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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 (2 350.39±367.02)pg/ml, which was significantly higher than (1 513.31±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.