Identification And Analysis Of The Retina Regeneration Related MiRNAs After Optic Nerve Injury In Zebrafish

As an important small molecule of the large non-coding RNA family which can regulate gene expression levels after transcription and after years of exploration since being found,miRNA has been gradually understood.An increasing amount of evidence shows that,miRNA plays an important role in different neurobiological processes,such as cell proliferation,growth,differentiation,apoptosis,and neural activities.The functions and roles of miRNA in neural regulation have been acknowledged in many aspects and also have been found to be related with a large amount of central nervous system diseases.Studies show that,after being injured,the regeneration ability of optic nerve of higher animal,such as mammals,and human beings is very limited and the expression of miRNAs undergo significant changes at different times of the regeneration of damaged central nervous system.Studies have shown that the central nervous system of fish and amphibians has the ability to regenerate,and as part of the central nervous system,the optic nerve is characteristically easy to progress in the experiments.Therefore,in this experiment,the zebrafish was used as a model to explore the associated miRNAs expression changes and regulatory functions during the regeneration process of the damaged optic nerve,which will help to understand the molecular regulation mechanism of miRNA in the central nervous system injury and regeneration process.As a new field of research in neurology,the relationship between miRNA and neurological diseases has attracted people's attention.According to current research,in order to further promote the understanding of the relationship between miRNA and regeneration function of damaged optic nerve,in this research,we constructed A,B,C,D four small RNA c DNA libraries,which are associated with the zebrafish optic nerve damage,(group A as the control group,group B,C,D as the three experimental groups,among which group B is the group after one-day optic nerve injury,group C is the group after four-day optic nerve injury,group D is the group after seven-day optic nerve injury).After conducting high-throughput sequencing and subsequent bioinformatics analysis on these four small RNA libraries,miRNAs in different periods of optic nerve damage were identified.Besides,identified miRNAs were selected,and their expression profiles at different stages of injury were further described by real-time PCR analysis,so as to describe the relationship between miRNA and the repair of nerve injury with variation in abundance expression.The principal research results are as follows:1.In four sRNA libraries of A,B,C and D groups,17,839,945 original sequences,15,607,126 original sequences,15,398,526 original sequences and 16,771,780 original sequence were respectively identified.After removal of the low-quality readings,linker sequence,pollution and so on,sequences of high-quality reading accounted for respectively98.74%,98.94%,98.94% and 98.87% of the original readings in the four libraries.In addition,among the high-quality readings of A,B,C and D four libraries,the clean reads,greater than or equal to 17 nt,respectively,accounting for 66.51%,75.37%,73.66%,72.72%.2.Compared with miRNA sequences of mature zebrafish,a total of 204 kinds of conserved miRNAs were identified in four libraries.193,199,196,199 kinds of conserved zebrafish miRNAs were respectively identified in the libraries of group A,B,C and D.After analysis,189 of them were shared by four libraries;three of them only existed in group A;one of them only existed in group B and two only in group C;one of them were shared in group A and B,group A and C,group A and D as well as group C and D;two of them were shared by group A,B and C;one shared by group A,B and D;two of them shared by group A,C and D.3.After analyzed by Mireap software,a total of 23 kinds of new miRNAs were found in four sRNA libraries.Seven of them were expressed in all of these four libraries,four were only expressed in the control group,three of them were only expressed in group B,one was only expressed in group C and one was only expressed in group D.Two of them were only expressed in group A,C and D,and other four kinds of miRNA were only expressed in different periods as different combinations.4.The novel miRNAs,identified in these four libraries,were of different amounts of expression in each library,as the expression of some miRNAs is evidently in the dominant position in the library.The abundance of different miRNAs varies greatly,which vary from the counts of a few rare miRNAs to millions of readings the most abundant miRNAs;for example,mi R-21,mi R-22 and mi R-99 showed a high-abundance expression of over150,000;the readings of mi R-124,mi R-129,mi R-184,mi R-203 and mi R-738 were only over 10,000,mi R-132;and the Reads of mi R-15,mi R-206,mi R-7,and mi R-92,were only a few thousand.5.After screening of the differential expressions of novel miRNAs in the four libraries,it was found that 48 miRNAs were expressed differently in libraries A and B,47 miRNAs were expressed differently in libraries A and C,a total of 55 miRNAs were expressed differently in libraries A and D,a total of 43 miRNAs were expressed differently in libraries B and C,37 miRNAs were expressed differently in libraries C and D,and a total of 42 miRNAs were expressed differently in libraries C and D.6.Compared to the referenced sequence of zebrafish,a total of 24 kinds of highly conserved miRNAs were forecast to be matched with 8085 target genes.After the GO enrichment analysis and KEGG pathway analysis for these predicted target genes,the correlation between the functions and pathways of these conserved miRNAs and the regeneration of optic nerve was verified.