Circular RNA CircRNA-22903 Sponge MiR Let-7i To Regulate The Function Of Dendritic Cells

Background and purposeDendritic cells(DCs)play a key role in regulating the immune response of transplantation,in which tolerogenic DC can induce transplant immune tolerance and prolong the survival time of grafts.Circular RNA(circRNA)is involved in the initiation and progression of immune-related diseases.However,the role of circRNA in heart transplantation and dendritic cells is rarely reported.In this study,the regulatory effect and molecular mechanism of transplant immune-related circRNA-22903 on dendritic cells were investigated.Methods(1)The transplant-related circRNA-22903 was screened by circRNA chip,Sanger sequencing verified the splice sites.qRT-PCR analysed the expression of circRNA-22903 in DC.The stability of circRNA-22903 was detected by actinomycin D and RNase R test.RNA quantitative qRT-PCR and fluorescence in situ hybridization(FISH)analysed the expression of circRNA-22903 in cytoplasm and nucleus of DC.(2)The circRNA-22903 overexpression plasmid or si RNA was transfected into DC,respectively.The expression of CD80、CD86 and MHC II and the number of Treg(CD4~+CD25~+Foxp3~+)cells were detected by flow cytometry.The secretion of cytokines TGF-β、IL-35 and IL-12 in the supernatant of DC was evaluated by ELISA.After DC co-cultured with T cells,Brdu assessed the ability of T cell proliferation(3)Bioinformatics analysis predicted that there were binding sites between miR let-7i and circRNA-22903.RIP,luciferase report test and FISH confirmed the binding of miR let-7i to circRNA-22903.(4)The function of DC was detected by regulating circRNA-22903,miR let-7i mimic and SOCS1 in vitro.Results(1)CircRNA-22903 is significantly down-regulated in mature DC and has the structure and characteristics of circular RNA.(2)Overexpression of circRNA-22903induced tolerogenic DC:the low expression surface markers CD80、CD86 and MHC II on DC,promoted the secretion of anti-inflammatory cytokines TGF-β、IL-35,inhibited the secretion of proinflammatory cytokines IL-12,increased the number of Tregs,while decreased the proliferation ability of T cell.(3)Knockdown of circRNA-22903 induced inflammatory DC:elevated the surface markers on DC,reduced the secretion of anti-inflammatory cytokines TGF-β、IL-35,increased the secretion of proinflammatory cytokines IL-12,reduced the number of Tregs and increased the ability of T cell proliferation.(4)The molecular mechanism of circRNA-22903 regulates DCs function by targeting miR let-7i/SOCS1 axis:circRNA-22903 sponge miR let-7i,the regulatory effect of circRNA-22903 on DC can be reversed by increasing let-7i or knocking down SOCS1.ConclusionsIn mature DC,the expression of circRNA-22903 is down-regulated,and it regulates the function of DC by the molecular mechanism of regulating miR let-7i/SOCS1 axis.