Zhang Bihong, You Ting, Liu Yu, Li Pei
Department of Emergency, The First Affiliated Hospital, Hengyang Medical School, University of South China, Hengyang, China.
Department of Clinical Laboratory, The First Affiliated Hospital, Hengyang Medical School, University of South China, Hengyang, China.
Shock. 2023 Mar 1;59(3):505-513. doi: 10.1097/SHK.0000000000002077. Epub 2023 Jan 4.
Background: Septic acute kidney injury (AKI) is a serious complication of sepsis, which greatly threatened the life safety of critically ill patients. Recently, circular RNA is considered to be implicated in sepsis-induced renal cell damage. However, the role of circ_0114428 in sepsis AKI is still unclear. Methods: LPS was used to establish a sepsis-related AKI cell model. The expression of circ_0114428, microRNA (miR)-370-3p, tissue inhibitor of metalloproteinase-2 (TIMP2), Proliferating cell nuclear antigen, Bax, and Bcl-2 were detected by quantitative real-time polymerase chain reaction and Western blot. Cell counting kit 8 and enzyme-linked immunosorbent assay were used to measure cell proliferation ability and the secretion of inflammatory factors (tumor necrosis factor α, interleukin 1β, and interleukin 6), respectively. Cell cycle and apoptosis rate were analyzed by flow cytometry. Caspase-3 assay kits were used to detect Caspase-3 activity. Interaction between miR-370-3p and circ_0114428 or TIMP2 was analyzed by bioinformatics analysis, a dual-luciferase reporter assay, and RNA immunoprecipitation assay. Results: Circ_0114428 was upregulated in septic AKI serum samples and LPS-induced HK2 cells. The knockdown of circ_0114428 notably promoted cell proliferation and cycle, whereas it restrained cell inflammation and apoptosis in LPS-stimulated HK2 cells. Subsequent mechanism analysis revealed that miR-370-3p was a target of circ_0114428, and miR-370-3p inhibition could rescue the effects of circ_0114428 downregulation on LPS-induced cell injury. Meanwhile, TIMP2 was a target gene of miR-370-3p. miR-370-3p mimic could attenuate LPS-induced cell injury, whereas these impacts were overturned by overexpressed TIMP2. Furthermore, circ_0114428 enhanced TIMP2 protein expression by sponging miR-370-3p. Conclusion: Our data demonstrated that circ_0114428 contributed to septic AKI progression by regulating miR-370-3p-mediated TIMP2 expression, which provided a promising target for septic AKI treatment.
脓毒症急性肾损伤（AKI）是脓毒症的一种严重并发症，极大地威胁着危重症患者的生命安全。近年来，环状RNA被认为与脓毒症诱导的肾细胞损伤有关。然而，circ_0114428在脓毒症AKI中的作用仍不清楚。方法：采用脂多糖（LPS）建立脓毒症相关AKI细胞模型。通过定量实时聚合酶链反应和蛋白质免疫印迹法检测circ_0114428、微小RNA（miR）-370-3p、金属蛋白酶组织抑制剂-2（TIMP2）、增殖细胞核抗原、Bax和Bcl-2的表达。分别使用细胞计数试剂盒8和酶联免疫吸附测定法检测细胞增殖能力和炎性因子（肿瘤坏死因子α、白细胞介素1β和白细胞介素6）的分泌。通过流式细胞术分析细胞周期和凋亡率。使用Caspase-3检测试剂盒检测Caspase-3活性。通过生物信息学分析、双荧光素酶报告基因测定法和RNA免疫沉淀测定法分析miR-370-3p与circ_0114428或TIMP2之间的相互作用。结果：circ_0114428在脓毒症AKI血清样本和LPS诱导的HK2细胞中上调。敲低circ_0114428显著促进细胞增殖和细胞周期进展，而抑制LPS刺激的HK2细胞中的细胞炎症和凋亡。随后的机制分析表明，miR-370-3p是circ_0114428的靶标，抑制miR-370-3p可挽救circ_0114428下调对LPS诱导的细胞损伤的影响。同时，TIMP2是miR-370-3p的靶基因。miR-370-3p模拟物可减轻LPS诱导的细胞损伤，而这些影响被过表达的TIMP2逆转。此外，circ_0114428通过海绵吸附miR-370-3p增强TIMP2蛋白表达。结论：我们的数据表明，circ_0114428通过调节miR-370-3p介导的TIMP2表达促进脓毒症AKI进展，这为脓毒症AKI的治疗提供了一个有前景的靶点。
