Long non-coding RNA MALAT1 contributes to cell apoptosis by sponging miR-124 in Parkinson disease

Abstract

Background: Parkinson disease (PD) is the most common movement disturbance characterized by the loss of dopaminergic (DA) neurons in midbrain. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is aberrantly expressed in neurons and is involved in the dendritic and synapse development. However, the role of MALAT1 and its underlying mechanism in PD remain to be defined.

Methods: The expressions of MALAT1 and miR-124 were evaluated by qRT-PCR. N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice and SH-SY5Y cells subjected to N-methyl-4-phenylpyridinium (MPP⁺) were utilized to investigate the effect of MALAT1 on PD. TUNEL assay was performed to detect apoptosis of DA neurons in PD mice. Flow cytometry analysis was carried out to measure apoptosis of SH-SY5Y cells. Caspase3 activity and Cleaved Caspase3 expression were tested by caspase3 assay kit and western blot, respectively. TargetScan software and luciferase reporter assay were used to explore the relationship between MALAT1 and miR-124.

Results: MALAT1 was up-regulated and miR-124 was down-regulated in MPTP-induced PD mice and MPP⁺-treated SH-SY5Y cells. MALAT1 knockdown attenuated MPTP-induced apoptosis of DA neurons in MPTP-induced PD mouse model. MALAT1 interacted with miR-124 to negatively regulate its expression. MALAT1 knockdown suppressed MPP⁺-induced apoptosis in SH-SY5Y cells, while miR-124 downregulation abrogated this effect. Moreover, MALAT1 knockdown improved miR-124 expression in MPTP/MPP⁺ induced models of PD.

Conclusions: MALAT1 promotes the apoptosis by sponging miR-124 in mouse models of PD and in vitro model of PD, providing a potential theoretical foundation for the clinical application of MALAT1 against PD.

Keywords: Apoptosis; MALAT1; Parkinson disease; miR-124.

Fig. 1

Expressions of MALAT1 and miR-124 in MPTP-induced PD mouse model and in MPP⁺-intoxicated SH-SY5Y cells. a, b The expressions of MALAT1 and miR-124 were examined by qRT-PCR in MPTP-induced PD mouse model. c, d The levels of MALAT1 and miR-124 were assessed by qRT-PCR in SH-SY5Y cells treated with 1 mM MPP⁺ for 24 h. *P < 0.05 vs. control group

Fig. 2

Effects of MALAT1 knockdown on apoptosis of DA neurons in MPTP-induced PD mouse model. Recombinant lentivirus vectors with sh-MALAT1 or sh-control were constructed and administered into the midbrain of mice. Two days post transfection, mice were treated with MPTP-HCl (30 mg/kg) by intraperitoneal injection for 4 days. Seven days after the last MPTP injection, mice were killed and ventral midbrain with SNpc was removed. a The expression of MALAT1 in sh-MALAT1- or sh-control-injected mice was detected by qRT-PCR. b TUNEL assay was carried out to analyze the apoptotic cells in treated mice (scale bar 50 μm). c Western blot was performed to assess the level of Cleaved Caspase3 in treated mice. β-actin was used as the internal control. *P < 0.05 vs. control group

Fig. 3

Interaction between MALAT1 and miR-124. a The putative recognition sequence of miR-124 in MALAT1 was predicted by Targetscan. b Luciferase activity was detected in HEK293 cells co-transfected with pGL3-MALAT1-WT or pGL3-MALAT1-MUT(1+2) and miR-124 or miR-control. c qRT-PCR was performed to evaluate miR-124 expression in SH-SY5Y cells transfected with si-MALAT1, pcDNA-MALAT1 or matched controls. *P < 0.05 vs. control group

Fig. 4

Effects of si-MALAT1 and anti-miR-124 on apoptosis of MPP⁺-intoxicated SH-SY5Y cells. SH-SY5Y cells were transfected with si-MATAL1 or in combination with anti-miR-124, followed by the treatment of 1 mM MPP⁺ solution for 24 h. a Flow cytometry analysis was performed to examine apoptosis in treated SH-SY5Y cells. b Caspase3 activity was determined by the caspase3 assay kit in treated SH-SY5Y cells. c Western blot was carried out to detect the level of Cleaved Caspase3 in treated SH-SY5Y cells. β-actin was used as the internal control. d Quantification analysis of Cleaved Caspase3. *P < 0.05 vs. control group

Fig. 5

Effects of MALAT1 knockdown on expression of miR-124 in MPTP-induced PD mouse model and in MPP⁺-intoxicated SH-SY5Y cells. The expression of miR-124 was examined by qRT-PCR in MPTP-induced PD mouse model transfected with sh-MALAT1 or sh-control (a) and in MPP⁺-intoxicated SH-SY5Y cells transfected with si-MALAT1 or si-control (b). *P < 0.05 vs. control group