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Circ_0091702 通过上调 THBS2 作为 miR-545-3p 的海绵减轻脓毒症相关急性肾损伤。

Circ_0091702 serves as a sponge of miR-545-3p to attenuate sepsis-related acute kidney injury by upregulating THBS2.

机构信息

Department of Emergency Medicine, People's Hospital of Tiantai County, Taizhou, Zhejiang, China.

Department of Emergency Medicine, Taizhou First People's Hospital, No. 218 Hengjie Road, Huangyan District, Taizhou, 318020, Zhejiang, China.

出版信息

J Mol Histol. 2021 Aug;52(4):717-728. doi: 10.1007/s10735-021-09991-z. Epub 2021 Jun 8.

Abstract

Circular RNA (circRNA) has been shown to play an important function in the progression of human diseases, including sepsis with acute kidney injury (AKI). However, the role and mechanism of circ_0091702 in sepsis-induced AKI have yet to be confirmed. Lipopolysaccharide (LPS) was used to induce HK2 cells to construct AKI cell models in vitro. Quantitative real-time PCR was used to measure the expression of circ_0091702, inflammatory cytokines, microRNA (miR)-545-3p and thrombospondin 2 (THBS2). Cell counting kit 8 assay and flow cytometry were used to assess cell viability and apoptosis. Besides, the protein levels of apoptosis markers and THBS2 were evaluated by western blot analysis. In addition, the concentrations of inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA). Cell oxidative stress was determined by detecting the contents of oxidative stress markers. Dual-luciferase reporter assay and RIP assay were used to confirm the relationship between miR-545-3p and circ_0091702 or miR-545-3p and THBS2. Circ_0091702 was downregulated in septic AKI patients and LPS-induced HK2 cells. Circ_0091702 could attenuate LPS-induced HK2 cell injury, while its silencing had an opposite effect. In the terms of mechanism, circ_0091702 could act as a sponge of miR-545-3p, and miR-545-3p could directly target THBS2. Functional experiments revealed that miR-545-3p could reverse the alleviating effect of circ_0091702 on LPS-induced HK2 cell injury, and THBS2 knockdown also could overturn the suppressing effect of miR-545-3p inhibitor on LPS-induced HK2 cell injury. Additionally, we also suggested that circ_0091702 could sponge miR-545-3p to regulate THBS2 expression. In conclusion, our results showed that circ_0091702 could suppress LPS-induced HK2 cell injury via the miR-545-3p/THBS2 axis, indicating that circ_0091702 might be an important biomarker for relieving sepsis-related AKI.

摘要

环状 RNA(circRNA)已被证明在包括伴有急性肾损伤(AKI)的脓毒症在内的人类疾病的进展中发挥重要作用。然而,circ_0091702 在脓毒症诱导的 AKI 中的作用和机制尚未得到证实。脂多糖(LPS)用于体外诱导 HK2 细胞构建 AKI 细胞模型。采用实时定量 PCR 检测 circ_0091702、炎症细胞因子、微小 RNA(miR)-545-3p 和血小板反应蛋白 2(THBS2)的表达。细胞计数试剂盒 8 检测和流式细胞术用于评估细胞活力和凋亡。此外,通过 Western blot 分析评估凋亡标志物和 THBS2 的蛋白水平。此外,通过酶联免疫吸附测定(ELISA)检测炎症细胞因子的浓度。通过检测氧化应激标志物的含量来确定细胞氧化应激。双荧光素酶报告基因检测和 RIP 检测用于证实 miR-545-3p 与 circ_0091702 或 miR-545-3p 与 THBS2 之间的关系。在脓毒症 AKI 患者和 LPS 诱导的 HK2 细胞中下调 circ_0091702。Circ_0091702 可减轻 LPS 诱导的 HK2 细胞损伤,而其沉默则产生相反的效果。就机制而言,circ_0091702 可以作为 miR-545-3p 的海绵,miR-545-3p 可以直接靶向 THBS2。功能实验表明,miR-545-3p 可以逆转 circ_0091702 对 LPS 诱导的 HK2 细胞损伤的缓解作用,而 THBS2 敲低也可以推翻 miR-545-3p 抑制剂对 LPS 诱导的 HK2 细胞损伤的抑制作用。此外,我们还表明 circ_0091702 可以通过海绵 miR-545-3p 来调节 THBS2 表达。总之,我们的结果表明,circ_0091702 可以通过 miR-545-3p/THBS2 轴抑制 LPS 诱导的 HK2 细胞损伤,表明 circ_0091702 可能是缓解脓毒症相关 AKI 的重要生物标志物。

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