Emergency Department, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, No.600 Yishan Road, Shanghai, 200233, People's Republic of China.
Department of Trauma-Emergency & Critical Care Medicine, Shanghai Fifth People's Hospital, Fudan University, Shanghai, 200240, PR China.
Biochem Genet. 2022 Dec;60(6):2000-2014. doi: 10.1007/s10528-022-10194-8. Epub 2022 Feb 22.
This study aims to investigate the effects of miR-29b-3p on the inflammation injury of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS) and explore the underlying mechanisms. The effects of different concentrations of LPS (0, 1, 5 and 10 μg/mL) on inflammation injury in HUVECs are detected by ELISA, CCK-8, EdU, flow cytometry and western blot analyses to determine the optimal stimulus concentration. After stimulating HUVECs with 10 μg/mL LPS, the expression levels of miR-29b-3p are detected, and the effects of miR-29b-3p on inflammation injury are detected by ELISA, CCK-8, EdU, flow cytometry and western blot analyses. Bioinformatic analysis, luciferase reporter assay and confirmatory experiments are applied to identify the target gene bound with miR-29b-3p. Rescue experiments have verified the roles of miR-29b-3p and the target gene in inflammation injury. We found that pro-inflammatory factor was increased, apoptosis was promoted, and cell proliferation was inhibited after the treatment of LPS in HUVECs. Overexpression of miR-29b-3p inhibited LPS-induced inflammatory response and apoptosis while promoting proliferation in HUVECs. Besides, bioinformatics analysis indicated that SEC23A was the target gene of miR-29b-3p and the confirmatory experiments showed that SEC23A was negatively correlated with miR-29b-3p and positively correlated with LPS concentration. Rescue experiments revealed that overexpression of SEC23A partially enhanced the inflammation injury effects in LPS-induced HUVECs with overexpression of miR-29b-3p. Hence, miR-29b-3p repressed inflammatory response, cell apoptosis and promoted cell proliferation in LPS-induced HUVECs by targeting SEC23A, providing a potential target for treating sepsis.
本研究旨在探讨 miR-29b-3p 对脂多糖（LPS）诱导的人脐静脉内皮细胞（HUVEC）炎症损伤的影响，并探讨其潜在机制。通过 ELISA、CCK-8、EdU、流式细胞术和 Western blot 分析检测不同浓度 LPS（0、1、5 和 10μg/mL）对 HUVEC 炎症损伤的影响，确定最佳刺激浓度。用 10μg/mL LPS 刺激 HUVEC 后，检测 miR-29b-3p 的表达水平，并通过 ELISA、CCK-8、EdU、流式细胞术和 Western blot 分析检测 miR-29b-3p 对炎症损伤的影响。通过生物信息学分析、荧光素酶报告实验和验证实验确定与 miR-29b-3p 结合的靶基因。通过 rescue 实验验证了 miR-29b-3p 和靶基因在炎症损伤中的作用。结果发现，LPS 处理后 HUVEC 中促炎因子增加，促进细胞凋亡，抑制细胞增殖。过表达 miR-29b-3p 抑制 LPS 诱导的 HUVEC 炎症反应和细胞凋亡，促进细胞增殖。此外，生物信息学分析表明 SEC23A 是 miR-29b-3p 的靶基因，验证实验表明 SEC23A 与 miR-29b-3p 呈负相关，与 LPS 浓度呈正相关。Rescue 实验表明，过表达 miR-29b-3p 可部分增强 LPS 诱导的 SEC23A 过表达 HUVEC 中的炎症损伤效应。因此，miR-29b-3p 通过靶向 SEC23A 抑制 LPS 诱导的 HUVEC 炎症反应、细胞凋亡，促进细胞增殖，为治疗脓毒症提供了一个潜在的治疗靶点。
