Dynamic Expression And Function Prediction Analysis Of CircRNA And MRNA During Follicle Development In Chicken

The reproduction performance of hen is dependent on the ovary development,while the ovary function exertion is dependent on the follicular development.Once the sexual maturity onset,a series of development including follicle recruitment,selection hierarchical follicle growth,and then ovulation lastly.Follicle structure consists of granulosa cell（GC）,theca cell（TC）and oocyte.To study the transcriptome expression level of follicle,we performed an RNA-seq to screen the expression profile of circRNA and mRNA in GC and TC during follicle development in Jianghai Yellow Chicken and carried out bioinformatic to analyze the circRNA and mRNA.The main results are as follow:Section one:The granulosa cells of small yellow follicle（SYF）,the smallest hierarchical follicle（F6）and the largest hierarchical follicle（F1）were separated from the same physiological condition of half-sib hens.Then we implemented RNA-seq to screen circRNA and mRNA in GCs3 identified the novel circRNA,performed differentially expressed（DE）analysis and annotated functional enrichment analysis of genes,to predict the putative function that transcripts may involve in.1.We identified a total of 11,641 circRNAs,with 3,742 pervasively distributed on all GCs.The circRNAs distributed on all chromosome,with the longer length of chromosome the more number distributed.The feature of circRNA including genomic distance,flank intron showed the coumon feature,while the other part feature of circRNA length,the main host genes type,number of exons displayed the species or tissue specificity.2.The number of DE circRNAs and mRNA showed different.The number of DE circRNA were from 77 to 380 in different comparison group,while the number of DE mRNA was from 490 to 1,521 in SYFG.vs.F6G and F6G.vs.FlG group.There were 443 genes overlapped from the host genes of DE circRNA and DE mRNA.The results suggest that the two transcripts may perform different regulation model.3.Functional enrichment analysis were carried out to analyzed DE transcripts,which showed that the host genes of DE circRNA may participate in cell growth,metabolism,phosphatidylinositol signaling system,MAPK signaling pathway,the DE mRNA may involve in cell cycle,steroid metabolic pathway,and several pathways related to reproduction.4.According to the competing endogenous RNA（ceRNA）hypothesis,we constructed the circRNA-miRNA network and screened out gga-miR-15a as the target miRNA.The targeting relationship of gga-miR-15a was predicted and the target genes may be involved in cell growth and oocyte meiosis.Candidate gene YWHAH was identified.Section two:Transcriptome analysis was performed to analyze the circRNA and mRNA in theca cells.The experiment animals and samples were from Section one.1.We identified a total of 14,503 circRNAs,with 5,622 pervasively distributed on all development stage.The feature of circRNAs in theca clls was in line with that in granulosa cells.2.The number or type of DE circRNA and DE mRNA showed difference.The number of DE circRNAs was 5,75 and 48 in SYFT.vs.F6T,SYFT.vs.FIT and F6T.vs.F1T,respectively.The number of DE mRNAs was 672,1,214 and 2 in the three group,respectively.There were none genes overlapped from host genes of DE circRNAs and DE mRNA.The results suggested that these two transcripts may exert different regulation function.The expression level of mRNA in SYFT was higher than that in F6T and FIT,illustrating the more complexity of SYFT.3.Enrichment analysis showed that,the DE circRNA may involve in negative regulation of response to cytokine stimulus,lipid localization,and several reproduction pathways and inflammatory reaction.The DE mRNA may play a role in cell cycle,cell development,ECM-receptor interaction,oocyte meiosis and several process that related to reproduction.Section three:The results of high throughput sequencing and DE analysis showed a hot-spot gene,RalGPS2,produced 20 isoforms circRNA,three of them（novel_circ₀027214,novel_circ₀027217 and novel_circ₀027220）showed DE during granulosa cell development,and two of them（novel_circ₀027214 and novel_circ₀027217）showed DE during theca cell growth,all of them was named as circRalGPS2.We run the blast method to the sequence of circRalGPS2 and found that circRalGPS2s consist of public sequence from exon2,exon8 and exon9.Then researches on the polymorphism analysis,expression profile,function prediction and potential mechanism of action were studied.1.We found none mutation in exon2,exon8 or exon9 by PCR-SSCP method,which suggested that circRalGPS2 may involve in biological process through acting as a miRNA sponge.2.The expression level of circRalGPS2 were detected by RT-qPCR,which showed that three circRalGPS2 displayed similar trend in all reproductive tissue with the highest level in ovary stoma and decreased with the ovary advanced.3.We constructed the circRNA-miRNA network and predicted target genes by bioinformatics.A total of 538 potential target genes were obtained.circRalGPS2 may play a role in protein modification process,regulation of signaling,oocyte meiosis,regulation of actin cytoskeleton,ErbB signaling pathway biological process.The putative target genes were ADCY3,PLCB1,CAMK2G,PIK3R1,etc.4.The target site between circRalGPS2 and gga-miR-200a-3p were validated by dual luciferase reporter system.The results showed that the site may be combined but not significant,it is speculated that circRalGPS2 may play an indirect role on gga-miR-200a-3p to regulate follicle development.Section four:Follicle development is dependent on granulosa cell and theca cell and the interdependent of them.In this part,we first studied the DE of circRNA and mRNA in granulosa cell and theca cell,and then performed WGCNA employed the top 25%variance expression data to find the specific module or hub genes related to follicle development.1.A total of 8,127 circRNAs were widely expressed both in granulosa cell and theca cell,with 3,514 and 6,375 expressed specifically,respectively.The distribution and feature of circRNAs in these two cells were similar.A total of 23,769 mRNAs distributed on granulosa cell and theca cell,with 587 and 1,425 expressed specifically,respectively.The GC content of circRNAs in granulosa cell and theca cell were above 60%?and the GC content in theca cells were higher than that in granulosa cell.2.There were large significant of number or type of between circRNA and mRNA in different granulosa cell.vs.theca cell comparative group.A lot of DE circRNAs were obtained in different comparative group while only some DE mRNA detected in SYFG.vs.SYFT group.The up-regulated circRNAs in granulosa eell and theea cell mainly come from protein coding genes,A total of 207 circRNAs were DE transcripts in all comparative group.The highest expression DE circRNA of novel_circ₀002934 is produced from gene RAB11A.The DE mRNA in SYFG.vs.SYFT group were including KCNA1,RPL4,IL7R,etc.3.GO analysis for the DE transcripts showed that the DE circRNA in SYF and FI group were annotated to cell process,cell differentiation,cell metabolism,while the DE circRNA in F6 group were annotated to cellular response to stimulus,biological adhesion biological process.KEGG analysis showed that the DE circRNA mainly involved in FoxO signaling pathway,GnRH signaling pathway,ECM-signaling pathway,progesterone-mediated oocyte maturation.The DE mRNA play a role in cell-cell adhesion,FoxO signaling pathway,PPAR signaling pathway,apoptosis process.4.We performed WGCNA analysis for transcripts with top 25%variance of expression and identified 18 modules.Lastly,six specific modules association with 6 specific stage or cells were obtained by rigorous analysis.The specific modules involve in different GO and KEGG process,including genes ORC5,PIK3CA,PAK1,SOD1,ITGB1,CDC26,PAK2 and MCM6,and etc.A series of genes related to follicle development including MAPK1,GDF9,STAR,DST,SOD2,PSMC1,DH1,ACTC1 and nove1_circ₀004730 were detected by hub gene network.These genes may participate in cell proliferation,differentiation,and apoptosis biological process during follicular development.