Department of Anesthesiology, The Affiliated Cancer Hospital of Nanjing Medical University, Nanjing City, China.
Department of Thoracic Surgery, The Affiliated Cancer Hospital of Nanjing Medical University, Nanjing City, China.
Environ Toxicol. 2024 Jun;39(6):3578-3596. doi: 10.1002/tox.24201. Epub 2024 Mar 15.
Endothelial glycocalyx (EG) maintains vascular homeostasis and is destroyed after one-lung ventilation (OLV)-induced lung injury. Long noncoding RNAs (lncRNAs) are critically involved in various lung injuries. This study aimed to investigate the role and regulatory mechanism of KCNQ1 overlapping transcript 1 (KCNQ1OT1) in OLV-induced lung injury and LPS-induced type II alveolar epithelial cell (AECII) apoptosis.
The rat OLV model was established, and the effects of KCNQ1OT1 on OLV-induced ALI in vivo were explored. Bax and Caspase-3 expression in rat lung tissues was measured by immunochemistry (IHC). AECIIs were isolated from rat lungs and treated with LPS or normal saline (control) for in vitro analysis. The expression of KCNQ1OT1, miR-129-5p, and HMGB1 was measured by quantitative real-time PCR (qRT-PCR) or Western blot (WB). Cell proliferation and apoptosis were examined by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) and flow cytometry. The downstream targets of KCNQ1OT1 were predicted by bioinformatics, and the binding relationship between KCNQ1OT1 and miR-129-3p was verified by dual-luciferase reporter assays. The potential target of miR-129-5p was further explored on the Targetscan website and revealed to target HMGB1. Enzyme-linked immunosorbent assay (ELISA) or WB was adopted to determine the levels of IL-1β, TNF-α, MDA, SOD, heparanase (HPA), matrix metalloproteinase 9 (MMP9), heparan sulfate (HS) and syndecan-1 (SDC-1).
KCNQ1OT1 and HMGB1 were up-regulated during OLV-induced lung injury, and their expression was positively correlated. KCNQ1OT1 knockdown reduced OLV-induced pulmonary edema and lung epithelial cell apoptosis, increased vascular permeability, reduced IL-1β, TNF-α, MDA, and SOD levels and glycocalyx markers by targeting miR-129-5p or upregulating HMGB1. Overexpressing KCNQ1OT1 promoted cell apoptosis, reduced cell proliferation, aggravated inflammation and oxidative stress, and up-regulated HMGB1, HPA and MMP9 in LPS-treated AECIIs, while the HMGB1 silencing showed the opposite effects. MiR-129-5p mimics partially eliminated the KCNQ1OT1-induced effects, while recombinant HMGB1 restored the effects of miR-129-5p overexpression on AECIIs. Additionally, KCNQ1OT1 was demonstrated to promote the activation of the p38 MAPK/Akt/ERK signaling pathways in AECIIs via HMGB1.
KCNQ1OT1 knockdown alleviated AECII apoptosis and EG damage during OLV by targeting miR-129-5p/HMGB1 to inactivate the p38 MAPK/Akt/ERK signaling. The findings of our study might deepen our understanding of the molecular basis in OLV-induced lung injury and provide clues for the targeted disease management.
内皮糖萼（EG）维持血管内稳态，在单肺通气（OLV）引起的肺损伤后被破坏。长非编码 RNA（lncRNA）在各种肺损伤中起着至关重要的作用。本研究旨在探讨 KCNQ1 重叠转录本 1（KCNQ1OT1）在 OLV 诱导的肺损伤和 LPS 诱导的 II 型肺泡上皮细胞（AECII）凋亡中的作用及其调控机制。
建立大鼠 OLV 模型，探讨 KCNQ1OT1 对体内 OLV 诱导的 ALI 的影响。采用免疫组织化学（IHC）测定大鼠肺组织中 Bax 和 Caspase-3 的表达。分离大鼠肺中的 AECII，并用 LPS 或生理盐水（对照）进行体外处理。采用实时定量 PCR（qRT-PCR）或 Western blot（WB）检测 KCNQ1OT1、miR-129-5p 和 HMGB1 的表达。通过 3-（4,5）-二甲基噻唑（-z-y1）-3,5-二苯基四唑溴盐（MTT）和流式细胞术检测细胞增殖和凋亡。通过生物信息学预测 KCNQ1OT1 的下游靶点，并通过双荧光素酶报告基因实验验证 KCNQ1OT1 与 miR-129-3p 的结合关系。进一步在 Targetscan 网站上探索 miR-129-5p 的潜在靶标，并揭示其靶向 HMGB1。采用酶联免疫吸附试验（ELISA）或 WB 测定 IL-1β、TNF-α、MDA、SOD、肝素酶（HPA）、基质金属蛋白酶 9（MMP9）、肝素硫酸（HS）和 syndecan-1（SDC-1）的水平。
KCNQ1OT1 和 HMGB1 在 OLV 诱导的肺损伤过程中上调，且表达呈正相关。KCNQ1OT1 敲低可减少 OLV 诱导的肺水肿和肺上皮细胞凋亡，增加血管通透性，降低 IL-1β、TNF-α、MDA 和 SOD 水平以及糖萼标志物，其机制是通过靶向 miR-129-5p 或上调 HMGB1。过表达 KCNQ1OT1 可促进 AECII 凋亡，减少细胞增殖，加重炎症和氧化应激，并上调 LPS 处理的 AECII 中的 HMGB1、HPA 和 MMP9，而 HMGB1 沉默则显示出相反的效果。miR-129-5p 模拟物部分消除了 KCNQ1OT1 诱导的作用，而重组 HMGB1 恢复了 miR-129-5p 过表达对 AECII 的作用。此外，KCNQ1OT1 通过 HMGB1 促进 AECII 中 p38 MAPK/Akt/ERK 信号通路的激活。
KCNQ1OT1 敲低通过靶向 miR-129-5p/HMGB1 使 p38 MAPK/Akt/ERK 信号失活，减轻 OLV 时 AECII 凋亡和 EG 损伤。本研究结果可能加深我们对 OLV 诱导的肺损伤分子基础的理解，并为靶向疾病管理提供线索。
