Based On The Whole Transcriptomic Firstly Screening Retinal Plasticity Expression Profiles Of The Form Deprivation Amblyopia In Tree Shrews

Objectives:We analyzed the changes and trends of non-coding RNA expression in amblyopia formation and recovery during the visual developmental period in an animal model of amblyopia by high-throughput whole-transcriptome sequencing of retinas of tree shrews with form deprivation amblyopia.In this way,the link between non-coding RNAs and visual plasticity can be investigated to further explore the plasticity regulation mechanism of amblyopic retina and provide positive impact for early diagnosis and molecular targeting therapy of amblyopia.Methods:1.Model establishment and evaluation: 36 tree shrew pups with unopened eyes at 18 days were selected and randomly divided into 3 groups:（1）monocular form deprivation amblyopia model group: upper and lower eyelids of the right eye were sutured for 2 months;（2）form deprivation amblyopia recovery model group: upper and lower eyelids of the right eye were sutured for 1 month and then opened,and then the left eye was reversed and sutured for 1 month;（3）normal control group: both eyes were kept normally for 2 months without any treatment;After the model was established,the material was taken and examined by HE staining and transmission electron microscopy.2.Whole-transcriptome sequencing and data analysis: After the model was successfully established,retinal tissues from three groups of samples were collected for whole-transcriptome sequencing to screen out m RNAs,lncRNAs,as well as mi RNAs and circRNAs involved in the formation and recovery of amblyopia,and then the top 10 differentially expressed RNA target genes were analyzed for GO/KEGG functional enrichment.3.q RT-PCR quantification: Real-time quantitative polymerase chain reaction（q RT-PCR）was performed to verify the relative expression levels of some m RNAs,lncRNAs and mi RNAs with significant differences.The 2-ΔΔCt method was applied to calculate the fold of difference between the three groups of samples.Results:1.Model stablishment and evaluation: HE staining results showed that the number of retinal ganglion cells and nerve fiber layer thickness were significantly reduced in the amblyopic group with suture for 2 months compared with the normal control group,and the number of ganglion cells and nerve fiber layer thickness increased in the amblyopic recovery group with reversed suture,but could not be restored to normal level.The results of transmission electron microscopy showed that the ganglion cells in the amblyopic group had irregular nuclear membrane depression,reduced cytoplasmic density,significantly reduced number of mitochondria,and some mitochondria were swollen compared with the normal group;the ultrastructure of retinal ganglion cells in the recovery group was similar to that of the normal group,with slightly irregular nuclear membrane and some mitochondria were mildly swollen.The ultrastructure of retinal ganglion cells in the recovery group was similar to that of the normal group,with slightly irregular nuclear membrane and some mitochondria swelling.2.Analysis of whole transcriptome sequencing results:m RNA: 2 months after suture,995 m RNAs were identified in the amblyopic group compared with the normal group,among which 539 m RNAs were up-regulated and 456 m RNAs were down-regulated;874 m RNAs were identified in the amblyopic group compared with the recovery group,among which 500 m RNAs were up-regulated and 374 m RNAs were down-regulated.The total number of up-and down-regulated m RNAs in the three groups of samples analyzed by Wayne diagram was 282,and this m RNA was involved in both amblyopia formation and amblyopia recovery.The most significantly different m RNA was HAUS7,whose expression level was down-regulated in the amblyopic group and up-regulated in the recovery group.lncRNA: After 2 months of suturing,803 lncRNAs were identified in the amblyopic group compared with the normal group,of which 444 lncRNAs were up-regulated and 359 lncRNAs were down-regulated.795 lncRNAs were identified in the amblyopic group compared with the recovery group,of which 469 lncRNAs were up-regulated and 326 lncRNAs were down-regulated.The total number of up-and down-regulated lncRNAs in the three groups of samples by Wayne diagram analysis was 258,and this lncRNA was involved in both amblyopia formation and amblyopia recovery.The most significantly different lncRNAs were TCONS₀0196603 and TCONS₀0165200,whose expression levels were down-regulated in the amblyopia group and up-regulated in the recovery group.mi RNA: After 2 months of suturing,18 mi RNAs were identified in the amblyopic group compared with the normal group,of which 11 mi RNAs were up-regulated and 7 mi RNAs were down-regulated.17 mi RNAs were identified in the amblyopic group compared with the recovery group,of which 10 mi RNAs were up-regulated and 7 mi RNAs were down-regulated.The total number of up-and down-regulated mi RNAs in the three groups of samples by Wayne diagram analysis was 5.This mi RNA was involved in both amblyopia formation and amblyopia recovery.The most significantly different mi RNAs were tch-mi R-200b-3p,whose expression levels were up-regulated in the amblyopic group and down-regulated in the recovery group.circRNA: 2 months after suturing,185 circRNAs were identified in the amblyopic group compared with the normal group,of which 103 circRNAs were up-regulated and 82 circRNAs were down-regulated.231 circRNAs were identified in the amblyopic group compared with the recovery group,of which 101 circRNAs were up-regulated and 130 circRNAs were down-regulated.The total number of up-and down-regulated circRNAs in the three groups of samples by Wayne diagram analysis was 32,which were involved in both amblyopia formation and amblyopia recovery.The most significantly different circRNAs were novel_circ₀030400 and novel_circ₀020768,and these circRNAs were down-regulated in the amblyopia group and up-regulated in the recovery group.Target gene GO/KEGG enrichment analysis: The results showed that the formation and recovery of amblyopia may be related to the cytoplasmic membrane structure and protease activity.3.q RT-PCR quantification: Selected differentially expressed non-coding RNAs were subjected to q RT-PCR,and those with expression trends consistent with the sequencing results were as follows:The expression level of m RNA: HAUS7 in the retinal tissue of form-deprived amblyopia was significantly lower than that of the normal group（P < 0.05）;and its expression water would rebound in the amblyopic recovery group with reversed suture（P < 0.01）.The expression level of mi RNA: tch-mi R-200b-3p in the retinal tissue of form-deprived amblyopia was significantly higher than in the retinal tissue of normal group（P < 0.01）;its expression level decreased in the amblyopia recovery group with reversed suture（P < 0.05）.The expression level of lncRNA : TCONS₀0196603 and TCONS₀0165200 in the retinal tissues of form-deprived amblyopia were significantly lower than those in the normal group（P < 0.01）;their expression levels were elevated in the amblyopic recovery group with reversed sutures（P < 0.01）.Among them,TCONS₀0196603recovered beyond normal levels,while TCONS₀0165200 failed to recover to normal levels.Conclusions:1.The retina of form-deprived amblyopia has a certain degree of plasticity during the critical period of visual development.2.Functional enrichment analysis involved in amblyopia formation and recovery revealed that a large number of cytoplasmic membrane structures,and related protease activities may play a critical role in the development and recovery of amblyopia.3.The screened HAUS7 m RNA,tch-mi R-200b-3p,and TCONS₀0196603/TCONS₀0165200 may be involved in the activation and inhibition of neuronal and synaptic plasticity,which are closely related to the development of amblyo pia disease.