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NEAT1 knockdown suppresses endothelial cell proliferation and induces apoptosis by regulating miR?638/AKT/mTOR signaling in atherosclerosis.

ABSTRACT: Long non?coding RNAs (lncRNAs) have been validated to mediate the development of atherosclerosis (AS). In the present study, the molecular mechanisms and functions of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in the advancement of human aortic endothelial cells (HAECs) were investigated. The levels of lncRNA?NEAT1 and miR?638 expression in clinical samples and cells were explored via quantitative reverse transcription polymerase chain reaction. Colony formation and CCK?8 assays were performed to determine the proliferative capacity of cells, and the apoptotic capacity of cells was analyzed on the basis of apoptotic cell proportion and caspase?3 activity. Then, the proportion of cells and correlations among phosphoglycerate kinase 1 (PGK1), NEAT1, and miR?638 were determined through RNA immunoprecipitation and luciferase assays and bioinformatics analysis. Moreover, the expression levels of Ki?67, proliferating cell nuclear antigen, PGK1, Bax, Bcl?2, (p)?mTOR, (p)?AKT, and ??catenin were analyzed via western blot analysis. In the serum of patients with AS and HAECs induced by oxidized low?density lipoprotein (ox?LDL), the expression level of miR?638 was decreased, whereas that of NEAT1 was increased. After ox?LDL therapy, NEAT1 knockdown suppressed HAEC proliferation and stimulated HAEC apoptosis, which could be reversed by the miR?638 inhibitor. NEAT1 inhibited miR?638 expression through direct mutual action. The following mechanical investigations revealed that PGK1 was a miR?638 target, whose expression was increased by NEAT1, a competing endogenous RNA of miR?638. Additionally, the miR?638 inhibitor contributed to proliferation and suppressed apoptosis through the activation of the AKT/mTOR signaling pathway in ox?LDL?induced HAECs. NEAT1 adjusted the AKT/mTOR signaling pathway via miR?638 in ox?LDL?induced HAECs to accelerate their proliferation and impede their apoptosis. This result revealed that NEAT1 may be valuable in the treatment of AS.

SUBMITTER: Zhang X

PROVIDER: S-EPMC7251762 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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NEAT1 knockdown suppresses endothelial cell proliferation and induces apoptosis by regulating miR‑638/AKT/mTOR signaling in atherosclerosis.

Zhang Xia X Guan Ming-Xiu MX Jiang Qiu-Hong QH Li Sai S Zhang Hong-Yu HY Wu Zhi-Guo ZG Cong Hong-Liang HL Qi Xiu-Hui XH

Oncology reports 20200507 1

Long non‑coding RNAs (lncRNAs) have been validated to mediate the development of atherosclerosis (AS). In the present study, the molecular mechanisms and functions of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in the advancement of human aortic endothelial cells (HAECs) were investigated. The levels of lncRNA‑NEAT1 and miR‑638 expression in clinical samples and cells were explored via quantitative reverse transcription polymerase chain reaction. Colony formation and CCK‑8 assays we ...[more]

PMID: 32377692

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Project description:Atherosclerosis is the leading cause of death from vascular diseases worldwide, and endothelial cell (EC) dysfunction is the key cause of atherosclerosis. miR-155 was found to induce endothelial injury and to trigger atherosclerosis. In addition, brain and muscle ARNT-like protein-1 (Bmal1) has been found to be closely related to EC function. Therefore, the present study aimed to explore the mechanism underlying the regulation of Bmal1 by miR-155 in the induction of EC apoptosis and inflammatory response in atherosclerosis. The atherosclerosis model in apolipoprotein E (ApoE)- / - mice was established. miR-155 and Bmal1 expression was quantified by RT-qPCR and western blot analysis, respectively. The role of miR-155 and Bmal1 in atherosclerosis was evaluated through changes in cardiac function, plaque area, cardiomyocyte apoptosis, and inflammatory factor levels in mice. Moreover, the regulatory relationship between them was identified by dual-luciferase reporter gene assay to explore the mechanism of action of miR-155. After the modeling, the expression of miR-155 was upregulated and Bmal1 was downregulated in aorta, and there was a significant linear correlation between them. Upregulation of miR-155 increased the atherosclerotic plaque area, cell apoptosis, total cholesterol (TC) and triglyceride (TG), as well as weakened aortic diastolic function. However, opposite changes occurred after downregulation of miR-155 or an increase in Bmal1. In addition, the microRNA.org website predicted that there were targeted binding sites between miR-155 and Bmal1, which was verified with a dual-luciferase reporter gene assay. miR-155 was able to inhibit the expression by targeting Bmal1. Moreover, a rescue experiment showed that Bmal1 hindered the promotion of miR-155 in regards to atherosclerosis. In conclusion, miR-155 induces EC apoptosis and inflammatory response, weakens aortic diastolic function, and promotes the progression of atherosclerosis through targeted inhibition of Bmal1.

Project description:BackgroundLipid droplets (LD), lipid-storing organelles containing neutral lipids like glycerolipids and cholesterol, are increasingly accepted as hallmarks of inflammation. The nuclear paraspeckle assembly transcript 1 (NEAT1), a long non-coding RNA with over 200 nucleotides, exerts an indispensable impact on regulating both LD agglomeration and autophagy in multiple neurological disorders. However, knowledge as to how NEAT1 modulates the formation of LD and associated signaling pathways is limited.MethodsIn this study, primary microglia were isolated from newborn mice and exposed to oxygen-glucose-deprivation/reoxygenation (OGD/R). To further explore NEAT1-dependent mechanisms, an antisense oligonucleotide (ASO) was adopted to silence NEAT1 under in vitro conditions. Studying NEAT1-dependent interactions with regard to autophagy and LD agglomeration under hypoxic conditions, the inhibitor and activator of autophagy 3-methyladenine (3-MA) and rapamycin (RAPA) were used, respectively. In a preclinical stroke model, mice received intraventricular injections of ASO NEAT1 or control vectors in order to yield NEAT1 knockdown. Analysis of readout parameters included qRT-PCR, immunofluorescence, western blot assays, and behavioral tests.ResultsMicroglia exposed to OGD/R displayed a temporal pattern of NEAT1 expression, peaking at four hours of hypoxia followed by six hours of reoxygenation. After effectively silencing NEAT1, LD formation and autophagy-related proteins were significantly repressed in hypoxic microglia. Stimulating autophagy in ASO NEAT1 microglia under OGD/R conditions by means of RAPA reversed the downregulation of LD agglomeration and perilipin 2 (PLIN2) expression. On the contrary, application of 3-MA promoted repression of both LD agglomeration and expression of the LD-associated protein PLIN2. Under in vivo conditions, NEAT1 was significantly increased in mice at 24 h post-stroke. Knockdown of NEAT1 significantly alleviated LD agglomeration and inhibited autophagy, resulting in improved cerebral perfusion, reduced brain injury and increased neurological recovery.ConclusionNEAT1 is a key player of LD agglomeration and autophagy stimulation, and NEAT1 knockdown provides a promising therapeutic value against stroke.

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NEAT1 knockdown suppresses endothelial cell proliferation and induces apoptosis by regulating miR?638/AKT/mTOR signaling in atherosclerosis.

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NEAT1 knockdown suppresses endothelial cell proliferation and induces apoptosis by regulating miR‑638/AKT/mTOR signaling in atherosclerosis.

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