Abstract：
BACKGROUND: Infantile pneumonia (IP) is a common inflammatory disease, which brings a heavy burden to young children\'s health. Previous studies suggested that circular RNA (circRNA) hsa_circ_0026579 (also called circESPL1) was significantly upregulated in pneumonia patients, which was associated with the disease severity. This subject aimed to explore the functional effects and potential regulatory mechanism of circESPL1 on lipopolysaccharide (LPS)-induced lung cell injury.
METHODS: WI-38 and MRC-5 cells were stimulated by LPS to mimic the inflammatory injury model. CircESPL1, microRNA-326 (miR-326), and Mitogen-Activated Protein Kinase 14 (MAPK14)levels were measured using real-time quantitative polymerase chain reaction (RT-qPCR). Cell Counting Kit-8 (CCK-8), 5-ethynyl-2\'-deoxyuridine (EdU), and flow cytometry assays were performed to assess cell proliferation and apoptosis. Western blot analysis of B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), C-caspase 3, and MAPK14 protein levels. Tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), and IL-1β levels were examined using an Enzyme-linked immunosorbent assay (ELISA). Using Starbase analysis, the binding between miR-326 and circESPL1 or MAPK14 was predicted, followed by confirmation using a dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays.
RESULTS: Increased circESPL1 and MAPK14, and reduced miR-326 were observed in serum samples from preeclampsia sufferers and LPS-treated lung cells (P < 0.05). Furthermore, circESPL1 deficiency overturned LPS-mediated cell proliferation, apoptosis, and inflammatory response in vitro (P < 0.05). In terms of molecular mechanisms, circESPL1 worked as a sponge of miR-326, and miR-326 absence reversed the protective role of circESPL1 silencing on LPS-triggered lung cell injury (P < 0.05). Also, miR-326 directly targeted MAPK14, and MAPK14 overexpression abolished miR-326-mediated impacts under LPS treatment (P < 0.05).
CONCLUSIONS: CircESPL1 knockdown might attenuate LPS-caused lung cell injury by regulating the miR-326/ MAPK14 axis, providing useful insight for exploring a novel therapeutic approach for IP.
METHODS: WI-38 and MRC-5 cells were stimulated by LPS to mimic the inflammatory injury model. CircESPL1, microRNA-326 (miR-326), and Mitogen-Activated Protein Kinase 14 (MAPK14)levels were measured using real-time quantitative polymerase chain reaction (RT-qPCR). Cell Counting Kit-8 (CCK-8), 5-ethynyl-2\'-deoxyuridine (EdU), and flow cytometry assays were performed to assess cell proliferation and apoptosis. Western blot analysis of B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), C-caspase 3, and MAPK14 protein levels. Tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), and IL-1β levels were examined using an Enzyme-linked immunosorbent assay (ELISA). Using Starbase analysis, the binding between miR-326 and circESPL1 or MAPK14 was predicted, followed by confirmation using a dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays.
RESULTS: Increased circESPL1 and MAPK14, and reduced miR-326 were observed in serum samples from preeclampsia sufferers and LPS-treated lung cells (P < 0.05). Furthermore, circESPL1 deficiency overturned LPS-mediated cell proliferation, apoptosis, and inflammatory response in vitro (P < 0.05). In terms of molecular mechanisms, circESPL1 worked as a sponge of miR-326, and miR-326 absence reversed the protective role of circESPL1 silencing on LPS-triggered lung cell injury (P < 0.05). Also, miR-326 directly targeted MAPK14, and MAPK14 overexpression abolished miR-326-mediated impacts under LPS treatment (P < 0.05).
CONCLUSIONS: CircESPL1 knockdown might attenuate LPS-caused lung cell injury by regulating the miR-326/ MAPK14 axis, providing useful insight for exploring a novel therapeutic approach for IP.
摘要：
背景：婴儿肺炎（IP）是一种常见的炎症性疾病，这给幼儿的健康带来了沉重的负担。以前的研究表明，环状RNA（circRNA）hsa_circ_0026579（也称为circESPL1）在肺炎患者中显著上调，这与疾病的严重程度有关。本课题旨在探讨circESPL1对脂多糖(LPS)诱导的肺细胞损伤的功能影响及潜在调控机制。
方法：用LPS刺激WI-38和MRC-5细胞，模拟炎症损伤模型。CircESPL1,microRNA-326(miR-326),使用实时定量聚合酶链反应(RT-qPCR)测量丝裂原活化蛋白激酶14(MAPK14)水平。细胞计数套件-8（CCK-8），5-乙炔基-2'-脱氧尿苷（EdU），并进行流式细胞术测定以评估细胞增殖和凋亡。B细胞淋巴瘤-2(Bcl-2)的Westernblot分析,Bcl-2相关X蛋白(Bax),C-caspase3和MAPK14蛋白水平。肿瘤坏死因子-α(TNF-α),白细胞介素-6(IL-6),使用酶联免疫吸附测定(ELISA)检查IL-1β水平。使用Starbase分析，预测了miR-326与circESPL1或MAPK14之间的结合，然后使用双荧光素酶报告基因和RNA免疫沉淀(RIP)测定进行确认。
结果：在先兆子痫患者的血清样本和LPS处理的肺细胞中观察到circESPL1和MAPK14增加，miR-326减少（P<0.05）。此外，circESPL1缺乏推翻了LPS介导的细胞增殖，凋亡，和体外炎症反应(P<0.05)。从分子机制来看,circESPL1作为miR-326的海绵，miR-326缺失逆转了circESPL1沉默对LPS触发的肺细胞损伤的保护作用（P<0.05）。此外，miR-326直接靶向MAPK14，MAPK14过表达消除了LPS治疗下miR-326介导的影响（P<0.05）。
结论：CircESPL1敲低可能通过调节miR-326/MAPK14轴减轻LPS引起的肺细胞损伤，为探索新的IP治疗方法提供有用的见解。
方法：用LPS刺激WI-38和MRC-5细胞，模拟炎症损伤模型。CircESPL1,microRNA-326(miR-326),使用实时定量聚合酶链反应(RT-qPCR)测量丝裂原活化蛋白激酶14(MAPK14)水平。细胞计数套件-8（CCK-8），5-乙炔基-2'-脱氧尿苷（EdU），并进行流式细胞术测定以评估细胞增殖和凋亡。B细胞淋巴瘤-2(Bcl-2)的Westernblot分析,Bcl-2相关X蛋白(Bax),C-caspase3和MAPK14蛋白水平。肿瘤坏死因子-α(TNF-α),白细胞介素-6(IL-6),使用酶联免疫吸附测定(ELISA)检查IL-1β水平。使用Starbase分析，预测了miR-326与circESPL1或MAPK14之间的结合，然后使用双荧光素酶报告基因和RNA免疫沉淀(RIP)测定进行确认。
结果：在先兆子痫患者的血清样本和LPS处理的肺细胞中观察到circESPL1和MAPK14增加，miR-326减少（P<0.05）。此外，circESPL1缺乏推翻了LPS介导的细胞增殖，凋亡，和体外炎症反应(P<0.05)。从分子机制来看,circESPL1作为miR-326的海绵，miR-326缺失逆转了circESPL1沉默对LPS触发的肺细胞损伤的保护作用（P<0.05）。此外，miR-326直接靶向MAPK14，MAPK14过表达消除了LPS治疗下miR-326介导的影响（P<0.05）。
结论：CircESPL1敲低可能通过调节miR-326/MAPK14轴减轻LPS引起的肺细胞损伤，为探索新的IP治疗方法提供有用的见解。
