Department of Neurology, Xiangya Hospital, Central South University, Changsha, China.
Front Endocrinol (Lausanne). 2021 Mar 12;12:573143. doi: 10.3389/fendo.2021.573143. eCollection 2021.
Astrocyte 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.
In 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.
XIST 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.
LncRNA XIST promotes the secretion of inflammatory cytokines in LPS- treated CTX-TNA2 sponging miR-29c-3p and regulating NFAT5 expression.
星形胶质细胞的激活会促进谷氨酸的积累和炎症因子的分泌，这主要负责癫痫的发生。长链非编码 RNA（lncRNA）X 染色体失活特异性转录物（XIST）调节炎症；然而，XIST 在星形胶质细胞激活过程中的生物学作用和调控机制尚不清楚。
本研究建立了大鼠癫痫模型和脂多糖（LPS）处理的 CTX-TNA2。使用实时定量聚合酶链反应评估 XIST 和 miR-29c-3p 的表达。使用 Western blot 分析测定核因子活化 T 细胞 5（NFAT5）。使用酶联免疫吸附试验评估培养上清液中白细胞介素（IL）-1β、IL-6、肿瘤坏死因子-α和 L-谷氨酸的水平。使用双荧光素酶报告、RNA 结合蛋白免疫沉淀（RIP）和生物素下拉实验分析 XIST 与 miR-29c-3p 以及 miR-29c-3p 与 NFAT5 的 3'UTR 之间的结合。使用 CCK8 和流式细胞术分别评估细胞增殖和细胞凋亡。
在癫痫大鼠模型和 LPS 处理的 CTX-TNA2 细胞中，XIST 表达和 NFAT5 蛋白水平升高，而 miR-29c-3p 表达降低。沉默 XIST 表达、过表达 miR-29c-3p 或沉默 NFAT5 表达抑制 LPS 处理的 CTX-TNA2 细胞中 IL-1β、IL-6 和 TNF-α的分泌，并促进谷氨酸转运。miR-29c-3p 是 XIST 的潜在 miRNA 海绵。NFAT5 是 miR-29c-3p 的直接结合靶标。沉默 miR-29c-3p 表达或过表达 NFAT5 可逆转沉默 XIST 表达对 LPS 处理的 CTX-TNA2 的影响。XIST 和 miR-29c-3p 处理不影响 NFAT5 mRNA 表达，但影响 NFAT5 蛋白水平。此外，在 LPS 刺激的 CTX-TNA2 中下调 XIST 或过表达 miR-29c-3p 可减轻 LPS 刺激的 CTX-TNA2 诱导的神经元凋亡。
lncRNA XIST 通过海绵 miR-29c-3p 促进 LPS 处理的 CTX-TNA2 中炎症因子的分泌，并调节 NFAT5 的表达。
