lncRNA EGOT靶向miR-320a对LPS诱导肺泡上皮细胞炎症反应和细胞凋亡的影响

吴云飞; 钟海; 张智慧; 梁鸿章; 陈旭源; 李想; 吴旭

doi:10.13898/j.cnki.issn.1000-2200.2021.10.001

lncRNA EGOT靶向miR-320a对LPS诱导肺泡上皮细胞炎症反应和细胞凋亡的影响

Effect of lncRNA EGOT on LPS-induced inflammation and apoptosis of alveolar epithelial cells by targeting miR-320a

摘要

摘要:
目的探讨长链非编码RNA (lncRNA)嗜酸性粒细胞转录因子(EGOT)对脂多糖(LPS)诱导的肺泡上皮细胞A549凋亡、炎症反应的影响及可能机制。
方法将A549细胞分为对照组(Con)、LPS组、LPS+pcDNA组、LPS+pcDNA-EGOT组、LPS+anti-miR-NC组、LPS+anti-miR-320a组、LPS+pcDNA-EGOT+miR-NC组、LPS+pcDNA-EGOT+miR-320a组。实时荧光定量PCR (RT-qPCR)检测EGOT和miR-320a表达水平；流式细胞术检测细胞凋亡；试剂盒检测白细胞介素(IL)-6、IL-1β水平。双荧光素酶报告基因实验和RT-qPCR确定EGOT和miR-320a之间靶向作用。
结果与Con组比较，LPS组A549细胞凋亡率、IL-6和IL-1β水平均升高(P < 0.05)，EGOT表达水平降低(P < 0.01)，miR-320a表达水平明显升高(P < 0.01)。与LPS+pcDNA组比较，LPS+pcDNA-EGOT组A549细胞凋亡率、Bax蛋白表达、IL-6和IL-1β水平均降低(P < 0.05)，Bcl-2蛋白表达升高(P < 0.05)。与LPS+anti-miR-NC组比较，LPS+anti-miR-320a组A549细胞凋亡率、Bax蛋白表达、IL-6和IL-1β水平均明显降低(P < 0.01)，Bcl-2蛋白表达明显升高(P < 0.01)。与LPS+pcDNA-EGOT+miR-NC组比较，LPS+pcDNA-EGOT+miR-320a组A549细胞凋亡率、Bax蛋白表达、IL-6和IL-1β水平均明显升高(P < 0.01)，Bcl-2蛋白表达明显降低(P < 0.01)。双荧光素酶报告实验显示，EGOT靶向负性调控miR-320a表达。
结论lncRNA EGOT通过靶向miR-320a可减轻LPS诱导肺泡上皮细胞炎症反应和细胞凋亡。

Abstract:
ObjectiveTo explore the effect of long-chain non-coding RNA eosinophilic transcription factor (EGOT) on lipopolysaccharide (LPS) -induced apoptosis and inflammation of alveolar epithelial A549 cells and its possible mechanism.
MethodsA549 cells were divided into control (Con) group, LPS group, LPS+pcDNA group, LPS+pcDNA-EGOT group, LPS+anti-miR-NC group, LPS+anti-miR-320a group, LPS+pcDNA-EGOT+miR-NC group, and LPS+pcDNA-EGOT+miR-320a group.Real-time quantitative PCR (RT-qPCR) was used to detect the expression levels of EGOT and miR-320a, flow cytometry was applied to detect apoptosis, and the kit was used to detect the levels of interleukin (IL) -6 and IL-1β.Dual luciferase reporter gene assay and RT-qPCR was employed to confirm the targeting relationship between EGOT and miR-320a.
ResultsCompared with the Con group, the apoptosis rate, IL-6 and IL-1β levels of A549 cells in the LPS group were increased(P < 0.01), the EGOT expression level was reduced(P < 0.05), and the miR-320a expression level was significantly increased (P < 0.01).Compared with the LPS+pcDNA group, the apoptosis rate, Bax protein expression, IL-6 and IL-1β levels of A549 cells in the LPS+pcDNA-EGOT group were reduced(P < 0.05), and the Bcl-2 protein expression were increased(P < 0.05).Compared with the LPS+anti-miR-NC group, the apoptosis rate, Bax protein expression, IL-6 and IL-1β levels of A549 cells in the LPS+anti-miR-320a group were significantly reduced(P < 0.01), and the Bcl-2 protein expression were significantly increased(P < 0.01).Compared with the LPS+pcDNA-EGOT+miR-NC group, the apoptosis rate, Bax protein expression, IL-6 and IL-1β levels of A549 cells in the LPS+pcDNA-EGOT+miR-320a group were significantly increased (P < 0.01), and the Bcl-2 protein expression were significantly reduced (P < 0.01).The results of dual luciferase reporter gene assay showed that EGOT targeted and negatively regulated the miR-320a expression.
ConclusionsLncRNA EGOT can alleviate LPS-induced inflammation and apoptosis of alveolar epithelial cells by targeting miR-320a.

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