Role Of LncRNA MEG3 In The Pathogenises In Experimental Optic Neuritis

BackgroundOptic neuritis is inflammatory disease of optic nerve and is the most common optic neuropathy in young adults.Among optic neuritis,the most common type is idiopathic demyelinating optic neuritis and the etiology and pathogenesis remains to be unclear.Clinical characteristics include sudden visual loss,sometimes visual acuity may decrease to no light perception,with color vision and visual field defects.Patients with optic neuritis in China have worse visual impairment and poorer recovery,and bilateral disease is more common compared to the western countries.The pathological features of idiopathic demyelinating optic neuritis include perivascular inflammatory infiltration,oligodendroglia cells loss,demyelination,axonal injury,astrogliosis and neuronal degeneration.Experimental autoimmune encephalomyelitis(EAE)model is similar to human idiopathic demyelinating optic neuritis in the aspect of pathological features,imaging manifestations and visual electrophysiology change,and is an ideal animal model of optic neuritis.EAE model induced by myelin oligodendrocyte glycoprotein(MOG)35-55 in C57BL/6 mice is the most similar to human multiple sclerosis(MS)and is the preferred animal model in most research of MS and optic neuritis.Apoptosis of retinal ganglion cells(RGCs)occur during the course of optic neuritis and this can lead to thinning of retinal nerve fiber layer and affect the recovery of visual acuity.Apoptosis of RGCs is related to immune responeses and oxidative damage.The imbalance of Th1/Th2/Th17/Treg,the main four subtypes of CD4+T cells,and associated cytokine occur in the immune pathogenesis of EAE.In recent years,many studies verified that the imlalance of Th17/Treg plays an important part in the pathogenesis of demyelinating disease in central nervous system and EAE.Long noncoding RNA(lncRNA)is a kind of RNA molecule longer than 200 nt located in cytoplasm or nucleus.Lnc RNAs can not code protein and their expression have high tissue specificity.Recent researches found that lncRNAs take part in most biological process in vivo by chromosomal modification,controllong the maturity and transportation of m RNA,regulating protein synthesis.In the immune system,lncRNAs can regulate the differentiation,activation,proliferation,apoptosis and cytokine expression of T lymphocytes and B lymphocytes,and take part in the occurrence and development of many autoimmune diseases.In recent reports,several lncRNAs(eg.MEG3,TUG1,IFNG-AS1,ZFAS1,HOTAIR)were involved in T lymphocytes differentiation,neuronal degeneration and demyelinating disease in central nervous system.Lnc RNA expression profile and the following bioinformatics analysis of MS and neuromyelitis optica(NMO)showed that there were upregulative or downregulative expression of different lncRNAs in peripheral blood and these lncRNAs were associated with inflammatory response,activity of cytokine and neuronal apoptosis.However,the mechanism of lncRNAs in optic neuritis and EAE have not be reported.We will select and verify several lncRNAs associated with autoimmune disease in peripheral blood mononuclear cells(PBMCs)of optic neuritis patients and choose the most differently expressive lncRNA and then study the impact of this lncRNA in the occurrence and development of disease,optic nerve damage and immune system of EAE by upregulating or downregulating its expression level in EAE model of C57BL/6 mice.So that we can state the pathogenesis of lncRNA in experimental optic neuritis and provide new directions for the diagnose and treatment of optic neuritis in the future.Part ? Expression of autoimmune disease associated lncRNAs in peripheral blood of optic neuritis patientsObjective: To study the expression of autoimmune disease associated lncRNAs(MEG3?TUG1?IFNG-AS1)in PBMCs of optic neuritis patients,and select a lncRNA for further research.Methods: We collected peripheral venous blood from patients with optic neuritis and healthy person,isolated PBMCs by density gradient centrifugation,extracted total RNA and conducted reverse transcription,measured the relative expression level of lncRNA MEG3,TUG1 and IFNG-AS1 by real-time polymerase chain reaction(q PCR).Results: The expression level of lncRNA MEG3?TUG1?IFNG-AS1 were upregulated in PBMCs of optic neuritis patients compared to healthy person.Relative lncRNA MEG3 expression level of PBMCs in patients with optic neuritis was 6.87 folds compared to healthy persons(P=0.024).Relative lncRNA TUG1 expression level was 2.09 folds compared to healthy persons(P=0.109)and relative lncRNA IFNG-AS1 expression level was 3.48 folds compared to healthy persons(P=0.087).Expression level was higher than the other two lncRNAs.Conclusion: The expression level of lncRNA MEG3 in PBMCs of patients with optic neuritis was upregulated.Lnc RNA MEG3 is expected to be a biomarker helpful for the diagnose of optic neuritis.Part ? The effects of lncRNA MEG3 on optic nerve damage in experimental optic neuritisObjective: To study the effects of lncRNA MEG3 on optic nerve damage in experimental optic neuritis by upregulating or downregulating the expression level of lncRNA MEG3.Methods: We established EAE models induced by MOG35-55 in C57BL/6 mice.We divided the mice into 6 groups: normal group,EAE group,MEG3-CON group,MEG3-RNAi-CON group,MEG3 group and MEG3-RNAi group.We recorded the weight and neurological score of all the mice everyday after immunization.When we established AAV vector,we upregulated lncRNA MEG3 by establishing total length of the sequence of lncRNA MEG3.And we downregulated lncRNA MEG3 by si RNA.We transfected AAV via tail vein injection to upregulate or downregulate the expression level of lncRNA MEG3 in mice 3 days after immunization.At the peak of disease,we conducted flash visual evoked potential(VEP)for mice of all the groups and recorded the latency and amplitude of P1 and P2 wave.At the same time,we conducted optical coherence tomography(OCT)to measured the peripapillary retinal nerve fiber layer(RNFL)thickness.Optic nerves and eyeballs were obtained from mice of all the groups and made into histological sections.Sections were stained by HE to observe the infiltration of inflammatory cells and demyelination in the optic nerve and the structure of retina.Results: After immunization,weights of mice in all groups increased with time,and weights of EAE mice had gone up and down during observation and mice got sick 9-13 days after immunization.Neurological score reached the maximum values several days after onset,maintained for several days and then declined.In histological examination of optic nerve,we found that compared to MEG3-CON group,in MEG3 group the optic nerve fibers were more disordered and there were more inflammatory cells infiltrated among optic nerve fibers and around optic nerve,RNFL swelling was more obvious.On the contrary,compared to MEG3-RNAi-CON group,there were milder swelling of RNFL and less inflammatory cells infiltrated in MEG3-RNAi group.In flash VEP,compared to normal group,P2 latency prolonged(39.15±5.52 ms vs 64.52±8.0ms,P=0.025)in EAE group.Histological and VEP changes showed optic neuropathy in EAE mice and we established experimental optic neuritis model successfully.Compared to MEG3-CON group,P2 latency prolonged significantly in MEG3 group(65.31±13.55 ms vs 93.29±29.94 ms,P=0.003).compared to MEG3-RNAi-CON group,P1 latency(38.73±5.8ms vs 29.12±10.25 ms,P=0.029)and P2 latency shortened significantly(72.85±19.32 ms vs 56.31±7.0ms,P=0.036)in MEG3-RNAi group.In OCT,compared to normal group,temporal RNFL thickness(33.0±1.73?m vs 20.75±6.08?m,P=0.043)were thinner in EAE group,but nasal,superior,inferior and average RNFL thickness had no significant differences.Compared to MEG3-CON group,temporal,nasal,superior,inferior and average RNFL thickness in MEG3 group had no significant differences.Compared to MEG3-RNAi-CON group,temporal,nasal,superior,inferior and average RNFL thickness in MEG3-RNAi group had no significant differences.Conclusion: In EAE mouse model,overexpression of lncRNA MEG3 can aggravate optic nerve and RNFL swelling and inflammatory cell infiltration,prolong P2 latency in flash VEP.Inhibition of lncRNA MEG3 can alleviate optic nerve and RNFL swelling and inflammatory cell infiltration,shorten P1 and P2 latency.The present research illustrates that lncRNA MEG3 can aggravate the optic nerve damage in experimental optic neuritisPart ? The effects of lncRNA MEG3 on differentiation of lymphocytes in experimental optic neuritisObjective: To study the effects of lncRNA MEG3 on differentiation of lymphocytes in experimental optic neuritis by upregulating or downregulating the expression level of lncRNA MEG3.Methods: We established EAE models induced by MOG35-55 in C57BL/6 mice.We divided the mice into 6 groups: normal group,EAE group,MEG3-CON group,MEG3-RNAi-CON group,MEG3 group and MEG3-RNAi group.We recorded the weight and neurological score of all the mice everyday after immunization.When we established AAV vector,we upregulated lncRNA MEG3 by establishing total length of the sequence of lncRNA MEG3.And we downregulated lncRNA MEG3 by si RNA.We transfected AAV via tail vein injection to upregulate or downregulate the expression level of lncRNA MEG3 in mice 3 days after immunization.At the peak of disease We measured the distribution of CD4+ T lymphocytes,CD8+ T lymphocytes,Th1 cells,Th2 cells,Th17 cells,Treg cells in spleen lymphocytes by flow cytometry.Then we calculated the Th1/Th2 ratio and Th17/Treg ratio.Results: Compared to MEG3-CON group,Th17/Treg ratio was higher(0.17 ± 0.19 vs 0.65 ± 0.41,P=0.042)in MEG3 group.Percents of T lymphocytes in spleen lymphocytes,percents of CD4+T/CD8+T lymphocytes in T lymphocytes,percents of Th1/Th2/Th17/Treg cells in CD4+T lymphocytes had no significant differences.Compared to MEG3-RNAi-CON group,percents of Th1 cells in CD4+ T lymphocytes was lower in MEG3-RNAi group(8.05±2.60% vs 2.32 ± 0.96%,P=0.001).Percents of T lymphocytes in spleen lymphocytes,percents of CD4+T/CD8+T lymphocytes in T lymphocytes,percents of Th2/Th17/Treg cells in CD4+T lymphocytes had no significant differences.Conclusion: Overexpression of lncRNA MEG3 can lead to Th17/Treg imbalance.Inhibition of lncRNA MEG3 can inhibit differentiation of spleen CD4+ T lymphocytes towards Th1 cells.The present research illustrates that lncRNA MEG3 can induce Th17/Treg imbalance in experimental optic neuritis.