected
21
cell medium were mixed with the lactate dehydrogenase (LDH) assay reagent
22
(Nanjing Jiancheng Bioengineering Institute, Nanjing, China) for 1 h. LDH activity
23
was determined via detection of absorbance at 450 nm.
24
Flow cytometry
25
An annexin V/propidium iodide (PI) kit (BD Bioscience, Franklin Lakes, NJ, USA)
26
was used to determine apoptosis. After incubation at different experimental conditions,
27
the neurons were plated in 6-well plates (5×105 cells/well) and suspended in binding
28
buffer with 10 µL of Annexin V-FITC and 10 µL of PI in the dark for 15 minutes. 5
1
Afterwards, flow cytometry was conducted to determine apoptotic cells.
2
qRT-PCR
3
Total RNA was extracted from TRIzol reagent (Invitrogen), and cDNA synthesis
4
was conducted using a miRNA reverse transcription kit (Applied Biosystems, Foster
5
City, CA, USA) for miR-340-5p detection. Real-time PCR was performed using a
6
SYBR Green qPCR Master Mix Kit (Applied Biosystems). Real-time PCR was
7
performed using the ABI PRISM 7900 system (Applied Biosystems). The primers
8
were as follows: miR-340-5p forward 5’-GCG GTT ATA AAG CAA TG AGA-3’ and
9
reverse 5’-GTG CGT GTC GTG GAG TCG-3’; U6 forward 5’-CTC GCT TCG GCA
10
GCA CA-3’ and reverse 5’-AAC GCT TCA CGA ATT TG CGT-3’. The relative
11
abundance of miR-340-5p was normalized to U6. Data were quantified using the
12
2−∆∆Ct method.
13
Western blot
14
Hippocampal neurons were lysed in lysis buffer for 0.5 h. After cells were
15
centrifuged, the protein concentrations in lysates were measured using a bicinchoninic
16
acid (BCA) assay (Beyotime Biotechnology, Haimen, China). Equivalent amounts of
17
protein were separated using 10%–15% SDS-PAGE and transferred to polyvinylidene
18
difluoride (PVDF) membranes (Millipore, Boston, MA, USA), which were blocked
19
and incubated at 4 °C overnight with primary antibodies, including PDCD4 (1:1000),
20
Bcl-2 (1:1000), Bax (1:1000), p-PI3K (1:1000), PI3K (1:1000), p-Akt phospho
21
Ser473 (1:5000), Akt (1:500), and internal control β-actin (1:1000), all from Abcam
22
(Cambridge, MA, USA). After washing with TBST, the membranes were probed with
23
secondary antibody IgG (1:5000; ZSGB-BIO, Beijing, China) for 2 h. Blots were
24
processed using an enhanced chemiluminescence reaction system (Amersham, Little
25
Chalfont, UK). Gray scale was quantified using Quantity One Software (Bio-Rad,
26
Hercules, CA, USA).
27
Dual luciferase activity assay
28
A 350 bp sequence from the 3’-UTR of PDCD4 containing the miR-340-5p binding 6
1
site (Wt) was amplified by PCR. The sequence for mutant PDCD4 (Mut) was
2
amplified by point mutation method using KOD-Plus mutagenesis Kit (TOYOBO,
3
Osaka, Japan). The fragments from the Wt or Mut PDCD4 3’-UTR were then
4
subcloned into the pGL3 luciferase promoter vector (Promega, Madison, WI, USA).
5
HEK 293 cells or OGDR induced hippocampal neurons were co-transfected with the
6
constructs and miR-340-5p mimic or mimic control using Lipofectamine 2000
7
(Invitrogen) for 48 h. Luciferase activities were detected using the dual-luciferase
8
assay system (Promega).
9
Statistical analysis
10
Data are expressed as mean ± standard derivations (SD) and analyzed with SPSS
11
22.0 (SPSS Inc., Chicago, IL, USA). Differences between two groups were compared
12
via t test and differences among multiple groups were examined using ANOVA
13
followed by Bonferroni test.
14
Results
15
Low abundance of miR-340-5p in hippocampal neurons exposed to OGDR
16
To assess the effect of miR-340-5p in OGDR-injured hippocampal neurons, we first
17
used qRT-PCR to detect the level of miR-340-5p. The results showed significantly
18
downregulated abundance of miR-340-5p in OGDR-injured hippocampal neurons
19
(0.34±0.07), compared with the control (1.00±0.06) (t=12.61, P<0.05, Fig.1A),
20
implying that miR-340-5p may play an important role against OGDR injury of
21
hippocampal neurons.
22
MiR-340-5p had protective effect against neuronal injury caused by OGDR
23
To investigate the contribution of miR-340-5p in OGDR-induced hippocampal
24
neuron injury, neurons were transfected with a miR-340-5p mimic or miR-340-5p
25
inhibitor for 24 h and then exposed to OGDR. The miR-340-5p mimic upregulated
26
miR-340-5p abundance, and the miR-340-5p inhibitor significantly downregulated
27
miR-340-5p abundance in the injured neurons (F=170.1, P<0.05, Fig. 1B). 7
1
MiR-340-5p overexpression resulted in increased cell viability (F=54.94, P<0.05, Fig.
2
2A), and significantly decreased LDH activity (F=78.45, P<0.05, Fig. 2B) and
3
apoptosis (F=40.87, P<0.05, Fig. 2C) in OGDR treated cells. In contrast, miR-340-5p
4
suppression showed the opposite effect. Moreover, the upregulation of Bax (F=62.36,
5
P<0.05, Fig. 2D), the downregulation of Bcl-2 (F=41.63, P<0.05, Fig. 2D), and the
6
increased ratio of Bax/Bcl-2 (F=37.34, P<0.05, Fig. 2E), which induced by OGDR,
7
were weakened by miR-340-5p overexpression, whereas miR-340-5p suppression
8
produced the opposite effects. Overall, these results suggest that miR-340-5p had a
9
neuroprotective effect in OGDR-induced neuronal injury.
10
MiR-340-5p directly regulated PDCD4 expression
11
To explore the mechanism underlying the neuroprotective role of miR-340-5p, we
12
conducted a bioinformatic analysis of its targets. We identified PDCD4, a
13
pro-apoptotic gene, as a putative target of miR-340-5p (Fig. 3C). We also found that
14
co-transfection with the miR-340-5p mimic and the PDCD4-wt 3’-UTR significantly
15
decreased luciferase activity as compared with cells co-tranfected with miR-340-5p
16
mimic control and PDCD4-wt (t=7.933, P<0.05). However, co-transfection of the
17
miR-340-5p mimic and PDCD4-mut 3’-UTR showed no obvious effect on luciferase
18
activity in HEK 293 cells (Fig. 3A). Similar results was also detected in hippocampal
19
neurons exposed to OGDR (Fig. 3B). Furthermore, miR-340-5p inhibition markedly
20
upregulated the protein content of PDCD4 during OGDR, whereas its overexpression
21
restrained PDCD4 content (F=187.5, P<0.05, Fig. 3D and E). These results
22
demonstrate that miR-340-5p directly targeted PDCD4.
23
MiR-340-5p downregulated PDCD4 to protect hippocampal neurons against
24
injury caused by OGDR
25
To further investigate the contribution of PDCD4 to the protective effects of
26
miR-340-5p in OGDR-induced neuron injury, neurons were co-transfected with a
27
PDCD4 vector and miR-340-5p mimic. The data showed that PDCD4 overexpression
28
impeded the ability of miR-340-5p to repress PDCD4’s protein content. (F=33.73, Fig. 8
1
4A). In neurons exposed to OGDR, the miR-340-5p mimic improved cell viability
2
(F=19.48, Fig. 4B), reduced LDH (F=14.58, Fig. 4C) and apoptosis (F=23.96, Fig.
3
4D) levels. PDCD4 overexpression inhibited these effects (P<0.05). Additionally, The
4
miR-340-5p mimic led to upregulation of Bcl-2 (F=31.04, P<0.05, Fig. 4E),
5
downregulation of Bax
6
ratio in OGDR-injured neuron (F=24.04, P<0.05, Fig. 4F)s. PDCD4 overexpression
7
blocked these effects (P<0.05, Fig. 4E and 4F). These data indicate that PDCD4
8
overexpression abrogated the neuroprotective role of miR-340-5p in OGDR-injured
9
neurons.
(F=17.64, P<0.05, Fig. 4E), and an increased Bax/Bcl-2
10
MiR-340-5p overexpression activated the PI3K/Akt signaling pathway in
11
neurons exposed to OGDR
12
The PI3K/Akt pathway is an important cell survival signaling pathway that protects
13
against cerebral IR injury(Breivik et al., 2015). We explored its involvement in
14
modulating miR-340-5p in OGDR-injured neurons. Western blot results showed that
15
the phosphorylation of p-PI3K (F=61.95, Fig. 5B) and p-Akt (F=54.35, Fig. 5C),
16
which was inhibited by OGDR, was remarkably increased by miR-340-5p
17
overexpression, whereas it was significantly reduced by miR-340-5p suppression
18
(P<0.05, Fig.5).
19
MiR-340-5p targeted PDCD4 to influence PI3K/Akt signaling
20
Finally, we tested whether the PI3K/Akt pathway helped regulate miR-340-5p and
21
PDCD4 in OGDR-injured hippocampal neurons. Cells were co-transfected with
22
miR-340-5p mimic and PDCD4 vector and then exposed to OGDR. As shown in Fig.
23
6, miR-340-5p overexpression upregulated the phosphorylation of p-PI3K (F=61.95,
24
Fig. 6B) and p-Akt (F=61.95, Fig. 6C), whereas this upregulation was partly reversed
25
by PDCD4 overexpression (P<0.05). These results indicate that miR-340-5p could
26
activate the PI3K/Akt pathways by downregulating PDCD4.
27
Discussion
28
After cerebral IR injury, nerve cells undergo complex pathophysiological 9
1
processes. Treating cerebral IR injury requires alleviating this damage to save dying
2
nerve cells. Therefore, it is important to identify the targets in IR injury. MiRNAs
3
serve a crucial role in cerebral IR injury. For example, miR-340 has a role in nerve
4
injury(Cosset et al., 2016), and miR-340-5p is downregulated in peripheral blood
5
from patients with acute stroke (Yoo et al., 2018). However, the function of
6
miR-340-5p in IR-induced cerebral neural injury is unknown.
7
In the current study, we used primary rat hippocampal neurons to establish an in
8
vitro OGDR injury model that mimics cerebral IR injury. We found miR-340-5p to be
9
downregulated in primary hippocampal neurons that were injured by OGDR.
10
MiR-340-5p overexpression targeted PDCD4 to enhance cell viability and reduce
11
LDH activity and apoptosis, whereas miR-340-5p inhibition showed the opposite
12
effects. Our study confirmed the contribution of miR-340-5p in regulating OGDR
13
injury, revealing a potential target for treating cerebral ischemic stroke. In future
14
researches, it is needed to explore the function of miR-340-5p in other brain cells,
15
such as glial cells, to further understand the role of miR-340-5p in cerebral IR injury
16
and in cerebral diseases.
17
PDCD4 is a crucial regulator of apoptosis(Singh et al., 2019). It reportedly
18
participates in translation, transcription, and cell growth regulation(Matsuhashi et al.,
19
2019). Numerous studies have found low expression of PDCD4 in various cancers,
20
and it is associated with the progression of prostate cancer (Zennami et al., 2019),
21
colon carcinoma (Wang et al., 2017), and glioma(Monfared et al., 2019). Recently,
22
PDCD4 was identified as a crucial gene in the regulation of IR injury. A study has
23
shown that by regulating the miR-21/PDCD4 signaling pathway, serum-derived
24
extracellular vesicles protect mouse hearts against acute myocardial infarction (Gu et
25
al., 2018). Moreover, the lncRNA MEG3 regulates ischemic neuronal death by
26
targeting the miR-21/PDCD4 signaling pathway (Yan et al., 2017). In rat cardiac cells
27
exposed to IR injury, miR-21 regulates PDCD4, which exerts an anti-apoptotic effect
28
(Cheng et al., 2010). By targeting PDCD4, miR-499 inhibits cardiomyocyte apoptosis
29
and is involved in ischemic postconditioning-mediated cardiac protection against IR 10
1
injury (Zhu et al., 2016). In our study, it was revealed that miR-340-5p targeted and
2
negatively modulated the PDCD4 gene. PDCD4 protein levels markedly increased in
3
rat primary hippocampal neurons that were exposed to OGDR. Furthermore, we
4
confirmed that PDCD4 participated in the neuroprotective effect of miR-340-5p on
5
hippocampal neurons that were injured by OGDR.
6
The PI3K/Akt signaling pathway is an important survival signal in various nerve
7
cells. This pathway mainly involves PI3K, Akt, and their downstream molecules. It is
8
an important pathway for intracellular transduction of membrane receptor signals and
9
for various cellular processes, such as proliferation, differentiation, and apoptosis.
10
PI3K/Akt signaling plays a crucial role in cerebral IR injury(Kamel et al., 2019).
11
MiR-375 and Ctgf protect against cerebral IR injury by regulating the p21/PI3K/Akt
12
signaling pathways (Ou et al., 2017). MiR-19b exerts neuronal protective effects
13
during hypoxia/reoxygenation-induced injury by targeting PTEN via the PI3K/Akt
14
pathway (Liu et al., 2019). MiR-340 may impede laryngeal squamous cell carcinoma
15
progression by targeting EZH2 via p27 expression enhancement and PI3K/Akt
16
inactivation (Yu et al., 2016). MiR-340 suppression also has been shown to induce
17
activation of PI3K and Akt in ovarian cancer SKOV3 cells (Qu and Wang, 2017).
18
In this study, we found that OGDR injury blocked PI3K/Akt signaling and that
19
miR-340-5p mimics could activate this signaling. This result is contrary to the effects
20
of miR-340-5p on PI3K and Akt that have been previously described by (Yu et al.,
21
2016) and (Qu and Wang, 2017). These contradictory findings indicate that the effects
22
of miR-340-5p on the PI3K/Akt pathway might depend on the experimental
23
conditions. Moreover, there may be more than one target gene for miR-340-5p.
24
Therefore, miR-340a-5p may influence different target gene and then have
25
contradictory effect on PI3K/Akt signaling in different disease. Previous research
26
indicates that PDCD4 may impede Akt activation(Cristofoletti et al., 2019). Our data
27
reveal that miR-340-5p activated suppression of the PI3K/Akt pathway in
28
OGDR-injured hippocampal neurons and that PDCD4 overexpression partly reversed
29
this effect, which suggested miR-340-5p regulated OGDR-induced neurons injury 11
1
partially by modulating PI3K/Akt pathway. In future research, we should explore
2
other target gene of miR-340-5p apart from in ischemic stroke, and further understand
3
the underlying mechanism of miR-340-5P in brain injury. Additionally, the
4
down-stream of PDCD4 should also be investigated, thus other I/R related signaling
5
pathways regulated by PDCD4 should be explored.
6
In summary, our study suggests that miR-340-5p alleviated OGDR-induced
7
injury to neurons by targeting PDCD4 to activate the PI3K/Akt pathway. We
8
identified miR-340 as a promising target for the prevention of cerebral ischemic injury.
9
Our in vitro experiment examined the role of miR-340-5p in hippocampal neurons
10
that were exposed to OGDR injury. Its role in cerebral IR injury needs to be further
11
investigated in vivo.
12
Conflicts of interest
13
No conflict of interest.
14
12
1
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Postconditioning-Regulated
miR-499 16
Protects
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1
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2
39, 2364-2380.
3 4
17
1
Figure legends
2
Figure 1. Downregulation of miR-340-5p in hippocampal neurons after
3
oxygen-glucose deprivation/reoxygenation (OGDR). (A) The qRT-PCR results of
4
miR-340-5p in primary hippocampal neurons after exposure to OGDR. (B) The
5
relative miR-340-5p abundance in OGDR-injured hippocampal neurons after
6
transfection with miR-340-5p mimics or miR-340-5p inhibitors for 24 h. N=3,
7
#
8
Figure 2. MiR-340-5p relieved OGDR-induced neuronal injury. (A) Cell viability
9
was detected using a CCK-8 assay. (B) LDH release in culture medium. (C) Apoptosis
10
analyzed by flow cytometry, apoptosis cell rate were represented in right upper
11
quadrant. (D) Western blot analysis of protein content of Bax and Bcl-2 in
12
hippocampal neurons. (E) Ratio of Bax/Bcl-2. N=3, #P<0.05 vs. control; *P<0.05 vs.
13
OGDR.
14
Figure 3. PDCD4 was a target of miR-340-5p. (A) Dual-Luciferase activity assay
15
results of the interaction between miR-340-5p and the 3’-UTR of PDCD4 in HEK293
16
cells. (B) Dual-Luciferase activity assay results in OGDR induced hippocampal
17
neurons. (C) The putative binding sequences of the 3’-UTR of PDCD4 with
18
miR-340-5p. (D and E) The protein content of PDCD4, tested by western blot. N=3,
19
#
20
Figure 4. MiR-340-5p alleviated injury to hippocampal neurons exposed to OGDR by
21
downregulating PDCD4. OGDR stimulated Hippocampal neurons were co-transfected
22
with miR-340-5p mimics and a PDCD4 vector. (A) Relative PDCD4 protein content.
23
(B) Cell viability. (C) LDH activity. (D) Apoptosis. (E) Relative protein content of
24
Bax and Bcl-2. (F) Ratio of Bax and Bcl-2. N=3, #P<0.05 vs. OGDR; *P<0.05 vs.
25
OGDR+miR-340-5p mimic.
26
Figure 5. MiR-340-5p alleviated injury to hippocampal neurons exposed to OGDR by
27
activating PI3K/Akt signaling pathway. (A) Western blot analysis of p-PI3K, PI3K,
28
p-Akt, and Akt protein content. (B) Quantitative analysis of relative protein content of
P<0.05 vs. control; *P<0.05 vs. OGDR.
P<0.05 vs. mimic control.
18
1
p-PI3K and PI3K. (C) Quantitative analyses of relative protein content of p-Akt and
2
Akt. N=3, #P<0.05 vs. control; *P<0.05 vs. OGDR.
3
Figure 6. MiR-340-5p activated PI3K/Akt signaling pathway by targeting PDCD4 in
4
OGDR induced neurons. (A) Western blot analysis of p-PI3K, PI3K, p-Akt, and Akt
5
protein content. (B) Quantitative analysis of relative protein content of p-PI3K and
6
PI3K. (C) Quantitative analysis of relative protein levels of p-Akt and Akt. N=3,
7
#
P<0.05 vs. control; *P<0.05 vs. OGDR. (D) Schematic diagram.
19
Highlights 1.
Low expression of miR-340-5p in hippocampal neurons exposed to OGDR
2.
MiR-340-5p had protective effect against neuronal injury caused by OGDR
3.
MiR-340-5p directly regulated PDCD4 expression
4.
MiR-340-5p inhibited PDCD4 to protect hippocampal neurons against OGDR injury
5.
MiR-340-5p targeted PDCD4 to influence PI3K/Akt signaling
Yake Zheng: Design, Experiment perform, Writing original draft. Peng Zhao: Experiment perform, Data analyze Yajun Lian: Design, Supervision, Paper revise Shuang Li: Experiment perform, Data analyze Yuan Chen: Experiment perform, Paper revise Lihao Li: Experiment perform,
S0197-0186(19)30441-3
DOI:
https://doi.org/10.1016/j.neuint.2019.104650
Reference:
NCI 104650
To appear in:
Neurochemistry International
Received Date: 8 August 2019 Revised Date:
28 November 2019
Accepted Date: 19 December 2019
Please cite this article as: Zheng, Y., Zhao, P., Lian, Y., Li, S., Chen, Y., Li, L., MiR-340-5p alleviates oxygen-glucose deprivation/reoxygenation-induced neuronal injury via PI3K/Akt activation by targeting PDCD4, Neurochemistry International (2020), doi: https://doi.org/10.1016/j.neuint.2019.104650. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
MiR-340-5p
alleviates
oxygen-glucose
deprivation/reoxygenation-induced
2
neuronal injury via PI3K/Akt activation by targeting PDCD4
3
Yake Zheng1, Peng Zhao2, Yajun Lian1,*, Shuang Li1, Yuan Chen1, Lihao Li1
4
1
5
Zhengzhou, China, 450052;
6
2
7
China, 450004;
8
* Corresponding author: Yajun Lian
9
Jianshe East Road, The First Affiliated Hospital of Zhengzhou University, Zhengzhou,
Department of Neurology, The First Affiliated Hospital of Zhengzhou University,
Department of Neurology, The First People’s Hospital of Zhengzhou, Zhengzhou,
10
China, 450052;
11
Email: [email protected]
12
Tel: 13838367143
13
1
1
Abstract
2
MicroRNA-340-5p (miR-340-5p), a suppressor of certain target genes in brain,
3
reportedly is decreased in peripheral circulation of acute stroke patients. However,
4
little is known regarding its role in regulating cerebral ischemia/reperfusion injury.
5
This study explores the effect of miR-340-5p on ischemia/reperfusion insults by
6
exposing rat hippocampal neurons to oxygen-glucose deprivation/reoxygenation
7
(OGDR) in vitro. We found miR-340-5p to be poorly expressed in these neurons after
8
OGDR stimulation. OGDR stimulation decreased cell viability, increased lactate
9
dehydrogenase (LDH) activity and cell apoptosis, all of which were significantly
10
inhibited by miR-340-5p overexpression and enhanced by miR-340-5p inhibition.
11
Using bioinformatics analysis, we identified mRNA encoding the pro-apoptotic factor,
12
programmed cell death 4 (PDCD4) as a putative target of miR-340-5p. A
13
dual-luciferase reporter assay suggested that miR-340-5p targeted the 3’-UTR of
14
PDCD4. PDCD4 was upregulated in cells exposed to OGDR, and miR-340-5p
15
negatively modulated expression of PDCD4. PDCD4 overexpression partly reversed
16
the neuroprotective effect of miR-340-5p during OGDR-induced injury. MiR-340-5p
17
overexpression significantly promoted the activation of PI3K/Akt signaling pathway
18
(P<0.05) in OGDR-exposed cells, and PDCD4 overexpression attenuated this effect
19
(P<0.05). Collectively, our results indicate that miR-340-5p might exerted
20
neuroprotective effects during OGDR injury by targeting PDCD4 and then activating
21
the PI3K/Akt pathway. These results indicated a novel target for treating cerebral
22
ischemic injury.
23
Keywords: miR-340-5p, oxygen glucose deprivation/reoxygenation (OGDR),
24
neuroprotection, PDCD4, PI3K/Akt
25 26
Abbreviations
27
miRNAs, microRNAs; IR, ischemia/reperfusion; PDCD4, programmed cell death 4;
28
OGDR, oxygen-glucose deprivation/reoxygenation; FBS, fetal bovine serum;
29
HEK293, human embryonic kidney 293 cells; LDH, lactate dehydrogenase; PI,
30
propidium iodide; PVDF, polyvinylidene difluoride 2
1
Introduction
2
Ischemic stroke is a brain disease caused by temporary cessation or insufficient
3
blood flow to brain. It is associated with high disability and mortality rates(Stegner et
4
al., 2019). The subsequent reperfusion and reoxygenation of blood can further
5
aggravate tissue injury and cell death(Galkin, 2019). Due to its high disability and
6
mortality, ischemic stroke brings heavy burden to the patients and the society(Patel
7
and McMullen, 2017). Understanding the complex pathophysiological mechanism
8
underlying stroke-related neuron damage may help identify effective treatments for
9
ischemic stroke.
10
MicroRNAs (miRNAs) are small (18–28 nucleotides), noncoding RNAs that
11
negatively modulate expression of their target genes by binding to the 3’-UTRs of
12
target mRNA. MiRNAs help modulate pathophysiological processes in cerebral
13
ischemia/reperfusion (IR) injury. For example, miR-34b targets Keap1 to protect
14
against focal cerebral IR injury in rats with middle cerebral artery occlusion (Huang et
15
al., 2019). MiR-424 inhibits oxidative stress to ameliorate focal cerebral IR injury in
16
mice (Liu et al., 2015). MiR-99a reduces cerebral IR-induced neuronal damage by
17
regulating the cell cycle and apoptosis (Tao et al., 2015). MiR-200a silencing protects
18
neural stem cells during cerebral IR injury (Ma et al., 2017). Ou et al. reports that
19
miR-375 exhibits protective effects against cerebral IR injury by targeting Ctgf (Ou et
20
al., 2017). Additionally, miR-182-5p mitigates neuronal injury after cerebral IR injury
21
by targeting Toll-like receptor 4 (Wang et al., 2018). In rats with sciatic nerve crush
22
injury, miR-340 dysregulation occurs in the injured nerves, which affects the removal
23
of cell debris and axon regeneration (Li et al., 2017). Furthermore, miR-340-5p is
24
downregulated in the peripheral blood of patients with acute stroke (Yoo et al., 2018).
25
These findings suggest that miRNAs are potential targets for cerebral IR injury.
26
However, the specific function of miR-340-5p in cerebral IR-induced neural injury
27
remains unknown.
28
Apoptosis is a critical mechanism in cerebral IR injury, and the programmed cell
29
death 4 (PDCD4) protein mediates this process. PDCD4 knockdown attenuates mouse
30
myocardial injury by inhibiting apoptosis(Xu et al., 2017). It also inhibits protein 3
1
translation and accelerates apoptosis (Colburn et al., 2006). A study reveals that
2
miR-499-5p protects against cardiomyocyte ischemic injury by directly targeting
3
PDCD4 (Li et al., 2016). Other studies shows that PDCD4 participates in regulation
4
of acute myocardial infarction (Gu et al., 2018). PDCD4 also directly binds to miR-21
5
and helps mediate ischemic neuron death (Yan et al., 2017). Therefore, targeting
6
PDCD4 may be a novel therapeutic strategy for cerebral IR injury.
7
In
this
study,
we
used
an
in
vitro
model
of
oxygen-glucose
8
deprivation/reperfusion (OGDR) injury in primary rat hippocampal neurons to
9
simulate cerebral IR injury. We explored the expression, effect, and potential
10
mechanism of miR-340-5p in OGDR-injured hippocampal neurons.
11
Materials and methods
12
Cell culture
13
Primary hippocampal neurons were isolated, as previously described (Krohn et al.,
14
1998) (provided by the Committee of Animal Care and Use at Zhengzhou University).
15
Briefly, the hippocampal tissues were dissected from neonatal SD rats and dissociated
16
using trypsin-EDTA (0.25%). The isolated cells were seeded on neurobasal media
17
(Life Technologies, Carlsbad, CA, USA) using GlutaMAX, B27, and glucose (4.5
18
mg/ml) for 7 days. Cells then were cultured in neurobasal medium with 5% fetal
19
bovine serum (FBS) and glucose (4.5 mg/ml) for 14 days. Animal experiments were
20
approved by The First Affiliated Hospital of Zhengzhou University. Human
21
embryonic kidney 293 (HEK293) cells (ATCC, Manassas, VA, USA) were grown in
22
DMEM (Life Technologies, Carlsbad, CA, USA). Upon reaching 70% to 80%
23
confluency, the HEK293 cells were collected for experiments. All cells were cultured
24
in an incubator containing 5% CO2 at 37 °C.
25
Oxygen-glucose deprivation/reperfusion model
26
Hippocampal neurons were cultured in OGDR conditions to mimic cerebral IR in
27
vitro. The OGDR model was prepared as previously described (Yang et al., 2015).
28
Briefly, hippocampal neurons were subjected to glucose-free DMEM (Gibco, Grand
29
Island, NY, USA) and incubated at 37 °C in hypoxic conditions (1% O2, 94% N2, 5%
30
CO2) for 2 h. Then, the cells were cultured in normal DMEM containing glucose in a 4
1
normoxic atmosphere (95% air, 5% CO2) for 24 h.
2
Cell transfection
3
Hippocampal neurons were transfected with a miR-340-5p mimic, miR-340-5p
4
inhibitor, and their respective negative controls, mimic Ctrl and inhibitor Ctrl
5
(GenePharma, Shanghai, China), using Lipofectamine 2000 (Invitrogen, Carlsbad,
6
CA, USA). MiR-340-5p mimic sequences: 5’-UUA UAA AGC AAU GAG ACU GA
7
UU-3’; mimic control sequences are: 5’- UUC UCC GAA CGU GUC ACG UTT-3’;
8
miR-340-5p inhibitor sequences: 5’-AAU CAG UCU CAU UGC UUU AU AA-3’;
9
inhibitor control sequences: 5’-CAC UAC UUU UGU GU AGUA CAA-3’. The
10
full-length of PDCD4 was amplified and cloned into the pcDNA3.1 plasmid to
11
construct pcDNA3.1-PDCD4. The pcDNA3.1-PDCD4 or pcDNA3.1 plasmid was
12
transfected into hippocampal neurons using Lipofectamine 2000.
13
Cell viability
14
MiRNA-transfected hippocampal neurons were seeded in 96-well plates (5×104
15
cells/well).
After
OGDR
exposure,
cells
were
incubated
with
3-(4,
16
5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 5 mg/ml; Sigma St.
17
Louis, MO, USA) for 4 h. Dimethyl sulfoxide (Sigma) was used to dissolve the
18
formazan crystals. Absorbance at 490 nm was detected to determine cell viability.
19
Lactate dehydrogenase assay
20
Neuronal cells in each group were cultured into 96-well plates, and the collected
21
cell medium were mixed with the lactate dehydrogenase (LDH) assay reagent
22
(Nanjing Jiancheng Bioengineering Institute, Nanjing, China) for 1 h. LDH activity
23
was determined via detection of absorbance at 450 nm.
24
Flow cytometry
25
An annexin V/propidium iodide (PI) kit (BD Bioscience, Franklin Lakes, NJ, USA)
26
was used to determine apoptosis. After incubation at different experimental conditions,
27
the neurons were plated in 6-well plates (5×105 cells/well) and suspended in binding
28
buffer with 10 µL of Annexin V-FITC and 10 µL of PI in the dark for 15 minutes. 5
1
Afterwards, flow cytometry was conducted to determine apoptotic cells.
2
qRT-PCR
3
Total RNA was extracted from TRIzol reagent (Invitrogen), and cDNA synthesis
4
was conducted using a miRNA reverse transcription kit (Applied Biosystems, Foster
5
City, CA, USA) for miR-340-5p detection. Real-time PCR was performed using a
6
SYBR Green qPCR Master Mix Kit (Applied Biosystems). Real-time PCR was
7
performed using the ABI PRISM 7900 system (Applied Biosystems). The primers
8
were as follows: miR-340-5p forward 5’-GCG GTT ATA AAG CAA TG AGA-3’ and
9
reverse 5’-GTG CGT GTC GTG GAG TCG-3’; U6 forward 5’-CTC GCT TCG GCA
10
GCA CA-3’ and reverse 5’-AAC GCT TCA CGA ATT TG CGT-3’. The relative
11
abundance of miR-340-5p was normalized to U6. Data were quantified using the
12
2−∆∆Ct method.
13
Western blot
14
Hippocampal neurons were lysed in lysis buffer for 0.5 h. After cells were
15
centrifuged, the protein concentrations in lysates were measured using a bicinchoninic
16
acid (BCA) assay (Beyotime Biotechnology, Haimen, China). Equivalent amounts of
17
protein were separated using 10%–15% SDS-PAGE and transferred to polyvinylidene
18
difluoride (PVDF) membranes (Millipore, Boston, MA, USA), which were blocked
19
and incubated at 4 °C overnight with primary antibodies, including PDCD4 (1:1000),
20
Bcl-2 (1:1000), Bax (1:1000), p-PI3K (1:1000), PI3K (1:1000), p-Akt phospho
21
Ser473 (1:5000), Akt (1:500), and internal control β-actin (1:1000), all from Abcam
22
(Cambridge, MA, USA). After washing with TBST, the membranes were probed with
23
secondary antibody IgG (1:5000; ZSGB-BIO, Beijing, China) for 2 h. Blots were
24
processed using an enhanced chemiluminescence reaction system (Amersham, Little
25
Chalfont, UK). Gray scale was quantified using Quantity One Software (Bio-Rad,
26
Hercules, CA, USA).
27
Dual luciferase activity assay
28
A 350 bp sequence from the 3’-UTR of PDCD4 containing the miR-340-5p binding 6
1
site (Wt) was amplified by PCR. The sequence for mutant PDCD4 (Mut) was
2
amplified by point mutation method using KOD-Plus mutagenesis Kit (TOYOBO,
3
Osaka, Japan). The fragments from the Wt or Mut PDCD4 3’-UTR were then
4
subcloned into the pGL3 luciferase promoter vector (Promega, Madison, WI, USA).
5
HEK 293 cells or OGDR induced hippocampal neurons were co-transfected with the
6
constructs and miR-340-5p mimic or mimic control using Lipofectamine 2000
7
(Invitrogen) for 48 h. Luciferase activities were detected using the dual-luciferase
8
assay system (Promega).
9
Statistical analysis
10
Data are expressed as mean ± standard derivations (SD) and analyzed with SPSS
11
22.0 (SPSS Inc., Chicago, IL, USA). Differences between two groups were compared
12
via t test and differences among multiple groups were examined using ANOVA
13
followed by Bonferroni test.
14
Results
15
Low abundance of miR-340-5p in hippocampal neurons exposed to OGDR
16
To assess the effect of miR-340-5p in OGDR-injured hippocampal neurons, we first
17
used qRT-PCR to detect the level of miR-340-5p. The results showed significantly
18
downregulated abundance of miR-340-5p in OGDR-injured hippocampal neurons
19
(0.34±0.07), compared with the control (1.00±0.06) (t=12.61, P<0.05, Fig.1A),
20
implying that miR-340-5p may play an important role against OGDR injury of
21
hippocampal neurons.
22
MiR-340-5p had protective effect against neuronal injury caused by OGDR
23
To investigate the contribution of miR-340-5p in OGDR-induced hippocampal
24
neuron injury, neurons were transfected with a miR-340-5p mimic or miR-340-5p
25
inhibitor for 24 h and then exposed to OGDR. The miR-340-5p mimic upregulated
26
miR-340-5p abundance, and the miR-340-5p inhibitor significantly downregulated
27
miR-340-5p abundance in the injured neurons (F=170.1, P<0.05, Fig. 1B). 7
1
MiR-340-5p overexpression resulted in increased cell viability (F=54.94, P<0.05, Fig.
2
2A), and significantly decreased LDH activity (F=78.45, P<0.05, Fig. 2B) and
3
apoptosis (F=40.87, P<0.05, Fig. 2C) in OGDR treated cells. In contrast, miR-340-5p
4
suppression showed the opposite effect. Moreover, the upregulation of Bax (F=62.36,
5
P<0.05, Fig. 2D), the downregulation of Bcl-2 (F=41.63, P<0.05, Fig. 2D), and the
6
increased ratio of Bax/Bcl-2 (F=37.34, P<0.05, Fig. 2E), which induced by OGDR,
7
were weakened by miR-340-5p overexpression, whereas miR-340-5p suppression
8
produced the opposite effects. Overall, these results suggest that miR-340-5p had a
9
neuroprotective effect in OGDR-induced neuronal injury.
10
MiR-340-5p directly regulated PDCD4 expression
11
To explore the mechanism underlying the neuroprotective role of miR-340-5p, we
12
conducted a bioinformatic analysis of its targets. We identified PDCD4, a
13
pro-apoptotic gene, as a putative target of miR-340-5p (Fig. 3C). We also found that
14
co-transfection with the miR-340-5p mimic and the PDCD4-wt 3’-UTR significantly
15
decreased luciferase activity as compared with cells co-tranfected with miR-340-5p
16
mimic control and PDCD4-wt (t=7.933, P<0.05). However, co-transfection of the
17
miR-340-5p mimic and PDCD4-mut 3’-UTR showed no obvious effect on luciferase
18
activity in HEK 293 cells (Fig. 3A). Similar results was also detected in hippocampal
19
neurons exposed to OGDR (Fig. 3B). Furthermore, miR-340-5p inhibition markedly
20
upregulated the protein content of PDCD4 during OGDR, whereas its overexpression
21
restrained PDCD4 content (F=187.5, P<0.05, Fig. 3D and E). These results
22
demonstrate that miR-340-5p directly targeted PDCD4.
23
MiR-340-5p downregulated PDCD4 to protect hippocampal neurons against
24
injury caused by OGDR
25
To further investigate the contribution of PDCD4 to the protective effects of
26
miR-340-5p in OGDR-induced neuron injury, neurons were co-transfected with a
27
PDCD4 vector and miR-340-5p mimic. The data showed that PDCD4 overexpression
28
impeded the ability of miR-340-5p to repress PDCD4’s protein content. (F=33.73, Fig. 8
1
4A). In neurons exposed to OGDR, the miR-340-5p mimic improved cell viability
2
(F=19.48, Fig. 4B), reduced LDH (F=14.58, Fig. 4C) and apoptosis (F=23.96, Fig.
3
4D) levels. PDCD4 overexpression inhibited these effects (P<0.05). Additionally, The
4
miR-340-5p mimic led to upregulation of Bcl-2 (F=31.04, P<0.05, Fig. 4E),
5
downregulation of Bax
6
ratio in OGDR-injured neuron (F=24.04, P<0.05, Fig. 4F)s. PDCD4 overexpression
7
blocked these effects (P<0.05, Fig. 4E and 4F). These data indicate that PDCD4
8
overexpression abrogated the neuroprotective role of miR-340-5p in OGDR-injured
9
neurons.
(F=17.64, P<0.05, Fig. 4E), and an increased Bax/Bcl-2
10
MiR-340-5p overexpression activated the PI3K/Akt signaling pathway in
11
neurons exposed to OGDR
12
The PI3K/Akt pathway is an important cell survival signaling pathway that protects
13
against cerebral IR injury(Breivik et al., 2015). We explored its involvement in
14
modulating miR-340-5p in OGDR-injured neurons. Western blot results showed that
15
the phosphorylation of p-PI3K (F=61.95, Fig. 5B) and p-Akt (F=54.35, Fig. 5C),
16
which was inhibited by OGDR, was remarkably increased by miR-340-5p
17
overexpression, whereas it was significantly reduced by miR-340-5p suppression
18
(P<0.05, Fig.5).
19
MiR-340-5p targeted PDCD4 to influence PI3K/Akt signaling
20
Finally, we tested whether the PI3K/Akt pathway helped regulate miR-340-5p and
21
PDCD4 in OGDR-injured hippocampal neurons. Cells were co-transfected with
22
miR-340-5p mimic and PDCD4 vector and then exposed to OGDR. As shown in Fig.
23
6, miR-340-5p overexpression upregulated the phosphorylation of p-PI3K (F=61.95,
24
Fig. 6B) and p-Akt (F=61.95, Fig. 6C), whereas this upregulation was partly reversed
25
by PDCD4 overexpression (P<0.05). These results indicate that miR-340-5p could
26
activate the PI3K/Akt pathways by downregulating PDCD4.
27
Discussion
28
After cerebral IR injury, nerve cells undergo complex pathophysiological 9
1
processes. Treating cerebral IR injury requires alleviating this damage to save dying
2
nerve cells. Therefore, it is important to identify the targets in IR injury. MiRNAs
3
serve a crucial role in cerebral IR injury. For example, miR-340 has a role in nerve
4
injury(Cosset et al., 2016), and miR-340-5p is downregulated in peripheral blood
5
from patients with acute stroke (Yoo et al., 2018). However, the function of
6
miR-340-5p in IR-induced cerebral neural injury is unknown.
7
In the current study, we used primary rat hippocampal neurons to establish an in
8
vitro OGDR injury model that mimics cerebral IR injury. We found miR-340-5p to be
9
downregulated in primary hippocampal neurons that were injured by OGDR.
10
MiR-340-5p overexpression targeted PDCD4 to enhance cell viability and reduce
11
LDH activity and apoptosis, whereas miR-340-5p inhibition showed the opposite
12
effects. Our study confirmed the contribution of miR-340-5p in regulating OGDR
13
injury, revealing a potential target for treating cerebral ischemic stroke. In future
14
researches, it is needed to explore the function of miR-340-5p in other brain cells,
15
such as glial cells, to further understand the role of miR-340-5p in cerebral IR injury
16
and in cerebral diseases.
17
PDCD4 is a crucial regulator of apoptosis(Singh et al., 2019). It reportedly
18
participates in translation, transcription, and cell growth regulation(Matsuhashi et al.,
19
2019). Numerous studies have found low expression of PDCD4 in various cancers,
20
and it is associated with the progression of prostate cancer (Zennami et al., 2019),
21
colon carcinoma (Wang et al., 2017), and glioma(Monfared et al., 2019). Recently,
22
PDCD4 was identified as a crucial gene in the regulation of IR injury. A study has
23
shown that by regulating the miR-21/PDCD4 signaling pathway, serum-derived
24
extracellular vesicles protect mouse hearts against acute myocardial infarction (Gu et
25
al., 2018). Moreover, the lncRNA MEG3 regulates ischemic neuronal death by
26
targeting the miR-21/PDCD4 signaling pathway (Yan et al., 2017). In rat cardiac cells
27
exposed to IR injury, miR-21 regulates PDCD4, which exerts an anti-apoptotic effect
28
(Cheng et al., 2010). By targeting PDCD4, miR-499 inhibits cardiomyocyte apoptosis
29
and is involved in ischemic postconditioning-mediated cardiac protection against IR 10
1
injury (Zhu et al., 2016). In our study, it was revealed that miR-340-5p targeted and
2
negatively modulated the PDCD4 gene. PDCD4 protein levels markedly increased in
3
rat primary hippocampal neurons that were exposed to OGDR. Furthermore, we
4
confirmed that PDCD4 participated in the neuroprotective effect of miR-340-5p on
5
hippocampal neurons that were injured by OGDR.
6
The PI3K/Akt signaling pathway is an important survival signal in various nerve
7
cells. This pathway mainly involves PI3K, Akt, and their downstream molecules. It is
8
an important pathway for intracellular transduction of membrane receptor signals and
9
for various cellular processes, such as proliferation, differentiation, and apoptosis.
10
PI3K/Akt signaling plays a crucial role in cerebral IR injury(Kamel et al., 2019).
11
MiR-375 and Ctgf protect against cerebral IR injury by regulating the p21/PI3K/Akt
12
signaling pathways (Ou et al., 2017). MiR-19b exerts neuronal protective effects
13
during hypoxia/reoxygenation-induced injury by targeting PTEN via the PI3K/Akt
14
pathway (Liu et al., 2019). MiR-340 may impede laryngeal squamous cell carcinoma
15
progression by targeting EZH2 via p27 expression enhancement and PI3K/Akt
16
inactivation (Yu et al., 2016). MiR-340 suppression also has been shown to induce
17
activation of PI3K and Akt in ovarian cancer SKOV3 cells (Qu and Wang, 2017).
18
In this study, we found that OGDR injury blocked PI3K/Akt signaling and that
19
miR-340-5p mimics could activate this signaling. This result is contrary to the effects
20
of miR-340-5p on PI3K and Akt that have been previously described by (Yu et al.,
21
2016) and (Qu and Wang, 2017). These contradictory findings indicate that the effects
22
of miR-340-5p on the PI3K/Akt pathway might depend on the experimental
23
conditions. Moreover, there may be more than one target gene for miR-340-5p.
24
Therefore, miR-340a-5p may influence different target gene and then have
25
contradictory effect on PI3K/Akt signaling in different disease. Previous research
26
indicates that PDCD4 may impede Akt activation(Cristofoletti et al., 2019). Our data
27
reveal that miR-340-5p activated suppression of the PI3K/Akt pathway in
28
OGDR-injured hippocampal neurons and that PDCD4 overexpression partly reversed
29
this effect, which suggested miR-340-5p regulated OGDR-induced neurons injury 11
1
partially by modulating PI3K/Akt pathway. In future research, we should explore
2
other target gene of miR-340-5p apart from in ischemic stroke, and further understand
3
the underlying mechanism of miR-340-5P in brain injury. Additionally, the
4
down-stream of PDCD4 should also be investigated, thus other I/R related signaling
5
pathways regulated by PDCD4 should be explored.
6
In summary, our study suggests that miR-340-5p alleviated OGDR-induced
7
injury to neurons by targeting PDCD4 to activate the PI3K/Akt pathway. We
8
identified miR-340 as a promising target for the prevention of cerebral ischemic injury.
9
Our in vitro experiment examined the role of miR-340-5p in hippocampal neurons
10
that were exposed to OGDR injury. Its role in cerebral IR injury needs to be further
11
investigated in vivo.
12
Conflicts of interest
13
No conflict of interest.
14
12
1
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3 4
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1
Figure legends
2
Figure 1. Downregulation of miR-340-5p in hippocampal neurons after
3
oxygen-glucose deprivation/reoxygenation (OGDR). (A) The qRT-PCR results of
4
miR-340-5p in primary hippocampal neurons after exposure to OGDR. (B) The
5
relative miR-340-5p abundance in OGDR-injured hippocampal neurons after
6
transfection with miR-340-5p mimics or miR-340-5p inhibitors for 24 h. N=3,
7
#
8
Figure 2. MiR-340-5p relieved OGDR-induced neuronal injury. (A) Cell viability
9
was detected using a CCK-8 assay. (B) LDH release in culture medium. (C) Apoptosis
10
analyzed by flow cytometry, apoptosis cell rate were represented in right upper
11
quadrant. (D) Western blot analysis of protein content of Bax and Bcl-2 in
12
hippocampal neurons. (E) Ratio of Bax/Bcl-2. N=3, #P<0.05 vs. control; *P<0.05 vs.
13
OGDR.
14
Figure 3. PDCD4 was a target of miR-340-5p. (A) Dual-Luciferase activity assay
15
results of the interaction between miR-340-5p and the 3’-UTR of PDCD4 in HEK293
16
cells. (B) Dual-Luciferase activity assay results in OGDR induced hippocampal
17
neurons. (C) The putative binding sequences of the 3’-UTR of PDCD4 with
18
miR-340-5p. (D and E) The protein content of PDCD4, tested by western blot. N=3,
19
#
20
Figure 4. MiR-340-5p alleviated injury to hippocampal neurons exposed to OGDR by
21
downregulating PDCD4. OGDR stimulated Hippocampal neurons were co-transfected
22
with miR-340-5p mimics and a PDCD4 vector. (A) Relative PDCD4 protein content.
23
(B) Cell viability. (C) LDH activity. (D) Apoptosis. (E) Relative protein content of
24
Bax and Bcl-2. (F) Ratio of Bax and Bcl-2. N=3, #P<0.05 vs. OGDR; *P<0.05 vs.
25
OGDR+miR-340-5p mimic.
26
Figure 5. MiR-340-5p alleviated injury to hippocampal neurons exposed to OGDR by
27
activating PI3K/Akt signaling pathway. (A) Western blot analysis of p-PI3K, PI3K,
28
p-Akt, and Akt protein content. (B) Quantitative analysis of relative protein content of
P<0.05 vs. control; *P<0.05 vs. OGDR.
P<0.05 vs. mimic control.
18
1
p-PI3K and PI3K. (C) Quantitative analyses of relative protein content of p-Akt and
2
Akt. N=3, #P<0.05 vs. control; *P<0.05 vs. OGDR.
3
Figure 6. MiR-340-5p activated PI3K/Akt signaling pathway by targeting PDCD4 in
4
OGDR induced neurons. (A) Western blot analysis of p-PI3K, PI3K, p-Akt, and Akt
5
protein content. (B) Quantitative analysis of relative protein content of p-PI3K and
6
PI3K. (C) Quantitative analysis of relative protein levels of p-Akt and Akt. N=3,
7
#
P<0.05 vs. control; *P<0.05 vs. OGDR. (D) Schematic diagram.
19
Highlights 1.
Low expression of miR-340-5p in hippocampal neurons exposed to OGDR
2.
MiR-340-5p had protective effect against neuronal injury caused by OGDR
3.
MiR-340-5p directly regulated PDCD4 expression
4.
MiR-340-5p inhibited PDCD4 to protect hippocampal neurons against OGDR injury
5.
MiR-340-5p targeted PDCD4 to influence PI3K/Akt signaling
Yake Zheng: Design, Experiment perform, Writing original draft. Peng Zhao: Experiment perform, Data analyze Yajun Lian: Design, Supervision, Paper revise Shuang Li: Experiment perform, Data analyze Yuan Chen: Experiment perform, Paper revise Lihao Li: Experiment perform,