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.
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。