miR-141-3p靶向调控HMGB1对LPS诱导的A549细胞损伤的影响

Effect of miR-141-3p targeting HMGB1 on LPS-induced A549 cell injury

目的探讨miR-141-3p通过靶向调控高迁移率族蛋白1(HMGB1)对脂多糖(LPS)诱导的A549细胞损伤的影响。方法以Ⅱ型肺泡上皮细胞来源的A549细胞作为研究对象,将miR-141-3p mimics、mimics NC、HMGB1基因过表达质粒(pcDNA3.1-HMGB1)和空载质粒(Vector)分别或共转染至A549细胞中,再采用10μg/ml LPS处理24 h。细胞计数试剂盒8(CCK-8)检测各组细胞增殖活性;比色法检测各组细胞培养上清液中乳酸脱氢酶(LDH)活性;流式细胞术检测各组细胞凋亡水平;酶联免疫吸附测定法(ELISA)检测各组细胞中白介素(IL)-1β、IL-6和肿瘤坏死因子α(TNF-α)水平;双荧光素酶报告基因实验验证miR-141-3p与HMGB1之间的靶向调控关系。结果LPS干预后,A549细胞增殖活性及细胞中miR-141-3p表达水平降低(P<0.05),细胞凋亡率升高(P<0.05),细胞中IL-1β、IL-6、TNF-α水平及上清液中LDH活性升高(P<0.05)。过表达miR-141-3p可增强LPS处理后的A549细胞增殖活性(P<0.05),降低细胞凋亡率及细胞中IL-1β、IL-6、TNF-α水平和上清液中LDH活性(P<0.05)。然而,HMGB1基因过表达可逆转miR-141-3p对LPS诱导A549细胞损伤的改善作用。双荧光素酶报告基因实验证实,HMGB1是miR-141-3p下游靶基因。结论miR-141-3p可抑制LPS诱导的A549细胞凋亡,降低炎症因子表达水平,改善A549细胞损伤,其作用机制可能与靶向调控HMGB1表达有关。

Objective To investigate the effect of miR-141-3p on LPS induced A549 cell injury by targeting high mobility group protein 1(HMGB1).Methods A549 cells derived from typeⅡalveolar epithelial cells were taken as the study object,miR-141-3p mimics,mimics NC,HMGB1 gene overexpression plasmid(pcDNA3.1-HMGB1)and empty Vector were transfected into A549 cells respectively or co-transfected,then 10μg/ml LPS was used for 24 h.Cell proliferation activity was detected by cell counting kit-8(CCK-8).The activity of lactate dehydrogenase(LDH)in the supernatant of cell culture was detected by colorimetry.The apoptosis level of each group was detected by flow cytometry.The levels of interleukin(IL)-1β,IL-6 and tumor necrosis factorα(TNF-α)were detected by enzyme-linked immunosorbent assay(Elisa).Dual luciferase reporter gene assay verified the targeted regulatory relationship between miR-141-3p and HMGB1.Results After treatment with LPS,the proliferative activity of A549 cells and the expression level of miR-141-3p decreased(P<0.05),the apoptosis rate increased(P<0.05),the levels of IL-1β,IL-6,TNF-αand the activity of LDH in supernatant increased(P<0.05).Overexpression of miR-141-3p increased the proliferation activity of A549 cells treated with LPS(P<0.05),and decreased the apoptosis rate and the levels of IL-1β,IL-6,TNF-αin cells and LDH activity in supernatant(P<0.05).However,overexpression of HMGB1 gene could reverse the ameliorative effect of miR-141-3p on LPS-induced A549 cell injury.Dual luciferase reporter gene experiment confirmed that HMGB1 was the downstream target gene of miR-141-3p.Conclusion miR-141-3p can inhibit LPS-induced apoptosis,reduce the expression level of inflammatory factors,and improve the damage of A549 cells,which may be related to the targeted regulation of HMGB1 expression.