Screening And Identification Of CircRNA In Xiang Pig Ovary

circular RNAs(circular RNAs,circRNAs)are a large class of non coding endogenous RNAs and covalently closed single-stranded RNAs,which originate from reverse splicing of pre-mRNAs during the transcription process,without 5' caps and 3'ploy(A)tails.It is not easily degraded by RNase and high stability since its special covalent closed structure.circRNAs can served as molecular sponges of miRNAs,which regulate the silencing effect of target genes.circRNAs not only cis-regulate the expression of parental genes and alternative splicing,but also act as protein translators.At present,many studies have shown that circRNAs regulated follicle maturation,the proliferation and apoptosis of granulosa cells,and the synthesis of progesterone through the circRNAs-miRNAs-mRNAs axis,thereby regulating the estrus cycle of pigs.However,it is still unclear that circRNAs play a role in regulation the estrus of Xiang pigs in the ovarian tissues.In order to analyze the mechanism of circRNAs of regulating ovarian function,we selected four head of Xiang pigs with estrus and diestrus,which were around six month as the research materials.We selected candidate circRNAs through RNA-seq technology and bioinformatics methods,and analyzed the differentially expressed circRNAs.Then we predicted and analyzed the miRNAs binding sites of the differentially expressed circRNAs and the target genes of the differentially expressed of miRNAs.We perfomed for differentially expressed target genes enrichment of function analysis to obtain key target genes related to ovarian function.Therefore,a circRNAs-miRNAs-mRNAs network interaction of regulation diagram was constructed with differentially expressed target genes related to ovarian function.Further,it was possible that we realized the mechanism of circRNAs regulating the estrus of Xiang pigs.Our results provided a novel perspective for the mechanism of estrus of Xiang pigs.The main results of this study are as follows:(1)In this study,we selected 8937 candidate circRNAs,which were mainly derived from exons,and partly derived from introns and intergenic regions.circRNAs were distributed on each chromosome,with the most on chromosome 1.Many of the circRNAs have a low cyclization rate,mainly between 1%-10%.The average length of circRNAs of estrus group and diestrus group was 258 nt and 234 nt in the ovarian tissues of Xiang pigs,respectively,the length distribution of each stage of circRNAs were consistent.Most genes could produce only one circRNA.(2)The functions of host genes of candidate circRNAs were significantly enriched in ATP binding,ubiquitin-dependent protein catabolic process,metal ion binding,protein serine/threonine kinase activity,RNA binding,GTPase activator activity,protein phosphorylation,intracellular signal transduction,microtubule binding,histone binding,cytoplasm,positive regulation of GTPase activity and other terms.Further,it was associated with cell cycle,cellular senescence,steroid biosynthesis,prolactin signaling pathway,Hippo signaling pathway,TGF-beta signaling pathway,ErbB signaling pathway,phosphatidylinositol signaling system,Wnt signaling pathway,Ubiquitin mediated proteolysis,mTOR signaling pathway and so on.(3)We analyzed the expression values(RPM values)of circRNAs in the ovarian tissues of Xiang pigs with the estrus group and diestrus group.Respectively,it was considered be highly expressed that the circRNAs of RPM values greater than 1000.Among the top 20 circRNAs expressed in the ovarian tissues of Xiang pigs of the estrus and diestrus,12 circRNAs co-existed between the two groups,including circ?0412,circ?1114,circ?1952,circ?2414,circ?2785,circ—3095,circ?4508,circ?5579,circ?5597,circ?5843,circ?6967,circ?8670.It was mainly related to gamete production,cell cycle regulation,cell proliferation,cell cycle binding protein,prostaglandin biosynthesis,protein phosphorylation,etc.(4)Differential analysis showed that 80 circRNAs were differentially expressed,among which 76 circRNAs were up-regulated and 4 circRNAs were down-regulated in the ovarian tissues of the estrus of Xiang pigs.The miRNA binding sites were predicted and analyzed that were the differentially expressed circRNAs.Among the differentially expressed circRNAs,33 differentially expressed circRNAs had binding sites with 14 differentially expressed miRNAs,and 14 differentially expressed miRNAs had 296 differentially expressed target genes.Functional enrichment analysis of 296 differentially expressed target genes mainly included positive regulation of cell migration,cytokine-mediated signaling pathway,metallopeptidase activity,peptide hormone binding,mitotic spindle organization,mitotic DNA replication checkpoint,intracellular signal transduction,calcium ion binding,cadherin binding,ATP binding,positive regulation of MAPK cascade,mitotic G2 DNA damage checkpoint,protein kinase C binding,intracellular receptor signaling pathway,positive regulation of G2/M transition of mitotic cell cycle,positive regulation of cell cycle,cell division,positive regulation of cell division,hormone-mediated signaling pathway and so on.Further,it was associated with cell cycle,cell apoptosis,oocyte meiosis,progesterone-mediated oocyte maturation,PI3K-AKT signaling pathway,Foxo signaling pathway,MAPK signaling pathway,cAMP signaling pathway,AMPK signaling pathway,calcium signaling pathway,cellular senescence,Hippo signaling pathway,oxytocin signaling pathway,p53 signaling pathway,prolactin signaling pathway,folate biosynthesis,mTOR signaling pathway,steroid biosynthesis and other paythways.(5)The differentially expressed genes with interaction relationships were analyzed and enriched into the pathways related to ovarian function in the constructed circRNAsmiRNAs-mRNAs regulatory network,so we obtained the crucial target genes,which were differentially expressed related to ovarian function,and then the differentially expressed circRNAs-miRNAs-mRNAs regulatory network interaction diagram was constructed that related to ovarian function of Xiang pigs.It was included 32 differentially expressed circRNAs,13 differentially expressed miRNAs,and 30 differentially expressed genes.The constructed regulatory axis preliminarily indicated that circRNAs were involved in multiple signaling pathways to regulate multiple biological functions in the ovarian tissues of Xiang pigs,and furtherly regulate the estrus cycle of Xiang pigs.(6)Seven randomly selected circRNAs were identified in the ovarian tissues of Xiang pigs,including circ?CSPP1,circ?PAN3,circ?CDK13,circ?FANCL,circ?IGF 1R,circ?SUGCT,and circ?ELF2,which was consistent with analysis results of RNA-seq.