| Objective:Based on the high-throughput sequencing data of peripheral blood of patients with non-arteritic anterior ischemic optic neuropathy(Non-arteritic ischemic optic neuropathy,NAION),bioinformatics was used to analyze the changes of miRNAs expression profile and verify the role and molecular mechanism of miR-216a-5p in anoxic RGC-5 apoptosis.Methods: 1.Elbow vein blood samples from 8 patients with NAION and 6 healthy controls were collected.RNA was extracted from peripheral blood cells and analyzed by high-throughput sequencing to screen differentially expressed genes.2.Bioinformatics analysis was used to analyze transcripts and gene expression,differential expression analysis,differential gene enrichment analysis and interaction network analysis,and miRNA length,base preference,expression analysis,differential expression analysis and target gene enrichment analysis.3.The expression levels of differential genes in blood of12 patients with NAION and 12 healthy controls were verified by real-time quantitative polymerase chain reaction(QRT-PCR).4.The hypoxic injury model of RGC-5 was established.The effect of hypoxia on cell viability was detected by cell counting kit(CCK-8),and the expression level of differential miRNA was detected by QRT-PCR technique.5.After transfection to inhibit the expression of miR-216a-5p,apoptosis was detected by MTT assay,and the mechanism of the effect of down-regulation of miR-216a-5p on apoptosis of hypoxic RGC-5 was analyzed by Western blotting(WB).6.The plasmid was constructed,and the targeted regulation of miR-216a-5p and target gene Dazap1 3’UTR region was verified by double luciferase gene reporting experiment.Results: 1.A total of 60756 genes were obtained by high-throughput sequencing,including 57952 known genes and 2804 predicted new genes,accounting for 95.4% and4.6% of the total,respectively.The number of GO annotated by new genes was 369 and the number of KEGG was 17.There were 608 significant differential genes,including306 up-regulated and 302 down-regulated,and 1533 differential transcripts,including836 up-regulated and 697 down-regulated.In the differential gene enrichment analysis,the number of up-regulated GO was 23 and down-regulated GO was 85,up-regulated KEGG was 22 and down-regulated KEGG was 2.The enrichment analysis of differential genes was mainly concentrated in cell composition,metabolism,development,immunity,localization and catalytic activity,transporter activity,molecular sensor activity and so on.KEGG analysis showed that differentially expressed genes were mainly involved in metabolic pathways such as MAPK signal pathway and Chagas disease.2.A total of 149 differentially expressed miRNA were screened,of which 18 were up-regulated and 15 were down-regulated.In GO enrichment analysis,differentially expressed miRNAs is mainly enriched in organelle bioadhesion,biological regulation,cell development,growth and immune system,synaptic partial binding,transcription factor activity,catalytic activity,transporter activity and so on.KEGG pathway enrichment analysis explores a variety of metabolic processes,including vasopressin regulation,aldosterone synthesis,adrenergic signal transduction system,epithelial signal transduction,sphingomyelin signal transduction,osteoclast differentiation and MAPK signal transduction.3.The results of QRT-PCR verification showed that miR-6859-5p and miR-216a-5p were significantly up-regulated and miR-3607-5p and miR-4690-3p were significantly down-regulated in peripheral blood of patients with NAION.4.Cobalt chloride could cause hypoxia and increase the apoptosis rate of RGC-5,which was significantly higher than that of the blank control group(P < 0.05).The results of MTT showed that the degree of hypoxia and apoptosis rate increased with the increase of cobalt chloride concentration,and the apoptosis rate was positively correlated with the concentration of cobalt chloride.QPCR results showed that the expression level of miR-216a-5p increased significantly with the increase of cobalt chloride concentration in the range of 0 ～ 700 μ M,which was significantly higher than that in the control group(P< 0.05).5.After transfection of miRNA inhinbitor,the expression of p Trk B,p Akt,p Erk1/2 protein increased,the expression of caspase-3 and caspase-9 down-regulated,the expression of anti-apoptotic protein Bcl-2 up-regulated,the expression of pro-apoptotic protein Bax down-regulated,and the apoptosis rate of RGC-5 decreased.6.After transfection of miR-216a-5p mimic,the activity of fluorescein in 3’UTR region of wild-type Dazap1 decreased significantly,but there was no significant change in mutant and other groups,which confirmed that Dazap1 was the target gene of miR-216a-5p.Conclusion: 1.The results of high-throughput sequencing showed that the gene expression profile in peripheral blood of patients with NAION changed significantly compared with the control group,and the differentially expressed genes may be involved in multiple signal pathways in the occurrence and development of NAION.2.In a certain range,the degree of anoxic apoptosis and the expression of miR-216a-5p in RGC-5increased with the increase of cobalt chloride concentration.3.Down-regulation of miR-216a-5p can inhibit RGC-5 apoptosis and protect cells by activating Trk B pathway and Caspase pathway.Dazap1 is the target gene of miR-216a-5p. |
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