miR-21a-5p靶向MATN2抑制LPS诱导的巨噬细胞损伤的实验研究

miR-21a-5p Inhibits LPS Induced Macrophage Injury by Targeting MATN2

目的miR-21a-5p对脂多糖(LPS)处理的RAW264.7巨噬细胞的调控作用及分子机制研究。方法实时荧光定量PCR(qRT-PCR)检测不同浓度LPS处理的巨噬细胞中miR-21a-5p水平;应用miR-21a-5p模拟物(mimics)和阴性对照(miRNA-NC)分别转染LPS处理的巨噬细胞,MTT法检测细胞活力,流式细胞术检测细胞凋亡比例;ELISA检测炎性细胞因子水平;TargetScan数据库分析miR-21a-5p的潜在靶基因,蛋白免疫印迹技术(Western blot法)检测MATN2蛋白表达水平;双荧光素酶实验验证miR-21a-5p和MATN2之间的关系。结果随着LPS处理浓度的增加,miR-21a-5p水平逐渐降低。miR-21a-5p mimics转染RAW264.7巨噬细胞后miR-21a-5p水平显著升高。上调LPS处理的巨噬细胞miR-21a-5p水平后,细胞活力显著升高,细胞凋亡比例降低;促炎性细胞因子TNF-α、IL-6和IL-1β水平降低。qRT-PCR和Western blot法结果表明,上调miR-21a-5p水平后,MATN2蛋白表达和mRNA水平明显降低,而下调miR-21a-5p水平后,MATN2蛋白表达和mRNA明显升高。双荧光素酶报告实验显示miR-21a-5p靶向MATN2的mRNA的3′非编码区(3′UTR)。结论miR-21a-5p可以靶向MATN2抑制LPS诱导的巨噬细胞损伤。

Objective To investigate the regulatory effect and molecular mechanism of miR-21a-5p on lipopolysaccharide(LPS)-treated RAW264.7 cells.Methods The levels of miR-21a-5p in macrophages treated with different concentrations of LPS were detected by real-time quantitative PCR(qRT-PCR).LPS treated macrophages were transfected with miR-21a-5p mimics and negative control(miRNA-NC)respectively.The cell viability was measured by MTT method,and the apoptosis rate was detected by flow cytometry,and the level of inflammatory factors was detected by ELISA.The potential target genes of miR-21a-5p were analyzed by TargetScan database,and the expression level of MATN2 protein was detected by Western blot.The relationship between miR-21a-5p and MATN2 was verified by double luciferase assay.Results With the increase of LPS concentration,the level of miR-21a-5p decreased gradually.After transfection of miR-21a-5p mimics into RAW264.7 cells,the level of miR-21a-5p increased significantly.After up regulating the level of miR-21a-5p in LPS treated macrophages,the cell viability was significantly increased,the apoptosis rate was decreased,and the levels of pro-inflammatory factors TNF-α,IL-6 and IL-1βwere decreased.The results of qRT-PCR and Western blot showed that the protein expression and mRNA level of MATN2 were significantly decreased after up regulating miR-21a-5p,while the protein expression and mRNA level of MATN2 were significantly increased after down regulating miR-21a-5p.Double luciferase reporter assay showed that miR-21a-5p targeted the 3′untranslated region(3′UTR)of MATN2 mRNA.Conclusion miR-21a-5p can target MATN2 to inhibit LPS induced macrophage injury.