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Metabolic Brain Disease (2023) 38:2077–2091

https://doi.org/10.1007/s11011-023-01219-1

ORIGINAL ARTICLE

miR‑181a targets PTEN to mediate the neuronal injury caused

by oxygen‑glucose deprivation and reoxygenation
Shengnan Li1,2,3 · Peiyi Zhu1,2 · Yajun Wang4 · Shaoting Huang1,2,3 · Zhaochun Wu1,2 · Jiawen He1,2 ·
Xingjuan Hu1,2 · Ying Wang1,2 · Yanquan Yuan1,2 · Bin Zhao1,2,3 · Guoda Ma4 · You Li1,2,3

Received: 2 December 2022 / Accepted: 18 April 2023 / Published online: 13 May 2023
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023

Abstract
Evidence suggests that the microRNA-181 (miR-181) family performs various roles in the pathophysiology of cerebral
ischemia and reperfusion injury (CIRI). MiR-181a has been identified as a critical determinant of neuronal survival. Moreo-
ver, the significance of miR-181a in controlling neuronal death after CIRI has received little attention. The objective of this
study was to assess the role of miR-181a in neuronal cell injury after CIRI. To mimic the in-vitro and in-vivo CIRI, we
developed an oxygen-glucose deficiency/reoxygenation (OGD/R) model in SH-SY5Y cells and a transient middle cerebral
artery occlusion model in rats. MiR-181a expression was significantly higher in both in-vivo and in-vitro CIRI models. The
overexpression of miR-181a increased cell damage and oxidative stress caused by OGD/R, whereas inhibition of miR-181a
reduced both. PTEN has also been found to be a direct miR-181a target. PTEN overexpression reduced cell apoptosis and
oxidative stress induced by miR-181a upregulation under an OGD/R condition. Furthermore, we found that the rs322931 A
allele was related to increased miR-181a levels in IS peripheral blood and higher susceptibility to IS. The current results offer
new insights into the understanding of the molecular pathophysiology of CIRI, as well as possible new treatment candidates.

Keywords Ischemic stroke · miR-181a · PTEN · Oxygen-glucose deprivation and reoxygenation · Polymorphism

Introduction recanalization of blood vessels through thrombolysis or

Ischemic stroke (IS) is defined by an abrupt interruption in
blood flow produced by embolic or thrombotic blockage of
major arteries in the brain (Cuartero et al. 2016). Timely
Shengnan Li, Peiyi Zhu and Yajun Wang contributed equally to this
work.
* Guoda Ma
sihan1107@126.com
* You Li
youli805@163.com
1
Guangdong Key Laboratory of Age‑Related Cardiac
and Cerebral Diseases, Affiliated Hospital of Guangdong
Medical University, Zhanjiang 524001, China
2
Department of Neurology, Affiliated Hospital of Guangdong
Medical University, Zhanjiang 524001, China
3
Institute of Neurology, Affiliated Hospital of Guangdong
Medical University, Zhanjiang 524001, China
4
Shunde Maternal and Children’s Hospital, Maternal
and Children’s Health Research Institute, Guangdong
Medical University, Shunde 528300, China
mechanical thrombectomy is essential for treating IS. On the
other hand, blood reperfusion may aggravate the neuronal
injury induced by ischemia, a condition known as cerebral
ischemia-reperfusion injury (CIRI) (Jean et al. 1998). The
CIRI pathogenesis is complex. It is characterized by a suc-
cession of neurologic disturbances such as excitatory toxic-
ity, necrosis, apoptosis, inflammation, oxidative stress, and
calcium dysregulation, leading to irreparable brain tissue
damage (Moskowitz et al. 2010). As a result, it is critical to
understand CIRI-induced neuronal injury to develop effi-
cient neuroprotective therapies for IS.
MiR-181a, one of the most abundant members of the
miR-181 family, is a brain-enriched miRNA that contrib-
utes to the pathogenesis of CIRI. MiR-181a expression was
increased in the infarct core but decreased in the penumbra
following transient focal ischemia (Ouyang et al. 2012). Fur-
thermore, post-treatment with a miR-181a blocker in mice
reduced infarction size, improved neurological impairments,
and reduced forebrain ischemia-induced neuronal loss (Xu
et al. 2015). A genome-wide association study (GWAS)
found that a genetic variant of rs322931 might influence

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positive emotion via the nucleus accumbens and miR-181
(Wingo et al. 2017). Despite these advances, the exact
regulatory mechanism of miR-181a in CIRI remains to be
investigated.
Phosphatase and tensin homologs (PTEN), also known as
tumor suppressors, work by inhibiting the activation of the
PI3K/Akt signaling pathway. PTEN mediates multiple cellu-
lar processes such as cellular configuration, cell proliferation
and survival, energy homeostasis, and inflammation (Song
et al. 2012). Furthermore, through the PI3K/AKT signaling
cascade, PTEN may modulate caspase activation in mitochon-
dria-mediated apoptosis (Ghafouri-Fard et al. 2021). PTEN was
also observed to be significantly reduced in the infarct areas
of MCAO-treated mice (Pan et al. 2022), and OGD/R treated
neuronal cells (Guo et al. 2019) in recent studies. PTEN is a
target of miRNAs in various human disorders, such as CIRI.
Notably, the bioinformatic study revealed possible bind-
ing sites for PTEN and miR-181a. However, whether the
miR-181a/PTEN axis plays a regulatory role in CIRI was
uninvestigated. This work aims to look at the roles of miR-
181a and PTEN in CIRI-induced neuronal cell injury and
the underlying regulatory mechanisms. In addition, the
study will examine the relationship between the miR-181a
rs322931 variant and IS risk in a southern Chinese commu-
nity. This could lead to the identification of new targets for
the treatment of IS.
Materials and methods
Occlusion of the middle cerebral artery (MCAO)
in rats
All experimental procedures were approved by the Institu-
tional Animal Care and Use Committee of the Guangdong
Medical University, and carried out in accordance with the
recommendations in the Guide for the Care and Use of Lab-
oratory Animals of the National Institutes of Health. The
male Sprague-Dawley (SD) rats (weight = 250-300 g) were
obtained from Guangdong Experimental Animal Center,
China. CIRI was induced by middle cerebral artery (MCA)
occlusion (MCAO) for 2 h with subsequent reperfusion for
24 h as we have described previously (Li et al. 2021).
Cell culture and OGD/R modeling
SH-SY5Y cell was obtained from American Type Culture
Collection (ATCC, Rockville, MD, USA). Cells were main-
tained in DMEM (Thermo, Waltham, MA, USA) containing
10% fetal bovine serum (FBS, Thermo, Beijing, China) and
100 U/mL antibiotics (penicil-lin–streptomycin) at 37 °C
with 5% ­CO2. An in-vitro CIRI model was developed using
an OGD/R injury. For 2 h, SH-SY5Y cells were cultured
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Metabolic Brain Disease (2023) 38:2077–2091
in glucose-free DMEM at 3­ 7oC with 95% N ­ 2 and 5% C
­ O 2.
The SH-SY5Y cells were reoxygenated after 3 h, 6 h, 12 and
24 h of being returned to normal conditions using glucose-
containing DMEM instead of glucose-free DMEM.
Cell transfection
The Lipofectamine 2000 was applied to transfect the SH-
SY5Y cells with miR-181a mimic, inhibitors, correspond-
ing negative controls (miR-NC and inhibitor NC), and
full-length PTEN (GeneChem Corp., Shanghai, China).
Quantitative real-time polymerase chain reaction (qRT-PCR)
confirmed that the transfection was successful 48 h after the
procedure.
qRT‑PCR
Through the Trizol reagent (Qiagen, CA, USA), the total
RNA from the brain, blood, and cultured SH-SY5Ys was
extracted. Then, RNA was converted into cDNA via a
SuperScript II First-Strand cDNA Synthesis Kit (Thermo,
CA, USA). SYBR Premix ExTag TMII kit (Takara, Japan)
was utilized in a quantitative real-time RT-PCR on an ABI
7500 Fast Real-time PCR system (Applied Biosystems, CA,
USA). GAPDH and U6 were employed as internal controls
for the ­2−ΔΔCt method (Livak and Schmittgen 2001), which
calculates the relative expression of targets. The correspond-
ing primers were listed in Table S1.
Cell viability assay
Transfected or untransfected SH-SY5Ys with or without
OGD were treated and seeded into 96-well plates at a rate
of ~ 1 × ­104. A further 24 h of incubation was required before
OGD/R treatment after the cells had been transfected with
the appropriate miRNA, inhibitor, controls, and/or full-
length PTEN constructs. The cells were then incubated
for an additional 4 h with fresh media supplemented with
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) (Sigma, CA, USA). The optical density (OD)
was detected at an absorbance of 450 nm and was recorded
to calculate cell viability.
Lactate dehydrogenase (LDH) assay
The LDH Cytotoxicity Assay Kit (Promega Corporation,
USA) was used to detect cell death in SH-SY5Ys. An LDH
reaction substrate was utilized for 1 h at 37 °C after transfec-
tion or OGD treatment. The supernatants were then mixed
for 30 min with LDH detection reagent, and the OD values
at 490 nm were measured.
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Metabolic Brain Disease (2023) 38:2077–2091
Dichlorofluoresceindiacetate (DCFH‑DA) assay
SH-SY5Ys were treated for 20 min at 37 °C with the DCFH-
DA probe to facilitate ROS detection. Supernatants were
then removed, and through flow cytometry (FACSCanto II,
Becton, Dickinson and Company, USA), the collected cells
were assessed for DCFH-DA staining.
Apoptosis assay
An Annexin-V/propidium iodide apoptosis detection kit
(vazyme, Nanjing, China) was used to evaluate the cell apop-
tosis of SH-SY5Ys. SH-SY5Ys were harvested and stained
with Annexin V-FITC/PI for 15 min after being transfected
with appropriate miRNA, inhibitor, and/or constructs for
24 h. Cell apoptosis was then detected using a FACSCanto
II flow cytometer (Becton Dickinson and Company). The
apoptosis rate was calculated through the total percentage
of early and late apoptotic cells.
Dual‑luciferase reporter assay
From wild-type and mutant PTEN, the seed sequence of the
miR-181a-binding site was cloned into the pmirGLO dual-
luciferase reporter vector. These luciferase reporter vectors
were co-transfected into SH-SY5Ys with miR-181a mimics
or equivalent controls using Lipofectamine 2000 (Invitro-
gen, CA, USA). The Dual-luciferase reporter assay system
(Promega, WI, USA) detected the relative luciferase activity
48 h after transfection.
Western blot
RIPA (Radioimmunoprecipitation assay) lysis buffer was used
to extract total proteins from cultured SH-SY5Ys. After BCA
(Bicinchoninic acid) quantification, equal amounts of protein
were separated on a 12% SDS-PAGE and deposited onto PVDF
membranes. The membranes were incubated at 4 °C overnight
after blocking with 5% skim milk with specific primary anti-
bodies, including anti-GAPDH (Abcam; 1:1000), anti-Bax
(Abcam; 1:1000), anti-Bcl-2 (Abcam; 1:1000), anti-cleaved
caspase-3 (Abcam; 1:1000), and anti-PTEN (Abcam; 1:500).
After that, with secondary HRP-conjugated goat anti-rabbit
IgG (Abcam; 1:2000), the blots were probed for 2 h. Enhanced
chemiluminescence reagents (Pierce, Thermo, USA) were uti-
lized to observe the protein signals, and Image-Pro Plus 6.0
software was applied to calculate the grey values.
Patient characteristics
The Ethics Committee of Guangdong Medical University’s
Affiliated Hospital approved the current study. The informed
consent letter was obtained from each member enlisting before
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the investigation. Between 2015 and 2019, 1061 age-matched
healthy controls and 1035 IS patients were tested at Guang-
dong Medical University’s Affiliated Hospital. Magnetic reso-
nance imaging, computed tomography scans, and clinical signs
and symptoms tests were used to examine patients with IS
independent. Patients with a history of CTI (cerebral, tran-
sient ischemia), or subarachnoid hemorrhage, auto-immune
illnesses (such as malignant tumors, chronic infections, hema-
tological, systemic inflammatory, or coronary artery disease)
were excluded from the investigations. The controls in this
study did not have tumors with malignant status, autoimmun-
ity, chronic inflammation, or an IS history.
Genotyping of rs322931 variants
According to past studies (Lancaster et al. 2017; Wingo et al.
2017), the rs322931 SNP may affect the expression of miR-
181a in blood and brain, therefore, we are interested in whether
the rs322931 variant of miR-181a affects the risk of IS. A
DNA purification kit was used to extract genomic DNA from
peripheral blood leukocyte samples. The rs322931 genotyping
was accomplished with the primers listed in Table S1 using
an Improved Multiplex, Ligase-Detection Reaction technique.
Statistical analysis
The statistical data, presented as means and standard devia-
tion (± SD) from triplicate experiments, was examined
using GraphPad Prism 5.0 (GraphPad, CA,USA). One-way
ANOVA or Student’s t-tests were utilized to compare data
between groups, with a P < 0.05 as a significance threshold.
The categorical variables and HWE were related using the
X 2 test for the gene polymorphism analysis. Student’s t-tests
have given consecutive data. The Mann-Whitney U-test was
applied if the information was not distributed as expected.
The additive dominant and recessive models were used to
link genotype distribution between patients and controls.
The connection between definite variants and IS risk was
assessed through OR (odds ratios) with a 95% CI (confi-
dence interval) after modifying several criteria, i.e., hyper-
lipidaemia, diabetes mellitus, smoking, high blood pressure,
sex, and age. Bonferroni correction was used in the statis-
tical analysis to account for differences when considering
control type-1 error.
Results
miR‑181a and PTEN are involved in CIRI
In both in-vivo and in-vitro CIRI models, the expression
profiles of miR-181a and PTEN were detected; to investi-
gate the possible relevance of miR-181a and PTEN in CIRI.
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2080 Metabolic Brain Disease (2023) 38:2077–2091

In rats treated with MCAO-2 h/reperfusion-24 h, RT-PCR
results revealed that miR-181a levels were significantly
upregulated (Fig. 1a), while PTEN expression was signifi-
cantly downregulated (Fig. 1b). Western blotting also con-
firmed that PTEN was considerably decreased in the MCAO
model (Fig. 1c, d). Similarly, miR-181a levels in SH-SY5Y
cells increased significantly, with expression increasing as
the reoxygenation step was extended from 3 to 12 h, but
down-regulation at 24 h of reoxygenation (Fig. 1e). The
PTEN levels decreased significantly in SH-SY5Y cells,
with expression dropping when the reoxygenation phase was
extended from 3 to 12 h but increasing at 24 h of reoxygena-
tion (Fig. 1f-h). Based on our findings, we carefully selected
the 12 h reoxygenation time point for the supplementary

Fig. 1  The expression of

miR-181a and PTEN in in invo
and in vitro models of IS. a, b
Rats were subjected to sham
and MCAO for 2 h, followed
by 24 h of reperfusion. Then,
brain tissues were harvested and
detected with qRT-PCR. The
whiskers indicate error bars.
c, d The expression of PTEN in
brain tissues was quantified by
western blotting. e, f SH-SY5Y
cells were exposed to OGD for
4 h and reoxygenation for 3 h,
6 h, 12 h, 24 h. The expression
of miR-181a and PTEN was
determined by qRT-PCR. g, h
The expression of PTEN in SH-
SY5Y cells exposed to OGD for
4 h and reoxygenation for 3 h,
6 h, 12 h, 24 h, was quanti-
fied by western blotting. Cells
cultured in normal medium and
normoxic conditions served
as a control (n.s., not sig-
nificant, *P < 0.05, **P < 0.01,
***P < 0.001)

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Metabolic Brain Disease (2023) 38:2077–2091
OGD/R model investigation. These results indicate that miR-
181a and PTEN deregulation may be closely linked to CIRI
pathogenesis.
OGD/R‑induced cell injury is reduced
when miR‑181a is inhibited
To elucidate the specific biological function of miR-181a
in OGD/R-treated SH-SY5Y cells, we performed loss-of-
function and gain-of-function investigations using miR-181a
inhibitors or miR-181a mimics (Fig. 2a) and assessed their
impact on OGD/R-induced cell injury. The MTT assays
revealed that silencing miR-181a enhanced cell viability in
SH-SY5Y cells treated with OGD/R (Fig. 2b). While the
lactate dehydrogenase (LDH) assay showed that silencing
Fig. 2  Inhibition of miR-181a alleviates OGD/R-induced cell injury.
a Relative expression of miR-181a in SH-SY5Y cells transfected with
miR-181a mimics ,inhibitor or respective controls was detected by
qRT-PCR. b MTT assay showed that cell viability was significantly
decreased in SH-SY5Y cells transfected with miR-181a mimics
and increased in SH-SY5Y cells transfected with miR-181a inhibi-
tor (antagomir) after OGD/R treatment. c LDH assay showed that
overexpression of miR-181a substantially aggravated the cell death,
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miR-181a lowered OGD/R-induced cell damage signifi-
cantly (Fig. 2c). Additionally, ROS levels were dramatically
enhanced by OGD/R therapy, whereas miR-181a suppres-
sion significantly decreased ROS production (Fig. 2d). This
evidence collectively demonstrated that miR-181a suppres-
sion abates OGD/R induced cell injury.
OGD/R‑induced cell apoptosis is reduced
by inhibiting miR‑181a
Additionally, we evaluate how miR-181a expression
affects cellular apoptosis using Annexin V-FITC/PI
staining, suggesting that cellular apoptosis was elevated
following OGD/R treatment. Notably, overexpression
or suppression of miR-181a significantly increased or
while inhibition of miR-181a suppressed cell death induced by 4 h
OGD/12 h reoxygenation. d After transfection with appropriate mim-
ics, inhibitor, and respective controls for 24 h as appropriate followed
by a 4 h OGD/12 h reoxygenation exposure, flow cytometry was uti-
lized to measure ROS production in SH-SY5Y cells. Overexpression
of miR-181a substantially facilitated ROS production, while inhibi-
tion of miR-181a reduced ROS production in SH-SY5Y cells after
OGD/R treatment (*P < 0.05, **P < 0.01, ***P < 0.001)
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2082 Metabolic Brain Disease (2023) 38:2077–2091

reduced the rate of OGD/R-induced apoptosis, respec- in the apoptosis-related proteins. We showed that miR-
tively (Fig. 3a, b). We also used western blot to deter- 181a inhibition leads to decreased cleaved caspase-3
mine if Bax, Bcl-2, and cleaved caspase-3 were involved and Bax levels and increased Bcl2 expression, whereas

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◂Fig. 3  Inhibition of miR-181a leads to reduced SH-SY5Y cell apop-
tosis in response to OGD/R conditions. a After transfection with
appropriate mimics, inhibitor, and respective controls for 24 h as
appropriate followed by a 4 h OGD/12 h reoxygenation exposure,
cell apoptosis was determined by Annexin V-FITC/PI staining in SH-
SY5Y cells. b The apoptotic cell rate was also shown in each group.
c After transfection with appropriate mimics, inhibitor, and respec-
tive controls for 24 h as appropriate followed by a 4 h OGD/12 h
reoxygenation incubation, western blotting was explored to assay the
expression of apoptosis-related caspase3, Bcl-2 and Bax in SH-SY5Y
cells. d-f Relative expression levels of caspase3, Bcl-2 and Bax were
quantified and normalized (*P < 0.05, **P < 0.01)
miR-181a overexpression resulted in the opposite phe-
notype (Fig. 3c-f).
miR‑181a directly targets PTEN
As per the bioinformatics analysis, PTEN is a potential tar-
get gene for miR-181a (Fig. 4a). Overexpression of miR-
181a significantly inhibited the luciferase reporter contain-
ing wild-type PTEN 3’-UTR but did not affect the luciferase
reporter containing mutant PTEN 3’-UTR according to the
dual-luciferase reporter assay (Fig. 4b). Moreover, miR-
181a inhibition or overexpression significantly increased
or decreased PTEN expression, suggesting that PTEN is a
miR-181a target gene (Fig. 4c-e).
Fig. 4  miR-181a directly targets PTEN. a Alignment of miR-181a
with WT PTEN-3′UTR and MUT-PTEN-3′UTR showing the com-
plementary pairing. b Dual luciferase assay was performed in SH-
SY5Y cells by co-transfection with the WT PTEN-3′UTR or MUT-
PTEN-3′UTR and with miR-181a mimics or corresponding controls.
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miR‑181a targets PTEN to aggravate OGD/R‑induced
cell injury
The next step was to explore if miR-181a’s ability to increase
cell I/R injury was associated with the ability to inhibit PTEN
expression. First, we constructed plasmids that represented
the entire length of PTEN. Transfected cells with full-length
PTEN led to a significant increase in PTEN expression in con-
trols (Fig. 5a-c). Furthermore, miR-181a mimic transfection in
SH-SY5Y cells decreased PTEN expression in the framework
of OGD/R treatment, whereas miR-181a inhibition caused the
opposite phenotype. In PTEN overexpressed cells, the ability
of miR-181a overexpression (enhanced cell death, increased
ROS production and reduced cell viability) was significantly
reversed (Fig. 5f-h). This revealed that PTEN overexpression
hampered the ability of miR-181a upregulation to cause neu-
ron damage in the event of CIRI.
Overexpressing of PTEN abolishes miR‑181a
upregulation’s ability to cell apoptosis
We found that the reliance on miR-181a-mediated con-
trol of SH-SY5Y cell apoptosis induced by OGD/R dam-
aged PTEN expression. When compared to control cells,
PTEN overexpression significantly reduces OGD/R-induced
qRT-PCR (c) and western blotting (d) was used to detect the expres-
sion of PTEN in SH-SY5Y cells transfected with miR-181a mimics,
inhibitor or and respective controls. (e) Relative protein expression of
PTEN was quantified and normalized (n.s., not significant, *P < 0.05,
***P < 0.001)
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Fig. 5  miR-181a targets PTEN to aggravate cell injury in response
to OGD/R. Constructs harbouring the full-length PTEN (PTEN-FL)
were prepared. PTEN-FL efficiently overexpresses the expression
of PTEN both in mRNA level (a) and protein level (b and c). d The
expression of PTEN in SH-SY5Y cells transfected with miR-181a
mimics, inhibitor or and respective controls after OGD/R treatment; e
apoptosis. When miR-181a was overexpressed, it accelerated
OGD/R-induced apoptosis, whereas PTEN overexpression
reversed this effect (Fig. 6a, b). According to this, in OGD/
R-exposed cells, cleaved caspase-3 and Bax levels were
improved, followed by PTEN overexpression, and Bcl-2
levels were increased. Upregulating miR-181a increased
Bax and cleaved caspase 3 levels while decreasing Bcl2,
whereas PTEN overexpression cells had the opposite effect
(Fig. 6c-e). As previously stated, miR-181a targets PTEN to
cause cellular death in OGD/R-induced cells.
miR‑181a rs322931 variants and the morbidity of IS
We next explored the association between miR-181a
rs322931 variants and IS in a Southern Chinese population.
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Metabolic Brain Disease (2023) 38:2077–2091
Relative protein expression of PTEN was quantified and normalized;
f MTT assay showed PTEN-FL improved cell viability decreased by
miR-181a overexpression; g LDH assay indicated that PTEN-FL alle-
viated cell injury caused by miR-181a overexpression; h PTEN-FL
reduced the production of ROS caused by miR-181a overexpression
(*P < 0.05, **P < 0.01, ***P < 0.001)
We collected blood samples from 1035 IS patients and 1061
healthy controls. Table 1 is supplemented with basic infor-
mation on stroke patients and healthy controls. There were
no substantial differences between the two groups in age,
serum uric acid, low-density lipoprotein, and total choles-
terol (P > 0.05). However, there were differences between the
IS and the controls regarding gender, hypertension, diabetes,
and smoking status (P < 0.001). The IS group had higher tri-
glyceride and homocysteine levels than the control group but
lower high-density lipoprotein cholesterol levels (P < 0.001).
The genotype distribution and allelic frequency of the
rs322931 variants are shown in Table 2. The tested varia-
tions do not vary from Hardy Weinberg equilibrium in both
groups (P > 0.05). A genotypic study of IS patients and
controls, on the other hand, found a statistical correlation
between the rs322931 variations and IS risk (P = 5.6 × ­10− 3).
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Metabolic Brain Disease (2023) 38:2077–2091
Fig. 6  Overexpressing PTEN reverses the ability of miR-181a upreg-
ulation to cell apoptosis. a Cell apoptosis was detected by Annexin
V-FITC/PI double staining in SH-SY5Y cells transfected with differ-
ent groups following 4 h OGD/12 h reoxygenation exposure; b The
apoptotic cell rate was also shown in each group. c Western blotting
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was used to determine the expression of apoptosis-related caspase3,
Bcl-2, and Bax as well as PTEN in SH-SY5Y cells transfected with
different groups following 4 h OGD/12 h reoxygenation exposure.
d-g Relative protein expression of PTEN, caspase3, Bcl-2, and Bax
was quantified and normalized (*P < 0.05, **P < 0.01, ***P < 0.001)
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Table 1  Characteristics of IS Variables IS patients Controls P value P value*

cases and controls (n = 1035) (n = 1061)

Mean age (years) 65.5 ± 9.89 65.3 ± 8.90 0.63 0.65

Gender (%)
Male 700(67.6) 522(49.2) < 0.001 < 0.001
Female 335(32.4) 539(50.8)
Smoking (%)
Yes 288(27.8) 131(12.3) < 0.001 < 0.001
No 747(72.2) 930(87.7)
Hypertension (%)
Yes 785(75.8) 352(33.2) < 0.001 < 0.001
No 250(24.2) 709(66.8)
Diabetes (%)
Yes 341(32.9) 121(11.4) < 0.001 < 0.001
No 694(67.1) 940(88.6)
Uric acid (mmol/L) 315.8 ± 88.4 313.1 ± 90.3 0.49 0.72
Total cholesterol (mmol/L) 5.08 ± 1.08 5.09 ± 0.99 0.83 0.94
Triglycerides (mmol/L) 1.62 ± 1.09 1.43 ± 0.97 < 0.001 < 0.001
LDL-cholesterol (mmol/L) 3.07 ± 1.03 3.02 ± 0.92 0.24 0.42
HDL-cholesterol (mmol/L) 1.35 ± 0.64 1.47 ± 0.42 < 0.001 < 0.001
HCY(mmol/L) 11.05 ± 6.06 9.93 ± 3.33 < 0.001 < 0.001

HCY: Homocysteine; HDL: high-density lipoprotein; IS: ischemic stroke; LDL: low-density lipoprotein;
Continuous data are presented as the mean ± standard deviation, median (range) or n (%)
P < 0.05 is indicated in bold font
* False discovery rate-adjusted P value for multiple hypotheses testing using the Benjamini-Hochberg
method

Significant differences in rs322931 frequency in IS patients
compared to the control group were seen both in a domi-
nant (GG + GA/AA) (P = 5.4 × ­10− 3) and recessive model
(GA + AA/GG) (P = 7.2 × ­1 0 − 4). Substantially, the IS
patients harbour higher frequencies of the variant A allele at
rs322931 than the controls (P = 3.8 × ­10− 4). We also looked
to see if the rs322931 variants were restricted to a specific
subset, and we divided the IS patients into stroke subsets
based on the Trial of Org 10,172 in Acute Stroke Treat-
ment (TOAST) classification. Carriers of the rs322931 A
allele (P = 2.9 × ­10− 4) had a higher risk of stroke in the LAA
subset than controls, as shown in Table 3. Following that,
we looked at the relationship between the rs322931 variants
that were studied and the demographic features of IS patients
and controls. We observed the rs322931 after categorizing
participants based on age, gender, smoking status, diabetes,
and hypertension. The allele A was associated with a greater
risk of IS in both ≥ 65-year-old individuals (P = 1.5 × ­10− 3),
males (P = 1.5 × ­10− 3), smokers (P = 1.5 × ­10− 3), hyper-
tensive patients (P = 1.5 × ­10− 3) and non-diabetic patients
(P = 1.6 × ­10− 3) (as shown in Table 4).

Table 2  Frequencies of miR- rs322931 IS patients Controls AOR (95% CI) P value P ­value*
181a rs322931 genotypes n = 1035(%) n = 1061(%)
and alleles in IS patients and
controls GG 654(63.2) 746(70.3) 4.1 × 10− 4 8.2 × 10− 4
Genotypes GA 331(32.0) 288(27.1)
AA 50(4.8) 27(2.5)
Dominant model GG/GA vs. AA 985(95.2) 1034(97.5) 0.51(0.32–0.83) 5.4 × 10− 3 5.4 × 10− 3
Recessive model GG vs. GA/AA 381(36.8) 315(29.7) 1.38(1.15–1.66) 5.4 × 10− 4 7.2 × 10− 4
Allele G 1639(79.2) 1780(83.9)
A 431(20.8) 342 (16.1) 1.37(1.17–1.60) 9.4 × 10− 5 3.8 × 10− 4

P < 0.05 is indicated in bold font

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Table 3  The relationship between miR-181a rs322931 variant and stroke subtypes in IS patients
rs322931 Genotype (%) P value
GG GA AA
Control (n = 1061) 746(70.3) 288(27.2) 27(2.5)
LAA (n = 656) 410(62.5) 209(31.9) 37(5.6) 2.1 × 10− 4
SAA (n = 282) 182(64.5) 88(31.2) 12(4.3) 0.096
CE (n = 35) 22(62.9) 13(37.1) 0(0) 0.41
UE (n = 62) 40(64.5) 21(33.9) 1(1.6) 0.53
LAA: Large-artery atherosclerosis; SAO: Small-artery occlusion; CE: Cardioembolic; UE: Unspecified aetiology
*
False discovery rate-adjusted P value for multiple hypotheses testing using the Benjamini-Hochberg method. P < 0.05 is indicated in bold font
Effect of rs322931 variants on miR‑181a expression
MiR-181a expression levels were assessed in the peripheral
blood mononuclear cells of 89 IS patients and 94 controls.
We discovered that the mean value of miR-181a levels in IS
patients was significantly higher than in controls (P < 0.001)
(Fig. 7a). Furthermore, when IS patients were subdivided
based on rs322931 genotypes, we found that those with the
rs322931 GA + AA genotype had significantly higher miR-
181a expression than those with the rs322931 AA genotype
(P = 0.016) (Fig. 7b). When the rs322931 GA + AA and GG
genotypes were compared, there was no significant differ-
ence in miR-181a expression in control samples (P > 0.05).
Discussion
Apoptosis of neuronal cells plays a significant part in
the pathophysiology of cerebral stroke, and miRNAs
have been implicated as essential regulators of neu-
ronal death during CIRI. In the current investigation,
we discovered that miR-181a expression was elevated
in both in vivo and in vitro CIRI experimental models.
Suppressing miR-181a reduced oxidative stress and the
OGD/R-induced cell injury, whereas overexpression of
miR-181a had the opposite effects. Furthermore, inhibit-
ing miR-181a reduces OGD/R-induced cell damage by
directly targeting PTEN. In addition, we discovered that
the miR-181a rs322931 variations were linked to a high
IS risk and increased miR-181a expression in a Southern
Chinese population. These results indicated that the miR-
181a/PTEN axis is involved in CIRI and plays an impor-
tant role in OGD/R-induced neuronal cell apoptosis.
MiR-181a is the most common member of the miR-
181 family and is found in the brain. MiR-181a expres-
sion level was higher in the ischemic core of MCAO-
treated mice and OGDR-induced neurons in previous
research (Song et al. 2021). Furthermore, inhibiting
miR-181a reduced cerebral ischemia in MCAO mice
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P ­value* Allele (%) P value P ­value* OR (95% CI)
G A
1780(83.9) 342 (16.1)
8.4 × 10− 4 1029(78.5) 283(21.5) 7.3 × 10− 5 2.9 × 10− 4 1.43(1.20–1.71)
0.19 452(79.8) 112(20.2) 0.037 0.074 1.29(1.02–1.63)
0.53 57(81.4) 13(18.6) 0.62 0.60 1.19(0.64–2.19)
0.53 101(82.3) 23(17.7) 0.45 0.62 1.19(0.74–1.89)
by reducing apoptosis, neurological deficit, and infarct
volume (Zhang et al. 2019). Similarly, as previously
reported, miR-181a overexpression facilitated cellular
injury in primary cortical neurons following OGD by
decreasing cell viability, enhancing LDH release, and
increasing apoptosis. Though, the precise mechanism
of miR-181a in CIRI is still unknown. As a result, we
found that miR-181a was upregulated in MCAO rats and
OGD/R cells. Further, the downregulation of miR-181a
could ameliorate OGD/R-induced neuronal cell injury.
After that, we examined the miR-181a’s downstream
pathway in CIRI and identified that PTEN is a direct miR-
181a target gene. Surprisingly, the miR-181a/PTEN axis
has recently been found to be associated with osteosarcoma
(Sun et al. 2022), pancreatitis (Li et al. 2020a), asthma
(Lv et al. 2019), and type 2 diabetes mellitus (Song et al.
2021), implying that this axis may be a key contributor to
disease. Our findings are the first to demonstrate that miR-
181a directly suppresses PTEN levels in SH-SY5Y cells in
response to OGD/R treatment.
PTEN is a tumor inhibitor required for CIRI because it
acts as a suppressor of the phosphatidylinositol 3-kinase/
AKT/mTOR pathway (Ghafouri-Fard et al. 2021). Numer-
ous microRNAs (miRNAs) that inhibit PTEN expression
at the post-transcriptional level have been identified.
Through suppressing PTEN, miR-188-5p prevents neu-
ronal cell apoptosis during PGD/R-induced stroke (Li et al.
2020b). By downregulating PTEN, MiR-217-5p protects
neurons from OGD/R-induced injury (Yi et al. 2020).
MiR-130a mitigates CIRI-induced damage by inhibiting
PTEN and activating the PI3K/AKT axis (Zheng et al.
2019). In addition, several studies have shown that PTEN
plays a protective role in nervous system diseases. Huang
et al. (Huang et al. 2015) reported that PTEN plays a
protective role in neuropathic pain, and overexpression
of PTEN significantly alleviates different phenotypes of
neuropathic pain, and inhibits microglia and astrocyte
activation, as well as tumor necrosis factor-α expression.
Shabanzadeh et al. (Shabanzadeh et al. 2019) revealed
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Table 4  Stratified analysis between the genotypes and alleles of miR-181a rs322931 variant among IS patients and control group
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Characteristics IS patient group Control group P value

Genotype n (%) Allele n (%) Genotype n (%) Allele n (%)
GG GA AA G A GG GA AA G A PG PG* PA PA*

Age
≥65 355(60.6) 199(34.0) 32(5.4) 909(77.6) 263(22.4) 400(70.0) 157(27.4) 15(2.6) 957(83.7) 187(16.3) 1.1 × 10− 3 5.8 × 10− 3 2.3 × 10− 4 1.5 × 10− 3
<65 299(66.6) 132(29.4) 18(4.0) 730(81.3) 168(18.7) 346(70.8) 131(26.8) 12(2.4) 823(84.2) 155(15.8) 0.24 0.30 0.11 0.14
Gender
Male 430(61.5) 234(33.4) 36(5.1) 1094(78.1) 306(21.9) 368(70.5) 141(27.0) 13(2.5) 877(84.0) 167(16.0) 1.4 × 10− 3 5.8 × 10− 3 2.9 × 10− 4 1.5 × 10− 3
Female 224(66.8) 97(29.0) 14(4.2) 545(81.3) 125(18.7) 378(70.1) 147(27.3) 14(2.6) 903(83.8) 175(16.2) 0.34 0.37 0.19 0.19
Hypertension
Yes 490(62.4) 255(32.5) 40(5.1) 1235(78.7) 335(21.3) 253(71.9) 91(25.8) 8(2.3) 597(84.8) 107(15.2) 3.0 × 10− 3 6.0 × 10− 3 5.8 × 10− 4 1.5 × 10− 3
No 164(65.6) 76(30.4) 10(4.0) 404(80.8) 96(19.2) 493(69.5) 197(27.8) 19(2.7) 1183(83.4) 235(16.6) 0.37 0.37 0.19 0.19
Diabetes
Yes 213(62.5) 114(33.4) 14(4.1) 540(79.2) 142(20.8) 87(71.9) 31(25.6) 3(2.5) 205(84.7) 37(15.3) 0.19 0.27 0.072 0.11
No 441(63.5) 217(31.3) 36(5.2) 1099(79.2) 289(20.8) 659(70.1) 257(27.3) 24(2.6) 1575(83.8) 305(16.2) 2.3 × 10− 3 5.8 × 10− 3 8.1 × 10− 4 1.6 × 10− 3
Smoking
Yes 153(53.2) 115(39.9) 20(6.9) 421(73.1) 155(26.9) 93(71.0) 34(26.0) 4(3.0) 220(84.0) 42(16.0) 2.0 × 10− 3 5.8 × 10− 3 5.8 × 10− 4 1.5 × 10− 3
No 501(67.1) 216(28.9) 30(4.0) 1218(81.5) 276(18.5) 653(70.2) 254(27.3) 23(2.5) 1560(83.9) 300(16.1) 0.13 0.22 0.080 0.11

PG: P value of the difference in alleles between the case and control groups; PA: P value of the difference in genotype between the case and control groups;
*
False discovery rate-adjusted P value for multiple hypotheses testing using the Benjamini-Hochberg method. P < 0.05 is indicated in bold font
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Metabolic Brain Disease (2023) 38:2077–2091
Fig. 7   a Relative miR-181a expression in PBMCs from IS patients
(n = 89) and healthy controls (n = 94). b Relative miR-181a expres-
sion in IS patients and control subjects with rs322931 GG and
that human PTEN peptide could promote functional
improvement after MCAO or retinal ischemia. Zhao et al.
(Zhao et al. 2021) found that nuclear PTEN translocation
increases significantly following OGD treatment, and
inhibition of PTEN nuclear translocation could protects
against ischemic brain damage. We observed that miR-
181a targets PTEN to increase OGD/R-induced cell injury
and that PTEN overexpression temporarily inhibits miR-
181a’s ability to trigger cell apoptosis. PTEN suppression,
on the other hand, has earlier been shown to suppress IS
(Mu et al. 2020). As a result, the effects of PTEN on cer-
ebral ischemia are complex. Given the conflicting role of
PTEN in our and previous research, more investigation is
necessary to thoroughly analyze the connection between
miR-181a and PTEN during IS development.
A Genome-wide association study reported that the miR-
181 rs322931 polymorphism was associated with positive
emotion (Wingo et al. 2017). Recently, He et al. stated that
the rs322931 T allele was linked with lower miR-181b lev-
els and an increased risk of SLE (Mu et al. 2020). Han et al.
also found that miR-181b rs322931 variant contributed to
the risk of IS, partly caused by reducing transcriptional
activity (Han et al. 2018). In our current investigation,
we discovered a link between the rs322931 variants and
an increased risk of IS and LAA. Moreover, we observed
elevated miR-181a expression in IS patients carrying the
rs322931 GA + AA genotypes. Wingo et al. discovered that
the rs322931 SNP affects miR-181b and miR-181a expres-
sion in blood and brain (Wingo et al. 2017). Overexpression
of miR-181a in rat hippocampal neurons inhibits dendritic
growth and reduces the size and number of dendritic spines
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2089
GA + AA genotypes. U6 served as the normalization control. Data
are expressed as medians with interquartile ranges (*P < 0.05,
***P < 0.001, n.s., not significant)
(Saba et al. 2012). Our conclusions and previous findings
suggest that the rs322931 A allele is linked with increased
miR-181a expression, which may downregulate the PTEN
signaling and reduce the synaptic plasticity of neurons,
leading to increased susceptibility to IS.
Conclusion
The current work revealed that inhibiting miR-181a
enhances neuron endurance following OGD/R by targeting
the PTEN directly. As a result, targeting PTEN with miR-
181a could be a novel and appealing therapeutic approach
for IS treatment. The rs322931 variants increase the risk
of IS, and the rs322931 A allele may influence the genetic
predisposition to this disease by modulating miR-181a
expression. More research is certain to confirm our find-
ings and shed light on the precise mechanisms underlying
the miR-181a/PTEN axis in the onset and progression of
IS.
Supplementary information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 11011-0​ 23-0​ 1219-1.
Acknowledgements We’d like to thank MJEditor (www.m ​ jedit​ or.c​ om)
for its linguistic assistance during the prepare of this manuscript. We
also thank the participants in this study.
Author contribution Study design were performed by Y.L. and G.M.;
Material preparation and data collection were performed by S.L., P.Z.
and Y.W.; Data analysis were finished by X.H., Y.W., Z.W., S.H.,
13
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2090
J.H. and Y.Y.; The first draft was edited by B.Z.; All authors read and
approved the final manuscript.
Funding This work was supported by funding from the National
Nature Science Foundation of China (grant numbers 81571157,
81300929 and 81670252) and the Natural Science Foundation
of Guangdong Province (grant numbers 2019A1515011424 and
2023A1515012750), the Youth Cultivation Foundation (grant num-
ber GDMUQ2021006) and One Hundred Youth Research Projects
(grant number GDMUD2022010) of Guangdong Medical Univer-
sity, the Medical Scientific Research Foundation of Guangdong
Province, China (grant number: A2023193 and A2022139) and
the Non-funded Science and Technology Research Foundation of
Zhanjiang City (grant number: 2021B01370).
Data availability The datasets used in this study are available from the
corresponding author upon reasonable request.
Declarations
Research involving human participants and/or animals This study was
performed in line with the principles of the Declaration of Helsinki.
This study was approved via the Ethics Committee of the Affiliated
Hospital of Guangdong Medical University and the animal ethics com-
mittee of Guangdong Medical University.
Informed consent The letter of informed consent was gained from each
participant prior to investigation enlistment.
Consent for publication The authors confirm that informed consent
was obtained from the human study participants for publication of the
data involved in this study.
Competing interests The authors have no relevant financial or non-
financial interests to disclose.
Conflict of interest The authors declare no conflicts of interest.
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