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Inhibition of JAK1 by microRNA-708 promotes SH-SY5Y neuronal cell survival after oxygen and glucose deprivation and reoxygenation
Accepted Manuscript Title: Inhibition of JAK1 by microRNA-708 promotes SH-SY5Y neuronal cell survival after oxygen and glucose deprivation and reoxygenation Authors: Chao Huang, Haitao Zhou, Xiangyang Ren, Junfang Teng PII: DOI: Reference:
S0304-3940(17)30915-1 https://doi.org/10.1016/j.neulet.2017.11.017 NSL 33225
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
20-5-2017 6-11-2017 7-11-2017
Please cite this article as: Chao Huang, Haitao Zhou, Xiangyang Ren, Junfang Teng, Inhibition of JAK1 by microRNA-708 promotes SH-SY5Y neuronal cell survival after oxygen and glucose deprivation and reoxygenation, Neuroscience Letters https://doi.org/10.1016/j.neulet.2017.11.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
Inhibition of JAK1 by microRNA-708 promotes SH-SY5Y neuronal cell survival after oxygen and glucose deprivation and reoxygenation Running Title: miR-708 and JAK1 in SH-SY5Y neuron cell survival. Chao Huang1,2, Haitao Zhou2, Xiangyang Ren2, Junfang Teng1*
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1) Department of Neurology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, P.R. China
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2) Department of Neurology, Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang 471000, Henan, P.R. China
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*Correspondence to: Dr Junfang Teng, Department of Neurology, the First Affiliated Hospital of
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Zhengzhou University, No. 1 Jianshe East Road, Zhengzhou 450052, Henan, P.R. China.
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Email: [email protected]. [email protected].
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Tel: +86 13838210077
Highlights
Human neuroblastoma cells (SH-SY5Y) downregulated miR-708 and upregulated JAK after OGD/R treatment.
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Silence of miR-708 can promote OGD/R SH-SY5Y cell proliferation migration and invasion.
JAK1 as a direct target of miR-708.
A reversed correlation existed between miR-708 and JAK1 mRNA levels in OGD/R SH-SY5Y.
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Abstract
MicroRNAs mediates gene expression in various diseases. Studies have shown that aberrant
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expression of miRNAs affected cerebral protection. In this study, we have investigated the effects of microRNA-708 (miR-708) on cell survival of oxygen and glucose-deprived reoxygenation (OGD/R) human neuroblastoma cells (SH-SY5Y) and explored whether miR-708 inhibited neuronal death by targeting JAK1. In vitro model of ischemia was used to investigate the neuroprotective functions of miR-708. MiR-708 mimics/siJAK1 transfected SH-SY5Y cells were treated with OGD. After 48 h of reoxygenation, cell viability and cell survival were determined by EdU and FITC/PI double staining 1
flow cytometry, respectively. Luciferase activity assay was performed to validate the role of JAK1 as a direct target of miR-708. qRT-PCR and Immunofluorescence assays were used to determine the expression of JAK1, MAP2 and NEUN in miR-708 mimics transfected SH-SY5Y cells. To explore the mechanisms involved in cell growth promotion by JAK1, morphological changes in cells were detected upon knockdown of JAK1 , and the expression levels of JAK1, Bax, Bcl-2, cleaved-caspase-3, STAT3 and Mcl-1 were determined by Western blotting. The expression of miR-708 significantly decreased in cells treated with OGD/R. MiR-708 directly targeted JAK1
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3’UTR to down-regulate JAK1 mRNA expression, whereas the expression of MAP2 and NEUN was upregulated. Previous studies have demonstrated that the suppression of JAK1 inhibited apoptosis
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phenocopied function of the miR-708 overexpression in OGD/R SH-SY5Y cells. miR-708 decreased the rate of apoptosis of OGD/R SH-SY5Y cells by suppressing the expression of JAK1.
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Key words: MiR-708; JAK1; OGD/R; Cell apoptosis; SH-SY5Y
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Introduction
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Ischemic stroke is associated with high rate of mortality and disability worldwide [22]. The increase in the number of new stroke cases and ageing population is directly proportional [9]. Cerebral
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ischemia/reperfusion injury regulates multiple cell apoptotic pathway. Reactive oxygen regulates cell apoptosis by activating multiple cell signaling pathways, such as p38, c-Jun N-terminal kinases and
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JAK/STAT pathway [27, 28]. MicroRNAs (miRNAs) are 21 nucleotides long and belong to the class of noncoding RNA. It directly integrates with the 3′-UTR of target mRNA and decreases target
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mRNA expression level or inhibit translation in different biological processes [25]. Previous study has showed that miRNAs were involved in different cellular processes, including cell proliferation, apoptosis, migration, invasion, cell cycle and stem cell renewal [4, 8, 14]. The study of miRNAs is
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critical in demonstrating a novel direction of disease research for studying the complexity of cancer biology [5]. MiR-708 is a newly found miRNA that plays an important role in various diseases. Aberrant miR-708 level also has an effect on cell proliferation and apoptosis [23].
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Previous studies have shown that activated JAK1 and STAT3 are up-regulated in neurons,
astrocytes and microglia after cerebral infarction [17, 21], and may provide neuroprotection in the acute phase of ischemia [7, 15, 16]. Following the injury to nerve cells, overexpressed and activated STAT3 has been found to contribute to neuronal survival and axon regeneration [2, 3, 29]. Also, it has been reported that JAK2/STAT3 activation significantly contributed to cell apoptosis after ischemia/reperfusion injury [13]. In this study, we investigated the role of miR-708 in oxidative stress-induced cell death and the 2
possible mechanism. An in vitro model of ischemia in SH-SY5Y cells was used to study the neuroprotective functions of miR-708. The results suggested that miR-708 rescued OGD-induced SH-SY5Y cell injury by suppressing the JAK1 apoptotic pathway. The results have shown that miR-708 is a novel therapeutic target for ischemic brain injury.
Materials and methods Cell culture
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SH-SY5Y cells (purchased from ATCC) were incubated in Dulbecco's Modified Eagle's medium (DMEM; Hyclone, USA) supplemented with 10 % fetal bovine serum (FBS; Gibco, USA), 100
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units/mL streptomycin and 100 units/mL penicillin (Sigma, USA) at 37 °C in an atmosphere of 5% CO2. For OGD model, the SH-SY5Y cells were incubated in serum and sugar-free artificial cerebrospinal fluid at 37°C in an atmosphere of 1% O2, 94% N2 and 5% CO2 for 2–6 h, and then
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incubated in DMEM medium in normoxic atmosphere for 48 h.
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Antibodies
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Commercially available antibodies were used for all the immunoblotting and immunofluorescence studies. Anti-JAK1, Bax, Bcl-2 and anti-cleaved caspase-3 antibodies were obtained from Abcam Company (UK). Anti-GAPDH, anti-MAP2, anti-STAT3 and anti-Mcl-1 were obtained from Shanghai Kangcheng Biotech Company, China. All the secondary antibodies used were obtained from Boster, China. qRT-PCR for miRNA and mRNA expression
Quantitative polymerase chain reaction (qPCR), is a laboratory technique of molecular biology based
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on the polymerase chain reaction (PCR). Real-time PCR can be used quantitatively (quantitative real-time PCR), and semi-quantitatively, i.e. above/below a certain amount of DNA molecules (semi
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quantitative real-time PCR).Total RNA was extracted using Trizol reagent (Takara, Japan). RNA was reverse transcribed with a Bestar qPCR RT Kit (DBI Bioscience, Germany) according to the manufacturer’s instructions. Expression values were normalized to the control endogenous small
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RNA U6 or β-actin. Primer sequences used are as follows: β-actin (forward: 5′-ATC GTG CGT GAC ATT AAG GAG AAG -3′; reverse: 5′- AGG AAG GAA GGC TGG AAG AGT G -3′), JAK1 (forward: 5′- ATT GAG AAC GAG TGT CTA GGG A -3′; reverse: 5′-CCT TCA GGT CAT GCG TGG AC -3′), miR-708 (forward: 5′- ACA CTC CAG CTG GGA AGG AGC TTA CAA TCT AG -3′; reverse: 5′- CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG AGC TGG G-3′), U6 (forward: 5′- CTC GCT TCG GCA GCA CA -3′; reverse: 5′- AAC GCT TCA CGA ATT TGC GT -3′), NEUN (forward: 5′- CCC TCC GAC CCT ACA GAG AA -3′; reverse: 5′- AAT TCA GGC CCG 3
TAG ACT GC -3′). The data was analyzed using 2–△△Ct calculation.
Western blotting for protein expression Western blotting is a widely used analytical technique used to detect specific proteins in a sample of tissue homogenate or extract. Cells were grown to 80–90 % confluence and were washed with ice-cold PBS. After 48 h of transfection, lysed cells with protein extraction buffer (Beyotime, China)
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were added with PMSF (Genebase, China). For western blotting, the proteins were mixed with SDS loading buffer (pH = 6.8 250mM Tris-HCL, 10% SDS, 0.5% bromophenol blue, 5% β-mercaptoethanol, 50% glycerol) and incubated in 98°C for 8 min. The proteins were separated with
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5–15% SDS-PAGE and were transferred to PVDF membranes (Millipore, USA) before detection with primary antibodies. PVDF membranes were observed using chemiluminescent HRP substrate (Millipore, USA). Primary antibodies used in this study are follows: for JAK1 detection, anti-JAK1 antibody at 1:1000 dilution were used along with JAK1 transfer membrane in a constant current of
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300 mA for 120 min; for cleaved caspase-3 detection, anti-cl.caspase-3 antibody was used at 1:1500
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dilution along with cl.caspase-3 transfer membrane in a constant current of 300 mA for 20 min; for
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Bax detection, anti-Bax antibody was used at 1:1000 dilution along with Bax transfer membrane in a constant current of 300 mA for 21 min; for Bcl-2 detection, anti-Bcl-2 antibody was used at 1:500
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dilution along with Bcl-2 transfer membrane in a constant current of 300 mA for 25 min; for GAPDH detection, anti-GAPDH antibody was used at 1:10000 dilution along with GAPDH transfer
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membrane in a constant current of 300 mA for 40min; for STAT3 detection, anti-STAT3 antibody was used at 1:10000 dilution along with STAT3 transfer membrane in a constant current of 300 mA
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for 40 min; for Mcl-1 detection, anti-Mcl-1 antibody was used at 1:10000 dilution along with Mcl-1
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transfer membrane in a constant current of 300 mA for 20 min.
Scratch assay for the study of the cell movement ability The scratch assay is a simple, reproducible assay commonly used to measure basic cell migration parameters such as speed, persistence, and polarity. Eighty percent of the SH-SY5Y cells were
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blotted out from old culture medium and were washed with PBS. Trypsin solution was added to the cells before washing. The cells were observed under inverted microscope. When the cells were separated, fresh medium was added and the cell density was adjusted to 5×105 /mL. The 6-well plate was removed and inoculated with 1 mL cell suspension at a density of 5×105 /mL and was left to incubate
overnight
at
37℃ at
5%
CO2.
The
sequences
of
miR-708
mimic
were
5’-AAGGAGCUUACAAUCUAGCUGGG-3’. After 4 h of OGD treatment, miR-708 mimics were 4
dyed. Following this, 10 µL of miR-708 mimics/NC was added to 250 µL of serum-free medium. Five µL of LipofectamineTM 2000 was added to 250 µL of serum-free medium at 25℃ for 5 min. The mixture was diluted with miR-708 with LipofectamineTM 2000 and mixed gently for 20 min at 25℃ to form the miR-708/LipofectamineTM 2000 complex. The miR-708/ LipofectamineTM 2000 complex was added to the wells of the culture medium and mixed gently. After incubation for 4-6 h, the complex was removed and replaced with fresh medium. The cells were scratched with sterile 200 mL pipette tips and were washed 3 times with PBS. The cells were removed, and serum-free medium
photographed with MOTIC inverted microscope and analyzed by IPP software.
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Transwell migration assay for the study of the cell migration ability
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was added and incubated at 37℃ at 5% CO2 for 48 h. The cell movement ability was observed and
The transwell assay is a commonly used test to study the migratory response of cells in vitro. The cells were treated with OGD for 4 h and then transplanted into complete culture medium for 24 h for
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Transwell assay. After 12 h, the cells were cultured with serum-free medium. The cells were washed
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with PBS and then resuspended with PBS containing 0.2% BSA. The cells were counted and the cell density was adjusted to 2 × 105/mL. 100 μl of the cell suspension was added to the Transwell
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chamber. 700 uL of complete medium containing 10 % FBS serum was added to the lower chamber.
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The cells were cultured in 5% CO2 at 37℃ 48 h. The cells were removed in the middle of the incubation with gentle wiping of the cells in the upper chamber with a cotton swab. The cells were
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washed 3 times with PBS and were fixed in 4% paraformaldehyde for 20 min. The cells were washed with PBS 3 times and were placed in a vial containing crystal violet dye solution for 5-10 min. Scalpel was used to remove the mold from the chamber. The cells were observed and photographed
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with OLYMPUS CX41 positive microscope, taking four fields, and were counted with IPP software.
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Transwell invasion assay for the study of the cell invasive ability The transwell assay is a commonly used test to study the invasive response of cells in vitro. The cells were treated with OGD for 4 h and were transferred to complete culture medium for 24 h for
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Transwell assay. The Matrigel stock solution was moved from -20℃ to 4℃ refrigerator and was left overnight. The Matrigel stock solution and the pre-cold serum-free high glucose DMEM medium were mixed in a pre-cooled sterile centrifuge tube at a ratio of 1:15 (0.8 μg/μL) Gel solution (100 μL) was added to each of the Transwell assay chamber. After 2 h, the residual liquid in the chamber was aspirated and 50 μL of sterile PBS was added to each well. The assay chamber was placed at 37℃ for 30 min to remove any unfixed Matrigel. The cells were cultured with serum-free medium 12 h before migration. The cells were washed with PBS for 2 times and then resuspended with PBS containing 5
0.2 % BSA. The cells were counted and the cell density was adjusted to 2 × 105/mL. 100 μL of the cell suspension was added to the Transwell chamber. 700 uL of complete medium containing 10% FBS serum was added to the lower chamber. The chamber was cultured in 5% CO2 at 37℃ in an incubator for 48 h. The cells were removed in the middle of the incubation and were gently wiped from the upper chamber using a cotton swab. The cells were washed with PBS 3 times and were fixed in 4% paraformaldehyde for 20 min. The chamber was placed in a vial containing crystal violet dye solution for 5-10 min. Scalpel was used to remove the mold from the chamber. The cells were
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observed and photographed with OLYMPUS CX41 positive microscope, taking four fields, and were
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counted with IPP software.
Luciferase reporter assay for transcriptional ability
The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. The target genes of miR-708 were determined using miRTarBase
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(http://mirtarbase.mbc.nctu.edu.tw/). The wild-type 3’-UTR segment of the JAK1 mRNA (not the
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full length of JAK1 3’-UTR) containing miR-708 binding sites was cloned into the dual-luciferase reporter pGL3 vector, Wt-JAK1-3’UTR (Promega, USA). A mutant construct in miR-708 binding
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sites of JAK1 3’UTR region also was syntheszed and subcloned into pGL3-control vector,
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Mut-JAK1-3’UTR (Ambion). For dual-luciferase reporter assay, SH-SY5Y cells were transfected with miR-708 or normal control for 24 h, and the cells were transfected with Wt/Mut- JAK1-3’UTR
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reporter plasmid using Lipofectamine 2000 (Invitrogen). After 48 h, luciferase activity was determined using a Dual-Luciferase Reporter Assay Kit (Promega, USA) according to the
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manufacturer’s instructions. Renilla-luciferase was used for normalization.
5-ethynyl-2-deoxyuridine cell proliferation assay
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5-ethynyl-2’-deoxyuridine is a novel alternative for BrdU (5-bromo-2’-deoxyuridine) assay to directly measure active DNA synthesis or S-phase synthesis of the cell cycle. The cells were seeded onto 96-well plates. Cell proliferation ability was detected by 5-ethynyl-2’-deoxyuridine (EdU) kit
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(Molecular Probes, USA) according to the manufacturer’s instructions. Each well was added with 100 µL culture medium and 5 mol/L EdU and was incubated for 2 h. After 30 min 4% paraformaldehyde was treated, and the plates were treated with 0.5% Triton, Hoechst 33342 (10 μg/mL) reaction solution (Sigma, USA). The plates were stained with 4,6-diamidino-2-phenylindole (DAPI). After washing with PBS for three times, the cells were detected under fluorescence microscopy (Olympus, Japan) at 40× magnification. Condensed or fragmented nuclei were considered as apoptotic. 6
Immunofluorescence The cells were fixed in 4% formaldehyde for 10 min and were permeabilized in 0.5% Triton X-100 for 10 min and blocked in 1% BSA solution for 45 min. The cells were incubated with primary antibody, JAK1 (1:1000) or MAP2 (1:500) at 4°C overnight and Cy3-labelled or FITC-labelled secondary antibodies (Beyotime, China) at room temperature for 1 h. The cells were counterstained
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with DAPI (5 g/L) for 5 min and observed under fluorescence microscope (Olympus, Japan).
Apoptosis assay
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A Coulter Epics XL flow cytometer (Beckman Coulter, CA) was used to analyze cell apoptotic rates using FITC/PI apoptosis detection kit (Ebioe, China). Annexin V-Alexa Fluor/PI staining was used according to the manufacturer’s instructions.
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Statistical analysis
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SPSS 20.0 software HPACkage (Chicago, USA) was used to analyze the data with independent
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samples t-test. All the values were represented as mean ± standard deviation (SD). Each experiment
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was repeated three times. P < 0.05 was considered statistically significant..
Results
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Oxygen and glucose-deprived (OGD)/ reoxygenation promoted SH-SY5Y cell apoptosis To establish an in vitro model of ischemic stroke, oxygen and glucose were removed from human
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neuroblastoma SH-SY5Y cell culture. Cell survival was detected using a flow cytometer after 48 h of reoxygenation. The results showed that SH-SY5Y cells survival was remarkably decreased with the increase in duration of OGD (Figure 1). As shown in Figure 2, the OGD/R treatment reduced
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miR-708 and JAK1 mRNAs expression (Figure 2A), while JAK1 protein expression was upregulated (Figure 2B and 2C).
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miR-708 mimics promoted OGD/R SH-SY5Y cell proliferation and SH-SY5Y neuronal cell survival To examine the functional significance of miR-708 in OGD/R SH-SY5Y, the cells were transfected with miR-708 mimics. The qRT-PCR results showed that miR-708 mimic transfection could significantly upregulate miR-708 mRNA expression in SH-SY5Y cells by 3-folds (Figure 3A). Cell proliferation was significantly increased in OGD/R SH-SY5Y cells after transfection with miR-708 mimics as compared to control transfected cells (Figure 3B). We next examined the effect of miR-708 on cell cycle. The Annexin V apoptosis assay showed that miR-708 mimics reduced cell 7
apoptosis rate from 29.19% to 16.19% as compared to control (Figure 4C, 4D). Cell cycle analysis showed that miR-708 mimics resulted in a significant increase of S-phase and G2-phase of OGD/R SH-SY5Y cells from 11% to 17% (Figure 4E, 4F), and decreased G1-phase cells. Our results suggested that the expression of miR-708 significantly increased the proliferation of human neuroblastoma cells.
miR-708 mimics increase the migration and invasion ability of OGD/R SH-SY5Y cells
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Next, we investigated the migration and invasive roles of miR-708 in neuroblastoma cells. OGD/R SH-SY5Y cells were transfected with miR-708 mimics. Transwell migration and invasion assay was
transfected cells in vitro. The results showed
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performed to investigate the cell mobility and cell invasive ability of miR-708 mimics or control that miR-708 overexpression in SH-SY5Y cells
significantly promoted cell migration and invasion (Figure 3C, 3D, 3E). The scratch assay analysis was performed to further indicate the cell mobility of miR-708 transfected cells. The results showd
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thatoverexpression of miR-708 significantly increased cell motility as compared to control (1.0 vs.
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0.62. P = 0.03; Figure 4A, 4B).
JAK1 acts as a target for miR-708 in SH-SY5Y cells
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The target of miR-708 in human neuroblastoma cells was determined using bioinformatics software (miRTarBase and Target-Scan). It was observed that JAK1 3’UTRs exhibited binding sequences for
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miR-708 at position, 594-600 (Figure 5A). To further verify the role of JAK1 as a target of miR-708, luciferase activity assay was performed. It was observed that miR-708 significantly inhibited the
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luciferase activity of 3’-UTR of JAK1 in SH-SY5Y cells (Figure 5B). Further experiments were performed to investigate the expression of JAK1 in miR-708 mimics transfected SH-SY5Y cells. The results showed that the mRNA and protein levels of JAK1 expression were downregulated with
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exogenous miR-708 expression (Figure 5C, 5D). To further test the protective effects of miR-708 on neuron, we measured the expression of neuron markers NEUN and MAP2, we found that the mRNAexpression of NEUN and the protein expression of MAP2 were upregulated as measured by
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qRT-PCR and immunofluorescence assay (Figure 5C, 5E). These findings suggested that miR-708 mimics played a protective role in OGD/R SH-SY5Y cells and JAK1 was a target of miR-708.
Loss of JAK1 expression inhibited apoptosis of OGD/R SH-SY5Y cells To explore the mechanisms involved in the cell growth promotion of JAK1, we studied the apoptotic and morphological changes in the cells upon knockdown of JAK1 mRNA. Knockdown
of JAK1
mRNA decreased JAK1 expression and increased the expression of NEUN in mRNA level, 8
respectively (Figure 6A). As shown in Figure 6B, knockdown of JAK1 mRNA significantly decreased the apoptosis of cultured OGD/R SH-SY5Y cells, thereby indicating that JAK1 reduced SH-SY5Y cancer cell growth by inducing apoptosis. Additionally, immunofluorescence showed that knockdown of JAK1 protein upregulated the expression of neuron marker MAP2 in siJAK1 transfected OGD/R SH-SY5Y cells (Figure 6C), suggesting that inhibition of JAK1 played a protective role in OGD/R. Also, the Western blotting assay showed that overexpression of miR-708 or knockdown of JAK1 protein down-regulated the expression of JAK1, STAT3, Bax and
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cl.caspase-3, which, in turn, promoted Mcl-1 and Bcl-2 level, which is directly associated with cell
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apoptosis (Figure 6D, 6E).
Discussion
Ischemic stroke is associated with high incidence of long-term cognitive impairment, which, in turn, leads to loss of hippocampal neurons and synapses [12]. Multiple evidences have demonstrated that
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neuronal damage, including apoptosis and necrosis, is usually observed after stroke [6, 18].
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Neuroprotection usually involves protection of neurons from apoptosis by cellular defense
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mechanisms [1]. Here, we investigated the biological functions of miR-708 in cerebral ischemia/reperfusion injury model.
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Investigation of pathways of miRNAs regulating the neuronal death and apoptosis is critical in designing a therapeutic strategy. Previous study showed that multiple biological events were
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associated with miRNAs, which resulted in cerebral damage [30]. Knockdown of miR-181b reduces the ischemic damage by upregulating expressions of UCHL1 and HSPA5 [20]. miR-455 is markedly
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down-regulated in ischemic stroke [11]. In this study, we have demonstrated that the expression level of miR-708 was reduced in neuronal cells after OGD/R. Importantly, down-expression of miR-708 suppressed neuronal cell apoptosis, thereby indicating that miR-708 may be a promising therapeutic
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target for cerebral ischemia/reperfusion injury. JAK1 has been found to be a critical regulator of ischemic signaling cascades, including neuronal death, oxidative stress and neuroapoptosis [7, 17], and thus may provide neuroprotection during
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acute phase of ischemia [29]. JAK kinase inhibitor CP-690550 reduced the degree of cerebral ischemia injury in the C57BL/6 mouse model of cerebral ischemia [19]. In addition, after 6-72 h of transient middle cerebral artery occlusion reperfusion, intracerebroventricular injection of JAK2 phosphorylation inhibitor AG490 resulted in the suppression of STAT3 expression and reduced in volume and the number of apoptotic cells in the adult rat cerebral infarction [24]. Our results found that the expression of JAK1 decreased significantly in the OGD/R cell model. JAK1 siRNA significantly decreased the apoptosis level of OGD/R cells and inhibition of JAK1 expression 9
reduced the expression of STAT3 and Mcl-1 and induced the expression of cleaved caspase3. Moreover, miR-708 mimics and suppression of JAK1 significantly increased NEUN and MAP2 expression in OGD/R cells. Our results were comsistent with previous studies. It has been found that activated STAT3 increased the expression of synaptophysin in the hippocampal formation [26]. STAT3 plays a neuroprotective role by inducing neuroprotective genes, such as Bcl2. Inhibition of STAT3 increases the infarct size [10]. Previous reports have found that multiple growth factors and cytokines promote neuronal survival through the STAT3 signaling
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pathway. Inhibition of interleukin 6 signaling pathway reduces the phosphorylation of STAT3, leading to increase the volume of cerebral infarction [31]. However, we found that suppression the
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expression of JAK1 reduced the level of apoptosis, and played a protective role in ischemia-reperfusion injury. The possible reason was arised from the cells and treatments used in previous studies and our work were different. In addition, the protective effect of previous reports were focused on STAT3, the downstream signal molecule of JAK1 pathway. We found miR-708 by
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direct binding to the promoter of JAK1, inhibited the expression of JAK1 and played a protective
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role in cerebral ischemia reperfusion injury.
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Summary
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Luciferase reporter assay results have showed that JAK1 was a functional target of miR-708. JAK1 upregulation was involved in inhibiting neuronal cell death and suppressing ischemic brain infarction.
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JAK1 knockdown impaired the protective functions of miR-708 mimics on neurons. These findings
Conflict of interest
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suggested that miR-708 could inhibit neuronal cell apoptosis by suppressing JAK1 expression.
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The authors declare that they have no conflict of interest.
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Figure Legends Figure 1. Effect of OGD/R on SH-SY5Y neuroblastoma injury using flow cytometry assay A. SH-SY5Y cells were exposed to OGD for 0, 2, 4, 6 h, and reperfusion for 48 h (OGD/R). Annexin V/PI labeling assessed by flow cytometry to evaluate apoptosis in SH-SY5Y cells. B. Apoptosis data
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is expressed as mean ± SD. Results were analyzed using one-way ANOVA.
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Figure 2. Correlation between OGD/R treatment and expression of miR-708 and JAK1 A. qRT-PCR analysis of miR-708 and JAK1 after OGD/R injury. B and C. The level of JAK1 in
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mRNA and protein expression.
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Figure 3. Effect of miR-708 mimics on cell viability, migration and invasion in SH-SY5Y cells exposed to OGD/R A. miR-708 expression level in ODG/R SH-SY5Y cells transfected with miR-708 mimics and control by qRT-PCR. B. Cell proliferation in miR-708 mimics and control transfected ODG/R SH-SY5Y cells detected by EdU and Hoechst staining assay. C. Migration and invasion were determined in ODG/R SH-SY5Y cells transfected with miR-708 mimics and control. D and E. The number of cells in the established field of view for migration and invasion. Data is expressed as
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mean ± SD. **
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indicates P < 0.01.
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Figure 4. Effect of overexpression of miR-708 on cell migration, apoptosis and cell cycle arrest in SH-SY5Y cells exposed to OGD/R A. In vitro scratch assay at 0 and 48 h in ODG/R SH-SY5Y cells. B. Quantification of gap closure of in vitro scratch assay as described in (A). C. Apoptosis was determined in ODG/R SH-SY5Y cells transfected with miR-708 mimics analyzed using flow cytometry. D. Quantification of apoptosis rate as described in (C). E and F. Cell cycle in ODG/R SH-SY5Y cells transfected with miR-708 mimics analyzed using flow cytometry. **
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indicates P < 0.01.
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Figure 5. Targeting JAK1 by MiR-708 A. MiR-708 target site in JAK1 3′UTR. B. Detection of dual luciferase reporter gene activity: ODG/R SH-SY5Y cells co-transfected with miR-708 mimics and JAK1 3′UTR Wt/Mut recombinant plasmids. Luciferase activity assay indicates that miR-708 could inhibit luciferase activity of Wt plasmid. However, no change in the activity of Mut plasmid was observed. *P < 0.01. C. Overexpression of miR-708 downregulated JAK1 expression and upregulated NEUN expression at mRNA level . *indicates P < 0.05. D and E. MiR-708 and control transfected ODG/R SH-SY5Y cells
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were harvested for determining JAK1 and MAP2 expression using immunofluorescence staining.
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Figure 6. Effect of JAK1 expression on inhibition of apoptosis of OGD/R SH-SY5Y cells A. qRT-PCR analysis of JAK1 and NEUN expression in si-JAK1 transfected ODG/R SH-SY5Y cells. *
indicates P < 0.05. B and C. Hoechst staining of si-JAK1 and control transfected ODG/R SH-SY5Y
cells for JAK1 and MAP2 expression analysis using immunofluorescence staining.
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indicates P <
0.01. D and E. Western blot analysis of JAK1, STAT3, Mcl-1, Bax, Bcl-2 and cl.caspase-3
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expression in miR-708 mimics or si-JAK1 transfected ODG/R SH-SY5Y cells.
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S0304-3940(17)30915-1 https://doi.org/10.1016/j.neulet.2017.11.017 NSL 33225
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
20-5-2017 6-11-2017 7-11-2017
Please cite this article as: Chao Huang, Haitao Zhou, Xiangyang Ren, Junfang Teng, Inhibition of JAK1 by microRNA-708 promotes SH-SY5Y neuronal cell survival after oxygen and glucose deprivation and reoxygenation, Neuroscience Letters https://doi.org/10.1016/j.neulet.2017.11.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
Inhibition of JAK1 by microRNA-708 promotes SH-SY5Y neuronal cell survival after oxygen and glucose deprivation and reoxygenation Running Title: miR-708 and JAK1 in SH-SY5Y neuron cell survival. Chao Huang1,2, Haitao Zhou2, Xiangyang Ren2, Junfang Teng1*
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1) Department of Neurology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, P.R. China
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2) Department of Neurology, Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang 471000, Henan, P.R. China
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*Correspondence to: Dr Junfang Teng, Department of Neurology, the First Affiliated Hospital of
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Zhengzhou University, No. 1 Jianshe East Road, Zhengzhou 450052, Henan, P.R. China.
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Email: [email protected]. [email protected].
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Tel: +86 13838210077
Highlights
Human neuroblastoma cells (SH-SY5Y) downregulated miR-708 and upregulated JAK after OGD/R treatment.
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Silence of miR-708 can promote OGD/R SH-SY5Y cell proliferation migration and invasion.
JAK1 as a direct target of miR-708.
A reversed correlation existed between miR-708 and JAK1 mRNA levels in OGD/R SH-SY5Y.
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Abstract
MicroRNAs mediates gene expression in various diseases. Studies have shown that aberrant
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expression of miRNAs affected cerebral protection. In this study, we have investigated the effects of microRNA-708 (miR-708) on cell survival of oxygen and glucose-deprived reoxygenation (OGD/R) human neuroblastoma cells (SH-SY5Y) and explored whether miR-708 inhibited neuronal death by targeting JAK1. In vitro model of ischemia was used to investigate the neuroprotective functions of miR-708. MiR-708 mimics/siJAK1 transfected SH-SY5Y cells were treated with OGD. After 48 h of reoxygenation, cell viability and cell survival were determined by EdU and FITC/PI double staining 1
flow cytometry, respectively. Luciferase activity assay was performed to validate the role of JAK1 as a direct target of miR-708. qRT-PCR and Immunofluorescence assays were used to determine the expression of JAK1, MAP2 and NEUN in miR-708 mimics transfected SH-SY5Y cells. To explore the mechanisms involved in cell growth promotion by JAK1, morphological changes in cells were detected upon knockdown of JAK1 , and the expression levels of JAK1, Bax, Bcl-2, cleaved-caspase-3, STAT3 and Mcl-1 were determined by Western blotting. The expression of miR-708 significantly decreased in cells treated with OGD/R. MiR-708 directly targeted JAK1
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3’UTR to down-regulate JAK1 mRNA expression, whereas the expression of MAP2 and NEUN was upregulated. Previous studies have demonstrated that the suppression of JAK1 inhibited apoptosis
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phenocopied function of the miR-708 overexpression in OGD/R SH-SY5Y cells. miR-708 decreased the rate of apoptosis of OGD/R SH-SY5Y cells by suppressing the expression of JAK1.
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Key words: MiR-708; JAK1; OGD/R; Cell apoptosis; SH-SY5Y
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Introduction
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Ischemic stroke is associated with high rate of mortality and disability worldwide [22]. The increase in the number of new stroke cases and ageing population is directly proportional [9]. Cerebral
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ischemia/reperfusion injury regulates multiple cell apoptotic pathway. Reactive oxygen regulates cell apoptosis by activating multiple cell signaling pathways, such as p38, c-Jun N-terminal kinases and
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JAK/STAT pathway [27, 28]. MicroRNAs (miRNAs) are 21 nucleotides long and belong to the class of noncoding RNA. It directly integrates with the 3′-UTR of target mRNA and decreases target
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mRNA expression level or inhibit translation in different biological processes [25]. Previous study has showed that miRNAs were involved in different cellular processes, including cell proliferation, apoptosis, migration, invasion, cell cycle and stem cell renewal [4, 8, 14]. The study of miRNAs is
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critical in demonstrating a novel direction of disease research for studying the complexity of cancer biology [5]. MiR-708 is a newly found miRNA that plays an important role in various diseases. Aberrant miR-708 level also has an effect on cell proliferation and apoptosis [23].
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Previous studies have shown that activated JAK1 and STAT3 are up-regulated in neurons,
astrocytes and microglia after cerebral infarction [17, 21], and may provide neuroprotection in the acute phase of ischemia [7, 15, 16]. Following the injury to nerve cells, overexpressed and activated STAT3 has been found to contribute to neuronal survival and axon regeneration [2, 3, 29]. Also, it has been reported that JAK2/STAT3 activation significantly contributed to cell apoptosis after ischemia/reperfusion injury [13]. In this study, we investigated the role of miR-708 in oxidative stress-induced cell death and the 2
possible mechanism. An in vitro model of ischemia in SH-SY5Y cells was used to study the neuroprotective functions of miR-708. The results suggested that miR-708 rescued OGD-induced SH-SY5Y cell injury by suppressing the JAK1 apoptotic pathway. The results have shown that miR-708 is a novel therapeutic target for ischemic brain injury.
Materials and methods Cell culture
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SH-SY5Y cells (purchased from ATCC) were incubated in Dulbecco's Modified Eagle's medium (DMEM; Hyclone, USA) supplemented with 10 % fetal bovine serum (FBS; Gibco, USA), 100
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units/mL streptomycin and 100 units/mL penicillin (Sigma, USA) at 37 °C in an atmosphere of 5% CO2. For OGD model, the SH-SY5Y cells were incubated in serum and sugar-free artificial cerebrospinal fluid at 37°C in an atmosphere of 1% O2, 94% N2 and 5% CO2 for 2–6 h, and then
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incubated in DMEM medium in normoxic atmosphere for 48 h.
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Antibodies
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Commercially available antibodies were used for all the immunoblotting and immunofluorescence studies. Anti-JAK1, Bax, Bcl-2 and anti-cleaved caspase-3 antibodies were obtained from Abcam Company (UK). Anti-GAPDH, anti-MAP2, anti-STAT3 and anti-Mcl-1 were obtained from Shanghai Kangcheng Biotech Company, China. All the secondary antibodies used were obtained from Boster, China. qRT-PCR for miRNA and mRNA expression
Quantitative polymerase chain reaction (qPCR), is a laboratory technique of molecular biology based
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on the polymerase chain reaction (PCR). Real-time PCR can be used quantitatively (quantitative real-time PCR), and semi-quantitatively, i.e. above/below a certain amount of DNA molecules (semi
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quantitative real-time PCR).Total RNA was extracted using Trizol reagent (Takara, Japan). RNA was reverse transcribed with a Bestar qPCR RT Kit (DBI Bioscience, Germany) according to the manufacturer’s instructions. Expression values were normalized to the control endogenous small
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RNA U6 or β-actin. Primer sequences used are as follows: β-actin (forward: 5′-ATC GTG CGT GAC ATT AAG GAG AAG -3′; reverse: 5′- AGG AAG GAA GGC TGG AAG AGT G -3′), JAK1 (forward: 5′- ATT GAG AAC GAG TGT CTA GGG A -3′; reverse: 5′-CCT TCA GGT CAT GCG TGG AC -3′), miR-708 (forward: 5′- ACA CTC CAG CTG GGA AGG AGC TTA CAA TCT AG -3′; reverse: 5′- CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG AGC TGG G-3′), U6 (forward: 5′- CTC GCT TCG GCA GCA CA -3′; reverse: 5′- AAC GCT TCA CGA ATT TGC GT -3′), NEUN (forward: 5′- CCC TCC GAC CCT ACA GAG AA -3′; reverse: 5′- AAT TCA GGC CCG 3
TAG ACT GC -3′). The data was analyzed using 2–△△Ct calculation.
Western blotting for protein expression Western blotting is a widely used analytical technique used to detect specific proteins in a sample of tissue homogenate or extract. Cells were grown to 80–90 % confluence and were washed with ice-cold PBS. After 48 h of transfection, lysed cells with protein extraction buffer (Beyotime, China)
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were added with PMSF (Genebase, China). For western blotting, the proteins were mixed with SDS loading buffer (pH = 6.8 250mM Tris-HCL, 10% SDS, 0.5% bromophenol blue, 5% β-mercaptoethanol, 50% glycerol) and incubated in 98°C for 8 min. The proteins were separated with
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5–15% SDS-PAGE and were transferred to PVDF membranes (Millipore, USA) before detection with primary antibodies. PVDF membranes were observed using chemiluminescent HRP substrate (Millipore, USA). Primary antibodies used in this study are follows: for JAK1 detection, anti-JAK1 antibody at 1:1000 dilution were used along with JAK1 transfer membrane in a constant current of
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300 mA for 120 min; for cleaved caspase-3 detection, anti-cl.caspase-3 antibody was used at 1:1500
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dilution along with cl.caspase-3 transfer membrane in a constant current of 300 mA for 20 min; for
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Bax detection, anti-Bax antibody was used at 1:1000 dilution along with Bax transfer membrane in a constant current of 300 mA for 21 min; for Bcl-2 detection, anti-Bcl-2 antibody was used at 1:500
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dilution along with Bcl-2 transfer membrane in a constant current of 300 mA for 25 min; for GAPDH detection, anti-GAPDH antibody was used at 1:10000 dilution along with GAPDH transfer
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membrane in a constant current of 300 mA for 40min; for STAT3 detection, anti-STAT3 antibody was used at 1:10000 dilution along with STAT3 transfer membrane in a constant current of 300 mA
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for 40 min; for Mcl-1 detection, anti-Mcl-1 antibody was used at 1:10000 dilution along with Mcl-1
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transfer membrane in a constant current of 300 mA for 20 min.
Scratch assay for the study of the cell movement ability The scratch assay is a simple, reproducible assay commonly used to measure basic cell migration parameters such as speed, persistence, and polarity. Eighty percent of the SH-SY5Y cells were
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blotted out from old culture medium and were washed with PBS. Trypsin solution was added to the cells before washing. The cells were observed under inverted microscope. When the cells were separated, fresh medium was added and the cell density was adjusted to 5×105 /mL. The 6-well plate was removed and inoculated with 1 mL cell suspension at a density of 5×105 /mL and was left to incubate
overnight
at
37℃ at
5%
CO2.
The
sequences
of
miR-708
mimic
were
5’-AAGGAGCUUACAAUCUAGCUGGG-3’. After 4 h of OGD treatment, miR-708 mimics were 4
dyed. Following this, 10 µL of miR-708 mimics/NC was added to 250 µL of serum-free medium. Five µL of LipofectamineTM 2000 was added to 250 µL of serum-free medium at 25℃ for 5 min. The mixture was diluted with miR-708 with LipofectamineTM 2000 and mixed gently for 20 min at 25℃ to form the miR-708/LipofectamineTM 2000 complex. The miR-708/ LipofectamineTM 2000 complex was added to the wells of the culture medium and mixed gently. After incubation for 4-6 h, the complex was removed and replaced with fresh medium. The cells were scratched with sterile 200 mL pipette tips and were washed 3 times with PBS. The cells were removed, and serum-free medium
photographed with MOTIC inverted microscope and analyzed by IPP software.
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Transwell migration assay for the study of the cell migration ability
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was added and incubated at 37℃ at 5% CO2 for 48 h. The cell movement ability was observed and
The transwell assay is a commonly used test to study the migratory response of cells in vitro. The cells were treated with OGD for 4 h and then transplanted into complete culture medium for 24 h for
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Transwell assay. After 12 h, the cells were cultured with serum-free medium. The cells were washed
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with PBS and then resuspended with PBS containing 0.2% BSA. The cells were counted and the cell density was adjusted to 2 × 105/mL. 100 μl of the cell suspension was added to the Transwell
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chamber. 700 uL of complete medium containing 10 % FBS serum was added to the lower chamber.
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The cells were cultured in 5% CO2 at 37℃ 48 h. The cells were removed in the middle of the incubation with gentle wiping of the cells in the upper chamber with a cotton swab. The cells were
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washed 3 times with PBS and were fixed in 4% paraformaldehyde for 20 min. The cells were washed with PBS 3 times and were placed in a vial containing crystal violet dye solution for 5-10 min. Scalpel was used to remove the mold from the chamber. The cells were observed and photographed
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with OLYMPUS CX41 positive microscope, taking four fields, and were counted with IPP software.
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Transwell invasion assay for the study of the cell invasive ability The transwell assay is a commonly used test to study the invasive response of cells in vitro. The cells were treated with OGD for 4 h and were transferred to complete culture medium for 24 h for
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Transwell assay. The Matrigel stock solution was moved from -20℃ to 4℃ refrigerator and was left overnight. The Matrigel stock solution and the pre-cold serum-free high glucose DMEM medium were mixed in a pre-cooled sterile centrifuge tube at a ratio of 1:15 (0.8 μg/μL) Gel solution (100 μL) was added to each of the Transwell assay chamber. After 2 h, the residual liquid in the chamber was aspirated and 50 μL of sterile PBS was added to each well. The assay chamber was placed at 37℃ for 30 min to remove any unfixed Matrigel. The cells were cultured with serum-free medium 12 h before migration. The cells were washed with PBS for 2 times and then resuspended with PBS containing 5
0.2 % BSA. The cells were counted and the cell density was adjusted to 2 × 105/mL. 100 μL of the cell suspension was added to the Transwell chamber. 700 uL of complete medium containing 10% FBS serum was added to the lower chamber. The chamber was cultured in 5% CO2 at 37℃ in an incubator for 48 h. The cells were removed in the middle of the incubation and were gently wiped from the upper chamber using a cotton swab. The cells were washed with PBS 3 times and were fixed in 4% paraformaldehyde for 20 min. The chamber was placed in a vial containing crystal violet dye solution for 5-10 min. Scalpel was used to remove the mold from the chamber. The cells were
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observed and photographed with OLYMPUS CX41 positive microscope, taking four fields, and were
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counted with IPP software.
Luciferase reporter assay for transcriptional ability
The luciferase reporter assay is commonly used as a tool to study gene expression at the transcriptional level. The target genes of miR-708 were determined using miRTarBase
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(http://mirtarbase.mbc.nctu.edu.tw/). The wild-type 3’-UTR segment of the JAK1 mRNA (not the
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full length of JAK1 3’-UTR) containing miR-708 binding sites was cloned into the dual-luciferase reporter pGL3 vector, Wt-JAK1-3’UTR (Promega, USA). A mutant construct in miR-708 binding
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sites of JAK1 3’UTR region also was syntheszed and subcloned into pGL3-control vector,
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Mut-JAK1-3’UTR (Ambion). For dual-luciferase reporter assay, SH-SY5Y cells were transfected with miR-708 or normal control for 24 h, and the cells were transfected with Wt/Mut- JAK1-3’UTR
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reporter plasmid using Lipofectamine 2000 (Invitrogen). After 48 h, luciferase activity was determined using a Dual-Luciferase Reporter Assay Kit (Promega, USA) according to the
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manufacturer’s instructions. Renilla-luciferase was used for normalization.
5-ethynyl-2-deoxyuridine cell proliferation assay
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5-ethynyl-2’-deoxyuridine is a novel alternative for BrdU (5-bromo-2’-deoxyuridine) assay to directly measure active DNA synthesis or S-phase synthesis of the cell cycle. The cells were seeded onto 96-well plates. Cell proliferation ability was detected by 5-ethynyl-2’-deoxyuridine (EdU) kit
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(Molecular Probes, USA) according to the manufacturer’s instructions. Each well was added with 100 µL culture medium and 5 mol/L EdU and was incubated for 2 h. After 30 min 4% paraformaldehyde was treated, and the plates were treated with 0.5% Triton, Hoechst 33342 (10 μg/mL) reaction solution (Sigma, USA). The plates were stained with 4,6-diamidino-2-phenylindole (DAPI). After washing with PBS for three times, the cells were detected under fluorescence microscopy (Olympus, Japan) at 40× magnification. Condensed or fragmented nuclei were considered as apoptotic. 6
Immunofluorescence The cells were fixed in 4% formaldehyde for 10 min and were permeabilized in 0.5% Triton X-100 for 10 min and blocked in 1% BSA solution for 45 min. The cells were incubated with primary antibody, JAK1 (1:1000) or MAP2 (1:500) at 4°C overnight and Cy3-labelled or FITC-labelled secondary antibodies (Beyotime, China) at room temperature for 1 h. The cells were counterstained
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with DAPI (5 g/L) for 5 min and observed under fluorescence microscope (Olympus, Japan).
Apoptosis assay
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A Coulter Epics XL flow cytometer (Beckman Coulter, CA) was used to analyze cell apoptotic rates using FITC/PI apoptosis detection kit (Ebioe, China). Annexin V-Alexa Fluor/PI staining was used according to the manufacturer’s instructions.
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Statistical analysis
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SPSS 20.0 software HPACkage (Chicago, USA) was used to analyze the data with independent
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samples t-test. All the values were represented as mean ± standard deviation (SD). Each experiment
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was repeated three times. P < 0.05 was considered statistically significant..
Results
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Oxygen and glucose-deprived (OGD)/ reoxygenation promoted SH-SY5Y cell apoptosis To establish an in vitro model of ischemic stroke, oxygen and glucose were removed from human
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neuroblastoma SH-SY5Y cell culture. Cell survival was detected using a flow cytometer after 48 h of reoxygenation. The results showed that SH-SY5Y cells survival was remarkably decreased with the increase in duration of OGD (Figure 1). As shown in Figure 2, the OGD/R treatment reduced
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miR-708 and JAK1 mRNAs expression (Figure 2A), while JAK1 protein expression was upregulated (Figure 2B and 2C).
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miR-708 mimics promoted OGD/R SH-SY5Y cell proliferation and SH-SY5Y neuronal cell survival To examine the functional significance of miR-708 in OGD/R SH-SY5Y, the cells were transfected with miR-708 mimics. The qRT-PCR results showed that miR-708 mimic transfection could significantly upregulate miR-708 mRNA expression in SH-SY5Y cells by 3-folds (Figure 3A). Cell proliferation was significantly increased in OGD/R SH-SY5Y cells after transfection with miR-708 mimics as compared to control transfected cells (Figure 3B). We next examined the effect of miR-708 on cell cycle. The Annexin V apoptosis assay showed that miR-708 mimics reduced cell 7
apoptosis rate from 29.19% to 16.19% as compared to control (Figure 4C, 4D). Cell cycle analysis showed that miR-708 mimics resulted in a significant increase of S-phase and G2-phase of OGD/R SH-SY5Y cells from 11% to 17% (Figure 4E, 4F), and decreased G1-phase cells. Our results suggested that the expression of miR-708 significantly increased the proliferation of human neuroblastoma cells.
miR-708 mimics increase the migration and invasion ability of OGD/R SH-SY5Y cells
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Next, we investigated the migration and invasive roles of miR-708 in neuroblastoma cells. OGD/R SH-SY5Y cells were transfected with miR-708 mimics. Transwell migration and invasion assay was
transfected cells in vitro. The results showed
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performed to investigate the cell mobility and cell invasive ability of miR-708 mimics or control that miR-708 overexpression in SH-SY5Y cells
significantly promoted cell migration and invasion (Figure 3C, 3D, 3E). The scratch assay analysis was performed to further indicate the cell mobility of miR-708 transfected cells. The results showd
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thatoverexpression of miR-708 significantly increased cell motility as compared to control (1.0 vs.
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0.62. P = 0.03; Figure 4A, 4B).
JAK1 acts as a target for miR-708 in SH-SY5Y cells
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The target of miR-708 in human neuroblastoma cells was determined using bioinformatics software (miRTarBase and Target-Scan). It was observed that JAK1 3’UTRs exhibited binding sequences for
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miR-708 at position, 594-600 (Figure 5A). To further verify the role of JAK1 as a target of miR-708, luciferase activity assay was performed. It was observed that miR-708 significantly inhibited the
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luciferase activity of 3’-UTR of JAK1 in SH-SY5Y cells (Figure 5B). Further experiments were performed to investigate the expression of JAK1 in miR-708 mimics transfected SH-SY5Y cells. The results showed that the mRNA and protein levels of JAK1 expression were downregulated with
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exogenous miR-708 expression (Figure 5C, 5D). To further test the protective effects of miR-708 on neuron, we measured the expression of neuron markers NEUN and MAP2, we found that the mRNAexpression of NEUN and the protein expression of MAP2 were upregulated as measured by
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qRT-PCR and immunofluorescence assay (Figure 5C, 5E). These findings suggested that miR-708 mimics played a protective role in OGD/R SH-SY5Y cells and JAK1 was a target of miR-708.
Loss of JAK1 expression inhibited apoptosis of OGD/R SH-SY5Y cells To explore the mechanisms involved in the cell growth promotion of JAK1, we studied the apoptotic and morphological changes in the cells upon knockdown of JAK1 mRNA. Knockdown
of JAK1
mRNA decreased JAK1 expression and increased the expression of NEUN in mRNA level, 8
respectively (Figure 6A). As shown in Figure 6B, knockdown of JAK1 mRNA significantly decreased the apoptosis of cultured OGD/R SH-SY5Y cells, thereby indicating that JAK1 reduced SH-SY5Y cancer cell growth by inducing apoptosis. Additionally, immunofluorescence showed that knockdown of JAK1 protein upregulated the expression of neuron marker MAP2 in siJAK1 transfected OGD/R SH-SY5Y cells (Figure 6C), suggesting that inhibition of JAK1 played a protective role in OGD/R. Also, the Western blotting assay showed that overexpression of miR-708 or knockdown of JAK1 protein down-regulated the expression of JAK1, STAT3, Bax and
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cl.caspase-3, which, in turn, promoted Mcl-1 and Bcl-2 level, which is directly associated with cell
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apoptosis (Figure 6D, 6E).
Discussion
Ischemic stroke is associated with high incidence of long-term cognitive impairment, which, in turn, leads to loss of hippocampal neurons and synapses [12]. Multiple evidences have demonstrated that
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neuronal damage, including apoptosis and necrosis, is usually observed after stroke [6, 18].
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Neuroprotection usually involves protection of neurons from apoptosis by cellular defense
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mechanisms [1]. Here, we investigated the biological functions of miR-708 in cerebral ischemia/reperfusion injury model.
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Investigation of pathways of miRNAs regulating the neuronal death and apoptosis is critical in designing a therapeutic strategy. Previous study showed that multiple biological events were
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associated with miRNAs, which resulted in cerebral damage [30]. Knockdown of miR-181b reduces the ischemic damage by upregulating expressions of UCHL1 and HSPA5 [20]. miR-455 is markedly
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down-regulated in ischemic stroke [11]. In this study, we have demonstrated that the expression level of miR-708 was reduced in neuronal cells after OGD/R. Importantly, down-expression of miR-708 suppressed neuronal cell apoptosis, thereby indicating that miR-708 may be a promising therapeutic
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target for cerebral ischemia/reperfusion injury. JAK1 has been found to be a critical regulator of ischemic signaling cascades, including neuronal death, oxidative stress and neuroapoptosis [7, 17], and thus may provide neuroprotection during
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acute phase of ischemia [29]. JAK kinase inhibitor CP-690550 reduced the degree of cerebral ischemia injury in the C57BL/6 mouse model of cerebral ischemia [19]. In addition, after 6-72 h of transient middle cerebral artery occlusion reperfusion, intracerebroventricular injection of JAK2 phosphorylation inhibitor AG490 resulted in the suppression of STAT3 expression and reduced in volume and the number of apoptotic cells in the adult rat cerebral infarction [24]. Our results found that the expression of JAK1 decreased significantly in the OGD/R cell model. JAK1 siRNA significantly decreased the apoptosis level of OGD/R cells and inhibition of JAK1 expression 9
reduced the expression of STAT3 and Mcl-1 and induced the expression of cleaved caspase3. Moreover, miR-708 mimics and suppression of JAK1 significantly increased NEUN and MAP2 expression in OGD/R cells. Our results were comsistent with previous studies. It has been found that activated STAT3 increased the expression of synaptophysin in the hippocampal formation [26]. STAT3 plays a neuroprotective role by inducing neuroprotective genes, such as Bcl2. Inhibition of STAT3 increases the infarct size [10]. Previous reports have found that multiple growth factors and cytokines promote neuronal survival through the STAT3 signaling
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pathway. Inhibition of interleukin 6 signaling pathway reduces the phosphorylation of STAT3, leading to increase the volume of cerebral infarction [31]. However, we found that suppression the
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expression of JAK1 reduced the level of apoptosis, and played a protective role in ischemia-reperfusion injury. The possible reason was arised from the cells and treatments used in previous studies and our work were different. In addition, the protective effect of previous reports were focused on STAT3, the downstream signal molecule of JAK1 pathway. We found miR-708 by
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direct binding to the promoter of JAK1, inhibited the expression of JAK1 and played a protective
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role in cerebral ischemia reperfusion injury.
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Summary
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Luciferase reporter assay results have showed that JAK1 was a functional target of miR-708. JAK1 upregulation was involved in inhibiting neuronal cell death and suppressing ischemic brain infarction.
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JAK1 knockdown impaired the protective functions of miR-708 mimics on neurons. These findings
Conflict of interest
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suggested that miR-708 could inhibit neuronal cell apoptosis by suppressing JAK1 expression.
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The authors declare that they have no conflict of interest.
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Figure Legends Figure 1. Effect of OGD/R on SH-SY5Y neuroblastoma injury using flow cytometry assay A. SH-SY5Y cells were exposed to OGD for 0, 2, 4, 6 h, and reperfusion for 48 h (OGD/R). Annexin V/PI labeling assessed by flow cytometry to evaluate apoptosis in SH-SY5Y cells. B. Apoptosis data
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is expressed as mean ± SD. Results were analyzed using one-way ANOVA.
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Figure 2. Correlation between OGD/R treatment and expression of miR-708 and JAK1 A. qRT-PCR analysis of miR-708 and JAK1 after OGD/R injury. B and C. The level of JAK1 in
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mRNA and protein expression.
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Figure 3. Effect of miR-708 mimics on cell viability, migration and invasion in SH-SY5Y cells exposed to OGD/R A. miR-708 expression level in ODG/R SH-SY5Y cells transfected with miR-708 mimics and control by qRT-PCR. B. Cell proliferation in miR-708 mimics and control transfected ODG/R SH-SY5Y cells detected by EdU and Hoechst staining assay. C. Migration and invasion were determined in ODG/R SH-SY5Y cells transfected with miR-708 mimics and control. D and E. The number of cells in the established field of view for migration and invasion. Data is expressed as
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mean ± SD. **
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indicates P < 0.01.
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Figure 4. Effect of overexpression of miR-708 on cell migration, apoptosis and cell cycle arrest in SH-SY5Y cells exposed to OGD/R A. In vitro scratch assay at 0 and 48 h in ODG/R SH-SY5Y cells. B. Quantification of gap closure of in vitro scratch assay as described in (A). C. Apoptosis was determined in ODG/R SH-SY5Y cells transfected with miR-708 mimics analyzed using flow cytometry. D. Quantification of apoptosis rate as described in (C). E and F. Cell cycle in ODG/R SH-SY5Y cells transfected with miR-708 mimics analyzed using flow cytometry. **
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indicates P < 0.01.
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Figure 5. Targeting JAK1 by MiR-708 A. MiR-708 target site in JAK1 3′UTR. B. Detection of dual luciferase reporter gene activity: ODG/R SH-SY5Y cells co-transfected with miR-708 mimics and JAK1 3′UTR Wt/Mut recombinant plasmids. Luciferase activity assay indicates that miR-708 could inhibit luciferase activity of Wt plasmid. However, no change in the activity of Mut plasmid was observed. *P < 0.01. C. Overexpression of miR-708 downregulated JAK1 expression and upregulated NEUN expression at mRNA level . *indicates P < 0.05. D and E. MiR-708 and control transfected ODG/R SH-SY5Y cells
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were harvested for determining JAK1 and MAP2 expression using immunofluorescence staining.
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Figure 6. Effect of JAK1 expression on inhibition of apoptosis of OGD/R SH-SY5Y cells A. qRT-PCR analysis of JAK1 and NEUN expression in si-JAK1 transfected ODG/R SH-SY5Y cells. *
indicates P < 0.05. B and C. Hoechst staining of si-JAK1 and control transfected ODG/R SH-SY5Y
cells for JAK1 and MAP2 expression analysis using immunofluorescence staining.
**
indicates P <
0.01. D and E. Western blot analysis of JAK1, STAT3, Mcl-1, Bax, Bcl-2 and cl.caspase-3
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expression in miR-708 mimics or si-JAK1 transfected ODG/R SH-SY5Y cells.
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