lncRNA 5033413D16Rik对实验性自身免疫性葡萄膜炎Th17细胞活性的抑制作用

Inhibitory effect of lncRNA 5033413D16Rik on the activity of T helper 17 cells in experimental autoimmune uveitis

目的探讨长链非编码RNA 5033413D16Rik(lncRNA 5033413D16Rik)对实验性自身免疫性葡萄膜炎(EAU)中光感受器间维生素A类结合蛋白1-20(IRBP_(1-20))特异性辅助性T17(Th17)细胞活性的影响。方法选取SPF级8~10周龄C57BL/6雌性小鼠12只,采用随机数字表法分为EAU组和正常对照组,每组各6只。正常对照组小鼠不接受任何处理,将IRBP_(1-20)和完全弗氏佐剂充分乳化后主动免疫EAU组小鼠。免疫后13 d,行眼底检查并进行炎症评分,苏木精-伊红染色观察视网膜组织病理学改变;分离EAU小鼠脾脏及淋巴结中T细胞,实时荧光定量PCR检测lncRNA 5033413D16Rik相对表达量。将分离培养的T细胞分为短发夹RNA(shRNA)-5033413D16Rik转染组和shRNA-阴性对照(NC)转染组,分别转染相应shRNA,实时荧光定量PCR验证shRNA-5033413D16Ri的敲低效率。将2个组细胞分别与骨髓源性树突状细胞在Th17极化条件下共培养,实时荧光定量PCR检测2个组共培养细胞中转录因子维甲酸相关核孤儿受体γt(RORγt)、白细胞介素17(IL-17)、IL-23受体(IL-23R)和粒细胞-巨噬细胞集落刺激因子(GM-CSF)mRNA相对表达量;ELISA试剂盒检测2个组共培养细胞上清中IL-17质量浓度;流式细胞仪检测2个组共培养细胞中Th17细胞百分比。结果成功建立小鼠EAU模型。实时荧光定量PCR检测结果显示,与正常对照组相比,EAU组小鼠T细胞中lncRNA 5033413D16Rik相对表达量显著降低,差异有统计学意义(t=-13.332,P<0.001)。与shRNA-NC转染组相比,shRNA-5033413D16Rik转染组T细胞中lncRNA 5033413D16Rik相对表达量显著降低,差异有统计学意义(t=-6.338,P<0.01)。shRNA-5033413D16Rik转染组共培养细胞中RORγt、IL-17、IL-23R和GM-CSF mRNA相对表达量分别为1.61±0.13、1.51±0.13、1.85±0.33和1.45±0.11,明显高于shRNA-NC转染组的1.00±0.01、1.00±0.01、1.00±0.01和1.00±0.01,差异均有统计学意义(t=-6.839、-8.221、-4.538、-4.189,均P<0.05);ELISA检测结果显示,shRNA-5033413D16Rik转染组共培养细胞上清中IL-17质量浓度为(2350.39±367.02)pg/ml,明显高于shRNA-NC转染组的(1513.31±310.37)pg/ml,差异有统计学意义(t=-3.016,P=0.039);流式细胞仪检测结果显示,shRNA-5033413D16Rik转染组共培养细胞中Th17细胞比例为(17.20±0.44)%,明显高于shRNA-NC转染组的(14.10±0.84)%,差异有统计学意义(t=-3.264,P=0.031)。结论lncRNA 5033413D16Rik可以抑制抗原特异性Th17细胞致病性相关基因的表达,负向调控IRBP_(1-20)特异性Th17细胞反应。

Objective To investigate the effect of long noncoding RNA(lncRNA)5033413D16Rik(lncRNA 5033413D16Rik)on the activity of interphotoreceptor retinoid-binding protein_(1-20)(IRBP)1-20-specific T helper 17(Th17)cells in experimental autoimmune uveitis(EAU).Methods Twelve SPF grade C57BL/6 female mice aged 8 to 10 weeks were selected and divided into EAU group and normal control group using the random number table method,with 6 mice in each group.Mice in the normal control group received no treatment.Mice in the EAU group were immunized with IRBP_(1-20)emulsified in complete Freund's adjuvant to induce EAU.On day 13 after immunization,fundus examination and inflammation scoring were performed,and retinal histopathological changes were observed with hematoxylin and eosin staining.T cells were isolated from the spleen and lymph nodes of EAU mice,and the relative expression of lncRNA 5033413D16Rik was detected by real-time fluorescence quantitative PCR(qRT-PCR).In addition,the isolated T cells were divided into short hairpin RNA(shRNA)-5033413D16Rik transfected group and shRNA-negative control(NC)transfected group,and transfected with corresponding shRNAs,respectively.The knockdown efficiency of shRNA-5033413D16Rik was verified by qRT-PCR.T cells in the two groups were co-cultured with bone marrow-derived dendritic cells(BMDCs)under Th17 polarizing conditions.The relative expression of retinoic acid-related orphan receptor(RORγt),interleukin 17(IL-17),IL-23 receptor(IL-23R),and granulocyte-macrophage colony-stimulating factor(GM-CSF)mRNA in co-cultured cells were analyzed by qRT-PCR.The IL-17 concentration in co-culture supernatants was assayed by ELISA and percentages of Th17 cells were determined by flow cytometry.The use and care of experimental animals complied with the Regulations for the Administration of Affairs Concerning Experimental Animals promulgated by the State Science and Technology Commission.The study protocol was reviewed and approved by the Experimental Animal Ethics Committee of Tianjin Medical University(No.TJYY2019110117).Results EAU model was established successfully.qRT-PCR analysis showed that the expression of lncRNA 5033413D16Rik was significantly decreased in T cells from EAU mice compared with normal control mice(t=-13.332,P<0.001).Compared with the shRNA-NC transfected group,the relative expression of lncRNA 5033413D16Rik in T cells of the shRNA-5033413D16Rik transfected group was significantly reduced(t=-6.338,P<0.01).In co-cultured T cells,the expression levels of RORγt,IL-17,IL-23R,and GM-CSF mRNA in shRNA-5033413D16Rik transfected group were 1.61±0.13,1.51±0.13,1.85±0.33 and 1.45±0.11,which were significantly higher than 1.00±0.01,1.00±0.01,1.00±0.01 and 1.00±0.01 in shRNA-NC-transfected group(t=-6.839,-8.221,-4.538,-4.189;all at P<0.05).ELISA analysis revealed that IL-17 concentraion in shRNA-5033413D16Rik transfected group was(2350.39±367.02)pg/ml,which was significantly higher than(1513.31±310.37)pg/ml(t=-3.016,P=0.039).Flow cytometry showed that the percentage of Th17 cells in shRNA-5033413D16Rik transfected group was(17.20±0.44)%,which was higher than(14.10±0.84)%in normal control group(t=-3.264,P=0.031).Conclusions LncRNA 5033413D16Rik can inhibit the expression of pathogenicity related genes in antigen-specific Th17 cells and negatively regulates IRBP_(1-20)-specific Th17 cell responses.