Long non-coding RNA NEAT1 mediates MPTP/MPP⁺-induced apoptosis via regulating the miR-124/KLF4 axis in Parkinson’s disease

2.1 Animals and treatment

Six-week-old female C57BL/6 mice were obtained from the Henan Experimental Animals Centre (Zhengzhou, China). A total of 14 mice were randomly divided into two groups (n = 7 per group). MPTP is a neurotoxin and has been widely used to establish PD animal models in many studies. To establish the mouse model of PD, one group received intraperitoneal injection of MPTP–HCl (30 mg/kg free base; Sigma-Aldrich, St Louis, MO, USA) per day. The control group received only the same volume of sterile saline solution (0.9%). Seven days after injection, all mice were sacrificed to remove the midbrains. Subsequently, the midbrains were stored at −80°C until experiments were carried out.

To explore the role of NEAT1 in PD mice, 2 days before the establishment of the PD mouse model, lentivirus-mediated short hairpin RNA (shRNA) targeting NEAT1 (sh-NEAT1) or its negative control (sh-NC) established by GeneCopoeia (Rockville, MD, USA) was injected into the midbrain of mice. Subsequently, the mice were treated as described above.

1. Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals, and has been approved by the ethics committee of Anning Branch of the 940th Hospital of Joint Logistic Support Force of PLA. All operations were carried out in accordance with the Guide for the Care and Use of Laboratory Animals [21].

2.2 Cell culture and transfection

Human neuroblastoma SH-SY5Y cells were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). The cells were cultured in DMEM (Hyclone, Logan, Utah, USA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) at 37°C in an incubator with 5% CO₂. For MPP⁺ treatment, SH-SY5Y cells were treated with 0.5, 1 or 2 mM MPP⁺ (Sigma-Aldrich) for 24 h.

ShRNA targeting NEAT1 (sh-NEAT1) and its negative control (sh-NC) were constructed by GenePharma (Shanghai, China). The KLF4 expression plasmid (pcDNA-KLF4), its negative control (pcDNA-control), miR-124 mimic (miR-124) and its negative control (miR-NC) were purchased from Hanbio Biotechnology Co., Ltd (Shanghai, China). SH-SY5Y cells were transfected with oligonucleotides or vectors using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

2.3 TH+ neurons

The midbrain sections that underwent cryostat-cut were incubated with a rabbit polyclonal anti-TH antibody (1:1,000; Cell Signaling Technology, Beverly, MA, USA) at 4°C overnight and then treated with biotinylated goat anti-rabbit IgG (1:2,000; Cell Signaling Technology) for 2 h. TH+ cells were counted using stereo investigator software.

2.4 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay

Total RNA was isolated from midbrain tissues or SH-SY5Y cells using Trizol reagent (Invitrogen). Complementary DNA (cDNA) was synthesized using a TaqMan Reverse Transcription Kit or TaqMan microRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). After reverse transcription, real-time PCR was carried out using a standard SYBR Green PCR kit (Thermo Fisher Scientific) on a CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA) according to the amplification instructions. All primers were purchased from Sangon Biotech (Shanghai, China), and the primer sequences were as follows: NEAT1 (forward 5′-CTTCCTCCCTTTAACTTATCCATTCAC-3′, reverse 5′-CTCTTCCTCCACCATTACCAACAATAC-3′), KLF4 (forward 5′-GAAATTCGCCCGCTCCGATGA-3′, reverse 5′-CTGTGTGTTTGCGGTAGTGCC-3′), miR-124 (forward 5′-GCTAAGGCACGCGGTG-3′, reverse 5′-GTGCAGGGTCCGAGGT-3′), GAPDH (forward 5′-TATGATGATATCAAGAGGGTAGT-3′, reverse 5′-TGTATCCAAACTCATTGTCATAC-3′), U6 (forward 5′-CTCGCTTCGGCAGCACATATACT-3′), reverse 5′-ACGCTTCACGAATTTGCGTGTC-3′). The relative expression levels of NEAT1, KLF4 and miR-124 were calculated using the 2^−ΔΔCt method and normalized to GAPDH or U6 serving as internal controls.

2.5 Western blot assay

The tissues and SH-SY5Y cells were lysed using RIPA lysis buffer (Sigma-Aldrich) containing a protease inhibitor (Sigma-Aldrich) for 20 min on ice. Subsequently, the cells were centrifuged at 12,000 rpm for 15 min to collect the protein supernatant. The concentration of protein was measured using a BCA protein assay kit (Beyotime, Shanghai, China). Total protein (30 µg) was separated by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane (Millipore Corp., Atlanta, GA, USA). Next, the membranes were blocked using Tris-buffered saline Tween supplemented with 5% non-fat milk for 2 h at room temperature. The membranes were then incubated with primary antibodies against cleaved-caspase-3 (cleaved-casp-3) (1:1,000; #9664), Bax (1:1,000; #14796), Bcl-2 (1:1,000; #3498), KLF4 (1:1,000; #12173) and GAPDH (1:1,000; #5174) (Cell Signaling Technology, Beverly, MA, USA) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sangon Biotech) at 1:4,000 dilution for 2 h. Finally, the membranes were examined with enhanced chemiluminescence (Tanon, Shanghai, China) and imaged using a chemiluminescence gel imaging system (Tanon). Protein levels were quantified using ImageJ software and normalized to GAPDH.

2.6 MTT assay

Cell viability was detected using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Briefly, SH-SY5Y cells were seeded in 96-well plates at a density of 2 × 10⁴ cells/mL and cultured overnight. Then, SH-SY5Y cells were treated with MPP⁺ (2 mM) for 24 h and transfected with sh-NC, sh-NEAT1, sh-NEAT1 + pcDNA-control, sh-NEAT1 + pcDNA-KLF4, sh-NEAT1 + miR-NC or sh-NEAT1 + miR-124. After transfection for 48 h, 20 µL of MTT solution (5 mg/mL) was added to each well. After further incubation for 4 h, the culture medium mixture was discarded, and the formazan crystals were dissolved by adding 150 µL of dimethyl sulfoxide (Sigma-Aldrich) to each well. Cell viability was assessed at 490 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

2.7 Apoptosis assay

The apoptosis rate was measured using flow cytometry with an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Sangon Biotech). SH-SY5Y cells were seeded in six-well plates and treated with MPP⁺ (2 mM) for 24 h and transfected with different transfection reagents. After 48 h, the cells were collected and stained with Annexin V-FITC and PI in a dark environment for 15 min. The percentage of apoptotic cells was examined using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

2.8 Luciferase reporter assay

The putative binding sites of miR-124 and NEAT1 or KLF4 were predicted using online software MiRcode Tool or TargetScan Tool. Partial fragments of wild-type NEAT1 (WT-NEAT1), mutant NEAT1 (MUT-NEAT1), 3′-untranslated regions of KLF4 wild type (KLF4 3′-UTR-WT) or 3′-untranslated regions of KLF4 mutant (KLF4 3′-UTR-MUT) were amplified and cloned into pGL3 plasmids (Promega, Madison, WI, USA). The reporter vectors with miR-NC, miR-124, miR-124 + pcDNA-control or miR-124 + pcDNA-NEAT1 were co-transfected into SH-SY5Y cells according to the manufacturer’s protocols. Luciferase activities were detected with a dual luciferase reporter assay kit (Promega) after transfection for 48 h.

2.9 Statistical analysis

In the present study, all data from at least three individual experiments were expressed as mean ± standard deviation. Differences between the two groups were analyzed using GraphPad Prism 5.0 software (GraphPad Prism, San Diego, CA). A P value of <0.05 was considered statistically significant.

3.1 Effects of MPTP and MPP⁺ on NEAT1 and KLF4 expression in vivo and in vitro

In order to detect the number of TH+ cells and the expression of NEAT1 and KLF4 in the brain of MPTP-treated mice (n = 7), MPTP–HCl was injected into a group of mice, and a sterile saline solution (0.9%) was injected into the control group. The number of TH+ cells was dramatically reduced in PD mice in contrast to the control group (P < 0.05, Figure 1a). However, the results of qRT-PCR showed that NEAT1 and KLF4 expression levels were upregulated in the midbrain of PD mice (P < 0.05, Figure 1b and c). Besides, KLF4 protein abundance was also increased in the midbrain of PD mice (P < 0.05, Figure 1d). In addition, the expression of apoptosis-related proteins was analyzed by western blot. Results showed that the levels of cleaved-casp-3 and Bax proteins were significantly elevated in the midbrain of PD mice, while the expression of Bcl-2 protein was significantly decreased (P < 0.05, Figure 1e). Furthermore, to explore the effects of MPP⁺ on neuronal cells, human SH-SY5Y cells were treated with different concentrations (0, 0.5, 1 and 2 mM) of MPP⁺ for 24 h, and the cells without MPP⁺ treatment were used for the control group. The results revealed that the expression levels of NEAT1 and KLF4 were increased in a dose-dependent manner in SH-SY5Y cells treated with MPP⁺ (P < 0.05, Figure 1f and g). Similarly, expression of KLF4 protein was also enhanced in a dose-dependent manner (P < 0.05, Figure 1h). These results indicated that NEAT1 and KLF4 might play vital roles in the development of PD.

Figure 1

Effects of MPTP on NEAT1 and KLF4 expression in vivo and in vitro. (a) The number of TH+ neurons was dramatically decreased in PD mice. (b) Abundance of NEAT1 detected in the midbrain of PD mice by qRT-PCR. (c and d) mRNA and protein levels of KLF4 detected by qRT-PCR and western blot, respectively. (e) Expression of apoptosis-related proteins analyzed by western blot. (f) Expression of NEAT1 measured in SH-SY5Y cells treated with MPP+. (g and h) mRNA and protein levels of KLF4 examined in MPP⁺-treated SH-SY5Y cells. *P < 0.05.

3.2 Effects of NEAT1 on cell viability and apoptosis in MPP⁺-treated SH-SY5Y cells

To investigate the effect of NEAT1 on PD progression, sh-NEAT1 or sh-NC was transfected into SH-SY5Y cells and then treated with MPP⁺ (2 mM). According to Figure 2a (P < 0.05), transfection of sh-NEAT1 led to an obvious reduction of NEAT1 expression in SH-SY5Y cells compared with the sh-NC group, suggesting that sh-NEAT1 was successfully transfected into SH-SY5Y cells. Cell viability and apoptosis were measured by MTT assay and flow cytometry analysis, respectively. Results showed that cell viability was evidently inhibited, while the apoptosis rate was conspicuously increased in SH-SY5Y cells treated with MPP⁺, which was reversed by the inhibition of NEAT1 (P < 0.05, Figure 2b and c). Western blot analysis displayed that cleaved-casp-3 and Bax protein levels were notably increased, while the Bcl-2 protein level was prominently suppressed in SH-SY5Y cells treated with MPP⁺, whereas these effects were reversed by transfection of sh-NEAT1 (P < 0.05, Figure 2d). Our data suggested that knockdown of NEAT1 elevated cell viability and restrained apoptosis in SH-SY5Y cells treated with MPP⁺.

Figure 2

Knockdown of NEAT1 increased cell viability and reduced apoptosis in SH-SY5Y cells treated with MPP⁺. (a) Expression of NEAT1 detected by qRT-PCR in SH-SY5Y cells transfected with sh-NC or sh-NEAT1. (b–d) SH-SY5Y cells were transfected with sh-NC or sh-NEAT1 and then treated with MPP⁺ (2 mM). (b and c) Cell viability and apoptosis analyzed by MTT assay or flow cytometry, respectively. (d) Expression of apoptosis-related proteins analyzed by western blot. *P < 0.05.

3.3 KLF4 overexpression reversed the effects of NEAT1 knockdown on cell viability and apoptosis in MPP⁺-treated SH-SY5Y cells

To explore the effect of NEAT1 on PD and the relationship between KLF4 and NEAT1, SH-SY5Y cells were transfected with sh-NC, sh-NEAT1, sh-NEAT1 + pcDNA-control or sh-NEAT1 + pcDNA-KLF4 and then exposed to MPP⁺. qRT-PCR and western blot analysis revealed that knockdown of NEAT1 evidently suppressed the mRNA and protein levels of KLF4 compared with the control group (P < 0.05, Figure 3a and b). Upregulation of KLF4 abolished the effects of NEAT1 knockdown on promotion of cell viability and inhibition of the apoptosis rate in MPP⁺-treated SH-SY5Y cells (P < 0.05, Figure 3c and d). Moreover, western blot analysis revealed that overexpression of KLF4 reversed the effects of NEAT1 knockdown on the decrease of cleaved-casp-3 and Bax protein expression and the increase of Bcl-2 protein expression (P < 0.05, Figure 3e). These data demonstrated that overexpression of KLF4 reversed the effects of NEAT1 knockdown on cell viability and apoptosis in MPP⁺-treated SH-SY5Y cells.

Figure 3

KLF4 overexpression reversed the effects of NEAT1 knockdown on cell viability and apoptosis in MPP⁺-treated SH-SY5Y cells. (a and b) mRNA and protein abundance of KLF4 analyzed in SH-SY5Y cells transfected with sh-NC or sh-NEAT1. (c–e) SH-SY5Y cells were transfected with sh-NC, sh-NEAT1, sh-NEAT1 + pcDNA-control or sh-NEAT1 + pcDNA-KLF4 and then exposed to MPP⁺. (c and d) Cell viability and apoptosis measured by MTT assay and flow cytometry, respectively. (e) Expression of apoptosis-related proteins analyzed by western blot. *P < 0.05.

3.4 NEAT1 targeted miR-124 to regulate KLF4 expression

The potential binding sites of NEAT1 and miR-124 were predicted by MiRcode Tool, and we found that miR-124 was a potential binding target of NEAT1 (P < 0.05, Figure 4a). Next, the prediction was confirmed by a dual luciferase reporter assay. Results showed that the transfection of miR-124 decreased the relative luciferase activity of the WT-NEAT1 reporter, whereas the effect was small with respect to the relative luciferase activity of the MUT-NEAT1 reporter (P < 0.05, Figure 4b). Subsequently, online software TargetScan Tool also showed that miR-124 might bind to KLF4 3′-UTR (P < 0.05, Figure 4c). Then, pGL3 luciferase reporter plasmids containing the 3′-UTR sequences of KLF4-WT and KLF4-MUT were established. We found that the luciferase activity of KLF4 3′-UTR-WT was reduced in SH-SY5Y cells transfected with miR-124, which was abrogated by the addition of pcDNA-NEAT1 (P < 0.05, Figure 4d). Similarly, the luciferase activity of KLF4 3′-UTR-MUT was unaffected by transfection of miR-124 and pcDNA-NEAT1. Moreover, qRT-PCR and western blot analysis indicated that overexpression of miR-124 markedly suppressed the mRNA and protein levels of KLF4 in contrast to the control group, which was abated by transfection of pcDNA-NEAT1 (P < 0.05, Figure 4e and f). Our preliminarily data suggest that NEAT1 regulated KLF4 expression by targeting miR-124.

Figure 4

NEAT1 targeted miR-124 to regulate KLF4 expression. (a) The potential binding sites of NEAT1 and miR-124 predicted by MiRcode Tool. (b) Luciferase activity measured in SH-SY5Y cells co-transfected with WT-NEAT1 or MUT-NEAT1 and miR-124 or miR-NC by a dual luciferase reporter assay. (c) The putative binding sites of miR-124 and KLF4 predicted by TargetScan Tool. (d) Luciferase activity detected in SH-SY5Y cells co-transfected with different reagents. (e and f) mRNA and protein expression of KLF4 evaluated in SH-SY5Y cells transfected with miR-NC, miR-124, miR-124 + pcDNA-control or miR-124 + pcDNA-NEAT1. *P < 0.05.

3.5 NEAT1 could regulate cell viability and apoptosis in MPP⁺-treated SH-SY5Y cells by sponging miR-124

To determine the regulatory network of NEAT1 and miR-124 in PD, SH-SY5Y cells were transfected with sh-NEAT1, sh-NC sh-NEAT1 + miR-124 or sh-NEAT1 + miR-NC and then treated with MPP⁺ (2 mM). As shown in Figure 5a and b (P < 0.05), transfection of sh-NEAT1 led to an obvious increase of cell viability and a decrease of apoptosis rate in MPP⁺-treated SH-SY5Y cells compared with the transfection of sh-NC, and these effects were improved by the addition of miR-124. Also, western blot analysis revealed that knockdown of NEAT1 resulted in a significant decrease in cleaved-casp-3 and Bax protein levels, while an increase in Bcl-2 protein expression in SH-SY5Y cells treated with MPP⁺ when compared with the sh-NC transfection group, and addition of miR-124 could enhance these effects (P < 0.05, Figure 5c). These findings suggest that NEAT1 targets miR-124 to regulate cell viability and apoptosis in SH-SY5Y cells treated with MPP⁺.

Figure 5

NEAT1 regulated cell viability and apoptosis in MPP⁺-treated SH-SY5Y cells by targeting miR-124. SH-SY5Y cells were transfected with sh-NC, sh-NEAT1, sh-NEAT1 + miR-NC or sh-NEAT1 + miR-124 and then treated with MPP⁺ (2 mM). (a) Cell viability assessed by MTT assay. (b) Cell apoptosis determined by flow cytometry. (c) Expression of apoptosis-related proteins detected by western blot. *P < 0.05.

3.6 Effects of NEAT1 knockdown on the PD mouse model

To explore the effects of NEAT1 on PD in vivo, sh-NC or lenti-sh-NEAT1 was injected into the midbrain of mice 2 days before the establishment of the PD mouse model. Results showed that the number of TH+ neurons was obviously increased in PD mice transfected with lenti-sh-NEAT1 (P < 0.05, Figure 6a). Moreover, qRT-PCR revealed that the expression of miR-124 was significantly increased in PD mice transfected with lenti-sh-NEAT1, whereas the mRNA expression of KLF4 was notably decreased (P < 0.05, Figure 6b and c). In addition, injection of lenti-sh-NEAT1 clearly reduced the levels of KLF4, cleaved-casp-3 and Bax proteins while elevating the expression of Bcl-2 protein (P < 0.05, Figure 6d). These results suggested that knockdown of NEAT1 might inhibit the development of PD in vivo.

Figure 6

The role of NEAT1 knockdown in the PD mouse model. Lenti-sh-NC or lenti-sh-NEAT1 was injected into the midbrain of mice 2 days before the establishment of the PD mice. (a) The number of TH+ neurons in the midbrain of PD mice. (b and c) Expression levels of miR-124 and KLF4 measured by qRT-PCR in the midbrain of PD mice. (d) The levels of KLF4, cleaved-casp-3, Bax and Bcl-2 proteins analyzed by western blot. *P < 0.05.