Regulatory roles of rno-miR-30b-5p in expressions of IL-10 and TLR4 in rats with experimental autoimmune uveitis / 眼科新进展

ABSTRACT

Objective To investigate the regulatory role of rno-miR-30b-5p in the expressions of interleukin-10 (IL-10) and toll-like receptor 4 (TLR4) in uveitis.Methods Both IL-10 and TLR4 gene 3'UTR lucfferase vectors and relevant binding site mutant vectors were constructed.Further,both rno-miR-30b-5p mimics and reporter gene vector were co-transferred into 293 T cells to validate the fluorescent alterations of the reporter gene expression to detect the interactions between rno-miR-30b-5p and the related target genes.Moreover,an experimental autoimmune uveitis (EAU) model was induced with IRBP peptide emulsion in rats,and both lymph node and spleen were isolated on day 12 after EAU induction.In order to measure rno-miR-30b-5p levels and IL-1 0,TLR4 expressions in spleen and lymph node,quantitative PCR and ELISA techniques were applied.Results The results of double lucfferase reporter gene expression analysis showed rno-miR-30b-5p mimic apparently down-regulated the fluorescence intensity of both IL-10 and TLR4 in wild type cells.After the mutation of the target site,the fluorescence intensity of the mutant vector was significantly reduced,accompanied by a significantly statistical difference (all P ＜ 0.01).Moreover,animal results revealed the expressions of rno-miR-30b-5p were apparently decreased,whereas IL-10 and TLR4 were markedly increased in both lymph node and spleen (all P ＜ 0.05).Conclusion Target identification shows that rno-miR-30b-5p can obviously regulate the expressions of 3'UTR gene with either IL-10 or TLR4 gene fragment,though its regulation might not be through the predicted site.The down-regulated expression of rno-miR-30b-5p in both spleen and lymph node in EAU rats result in the up-regulated expressions of both IL-10 and TLR4,further influence the development of uveitis.This study paves a way for the modulation of microRNA on the occurrence and development of uveitis,and will provide a new insight on treating uveitis.