Retracted Article: LncRNA MALAT1 aggravates MPP-induced neuronal injury by regulating miR-212 in SH-SY5Y cells

Abstract

Parkinson's disease (PD) is the most common neurodegenerative disease and its incidence is rising. Long noncoding RNAs (lncRNAs) have been reported to have essential roles in development of PD. LncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is dysregulated in PD, while the role of MALAT1 and its mechanism in PD remain poorly understood. In this study, SH-SY5Y cells were exposed to 1-methyl-4-phenylpyridinium (MPP⁺) to induce a PD model in vitro. Then we explored the effect of MALAT1 on cell viability, apoptosis and inflammatory response as well as its interaction with miR-212 in MPP⁺-treated SH-SY5Y cells. The results showed that MALAT1 was up-regulated in MPP⁺-treated SH-SY5Y cells compared with that in the normal group. Overexpression of MALAT1 exacerbated MPP⁺-induced neuronal injury, uncovered by inhibition of cell viability and increase of cell apoptosis as well as inflammatory cytokine expressions in SH-SY5Y cells. However, knockdown of MALAT1 exerted the opposite effect in MPP⁺-treated SH-SY5Y cells. Moreover, MALAT1 was bound to miR-212 and negatively regulated the miR-212 level. Furthermore, addition of miR-212 ablated the regulatory effect of MALAT1 on MPP⁺-induced neuronal injury, as indicated by restoration of cell viability and lower apoptotic rate along with inflammatory cytokine levels in SH-SY5Y cells. Therefore, we concluded that MALAT1 exacerbated MPP⁺-induced neuronal injury through regulating cell viability, apoptosis and inflammatory cytokines by sponging miR-212, providing a novel theoretical foundation for application of MALAT1 in PD.

Fig. 1. MALAT1 expression was enhanced in MPP⁺-treated SH-SY5Y cells. (A) The chemical structure of MPP⁺. (B) The expression of MALAT1 was measured by qRT-PCR in SH-SY5Y cells after treatment with different concentrations of MPP⁺ for 24 h. (C) The abundance of MALAT1 was detected by qRT-PCR in 1 mM MPP⁺-treated SH-SY5Y cells at different treatment times. *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 2. MALAT1 exacerbated MPP⁺-mediated regulatory effect on cell viability and apoptosis in SH-SY5Y cells. (A) The abundance of MALAT1 was detected by qRT-PCR in SH-SY5Y cells transfected with pcDNA, MALAT1, si-NC or si-MALAT1. (B) Cell viability was measured by MTT in SH-SY5Y cells transfected with pcDNA, MALAT1, si-NC or si-MALAT1 after treatment with MPP⁺. (C and D) Cell apoptosis was examined by flow cytometry in SH-SY5Y cells transfected with pcDNA, MALAT1, si-NC or si-MALAT1 after treatment with MPP⁺. **p < 0.01 and ***p < 0.001.

Fig. 3. MALAT1 exacerbated MPP⁺-induced production of inflammatory cytokines in SH-SY5Y cells. (A) The level of IL-1β was measured by ELISA in SH-SY5Y cells transfected with pcDNA, MALAT1, si-NC or si-MALAT1 after treatment of MPP⁺. (B) The level of IL-6 was detected by ELISA in SH-SY5Y cells transfected with pcDNA, MALAT1, si-NC or si-MALAT1 after exposure to MPP⁺. (C) The expression of TNF-α was examined by ELISA in SH-SY5Y cells transfected with pcDNA, MALAT1, si-NC or si-MALAT1 after stimulation of MPP⁺. **p < 0.01 and ***p < 0.001.

Fig. 4. MALAT1 was bound to miR-212 in SH-SY5Y cells. (A) The putative binding sites of miR-212 and MALAT1 were predicted by starBase online. (B) Luciferase activity was measured in SH-SY5Y cells co-transfected with MALAT1-WT or MALAT1-MUT and miR-212 or miR-NC. (C) RNA pull-down assay was conducted in SH-SY5Y cells transfected with bio-NC, bio-miR-212-WT or bio-miR-212-MUT and the level of MALAT1 was measured by qRT-PCR. (D) Ago2 RIP assay was performed in SH-SY5Y cells transfected with miR-212 or miR-NC and the abundance of MALAT1 was detected by agarose electrophoresis and qRT-PCR. (E) The expression of miR-212 was measured by qRT-PCR in SH-SY5Y cells transfected with pcDNA, MALAT1, si-NC or si-MALAT1. ***p < 0.001.

Fig. 5. Overexpression of miR-212 reversed the regulatory effect of MALAT1 on cell viability and apoptosis in MPP⁺-treated SH-SY5Y cells. (A) Cell viability was measured by MTT in SH-SY5Y cells transfected with MALAT1 + miR-NC or MALAT1 + miR-212 after treatment with MPP⁺. (B) Cell apoptosis was detected by flow cytometry in SH-SY5Y cells transfected with MALAT1 + miR-NC or MALAT1 + miR-212 after treatment with MPP⁺. (C) Cell viability was measured by MTT in SH-SY5Y cells transfected with si-MALAT1 + anti-miR-NC or si-MALAT1 + anti-miR-212 after stimulation of MPP⁺. (D) Cell apoptosis was detected by flow cytometry in SH-SY5Y cells transfected with si-MALAT1 + anti-miR-NC or si-MALAT1 + anti-miR-212 after exposure to MPP⁺. **p < 0.01 and ***p < 0.001.

Fig. 6. Addition of miR-212 attenuated MALAT1-promoted secretion of inflammatory cytokines in MPP⁺-treated SH-SY5Y cells. (A and B) The level of IL-1β was measured by ELISA in SH-SY5Y cells transfected with MALAT1 + miR-NC, MALAT1 + miR-212, si-MALAT1 + anti-miR-NC or si-MALAT1 + anti-miR-212 after treatment with MPP⁺. (C and D) The level of IL-6 was detected by ELISA in SH-SY5Y cells transfected with MALAT1 + miR-NC, MALAT1 + miR-212, si-MALAT1 + anti-miR-NC or si-MALAT1 + anti-miR-212 after exposure to MPP⁺. (E and F) The expression of TNF-α was examined by ELISA in SH-SY5Y cells transfected with MALAT1 + miR-NC, MALAT1 + miR-212, si-MALAT1 + anti-miR-NC or si-MALAT1 + anti-miR-212 after stimulation of MPP⁺. **p < 0.01 and ***p < 0.001.