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200b negatively regulate Notch1 signaling pathway to suppress CoCl2-induced apoptosis in PC12 cells
Toxicology in Vitro 65 (2020) 104787
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MiR-429/200a/200b negatively regulate Notch1 signaling pathway to suppress CoCl2-induced apoptosis in PC12 cells Chunxiao Yang1, Xiaochen Zhang1, Hongqiang Yin, Zhanqiang Du, Zhuo Yang
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College of Medicine, State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials for Ministry of Education, Nankai University, Tianjin 300071, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Apoptosis Cobalt chloride Hypoxia miR-429/200a/200b Notch1 PC12
Neuronal apoptosis is a central hallmark of cerebral ischemia, which is serious threats to human health. Notch1 signaling pathway and three members of miR-200 family, miR-429, miR-200a and miR-200b, are reported to have tight connection with hypoxia-induced injury. However, their mutual regulation relationship and their roles in neuronal apoptosis caused by hypoxia are rarely reported. In the present study, differentiated pheochromocytoma (PC12) cells were treated with chemical hypoxia inducer, cobalt chloride (CoCl2) to establish in vitro neuronal hypoxia model. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, Western blot assay and Hoechst staining indicated that CoCl2 caused apoptosis of PC12 cells along with the activation of Notch1 signallilng pathway. The treatment of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester (DAPT) inhibited Notch1 signaling pathway and attenuated the apoptosis induced by CoCl2. Real-time polymerase chain reaction (RT-PCR) showed that expressions of miR-429/200a/200b were dynamically changed during the treatment of CoCl2, and significantly decreased after 12-hour treatment of CoCl2. Overexpression of miR-429/200a/200b inhibited the Notch1 signaling pathway and suppressed CoCl2induced apoptosis in PC12 cells. These results may clarify the roles of miR-429/200a/200b and Notch1 signaling pathway in hypoxia-induced nerve injury and provide a new theoretical basis to relieve nerve injury.
1. Introduction
impaired the learning and memory ability, as well as synaptic plasticity of perforant path (PP) to dentate gyrus (DG) in hippocampus of C57BL mice (Zhang et al., 2017). In addition, neural oscillations in the hippocampus of mice were also impaired by Notch1 genetic deficiency (Li et al., 2018). More importantly, Notch1 receptor is also reported to be involved in cerebral ischemia induced neurogenesis (Wang et al., 2009), angiogenesis (Lou et al., 2012) and microglia-mediated inflammatory response (Wei et al., 2011). Nevertheless, reports about the role of Notch1 in hypoxia-induced nerve damage are not coincident. (Li et al., 2012; Wang et al., 2009). MicroRNA (miRNAs) are noncoding RNA molecules that modulating gene expression by translational inhibition and destabilization of mRNAs (Filipowicz et al., 2008). There are five members in miR-200 family including miR-200a, miR-200b, miR-200c, miR-429 and miR141, which lie in two different loci in the genome. MiR-429, miR-200a and miR-200b are clustered in chromosome 1p36, whereas miR-200c and miR-141 are located in chromosome 12p13 (Bracken et al., 2008). They can also be sub-divided into two subfamilies, with miR-429, miR200b and miR-200c as one group, miR-200a and miR-141 as another
Cerebrovascular diseases have high fatality and disability and bring great financial burden to patient families and society (Chen et al., 2014a; Hu et al., 2017). A central hallmark of these diseases is hypoxia, which induces increasing intracellular reactive oxygen species (ROS), damaging mitochondrial function and cell apoptosis in brain (Gong et al., 2017; Niatsetskaya et al., 2012). Accumulating evidences suggest that neuronal apoptosis leads to variety of neurological disorders, such as learning and memory disabilities, dementia and motor dysfunction (Giaccia et al., 2003; Yang et al., 2017a). Thus, it is necessary to elucidate the molecular mechanisms of hypoxic brain injury and seek novel therapy. Notch1 signaling pathway was reported to be crucial in nervous system and was participated in lots of important events in brain, including regulating neurogenesis, promoting radial glia-like identity, regulating cell cycle and governing self-renewal in embryonic NSCs (neural stem cells) (Breunig et al., 2007; Corbin et al., 2008; Favaro et al., 2009). In our previous studies, we found that Notch1 knockdown
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Corresponding author. E-mail address: [email protected] (Z. Yang). 1 CY, XZ have contributed equally to this work. https://doi.org/10.1016/j.tiv.2020.104787 Received 2 November 2019; Received in revised form 11 January 2020; Accepted 28 January 2020 Available online 29 January 2020 0887-2333/ © 2020 Elsevier Ltd. All rights reserved.
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Fig. 1. CoCl2 induces apoptosis in PC12 cells. (a) The viability of PC12 cells treated with different concentrations of CoCl2. n = 6/group, P < .001 vs. Control group at each time point. (b) The representative immunoreactive bands of Bax (21 kDa), Bcl-xl (30 kDa), PARP (116 kDa), cleaved-PARP (89 kDa) and β-actin (43 kDa) of control group, CoCl2 (0.2 mM) group and CoCl2 (0.4 mM) group. The treatment time of CoCl2 was 12 h. Δ represents cleaved fragment. (c) Quantitative analysis of Bax/β-actin in three groups. n = 3/group, **P < .01 vs. Control group; #P < .05 vs. 0.2 mM CoCl2 group. (d) Quantitative analysis of Bcl-xl/β-actin in three groups. n = 3/group, *P < .05, **P < .01 vs. Control group; #P < .05 vs. 0.2 mM CoCl2 group. (e) Quantitative analysis of cleaved-PARP/PARP in three groups. n = 3/group, *P < .05 vs. Control group.
Fig. 2. CoCl2 activates Notch1 signaling pathway in PC12 cells. The treatment time of CoCl2 was 12 h (a) The representative immunoreactive bands of Notch1 (125 kDa), Hes1 (30 kDa), Jagged1 (60 kDa) and β-actin (43 kDa) of control group, CoCl2 (0.2 mM) group and CoCl2 (0.4 mM) group. (b) Quantitative analysis of Notch1/β-actin in three groups. n = 3/group, *P < .05 vs. Control group; #P < .05 vs. 0.2 mM CoCl2 group. (c) Quantitative analysis of Hes1/β-actin in three groups. n = 3/group, **P < .01, ***P < .001 vs. Control group; ###P < .01, vs. 0.2 mM CoCl2 group. (d) Quantitative analysis of Jagged1/β-actin in three groups. n = 3/group.
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Fig. 3. DAPT inhibits the Notch1 signaling pathway activated by CoCl2. The treatment time of CoCl2 and DAPT was 12 h (a) The representative immunoreactive bands of Notch1 (125 kDa), NICD (120 kDa), Hes1 (30 kDa) and β-actin (43 kDa) of control group, DAPT (1 μM) group, DAPT (5 μM) group DAPT (10 μM) group, CoCl2 (0.4 mM) + DAPT (1 μM) group, CoCl2 (0.4 mM) + DAPT (5 μM) group and CoCl2 (0.4 mM) + DAPT (10 μM) group. (b) Quantitative analysis of Notch1/βactin in eight groups. n = 3/group, *P < .05 vs. Control group; #P < .05 vs. 0.4 mM CoCl2 group. (c) Quantitative analysis of NICD/β-actin in eight groups. n = 3/ group, *P < .05 vs. Control group; ##P < .01 vs. 0.4 mM CoCl2 group. (d) Quantitative analysis of Hes1/β-actin in eight groups. n = 3/group, **P < .01, ***P < .001 vs. Control group; #P < .05, ##P < .01 vs. 0.4 mM CoCl2 group. (e) The representative immunofluorescence photographs of NICD in PC12 cells of eight groups. Scale bar = 20 μm.
induced by hypoxia have not been fully investigated. It has been reported that cabalt chloride (CoCl2) can mimic hypoxia as a chemical hypoxia inducer (Chen et al., 2014b; Gong et al., 2017; Hartwig et al., 2014). Thus, in this study, differential pheochromocytoma (PC12) cells, which are morphologically and functionally similar with neurocytes, were treated with CoCl2 to establish a cell culture model of chemical hypoxia-induced neurocyte injury. We aim to investigate the role of Notch1 signaling pathway in CoCl2 induced apoptosis. Besides, whether and how the Notch1 is regulated by three representative miR-200 family members, miR-429/200a/200b, during chemical hypoxia-induced damage of PC12 cells are also investigated.
group (Gregory et al., 2008; Park et al., 2008). MiR-429, miR-200a and miR-200b are three important members of miR-200 family and are thought to play a crucial role in some important events that are related to hypoxia, such as angiogenic responses of human microvascular endothelial cells (HMECs) (Chan et al., 2011), differentiation of osteoblastic cells (Huang et al., 2016), epithelial-mesenchymal transition of lung cancer cells (Gao et al., 2015) and apoptosis of cardiomyocytes (Sun et al., 2016; Xu et al., 2016). Thus, we choose miR-429/200a/ 200b, which located in the same chromosome, as three representative miRNAs of the two subfamilies in this study. However, the expression and functions of miR429/200a/200b vary with different cell types. The precise functions and effects of these three members in neurocyte injury 3
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Fig. 4. DAPT diminishes CoCl2-induced apoptosis in PC12 cells. The treatment time of CoCl2 and DAPT was 12 h (a) The representative immunoreactive bands of Bax (21 kDa), cleaved-Caspase3 (17 kDa), PARP (116 kDa), cleaved-PARP (89 kDa) and β-actin (43 kDa) of control group, DAPT (1 μM) group, DAPT (5 μM) group, DAPT (10 μM) group, CoCl2 (0.4 mM) + DAPT (1 μM) group, CoCl2 (0.4 mM) + DAPT (5 μM) group and CoCl2 (0.4 mM) + DAPT (10 μM) group. Δ represents cleaved fragment. (b) Quantitative analysis of Bax/β-actin in eight groups. n = 3/group, *P < .05, **P < .01 vs. Control group; #P < .05 vs. 0.4 mM CoCl2 group. (c) Quantitative analysis of cleaved-Caspase3/β-actin in eight groups. n = 3/group, *P < .05, ***P < .001 vs. Control group; ##P < .01 vs. 0.4 mM CoCl2 group. (d) Quantitative analysis of cleaved-PARP/PARP in eight groups. n = 3/group, *P < .05 vs. Control group; #P < .05 vs. 0.4 mM CoCl2 group. (e) The representative photographs of Hoechst staining in PC12 cells of eight groups. Scale bar = 20 μm. (f) The cell viability of eight groups. n = 6/group, ***P < .001 vs. Control group; ## P < .01 vs. 0.4 mM CoCl2 group.
2. Materials and methods
difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester (DAPT), nerve growth factor (NGF), Hoechst 33258 and 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma, USA. RNAiso plus, PrimeScript™ RT reagent Kit, SYBR Green qPCR Master Mix and the primer for Notch1 were from TaKaRa, Japan. MiRNA-mimics, stem-loop primer and forward and reverse primer pairs
2.1. Reagents and antibodies RPMI 1640 cell culture medium and fetal bovine serum (FBS) were purchased from GIBCO Invitrogen. Cobalt chloride (CoCl2), N-[N-(3, 54
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Fig. 5. CoCl2 induces dynamic changes of expression profiles of miR-429 (a), miR-200a (b), miR-200b (c) in PC12 cells. *P < .05, **P < .01, ***P < .001 vs. Control group.
with miRNA-mimics and negative controls (20 nM; RiboBio Co., Ltd., Guangzhou, Guangdong, China), using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions.
for miR-429, miR-200a, miR-200b were designed by RiboBio Co., Ltd., China. Anti-Notch1 antibody (1:2000), anti-Hes1 antibody (1:2000), antiNICD antibody (1:1000 for Western blot assay and 1:500 for fluorescent staining), anti-Jagged1 antibody (1:2000) and anti-Bax antibody (1:2000) were purchased from Abcam, UK. Anti-PARP antibody (1:1000), anti-Bcl-xl antibody (1:2000) and anti-cleaved Caspase3 antibody (1:1000) were purchased from Cell Signaling Technology, USA. β-actin (1:5000) were purchased from Sangon Biotech (Shanghai, China). The anti-rabbit secondary peroxidase-conjugated antibodies (1:5000) were bought from Promega (Promega Co, USA). Alexa 594conjugated anti-rabbit IgG purchased from Invitrogen (New York, USA). The chemiluminescent horseradish peroxidase (HRP) substrate was purchased from Millipore, USA.
2.5. RNA isolation and real-time quantitative PCR Total RNA for detecting miR-429, miR-200a and miR-200b expressions was harvested from cells using RNAiso plus (TaKaRa, Ohtsu, Japan) according to the manufacturer's protocols. The harvested RNA was reverse transcribed into cDNA using PrimeScript™ RT reagent Kit (Takara, Japan). Then, miR-200a, miR-200b and miR-429 expressions were detected with U6 as an internal control. The stem-loop primer and forward and reverse primer pairs for miR-200a, miR-200b, miR-429 were designed by RiboBio Co., Ltd. (Guangzhou, Guangdong, China). All reactions were done in triplicate using Mastercycler ep realplex 2 (Eppendorf AG, Hamburg, Germany) and expression levels were calculated using the comparative CT method (2 −ΔΔCT).
2.2. Cell culture Rat pheochromocytoma PC12 cells were cultured in RPMI 1640 (Gibco, Gaithersburg, MD, USA) supplemented with 10% horse serum and 5% fetal bovine serum (FBS, Gibco, Gaithersburg, MD, USA) in a humidified atmosphere containing 5% CO2 at 37 °C. For differentiation, cells were kept in the medium supplemented with 1% FBS and 50 ng/m nerve growth factor (NGF, Sigma, St. Louis, MO, USA) for 7 days. The differentiation medium was replaced with fresh medium every second days.
2.6. Western blot analysis Preparation of cell lysates has been described in our previous studies (Li et al., 2016b). Briefly, cells were washed with phosphate-buffered saline (PBS, pH 7.4) at the end of the culture period and lysed in RIPA buffer (Beyotime Biotechnology, Shanghai, China). The lysates were centrifuged at 12,000 rpm at 4 °C for 20 min. The supernate was separated, mixed with loading buffer, and boiled at 100 °C for 20 min. Total protein concentration was determined with bicinchoninic acid assay (Beyotime biotechnology, China). Equal samples (20 μg) were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride transfer membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with 1% Tween-20 (TBST) and incubated with the primary antibodies diluted in blocking buffer overnight at 4 °C. After four washing steps with TBST, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, after washing four times, protein band intensities were detected with HRP substrate (Millipore, USA) by using the Tanon 5500 chemiluminescent imaging system (Tanon, Science and Technology, China). β-actin was used as an internal control and the quantitation analysis was performed by Image J (National Institutes of Health, Bethesda, MD, USA).
2.3. Cell viability assay The cell viability of PC12 was evaluated by 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay as previously described (Zhang et al., 2016). Briefly, cells were seeded in 96-well plates with 1 × 104 cells per well and cultured 24 h for the stabilization. Then, the medium containing different concentrations (0.2, 0.4, 0.6, 0.8 mM) of CoCl2 was added in each well for a certain time (6, 12, 18 and 24 h). In order to investigate the effect of DAPT on cell viability, PC12 cells were incubated with medium without drugs (control group), medium containing DAPT (1 μM, 5 μM and 10 μM), medium containing CoCl2 (0.4 mM) and medium containing CoCl2 (0.4 mM) with DAPT (1 μM, 5 μM and 10 μM) for 12 h. After incubation, the cell medium was replaced by the fresh medium containing MTT (20 μl, final concentration 5 mg/ml) and the cells were incubated for 4 h. After that, a total of 100 μl of DMSO was added to dissolve the formazan crystals in each well. Absorbance at 492 nm was measured using a microplate reader (Multiskan Mk3; Thermo Labsystems, Helsinki, Finland).
2.7. Fluorescent staining Fluorescent staining was performed following our previously description with some modifications (Li et al., 2016b). After appropriate treatment, cells cultured on glass coverslips were fixed with 4% paraformaldehyde in PBS for 15 min at 4 °C, and incubated in blocking
2.4. Transfection with miRNA-mimics To induce miR-200 family overexpression, the cells were transfected 5
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(caption on next page)
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Fig. 6. Overexpression of miR-429/200a/200b inhibits the Notch1 signaling pathway activated by CoCl2. MiR-429 (a), miR-200a (b) and miR-200b (c) were markedly increased by transfection with miRNA-mimics. The treatment time of CoCl2 was 12 h. (d) The representative immunoreactive bands of Notch1 (125 kDa), NICD (120 kDa), Hes1 (30 kDa) and β-actin (43 kDa) of negative control (NC) group, NC + CoCl2 (0.4 mM) group, miR-429 mimic group, miR-429 mimic + CoCl2 (0.4 mM) group, miR-200a mimic group, miR-200a mimic + CoCl2 (0.4 mM) group, miR-200b mimic group, miR-200b mimic + CoCl2 (0.4 mM) group. (e) Quantitative analysis of Notch1/β-actin in eight groups. n = 3/group, *P < .05, **P < .01, ***P < .001 vs. Control group; &&& P < .01 vs. miR-200a mimic group; $$$P < .001 vs. miR-200b mimic group; ++ P < .01, +++ P < .001 vs. NC + CoCl2 (0.4 mM) group; N.S. no significant difference. (f) Quantitative analysis of NICD/β-actin in eight groups. n = 3/group, *P < .05 vs. Control group; +P < .05, ++P < .01 vs. NC + CoCl2 (0.4 mM) group; N.S. no significant difference. (g) Quantitative analysis of Hes1/β-actin in eight groups. n = 3/group, ***P < .001 vs. Control group; &&&P < .01, vs. miR-200a mimic group; ++ + P < .001 vs. NC + CoCl2 (0.4 mM) group; N.S. no significant difference. (h) The representative immunofluorescence photographs of NICD in PC12 cells of eight groups. Scale bar = 20 μm.
3.2. CoCl2 activated Notch1 signaling pathway in PC12 cells
buffer (10% normal goat serum and 0.3% Triton X-100) for 1 h at room temperature. Then, cells were incubated with NICD antibody (1:500 dilution) diluted in 5% of normal goat serum, 0.3% Triton X-100 in PBS overnight at 4 °C. After three times of gentle wash with PBS, the cells were probed with fluorescence-labeled secondary antibody (1:1000 dilution) for 1 h at room temperature. Finally, after another three times of PBS washing, cells were stained with PBS containing 5 μg/ml DAPI (Sigma, St. Louis, MO, USA) for 5 min, rinsed in PBS for one time, and observed under a confocal laser scanning microscope (FV300, Olympus, Japan).
Western blot assay was performed to detect expressions of proteins in Notch1 signaling pathway. We incubated the cells in fresh medium (control group), medium containing CoCl2 (0.2 mM) and medium containing CoCl2 (0.4 mM) for 12 h. As shown in Fig. 2a and b, the expression of Notch1 was significantly increased after 0.4 mM CoCl2 treatment compared to those of control group (P < .05) and 0.2 mM treatment group (P < .05). Hes1, as a downstream effect protein in Notch1 signaling pathway, was also increased after different concentrations of CoCl2 treatment compared to control group (Fig. 2c, P < .01 for 0.2 mM treatment group and P < .001 for 0.4 mM treatment group). In addition, the expression of Hes1 was significantly higher in 0.4 mM CoCl2 treatment group compared to that of 0.2 mM CoCl2 treatment group (P < .001). The expression of Jagged1, a bound ligand of Notch1 receptor, showed an increasing trend after CoCl2 treatment with no significant difference. These results suggested that CoCl2 treatment activated Notch1 signaling pathway in PC12 cells.
2.8. Hoechst fluorescent staining Following the various treatments, PC12 were analyzed for apoptotic nuclei using Hoechst fluorescence staining. After the incubation period, the medium was removed and the PC12 cells were washed with PBS two times at room temperature. Then, the cells were stained with 1 μg/ ml Hoechst 33258 (Sigma, St. Louis, MO, USA) in medium for 30 min in dark at 37 °C to identify nuclear fragmentation, and then visualized under a confocal laser scanning microscope (FV300, Olympus, Japan).
3.3. DAPT inhibited CoCl2-induced Notch1 signaling activation To investigate the role of Notch1 signaling pathway in CoCl2 induced apoptosis, DAPT, the γ-secretase inhibitor as well as the inhibitor of Notch1 siganalling pathway, was used. Western blot assay was performed to investigate the effects of different treatment concentrations of DAPT on Notch1 signaling pathway. PC12 cells were incubated with medium without drugs (control group), medium containing DAPT (1 μM, 5 μM and 10 μM), medium containing CoCl2 (0.4 mM) and medium containing CoCl2 (0.4 mM) with DAPT (1 μM, 5 μM and 10 μM) for 12 h. As shown in Fig. 3a and b, the expression of Notch1 receptor was significantly increased after CoCl2 (0.4 mM) treatment for 12 h (P < .05). Co-treatment of CoCl2 and DAPT (1 μM and 5 μM) did not influence the expression of Notch1 compared with that of CoCl2 treatment group. However, co-treatment of CoCl2 and DAPT (10 μM) significantly decreased the expression of Notch1 compared with that of CoCl2 group (P < .05). The expression of Notch intracellular domain (NICD) was significantly up-regulated in CoCl2 treatment group (P < .05). Meanwhile, co-treatment with CoCl2 and DAPT (10 μM) significantly decreased the expression of NICD compared with that of CoCl2 treatment group (P < .01). In order to detect whether NICD was translocated into the nucleus and performed function, immunofluorescence staining was performed. We incubated the cells in fresh medium (control group), medium containing DAPT (10 μM), medium containing CoCl2 (0.4 mM) and medium containing both DAPT (10 μM) and CoCl2 (0.4 mM) for 12 h. As shown in Fig. 3e, the expression of NICD in nucleus was obviously increased after CoCl2 treatment compared with that of control group, whereas CoCl2 and DAPT co-treatment led to a visible decrease density of fluorescence compared to CoCl2 treatment group. Moreover, the expression of Hes1 was markedly higher in CoCl2 group (P < .001), CoCl2 + DAPT (1 μM) group (P < .01) and CoCl2 + DAPT (5 μM) group (P < .01) compared with that of control group. Co-treatment of CoCl2 and DAPT (P < .05 for 5 μM and P < .01 for 10 μM) significantly decreased the expression of Hes1 compared with that of CoCl2 group (Fig. 3d). These results suggested that the treatment of DAPT inhibited CoCl2-activated Notch1
2.9. Statistical analysis All data were expressed as mean ± S.E.M. Multiple samples were statistically analyzed for significance using one-way analysis of variances (One-Way ANOVA). Post hoc statistics were obtained using the LSD test (Fisher'sleast significant difference t-test) for multiple comparisons. All of the statistical analyses were performed using SPSS 22.0 software (IBM, Chicago, IL). P-values less than 0.05 were considered to be significant. All experiments were performed at least in triplicate.
3. Results 3.1. CoCl2 induced apoptosis in PC12 cells The cell viability of PC12 cells was determined by the MTT assay. As shown in Fig. 1a, the cell viability was decreased when treatment concentration increased at different time (P < .001 compared with control group). In addition, the results of Western blot assay revealed that CoCl2 promoted apoptosis in PC12 cells. As shown in Fig. 1b and c, the expression of Bax was significantly increased after 0.4 mM CoCl2 treatment for 12 h compared to those of control group (P < .01) and 0.2 mM CoCl2 treatment group (P < .05). The expression of Bcl-xl was markedly decreased in 0.2 mM treatment group (P < .05) and 0.4 mM treatment group (P < .01) compared to that of control group. The expression of Bcl-xl was significantly lower in 0.4 mM treatment group compared to that of 0.2 mM treatment group (P < .05, Fig. 1b and d). Beyond that, the ratio of cleaved PARP/PARP was significantly increased in 0.2 mM CoCl2 treatment group (P < .05) and 0.4 mM CoCl2 treatment group (P < .05) compared to that of control group (Fig. 1b and e). Taken together, these results suggested that CoCl2 induced apoptosis in PC12 cells. 7
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Fig. 7. Overexpression of miR-429/200a/200b reduces apoptosis induced by CoCl2. The treatment time of CoCl2 was 12 h. (a) The representative immunoreactive bands of Bax (21 kDa), cleaved-Caspase3 (17 kDa), PARP (116 kDa), cleaved-PARP (89 kDa) and β-actin (43 kDa) of negative control (NC) group, NC + CoCl2 (0.4 mM) group, miR-429 mimic group, miR-429 mimic + CoCl2 (0.4 mM) group, miR-200a mimic group, miR-200a mimic + CoCl2 (0.4 mM) group, miR-200b mimic group, miR-200b mimic + CoCl2 (0.4 mM) group. (b) Quantitative analysis of Bax/β-actin in eight groups. n = 3/group, **P < .01 vs. Control group; N.S. no significant difference. (c) Quantitative analysis of cleaved-Caspase3/β-actin in eight groups. n = 3/group, ***P < .001 vs. Control group; &&&P < .01 vs. miR-200a mimic group; +++P < .001 vs. NC + CoCl2 (0.4 mM) group; N.S. no significant difference. (d) Quantitative analysis of cleaved-PARP/PARP in eight groups. n = 3/ group, *P < .05, ***P < .001 vs. Control group; &&&P < .01, vs. miR-200a mimic group; $$P < .01 vs. miR-200b mimic group; +++P < .001 vs. NC + CoCl2 (0.4 mM) group; N.S. no significant difference. (e) The representative photographs of Hoechst staining in PC12 cells of eight groups. Scale bar = 20 μm.
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overexpress miR-429, miR-200a and miR-200b in PC12 cells. The expression of miR-200 family members were significantly higher in the miRNA-mimic groups than those in negative control (NC) group, respectively (Fig. 6a, b and c). These results demonstrated that the transfection with miRNA-mimics was effective in overexpressing miR200 family members in PC12 cells. Then, Western blot assay was performed to detect expressions of Notch1 signaling pathway proteins. As shown in Fig. 6d, e, f and g, expressions of Notch1, NICD and Hes1 were significantly increased after 12 h CoCl2 treatment (P < .001 for Notch1, P < .05 for NICD and P < .001 for Hes1). Notch1 expression was notably suppressed following miR-429, miR-200a and miR-200b overexpression (P < .01 for miR-429, P < .01 for miR-200a and P < .05 for miR-200b), but expressions of NICD and Hes1 were not affected by the miRNA-mimics. Beyond that, miR-429 overexpression abolished the higher expressions of Notch1, NICD and Hes1 induced by CoCl2 treatment; miR-200a overexpression abolished the higher expression of NICD induced by CoCl2 treatment; miR-200b overexpression abolished the higher expressions of NICD and Hes1 induced by CoCl2 treatment. In addition, immunofluorescence staining was also preformed to examine the expression of NICD in nucleus. Cells in each groups were also exposured to CoCl2 for 12 h. As shown in Fig. 6h, the overexpression of miR-200 family members reduced the higher expression of NICD in the nucleus induced by cobalt chloride. These results indicated that overexpression of miR-429/200a/200b effectively inhibited the activating of Notch1 signaling pathway induced by CoCl2.
signaling pathway. 3.4. DAPT diminished CoCl2-induced apoptosis in PC12 cells We subsequently investigated whether DAPT had influence on the apoptosis of PC12 cells induced by CoCl2 treatment. Western blot assay and Hoechst fluorescent staining were performed. For Western blot assay, PC12 cells were incubated with medium without drugs (control group), medium containing DAPT (1 μM, 5 μM and 10 μM), medium containing CoCl2 (0.4 mM) and medium containing CoCl2 (0.4 mM) with DAPT (1 μM, 5 μM and 10 μM) for 12 h. As shown in Fig. 4a, b, c and d, treatment of CoCl2 significantly increased the expressions of Bax (P < .01) and cleaved-Caspase3 (P < .001), as well as the ratio of cleaved-PARP/PARP (P < .05) compared with those of control group. The use of DAPT (10 μM) decreased the up-regulated expressions of apoptosis related proteins induced by CoCl2 treatment (P < .05 for Bax, P < .01 for cleaved-Caspase3 and P < .05 for cleaved-PARP/ PARP). For Hoechst staining, cells were treated with fresh medium (control group), medium containing DAPT (10 μM), medium containing CoCl2 (0.4 mM) and medium containing both DAPT (10 μM) and CoCl2 (0.4 mM) for 12 h. Hoechst staining showed that nuclei of control group were intact and exhibited dispersed blue fluorescent. On the contrary, the cells treated with CoCl2 showed obvious nuclear condensation and excessive blue fluoresent. The co-treatment of CoCl2 and DAPT (10 μM) decreased the number of cells with nuclear condensation obviously (Fig. 4e). In addition, we performed MTT assay to investigate the effect of DAPT on PC12 cell death induced by CoCl2. The results were shown in Fig. 4f. The cell viabilities of DAPT groups (1 μM, 5 μM and 10 μM) were comparable to that of control group. On the other hand, the cell viability of CoCl2 group was significantly decreased compared with that of control group, which was consistent with Fig. 1a. The cell viability in CoCl2 + DAPT (10 μM) group was significantly increased compared with that of CoCl2 group (P < .01) even though it was still lower than that of control group (P < .001). Taken together, these results suggested that the inhibition of Notch1 signaling pathway ameliorated apoptosis of PC12 cells induced by CoCl2.
3.7. Overexpression of miR-429/200a/200b attenuated apoptosis induced by CoCl2 in PC12 cells Subsequently, we further investigated the role of miR-200 family members in PC12 cell apoptosis, expressions of Bax, cleaved-Caspase3 and the ratio of cleaved-PARP/PARP were detected by Western blot assay. Cells in each groups were exposured to CoCl2 for 12 h. Compared with the NC group, the level of Bax, cleaved-Caspase3 and the ratio of cleaved-PARP/PARP were markedly increased after treatment of CoCl2 (P < .01 for Bax, P < .001 for cleaved-Caspase3 and P < .001 for the ratio of cleaved-PARP/PARP). However, miR-429 overexpression abolished the higher expressions of Bax, cleaved-Caspase3 and cleavedPARP/PARP induced by CoCl2 treatment; miR-200a overexpression abolished the higher expression of Bax induced by CoCl2 treatment; miR-200b overexpression abolished the higher expressions of Bax and cleaved-Caspase3 induced by CoCl2 treatment. Hochest staining was carried out to confirm these immunoblotting results. As shown in Fig. 7e. CoCl2 treatment led to a visible enhancement of apoptosis cells compared to that of NC group, whereas overexpression of miR-429/ 200a/200b dimished the apoptosis of cells in different degrees. Together, these results suggested that overexpression of miR-429/200a/ 200b attenuated apoptosis induced by CoCl2 in PC12 cells.
3.5. Expressions of miR-429/200a/200b dynamically changed during CoCl2 treatment in PC12 cells The dynamic expressions of miR-429, miR-200a and miR-200b during CoCl2 treatment were monitored during a time course by realtime quantitative PCR (qPCR) assay. The results indicated that expressions of miR-429, miR-200a and miR-200b were rapidly up-regulated after CoCl2 treatment and reached a maximum level in 6 h (Fig. 5, P < .001 for miR-429, miR-200a and miR-200b). After 12 h of CoCl2 treatment, relative expressions of miR-429, miR-200a and miR-200b decreased to below background levels (P < .001 for miR-429, P < .05 for miR-200a and P < .001 for miR-200b). The expressions of miR429, miR-200a and miR-200b were significantly lower than those of control group after 18 h CoCl2 treatment, respectively (P < .01 for miR-429, P < .05 for miR-200a and P < .001 for miR-200b). The expressions of miR-429 and miR-200b were significantly lower than those of control group after 24 h CoCl2 treatment (P < .01 for miR-429 and P < .001 for miR-200b), but the expression of miR-200a showed no significant difference compared with that of control group. Interestingly, we found that expressions of miR-429, miR-200a and miR200b were down-regulated, but expressions of Notch1 signaling pathway proteins were up-regulated after 12 h treatment of CoCl2. Therefore, we aimed to explore whether Notch1 signaling pathway was regulated by these three miR-200 family members.
4. Discussion CoCl2 has been widely used in PC12 cells to establish a chemical hypoxia in vitro cell model as a well-known hypoxia-mimetic agent. Our results in the present study showed that the cell viability and apoptosis of PC12 cells were significantly increased after CoCl2 treatment on a time-dependent and dose-dependent manner. Increasing evidence has supported with these results in our study (Chen et al., 2015; Gong et al., 2017; Hartwig et al., 2014; Yang et al., 2018). Notch1 receptor was reported to be crucial in nervous system. It regulated some important processes in nervous system, such as learning and memory, synaptic plasticity and neurogenesis (Costa et al., 2003; Johnson et al., 2009; Wang et al., 2004). In our previous study, we found that deficiency of Notch1 receptor led to the lower cell density in dentate gyrus (DG) region in C57BL mice compared with wildtype littermates (Zhang et al., 2017). These results indicated that Notch1 receptors played a promoting role on cell proliferation under
3.6. Notch1 signaling pathway was inhibited by miR-429/200a/200b overexpression To test this hypothesis, we used miRNA-mimics to artificially 9
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physiological conditions. On the contrary, in the present study, we demonstrated that CoCl2-induced hypoxia increased PC12 proliferation following Notch1 signaling pathway suppression by using the Notch signal inhibitor, DAPT. These results suggested that Notch1 played a pro-apoptosis role under cobalt-mimicked hypoxia. A recent publication verified our results to some extent even though they used osteoblasts as experimental subject (Li et al., 2016a). Therefore, we infer that Notch1 receptors may have different effects on cell proliferation or apoptosis under physiological and hypoxic conditions. Further studies that use in vivo hypoxia animal model are needed to clarify this point. But it is certain that the unbalanced expression level of Notch1 may result in nerve cell apoptosis and even damage the function of the nervous system. In the present study, we found that expressions of miR-429, miR200a and miR-200b were dynamically changes at different times of CoCl2 treatment. It has been reported that treatment of hypoxia for 24 h inhibited miR-200b expression in human dermal microvascular endothelial cells (HMECs) (Chan et al., 2011). Moreover, another research reported that miR-200b was reduced in H1299 cells under hypoxia for 24 h (Gao et al., 2015). These observations are in line with our data indicating that the treatment of CoCl2 for 12 h induces down-regulating of miR-200b. On the other hand, Huang et al. reported that miR-429 was up-regulated by CoCl2 treatment in MC3T3-E1 cells with the peak value at 4 h (Huang et al., 2016). Xu et al. suggested that miR-429 expression was significantly increased after human cardiomyocytes (AC-16) were subjected to culture under hypoxic conditions for 12 h (Xu et al., 2016). These results showed different trends with ours, in which the treatment with CoCl2 was for 12 h, but it was consistent with the results that treatment with CoCl2 for 6 h. Based on these results, we hypothesize that response to hypoxia of different cell types at different times is inconsistent. The changing pattern of miR-429/200a/200b in PC12 cells under hypoxia-mimetic agent treatment is specific. In the present study, we found that the effects of Notch1 signaling pathway in CoCl2 induced apoptosis of PC12 cells, a widely-used and very well-characterized model system of neuronal cells. Subsequently, we also explored how the Notch1 signaling pathway was regulated. Intriguingly, we found that the expression of Notch1 was increased, whereas the expressions of three miR-200 family members were decreased after CoCl2 treatment for 12 h. The overexpression of these three miRNAs inhibited the expression of Notch1 and attenuated the apoptosis induced by CoCl2. Notch1 receptor was identified as the targets gene of miR-429 (Xu et al., 2016) and miR-200b (Yang et al., 2013) in other studies. Generally, the target genes of miRNA were considered to be conserved between different species. Thus, we suggest that miR-429 and miR-200b reduce apoptosis in PC12 cells through inhibiting Notch1 receptor. For miR-200a, the underlying mechanism may be that miR-200a regulated its target genes, Sirt1 (Yang et al., 2017b) or ZEB2 (Xia et al., 2010), and then indirectly affected Notch1 expression. Therefore, the effect of miR-200a on inhibiting Notch1 signaling pathway and reducing apoptosis induced by CoCl2 were not obvious compared with the effects of miR-429 and miR-200b. The specific underlying mechanisms need further study. In conclusion, under CoCl2 treatment, expressions of Notch1 signaling pathway proteins were significantly up-regulated. Meantime, the apoptosis level of PC12 cells was also elevated, indicating that Notch1 signaling pathway played a vital role in this process. We then further explored the role of Notch1 in CoCl2-induced cell apoptosis. The results revealed that the inhibiting of Notch1 signaling pathway reduced the apoptosis ratio of PC12 cells, implying that the down-regulation of Notch1 provided cytoprotective effects. In addition, three miR-200 family members, miR-429, miR-200a and miR-200b, were proven to have inhibiting effects on Notch1 signaling pathway and be involved in the anti-apoptotic effects. Our findings may give some help to clarify the vital role of Notch1 signaling pathway and miR-429/200a/200b in hypoxia induced nerve injury and provide a new theoretical basis to relieve nerve injury.
The article does not contain any studies with human participants or animals performed by any of the authors. Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgements This study was supported by the National Natural Science Foundation of China (81771979, 81571804). References Bracken, C.P., Gregory, P.A., Kolesnikoff, N., Bert, A.G., Wang, J., Shannon, M.F., Goodall, G.J.J.C.r., 2008. A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition. Cancer Res. 68, 7846–7854. Breunig, J.J., Silbereis, J., Vaccarino, F.M., Šestan, N., Rakic, P., 2007. Notch regulates cell fate and dendrite morphology of newborn neurons in the postnatal dentate gyrus. Proc. Natl. Acad. Sci. 104, 20558–20563. Chan, Y.C., Khanna, S., Roy, S., Sen, C.K., 2011. miR-200b targets Ets-1 and is downregulated by hypoxia to induce angiogenic response of endothelial cells. J. Biol. Chem. 286, 2047–2056. Chen, J., Venkat, P., Zacharek, A., Chopp, M., 2014a. Neurorestorative therapy for stroke. Front. Hum. Neurosci. 8, 382. Chen, P.Y., Ho, Y.R., Wu, M.J., Huang, S.P., Chen, P.K., Tai, M.H., Ho, C.T., Yen, J.H., 2014b. Cytoprotective effects of fisetin against hypoxia-induced cell death in PC12 cells. Food Funct. 6, 286–295. Chen, P.-Y., Ho, Y.-R., Wu, M.-J., Huang, S.-P., Chen, P.-K., Tai, M.-H., Ho, C.-T., Yen, J.H., 2015. Cytoprotective effects of fisetin against hypoxia-induced cell death in PC12 cells. Food Funct. 6, 286–295. Corbin, J.G., Gaiano, N., Juliano, S.L., Poluch, S., Stancik, E., Haydar, T.F., 2008. Regulation of neural progenitor cell development in the nervous system. J. Neurochem. 106, 2272–2287. Costa, R.M., Honjo, T., Silva, A.J., 2003. Learning and memory deficits in notch mutant mice. Curr. Biol. 13, 1348–1354. Favaro, R., Valotta, M., Ferri, A.L., Latorre, E., Mariani, J., Giachino, C., Lancini, C., Tosetti, V., Ottolenghi, S., Taylor, V., 2009. Hippocampal development and neural stem cell maintenance require Sox2-dependent regulation of Shh. Nat. Neurosci. 12, 1248. Filipowicz, W., Bhattacharyya, S.N., Sonenberg, N., 2008. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet. 9, 102. Gao, X.-J., Liu, J.-W., Zhang, Q.-G., Zhang, J.-J., Xu, H.-T., Liu, H.-J., 2015. Nobiletin inhibited hypoxia-induced epithelial-mesenchymal transition of lung cancer cells by inactivating of Notch-1 signaling and switching on miR-200b. Die Pharmazie 70, 256–262. Giaccia, A., Siim, B.G., Johnson, R.S., 2003. HIF-1 as a target for drug development. Nat. Rev. Drug Discov. 2, 803. Gong, W., Qie, S., Huang, P., Xi, J., 2017. Protective effect of miR-374a on chemical hypoxia-induced damage of PC12 cells in vitro via the GADD45α/JNK signaling pathway. Neurochem. Res. 1–10. Gregory, P.A., Bert, A.G., Paterson, E.L., Barry, S.C., Tsykin, A., Farshid, G., Vadas, M.A., Khew-Goodall, Y., Goodall, G.J., 2008. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat. Cell Biol. 10, 593. Hartwig, K., Fackler, V., Jaksch-Bogensperger, H., Winter, S., Furtner, T., CouillardDespres, S., Meier, D., Moessler, H., Aigner, L., 2014. Cerebrolysin protects PC12 cells from CoCl2-induced hypoxia employing GSK3β signaling. Int. J. Dev. Neurosci. 38, 52–58. Hu, X., De Silva, T.M., Chen, J., Faraci, F.M., 2017. Cerebral vascular disease and neurovascular injury in ischemic stroke. Circ. Res. 120, 449–471. Huang, J., Peng, J., Cao, G., Lu, S., Liu, L., Li, Z., Zhou, M., Feng, M., Shen, H., 2016. Hypoxia-induced MicroRNA-429 promotes differentiation of MC3T3-E1 osteoblastic cells by mediating ZFPM2 expression. Cell. Physiol. Biochem. 39, 1177–1186. Johnson, M.A., Ables, J.L., Eisch, A.J., 2009. Cell-intrinsic signals that regulate adult neurogenesis in vivo: insights from inducible approaches. BMB Rep. 42, 245. Li, S., Zhang, X., Wang, Y., Ji, H., Du, Y., Liu, H., 2012. DAPT protects brain against cerebral ischemia by down-regulating the expression of notch 1 and nuclear factor kappa B in rats. Neurol. Sci. 33, 1257–1264. Li, C.T., Liu, J.X., Yu, B., Liu, R., Dong, C., Li, S.J., 2016a. Notch signaling represses hypoxia-inducible factor-1α-induced activation of Wnt/β-catenin signaling in osteoblasts under cobalt-mimicked hypoxia. Mol. Med. Rep. 14, 689–696. Li, Z., Yin, H., Hao, S., Wang, L., Gao, J., Tan, X., Yang, Z., 2016b. miR-200 family promotes podocyte differentiation through repression of RSAD2. Sci. Rep. 6, 27105. Li, Q., Zhang, X., Cheng, N., Yang, C., Zhang, T., 2018. Notch1 knockdown disturbed neural oscillations in the hippocampus of C57BL mice. Prog. NeuroPsychopharmacol. Biol. Psychiatry 84, 63–70.
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Lin, M.C., 2010. miR-200a regulates epithelial-mesenchymal to stem-like transition via ZEB2 and β-catenin signaling. J. Biol. Chem. 285, 36995–37004. Xu, H., Jin, L., Chen, Y., Li, J., 2016. Downregulation of microRNA-429 protects cardiomyocytes against hypoxia-induced apoptosis by increasing Notch1 expression. Int. J. Mol. Med. 37, 1677–1685. Yang, X., Ni, W., Lei, K., 2013. miR-200b suppresses cell growth, migration and invasion by targeting Notch1 in nasopharyngeal carcinoma. Cell. Physiol. Biochem. 32, 1288–1298. Yang, C., Zhang, X., Gao, J., Wang, M., Yang, Z., 2017a. Arginine vasopressin ameliorates spatial learning impairments in chronic cerebral hypoperfusion via V1a receptor and autophagy signaling partially. Transl. Psychiatry 7, e1174. Yang, J.-J., Tao, H., Liu, L.-P., Hu, W., Deng, Z.-Y., Li, J., 2017b. miR-200a controls hepatic stellate cell activation and fibrosis via SIRT1/Notch1 signal pathway. Inflamm. Res. 66, 341–352. Yang, L., Xu, F., Zhang, M., Shang, X., Xie, X., Fu, T., Li, J., Li, H., 2018. Role of LncRNA MALAT-1 in hypoxia-induced PC12 cell injury via regulating p38MAPK signaling pathway. Neurosci. Lett. 670, 41–47. Zhang, X., Yin, H., Li, Z., Zhang, T., Yang, Z., 2016. Nano-TiO2 induces autophagy to protect against cell death through antioxidative mechanism in podocytes. Cell Biol. Toxicol. 32, 513–527. Zhang, X., Yang, C., Gao, J., Yin, H., Zhang, H., Zhang, T., Yang, Z., 2017. Voluntary running-enhanced synaptic plasticity, learning and memory are mediated by Notch1 signal pathway in C57BL mice. Brain Struct. Funct. 1–19.
Lou, Y.-L., Guo, F., Liu, F., Gao, F.-L., Zhang, P.-Q., Niu, X., Guo, S.-C., Yin, J.-H., Wang, Y., Deng, Z.-F., 2012. miR-210 activates notch signaling pathway in angiogenesis induced by cerebral ischemia. Mol. Cell. Biochem. 370, 45–51. Niatsetskaya, Z.V., Sosunov, S.A., Matsiukevich, D., Utkina-Sosunova, I.V., Ratner, V.I., Starkov, A.A., Ten, V.S., 2012. The oxygen free radicals originating from mitochondrial complex I contribute to oxidative brain injury following hypoxia–ischemia in neonatal mice. J. Neurosci. 32, 3235–3244. Park, S.-M., Gaur, A.B., Lengyel, E., Peter, M.E., 2008. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev. 22, 894–907. Sun, X., Zuo, H., Liu, C., Yang, Y., 2016. Overexpression of miR-200a protects cardiomyocytes against hypoxia-induced apoptosis by modulating the kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 signaling axis. Int. J. Mol. Med. 38, 1303–1311. Wang, Y., Chan, S.L., Miele, L., Yao, P.J., Mackes, J., Ingram, D.K., Mattson, M.P., Furukawa, K., 2004. Involvement of notch signaling in hippocampal synaptic plasticity. Proc. Natl. Acad. Sci. U. S. A. 101, 9458–9462. Wang, X., Mao, X., Xie, L., Greenberg, D.A., Jin, K., 2009. Involvement of Notch1 signaling in neurogenesis in the subventricular zone of normal and ischemic rat brain in vivo. J. Cereb. Blood Flow Metab. 29, 1644–1654. Wei, Z., Chigurupati, S., Arumugam, T.V., Jo, D.-G., Li, H., Chan, S.L., 2011. Notch activation enhances the microglia-mediated inflammatory response associated with focal cerebral ischemia. Stroke 42, 2589–2594. Xia, H., Cheung, W.K., Sze, J., Lu, G., Jiang, S., Yao, H., Bian, X.-W., Poon, W.S., Kung, H.,
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MiR-429/200a/200b negatively regulate Notch1 signaling pathway to suppress CoCl2-induced apoptosis in PC12 cells Chunxiao Yang1, Xiaochen Zhang1, Hongqiang Yin, Zhanqiang Du, Zhuo Yang
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College of Medicine, State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials for Ministry of Education, Nankai University, Tianjin 300071, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Apoptosis Cobalt chloride Hypoxia miR-429/200a/200b Notch1 PC12
Neuronal apoptosis is a central hallmark of cerebral ischemia, which is serious threats to human health. Notch1 signaling pathway and three members of miR-200 family, miR-429, miR-200a and miR-200b, are reported to have tight connection with hypoxia-induced injury. However, their mutual regulation relationship and their roles in neuronal apoptosis caused by hypoxia are rarely reported. In the present study, differentiated pheochromocytoma (PC12) cells were treated with chemical hypoxia inducer, cobalt chloride (CoCl2) to establish in vitro neuronal hypoxia model. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, Western blot assay and Hoechst staining indicated that CoCl2 caused apoptosis of PC12 cells along with the activation of Notch1 signallilng pathway. The treatment of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester (DAPT) inhibited Notch1 signaling pathway and attenuated the apoptosis induced by CoCl2. Real-time polymerase chain reaction (RT-PCR) showed that expressions of miR-429/200a/200b were dynamically changed during the treatment of CoCl2, and significantly decreased after 12-hour treatment of CoCl2. Overexpression of miR-429/200a/200b inhibited the Notch1 signaling pathway and suppressed CoCl2induced apoptosis in PC12 cells. These results may clarify the roles of miR-429/200a/200b and Notch1 signaling pathway in hypoxia-induced nerve injury and provide a new theoretical basis to relieve nerve injury.
1. Introduction
impaired the learning and memory ability, as well as synaptic plasticity of perforant path (PP) to dentate gyrus (DG) in hippocampus of C57BL mice (Zhang et al., 2017). In addition, neural oscillations in the hippocampus of mice were also impaired by Notch1 genetic deficiency (Li et al., 2018). More importantly, Notch1 receptor is also reported to be involved in cerebral ischemia induced neurogenesis (Wang et al., 2009), angiogenesis (Lou et al., 2012) and microglia-mediated inflammatory response (Wei et al., 2011). Nevertheless, reports about the role of Notch1 in hypoxia-induced nerve damage are not coincident. (Li et al., 2012; Wang et al., 2009). MicroRNA (miRNAs) are noncoding RNA molecules that modulating gene expression by translational inhibition and destabilization of mRNAs (Filipowicz et al., 2008). There are five members in miR-200 family including miR-200a, miR-200b, miR-200c, miR-429 and miR141, which lie in two different loci in the genome. MiR-429, miR-200a and miR-200b are clustered in chromosome 1p36, whereas miR-200c and miR-141 are located in chromosome 12p13 (Bracken et al., 2008). They can also be sub-divided into two subfamilies, with miR-429, miR200b and miR-200c as one group, miR-200a and miR-141 as another
Cerebrovascular diseases have high fatality and disability and bring great financial burden to patient families and society (Chen et al., 2014a; Hu et al., 2017). A central hallmark of these diseases is hypoxia, which induces increasing intracellular reactive oxygen species (ROS), damaging mitochondrial function and cell apoptosis in brain (Gong et al., 2017; Niatsetskaya et al., 2012). Accumulating evidences suggest that neuronal apoptosis leads to variety of neurological disorders, such as learning and memory disabilities, dementia and motor dysfunction (Giaccia et al., 2003; Yang et al., 2017a). Thus, it is necessary to elucidate the molecular mechanisms of hypoxic brain injury and seek novel therapy. Notch1 signaling pathway was reported to be crucial in nervous system and was participated in lots of important events in brain, including regulating neurogenesis, promoting radial glia-like identity, regulating cell cycle and governing self-renewal in embryonic NSCs (neural stem cells) (Breunig et al., 2007; Corbin et al., 2008; Favaro et al., 2009). In our previous studies, we found that Notch1 knockdown
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Corresponding author. E-mail address: [email protected] (Z. Yang). 1 CY, XZ have contributed equally to this work. https://doi.org/10.1016/j.tiv.2020.104787 Received 2 November 2019; Received in revised form 11 January 2020; Accepted 28 January 2020 Available online 29 January 2020 0887-2333/ © 2020 Elsevier Ltd. All rights reserved.
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Fig. 1. CoCl2 induces apoptosis in PC12 cells. (a) The viability of PC12 cells treated with different concentrations of CoCl2. n = 6/group, P < .001 vs. Control group at each time point. (b) The representative immunoreactive bands of Bax (21 kDa), Bcl-xl (30 kDa), PARP (116 kDa), cleaved-PARP (89 kDa) and β-actin (43 kDa) of control group, CoCl2 (0.2 mM) group and CoCl2 (0.4 mM) group. The treatment time of CoCl2 was 12 h. Δ represents cleaved fragment. (c) Quantitative analysis of Bax/β-actin in three groups. n = 3/group, **P < .01 vs. Control group; #P < .05 vs. 0.2 mM CoCl2 group. (d) Quantitative analysis of Bcl-xl/β-actin in three groups. n = 3/group, *P < .05, **P < .01 vs. Control group; #P < .05 vs. 0.2 mM CoCl2 group. (e) Quantitative analysis of cleaved-PARP/PARP in three groups. n = 3/group, *P < .05 vs. Control group.
Fig. 2. CoCl2 activates Notch1 signaling pathway in PC12 cells. The treatment time of CoCl2 was 12 h (a) The representative immunoreactive bands of Notch1 (125 kDa), Hes1 (30 kDa), Jagged1 (60 kDa) and β-actin (43 kDa) of control group, CoCl2 (0.2 mM) group and CoCl2 (0.4 mM) group. (b) Quantitative analysis of Notch1/β-actin in three groups. n = 3/group, *P < .05 vs. Control group; #P < .05 vs. 0.2 mM CoCl2 group. (c) Quantitative analysis of Hes1/β-actin in three groups. n = 3/group, **P < .01, ***P < .001 vs. Control group; ###P < .01, vs. 0.2 mM CoCl2 group. (d) Quantitative analysis of Jagged1/β-actin in three groups. n = 3/group.
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Fig. 3. DAPT inhibits the Notch1 signaling pathway activated by CoCl2. The treatment time of CoCl2 and DAPT was 12 h (a) The representative immunoreactive bands of Notch1 (125 kDa), NICD (120 kDa), Hes1 (30 kDa) and β-actin (43 kDa) of control group, DAPT (1 μM) group, DAPT (5 μM) group DAPT (10 μM) group, CoCl2 (0.4 mM) + DAPT (1 μM) group, CoCl2 (0.4 mM) + DAPT (5 μM) group and CoCl2 (0.4 mM) + DAPT (10 μM) group. (b) Quantitative analysis of Notch1/βactin in eight groups. n = 3/group, *P < .05 vs. Control group; #P < .05 vs. 0.4 mM CoCl2 group. (c) Quantitative analysis of NICD/β-actin in eight groups. n = 3/ group, *P < .05 vs. Control group; ##P < .01 vs. 0.4 mM CoCl2 group. (d) Quantitative analysis of Hes1/β-actin in eight groups. n = 3/group, **P < .01, ***P < .001 vs. Control group; #P < .05, ##P < .01 vs. 0.4 mM CoCl2 group. (e) The representative immunofluorescence photographs of NICD in PC12 cells of eight groups. Scale bar = 20 μm.
induced by hypoxia have not been fully investigated. It has been reported that cabalt chloride (CoCl2) can mimic hypoxia as a chemical hypoxia inducer (Chen et al., 2014b; Gong et al., 2017; Hartwig et al., 2014). Thus, in this study, differential pheochromocytoma (PC12) cells, which are morphologically and functionally similar with neurocytes, were treated with CoCl2 to establish a cell culture model of chemical hypoxia-induced neurocyte injury. We aim to investigate the role of Notch1 signaling pathway in CoCl2 induced apoptosis. Besides, whether and how the Notch1 is regulated by three representative miR-200 family members, miR-429/200a/200b, during chemical hypoxia-induced damage of PC12 cells are also investigated.
group (Gregory et al., 2008; Park et al., 2008). MiR-429, miR-200a and miR-200b are three important members of miR-200 family and are thought to play a crucial role in some important events that are related to hypoxia, such as angiogenic responses of human microvascular endothelial cells (HMECs) (Chan et al., 2011), differentiation of osteoblastic cells (Huang et al., 2016), epithelial-mesenchymal transition of lung cancer cells (Gao et al., 2015) and apoptosis of cardiomyocytes (Sun et al., 2016; Xu et al., 2016). Thus, we choose miR-429/200a/ 200b, which located in the same chromosome, as three representative miRNAs of the two subfamilies in this study. However, the expression and functions of miR429/200a/200b vary with different cell types. The precise functions and effects of these three members in neurocyte injury 3
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Fig. 4. DAPT diminishes CoCl2-induced apoptosis in PC12 cells. The treatment time of CoCl2 and DAPT was 12 h (a) The representative immunoreactive bands of Bax (21 kDa), cleaved-Caspase3 (17 kDa), PARP (116 kDa), cleaved-PARP (89 kDa) and β-actin (43 kDa) of control group, DAPT (1 μM) group, DAPT (5 μM) group, DAPT (10 μM) group, CoCl2 (0.4 mM) + DAPT (1 μM) group, CoCl2 (0.4 mM) + DAPT (5 μM) group and CoCl2 (0.4 mM) + DAPT (10 μM) group. Δ represents cleaved fragment. (b) Quantitative analysis of Bax/β-actin in eight groups. n = 3/group, *P < .05, **P < .01 vs. Control group; #P < .05 vs. 0.4 mM CoCl2 group. (c) Quantitative analysis of cleaved-Caspase3/β-actin in eight groups. n = 3/group, *P < .05, ***P < .001 vs. Control group; ##P < .01 vs. 0.4 mM CoCl2 group. (d) Quantitative analysis of cleaved-PARP/PARP in eight groups. n = 3/group, *P < .05 vs. Control group; #P < .05 vs. 0.4 mM CoCl2 group. (e) The representative photographs of Hoechst staining in PC12 cells of eight groups. Scale bar = 20 μm. (f) The cell viability of eight groups. n = 6/group, ***P < .001 vs. Control group; ## P < .01 vs. 0.4 mM CoCl2 group.
2. Materials and methods
difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester (DAPT), nerve growth factor (NGF), Hoechst 33258 and 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma, USA. RNAiso plus, PrimeScript™ RT reagent Kit, SYBR Green qPCR Master Mix and the primer for Notch1 were from TaKaRa, Japan. MiRNA-mimics, stem-loop primer and forward and reverse primer pairs
2.1. Reagents and antibodies RPMI 1640 cell culture medium and fetal bovine serum (FBS) were purchased from GIBCO Invitrogen. Cobalt chloride (CoCl2), N-[N-(3, 54
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Fig. 5. CoCl2 induces dynamic changes of expression profiles of miR-429 (a), miR-200a (b), miR-200b (c) in PC12 cells. *P < .05, **P < .01, ***P < .001 vs. Control group.
with miRNA-mimics and negative controls (20 nM; RiboBio Co., Ltd., Guangzhou, Guangdong, China), using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions.
for miR-429, miR-200a, miR-200b were designed by RiboBio Co., Ltd., China. Anti-Notch1 antibody (1:2000), anti-Hes1 antibody (1:2000), antiNICD antibody (1:1000 for Western blot assay and 1:500 for fluorescent staining), anti-Jagged1 antibody (1:2000) and anti-Bax antibody (1:2000) were purchased from Abcam, UK. Anti-PARP antibody (1:1000), anti-Bcl-xl antibody (1:2000) and anti-cleaved Caspase3 antibody (1:1000) were purchased from Cell Signaling Technology, USA. β-actin (1:5000) were purchased from Sangon Biotech (Shanghai, China). The anti-rabbit secondary peroxidase-conjugated antibodies (1:5000) were bought from Promega (Promega Co, USA). Alexa 594conjugated anti-rabbit IgG purchased from Invitrogen (New York, USA). The chemiluminescent horseradish peroxidase (HRP) substrate was purchased from Millipore, USA.
2.5. RNA isolation and real-time quantitative PCR Total RNA for detecting miR-429, miR-200a and miR-200b expressions was harvested from cells using RNAiso plus (TaKaRa, Ohtsu, Japan) according to the manufacturer's protocols. The harvested RNA was reverse transcribed into cDNA using PrimeScript™ RT reagent Kit (Takara, Japan). Then, miR-200a, miR-200b and miR-429 expressions were detected with U6 as an internal control. The stem-loop primer and forward and reverse primer pairs for miR-200a, miR-200b, miR-429 were designed by RiboBio Co., Ltd. (Guangzhou, Guangdong, China). All reactions were done in triplicate using Mastercycler ep realplex 2 (Eppendorf AG, Hamburg, Germany) and expression levels were calculated using the comparative CT method (2 −ΔΔCT).
2.2. Cell culture Rat pheochromocytoma PC12 cells were cultured in RPMI 1640 (Gibco, Gaithersburg, MD, USA) supplemented with 10% horse serum and 5% fetal bovine serum (FBS, Gibco, Gaithersburg, MD, USA) in a humidified atmosphere containing 5% CO2 at 37 °C. For differentiation, cells were kept in the medium supplemented with 1% FBS and 50 ng/m nerve growth factor (NGF, Sigma, St. Louis, MO, USA) for 7 days. The differentiation medium was replaced with fresh medium every second days.
2.6. Western blot analysis Preparation of cell lysates has been described in our previous studies (Li et al., 2016b). Briefly, cells were washed with phosphate-buffered saline (PBS, pH 7.4) at the end of the culture period and lysed in RIPA buffer (Beyotime Biotechnology, Shanghai, China). The lysates were centrifuged at 12,000 rpm at 4 °C for 20 min. The supernate was separated, mixed with loading buffer, and boiled at 100 °C for 20 min. Total protein concentration was determined with bicinchoninic acid assay (Beyotime biotechnology, China). Equal samples (20 μg) were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride transfer membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline with 1% Tween-20 (TBST) and incubated with the primary antibodies diluted in blocking buffer overnight at 4 °C. After four washing steps with TBST, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, after washing four times, protein band intensities were detected with HRP substrate (Millipore, USA) by using the Tanon 5500 chemiluminescent imaging system (Tanon, Science and Technology, China). β-actin was used as an internal control and the quantitation analysis was performed by Image J (National Institutes of Health, Bethesda, MD, USA).
2.3. Cell viability assay The cell viability of PC12 was evaluated by 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay as previously described (Zhang et al., 2016). Briefly, cells were seeded in 96-well plates with 1 × 104 cells per well and cultured 24 h for the stabilization. Then, the medium containing different concentrations (0.2, 0.4, 0.6, 0.8 mM) of CoCl2 was added in each well for a certain time (6, 12, 18 and 24 h). In order to investigate the effect of DAPT on cell viability, PC12 cells were incubated with medium without drugs (control group), medium containing DAPT (1 μM, 5 μM and 10 μM), medium containing CoCl2 (0.4 mM) and medium containing CoCl2 (0.4 mM) with DAPT (1 μM, 5 μM and 10 μM) for 12 h. After incubation, the cell medium was replaced by the fresh medium containing MTT (20 μl, final concentration 5 mg/ml) and the cells were incubated for 4 h. After that, a total of 100 μl of DMSO was added to dissolve the formazan crystals in each well. Absorbance at 492 nm was measured using a microplate reader (Multiskan Mk3; Thermo Labsystems, Helsinki, Finland).
2.7. Fluorescent staining Fluorescent staining was performed following our previously description with some modifications (Li et al., 2016b). After appropriate treatment, cells cultured on glass coverslips were fixed with 4% paraformaldehyde in PBS for 15 min at 4 °C, and incubated in blocking
2.4. Transfection with miRNA-mimics To induce miR-200 family overexpression, the cells were transfected 5
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Fig. 6. Overexpression of miR-429/200a/200b inhibits the Notch1 signaling pathway activated by CoCl2. MiR-429 (a), miR-200a (b) and miR-200b (c) were markedly increased by transfection with miRNA-mimics. The treatment time of CoCl2 was 12 h. (d) The representative immunoreactive bands of Notch1 (125 kDa), NICD (120 kDa), Hes1 (30 kDa) and β-actin (43 kDa) of negative control (NC) group, NC + CoCl2 (0.4 mM) group, miR-429 mimic group, miR-429 mimic + CoCl2 (0.4 mM) group, miR-200a mimic group, miR-200a mimic + CoCl2 (0.4 mM) group, miR-200b mimic group, miR-200b mimic + CoCl2 (0.4 mM) group. (e) Quantitative analysis of Notch1/β-actin in eight groups. n = 3/group, *P < .05, **P < .01, ***P < .001 vs. Control group; &&& P < .01 vs. miR-200a mimic group; $$$P < .001 vs. miR-200b mimic group; ++ P < .01, +++ P < .001 vs. NC + CoCl2 (0.4 mM) group; N.S. no significant difference. (f) Quantitative analysis of NICD/β-actin in eight groups. n = 3/group, *P < .05 vs. Control group; +P < .05, ++P < .01 vs. NC + CoCl2 (0.4 mM) group; N.S. no significant difference. (g) Quantitative analysis of Hes1/β-actin in eight groups. n = 3/group, ***P < .001 vs. Control group; &&&P < .01, vs. miR-200a mimic group; ++ + P < .001 vs. NC + CoCl2 (0.4 mM) group; N.S. no significant difference. (h) The representative immunofluorescence photographs of NICD in PC12 cells of eight groups. Scale bar = 20 μm.
3.2. CoCl2 activated Notch1 signaling pathway in PC12 cells
buffer (10% normal goat serum and 0.3% Triton X-100) for 1 h at room temperature. Then, cells were incubated with NICD antibody (1:500 dilution) diluted in 5% of normal goat serum, 0.3% Triton X-100 in PBS overnight at 4 °C. After three times of gentle wash with PBS, the cells were probed with fluorescence-labeled secondary antibody (1:1000 dilution) for 1 h at room temperature. Finally, after another three times of PBS washing, cells were stained with PBS containing 5 μg/ml DAPI (Sigma, St. Louis, MO, USA) for 5 min, rinsed in PBS for one time, and observed under a confocal laser scanning microscope (FV300, Olympus, Japan).
Western blot assay was performed to detect expressions of proteins in Notch1 signaling pathway. We incubated the cells in fresh medium (control group), medium containing CoCl2 (0.2 mM) and medium containing CoCl2 (0.4 mM) for 12 h. As shown in Fig. 2a and b, the expression of Notch1 was significantly increased after 0.4 mM CoCl2 treatment compared to those of control group (P < .05) and 0.2 mM treatment group (P < .05). Hes1, as a downstream effect protein in Notch1 signaling pathway, was also increased after different concentrations of CoCl2 treatment compared to control group (Fig. 2c, P < .01 for 0.2 mM treatment group and P < .001 for 0.4 mM treatment group). In addition, the expression of Hes1 was significantly higher in 0.4 mM CoCl2 treatment group compared to that of 0.2 mM CoCl2 treatment group (P < .001). The expression of Jagged1, a bound ligand of Notch1 receptor, showed an increasing trend after CoCl2 treatment with no significant difference. These results suggested that CoCl2 treatment activated Notch1 signaling pathway in PC12 cells.
2.8. Hoechst fluorescent staining Following the various treatments, PC12 were analyzed for apoptotic nuclei using Hoechst fluorescence staining. After the incubation period, the medium was removed and the PC12 cells were washed with PBS two times at room temperature. Then, the cells were stained with 1 μg/ ml Hoechst 33258 (Sigma, St. Louis, MO, USA) in medium for 30 min in dark at 37 °C to identify nuclear fragmentation, and then visualized under a confocal laser scanning microscope (FV300, Olympus, Japan).
3.3. DAPT inhibited CoCl2-induced Notch1 signaling activation To investigate the role of Notch1 signaling pathway in CoCl2 induced apoptosis, DAPT, the γ-secretase inhibitor as well as the inhibitor of Notch1 siganalling pathway, was used. Western blot assay was performed to investigate the effects of different treatment concentrations of DAPT on Notch1 signaling pathway. PC12 cells were incubated with medium without drugs (control group), medium containing DAPT (1 μM, 5 μM and 10 μM), medium containing CoCl2 (0.4 mM) and medium containing CoCl2 (0.4 mM) with DAPT (1 μM, 5 μM and 10 μM) for 12 h. As shown in Fig. 3a and b, the expression of Notch1 receptor was significantly increased after CoCl2 (0.4 mM) treatment for 12 h (P < .05). Co-treatment of CoCl2 and DAPT (1 μM and 5 μM) did not influence the expression of Notch1 compared with that of CoCl2 treatment group. However, co-treatment of CoCl2 and DAPT (10 μM) significantly decreased the expression of Notch1 compared with that of CoCl2 group (P < .05). The expression of Notch intracellular domain (NICD) was significantly up-regulated in CoCl2 treatment group (P < .05). Meanwhile, co-treatment with CoCl2 and DAPT (10 μM) significantly decreased the expression of NICD compared with that of CoCl2 treatment group (P < .01). In order to detect whether NICD was translocated into the nucleus and performed function, immunofluorescence staining was performed. We incubated the cells in fresh medium (control group), medium containing DAPT (10 μM), medium containing CoCl2 (0.4 mM) and medium containing both DAPT (10 μM) and CoCl2 (0.4 mM) for 12 h. As shown in Fig. 3e, the expression of NICD in nucleus was obviously increased after CoCl2 treatment compared with that of control group, whereas CoCl2 and DAPT co-treatment led to a visible decrease density of fluorescence compared to CoCl2 treatment group. Moreover, the expression of Hes1 was markedly higher in CoCl2 group (P < .001), CoCl2 + DAPT (1 μM) group (P < .01) and CoCl2 + DAPT (5 μM) group (P < .01) compared with that of control group. Co-treatment of CoCl2 and DAPT (P < .05 for 5 μM and P < .01 for 10 μM) significantly decreased the expression of Hes1 compared with that of CoCl2 group (Fig. 3d). These results suggested that the treatment of DAPT inhibited CoCl2-activated Notch1
2.9. Statistical analysis All data were expressed as mean ± S.E.M. Multiple samples were statistically analyzed for significance using one-way analysis of variances (One-Way ANOVA). Post hoc statistics were obtained using the LSD test (Fisher'sleast significant difference t-test) for multiple comparisons. All of the statistical analyses were performed using SPSS 22.0 software (IBM, Chicago, IL). P-values less than 0.05 were considered to be significant. All experiments were performed at least in triplicate.
3. Results 3.1. CoCl2 induced apoptosis in PC12 cells The cell viability of PC12 cells was determined by the MTT assay. As shown in Fig. 1a, the cell viability was decreased when treatment concentration increased at different time (P < .001 compared with control group). In addition, the results of Western blot assay revealed that CoCl2 promoted apoptosis in PC12 cells. As shown in Fig. 1b and c, the expression of Bax was significantly increased after 0.4 mM CoCl2 treatment for 12 h compared to those of control group (P < .01) and 0.2 mM CoCl2 treatment group (P < .05). The expression of Bcl-xl was markedly decreased in 0.2 mM treatment group (P < .05) and 0.4 mM treatment group (P < .01) compared to that of control group. The expression of Bcl-xl was significantly lower in 0.4 mM treatment group compared to that of 0.2 mM treatment group (P < .05, Fig. 1b and d). Beyond that, the ratio of cleaved PARP/PARP was significantly increased in 0.2 mM CoCl2 treatment group (P < .05) and 0.4 mM CoCl2 treatment group (P < .05) compared to that of control group (Fig. 1b and e). Taken together, these results suggested that CoCl2 induced apoptosis in PC12 cells. 7
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Fig. 7. Overexpression of miR-429/200a/200b reduces apoptosis induced by CoCl2. The treatment time of CoCl2 was 12 h. (a) The representative immunoreactive bands of Bax (21 kDa), cleaved-Caspase3 (17 kDa), PARP (116 kDa), cleaved-PARP (89 kDa) and β-actin (43 kDa) of negative control (NC) group, NC + CoCl2 (0.4 mM) group, miR-429 mimic group, miR-429 mimic + CoCl2 (0.4 mM) group, miR-200a mimic group, miR-200a mimic + CoCl2 (0.4 mM) group, miR-200b mimic group, miR-200b mimic + CoCl2 (0.4 mM) group. (b) Quantitative analysis of Bax/β-actin in eight groups. n = 3/group, **P < .01 vs. Control group; N.S. no significant difference. (c) Quantitative analysis of cleaved-Caspase3/β-actin in eight groups. n = 3/group, ***P < .001 vs. Control group; &&&P < .01 vs. miR-200a mimic group; +++P < .001 vs. NC + CoCl2 (0.4 mM) group; N.S. no significant difference. (d) Quantitative analysis of cleaved-PARP/PARP in eight groups. n = 3/ group, *P < .05, ***P < .001 vs. Control group; &&&P < .01, vs. miR-200a mimic group; $$P < .01 vs. miR-200b mimic group; +++P < .001 vs. NC + CoCl2 (0.4 mM) group; N.S. no significant difference. (e) The representative photographs of Hoechst staining in PC12 cells of eight groups. Scale bar = 20 μm.
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overexpress miR-429, miR-200a and miR-200b in PC12 cells. The expression of miR-200 family members were significantly higher in the miRNA-mimic groups than those in negative control (NC) group, respectively (Fig. 6a, b and c). These results demonstrated that the transfection with miRNA-mimics was effective in overexpressing miR200 family members in PC12 cells. Then, Western blot assay was performed to detect expressions of Notch1 signaling pathway proteins. As shown in Fig. 6d, e, f and g, expressions of Notch1, NICD and Hes1 were significantly increased after 12 h CoCl2 treatment (P < .001 for Notch1, P < .05 for NICD and P < .001 for Hes1). Notch1 expression was notably suppressed following miR-429, miR-200a and miR-200b overexpression (P < .01 for miR-429, P < .01 for miR-200a and P < .05 for miR-200b), but expressions of NICD and Hes1 were not affected by the miRNA-mimics. Beyond that, miR-429 overexpression abolished the higher expressions of Notch1, NICD and Hes1 induced by CoCl2 treatment; miR-200a overexpression abolished the higher expression of NICD induced by CoCl2 treatment; miR-200b overexpression abolished the higher expressions of NICD and Hes1 induced by CoCl2 treatment. In addition, immunofluorescence staining was also preformed to examine the expression of NICD in nucleus. Cells in each groups were also exposured to CoCl2 for 12 h. As shown in Fig. 6h, the overexpression of miR-200 family members reduced the higher expression of NICD in the nucleus induced by cobalt chloride. These results indicated that overexpression of miR-429/200a/200b effectively inhibited the activating of Notch1 signaling pathway induced by CoCl2.
signaling pathway. 3.4. DAPT diminished CoCl2-induced apoptosis in PC12 cells We subsequently investigated whether DAPT had influence on the apoptosis of PC12 cells induced by CoCl2 treatment. Western blot assay and Hoechst fluorescent staining were performed. For Western blot assay, PC12 cells were incubated with medium without drugs (control group), medium containing DAPT (1 μM, 5 μM and 10 μM), medium containing CoCl2 (0.4 mM) and medium containing CoCl2 (0.4 mM) with DAPT (1 μM, 5 μM and 10 μM) for 12 h. As shown in Fig. 4a, b, c and d, treatment of CoCl2 significantly increased the expressions of Bax (P < .01) and cleaved-Caspase3 (P < .001), as well as the ratio of cleaved-PARP/PARP (P < .05) compared with those of control group. The use of DAPT (10 μM) decreased the up-regulated expressions of apoptosis related proteins induced by CoCl2 treatment (P < .05 for Bax, P < .01 for cleaved-Caspase3 and P < .05 for cleaved-PARP/ PARP). For Hoechst staining, cells were treated with fresh medium (control group), medium containing DAPT (10 μM), medium containing CoCl2 (0.4 mM) and medium containing both DAPT (10 μM) and CoCl2 (0.4 mM) for 12 h. Hoechst staining showed that nuclei of control group were intact and exhibited dispersed blue fluorescent. On the contrary, the cells treated with CoCl2 showed obvious nuclear condensation and excessive blue fluoresent. The co-treatment of CoCl2 and DAPT (10 μM) decreased the number of cells with nuclear condensation obviously (Fig. 4e). In addition, we performed MTT assay to investigate the effect of DAPT on PC12 cell death induced by CoCl2. The results were shown in Fig. 4f. The cell viabilities of DAPT groups (1 μM, 5 μM and 10 μM) were comparable to that of control group. On the other hand, the cell viability of CoCl2 group was significantly decreased compared with that of control group, which was consistent with Fig. 1a. The cell viability in CoCl2 + DAPT (10 μM) group was significantly increased compared with that of CoCl2 group (P < .01) even though it was still lower than that of control group (P < .001). Taken together, these results suggested that the inhibition of Notch1 signaling pathway ameliorated apoptosis of PC12 cells induced by CoCl2.
3.7. Overexpression of miR-429/200a/200b attenuated apoptosis induced by CoCl2 in PC12 cells Subsequently, we further investigated the role of miR-200 family members in PC12 cell apoptosis, expressions of Bax, cleaved-Caspase3 and the ratio of cleaved-PARP/PARP were detected by Western blot assay. Cells in each groups were exposured to CoCl2 for 12 h. Compared with the NC group, the level of Bax, cleaved-Caspase3 and the ratio of cleaved-PARP/PARP were markedly increased after treatment of CoCl2 (P < .01 for Bax, P < .001 for cleaved-Caspase3 and P < .001 for the ratio of cleaved-PARP/PARP). However, miR-429 overexpression abolished the higher expressions of Bax, cleaved-Caspase3 and cleavedPARP/PARP induced by CoCl2 treatment; miR-200a overexpression abolished the higher expression of Bax induced by CoCl2 treatment; miR-200b overexpression abolished the higher expressions of Bax and cleaved-Caspase3 induced by CoCl2 treatment. Hochest staining was carried out to confirm these immunoblotting results. As shown in Fig. 7e. CoCl2 treatment led to a visible enhancement of apoptosis cells compared to that of NC group, whereas overexpression of miR-429/ 200a/200b dimished the apoptosis of cells in different degrees. Together, these results suggested that overexpression of miR-429/200a/ 200b attenuated apoptosis induced by CoCl2 in PC12 cells.
3.5. Expressions of miR-429/200a/200b dynamically changed during CoCl2 treatment in PC12 cells The dynamic expressions of miR-429, miR-200a and miR-200b during CoCl2 treatment were monitored during a time course by realtime quantitative PCR (qPCR) assay. The results indicated that expressions of miR-429, miR-200a and miR-200b were rapidly up-regulated after CoCl2 treatment and reached a maximum level in 6 h (Fig. 5, P < .001 for miR-429, miR-200a and miR-200b). After 12 h of CoCl2 treatment, relative expressions of miR-429, miR-200a and miR-200b decreased to below background levels (P < .001 for miR-429, P < .05 for miR-200a and P < .001 for miR-200b). The expressions of miR429, miR-200a and miR-200b were significantly lower than those of control group after 18 h CoCl2 treatment, respectively (P < .01 for miR-429, P < .05 for miR-200a and P < .001 for miR-200b). The expressions of miR-429 and miR-200b were significantly lower than those of control group after 24 h CoCl2 treatment (P < .01 for miR-429 and P < .001 for miR-200b), but the expression of miR-200a showed no significant difference compared with that of control group. Interestingly, we found that expressions of miR-429, miR-200a and miR200b were down-regulated, but expressions of Notch1 signaling pathway proteins were up-regulated after 12 h treatment of CoCl2. Therefore, we aimed to explore whether Notch1 signaling pathway was regulated by these three miR-200 family members.
4. Discussion CoCl2 has been widely used in PC12 cells to establish a chemical hypoxia in vitro cell model as a well-known hypoxia-mimetic agent. Our results in the present study showed that the cell viability and apoptosis of PC12 cells were significantly increased after CoCl2 treatment on a time-dependent and dose-dependent manner. Increasing evidence has supported with these results in our study (Chen et al., 2015; Gong et al., 2017; Hartwig et al., 2014; Yang et al., 2018). Notch1 receptor was reported to be crucial in nervous system. It regulated some important processes in nervous system, such as learning and memory, synaptic plasticity and neurogenesis (Costa et al., 2003; Johnson et al., 2009; Wang et al., 2004). In our previous study, we found that deficiency of Notch1 receptor led to the lower cell density in dentate gyrus (DG) region in C57BL mice compared with wildtype littermates (Zhang et al., 2017). These results indicated that Notch1 receptors played a promoting role on cell proliferation under
3.6. Notch1 signaling pathway was inhibited by miR-429/200a/200b overexpression To test this hypothesis, we used miRNA-mimics to artificially 9
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Ethical standards
physiological conditions. On the contrary, in the present study, we demonstrated that CoCl2-induced hypoxia increased PC12 proliferation following Notch1 signaling pathway suppression by using the Notch signal inhibitor, DAPT. These results suggested that Notch1 played a pro-apoptosis role under cobalt-mimicked hypoxia. A recent publication verified our results to some extent even though they used osteoblasts as experimental subject (Li et al., 2016a). Therefore, we infer that Notch1 receptors may have different effects on cell proliferation or apoptosis under physiological and hypoxic conditions. Further studies that use in vivo hypoxia animal model are needed to clarify this point. But it is certain that the unbalanced expression level of Notch1 may result in nerve cell apoptosis and even damage the function of the nervous system. In the present study, we found that expressions of miR-429, miR200a and miR-200b were dynamically changes at different times of CoCl2 treatment. It has been reported that treatment of hypoxia for 24 h inhibited miR-200b expression in human dermal microvascular endothelial cells (HMECs) (Chan et al., 2011). Moreover, another research reported that miR-200b was reduced in H1299 cells under hypoxia for 24 h (Gao et al., 2015). These observations are in line with our data indicating that the treatment of CoCl2 for 12 h induces down-regulating of miR-200b. On the other hand, Huang et al. reported that miR-429 was up-regulated by CoCl2 treatment in MC3T3-E1 cells with the peak value at 4 h (Huang et al., 2016). Xu et al. suggested that miR-429 expression was significantly increased after human cardiomyocytes (AC-16) were subjected to culture under hypoxic conditions for 12 h (Xu et al., 2016). These results showed different trends with ours, in which the treatment with CoCl2 was for 12 h, but it was consistent with the results that treatment with CoCl2 for 6 h. Based on these results, we hypothesize that response to hypoxia of different cell types at different times is inconsistent. The changing pattern of miR-429/200a/200b in PC12 cells under hypoxia-mimetic agent treatment is specific. In the present study, we found that the effects of Notch1 signaling pathway in CoCl2 induced apoptosis of PC12 cells, a widely-used and very well-characterized model system of neuronal cells. Subsequently, we also explored how the Notch1 signaling pathway was regulated. Intriguingly, we found that the expression of Notch1 was increased, whereas the expressions of three miR-200 family members were decreased after CoCl2 treatment for 12 h. The overexpression of these three miRNAs inhibited the expression of Notch1 and attenuated the apoptosis induced by CoCl2. Notch1 receptor was identified as the targets gene of miR-429 (Xu et al., 2016) and miR-200b (Yang et al., 2013) in other studies. Generally, the target genes of miRNA were considered to be conserved between different species. Thus, we suggest that miR-429 and miR-200b reduce apoptosis in PC12 cells through inhibiting Notch1 receptor. For miR-200a, the underlying mechanism may be that miR-200a regulated its target genes, Sirt1 (Yang et al., 2017b) or ZEB2 (Xia et al., 2010), and then indirectly affected Notch1 expression. Therefore, the effect of miR-200a on inhibiting Notch1 signaling pathway and reducing apoptosis induced by CoCl2 were not obvious compared with the effects of miR-429 and miR-200b. The specific underlying mechanisms need further study. In conclusion, under CoCl2 treatment, expressions of Notch1 signaling pathway proteins were significantly up-regulated. Meantime, the apoptosis level of PC12 cells was also elevated, indicating that Notch1 signaling pathway played a vital role in this process. We then further explored the role of Notch1 in CoCl2-induced cell apoptosis. The results revealed that the inhibiting of Notch1 signaling pathway reduced the apoptosis ratio of PC12 cells, implying that the down-regulation of Notch1 provided cytoprotective effects. In addition, three miR-200 family members, miR-429, miR-200a and miR-200b, were proven to have inhibiting effects on Notch1 signaling pathway and be involved in the anti-apoptotic effects. Our findings may give some help to clarify the vital role of Notch1 signaling pathway and miR-429/200a/200b in hypoxia induced nerve injury and provide a new theoretical basis to relieve nerve injury.
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