Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Anhui Medical University, Hefei, China.
Department of Gynecology and Obstetrics, The First Affiliated Hospital of Anhui Medical University, Hefei, China.
J Gene Med. 2022 Apr;24(4):e3385. doi: 10.1002/jgm.3385. Epub 2022 Feb 9.
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening diseases and endothelial barrier injury is an important contributor to the pathogenesis of ALI/ARDS. Long non-coding (lncRNA) has been shown to participate in the progression of ALI/ARDS. The present study aimed to investigate the function of lncRNA opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) in lipopolysaccharide (LPS)-induced ALI/ARDS.
OIP5-AS1 and miR-223 levels were detected by a polymerase chain reaction in the serum of ALI/ARDS patients or healthy donors. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay was performed to detect the proliferation of human pulmonary microvascular endothelial cells (HPMECs). Flow cytometry were performed to detect the apoptosis of HPMECs. The protein levels of NLRP3, ASC, GSDMD-N and caspase-1 were measured by western blotting to detect the pyroptosis of HPMECs. Interleukin (IL)-1β, IL-6, IL-18 and IL-10 were detected by an enzyme-linked immunosorbent assay to measure the inflammatory response of HPMECs. The production of reactive oxygen species, superoxide dismutase and malondialdehyde was measured to determine the oxidative stress of HPMECs. Targets of OIP5-AS1 and miR-223 were predicted by StarBase and confirmed by a dual-luciferase reporter assay.
We found that OIP5-AS1 was up-regulated and miR-223 was down-regulated in the serum of ALI/ARDS patients and LPS-treated HPMECs. Functionally, knockdown of OIP5-AS1 induced proliferation and inhibited apoptosis, pyroptosis, inflammatory response and oxidative stress of LPS-treated HPMECs. Interestingly, miR-223 was a target of OIP5-AS1 and miR-223 inhibition abolished the effects of si-OIP5-AS1 on LPS-induced HPMECs. More importantly, miR-223 directly targeted NLRP3, and miR-223 overexpression promoted proliferation and inhibited apoptosis, pyroptosis, inflammatory response and oxidative stress of LPS-treated HPMECs, with this being abolished by NLRP3 overexpression. Finally, we found that OIP5-AS1 knockdown and miR-223 overexpression could both alleviate LPS-induced ALI/ARDS in vivo.
Taken together, we find that lncRNA OIP5-AS1 aggravates LPS-induced ALI/ARDS via miR-223/NLRP3 axis and provides new targets for ALI/ARDS therapy.
急性肺损伤（ALI）和急性呼吸窘迫综合征（ARDS）是危及生命的疾病，内皮屏障损伤是 ALI/ARDS 发病机制的重要因素。长链非编码（lncRNA）已被证明参与 ALI/ARDS 的进展。本研究旨在探讨 lncRNA opa 相互作用蛋白 5 反义 RNA 1（OIP5-AS1）在脂多糖（LPS）诱导的 ALI/ARDS 中的作用。
采用聚合酶链反应检测 ALI/ARDS 患者或健康供者血清中的 OIP5-AS1 和 miR-223 水平。采用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-2H-四唑溴盐法检测人肺微血管内皮细胞（HPMEC）的增殖。流式细胞术检测 HPMEC 的凋亡。采用 Western blot 法检测 NLRP3、ASC、GSDMD-N 和 caspase-1 蛋白水平，以检测 HPMEC 的焦亡。采用酶联免疫吸附试验检测白细胞介素（IL）-1β、IL-6、IL-18 和 IL-10，以测定 HPMEC 的炎症反应。测定活性氧、超氧化物歧化酶和丙二醛的产生，以确定 HPMEC 的氧化应激。通过 StarBase 预测 OIP5-AS1 和 miR-223 的靶标，并通过双荧光素酶报告基因检测进行验证。
我们发现，ALI/ARDS 患者血清和 LPS 处理的 HPMEC 中 OIP5-AS1 上调，miR-223 下调。功能上，敲低 OIP5-AS1 可诱导 LPS 处理的 HPMEC 增殖，并抑制其凋亡、焦亡、炎症反应和氧化应激。有趣的是，miR-223 是 OIP5-AS1 的靶标，而 miR-223 抑制可消除 si-OIP5-AS1 对 LPS 诱导的 HPMEC 的影响。更重要的是，miR-223 直接靶向 NLRP3，miR-223 过表达可促进 LPS 处理的 HPMEC 的增殖，并抑制其凋亡、焦亡、炎症反应和氧化应激，而过表达 NLRP3 则可消除这一作用。最后，我们发现，OIP5-AS1 敲低和 miR-223 过表达均可在体内减轻 LPS 诱导的 ALI/ARDS。
综上所述，我们发现 lncRNA OIP5-AS1 通过 miR-223/NLRP3 轴加重 LPS 诱导的 ALI/ARDS，并为 ALI/ARDS 治疗提供了新的靶点。
