微小RNA-9-5p调控脂多糖诱导小鼠树突状细胞免疫功能的机制研究

Study on the mechanism of mir-9-5p regulating lipopolysaccharide-induced the immune function of mouse dendritic cells

目的研究微小RNA-9-5p(miR-9-5p)对脂多糖炎性损伤小鼠树突状细胞(DCs)免疫功能的影响,并探讨其机制。方法该研究于2019年1—9月完成。小鼠树突状细胞DC2.4购于美国模式培养物中心。运用脂多糖(LPS)处理DC2.4细胞,建立炎性树突状细胞损伤模型;将miRNA模拟物阴性对照(miR-NC)、miR-9-5p模拟物(mimics)、miRNA抑制物阴性对照(antimiR-NC)、miR-9-5p抑制剂(anti-miR-9-5p)、小干扰RNA无序对照(si-NC)组、信号转导和转录激活因子6(STAT6)的小干扰RNA(si-STAT6)、miR-9-5p mimics+空载体质粒(pcDNA)、miR-9-5p mimics+STAT6过表达质粒(pcDNA-STAT6)均用脂质体法转染至LPS诱导的DC2.4细胞;噻唑蓝(MTT)法检测细胞增殖;实时荧光定量逆转录聚合酶链反应(qRT-PCR)法检测细胞中miR-9-5p、STAT6、白细胞分化抗原80(CD80)、白细胞分化抗原86(CD86)、白细胞介素-12(IL-12)的mRNA表达;蛋白质印迹法(Western blot)检测细胞中STAT6的蛋白表达;双荧光素酶报告基因实验检测细胞的荧光素酶活性。结果与正常DC2.4相比,脂多糖炎性损伤的DC2.4细胞中miR-9-5p表达(0.16±0.01)比(1.00±0.08)降低,STAT6表达(3.02±0.26)比(1.00±0.07)升高(P<0.05);过表达miR-9-5p可降低LPS诱导的DC2.4细胞中CD80(0.31±0.03)比(1.00±0.08)、CD86(0.29±0.02)比(1.01±0.06)、IL-12(0.46±0.04)比(1.00±0.05)的表达;敲减STAT6可降低LPS诱导的DC2.4细胞中CD80(0.42±0.04)比(1.00±0.08)、CD86(0.34±0.03)比(0.99±0.07)、IL-12(0.29±0.02)比(1.01±0.08)的表达;miR-9-5p明显的抑制野生型STAT6细胞的荧光活性,并负向调控STAT6的表达水平;重要的是,过表达STAT6可逆转过表达miR-9-5p对LPS诱导的DC2.4细胞中CD80[(1.68±0.13)比(1.00±0.06)]、CD86[(1.79±0.15)比(0.99±0.08)]、IL-12[(1.82±0.18)比(0.98±0.09)]的抑制作用。结论miR-9-5p抑制LPS诱导的DC2.4细胞的免疫功能,其机制可能与靶向STAT6有关,将可为炎性疾病的治疗提供新靶点。

Objective To study the effect of microRNA-9-5 p(miR-9-5 p)on the immune function of dendritic cells(DCs)in mice with lipopolysaccharide inflammatory injury,and explore its mechanism.Methods The study was completed from January2019 to September 2019.Mice dendritic cells DC2.4 were purchased from the American Model Culture Center.DC2.4 cells were treated with lipopolysaccharide(LPS)to establish a mice model of inflammatory dendritic cell(Nephritis DCs)injury.The miRNA mimic negative control(miR-NC),miR-9-5 p mimic(mimics),miRNA inhibitor negative control(anti-miR-NC),miR-9-5 p inhibitor(anti-miR-9-5 p),small interfering RNA disorder control(si-NC)group,signal transducer and activator of transcription 6(STAT6)small interfering RNA(si-STAT6),miR-9-5 p mimics+empty vector plasmid(pcDNA),miR-9-5 p mimics+STAT6 overexpression plasmid(pcDNA-STAT6)were transfected into LPS-induced DCs cells by liposome method;the proliferation was detected by thiazole blue(MTT)method;real-time fluorescence quantitative reverse transcription polymerase detected the expression of miR-9-5 p,STAT6,clusterdifferentiation 80(CD80),clusterdifferentiation 86(CD86),interleukin 12(IL-12)m RNA;the expression of STAT6 protein was detected by western blotting(western blot);the fluorescence activity of cells was detected by dual luciferase reporter gene assay.Results Compared with normal DC2.4 cells,the expression of miR-9-5 p(0.16±0.01)vs.(1.00±0.08)was significantly decreased,STAT6(3.02±0.26)vs.(1.00±0.07)was significantly increased in LPS-induced DCs cells(P<0.05).Overexpression miR-9-5 p significantly decreased the expression of CD80(0.31±0.03)vs.(1.00±0.08),CD86(0.29±0.02)vs.(1.01±0.06)and IL-12(0.46±0.04)vs.(1.00±0.05)in LPS-induced DC2.4 cells;STAT6 knockdown significantly reduced the expression of CD80(0.42±0.04)vs.(1.00±0.08),CD86(0.34±0.03)vs.(0.99±0.07),and IL-12(0.29±0.02)vs.(1.01±0.08)in LPS-induced DC2.4 cells;miR-9-5 p significantly inhibited the luciferase activity of wild-type STAT6 cells and negatively regulated the STAT6 expression;importantly,overexpression STAT6 could reverse the inhibition effect of overexpression miR-9-5 p on CD80(1.68±0.13)vs.(1.00±0.06),CD86(1.79±0.15)vs.(0.99±0.08)and IL-12(1.82±0.18)vs.(0.98±0.09)in LPS-induced DC2.4 cells.Conclusion MiR-9-5 p inhibits the immune function of LPS-induced DC2.4 cells,and its mechanism may be related to targeting STAT6,which will provide a new target for the treatment inflammatory diseases.