miR-148b靶向调控DcR3抑制LPS诱导的巨噬细胞极化

miR-148b inhibits M2 polarization of LPS-stimulated macrophages by targeting DcR3

目的探讨脓毒症中微小RNA-148b(miR-148b)靶向抑制诱骗受体3(DcR3)对巨噬细胞极化的影响。方法2019年12月至2022年12月期间,在上海交通大学医学院附属松江医院(筹)检验科,检测3例脓毒症患者及3名健康对照血清microRNA表达。采用佛波酯(PMA)诱导人急性单核细胞白血病细胞THP-1分化为巨噬细胞,继而加入脂多糖(LPS)刺激建立脓毒症细胞模型,检测miR-148b和DcR3表达变化。过表达DcR3检测LPS(100 ng/ml)刺激巨噬细胞TNF-α、CD163及IL-10的表达水平。过表达miR-148b观察巨噬细胞极化分子标志物的变化。采用双荧光素酶报告基因实验测定miR-148b对DcR3的靶向调控作用。通过t检验分析各组间是否具有统计学差异。结果LPS刺激THP-1巨噬细胞miR-148b表达下调(P<0.05),DcR3表达升高(P<0.01);过表达DcR3抑制TNF-α的表达(P<0.05),促进CD163(P<0.01)和IL-10(P<0.01)的表达,而加入miR-148b模拟物则相反;双荧光素酶报告基因实验证实miR-148b靶向结合DcR3,抑制其转录和表达,流式细胞检测结果显示DcR3可逆转miR-148b对巨噬细胞CD86/CD163比值的促进作用(P<0.05)。结论miR-148b靶向抑制DcR3的表达,从而抑制LPS诱导的巨噬细胞模型M2极化。

Objective To investigate the effect of microRNA(miR-148b)targeting decoy receptor 3(DcR3)on macrophage polarization in sepsis.Methods Experimental study.From December 2019 to December 2022,serum microRNA expression was detected in 3 patients with sepsis and 3 healthy controls in the clinical laboratory of Songjiang Hospital Affiliated to Shanghai Jiao Tong University School of Medicine.Phorbol 12-myristate 13-acetate(PMA)was used to induce the differentiation of human acute monocytic leukemia cells THP-1 into macrophages,and then lipopolysaccharide(LPS)was added to stimulate the establishment of a sepsis cell model,and the expression changes of miR-148b and DcR3 were detected by RT-PCR and Western blot.Overexpression of DcR3 was used to detect the expression levels of TNF-α,CD163 and IL-10 in macrophages stimulated by LPS(100 ng/ml).Overexpression of miR-148b was used to observe the changes of molecular markers of macrophage polarization.The targeting regulation effect of miR-148b on DcR3 was determined by dual-luciferase reporter assay.t test was used to analyze whether there were statistical differences among the groups.Results The expression of miR-148b was down-regulated(P<0.05)and the expression of DcR3 was up-regulated(P<0.01)in THP-1 macrophages stimulated by LPS.Overexpression of DcR3 inhibited the expression of TNF-α(P<0.05)and promoted the expression of CD163(P<0.01)and IL-10(P<0.01).When miR-148b mimics was added,the opposite effect was observed.The dual-luciferase reporter assay confirmed that miR-148b targets and binds to DcR3,inhibiting its transcription and expression.The results of flow cytometry showed that DcR3 could reverse the promoting effect of miR-148b on the CD86/CD163 ratio of macrophages(P<0.05).Conclusion miR-148b inhibits the expression of DcR3,thereby inhibiting M2 polarization in LPS-stimulated macrophage cells.