LncRNA NEAT1 regulates apoptosis in Parkinson's disease via sponging miR-132-3p

doi:10.21203/rs.3.rs-1518034/v1

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LncRNA NEAT1 regulates apoptosis in Parkinson's disease via sponging miR-132-3p

https://doi.org/10.21203/rs.3.rs-1518034/v1

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Emerging studies have indicated that dysregulation of long non-coding RNA nuclear-enriched assembly transcript 1 (lncRNA NEAT1) is closely associated with development of Parkinson’s disease (PD). But the potential mechanism of NEAT1 is unclear. SH-SY5Y cells were treated with MPP⁺ to mimic PD, transfected sh-NEAT1 to mediate NEAT1 level. Cell viability was detected by CCK-8 assays. Apoptotic cytokines were detected by qRT-PCR and western blot. TargetScan6.2 database and dual-luciferase reporter assay were conducted to validate the interaction between indicated molecules. The expression of Bim, Bax and cleaved caspase-3 was increased, while the expression of miR-132-3p, p-FOXO3a and Bcl-2 was reduced. Knockdown of NEAT1 retrain apoptosis and increased miR-132-3p expression. Besides, NEAT1 was verified as a direct target of miR-132-3p. NEAT1 knockdown suppressed apoptosis in PD through regulating the miR-132-3p/FOXO3a/Bim/caspase and miR-132-3p/Bax/Bcl-2/caspase 3 pathways, providing a promising therapeutic intervention strategy for PD.

parkinson’s disease

lncRNA NEAT1

miR-132-3p

apoptosis

Parkinson’s disease (PD) is a severe neurodegenerative disease of the central nervous system[1]. Due to the decrease of dopamine concentration, PD patients would experience a series of movement disorders, such as resting tremor and bradykinesia. The population of PD is increasing with aging, bring a heavy burden to the family and society[2]. Until now, the pathogenesis of PD is still uncertain, which may be connected to factors such as heredity, environment and aging. The pathophysiological mechanisms of PD is very complicated, including a-synuclein abnormal deposition, mitochondrial function impairment, oxidative damage and neuroinflammation, which lead to the death of dopaminergic neurons[3]. The current treatment methods could only improve the clinical symptoms[4, 5]. Therefore, exploring of the mechanism of PD and finding its potential intervention measures have important significance for controlling the development and progression of PD.

Long non-coding RNA (lncRNA) with a length of more than 200 nucleotides (nts), mediates the expression of genes which code related protein by regulating epigenetics and post-translational translation. Besides, many studies found lncRNAs play a vital role in neurodegenerative disorders[6, 7]. Five significantly differentially expressed lncRNAs were found in brain of PD patients[8]. Increasing expression of MALAT1 could improve the content of a-synuclein in PD mouse model[9]. Down-regulation of SNHG1 inhibited apoptosis of neurons by mediating miR-221/222[10]. Inhibiting HOTAIR expression could also inhibit apoptosis via decreasing the caspase-3 activity[11].

It is reported that expression of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in peripheral blood cells of PD patients is significantly increased[5]. NEAT1 could stabilize PINK1 protein to promote autophagy of dopaminergic neurons in PD models[12]. Besides, downregulation of NEAT1 also decreased the apoptosis-related protein in PD[13]. In addition, Xie et al. indicated that NEAT1 could act at miR-124 and improve inflammation and apoptosis in SH-SY5Y cells[14].

MicroRNAs (miRNAs) mediate hubs of gene expression and dysregulate in many neurodegenerative diseases[15, 16]. Increasing studies demonstrated that miR-153, miR-133b, and miR-34b/34c have a significant influence in PD progression[17–19]. The study indicated miR-132 expression significantly upregulated and could lead to cell apoptosis via the SIRT1/p53 pathway in animal and cell models of PD[20]. However, some investigations indicate that miR-132 plays neuroprotective role in AD and decreases in PD brains[21, 22].

The purpose of the study was to research the related molecular mechanisms of NEAT1 on apoptosis in MPP⁺-induced SH-SY5Y cells.

2.1 Cell culture and treatment

SH-SY5Y cells were presented as a gift from Basic Medicine of Qingdao University and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS: solarbio, china), 100 U/ml penicillin and 100µg/ml streptomycin in with 5% CO₂ at 37°C. We used 0.25, 0.5, 0.75, 1 or 1.25mM MPP⁺ (Sigma, St. Louis, MO, USA) to treat SH-SY5Y cells for 24h to construct PD model in vitro to get the suitable MPP⁺ concentration. Next, SH-SY5Y cells were divided into control and PD (MPP⁺); Then, SH-SY5Y cells were transfected with the short hairpin RNA (shRNA) targeting NEAT1 (sh-NEAT1) or shRNA scramble control (sh-NC) lentivirus, followed by treatment with 1mM MPP⁺ and divided cells into control, sh-NC + MPP⁺, sh-NC, sh-NEAT1 + MPP⁺group.

2.2 Cell viability assay

We utilize Cell Counting Kit-8 (CCK-8; Sigma, America) to detect cell viability. Place cells (100µL) in 96-well plates. CCK-8 reagent (10µL) was added to each well and then placed the cells in incubator for 3 hours after treatment or/and transfection. Finally, microplate reader (Bio-Rad, Hercules, CA, USA) was used to detect the absorbance value at 450nm.

2.3 RNA isolation and qRT-PCR

According to instructions, we extract total RNA with Trizol Reagent (Takara, Japan). Next, cDNAs were synthesized by cDNA Synthesis Kit (Takara, Japan). Real-time PCR for using to examine the level of NEAT1 and miR-132-3p which were performed using SYBR Green. The results were normalized to GAPDH expression and calculated by the 2^ΔΔCT method[23]. qRT-PCR primers were listed as follows:

GAPDH, F 5'-GGAGCGAGATCCCTCCAAAAT-3',

R 5'-GGCTGTTGTCATACTTCTCATGG-3';

NEAT1, F 5'-TGGCTAGCTCAGGGCTTCAG-3',

R 5'-TCTCCTTGCCAAGCTTCCTTC-3';

miR-132-3p, F 5'-ACGCACCACTAACAGTCTACAGC-3',

R 5'-TATGCTTGTTCTCGTCTCTGTGTC-3';

Bcl-2, F 5'-CATGTGTGTGGAGAGCGTCA-3',

R 5'-CACTTGTGGCTCAGATAGGCA-3';

Bcl-2-associated X protein (Bax), F 5'-CATGGGCTGGACATTGGACT-3',

R 5'-AAAGTAGGAGAGGAGGCCGT-3';

2.4 Western blot analysis

Total protein was separated with RIPA lysis buffer (Solarbio, China), then quantified by BCA protein detection kit. Using SDS-PAGE to isolate protein and then transferred protein to PVDF membranes. After blocking with 5% non-fat milk, add primary antibodies (β-actin: Biotech, 1:1000-1:10000; Phospho- the Forkhead box O 3a (p-FOXO3a), (Ser253), Abcam, 1:2000; Bcl-2-interacting mediator of cell death (Bim): Abcam, 1:2500; cleaved caspase-3: cell signaling technology, 1:1000; Bcl-2, Abcam, 1:2000; Bax, Abcam, 1:1000) into the membranes for 12 h at 4℃, then incubated with horseradish peroxidase-conjugated secondary antibody (1:2000). We utilize ECL reagent to display bands and Image J software to quantify the expression of protein.

2.5 Dual-Luciferase reporter assay

The coaction between miR-132-3p and NEAT1 was predicted through TargetScan6.2 online database. The wild-type (WT) and mutant-type (MUT)-NEAT1 3′UTR reporter vectors were synthesized by Hanbio Biotechnology. Using Lipofectamine 3000 to co-transfect cultured HEK293T cells with luciferase vectors and miR-212-3p mimic or miR-NC. Dual-luciferase reporter system (Promega) was utilized to detect luciferase activity after incubating cells for 48h.

2.6 Statistical analysis

All experiments were performed independently three times. Data was presented as mean ± standard deviation (mean ± SD). Statistical comparisons were performed using two-tailed Student t-test for two groups and one-way ANOVA for more than three groups. GraphPad Prism 5.0 (GraphPad, La Jolla, CA, USA) program was used to analyse data. statistically significant difference was set at P < 0.05.

3.1 The viability of cells was decreased while treated by MPP⁺

We used various concentrations MPP⁺ (0, 0.25, 0.5, 0.75, 1 or 1.25mM) to treat SH-SY5Y cells to ascertain the most effective condition for building the PD cell model. Just as shown in Figure 1A, the analyzed results of CCK8 assay showed the cell viability decreased as concentration increasing. When the concentration of MPP⁺reached 1mM, the results were statistically significant (P<0.005). In conclusion, MPP⁺ was used at a concentration of 1 mM to construct an in vitro simulated PD cell model for the subsequent experiment.

3.2 NEAT1 was increased and miR-132‑3p was decreased in PD cell model

To research possible role of NEAT1 and miR-132-3p in cell models of PD, we measured NEAT1 and miR-132-3p expression using qRT-PCR. The results indicated that MPP⁺ could increase NEAT1 expression (Figure 2A) and decrease miR-132-3p expression (Figure 2B). The relevant results reveal that abnormal expression of NEAT1 and miR-132-3p might be correlated with PD development.

3.3 MPP⁺ could enhance apoptosis in PD cell model

To study whether apoptosis leads to the cell death in PD cell model, we examined the apoptotic factors by qRT-PCR and western blot. As shown in Figure 3A, Bim/cleaved caspase-3 significantly increase and p-FOXO3a decreases in PD compared with control group. Besides, MPP⁺ treatment induced the increase of Bax expression and the decrease of Bcl-2 expression (Figure B-D). In consequence, apoptosis was enhanced in MPP⁺-treated SH-SY5Y cells, and consideration of the results of cell survival rate, we speculated that apoptosis plays an important role in PD development.

3.4 Knockdown of NEAT1 suppressed the expression of miR-132-3p in PD cell model

To elucidate the role of NEAT1 in neurons of PD, sh-NEAT1 and sh-NC were used to transfect into cells (Figure 4A). qRT-PCR revealed that the expression of miR-132-3p (Figure 5) was significantly increased in SH-SY5Y cells transfected with sh-NEAT1, whereas the NEAT1 expression was notably decreased, showing sh-NEAT1 was transfected successfully (Figure 4B).

3.5 Knockdown of NEAT1 suppressed apoptosis in PD cell model

Further experiments indicated that knockdown of NEAT1 alleviated apoptosis in the MPP⁺-treated SH-SY5Y cells through modulating different signaling pathway. Sh-NEAT1 treatment clearly reduced the levels of Bim, Bax and cleaved caspase-3 while elevating the expression of p-FOXO3a and Bcl-2 (Figure 6). These results suggested that knockdown of NEAT1 might suppress apoptosis of PD.

3.6 miR-132-3p was a target of NEAT1

The potential binding sites of NEAT1 and miR-132-3p were predicted by TargetScan6.2, and we found there is an underlying binding site between NEAT1 and miR-132-3p (Figure 7A). Next, dual luciferase reporter assay was used to confirm the prediction. Dual-luciferase reporter assay result indicated luciferase activity in the NEAT1-WT+miR-132-3p group was lower than NEAT1-MUT+miR-132-3p or NEAT1-WT+miR-NC group (Figure 7B). Therefore, we confirmed NEAT1 directly targeted to miR-132-3p.

PD is the commonest severe neurodegenerative disease of the central nervous system. Although the pathogenesis of PD is unclear, the crucial role of apoptosis has been demonstrated in PD models. Recently, it has been revealed that lncRNAs are used as a precise biomarker for PD[24–26]. In this study, MPP⁺ were used to establish PD models in vitro and we focused on the role and relationship of NEAT1, miR-132-3p and apoptotic factors in PD.

SH-SY5Y cells could express the tyrosine hydroxylase, dopamine transporter and dopamine 2B hydroxylase which produced by catecholaminergic neurons, it has been frequently used in neuroscientific research as an in vitro model for the investigation of pathogenesis of PD. Neurotoxin MPTP is a neurotoxin which could destroy dopamine neurons selectively through causing oxidative stress-mediated apoptotic death to mimic PD[27]. In the study, SH-SY5Y cells were treated with MPP⁺ at varying concentrations to determine the suitable concentration to construct PD cell models, and we selected 1mM MPP⁺ for further experiment.

Growing studies demonstrated that lncRNA played an essential role in PD development[28]. It has been reported that NEAT1 was upregulated and downregulation of NEAT1 could effectively suppress apoptosis and autophagy in PD mouse model[12, 13]. Apoptosis is indispensable for building an effective neural network in the developing brain. However, excessive apoptosis could accelerate disease progression, including PD. Proteins of the Bcl-2 family are known as crucial regulators of apoptosis, which includes anti-apoptotic Bcl-2 and pro-apoptotic Bax members. The significant increase of Bax/Bcl-2 could result in the collapse of the mitochondrial membrane potential and caspase-3 activation, ultimately leading to cell death[29, 30]. Previous studies have showed that Bax expression upregulates and Bcl-2 expression downregulation in the SNpc and striatum of mice of PD. Lacking of Bax could protect against MPP+-induced neuronal damage[31, 32]. In our study, the expression of Bcl-2 and Bax is consistent with previous studies. Knockdown the expression of NEAT1 significantly prevented the increase in Bax expression and the decrease in Bcl-2 expression, restoring the Bax/Bcl-2 ratio in MPP+-treated cells. Interestingly, we also found the increase of Bim/caspase 3 and decrease of p-Foxo3a expression in cells after MPP + treatment. Decrease NEAT1 expression could also downregulate the Bim/caspase 3 and upregulate p-Foxo3a expression. FoxO3a is highly expressed in the brain and mediates a range of pro-apoptotic genes related to neuronal cell death as an important transcription factor. It has been reported that activated FoxO3a could induce pro-apoptotic protein such as Bim in variety of neuron death paradigms[33]. P-FoxO3a could suppress FoxO3a activity via docking with 14-3-3 and excluding FoxO3a from the nucleus[34, 35]. Caspase-3 is classically known executioner of apoptosis and activated by Bim in response to related stimuli[36]. A study showed amyloid-β induced astrocytosis and astrocyte death via FoxO3a-Bim-caspase3 death signaling[37]. Based on these results, we speculate NEAT1 acts at apoptosis in PD pathogenesis through FOXO3a/Bim/caspase 3 and Bax/Bcl-2/caspase 3 signal pathway.

LncRNAs could reverse regulate miRNA-mediated suppression of target mRNA through competing with other genes for combining with miRNAs[38]. The lncRNA-miRNA-mRNA network has been shown to exert crucial influence in PD. NEAT1 could prompt autophagy and apoptosis through mediating miR-374c-5p in MPTP-induced PD[39]. Knockdown of NEAT1 inhibited PD development via regulating miR-212-3p/AXIN1 pathway[40]. In this study, we hypothesized that NEAT1 also acts as a miRNA sponge to regulate PD progression. TargetScan6.2 analysis and dual-luciferase reporter assay were preformed, and results showed NEAT1 contained binding sites for miR-132-3p. Recently, emerging studies indicated that miR-132-3p regulated apoptosis in PD. For instance, MIAT suppressed MPP⁺-induced apoptosis by regulating miR-132/SIRT1 axis in PC12 cells[41]. Initially, significant high expression of miR-132-3p was found in midbrain tissues in PD patients. MiR-132-3p may serve as the potential biomarkers for early diagnosis of PD[42]. A previous study revealed downregulation of miR-132-3p expression could dramatically attenuate neuroinflammation and degeneration of dopaminergic neurons induced by MPTP in PD mouse models[43]. Knockdown of NEAT1 inhibits glioma cell migration and invasion through modulation of SOX2 targeted by miR-132[44]. However, there is no evidence to support the regulatory network NEAT1/miR-132-3p/apoptotic factors in PD, and the underlying mechanism needs to be clarified. In this study, we revealed that NEAT1 could regulate apoptosis in SH-SY5Y cells treated by MPP⁺. Further studies indicated NEAT1 could bind directly to miR-132-3p. These results strongly demonstrated that knockdown the level of NEAT1 could reduce cell apoptosis through FOXO3a/Bim/caspase 3 and Bax/Bcl-2/caspase 3 pathways. Therefore, we speculated that NEAT1 exerted effect by regulating the miR-132-3p/FOXO3a/Bim/caspase 3 and miR-132-3p/Bax/Bcl-2/ caspase 3 pathways.

In conclusion, the study suggested MPP⁺ treatment could upregulate the levels of NEAT and apoptotic related factors, decreasing the expression of miR-132-3p and anti-apoptotic Bcl-2. Knockdown of NEAT1 could restrain apoptosis process of PD via mediating the miR-132-3p/FOXO3a /Bim/caspase 3 and miR-132-3p /Bax/Bcl-2/caspase 3 signal pathways. which might offer innovative ideas for alleviating the PD progression.

Funding

This work was supported by National Natural Science Foundation of China (81971192).

Competing Interests

All authors have no relevant financial or non-financial interests to disclose.

Author contributions

Yumei Liu，Weichao Yao conducted experiments, statistical analysis, and drafted the manuscript. Jing Gao, Liying Yang contributed to the experiment design. Anmu Xie: Conceptualization, Datacuration, Supervision, Funding acquisition, Project administration.

Data Availability

The original data will be provided if required.

Ethics approval

Compliance with ethical standards.

Acknowledgments

We would like to thank all the participants for their helpful comments on this paper. This work was supported by National Natural Science Foundation of China (81971192).

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LncRNA NEAT1 regulates apoptosis in Parkinson's disease via sponging miR-132-3p

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Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Cell culture and treatment

2.2 Cell viability assay

2.3 RNA isolation and qRT-PCR

2.4 Western blot analysis

2.5 Dual-Luciferase reporter assay

2.6 Statistical analysis

3. Results

3.1 The viability of cells was decreased while treated by MPP⁺

3.2 NEAT1 was increased and miR-132‑3p was decreased in PD cell model

3.3 MPP⁺ could enhance apoptosis in PD cell model

3.4 Knockdown of NEAT1 suppressed the expression of miR-132-3p in PD cell model

3.5 Knockdown of NEAT1 suppressed apoptosis in PD cell model

3.6 miR-132-3p was a target of NEAT1

4. Discussion

Declarations

References

Additional Declarations

Status:

Version 1

LncRNA NEAT1 regulates apoptosis in Parkinson's disease via sponging miR-132-3p

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Cell culture and treatment

2.2 Cell viability assay

2.3 RNA isolation and qRT-PCR

2.4 Western blot analysis

2.5 Dual-Luciferase reporter assay

2.6 Statistical analysis

3. Results

3.1 The viability of cells was decreased while treated by MPP+

3.2 NEAT1 was increased and miR-132‑3p was decreased in PD cell model

3.3 MPP+ could enhance apoptosis in PD cell model

3.4 Knockdown of NEAT1 suppressed the expression of miR-132-3p in PD cell model

3.5 Knockdown of NEAT1 suppressed apoptosis in PD cell model

3.6 miR-132-3p was a target of NEAT1

4. Discussion

Declarations

References

Additional Declarations

Status:

Version 1

3.1 The viability of cells was decreased while treated by MPP⁺

3.3 MPP⁺ could enhance apoptosis in PD cell model