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\begin{document}

\title{MicroRNA-196a-3p modulates neural stem cell proliferation against
cerebral ischemia/reperfusion injury by ARF4 signaling}



\author[1]{Hao Wang}%
\author[2]{Yuwu  Zhao}%
\author[2]{Feng Wang}%
\author[2]{Jiawen Yuan}%
\author[2]{Gang Zhu}%
\author[1]{Meng-shi  Dai}%
\author[3]{Jiachen  Li}%
\affil[1]{Fudan University}%
\affil[2]{Shanghai Jiao Tong University}%
\affil[3]{Michigan Heart Ann Arbor}%


\vspace{-1em}



  
  \date{May 16, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
MicroRNA-196a-3p (miR-196a-3p) is known to increase in rat models
subjected to middle cerebral artery occlusion (MCAO). In the present
work, we aimed to investigate the effects of miR-196a-3p in cerebral
ischemia/reperfusion (I/R) injury. Transient cerebral ischemia was
induced in C57/BL6 mice subjected to MCAO. Mice were also administered
miR-196a-3p antagomir by intracerebroventricular injection. MiR-196a-3p
and its target ARF4 levels were quantified and cerebral infarct volume
and neuronal apoptosis were evaluated. Primary neural stem cells (NSCs)
were activated by oxygen--glucose deprivation/reoxygenation. NSCs were
transfected with miR-196a-3p mimics, inhibitors, siARF4, or negative
control using Lipofectamine 2000 reagent. ARF4, Ki-67, and Nestin
expression levels were assessed using qRT-PCR and western blotting. The
proliferation of NSCs was detected by CCK-8 assay and EdU staining. We
found that levels of miR-196a-3p expression increased in vivo and in
vitro when expression levels of its target ARF4 were decreased. We also
found that miR-196a-3p aggravated cerebral I/R injury in vivo. We
established that ARF4 is the target of miR-196a-3p using a
dual-luciferase assay in vitro. Simultaneously, we observed that
miR-196a-3p overexpression or the inhibition of ARF4 inhibited NSC
proliferation. MiR-196a-3p inhibited NSC proliferation and aggravated
cerebral I/R injury by targeting ARF4.%
\end{abstract}%



\sloppy


\textbf{MicroRNA-196a-3p modulates neural stem cell proliferation
against cerebral ischemia/reperfusion injury by ARF4 signaling}

Jiawen Yuan \textsuperscript{1*}, Gang Zhu\textsuperscript{2*} ,Meng-shi
Dai\textsuperscript{4}, ~Jiachen Li~\textsuperscript{5}, Hao
Wang\textsuperscript{3}, Feng Wang\textsuperscript{1}, Yuwu
Zhao\textsuperscript{1}

Affiliations:

Jiawen Yuan: vigaro299@hotmail.com

Gang Zhu: zhugang1997@sina.com

Meng-shi Dai: 154184207@qq.com

Jiachen Li~: jiacheli@umich.edu

1. Department of Neurology, Shanghai Sixth People's Hospital affiliated
to , 200233,

2. Department of Neurology, , 200062,

3. Experimental Teaching Cener of Basic Medicine, , 200032,

4. Department of Geriatrics, , , 200040,

5. of , Science, and the Arts, Program in Biology~, of ,

E-mail: jiacheli@umich.edu

* co-first author

Correspondence to:

Yuwu Zhao

Department of Neurology, Shanghai Sixth People's Hospital affiliated to
, 200233,

Tel:+86-18930177606 , e-mail: zhaoyuwu2005@126.com

Hao Wang

Experimental of Basic Medicine, , 200032,

Tel:+86-15821872237 , e-mail:wh91351@126.com

Feng Wang

Department of Neurology, Shanghai Sixth People's Hospital affiliated to
, 200233,

Tel:+86-18930173523 , e-mail: wangfeng\_doctor@126.com

\textbf{Abstract}

MicroRNA-196a-3p (miR-196a-3p) is known to increase in rat models
subjected to middle cerebral artery occlusion (MCAO). In the present
work, we aimed to investigate the effects of miR-196a-3p in cerebral
ischemia/reperfusion (I/R) injury. Transient cerebral ischemia was
induced in C57/BL6 mice subjected to MCAO. Mice were also administered
miR-196a-3p antagomir by intracerebroventricular injection. MiR-196a-3p
and its target ARF4 levels were quantified and cerebral infarct volume
and neuronal apoptosis were evaluated. Primary neural stem cells (NSCs)
were activated by oxygen--glucose deprivation/reoxygenation. NSCs were
transfected with miR-196a-3p mimics, inhibitors, siARF4, or negative
control using Lipofectamine 2000 reagent. ARF4, Ki-67, and Nestin
expression levels were assessed using qRT-PCR and western blotting. The
proliferation of NSCs was detected by CCK-8 assay and EdU staining. We
found that levels of miR-196a-3p expression increased in vivo and in
vitro when expression levels of its target ARF4 were decreased. We also
found that miR-196a-3p aggravated cerebral I/R injury in vivo. We
established that ARF4 is the target of miR-196a-3p using a
dual-luciferase assay in vitro. Simultaneously, we observed that
miR-196a-3p overexpression or the inhibition of ARF4 inhibited NSC
proliferation. MiR-196a-3p inhibited NSC proliferation and aggravated
cerebral I/R injury by targeting ARF4.

Keywords: MicroRNA-196a-3p, Ischemia/reperfusion injury, ARF4, Middle
cerebral artery occlusion, Neural stem cells.

1. Introduction

Cerebral ischemia is the leading cause of permanent disability worldwide
{[}1{]}, which are a heavy burden to human health. Neural stem cells
(NSCs) have the capacity to differentiate towards astrocytes, neurons
and oligodendrocytes by their inherent multipotency {[}2{]}. During
development and post-injury (such as I/R) these NSCs are understood to
play significant roles through differentiation and migration from the
sub-ventricular zone to repopulate the damaged regions {[}3{]}. A study
by Tao et al., identified use of electroacupuncture post I/R could
enhance NSC proliferation and improve recovery {[}4{]}. Therefore, it
may be a promising therapeutic option to prevent cerebral
ischemia/reperfusion injury by promoting neural stem cell proliferation.

MicroRNAs (miRNAs) are a group of small non-coding RNAs that regulate
mRNA transcription and protein translation by targeting the
3'-untranslated regions (3'-UTRs) of their target genes {[}5{]}.In a rat
model of I/R injury, involving 24 h middle cerebral artery occlusion
(MCAO) followed by 48 h of reperfusion, 114 miRNAs were differentially
regulated in ischemic brain samples, 106 during MCAO, and 82 during
reperfusion {[}6{]}. In similar models, a significant change in the
expression of over 300 miRNAs was found to occur across the whole I/R
injury process {[}7,8{]}. Of these, miR-196a-3p has displayed increased
expression throughout all time points and has been implicated as a
potential marker of acute stroke {[}9,10,11{]}. However, the function of
miR-196a-3p in response to I/R is unclear. However, bioinformatics
analysis (http://www.targetscan.org/) has revealed that miR-196a-3p
targets ARF4. ADP-ribosylation factor 4 (ARF4), a member of the human
ADP-ribosylation factor family{[}12{]}. ADP-ribosylation is also
involved in cell proliferation and apoptosis and ARF4 is known to be a
suppressor of Bcl-2-associated X protein-induced cell death in yeast
{[}13{]}, indicating that ARF4 is involved in cell proliferation.

In this study, we adopted a mouse model of MCAO to assess the role of
miR-196a-3p in I/R injury in vivo. We also examined whether changes in
the expression of miR-196a-3p influences the expression of the ARF4
target gene. Additionally, we have elucidated the role of miR-196a-3p on
proliferation of NSCs by regulating the ARF4 gene on NSCs from OGDR
mouse model.

2. Materials and methods

2.1. Animals and ethics statement

Adult C57BL/6J mice (6 to 8-week-old males, weighing 20--25 g, (Shanghai
Sippr Bk Laboratory Animals Co., Ltd., Shanghai, China)) were housed
under diurnal lighting conditions (12 h light /12 h dark, humidity 60\%
\selectlanguage{ngerman}± 10\%, 23°C ± 2°C) and allowed access to food and water ad libitum. All
animal experiments were approved by the Ethics Committee of the
Institutional Animal Care and Use Committee of the Shanghai Sixth
People's Hospital affiliated to .

2.2. Intraluminal middle cerebral artery occlusion (MCAO) model

To generate the mouse MCAO model{[}14{]}, C57BL/6J mice were first
anesthetized with 200 mg/kg chloral hydrate intraperitoneally and body
temperature was maintained at 37°C. A midline incision was made in the
neck after disinfecting the area with povidone-iodine. The external
common carotid artery was located and temporarily occluded with a 5-0
silk suture. The internal common carotid artery was clipped with
tweezers and a silicone-coated filament was inserted. A suture was tied
around the filament to mimic the occlusion. The suture was removed after
90 min to simulate reperfusion and the wound was closed. The procedure
was repeated without the filament for sham-operated mice.

2.3. Oxygen--glucose deprivation and reoxygenation (OGDR) model

To generate an OGDR model, primary NSCs were seeded at 1 × 106/mL in
Dulbecco's modified Eagle medium (DMEM, Gibco, )/F12 supplemented with
10\% fetal bovine serum (Gibco) for 24 h. To achieve oxygen--glucose
deprivation, the cells were transferred into glucose-free DMEM and
placed in a sealed tank for 4 h with a constant flow of N2 (94\%), CO2
(5\%), and O2 (1\%). NSCs were transferred to normal DMEM and exposed to
ambient air to replicate reoxygenation.

2.4. Mouse NSC isolation and culture

To isolate and culture NSCs, mouse forebrains were first minced and
digested with a solution of papain, dispase II, and DNase I. Cells were
isolated using Percoll gradients and cultured in DMEM/F12 with 1 mM
L-glutamine, N2 supplement, epidermal growth factor (20 ng/mL), FGF2 (20
ng/mL), and heparin (5 mg/mL). NSCs were selected by using fluorescein
digalactoside.

2.5. MiR-196a-3p antagomir intracerebroventricular injection

For the implantation of a cannula in the left lateral cerebral
ventricle, each mouse was sedated (ketamine 100 mg/kg ip and xylazine 9
mg/kg ip). Each mouse's skull was positioned in a stereotaxic instrument
(942, David Kopf Instrument, ) so that the plane produced by the frontal
and parietal bones was parallel to the table top of the instrument. A
26-gauge stainless-steel guide cannula was implanted intracerebrally
using predetermined coordinates (anteroposterior -0.6 mm; lateral, 1.6
mm to the bregma; horizontal, 2.0 mm to the dura mater) and fixed with
two stainless steel screws and cranioplasticcement.

The miR-196a-3p antagomir and negative control (NC) were obtained from
Genechem (Shanghai, China). To administer the antagomir to mice, 2.5 \selectlanguage{greek}μ\selectlanguage{english}L
Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA,
USA) was added to miR-196a-3p antagomir (100 \selectlanguage{greek}μ\selectlanguage{english}mol/L) or NC (100 \selectlanguage{greek}μ\selectlanguage{english}mol/L)
and incubated at 37\selectlanguage{ngerman}°C for 30 min. The mixture (7 \selectlanguage{greek}μ\selectlanguage{english}L) was administered by
right intracerebroventricular injection 10 min before the MCAO
procedure.

2.6. Dual-luciferase reporter assay

To perform the dual-luciferase reporter assay, the full-length 3'UTR of
ARF4 was first amplified by PCR using the forward primer
5'-CCGCTCGAGATGAAATTGGATATCTAACCAAGG-3' and reverse primer
5'-TTGCGGCCGCCGTATTTTATCATTTTATTAGG-3'. The mutant 3'UTR of ARF4 was
created using a Mut Express-II Fast Mutagenesis Kit (Vazyme Biotech,
Nanjing, China). Full-length 3'UTR ARF4 and mutant 3'UTR ARF4 were both
cloned into the psi-CHECKTM-2 dual-luciferase vector. The
dual-luciferase reporter vectors (50 ng) were cotransfected into
HEK-293T cells with 60 nM mimics or antagomirs and incubated in 24-well
plates for 48 h. Cells were lysed and Firefly and Renilla luciferase
activity were measured using a dual-luciferase reporter assay system
(Promega, Madison, USA) following the manufacturer's instructions.

2.7. 2,3,5-Triphenyltetrazolium chloride (TTC) staining

After the mice were anesthetized to collect the brain, the brains were
dissected into four slices with a razor blade (2 mm in each slice). The
brain slices were put into TTC solution (2\%, Sigma, America) at 37 \selectlanguage{ngerman}°C
for 10 min. The brain tissue showed red when TTC reacted with
dehydrogenase in normal tissues. However, the brain tissue presented
white due to decreased dehydrogenase activity in ischemic tissues. After
TTC staining, the brain tissue was fixed with 4\% paraformaldehyde for
1-2 days. The images of the stained sections were scanned with a scanner
and the infarct volume was measured by the Image J software.

2.8. Real-time PCR

TRIzol reagent (Invitrogen) was used to isolate RNA according to the
instructions of the manufacturer. The ABI PRISM 7500 Sequence Detection
System (Applied Biosystems, Foster City, CA, USA), with a SYBR Premium
Ex Taq II kit (Takara, Dalian, China) and the following primers:
miR-196a-3p forward 5\selectlanguage{english}'-GCCGAGCGGCAACAAGAAAC-3\selectlanguage{english}' and reverse
5\selectlanguage{english}'-CCAGTGCAGGGTCCGAGGTATT-3\selectlanguage{english}'; ARF4 forward 5\selectlanguage{english}'-ACAGGATCTGCCAAACGCTA-3\selectlanguage{english}'
and reverse 5\selectlanguage{english}'-TGACAGCCAATCCAGTCCCT-3\selectlanguage{english}'. Relative mRNA or miRNA
expression was determined using the \selectlanguage{greek}ΔΔ\selectlanguage{english}Ct method and normalized against
GAPDH or U6.

2.9. Western blotting

Cells were first lysed with RIPA lysis buffer (Beyotime, Haimen, China).
Cell debris was removed by centrifugation and proteins in the
supernatant were separated using 12\% SDS-PAGE and transferred to PVDF
membranes. PVDF membranes were blocked in 5\% non-fat milk and then
incubated overnight at 4\selectlanguage{ngerman}°C with primary antibodies directed toward ARF4
(1:200 Origene USA), Ki-67 (1:200 Origene USA) and GAPDH (1:500 Origene
USA). Membranes were then incubated with secondary antibody for 1 h at
room temperatures and then visualized with SageBrightness West Pico Plus
ECL Solution (Pico Plus, Hong Kong, China).

2.10. TUNEL, CCK-8, and EdU assays

A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
assay was used to measure apoptotic cells in coronal sections using an
TUNEL Apoptosis Detection Kit (Abbkine, CA, USA) following the
manufacturer's instructions.

Cell viability and proliferation were assessed using a CCK-8 assay. NSCs
were seeded into 96-well plates (3 × 103/well) and CCK-8 reagent
(Dojindo, Japan) was added to each well (10 \selectlanguage{greek}μ\selectlanguage{english}L). After incubating the
cells for 1 h the optical density was measured at 450 nm.

A 5-ethynyl-2- deoxyuridine (EdU) assay was used to measure
proliferating NSCs. Cell cycles were synchronized by seeding cells in
serum-free medium for 24 h. They were then pulsed for 2 h with EdU using
Cell Proliferation EdU Image Kit (Abbkine, CA, USA). EdU detection was
carried out following the kit instructions. Cells were observed under a
confocal microscope and the percentage of EdU positive cells was
determined using Image J software.

2.11. Statistical analysis

All experiments were performed in triplicate. Statistics were calculated
by SPSS PASW Statistics 18.0 (SPSS Inc., Chicago, IL, USA) or GraphPad
Prism 5 (GraphPad, San Diego, CA, USA) by one-way analysis of variance
(one-way ANOVA) followed by Tukey's post hoc test. All results are
presented as means \selectlanguage{ngerman}± SD. P \textless{} 0.05 indicates a statistically
significant difference.

3. Results

3.1. Expression of miR-196a-3p and ARF4 after ischemic stroke in vitro
and in vivo

We quantified the expression of miR-196a-3p and ARF4 both in vitro and
in vivo using OGDR and MCAO models. The levels of miR-196a-3p in primary
mouse NSCs increased significantly after OGDR (Fig. 1A). In contrast,
the level of ARF4 decreased (Fig. 1B). Following OGDR, ARF4 protein
levels corresponded to the expression levels of mRNA (Fig. 1C).
Occlusion followed by reperfusion in vivo resulted in a significant
increase in the expression of miR-196a-3p after 6 days (P \textless{}
0.001 vs. sham) whereas ARF4 levels were significantly reduced (P
\textless{} 0.01 vs. sham). These results indicate that the expression
of miR-196a-3p is induced by OGDR and MCAO. These results indicate an
inverse correlation between the expression of miR-196a-3p and ARF4 in
OGDR and MCAO models.

3.2. MiR-196a-3p aggravates brain injury in response to I/R

To determine the function of miR-196a-3p following I/R, the cerebral
infarct volume was evaluated in coronal sections of brain tissue
subjected to MCAO with miR-196a-3p overexpressed (Fig. 2A). The extent
of cerebral infarct volume was significantly greater in brain tissue
overexpressing miR-196a-3p than in the I/R control NSC apoptosis in the
ipsilateral cortex was determined by a TUNEL assay (Fig. 2B). The
results indicate a significantly greater number of apoptotic cells in
response to I/R. However, the number of apoptotic cells was
significantly higher in tissue overexpressing miR-196a-3p compared with
the control following MCAO (P \textless{} 0.001), indicating that
miR-196a-3p contributes to the severity of I/R injury.

3.3. MiR-196a-3p inhibits proliferation in NSCs

To further investigate the aggravation of I/R injury by miR-196a-3p, we
assessed the effect of inhibiting its expression on NSC proliferation.
MiR-196a-3p levels in primary mouse neural stem cells (NSCs) were
measured by real-time PCR ,NSC proliferation measured by CCK-8 and EdU
staining was significantly reduced in cells overexpressing miR-196a-3p,
whereas the inhibition of miR-196a-3p increased proliferation to a rate
higher than that found in control cells (Fig. 3A--C). In this study, we
used Ki-67and Nestin markers to measure the extent of cell division and
proliferation in NSCs overexpressing miR-196a-3p (Fig. 3D--G). Ki-67 is
a marker that is often used to detect cell proliferation because it is
expressed in various stages of the cell cycle {[}33{]}. Nestin is a NSC
marker that is downregulated when stem cells differentiate into neurons
{[}34{]}. Levels of both Ki-67 and Nestin expression and protein levels
are lower than the NC when miR-196a-3p is overexpressedbut are increased
to a significant level when miR-196a-3p is inhibited. Overall, these
results indicate that miR-196a-3p has a significant role in preventing
the proliferation of NSCs.

3.4. MiR-196a-3p inhibits the proliferation of NSCs by targeting ARF4

To further investigate the involvement of miR-196a-3p in the inhibition
of cell proliferation in NSCs we sought to identify its interaction
partners with TargetScan. We found that the 3\selectlanguage{english}'-UTR of ARF4 has a
complementary binding sequence for miR-196a-3p (Fig. 4A). The luciferase
reporter activity of ARF4 with and without the miR-196a-3p binding site
mutated was determined in NSCs (Fig. 4B). A closer examination of ARF4
levels in NSCs with miR-196a-3p up or downregulated revealed that ARF4
mRNA expression and protein levels were highestwhen miR-196a-3p is
inhibited and significantly lower when miR-196a-3p is overexpressed
(Fig. 4C and D). These results indicate that ARF4 is suppressed by
miR-196a-3p in NSCs.

si-ARF4 inhibits the proliferation of NSCs

To examine the aggravation of I/R injury by ARF4, we assessed the effect
of inhibiting its expression on NSC proliferation. ARF4 levels in
primary mouse neural stem cells (NSCs) were measured by real-time PCR
,NSC proliferation measured by CCK8 and EdU staining was significantly
reduced in cells inhibiting ARF4, so the inhibition of ARF4 decreased
proliferation lower than that found in the scramble (Fig. 5A--C). In
this study, we used Ki-67and Nestin markers to measure the extent of
cell division and proliferation in NSCs inhibiting ARF4 (Fig. 5D--G).

4. Discussion

The disabilities that occur following ischemic stroke could be reduced
if the ability to protect the brain from injury before and after
perfusion was improved {[}15, 16{]}. Recently, attention has been drawn
toward characterizing the miRNome following cerebral ischemia and a
number of miRNAs associated with reperfusion have been identified {[}9,
17, 18{]}. From the miRNAs identified in these reports, we can confirm
that miR-196a-3p is upregulated in vitro and in vivo using OGDR and MCAO
ischemic models. Following OGDR, the levels of miR-196a-3p in primary
mouse NSCs increased significantly. Similar results were obtained in a
mouse model of occlusion followed by reperfusion, the levels of
miR-196a-3p expression increased significantly after 6 days. Moreover,
increased levels of miR-196a-3p expression are accompanied by an
increased level of cerebral infarct volume and a higher number of
apoptotic cells indicating that higher levels of miR-196a-3p expression
contribute to the severity of I/R injury.

Several studies implicate miR-196a in a reduction of cell proliferation
and increased cell differentiation {[}19-21{]}. For instance, Kim et al.
found that miR-196a could influence the osteogenic differentiation and
proliferation of human adipose tissue-derived mesenchymal stem cells
through its interaction with HOXC8 {[}19{]}. A reduction in the
expression of miR-196a is also implicated in the progression of cancer,
the lncRNA SNHG3 is thought to promote the proliferation of cancer cells
in osteosarcoma by inhibiting or sponging miR-196a-5p and preventing it
from interacting with HOXC8 {[}22{]}. A previous a study by Lu et al.,
identified upregulated miR-196a levels could increase apoptosis in GC-2
spermatogonia cell lines {[}23{]}. Recently, an increasing number of
studies indicate a connection between miR-196 and degenerative
neurological diseases such as Huntington's disease and Alzheimer's
disease {[}24, 26{]}. The upregulation of miR-196 is found in the brain
samples of patients with Huntington's disease, although it is not clear
whether it is upregulated in response to the disease or involved in the
pathogenesis {[}26{]}. This confirms our finding that the expression of
miR-196a-3 can be elevated in response to brain injury. However, we
found that increasing the expression of miR-196a-3 aggravates brain
injury and generates a higher number of apoptotic NSCs. Our results
correspond to the finding that increased miR-196a expression prevents
proliferation and promotes apoptosis {[}22{]}.

In the present study, we found that miR-196a-3p interacts with the
3\selectlanguage{english}'-UTR of ARF4 and that the overexpression of miR-196a-3p in NSCs
results in the downregulation of ARF4 and a reduction in cell
proliferation. ARF4 has been found to contribute to cell proliferation
and invasion in several cancers {[}27-29{]}. However, an interaction
with miR-196a-3p is not well-documented, although they are both strongly
associated with vertebrate development and ARF4 {[}30,31{]}. Moreover,
the overexpression of ARF4 was found to rescue the loss of primary
neurons in a mouse model of Alzheimer's disease {[}31{]}. Therefore,
Based on the literature and the results of our study, we found that
miR-196a-3p inhibits the proliferation of NSCs by targeting ARF4.

In conclusion, we have confirmed that miR-196a-3p is upregulated in the
NSCs following I/R injury in vitro and in vivo. Our results indicate
that a higher expression of miR-196a-3p is associated with greater
cerebral infarct volume, lower cell proliferation, and an increase in
the number of apoptotic cells. The inhibition of miR-196a-3p leads to
increased cell proliferation and a higher level of ARF4. This indicated
that the miR-196a-3p affected the NSC proliferation and its potential to
rescue post injury through the inhibition and targeting of ARF4.
Therefore, our results indicate that following ischemic stroke the
expression of miR-196a-3p leads to greater I/R injury.

Conflict of interest

The authors declare that they have no competing interests.

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\textbf{Figure Legends}

\textbf{Fig. 1. MiR-196a-3p aggravates cerebral ischemia/reperfusion
(I/R) injury in mice.} (A) MiR-196a-3p levels in mice cerebral cortex
0,12,24 and 48hours after OGDR were detected by qRT-PCR. (B) ARF4 levels
in mice cerebral cortex 0,12,24 and 48hrs after OGDR were detected by
qRT-PCR. (C) ARF4 levels in vitro 0,12,24 and 48hrs after OGDR were
detected by westernblot.(D) MiR-196a-3p levels in mice cerebral cortex
1,3, 6 and 9days after MACO were detected by qRT-PCR. (E) ARF4 levels in
mice cerebral cortex 1,3, 6 and 9 days after MACO were detected by
qRT-PCR.(F) ARF4 levels in mice cerebral cortex 1,3, 6 and 9 days after
MACO were detected by westernblot. The data are presented as mean ±
SD.** P \textless{} 0.01; *** P \textless{} 0.001.

\textbf{Fig.2} MiR-196a-3p aggravates brain injury in response to I/R in
mice\textbf{.}

(A) Cerebral infarct volume evaluated by 2, 3, 5-triphenyltetrazolium
chloride (TTC) staining of coronal brain sections. The relative infarct
area percentage was evaluated by observing the unstained infarcted
tissue zone (white) and the stained normal tissue zone (red). (B) Neural
stem cells apoptosis in the cerebral cortex as detected by TUNEL and
DAPI double staining. Scale bar =50 \selectlanguage{greek}μ\selectlanguage{english}m. N=10, the data are presented as
mean \selectlanguage{ngerman}± SD.** \emph{P} \textless{} 0.01; *** \emph{P} \textless{} 0.001.

\textbf{Fig. 3} \textbf{MiR-196a-3p inhibits proliferation in neural
stem cells.}

(A) MiR-196a-3p levels in primary mouse neural stem cells (NSCs) were
measured using quantitative real-time PCR. (B,C) CCK-8 and EdU staining
were used to detect the effect of miR-196a-3p on NSC proliferation.
Statistical data for relative EdU positive cell percentages are
shown.(D) The Ki-67 (an important marker for neural stem cells) mRNA
expression in NSCs was detected by qRT-PCR. (E) The Ki-67 protein
expression in NSCs was detected by westernblot (F) The Nestin (an
important marker for neural stem cells) mRNA expression in NSCs was
detected by qRT-PCR. (G) The Nestin protein expression in NSCs was
detected by westernblot.Scale bar =50 \selectlanguage{greek}μ\selectlanguage{english}m. The data are presented as mean
\selectlanguage{ngerman}± SD.** P \textless{} 0.01; *** P \textless{} 0.001.

\textbf{Fig. 4} \textbf{ARF4 is targeted by miR-196a-3p in neural stem
cells.} (A) TargetScan bioinformatics algorithm alignment of miR-196a-3p
(seed sequence in bold font) and its complementary binding sequence in
ARF4 3\selectlanguage{english}'-UTR revealed excellent evolutionary conservation across diverse
species, including human (hsa, Homo sapiens), mouse (mmu, Mus musculus),
and Rat (rno, Rattus norvegicus). The deletion and mutation locations in
the ARF4 3\selectlanguage{english}'-UTR of different species are highlighted in comparison to
human. (B) Luciferase reporter gene assays were used to investigate the
effect of miR-196a-3p or anti-miR-196a-3p on the reporter activities of
pGL3-ARF4-WT and pGL3-ARF4-MUT in neural stem cells. (C) NSCs were
transfected for 48 hours with mimics, miR-196a-3p mimics, inhibitors,
NC, or miR-196a-3p inhibitors, and ARF4 expression was determined using
qRT-PCR. (D) NSCs were transfected for 48 hours with mimics, miR-196a-3p
mimics, inhibitors, NC, or miR-196a-3p inhibitors, and ARF4 expression
was determined using western blotting. The data are presented as mean +-
SD.** P \textless{} 0.01; *** P \textless{} 0.001.

\textbf{Fig. 5. MiR-196a-3p inhibits proliferation by targeting ARF4 in
neural stem cells.}

(A) ARF4 expression in primary mouse neural stem cells (NSCs) was
measured using quantitative real-time PCR. (B,C) CCK-8 and EdU staining
were used to detect the effect of si-ARF4 on NSC proliferation.
Statistical data for relative EdU positive cell percentages are
shown.(D) The Ki-67 (an important marker for neural stem cells) mRNA
expression in NSCs was detected by qRT-PCR. (E) The Ki-67 protein
expression in NSCs was detected by westernblot (F) The Nestin (an
important marker for neural stem cells) mRNA expression in NSCs was
detected by qRT-PCR. (G) The Nestin protein expression in NSCs was
detected by westernblot.Scale bar =50 \selectlanguage{greek}μ\selectlanguage{english}m. The data are presented as mean
\selectlanguage{ngerman}± SD.** P \textless{} 0.01; *** P \textless{} 0.001.\selectlanguage{english}
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