Identification And Function Study Of CircRNA In Medaka Gonads

Medaka(Oryzias latipes)is a classic model animal in gonadal development and germ cell research due to its transparent embryos,short sexual maturity cycles(one generation at 3 months),small individuals(2-3 cm),fast growth,and small genomes.Unlike most fish,medaka has a diploid chromosome structure and sex-determining genes Foxl3 and Dmy.At present,embryonic stem cell lines have been successfully established,which has laid a solid foundation for subsequent research on gene function and non-coding RNA.As a new molecule for gene regulation,circRNAs have attracted the attention of researchers in recent years.CircRNAs splice from pre-m RNAs through reverse splicing to form covalently closed loops.Compared with linear RNAs(such as miRNAs and lncRNAs),circRNAs are more stable and not easily degraded by RNase R.CircRNAs are divided into three types: exon circRNAs,intron circRNAs(ci RNAs)and exonintron circRNAs(EIci RNAs).Most exon circRNAs act as miRNA sponges in the cytoplasm,while EIci RNAs and ci RNAs are regarded as regulators of host gene transcription in the nucleus.These circRNA categories are active molecules in gene expression regulatory networks.CircRNAs play an important role in the development of gonads and the occurrence of germ cells.Recently,a large number of circRNAs have been detected in Xenopus oocytes,mouse and human embryos,human granulosa cells and mouse sperm cells.Studies have found that most circRNAs expressions are developmental stage-specific and dynamically regulated,and some circRNAs have a regulatory role in the process of oocyte production.However,circRNA in medaka gonads has not been reported,and the function of circRNA in medaka gonads is unknown.In the previous research of our laboratory,the medaka gonadal miRNA library has been constructed and identified,and the regulation of ddx3 x expression by miR-26 has been reported.Therefore,the identification and functional study of circRNA in medaka gonads will provide new insights into the reproductive development of medaka.In this study,high-throughput sequencing technology was used to sequence the ovary and testis of medaka,1157 and 1570 circRNAs were obtained,respectively.Two types of circRNAs were identified,exon-type circRNAs and intron-type ci RNAs,among which exon-type circRNAs accounted for 93.1% of total circRNAs.Most circRNAs consisted of 2-4 exons and were 200-900 nt in length.There were 24 circRNAs differentially expressed in the ovary and testis,of which 12 circRNAs were up-regulated in the testis and 12 circRNAs were down-regulated.The differentially expressed parental genes of circRNAs participated in the oocyte meiosis signaling pathway,homologous recombination signaling pathway,Hippo signaling pathway and Ras signaling pathway.CircRNAs perform sponge function by adsorbing miRNAs is one of the recognized biological functions.Therefore,this study predicted the potential binding of circRNA-miRNA through Targetscan,Mi Randa and RNA22 constructed a circRNA-miRNA network through Cytoscape,and found that differentially expressed circRNAs contained 1-7 miRNA binding sites.9 differentially expressed circRNAs in gonads were randomly selected for q RT-PCR verification,which confirmed the reliability of the RNA-seq results.At the same time,it was found that circ452 was highly expressed in the testis and circ880 was highly expressed in the ovary.In addition,circ880 was predicted to participate in circ880/ miR-375-3p/dnd signal axis.Therefore,circ452 and circ880 were selected to further explore the cell location by gonadal in situ hybridization.It was found that the signal of circ452 was abundant in spermatocytes and weakened with the progress of meiosis.The signal of circ880 was abundant in oocytes and spermatogonia in stage I-IV,and decreased with the development of cells.Subsequently,the binding relationship between circ880 and miR-375-3p,miR-375-3p and dnd was verified by dual luciferase reporter gene detection technology.Next,si RNA interference was performed on circ880,and it was found that the expression of miR-375-3p increased and the expression of dnd decreased,indicating the existence of circ880/miR-375-3p/dnd signal axis.Overall,this study first supplied circRNA libraries in medaka gonads,which provided data support for exploring the reproductive and germ cells,and laid the foundation for the study of circRNA regulation and molecular mechanisms in gonads.