Abstract
BackgroundAstrocyte activation promotes glutamate accumulation and secretion of inflammatory factors, mainly responsible for epilepsy. Long noncoding RNA (lncRNA) X-inactive-specific transcript (XIST) regulates inflammation; however, the biological role and regulatory mechanism of XIST during astrocyte activation remain unclear.MethodsIn the present study, rat epilepsy model and lipopolysaccharide (LPS)-treated CTX-TNA2 were established. XIST and miR-29c-3p expression were evaluated using quantitative real-time polymerase chain reaction. Nuclear factor of activated T cells 5 (NFAT5) was measured using western blot analysis. Interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and L-glutamate levels in the culture supernatants were assessed using enzyme-linked immunosorbent assay. The binding between XIST and miR-29c-3p and between miR-29c-3p and the 3′-UTR of NFAT5 was analyzed using dual-luciferase reporter, RNA-binding protein immunoprecipitation (RIP), and Biotin pull-down assay. The proliferation and apoptosis were evaluated using CCK8 and flow cytometry, respectively.ResultsXIST expression and NFAT5 protein level was increased, whereas miR-29c-3p expression was decreased in the epilepsy rat model and LPS-treated CTX-TNA2 cells. Silenced XIST expression, miR-29c-3p overexpression, or silenced NFAT5 expression inhibited the secretion of IL-1β, IL-6, and TNF-α and promoted glutamate transport in LPS-treated CTX-TNA2 cells. miR-29c-3p was the potential miRNA sponged by XIST. NFAT5 acted as a direct binding target of miR-29c-3p. Silenced miR-29c-3p expression or NFAT5 overexpression reversed the effect of silenced XIST expression on LPS-treated CTX-TNA2.XIST and miR-29c-3p treatment does not affect NFAT5 mRNA expression, but affects NFAT5 protein level. Furthermore, underexpressed XIST or overexpressed miR-29c-3p in LPS-stimulated CTX-TNA2 can attenuate neuronal apoptosis induced by LPS-stimulated CTX-TNA2.ConclusionLncRNA XIST promotes the secretion of inflammatory cytokines in LPS- treated CTX-TNA2 via sponging miR-29c-3p and regulating NFAT5 expression.
Highlights
Epilepsy is a common chronic neurological disorder occurring worldwide, which poses patients insomnia, depression, and memory and mood impairments and the economic burden on patients, their families, and the society [1]
Silenced Xinactive-specific transcript (XIST) expression, miR-29c-3p overexpression, or silenced Nuclear factor of activated T cells 5 (NFAT5) expression inhibited the secretion of IL-1b, IL-6, and tumor necrosis factor- a (TNF-a) and promoted glutamate transport in LPS-treated CTX-TNA2 cells. miR-29c-3p was the potential miRNA sponged by XIST
LncRNA XIST promotes the secretion of inflammatory cytokines in LPStreated CTX-TNA2 via sponging miR-29c-3p and regulating NFAT5 expression
Summary
Epilepsy is a common chronic neurological disorder occurring worldwide, which poses patients insomnia, depression, and memory and mood impairments and the economic burden on patients, their families, and the society [1]. Astrocytes are the resident innate immune cells in the brain that provide trophic support to neurons, regulate glutamate transport and phagocytosis connectivity of neural circuits, control blood flow, and maintain brain homeostasis and blood–brain barrier [5] They are one of the crucial glial cell types that produce numerous proinflammatory cytokines in the central nervous system during brain inflammation induced by central nervous system injury and disease [6]. Activated astrocytes lose their functions, such as providing trophic support to neurons and maintaining brain homeostasis and blood–brain barrier [5]. Long noncoding RNA (lncRNA) Xinactive-specific transcript (XIST) regulates inflammation; the biological role and regulatory mechanism of XIST during astrocyte activation remain unclear
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