Regulatory Role Of Rno-mir-30b-5p In Interleukin-10 And Toll Like Receptor 4 Expressions In Experimental Autoimmune Uveitis Rats

Objective: 1.To study the m RNA and protein expression levels of mi R-30b-5p,interleukin 10(IL-10)and Toll like receptor 4(TLR4)in peripheral blood T lymphocytes of patients with uveitis,and to explore the effect of mi R-30b-5p on the occurrence and development of uveitis;to study the changes of IL-10 and TLR4 levels of spleen and lymph node T cells from experimental autoimmune uveitis(EAU)rats after treatment with rno-mi R-30b-5p mimic and inhibitor,and to analyze the relationship between the levels of both IL-10 and TLR4 positive cells and the incidence of EAU.2.To investigate the expressions of rno-mi R-30b-5p in the eyeball,spleen and lymph node tissues in EAU rats,and to analyze the expression levels of IL-10 and TLR4 m RNA and proteins.Methods: 1.Peripheral venous blood(4ml)of patients from either active anterior uveitis or healthy people was collected to isolate T lymphocytes to purify total RNA,respectively.After isolation of total RNA,changes of mi R-30b-5p,IL-10 and TLR4 m RNA were detected by polymerase chain reaction(PCR);meanwhile,changes of protein of IL-10 and TLR4 were further detected by Enzyme-Linked Immunosorbent Assay(ELISA).2.IL-10 and TLR4 gene sequences and vector sequences were analyzed,while 3’ UTR sequences containing IL-10 gene and TLR4 gene were obtained after treatment with Xho I,Not I restriction endonucleases by using PCR.Based on the wild type vector obtained,the mutant bases were introduced in both IL-10 and TLR4 primers using 3’ UTR sequences as template,and 3’ UTR sequences IL-10,TLR4 gene mutation were obtained by PCR.Further,both wild type(WT)and mutant(Mut)gene fragments were cloned intopmi R-RB-REPORTTM dual luciferase reporter vector to construct the related plasmid vectors containing luciferase.At the same time,both rno-mi R-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 whether there is interaction between rno-mi R-30b-5p and target genes.3.Female Lewis rats(6~8 weeks)were randomly divided into normal control group(n=6)and EAU group(n=6),resepctively.Rats in EAU group were immunized with Interphotoreceptor retinoid-binding protein(IRBP1177-1191)emulsion supplemented with complete freund adjuvant(CFA)and tuberculin(TB)via the injection of footpad,both napes and the back of the body1,resepctively.The same part of the control group was injected with the same amount of emulsified liquid without IRBP.Changes of ocular inflammation in EAU rats were observed with Genesis-D eye camera every day after immunization,and the clinical score was evaluated.When the EAU rats reached the peak of inflammation on day 12,the rat eyeball was extracted from the rats to perform the pathological analysis through haematoxylin and eosin(H&E)staining.At the same time,the eyeball,lymph nodes,and spleen tissues from sacrificed rats were removed.Meanwhile,the levels of rno-mi R-30b-5p in eye tissues,spleen and lymph nodes were measured using quantitative PCR(Q-PCR),and IL-10,TLR4 in spleen and lymph nodes were further separately determined by using Q-PCR and ELISA techniques.4.T lymphocytes from spleen and lymph nodes of EAU rats were isolated using rat lymphocyte separation solution.Rno-mi R-30b-5p mimic and its inhibitor were separately transfected into purified T cells.The alterations of IL-10,TLR4 levels were also determined by flow cytometry.The gene and protein levels of IL-10 and TLR4 were detected using PCR and ELISA.Results: 1.The mi R-30b-5p level in peripheral blood of patients with active anterior uveitis was increased,whereas the expressions of IL-10 and TLR4 m RNA was down-regulated.ELISA results showed that the changes of IL-10 and TLR4 proteins in serum were consistent with the trend of gene expressions.2.Based on the results of double luciferase reporter gene expression analysis,it was found that rno-mi R-30b-5p mimic has a clear down regulatory effect on the fluorescence intensity of both IL-10 and TLR4 wildtype cells.After the mutation of the target site,the fluorescence of the mutant vector was significantly reduced.3.The inflammation reached the peak on day 12 after immunization in EAU rats.The result of histopathological examination was consistent with that of ocular fundus camera observation.Animal experiments showed that the expression levels of rno-mi R-30b-5p in eyeball,spleen and lymph node tissues in EAU rats were significantly decreased,while the expressions of IL-10 and TLR4 m RNA and protein increased significantly.Compared with those in normal control group,the expression levels of IL-10 and TLR4 were statistically significant(all P<0.05).4.In vitro cell transfection experiments indicated that compared with those in inhibitor group,IL-10 and TLR4 levels at m RNA and protein levels had a marked down-regulation after treatment with rno-mi R-30b-5p mimic in spleen and lymph node T cells in EAU control group,mimic Negative group.The expressions of IL-10 and TLR4 m RNA were significantly increased in lymph nodes T cells after treatment with mi R-30b-5p inhibitor for 72h(P<0.05).By contrast,TLR4 m RNA expression significantly increased(P<0.05);whereas IL-10 m RNA expression was not significant(P > 0.05)in spleen T cells after treatment with rno-mi R-30b-5p inhibitor for 72 h.In addition,the up-regulated expressions of IL-10 and TLR4 proteins were not statistically significant compared with those in EAU control group,mimic Negative and inhibitor Negative(all P>0.05).In addition,flow cytometry analysis revealed that rno-mi R-30b-5p mimic could reduce the number of both IL-10 and TLR4 positive cells,whereas rno-mi R-30b-5p inhibitor could increase the number of IL-10 and TLR4 positive cells.Conclusions: 1.High level of mi R-30b-5p inhibits IL-10 and TLR4 expressions at m RNA and protein levels in peripheral blood of patients with active anterior uveitis,regulating the occurrence and development of uveitis.2.Target gene identification experiments further confirmed that rno-mi R-30b-5p can regulate the expressions of IL-10 and TLR4.This study lays a foundation on exploring the regulatory role of micro RNA in the occurrence and development of uveitis.3.After analysis of animal experiments and in vitro cell transfection,we found that inhibition of the expression of rno-mi R-30b-5p can up-regulate IL-10 and TLR4 gene and protein expressions in spleen and lymph nodes inEAU rats;it is also found that rno-mi R-30b-5p inhibits the development of uveitis by regulating the number of IL-10 and TLR4 positive cells.Our study demonstrates that rno-mi R-30b-5p influences the development of uveitis by regulating the level of both IL-10 and TLR4 positive cells,thereby playing a role in the pathogenesis of uveitis.