Articles in PresS. Am J Physiol Cell Physiol (February 10, 2016). doi:10.1152/ajpcell.00226.2015
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Down-regulation of miR-384-5p Attenuates Rotenone-induced Neurotoxicity in Dopaminergic
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SH-SY5Y Cells Through Inhibiting Endoplasmic Reticulum Stress
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Mingfang Jiang1,2,#, Qiang Yun3,#, Feng Shi4, Guangming Niu5, Yang Gao5, Shenghui Xie5, Shengyuan
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Yu1,*
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1
Department of Neurology, Chinese PLA General Hospital, Beijing 100853,China
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2
Department of Neurology, Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010059,
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China
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3
Department of Neurosurgery, Inner Mongolia People’s Hospital, Hohhot 010020, China
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4
Department of Radiology, Inner Mongolia Chinese Medicine Hospital, Hohhot 010020, China
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5
Department of Radiology, Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010059,
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China
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#
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*Corresponding author: Shengyuan Yu
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Correspondence to: Department of Neurology, Chinese PLA General Hospital, No. 28 Fuxing Road,
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Haidian District, Beijing 100853, China
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Tel. +86-010-13501171068; Fax: +86-010-66499020
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E-mail: [email protected]
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Running head: miR-384-5p regulates rotenone-induced neurotoxicity.
These authors (Mingfang Jiang and Qiang Yun) contributed equally to this work.
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AUTHORS’ CONTRIBUTIONS
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All authors participated in the design, interpretation of the studies and analysis of the data and
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review of the manuscript; MFJ, QY and SYY designed and prepared the experiments; MFJ and QY
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Copyright © 2016 by the American Physiological Society.
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performed the experiments; FS, GMN, YG and SHX contributed reagents/materials/analysis tools; MFJ
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and QY wrote the manuscript; SYY modified and revised the manuscript. All authors read and
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approved the final manuscript.
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Abstract
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Endoplasmic reticulum (ER) stress has been linked to the pathogenesis of Parkinson’s disease (PD).
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However, the role of microRNAs (miRNAs) in this process involved in PD remains poorly understood.
30
Recent studies indicate that miR-384-5p plays an important role for cell survival in response to
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different insults, but the role of miR-384-5p in PD-associated neurotoxicity remains unknown. In this
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study, we investigated the role of miR-384-5p in an in vitro model of PD using dopaminergic SH-SY5Y
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cells treated with rotenone. We found that miR-384-5p was persistently induced by rotenone in neurons.
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Also, the inhibition of miR-384-5p significantly suppressed rotenone-induced neurotoxicity, while
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overexpression of miR-384-5p aggravated rotenone-induced neurotoxicity. Through bioinformatics and
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the dual-luciferase reporter assay, miR-384-5p was found to directly target the 3’-UTR of glucose
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regulated protein 78 (GRP78), the master regulator of ER stress sensors. Quantitative polymerase chain
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reaction and Western blotting analysis showed that miR-384-5p negatively regulated the expression of
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GRP78. Inhibition of miR-384-5p remarkably suppressed rotenone-evoked ER stress which was
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evident by a reduction in the phosphorylation of activating transcription factor 4 (ATF4) and
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inositol-requiring enzyme 1 (IRE1α). The downstream target genes of ER stress including
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CCAAT/enhancer-binding protein-homologous protein (CHOP) and X box-binding protein-1 (XBP-1)
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were also decreased by the miR-384-5p inhibitor. In contrast, overexpression of miR-384-5p enhanced
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ER stress signaling. In addition, knockdown of GRP78 significantly abrogated the inhibitory effect of
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miR-384-5p inhibitors on cell apoptosis and ER stress signaling. Moreover, we observed a significant
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increase of miR-384-5p expression in primary neurons induced by rotenone. Taken together, our results
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suggested that miR-384-5p mediated ER stress by negatively regulating GRP78 and that miR-384-5p
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inhibition might be a novel and promising approach for the treatment of PD.
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49
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Keywords
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Endoplasmic reticulum stress; Parkinson's disease; miR-384-5p; GRP78
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53
Abbreviations
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PD, Parkinson’s disease; miRNAs, microRNAs; ER, endoplasmic reticulum; GRP78, glucose regulated
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protein 78; IRE1, inositol-requiring enzyme 1; ATF, activating transcription factor; PERK, pancreatic
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ER kinase-like ER kinase; eIF2α, eukaryotic initiation factor 2α; CHOP, CCAAT/enhancer-binding
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protein-homologous protein; XBP-1, X box-binding protein-1
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4
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INTRODUCTION
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Parkinson’s disease (PD) is one of the most common neurodegenerative diseases worldwide,
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characterized by the progressive loss of dopaminergic neurons in the substantia nigrapars compacta and
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a reduction of dopamine production in the striatum (6, 41, 43). The development and progress of the
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disease mainly depends on age and approximately 1% of the older population (>60 years) in
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industrialized countries are affected by PD (41). An increasing number of studies have suggested that
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numerous mechanisms, including mitochondrial defects, oxidative stress, dysimmunity, heredity and
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cell apoptosis, have been implicated in the pathogenesis of PD (22, 24, 46, 48). However, the precise
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mechanism underlying the pathogenesis of PD remains poorly understood, hampering the development
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of efficient treatments for PD. The current therapeutics only temporarily relieve the symptoms, thus
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improving patients’ life quality to a certain degree. Recently, increasing evidence has suggested that
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endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of PD, which indicates a
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potential therapeutic option for PD treatment (50).
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The hallmark of PD is the accumulation of misfolded proteins in the neurons (19, 36), which could
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evoke cellular stress in the ER (25, 30, 53). ER stress, also termed the unfolded protein response,
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contributes to protecting cells against toxic stimuli or cellular stress-induced accumulation of misfolded
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proteins (29, 36). However, the prolonged and excessive ER stress induces cell apoptosis (3).
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Glucose-regulated protein 78 (GRP78), also named binding protein (BiP), plays an important role in
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the initiation of ER stress. Under normal circumstances, GRP78 binds to three ER stress receptors,
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including inositol-requiring enzyme 1 (IRE1), activating transcription factor-6 (ATF6), and pancreatic
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ER kinase-like ER kinase (PERK), which becomes dissociated from complexes following the
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accumulation of misfolded proteins, resulting in the activation of IRE1-, ATF6- and PERK-mediated
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downstream signaling (38). The phosphorylation of PERK activates eukaryotic initiation factor 2α
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(eIF2α), thus inhibiting protein synthesis (15, 37). Activated eIF2α subsequently activates ATF4 which
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could
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protein-homologous protein) (14, 51, 55). The ATF6 pathway is activated by dissociation from GRP78
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followed by trafficking to the Golgi to produce cleaved ATF6, which regulates the expression of ER
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stress target genes such as CHOP and X box-binding protein-1 (XBP-1) (16, 54). The phosphorylation
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of IRE1α results in IRE1 pathway activation, which is associated with unconventional splicing of
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XBP-1mRNA, thereby activating the transcription of ER stress target genes (17, 26, 54). The ER stress
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signaling is activated to restore the ER function and enhance the folding capacity and degradation of
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protein aggregates. However, if the homeostasis is not re-established, the ER stress target genes
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including CHOP1, XBP-1 and caspase-12 promote cell apoptosis (47). An analysis using post mortem
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samples form PD patients revealed that phosphorylated PERK and eIF2α were expressed at a higher
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level in neuromelanin containing dopaminergic neurons in the substantia nigra, implying ER stress was
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activated in brain tissues (18). Furthermore, ER stress has been found to be involved in cellular model
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of PD (39, 44). It has been reported that the prolonged ER stress induced by toxic stimuli such as
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rotenone results in the apoptotic death of neurons (10). In rotenone rat model of PD, suppression of ER
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stress provides neuroprotection (49). Therefore, inhibiting the prolonged ER stress-induced cell
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apoptosis may provide a novel insight into the treatment of PD.
induce
cell
death
pro-apoptotic
genes
such
as
CHOP (CCAAT/enhancer-binding
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MicroRNAs (miRNAs) are a group of short, non-coding RNAs that negatively regulate gene
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expression, and thus participate in various pathological process (2). Increasing evidence has suggested
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that they may be implicated in pathogenesis of PD (7). However, their molecular mechanism has not
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been fully understood. In recent years, miR-384-5p has been reported to play an important role for cell
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survival in response to different insults. Ethanol exposure causes the expression changes of
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miR-384-5p in rat brain (20). Spinal cord injury significantly increases the expression of miR-384-5p
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in the serum (13). Importantly, miR-384-5p has been shown to be an indicator for trimethyltin-induced
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neurotoxicity (32) and is found to be differentially expressed in dopaminergic neurons following
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cocaine addiction (42). However, whether miR-384-5p is associated with neurotoxicity in PD remains
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unknown. In this study, we aimed to investigated the role and the potential underlying mechanism of
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miR-384-5p in an in vitro model of PD induced by rotenone. Through bioinformatics and expression
110
analysis, we found that miR-384-5p regulated ER stress in dopaminergic SH-SY5Y cells via negatively
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regulating GRP78 expression, the master regulator of ER stress sensors. Overall, our study for the first
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time revealed a direct link between miR-384-5p with ER stress in dopaminergic SH-SY5Y cells, and
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suggested that miR-384-5p-GRP78 axis might be served as potential molecular targets for preventing
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dopaminergic neuron loss in PD.
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MATERIALS AND METHODS
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Cell culture. Human dopaminergic neuroblastoma SH-SY5Y cells were obtained from the Type
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Culture Collection of the Chinese Academy of Sciences (Shanghai, China), and were maintained in
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Dulbecco's Modified Eagle Medium/Ham's Nutrient Mixture F-12 (DMEM/F12; Invitrogen, Carlsbad,
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CA, USA) that was supplemented with 10% fetal calf serum (FCS) plus penicillin and streptomycin
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(1%). The cells were cultured in humidified atmosphere containing 5% CO2 at 37°C. Human primary
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neurons were isolated from the postmortem brain tissues provided by Chinese PLA General Hospital.
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The cells were cultured in Neuronal Medium (ScienCell Research Laboratories, Carlsbad, CA, USA)
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supplemented with 1% neuronal growth supplement and 1% penicillin and streptomycin at 37 °C and
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5% CO2. Cells were used at passage zero. The experimental procedure was approved by Institutional
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Human Experiment and Ethic Committee of Chinese PLA General Hospital and carried out in
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accordance with Helsinki Declaration.
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Cell treatment. Cells were exposed to rotenone (Sigma, St. Louis, MO, USA) at various
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concentrations (1, 5, 10, 20, and 50 μM) (28) for the indicated times before being harvested for analysis.
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For miRNAs transfection, cells were transfected with miR-384-5p mimics or inhibitors using
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Lipofectamine 2000 (Invitrogen) at a final concentration of 50 nM for 24 h prior to rotenone (20 μM)
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exposure. For gene silencing, 50 nM of GRP78 siRNA or control siRNA (Santa Cruz Biotechnology,
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Santa Cruz, CA, USA) was transfected into SH-SY5Y cells in the presence or absence of miR-384-5p
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inhibitors using Lipofectamine 2000 (Invitrogen). All assays were performed in triplicate.
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MTT
assay.
Cell
growth
and
8
viability
were
measured
with
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were seeded
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into 96-well plates at a density of 1×104 cells/well and cultured for 24 h. Then, cells were transfected
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with miR-384-5p mimics or inhibitors for 24 h followed by exposure to 20 μM of rotenone for another
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24 h. Thereafter, the old medium was replaced by fresh medium plus 20 μl of MTT solution (0.5 mg/ml)
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and cultured for a further 4 h. Finally, the formazan dye crystals formed in the cell culture were
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dissolved with 150 µl of dimethylsulfoxide per well and the optical density value at 490 nm in the
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solution was measured by a microplate reader (ThermoElectron Corporation, Vantaa, Finland). The
145
results were expressed as the percentage of MTT reduction, assuming the absorbance of control cells to
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be 100%.
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Cell apoptosis assay. Cell apoptosis was evaluated by Annexin V/propidium iodide (PI) double
149
staining method. Briefly, cells were harvested and digested with 2.5 g/l trypsin. After washing with
150
phosphate buffered saline (PBS), cells were centrifuged, collected and re-suspended in 500 μl of PBS.
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Then, 5 μl of Annexin V-FITC and 5 μl of PI were added and co-incubated in the dark for 15 min at
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room temperature. Finally, apoptotic cells were quantified by fluorescence-activated cell sorting (BD
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Biosciences, San Jose, CA, USA) and then analyzed by Cell Quest software (BD Biosciences). The
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data were represented as cell percentage (%).
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Capase-3 activity assay. Capase-3 activity was measured using a commercial kit (BioVision,
157
Milpitas, CA, USA). Briefly, cells were lysed in ice-cold cell lysis buffer. The supernatant was
158
collected after centrifugation (10,000×g for 1 min) and the protein concentration was measured. Then,
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50 μl of cell lysis buffer containing 100 μg of protein was incubated with 5 μl of 4 mM DEVD-pNA
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160
substrate in 50 μl of reaction buffer for 2 h at 37 ºC. The OD value at 405 nm was determined using
161
microplate reader.
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Quantitative PCR analysis. Total RNA was isolated using miRNeasy Mini Kit (Qiagen, Dusseldorf,
164
Germany) which was used to synthesize cDNA using M-MLV reverse transcriptase (Clontech, Palo
165
Alto, CA, USA) for mRNA analysis, or using the one-step primescript miRNA cDNA synthesis kit
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(Takara, Dalian, China) for miRNA analysis according to the manufacturer’s instructions. The PCR
167
assay was performed using SYBR Green qPCR Master Mix (ThermoFisher, Shanghai, China). The
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primers used for qPCR were as follows: GRP78 forward, 5’-ggaaagaaggttacccatgc-3’ and reverse,
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5’-ggaacaggtccatgttcagc-3’; miR-384-5p forward, 5’-acactccagctgggattcctagaaattg-3’ and reverse,
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5’-tggtgtcgtggagtcg-3’;
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5’-tggttcacacccatgacgaa-3’; U6 SnRNA forward, 5’-gcttcggcagcacatatactaaaat-3’, and reverse,
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5’-cgcttcacgaatttgcgtgtcat-3’. Relative gene expression was calculated by comparison with the internal
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reference gene GAPDH or U6 SnRNA using the 2−ΔΔCt method.
GAPDH
forward,
5’-ggagtccactggcgtcttca-3’
and
reverse,
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175
Western blot analysis. Proteins in cell samples were extracted using a protein extraction kit
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(Applygen Technologies, Beijing, China) and the protein concentration was measured by a BCA
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Protein Assay Kit (Beyotime, Haimen, China) according to the manufacturers’ instructions. Equivalent
178
amounts of protein (50 µg) for each sample were run on 12.5% sodium dodecyl sulfate polyacrylamide
179
gel electrophoresis. The proteins were then electrophoretically transferred to a PVDF membrane which
180
was blocked by 2.5% nonfat milk for 1 h at 37°C. The membrane was blotted with anti-GRP78,
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anti-CHOP, anti-XBP-1 and anti-GAPDH antibodies purchased from Santa Cruz Biotechnology, Inc.
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182
(Santa Cruz, CA, USA), and anti-ATF4, anti-phospo-ATF4, anti-IRE1α and anti-phospho-IRE1α
183
antibodies purchased from Bioss (Beijing, China); the membrane was then incubated overnight at 4°C,
184
followed by exposure to horseradish peroxidase conjugated secondary antibodies (1:2,000; Bioss) for 1
185
h at room temperature. The immune-blot signals were developed using an enhanced
186
chemiluminescence method.
187
188
Dual-luciferase Reporter Assay. The 3’-UTR of human GRP78 gene containing seed regions of
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miR-384-5p (300 bp) were PCR amplified from human cDNA and then cloned into pGL3
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dual-luciferase miRNA target expression vector (Promega, Madison, WI, USA). The corresponding
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mutant constructs were created by mutating the seed regions of the miR-384-5p binding sites by using
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site-directed mutagenesis kit (Agilent Technologies Co. Ltd, Beijing, China). SH-SY5Y cells were
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seeded into 24-well plates for 24 h before transfection. For reporter assays, cells were transiently
194
co-transfected with pGL3-3’-UTR of GRP78 (WT) or pGL3-mutant 3’-UTR of GRP78 (MT) in the
195
presence of miR-384-5p mimics or inhibitors using Lipofectamine 2000 (Invitrogen). After incubation
196
of 48 h, the relative luciferase activity was analyzed using dual-luciferase system (Promega).
197
198
Statistical Analysis. The data were expressed as mean ± standard deviation. Statistical analysis was
199
performed by scientific statistic software SPSS version 11.5 (SPSS Inc., Chicago, IL, USA) using
200
one-way analysis of variance followed by Bonferroni post hoc. p < 0.05 was regarded statistically
201
significant.
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RESULTS
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The expression of miR-384-5p is significantly induced by rotenone in SH-SY5Y cells. To investigate
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whether miR-384-5p played a critical role in rotenone-induced neurotoxicity, we initially detected its
207
expression profile in SH-SY5Y cells treated with different concentrations of rotenone (0, 1, 5, 10, 20,
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and 50 μM) by the qPCR method. The results showed that miR-384-5p was dose-dependently increased
209
after rotenone treatment (Fig. 1A). Furthermore, its expression was persistently increased post-time
210
challenge (Fig. 1B). The results indicate that miR-384-5p plays an important role in regulating
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rotenone-induced neurotoxicity.
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Down-regulation of miR-384-5p inhibits rotenone-induced neurotoxicity. To explore the precise
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role of miR-384-5p in regulating rotenone-induced neurotoxicity, we detected the gain or loss of
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function of miR-384-5p on rotenone-induced neurotoxicity. According to the results of MTT (Fig. 2A),
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the viability of SH-SY5Y cells was significantly decreased by rotenone (20 μM) treatment relative to
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the control group. When the cells were pretreated with miR-384-5p inhibitor for 24 h, the cell viability
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was markedly increased compared with the rotenone-treated group (Fig. 2A). In contrast, cell viability
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was further decreased by treatment with miR-384-5p mimics (Fig. 2A). The results showed that
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down-regulation of miR-384-5p exhibited a protective effect against rotenone-induced neurotoxicity.
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To further verify the role of miR-384-5p on rotenone-induced neurotoxicity, we detected their effect on
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cell apoptosis flow cytometry. The apoptotic cells were markedly increased in rotenone-treated cells
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compared with the control group which could be effectively attenuated by miR-384-5p inhibitor or
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significantly aggravated by miR-384-5p mimics (Fig. 2 B and C). Furthermore, we detected cell
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225
apoptosis by measuring the activity of Caspase-3. The results showed that rotenone treatment
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significantly increased the activity of Caspase-3 which was decreased by miR-384-5p inhibitor or
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further increased by miR-384-5p mimics (Fig. 2 D). Again, the data suggest that down-regulation of
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miR-384-5p exhibits a protective effect against rotenone-induced neurotoxicity.
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miR-384-5p directly targets the 3’-UTR of GRP78. To explore the underlying mechanism of
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miR-384-5p, we sought to investigate its target gene. Using bioinformatics analysis, we found that
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GRP78 was one of the putative target genes of miR-384-5p (Fig. 3A). To confirm this relationship, we
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performed a dual luciferase reporter assay. The results showed that miR-384-5p mimics significantly
234
reduced the luciferase activity in pGL3- 3’-UTR of GRP78 (WT) transfected cells, which had no effect
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on pGL3-mutated 3’-UTR of GRP78 (MT) transfected group (Fig. 3B). In contrast, the miR-384-5p
236
inhibitor markedly increased luciferase activity in WT-transfected cells (Fig. 3B). Furthermore, we
237
detected the effect of rotenone treatment on the luciferase activity of WT- or MT-transfected cells. The
238
results showed that rotenone treatment significantly decreased the luciferase activity in WT-transfected
239
cells whereas it showed no obvious effect on MT-transfected cells (Fig. 3C). Taken together, these
240
results indicate that miR-384-5p directly targets the 3’-UTR of GRP78.
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miR-384-5p regulates the expression of GRP78 in SH-SY5Y cells. Due to the targeting relationship
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between miR-384-5p and the 3’-UTR of GRP78, the expression of GRP78 could be regulated by
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miR-384-5p. To test the hypothesis, we detected the expression of GRP78 upon treatment with
245
miR-384-5p inhibitors or mimics. The qPCR results demonstrated that the mRNA expression level of
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GRP78 was obviously increased by treatment with miR-384-5p inhibitors or decreased by treatment
13
247
with miR-384-5p mimics (Fig. 4A) which was consistent with the protein levels detected by Western
248
blot analysis (Fig. 4B). These results indicate that miR-384-5p directly regulates GRP78 pathway.
249
250
Down-regulation of miR-384-5p inhibits rotenone-evoked ER stress signaling. A previous report has
251
suggested that GRP78 is a master regulator for ER stress signaling (53). Thus, ER stress signaling
252
could be affected by modulating miR-384-5p expression. The results showed that the expression levels
253
of phosphorylated ATF4 (pATF4) and phosphorylated IRE1α (pIRE1α) were both significantly
254
increased by rotenone treatment (Fig. 5A and B), implying that rotenone treatment evoked ER stress
255
signaling, which is consistent with the findings of Goswami et al. (10). As expected, the expression
256
levels of pATF4 and pIRE1α were significantly decreased by miR-384-5p inhibitor treatment or further
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increased by treatment with miR-384-5p mimics (Fig. 4A and B). Furthermore, the downstream target
258
genes of ER stress signaling including CHOP and XBP1 were also decreased by miR-384-5p inhibitor
259
treatment or increased by treatment with miR-384-5p mimics (Fig. 5C and D). Taken together, our data
260
imply that miR-384-5p regulates ER stress signaling.
261
262
Silencing of GRP78 enhanced ER stress and abrogates the protective effect of miR-384-5p on
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rotenone-induced neurotoxicity. To further confirm that miR-384-5p regulated ER stress via GRP78,
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we used a knockdown of GRP78 and detected the effect of miR-384-5p down-regulation on ER stress
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and rotenone-induced neurotoxicity. The results exhibited that silencing of GRP78 (Fig. 6A)
266
significantly blocked the inhibitory effect of miR-384-5p inhibitors on ER stress signaling, as indicated
267
by the increased expression of ER stress signaling target gene including CHOP and XBP1 (Fig. 6B and
268
C). Furthermore, the protective effect of miR-384-5p inhibitor on rotenone-induced neurotoxicity was
14
269
also significantly attenuated by the knockdown of GRP78 (Fig. 6D). Overall, these results suggest that
270
miR-384-5p may regulate ER stress signaling via GRP78.
271
272
Detection of miR-384-5p in human primary neurons. To further determine the critical role of
273
miR-384-5p involved in PD, we used human primary neurons to validate the role of miR-384-5p in
274
regulating rotenone-induced neurotoxicity. The results showed that miR-384-5p was significantly
275
increased in primary neurons with treatment of rotenone (Fig. 7A). Furthermore, rotenone-induced
276
neurotoxicity was markedly reversed by miR-384-5p inhibition or further aggravated by miR-384-5p
277
overexpression (Fig. 7B). The results indicate that miR-384-5p functions as an important regulator for
278
neuron survival in PD.
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281
DISCUSSION
282
In the present study, we have demonstrated that miR-384-5p is strongly induced in dopaminergic
283
SH-SY5Y cells treated with rotenone. Also, the down-regulation of miR-384-5p significantly
284
attenuated rotenone-induced neurotoxicity by inhibiting ER stress. The direct link between miR-384-5p
285
and the 3’-UTR of GRP78 was confirmed by a dual-luciferase activity and miR-384-5p negatively
286
regulated the expression of GRP78. Thus, down-regulation of miR-384-5p elevated the expression of
287
GRP78, leading to the inhibition of rotenone-evoked ER stress. Our data were in line with the findings
288
of Gorbatyuk et al. (9), who reported that overexpression of GRP78 relieved α-Synuclein neurotoxicity
289
in a rat model of PD by down-regulating ER stress.
290
In this study, we used an in vitro model of PD induced by rotenone in human dopaminergic
291
SH-SY5Y cells. Rotenone can pass through the blood-brain barrier that induces neuronal dysfunction
292
and neurodegeneration (31, 35). A previous study has demonstrated that rotenone induces ER stress by
293
up-regulating the phosphorylation of PERK and eIF2α and that the knockdown of eIF2α significantly
294
blocked rotenone-induced ER stress and cytotoxicity in neuroblastoma cells (4). Wu et al. reported that
295
candesartan cilexetil inhibited rotenone-evoked ER stress by down-regulating the expression of ATF4
296
and CHOP) and protected rat dopaminergic neurons (52). A recent study revealed that ER stress played
297
an important role in rotenone-induced apoptosis of mouse neuroblastoma cells (10). In line with these
298
findings, our data also indicated that rotenone induced ER stress in human dopaminergic SH-SY5Y
299
cells which was evident by an increase in phosphorylation of ATF4 and IRE1α. Intriguingly, the
300
miR-384-5p significantly inhibited the phosphorylation of ATF4 and IRE1α leading to a reduction in
301
ER stress signaling.
302
In this study, we found that the expression of miR-384-5p was significantly increased in response to
303
rotenone treatment. Several studies have proposed miR-384-5p was served as potential biomarkers for
16
304
numerous pathological processes. It has been reported that the serum level of miR-384-5p was
305
significantly increased in mouse with acute spinal cord injury, and might be used as a promising
306
biomarker for predicting the severity of acute spinal cord injury (13). In rats treated with trimethyltin,
307
miR-384-5p was significantly up-regulated with the evolution of neural cell death that was regarded as
308
potential indicators of neurotoxicity (32). The decreased expression of miR-384-5p is required for spine
309
enlargement associated with long-term potentiation (12). Here, we demonstrated that miR-384-5p was
310
upregulated in response to rotenone treatment indicating miR-384-5p might has the potential to be used
311
as a biomarker for PD. Bao et al. reported that miR-384-5p was an important protecting factor for
312
cardioprotection
313
phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit delta (1). Here, we reported that
314
GRP78 was a target gene of miR-384-5p, both of which were involved in regulating rotenone-induced
315
neurotoxicity by affecting ER stress signaling.
in
myocardial
ischemia
by
regulating
the
expression
of
316
To uncover the underlying mechanism of miR-384-5p in regulating ER stress, we investigated its
317
target gene. Through bioinformatics analysis, dual-luciferase reporter assay and expression analysis, we
318
identified that miR-384-5p directly targeted the 3’-UTR of GRP78 and negatively regulated the mRNA
319
and protein expression of miR-384-5p. Several reports have suggested that numerous miRNAs regulate
320
the expression of GRP78 and its downstream signaling. It was reported that increased miR-181a
321
aggravated injury in the mouse stroke model by inhibiting GRP78 protein expression level, whereas
322
reduced levels of miR-181a were associated with increased GRP78 protein levels and reduced injury
323
(33). In several cancer cell lines, GRP78 was reported to be regulated by miR-199a-5p as well as
324
miR-30d and miR-181a and which cooperatively suppressed GRP78-mediated chemoresistance (45).
325
Similarly, Dai et al. (5) reported that miR-199a-5p was evaluated during hepatic ER stress and directly
17
326
regulated the expression of GRP78 in hepatocytes. Down-regulation of miR-181b produced
327
neuroprotection against ischemic injury via up-regulating GRP78 (34). Iwamune et al. reported that
328
miR-376a mediated the expression of GRP78 in rat granulosa cells (21). In the present study, we
329
identified miR-384-5p as a novel regulator of GRP78 and provided a novel option for modulating
330
GRP78 expression in related pathological processes.
331
GRP78 functions as the master regulator of ER stress, playing an important role in regulating many
332
diseases. It was reported that silencing of GRP78 increased the expression of CHOP and induced cell
333
apoptosis in retinal ischemia-reperfusion (27). Overexpression of GRP78 suppressed α-synuclein
334
aggregation by alleviating ER stress (23). Consistently, Gorbatyuk et al. found that overexpression of
335
GRP78 attenuated α-synuclein neurotoxicity in a rat model of PD by inhibiting ER stress (9). GRP78
336
overexpression reduces cell apoptosis through reprograming ER stress by reducing the protein levels of
337
cleaved pATF6 protein, phosphorylated eIF2α and the pro-apoptotic protein CHOP (8). Consistent with
338
these findings, our results demonstrated that overexpression of GRP78 by inhibiting miR-384-5p
339
significantly suppressed neuronal apoptosis induced by rotenone, and reprogrammed ER stress
340
signaling by inhibiting the protein levels of pATF4, pIRE1α, CHOP and XBP1. In addition, knockdown
341
of GRP78 significantly abrogated the inhibitory effect of miR-384-5p inhibitor on cell apoptosis and
342
ER stress signaling, further indicating the involvement of miR-384-5p and GRP78 in regulating ER
343
stress.
344
Previous study has suggested that GRP78 functions not only as the inhibitor for ER stress signaling
345
but also a bonafide target gene of ER stress (50). Therefore, there is a feedback loop for GRP78 in ER
346
stress signaling pathway. Here, we reported that rotenone treatment activated ER stress but the
347
expression of GRP78 had no obvious change in contrast to other ER stress target genes. The
18
348
rotenone-induced high expression of miR-384-5p might contribute to this discrepancies. We speculated
349
that miR-384-5p targeted and inhibited GRP78 expression induced by rotenone treatment, thus leading
350
to uncontrolled ER stress signaling activation. However, the expression of GRP78 in response to
351
rotenone remains to be further determined throughput over the different time interval.
352
In this study, we have demonstrated that miR-384-5p and ER stress were persistently induced in
353
neurons treated with rotenone. We demonstrated that miR-384-5p modulated ER stress by directly
354
targeting and regulating GRP78. Gorbatyuk et al. reported that overexpression of GRP78 diminished
355
α-synuclein neurotoxicity in a rat model of PD by downregulating ER stress (9). More recently,
356
Salganik et al. found that GRP78 protein overexpression protected aging nigral dopamine neurons in
357
the α- synuclein-induced rat model of PD (40). Rotenone has been reported to induce ER stress both in
358
cell model or animal model of PD (10, 11, 49). Here, we showed down-regulating miR-384-5p
359
increased the expression of GRP78 and reduced ER stress signaling induced by rotenone. The
360
inhibition of miR-384-5p showed a protective effect against rotenone-evoked neurotoxicity in human
361
SH-SY5Y cells. The expression pattern and effect of miR-384-5p on neuron survival were further
362
validated in human primary neurons. However, certain limitation of our study should be noted.
363
Whether miR-384-5p is dysregulated in PD patients remains unknown. In this study, we only
364
investigated the expression and effect of miR-384-5p using an in vitro cell model of PD induced by
365
rotenone. Whether miR-384-5p targets GRP78 to regulate ER stress and neuronal survival in in vivo
366
model of PD still needs to be further validated. Therefore, before concluding that miR-384-5p is an
367
important regulator for PD, more in vivo animal and clinical detection studies are needed.
368
369
GRANTS
19
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This study was supported by grants from Natural Science Foundation of Inner Mongolia Province of
371
China (2014MS0874) and Science and Technology One Million Engineering Project of Inner Mongolia
372
Medical University (YKD2013KJBW016).
373
374
375
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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20
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FIGURE LEGENDS
525
Fig. 1. Detection the expression of miR-384-5p in rotenone-treated cells. (A) miR-384-5p was
526
significantly increased in SH-SY5Y cells treated with different concentrations of rotenone (0, 1, 5, 10,
527
20, and 50 μM) for 24 h. *p<0.05. (B) miR-384-5p was persistently increased in SH-SY5Y cells treated
528
with 20 μM of rotenone for 12, 24, 48, 72 and 96 h. *p<0.05.
529
530
Fig. 2. Detection the effect of miR-384-5p on rotenone-induced neurotoxicity. (A) MTT method was
531
performed to detect the effect of miR-384-5p inhibitor or miR-384-5p mimics on cell viability treated
532
with rotenone. Cells were treated with miR-384-5p inhibitor, miR-384-5p mimics or miR-scrambled
533
control for 24 h prior to rotenone treatment. After incubation with rotenone (20 μM) for 24 h, cells
534
were harvested for analysis. Untreated cells were used as the control group. (B) Apoptotic cells were
535
measured by the Annexin V and PI double staining method using flow cytometry. (C) Representative
536
histograms show the percentage of apoptotic cells in different groups. (D) Cell apoptosis was examined
537
by Caspase-3 activity assay.*p<0.05.
538
539
Fig. 3. miR-384-5p directly targets the 3’-UTR of GRP78. (A) The miR-384-5p-binding site
540
predictions for GRP78. (B) Target validation using dual-luciferase reporter method in SH-SY5Y cells.
541
The relative luciferase activities of pGL3 luciferase vectors containing wild-type (WT) or mutant (MT)
542
transcripts were measured after co-transfection with the indicated miRNAs for 48 h. (C) The effect of
543
rotenone treatment on the relative luciferase activity in WT- or MT-transfected cells. *p<0.05.
544
545
Fig. 4. miR-384-5p regulates the expression of GRP78. (A) qPCR analysis of GRP78 mRNA
546
expression in cells transfected with miR-384-5p inhibitor or mimics. Cells were pretreated with
28
547
miRNAs for 24 h followed by exposure to rotenone for a further 24 h. *p<0.05. (B) GRP78 protein
548
expression was detected by Western blot analysis in different groups, and quantitatively analyzed using
549
Image-Pro Plus 6.0 and normalized to GAPDH. **p<0.01.
550
551
Fig. 5. miR-384-5p regulates ER stress signaling. The protein expression levels of pATF4 (A), pIRE1α
552
(B), CHOP (C) and XBP1 (D) in different groups were detected by Western blot analysis with the
553
indicated antibodies, and quantified using Image-Pro Plus 6.0. **p<0.01.
554
555
Fig. 6. Knockdown of GRP78 abolishes the effect of miR-384-5p inhibitors. (A) Detection of the
556
efficiency of GRP78 siRNA on GRP78 expression in different groups by Western blot analysis. Cells
557
were co-transfected with GRP78 siRNA and miR-354-5p inhibitor for 24 h and then exposed to
558
rotenone (20 μM) for a further 24 h and harvested for experimental analysis. **p<0.01. Western blot
559
analysis of GRP78 siRNA on CHOP (B) and XBP1 (C) protein expression in the presence of
560
miR-384-5p inhibitor and their quantitative analysis. **p<0.01. (D) Detection of the effect of GRP78
561
siRNA on the protective effect of miR-384-5p inhibitor against rotenone-induced neurotoxicity by the
562
MTT method. *p<0.05.
563
564
Fig. 7. Detection of miR-384-5p in primary human neurons. (A) miR-384-5p was significantly
565
increased in primary neurons treated with 20 μM of rotenone for 24 h. **p<0.01. (B) MTT method was
566
performed to detect the effect of miR-384-5p inhibitor or miR-384-5p mimics on cell viability of
567
primary neurons treated with rotenone. Cells were treated with miR-384-5p inhibitor, miR-384-5p
568
mimics or miR-scrambled control for 24 h prior to rotenone treatment. After incubation with rotenone
29
569
(20 μM) for 24 h, cells were harvested for analysis. *p<0.05.
30