Yonsei Med J.  2019 Jul;60(7):640-650. 10.3349/ymj.2019.60.7.640.

Long Noncoding RNA NEAT1 Aggravates Aβ-Induced Neuronal Damage by Targeting miR-107 in Alzheimer's Disease

• Ke S
• Yang Z
• Yang F
• Wang X
• Tan J
• Liao B

• KMID: 2450404
• DOI: http://doi.org/10.3349/ymj.2019.60.7.640

Abstract

PURPOSE
Alzheimer's disease (AD) is the most common neurodegenerative disease, with a rising prevalence worldwide. Long noncoding RNAs (lncRNAs) have been found to play important roles in the development and treatment of AD. However, the exact role of lncRNA nuclear enriched abundant transcript 1 (NEAT1) in neuronal damage in AD is largely unknown.
MATERIALS AND METHODS
The AD model was established in SH-SY5Y and SK-N-SH cells via treatment with amyloid β1−42 (Aβ). The expression of NEAT1 and microRNA-107 (miR-107) was measured by quantitative real-time polymerase chain reaction. Cell viability and apoptosis were detected by MTT assay, immunocytochemistry, and flow cytometry. The expression of phosphorylated tau protein (p-Tau) was measured by Western blot. The interaction between NEAT1 and miR-107 was explored by bioinformatics analysis, luciferase activity, and RNA immunoprecipitation assays.
RESULTS
NEAT1 expression was enhanced in Aβ-treated SH-SY5Y and SK-N-SH cells, and its knockdown attenuated Aβ-induced inhibition of viability and promotion of apoptosis and p-Tau levels. NEAT1 was indicated as a decoy of miR-107. miR-107 abundance was reduced in Aβ-treated cells, and its overexpression reversed Aβ-induced injury. Moreover, interference of miR-107 abated silencing of NEAT1-mediated inhibition of neuronal damage in Aβ-treated SH-SY5Y and SK-N-SH cells.
CONCLUSION
LncRNA NEAT1 aggravated Aβ-induced neuronal damage by sponging miR-107, indicating a novel avenue for treatment of AD.

Keyword

Alzheimer's disease; NEAT1; miR-107; neuronal damage

MeSH Terms

Alzheimer Disease*
Amyloid
Apoptosis
Blotting, Western
Cell Survival
Computational Biology
Flow Cytometry
Immunohistochemistry
Immunoprecipitation
Luciferases
Neurodegenerative Diseases
Neurons*
Prevalence
Real-Time Polymerase Chain Reaction
RNA
RNA, Long Noncoding*
tau Proteins
Amyloid
Luciferases
RNA
RNA, Long Noncoding
tau Proteins

Figure

• 

  Fig. 1 Expression of NEAT1 is elevated in Aβ-treated SH-SY5Y and SK-N-SH cells. (A and B) The expression of NEAT1 was measured in SH-SY5Y and SK-N-SH cells after treatment of different concentrations (0, 5, 10, and 20 µM) of Aβ for 24 h by qRT-PCR. (C and D) The levels of NEAT1 were detected in SH-SY5Y and SK-N-SH cells after treatment of 10 µM Aβ for different treatment times (0, 12, 24, and 48 h) by qRT-PCR. *p<0.05, **p<0.01, ***p<0.001. NEAT1, nuclear enriched abundant transcript 1; Aβ, amyloid β1–42.

• 

  Fig. 2 Knockdown of NEAT1 attenuates Aβ-induced neuronal damage in SH-SY5Y and SK-N-SH cells. (A and B) The expression of NEAT1 was measured in SH-SY5Y and SK-N-SH cells transfected with si-NEAT1 or si-NC after treatment of 10 µM Aβ for 24 h by qRT-PCR. Cell viability (C and D), Ki67 expression (E and F), and apoptosis (G and H) were detected in SH-SY5Y and SK-N-SH cells transfected with si-NEAT1 or si-NC after treatment of 10 µM Aβ for 24 h by MTT, immunocytochemistry, and flow cytometry. *p<0.05, **p<0.01, ***p<0.001. NEAT1, nuclear enriched abundant transcript 1; Aβ, amyloid β1–42.

• 

  Fig. 3 Interference of NEAT1 weakens Aβ-induced phosphorylation of Tau in SH-SY5Y and SK-N-SH cells. The protein levels of phosphorylation of Tau (p-Tau) were measured in SH-SY5Y (A) and SK-N-SH (B) cells transfected with si-NEAT1 or si-NC after treatment of 10 µM Aβ for 24 h by Western blot. *p<0.05, **p<0.01. NEAT1, nuclear enriched abundant transcript 1; Aβ, amyloid β1–42.

• 

  Fig. 4 miR-107 is bound to NEAT1. (A) The putative binding sites of NEAT1 and miR-107 were predicted by StarBase. (B) Luciferase activity was measured in 293T cells co-transfected with NEAT1-WT or NEAT1-MUT and miR-107 or miR-NC. (C and D) The enrichment of NEAT1 was detected in SH-SY5Y and SK-N-SH cells transfected with miR-107 or miR-NC after RIP assay. (E and F) The expression of miR-107 was measured in SH-SY5Y and SK-N-SH cells transfected with pcDNA, NEAT1, si-NC or si-NEAT1 by qRT-PCR. ***p<0.001. NEAT1, nuclear enriched abundant transcript 1.

• 

  Fig. 5 The levels of miR-107 are decreased in Aβ-treated SH-SY5Y and SK-N-SH cells. (A and B) The expression of miR-107 was measured in SH-SY5Y and SK-N-SH cells after treatment of different concentrations of Aβ for 24 h by qRT-PCR. (C and D) The abundance of miR-107 was detected in SH-SY5Y and SK-N-SH cells after treatment of 10 µM Aβ for different treatment times by qRT-PCR. *p<0.05, **p<0.01, ***p<0.001. Aβ, amyloid β1–42.

• 

  Fig. 6 Abrogation of miR-107 reverses the regulatory effect of NEAT1 knockdown on Aβ-induced neuronal damage in SH-SY5Y and SK-N-SH cells. (A and B) The expression of miR-107 was measured in SH-SY5Y and SK-N-SH cells transfected with miR-107, miR-NC, si-NEAT1, and anti-miR-NC or anti-miR-107 after treatment of 10 µM Aβ for 24 h by qRT-PCR. Cell viability (C and D) and apoptosis (E and F) were detected in SH-SY5Y and SK-N-SH cells transfected with miR-107, miR-NC, si-NEAT1, and anti-miR-NC or anti-miR-107 after treatment of 10 µM Aβ for 24 h by MTT or flow cytometry. *p<0.05, **p<0.01, ***p<0.001. NEAT1, nuclear enriched abundant transcript 1; Aβ, amyloid β1–42.

• 

  Fig. 7 miR-107 is involved in interference of NEAT1-mediated inhibition of phosphorylation of Tau (p-Tau) in Aβ-treated SH-SY5Y and SK-N-SH cells. The protein levels of p-Tau were detected in SH-SY5Y (A) and SK-N-SH (B) cells transfected with miR-107, miR-NC, si-NEAT1, and anti-miR-NC or anti-miR-107 after treatment of 10 µM Aβ for 24 h by Western blot. *p<0.05, **p<0.01, ***p<0.001. NEAT1, nuclear enriched abundant transcript 1; Aβ, amyloid β1–42.

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