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. 2022 Aug 26:13:824495.
doi: 10.3389/fgene.2022.824495. eCollection 2022.

LncRNA RP11-59J16.2 aggravates apoptosis and increases tau phosphorylation by targeting MCM2 in AD

Fulin Guan  1 , Qichang Gao  2 , Xinghua Dai  3 , Lei Li  4 , Rui Bao  5 , Jiaao Gu  2
Affiliations

LncRNA RP11-59J16.2 aggravates apoptosis and increases tau phosphorylation by targeting MCM2 in AD

Fulin Guan et al. Front Genet. .

Abstract

Alzheimer's disease (AD) is a degenerative disease of central nervous system with unclear pathogenesis, accounting for 60%-70% of dementia cases. Long noncoding RNAs (LncRNAs) play an important function in the development of AD. This study aims to explore the role of differentially expressed lncRNAs in AD patients' serum in the pathogenesis of AD. Microarray analysis was performed in the serum of AD patients and healthy controls to establish lncRNAs and mRNAs expression profiles. GO analysis and KEGG pathway analysis revealed that G1/S transition of mitotic cell cycle might be involved in the development of AD. The result showed that RP11-59J16.2 was up-regulated and MCM2 was down-regulated in serum of AD patients. SH-SY5Y cells were treated with Aβ 1-42 to establish AD cell model. Dual luciferase reporter gene analysis verified that RP11-59J16.2 could directly interact with 3'UTR of MCM2 and further regulate the expression of MCM2. Inhibition of RP11-59J16.2 or overexpression of MCM2, CCK-8 assay and Annexin V FITC/PI apoptosis assay kit results showed that RP11-59J16.2 could reduce cell viability, aggravate apoptosis and increase Tau phosphorylation in AD cell model by inhibiting MCM2. In short, our study revealed a novel lncRNA RP11-59J16.2 that could promote neuronal apoptosis and increase Tau phosphorylation by regulating MCM2 in AD model, and indicated that lncRNA RP11-59J16.2 might be a potential target molecule for AD development.

Keywords: Alzheimer disease; MCM2; P-tau; RP11-59J16.2; apoptosis; microarray analysis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Differentially expression of lncRNAs and mRNAs in AD patients. (A) Cluster heat map of the expression profiles of lncRNAs and mRNAs between 3 pairs of AD patients and controls. Red represents up-regulated genes and green represents down-regulated genes. (B) Scatter plot of lncRNAs and mRNAs in AD and control. Red represents up-regulated genes and green represents down-regulated genes. (C) Volcano plot of mRNA and lncRNA in AD and control. X-axis parallel line: p = 0.05; Y-axis parallel line: FC value = 2. Red area, p < 0.05, FC ≥ 2 differential genes; Green area, p < 0.05, FC ≤ 0.5 differential genes.
FIGURE 2
FIGURE 2
Enrichment biological process of the differentially expression genes. (A) GO analysis of differentially expressed mRNA between AD and control group. (B) KEGG analysis of differentially expressed mRNA between AD and control group. (C) PPI network analysis of mRNA.
FIGURE 3
FIGURE 3
RP11-59J16.2 was overexpressed in serum of AD patients and Aβ treated SH-SY5Y cell model. (A) RP11-59J16.2 was highly expressed in serum of AD patients. (B) Expression of RP11-59J16.2 was detected in SH-SY5Y after qRT-PCR treatment at different concentrations of Aβ1-42 (0, 5, 10, 20 μM) for 24 h. (C). Expression of RP11-59J16.2 was detected in SH-SY5Y after treatment with 10 μM Aβ1-42 at different times (0, 12, 24, 48 h). * p < 0.05; ** p < 0.01; *** p < 0.001.
FIGURE 4
FIGURE 4
RP11-59J16.2 target regulate the expression of MCM2 which was low expressed AD serum and cell model. (A) Bioinformatics analysis of the binding sites of RP11-59J16.2 and MCM2. (B) Double luciferase reporter gene assay verified the binding sites of RP11-59J16.2 and MCM2. (C,D) Low expression of MCM2 in serum of AD patients by qRT-PCR and Western blot. (E,F) MCM2 was low expressed and inhibited by RP11-59J16.2 in AD cell model. (G) qRT-PCR confirmed high expression of RP11-59J16.2 and low expression of MCM2 with positive correlation in AD cell model. The experiment was repeated three times in each group, * p < 0.05; ** p < 0.01, *** p < 0.001.
FIGURE 5
FIGURE 5
RP11-59J16.2 affect the neuronal damage of SH-SY5Y cells induced by Aβ by targeting MCM2. After treatment with 10 μM Aβ1-42 for 24h, SH-SY5Y cells were transfected with pcDNA-NC, si-RP11-59J16.2, pcDNA-MCM2, si-RP11-59J16.2 + si-NC and si-RP11-59J16.2 + si-MCM2 to detect cell viability (A) and apoptosis (B,C). (A) CCK-8 assay was used to detect the changes of cell viability in each group. Cell viability increased significantly in si-RP11-59J16.2 and MCM2 transfection group compared to Aβ treatment group. It reduced significantly after transfection of si-RP11-59J16.2+ si-MCM2. (B,C) Flow cytometry was used to explore the effects of RP11-59J16.2 and MCM2 on apoptosis of SH-SY5Y cells. Transfection of si-RP11-59J16.2 or MCM2 significantly reduced the apoptosis of SH-SY5Y cells after Aβ treatment. The cell apoptosis in si-RP11-59J16.2+ si-MCM2 group was significantly increased compared with si-RP11-59J16.2 group. The experiment was repeated three times in each group, * p < 0.05; ** p < 0.01; *** p < 0.001.
FIGURE 6
FIGURE 6
RP11-59J16.2 regulate p-Tau expression by inhibiting MCM2. (A) Western blot was used to detect p-Tau and MCM2 expression in each group. p-Tau expression was significantly decreased after transfection with si-RP11-59J16.2 or MCM2. The expression of p-Tau increased significantly in si-RP11-59J16.2 + si-MCM2 transfection group compared with si-RP11-59J16.2 or MCM2 group. The expression of p-Tau and MCM2 was negatively correlated. (B,C) Quantitative results of Western blot about the relative expression of MCM2 and p-Tau. The experiment was repeated three times in each group, *** p < 0.001.
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