| Background: Diabetic retinopathy(DR)is one of the most common complication of diabetes mellitus(DM).DR is also one of the leading causes of blindness in developed countries.As the first symptom of DM complications,DR is used as an indicator of DM complications.DR progression is under strict control at early stage.Mild DR patients rapidly progress to the late stage of DR.The screening of highly sensitive and specific biomarkers of DR initiation and progression is particularly important,which is instructive to the clinical evaluation and precise intervention of DR.Mounting studies have shown that diabetic retinal degeneration(DRN)is an early pathological event in DR.Identifying the key molecules mediating DRN is of great significance for developing new therapeutic targets for DRN.From the clinical point of view,it is also necessary to prevent the occurrence of diabetic macular edema(DME)and proliferative diabetic retinopathy(PDR)for the timely application of neuroprotective drugs in DR patients.DRN is characterized by reactive gliosis of Müller cells and apoptosis of retinal neurons.The published animal studies on the mechanism of DR mostly use streptozotocin(STZ)induced T1 DM rat after DM onset 4-10 weeks.At the very early stage of DM(within one week),there is no relevant report on the molecules that can trigger DRN induced by hyperglycemic stress.micro RNAs(miRNAs)are a class of evolutionarily conserved non-coding small RNA molecules with a size of about 22 nucleotides.miRNAs regulate gene expression by inhibiting m RNA translation or inducing m RNA degradation at the post transcriptional level.miRNAs are involved in the birth,growth,apoptosis,differentiation,metabolism and immune response of organisms in specific tissues and stages.miRNAs as a biomarker of DR are based on microarray data of the serum,aqueous humor and vitreous from DR patients.Due to most of the samples from DR、PDR patients,the diagnostic value of miRNAs for DRN is limited.There also are many studies focused miRNAs on the pathogenesis of DR model,including DR cell model with high glucose,glyoxal and hypoxia treatment,oxygen induced retinopathy model simulating partial DR vascular disease and DM model(spontaneous T1 DM mice(Ins2Akita)and T2 DM mice(db/db).Currently only one study found miR-200 b targeted oxidation resistance 1(OXR1)and mediated the pathological process of DR in Müller cells.miR-7 is one of the most abundant miRNAs in brain and retina.The mature miR-7 sequences in different species are completely conserved.Interestingly,there are only 9 target genes of miR-7 in the intersection of drosophila and human,suggesting that miR-7 has distinct functions in mammals.miR-7 promotes the differentiation of photoreceptor cells in drosophila melanogaster,and miR-7 in late retinal progenitor cells negatively regulates the Müller differentiation.We propose that miR-7 may have an important role in regulating Müller’s gliosis.Objective: Whether miR-7 is involved in DRN has not been reported.Taking the hypothesis driven approach,we systematically studied the role of miR-7 in DRN rat with STZ induction and explored its molecular mechanism.This work may provide novel biomarker and novel therapeutic targets for DRN.Methods: In the first part,the role of miR-7 in T1 DM retina and its functional target gene was identified.In vivo experiment: the expression pattern of miR-7 in DR retina was detected by laser capture microdissection(LCM),in situ hybridization and quantitative reverse transcription polymerase chain reaction(q RT-PCR).AAV2/8-miR-7 was injected into the subretinal space to intervene DR(Gain of Function)and AAV2/8-miR-7 was injected into the subretinal space of normal SD rats(Loss of Function).The retinal structure and visual function were analyzed by Hematoxylin & Eosin(HE)staining,Western blot,real-time q RT-PCR,immunofluorescence and electroretinogram(ERG).Finally,miR-7 was subretinally injected into the rd1 mice and to clarify the universality of the inhibiting gliosis of miR-7.The protective effects of AAV2/8-sh Gmfb on DR rats were detected by ERG,immunofluorescence and Western blot.In vitro experiment: q RT-PCR was performed to detect the expression of miR-7 in different retinal cell lines and primary cells.p Surper-miR-7 was transfected into r MC-1 and differentially expressed genes(DEGs)were analyzed by microarray analysis,gene ontology(GO),signal pathway analysis and q RT-PCR validation of some DEGs of interest.By cloning the 3 ’untranslated region of glia maturation factor beta(Gmfb),dual luciferase reporter gene assay,q RT-PCR and Western blotting,the interaction between miR-7 and the target gene Gmfb was confirmed.Using q RT-PCR the expression of miR-7 and Gmfb m RNA were examined with high-glucose(HG) treatment in r MC-1 cells.Western Blot was performed to detect the protein levels of GMFB in r MC-1 with high-glucose treatment.The mitogen-activated protein kinase(MAPK)signaling pathway was detected in r MC-1 with high-glucose treatment.The expression of miR-7 and Gmfb m RNA were examined in r MC-1 with U0126 treatment.Based on the fact that one gene is regulated by multiple miRNAs,Targetscan is used to predict other miRNAs regulating Gmfb.Due to the abundance of miR-194-5p in retina,agomir-194-5p was subretinally injected into DR rats to examine the visual function and the gliosis of Müller cells.In the second part,the role of GMFB in DR and its molecular mechanism were investigated.In vivo experiment: Western blot,Enzyme-Linked Immunosorbent Assay(ELISA)and immunofluorescence were used to detect the expression pattern of GMFB in retina and the content of GMFB in vitreous of DR rats.AAV2/8-GMFB was injected into the subretinal space in normal SD rats.The changes of retinal structure and function were detected by TUNEL,Western blot,q RT-PCR,immunofluorescence and ERG.ELISA was carried out to detect the content of GMFB in different stages of DR.In vitro experiment: r MC-1 was treated with high glucose(HG),and the expression of GMFB was detected by q RT-PCR,Western blot and immunofluorescence,respectively.The concentrated supernatants of r MC-1 with high glucose treatment were assayed for GMFB secretion.Overexpression of Gmfb in Müller cells was assayed for effects on cell viability,MAPK signaling pathway and glial fibrillary acidic protein(GFAP)expression.GMGB treated r MC-1 to detect the expression of solute carrier family 1 member 3(Slc1a3)and glutamine synthetase(GS).m RFP-GFP-LC3 virus infected 661 W cells.661 W cells were treated with GMFB.Phosphorylated protein chip assay,immunofluorescence and Western blotting were performed to detect the changes of signaling pathways possibly caused by GMFB treatment.Results: In the first part,the expression level of miR-7 reached its peak at 3 weeks after birth,and then decreased.In situ hybridization showed that the expression of miR-7 was high in both adult rat and mouse retinas.miR-7 was significantly down-regulated at the early stage of STZ induced T1 DM,and maintained at a low level with the progression of the disease.The overexpression of miR-7 effectively protected the structure and function of diabetic retina.The subretinal injection of miR-7 sponge in normal SD rats remarkably induced reactive gliosis of Müller cells.From down-regulated DEG profile in miR-7 expressed Müller cells,GMFB was selected as candidate target of miR-7.Using dual luciferase reporter gene assay,Western Blot,q RT-PCR,immunofluorescence and computing algorithm using Miranda,Gmfb was identified and confirmed to be direct functional target of miR-7.In vitro,the expression of miR-7 decreased in the early stage(within 2 hours)of r MC-1 with HG treatment,and the expression of Gmfb m RNA was up-regulated,showing a negative correlation in expression pattern between miR-7 and Gmfb in response to HG treatment of r MC-1 inhibited the ERK signaling pathway,the inhibition of ERK signaling pathway in r MC-1 led to a decreased expression of miR-7 and increased expression of Gmfb m RNA.At the early stage of DR,miR-7 inhibited reactive gliosis of Müller cells by targeting Gmfb to protect diabetic retina.The rapidly degenerative retina of rd1 mice showed significant gliosis of Müller cells,and the subretinal administration of miR-7 significantly inhibited gliosis of Müller cells,suggesting that the anti-gliosis of miR-7 was universal.miR-194-5p was predicted to target Gmfb using Targetscan software.However,the subretinal injection of agomir-194 failed to protect visual function in DR rat.We speculated that Gmfb was not the target of miR-194-5p and the interaction between miR-194-5p and Gmfb was not further investigated.In the second part,the immunoreactivity of GMFB reached the peak at 3 weeks after birth and decreased at 4 weeks after birth.The intensity of immunofluorescence was limited to GCL.In diabetic retina,GMFB increased significantly in the first week of DM onset,then maintained at a high level,and decreased in the eighth week,while GFAP expression began to increase in the second week of DM,suggesting that GMFB was more sensitive than GFAP to reflect reactive gliosis of Müller cells.ELISA was used to detect the GMFB in the vitreous of DR rats at different stages of onset.The results showed that the content of GMFB increased significantly on the first day of DM onset,and the high level of GMFB in the vitreous maintained until the 6th week after DM onset,and then decreased.There was no significant difference between the normal control group and the group at the 8th week after onset.The vitreous samples from DR patients at different clinical stages were detected by ELISA.The result showed that the content of GMFB increased in patients with mild DR and decreased in PDR vitreous.The results from the animal model of DR and DR patient samples support that GMFB can be used as a biomarker for the initiation and development of DR.The overexpression of GMFB by subretinal injection of AAV2/8-Gmfb virus caused retinal thickness thinning,visual function decline and retinal ganglion cell apoptosis.GMFB treatment of r MC-1 cells resulted in the down-regulation of GS and Scl1a3,which would increase the content of extracellular glutamate.Ganglion cell apoptosis was related to the elevated glutamate.In GMFB overexpressed retina,outer nuclear layer(ONL)thickness was markedly thinner when compared with empty virus group,and visual function decreased.GMFB secretion in concentrated supernatant from HG treated r MC-1 was detected.The overexpression of Gmfb in Müller cells significantly reduced cell viability,up-regulated GFAP expression,inhibited ERK pathway and activated p38 pathway.Knockdown of Gmfb significantly reduced high glucose-induced GFAP expression and increased p-ERK levels.r MC-1 cells were treated with GMFB for protein chip detection and m RFP-GFP-LC3 adenovirus labeled 661 W.Combined with the differentially regulated phosphorylation sites and signal intensity,m TOR pathway ranked first.The autophagy flux was impaired and p62 increased after GMFB treatment in 661 w cells.GMFB treatment led to the autophagy in photoreceptor cells via activating m TOR signal pathway,resulting in photoreceptor cell death.The detailed signaling pathway merits deep exploration.Conclusion: miR-7 inhibited gliosis of Müller cells by targeting Gmfb to protect the structure and function of diabetic retina.GMFB can be used as a biomarker for the initiation and development of DR.The overexpression of Gmfb can induce retinal degeneration.Intervention of GMFB can protect the structure and function of diabetic retina.This work provides a novel biomarker and therapeutic target for the diagnosis and treatment of DR.Small molecule inhibitors of GMFB deserve further investigation. |
