LINC00987 通过 Let-7b-5p/SIRT1 轴调控 LPS 诱导的细胞凋亡、氧化应激、炎症和自噬来改善 COPD。

LINC00987 Ameliorates COPD by Regulating LPS-Induced Cell Apoptosis, Oxidative Stress, Inflammation and Autophagy Through Let-7b-5p/SIRT1 Axis.

机构信息

Graduate School, Anhui University of Chinese Medicine, Anhui, Hefei 230012, People's Republic of China.

Department of Respiratory Medicine, The First Affiliated Hospital of Anhui University of Chinese Medicine, Anhui, Hefei, 230031, People's Republic of China.

出版信息

Int J Chron Obstruct Pulmon Dis. 2020 Dec 4;15:3213-3225. doi: 10.2147/COPD.S276429. eCollection 2020.

BACKGROUND

Chronic obstructive pulmonary disease (COPD) is the third cause of disease-related death and brings a heavy burden to human health. Long non-coding RNA (lncRNA) was revealed to participate in COPD pathogenesis. This study aims to establish the effects and regulatory mechanism of lncRNA long intergenic non-coding 00987 (LINC00987) in lipopolysaccharide (LPS)-induced apoptosis, oxidative stress, inflammation and autophagy in BEAS-2B cells.

METHODS

The expression levels of LINC00987 and let-7b-5p were detected by real-time quantitativepolymerase chain reaction (RT-qPCR). The expression of apoptosis-associated proteins, oxidative stress (ROS)-related proteins, autophagy-related proteins and sirtuin1 (SIRT1) protein was determined by Western blot. Cell viability was illustrated by cell counting kit-8 (CCK-8) assay. Cell apoptosis was investigated by caspase3 activity and apoptosis analysis assays. ROS, inflammation and autophagy were demonstrated by detecting reactive ROS level and superoxide dismutase (SOD) activity, enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, respectively. The binding sites between let-7b-5p and LINC00987 or SIRT1 were predicted by lncBase or miRWalk online database, and identified by dual-luciferase reporter assay.

RESULTS

LINC00987 expression was strikingly downregulated and let-7b-5p expression was obviously upregulated in COPD tissues and LPS-induced BEAS-2B cells compared with control groups. LINC00987 overexpression promoted BEAS-2B cells against LPS-mediated viability, apoptosis, oxidative stress, inflammation and autophagy, whereas these effects were attenuated by let-7b-5p mimic or SIRT1 knockdown. Furthermore, LINC00987 sponged let-7b-5p and let-7b-5p bound to SIRT1.

CONCLUSION

LINC00987 ameliorated COPD through modulating LPS-induced cell apoptosis, oxidative stress, inflammation and autophagy via sponging let-7b-5p to associate with SIRT1. This finding will provide a theoretical basis for the research of LncRNA-mediated treatment in COPD.

背景

慢性阻塞性肺疾病（COPD）是疾病相关死亡的第三大原因，给人类健康带来了沉重的负担。长链非编码 RNA（lncRNA）被揭示参与了 COPD 的发病机制。本研究旨在探讨长链非编码 RNA 长基因间非编码 00987（LINC00987）在脂多糖（LPS）诱导的 BEAS-2B 细胞凋亡、氧化应激、炎症和自噬中的作用及其调控机制。

方法

采用实时定量聚合酶链反应（RT-qPCR）检测 LINC00987 和 let-7b-5p 的表达水平。采用 Western blot 检测凋亡相关蛋白、氧化应激（ROS）相关蛋白、自噬相关蛋白和 SIRT1 蛋白的表达。通过细胞计数试剂盒-8（CCK-8）检测细胞活力。通过 caspase3 活性和凋亡分析检测细胞凋亡。通过检测活性 ROS 水平和超氧化物歧化酶（SOD）活性、酶联免疫吸附试验（ELISA）和 Western blot 分析分别检测 ROS、炎症和自噬。通过 lncBase 或 miRWalk 在线数据库预测 let-7b-5p 与 LINC00987 或 SIRT1 的结合位点，并通过双荧光素酶报告基因实验进行验证。

结果

与对照组相比，COPD 组织和 LPS 诱导的 BEAS-2B 细胞中 LINC00987 表达明显下调，let-7b-5p 表达明显上调。LINC00987 过表达促进了 BEAS-2B 细胞对 LPS 介导的活力、凋亡、氧化应激、炎症和自噬的抵抗，而 let-7b-5p 模拟物或 SIRT1 敲低则减弱了这些作用。此外，LINC00987 海绵吸附 let-7b-5p，let-7b-5p 结合 SIRT1。