MicroRNA-155在诱导RAW264.7细胞分泌CXCL10间接促进Th1细胞分化中作用研究

Study on the role of microRNA-155 in inducing RAW264.7 cells to secrete CXCL10 and indirectly promoting Th1 cells differentiation

目的探讨微小RNA(microRNA,miR)-155对氧化型低密度脂蛋白(oxidized-LDL particles,oxLDL)活化的RAW264.7细胞分泌的趋化因子的调控作用及其对辅助性T细胞1(helper T cell 1,Th1)分化的间接调控作用。方法培养RAW264.7细胞,向细胞内转染寡聚核苷酸(miR-ctrl、miR-155 mimics、miR-155 inhibitor、miR-155 mimics+siRNA-ctrl和miR-155 mimics+CXCL10 siRNA),并以此对细胞进行分组。oxLDL刺激所有细胞活化,实时定量反转录聚合酶链反应(quantitative reverse transcription polymerase chain reaction,qRT-PCR)和酶联免疫吸附测定(enzyme-linked immuno sorbent assay,ELISA)方法检测趋化因子表达情况,将上述细胞与纯化并活化的CD4^+T细胞共培养后,流式细胞术检测Th1细胞的分化情况。结果miR-155促进oxLDL活化的RAW264.7细胞分泌CXCL10(mRNA水平:oxLDL刺激细胞6、12和24 h的F值分别为44.86、214.91、688.88;蛋白水平:oxLDL刺激细胞6、12和24 h的F值分别为284.57、144.66、60.95,P值均<0.05);在RAW264.7中上调miR-155的表达可促进CD4^+T细胞分化为Th1细胞(F=26.72,P<0.05);阻断RAW264.7细胞中CXCL10的表达则抑制miR-155对Th1细胞的间接调控作用(t=3.78,P<0.05)。结论miR-155可通过促进oxLDL活化的RAW264.7细胞分泌CXCL10间接促进Th1细胞分化。

Objective To illustrate the role of microRNA(miR)-155 in regulating the secretion of CXCL10 by oxidized-LDL particles(oxLDL)-activated RAW264.7 cells and its indirect role in regulating helper T cell 1(Th1)cells differentiation.Methods RAW264.7 cells were divided into 5 groups by transfected with different oligonucleotides(including miR-ctrl,miR-155 mimics,miR-155 inhibitor,miR-155 mimics+siRNA-ctrl,and miR-155 mimics+CXCL10 siRNA),then cells were treated with oxLDL,and the level of chemokines were detected with quantitative reverse transcription polymerase chain reaction(qRT-PCR)and enzyme-linked immuno sorbent assay(ELISA).Then these cells were co-cultured with purified and activated CD4+T cells,Th1 differentiation was examined with flow cytometry.Results miR-155 enhances the secretion of CXCL10 in oxLDL-activated RAW264.7 cells(mRNA level:F values of oxLDL stimulated cells for 6,12,and 24 h were 44.86,214.91,688.88,respectively;protein level:F values of oxLDL stimulated cells for 6,12,and 24 h were 284.57,144.66,60.95,respectively,all P values<0.05).Up-regulated miR-155 in oxLDL-activated RAW264.7 cells can promote the differentiation of CD4+T cells into Th1 cells(F=26.12,P<0.05).Knock-down CXCL10 expression in RAW264.7 cells can suppress the indirect function of miR-155 in regulating Th1 cells differentiation(t=3.78,P<0.05).Conclusions miR-155 can indirectly enhance Th1 cell differentiation through promoting CXCL10 expression which secreted by oxLDL-activated RAW264.7 cells.