Role Of MicroRNA-125a-5p In Regulating The Differentiation Of Treg Cells In The Pathogenesis Of Optic Neuritis

Part I Correlation study between micro RNA-125a-5p and optic neuritisObjective To screen micro RNAs which differentially expressed in peripheral blood mononuclear cells(PBMC)between patients with optic neuritis and normal control people,and to study their correlation with visual evoked potential(VEP)and optical coherence tomography(OCT)in patients with optic neuritis.Methods(1)In order to screen differentially expressed genes,q PCR was used to detect the relative expression of micro RNA-145a-5p,micro RNA-146a-5p,micro RNA-155 and micro RNA-326 in peripheral blood PBMCs of optic neuritis group and normal control group.(2)F-VEP was used to measure the peak time and amplitude of the P2 wave in both groups of selected subjects.(3)OCT was used to measure the full-thickness of retinal in four quadrants of optic disc in selected subjects of both groups.Results(1)The expression of miR-125a-5p was significantly decreased in peripheral blood PBMCs from patients with optic neuritis compared with other selectedmiRNAs(P<0.05).(2)Compared with the normal control group,the F-VEP P2 peak time was significantly delayed(P<0.05).and the amplitude was significantly decreased(P<0.05).in patients with optic neuritis,and no significant correlation was observed with miR-125a-5p expression.(3)Compared with the normal control group,OCT revealed a significant increase in retinal thickness in optic disc of the nasal,inferior,and superior quadrants in patients with optic neuritis(P<0.05).Among them,there was a moderate negative correlation between inferior retinal thickness values and retinal miR-125a-5p expression(R =-0.425,P = 0.049).Conclusion The expression of Mi R-125a-5p is low in peripheral blood PBMCs of patients with optic neuritis,and is negatively correlated with optic disc retinal thickness,its pathway of action in optic neuritis requires further mechanistic studies to clarify.Part II Effect of miR-125a-5p on experimental autoimmune encephalomyelitis by regulating Treg cell differentiationObjective To investigate the effect of miR-125a-5p expression on optic neuritis and its pathway of action through experimental autoimmune encephalomyelitis mouse model and lymphocyte immunity model.Methods1.Experiment in vitro(1)Intrasplenic lymphocytes of EAE mice were isolated and divided into blank group,EAE group,EAE+miR-125a-5p overexpression intervention group(miR-125 group),EAE+miR-125a-5p overexpression unloaded control intervention group(miR-125-Ctrl group),EAE+miR-125a-5p-inhibitor intervention group(miR-125 i group)and EAE+miR-125a-5p-inhibitor unloaded control group(miR-125i-Ctrl group),in which EAE group was constructed by MOG35-55 induced immune splenic lymphocytes,and miR-125 group,miR-125-Ctrl group,and miR-125 i miR-125i-Ctrl group were transfected with lentiviruses containing corresponding miRNA fragment plasmids,respectively.(2)Fluorescence microscopy and flow cytometry was performed 72 hours after the second transfection in each group to assess the transfection efficiency.Flow cytometry was performed to compare the changes in the proportion of T lymphocytes of various subtypes and B lymphocytes.ELISA was performed to assess the expression of INF-γ,IL-4 and IL-17 inflammatory factors in the cell culture medium.2.Experiment in vivo(1)8-week-old C57BL/6 mice were divided into blank group,EAE group,EAE+miR-125a-5p overexpression intervention group(miR-125 group),EAE+miR-125a-5p overexpression no-load control intervention group(miR-125-Ctrl group),EAE+miR-125a-5p-inhibitor intervention group(miR-125 i group)and EAE+miR-125a-5p-inhibitor no-load control group(miR-125i-Ctrl group).Among them,EAE models were constructed by MOG35-55 inducing immunity in C57BL/6 mice.Adeno-associated viruses which containing plasmids of the corresponding miRNA fragments were used for transfection in miR-125,miR-125-Ctrl,miR-125 i and miR-125i-Ctrl groups respectively.(2)Body weight and neurological dysfunction scores of mice in each group were recorded every other day from the time of immunization until 30 days after immunization.(3)F-VEP was performed in mice at the peak of disease to assess the implicit time and amplitude of P2 wave in each group;and OCT was performed to assess the changes in retinal thickness in the optic disc of mice in each group.Results1.Experiment in vitro(1)Spleen lymphocytes culture experiments in vitro showed that the proportion of CD4+CD25+Foxp3 + cell cluster was significantly increased in the miR-125-group compared with the EAE group(P<0.05).(2)ELISA examination found that the expressions of inflammatory factors including INF-γ,IL-4 and IL-17 were down-regulated in the cell culture medium in every group,especially the expressions of INF-γ were decreased obviously(P<0.05).2.Experiment in vivo(1)During the observation period,the body weight of mice in each experimental group increased gradually,and the body weight amplification of mice in each experimental group was smaller than that in the blank group.The disease was onset in mice of each experimental group on 9–13 days after immunization,and the neurological dysfunction score reached the peak around 7days after disease onset.(2)Compared with the blank group,the implicit time of F-VEP P2 wave was delayed and the amplitude was significantly decreased in mice of each group at the peak of disease(P<0.05).Compared with the EAE group,the implicit time of the P2 wave was delayed less in the miR-125 group mouse,and there was significant difference between the two groups(P<0.05).OCT results showed that the retinal thickness in the optic disc was significantly thicker at the peak of disease in each experimental group compared with the blank group.Among the experimental groups,the retinal thickness in the optic disc was significantly thiner in the miR-125-group at the peak of disease compared with the EAE group(P<0.05),and there was no significant difference among miR-125-Ctrl,miR-125 i,miR-25i-Ctrl and EAE group.(3)HE staining of optic nerve and retina revealed that inflammatory cell infiltration was significantly increased in each experimental group compared with the blank group.Inflammatory cell infiltration in the miR-125-group was relatively less,optic nerve fibers were relatively more neatly arranged.Flow cytometry of splenocytes revealed that the proportion of CD4+CD25+Foxp3+cell cluster was significantly increased in the miR-125-group compared with the EAE group(P<0.05).Conclusion Mi R-125a-5p can inhibit the nervous system immune response in mice with experimental neuritis,which alleviates the process of experimental autoimmune optic neuritis mainly by regulating Treg cell differentiation and down-regulating the expression of inflammatory factors.Part III Bioinformatics prediction of miR-125a-5p target genesObjective Predicted target gene intersection as well as gene function enrichment analysis and signal transduction pathway enrichment analysis were performed through the online target gene prediction website to explore the target genes of miR-125a-5p.So as to provide a scientific basis for further study on how miR-125a-5p promotes Treg cell differentiation and regulates inflammatory processes in the internal environment of optic neuritis.Methods(1)Online target gene prediction databases miRanda,Target Scan,and Pic Tar were used to predict the target genes of miR-125a-5p,and their intersection genes were extracted using the R language packet.(2)DAVID online software was used for GO functional enrichment analysis and KEGG signal pathway enrichment analysis on intersection target genes of miR-125a-5p.(3)Target Scan was used to align the target gene sequence and the miR-125a-5p seed sequence.Results(1)The target genes predicting from the target gene prediction databases of miRanda,Target Scan and Pic Tar were intersected,and 178 target genes of miR-125a-5p were obtained.(2)GO analysis predicts showed that the most significant biological processes of target gene cross enrichment are negatively regulating the transcriptional function of the RNA polymerase II promoter,negative regulation of chemotaxis,regulation of multicellular organism growth,regulation of semaphorin-plexin signaling pathway,negative regulation of axon elongation in axon guidance,templated of transcription and DNA,post-embryonic development,positive regulation of autophagy,acetylation of tissue protein H3,hematopoiesis,and polyubiquitination of proteins.KEGG signal transduction pathway enrichment analysis prediction showed that miR-125a-5p target genes were significantly enriched in micro RNA action pathways of tumor,m RNA surveillance pathway,stem cell pluripotency regulation pathway and Epstein-Barr virus infection pathway.(3)SEMA4D was involved in multiple negatively regulated biological processes,and Target Scanc search results indicated that Aggregate PCT = 0.97,its base in 3 ′UTR region matches with the seed sequence of miR-125a-5p.Conclusion Bioinformatics prediction analysis obtained 178 target genes of miR-125a-5p.Among them,SEMA4 D gene which base of the 3 ′ UTR region matched with the seed sequence of miR-125a-5p was involved in multiple negatively regulated biological processes and could be used as a subsequent study target gene.Part IV Mechanism of miR-125a-5p target regulating SEMA4 D in experimental optic neuritisObjective Dual-luciferase reporter gene was used as the primary measure to further discuss whether miR-125a-5p exerts its regulatory effect on inflammation in experimental optic neuritis through SEMA4 D,so as to provide a scientific basis for investigating the immunoregulatory role of miR-125a-5p in experimental optic neuritis.Methods(1)Dual-luciferase reporter plasmid which containing the SEMA4 D 3 ′-UTR fragment and its variant sequence was built and verified.(2)Mi R-125a-5p mimics and SEMA4 D dual-luciferase reporter plasmids were transfected into 293 T cells for miR-125a-5p mimics target gene validation experiments.(3)Splenocyte-sorted Treg cells were divided into EAE+miR-125a-5p overexpression intervention group(miR-125-group),EAE+miR-125a-5p overexpression unloaded control intervention group(miR-125-Ctrl-group),and EAE miR-125a-5p-inhibitor intervention group(miR-125i-group).Among them,EAE was constructed using immune Treg cells which was induced by MOG35-55,and lentiviruses containing plasmids of the corresponding miRNA fragments were used for transfection intervention in the miR-125-group and miR-125-Ctrl-groups,respectively.Mi R-125a-5p and SEMA4 D gene expression was measured by q PCR in each group.(4)PBMCs were isolated from the normal control group people and optic neuritis group patient,and miR-125a-5p and SEMA4 D gene expression was detected by q PCR in each group.Results(1)The results of dual luciferase reporter assay showed compared with the control group transfected with only the pmir GLO Vector-SEMA4D-3’UTR(Wt)plasmid,the reporter gene fluorescence value of SEMA4D(Wt)+miR-125a-5p group which co-transfected by miR-125a-5p mimics and pmir GLO Vector-SEMA4D-3 ’UTR(Wt)plasmid was significantly reduced(P<0.05).(2)Compare with the control group,the the expression of miR-125a-5p was significantly increased as well as the expression of SEMA4 D m RNA was significantly decreased in the miR-125 group(P<0.05).To the contrast,the expression of miR-125a-5p was significantly decreased and the expression of SEMA4 D m RNA was significantly increased in the miR-125 i group(P<0.05).(3)The expression of SEMA4 D mRNA was significantly increased in PBMCs from patients with optic neuritis compared with normal controls(P<0.05).Conclusion In the Treg cells,miR-125a-5p plays a regulatory role on its target gene SEMA4 D,but its mechanism still unclarified.Mechanism experiment of miR-125a-5p/SEMA4 D pathway should be further designed,so as to clarify its effect on Treg cell differentiation and the pathway of action.