miR-155调控THP-1巨噬细胞炎症的作用机制

The role and mechanism of miRNA-155 in the inflammation of THP-1-derived macrophages

目的miR-155在巨噬细胞炎症反应中被诱导,但其调控巨噬细胞炎症的作用尚未完全探索,文章旨在探讨miR-155在脂多糖(LPS)诱导的THP-1巨噬细胞炎症反应中的调控作用及其机制。方法使用LPS诱导THP-1来源的巨噬细胞为体外炎症细胞模型。细胞进行分组:①miRNA转染:空白组、LPS组、miR-155 mimics+LPS组、mimics NC+LPS组;②siRNA转染:对照组、LPS组、siRNA-SOCS1+LPS组、siRNA-NC+LPS组。qRT-PCR检测miR-155在THP-1巨噬细胞炎症中的表达;转染miR-155 mimics/mimics NC后用qRT-PCR和ELISA检测miR-155对巨噬细胞炎症因子TNF-α和IL-8转录/蛋白的影响;Targetscan数据库预测miR-155作用的靶基因,并用qRT-PCR验证miR-155和SOCS1之间的靶向关系;siRNA沉默靶基因SOCS1,qRT-PCR和Western Blot检测沉默效率;qRT-PCR检测沉默靶基因SOCS1对巨噬细胞炎症因子TNF-α和IL-8转录的影响;Western blot验证miR-155通过靶基因SOCS1调控THP-1巨噬细胞炎症反应。结果qRT-PCR结果显示,与空白组相比,THP-1巨噬细胞在LPS诱导后miR-155表达明显上调(P<0.05)。miR-155 mimics+LPS组较LPS组TNF-α、IL-8升高(P<0.05)。转染miR-155 mimics后,THP-1巨噬细胞中miR-155 mRNA明显升高(P<0.05),而SOCS1 mRNA显著下调(P<0.05)。Western Blot结果显示,与LPS组相比,miR-155 mimics+LPS组中SOCS1蛋白表达明显降低(P<0.05)。结论miR-155可上调THP-1巨噬细胞炎症中TNF-α、IL-8的表达,其调控机制可能是通过靶向降低SOCS1蛋白的表达,促进LPS所致的THP-1巨噬细胞炎症反应及免疫应答。

Objective miR-155 is induced in macrophage inflammation,but its regulation of macrophage inflammation has not been fully explored.This study aims to investigate the regulatory role of miR-155 in lipopolysaccharide(LPS)-induced THP-1 macrophage inflammation and its mechanism.Methods Macrophages derived from THP-1 were induced by LPS as inflammatory cell models in vitro.Cells were grouped as follows:①miRNA transfection:blank group,LPS group,miR-155 mimics+LPS group,mimics NC+LPS group;②siRNA transfection:control group,LPS group,siRNA-SOCS1+LPS group,siRNA-NC+LPS group.The expression of miR-155 in THP-1 macrophage inflammation was detected by qRT-PCR.After transfection with miR-155 mimics/mimics NC,the effects of miR-155 on transcription/protein of macrophage inflammatory factors TNF-αand IL-8 were detected by qRT-PCR and ELISA.The target genes of miR-155 were predicted by Targetscan database,and the targeting relationship between miR-155 and SOCS1 was verified by qRT-PCR.siRNA silencing target gene SOCS1,qRT-PCR and Western Blot were used to detect silencing efficiency.qRT-PCR was used to detect the effect of SOCS1 silencing target gene on the transcription of macrophage inflammatory factors TNF-αand IL-8.Western Blot verified that miR-155 regulates THP-1 macrophage inflammation through target gene SOCS1.Results qRT-PCR results showed that miR-155 expression was significantly up-regulated in THP-1 macrophages after LPS induction compared with the blank group(P<0.05).TNF-αand IL-8 in miR-155 mimics+LPS group were higher than those in LPS group(P<0.05).After transfection with miR-155 mimics,miR-155 mRNA in THP-1 macrophages was significantly increased(P<0.05),while SOCS1 mRNA was significantly down-regulated(P<0.05).Western blot results showed that SOCS1 protein expression was significantly decreased in miR-155 mimics+LPS group compared with LPS group(P<0.05).Conclusion In all,our data suggest that the expression of TNF-αand IL-8 were promoted by miR-155 in THP-1 macrophages,by downregulating the expression of SOCS1,miR-155 wires macrophage inflammatory response.