Preliminary Study On The Regulation Mechanism Of Circular RNA On The Ovary Maturation Of Cynoglossus Semilaevis

With the rapid development of flatfish farming,the half-smooth tongue sole Cynoglossus semilaevis has gradually become one of the ideal marineculture fish in the northern coastal areas of China.In recent years,the significant breakthroughs have been made in the research of artificial breeding technology and reproductive regulation of Cynoglossus semilaevis.However,the reproduction dysfunction of half-smooth tongue sole often occurs in the actual production practice,which affects the healthy development of seedling production and aquaculture.Therefore,it is of great significance and application value to further study the reproductive regulation mechanism of Cynoglossus semilaevis.Circular RNA(cicular RNA,circ RNA)was first discovered in the 1990 s.However,due to the limited technology and knowledge for circular RNA,it was regarded as a type of junk RNA molecule at that time.Therefore,circular RNA has not been fully and thoroughly studied.In 2012,circular RNA was first demonstrated in gene expression programs in human cells.And the circular RNA molecule is not a linear RNA molecule with universal characteristics.With the development and wide application of high-throughput technology,researchers are finding circRNAs in a growing number of species in recent years,but there are still few reports in the aquaculture.In this study,high-throughput sequencing technology was used to construct the c DNA library of the brain,pituitary,liver and ovary,which play an important role in the growth axis and the reproductive axis of the Cynoglossus semilaevis at IV,V,and VI stages of ovarian development.The sequencing data were quality controlled,filtered,compared and identified.Then,analysis in circ RNA type,expression amount calculation,difference analysis,database annotation and targeting prediction were conducted.Also,designing the convergent primers and divergent primers of circRNAs,the sanger sequencing of circRNAs,the Rnase R resistance of circRNAs and the verification of fluorescence quantitative RT-PCR were done in present study.The mechanism of circRNAs involved in the regulation of ovarian maturation in half-smooth tongue sole was preliminarily revealed.The above research has the following important results:(1)RNA seq data of four tissue samples of brain,pituitary,liver and ovary of Cynoglossus semilaevis were mined for circRNAs.And 11276 circRNAs were detected.It was found that not all circRNAs were expressed in all detected tissues,and the number of circRNAs increased during the sexual maturation of Cynoglossus semilaevis.And the number of cross-repeated circRNAs between different tissues also increased.Those suggest that circRNAs play an important physiological role in the sexual maturation of Cynoglossus semilaevis.Most circRNAs are between 101 and 400 bp in length,which is shorter than the average transcriptional length of common protein-coding genes.Circular RNA molecules were detected,of which the number of exons of the coding gene for 58.42%.Through the calculation of expression between different groups,the p < 0.05 was selected,concurrently,circRNAs of |log2FC| > 1 were analyzed for differential expression between groups.It was found that the number of up-regulated and down-regulated circRNAs in the four tissues was also significantly different among the IV-V,IV-VI,and V-VI groups.It was found that the total number of up-regulated circRNAs in the three comparison groups showed an increasing trend,indicating that circRNAs played an important role in the ovarian maturation process of Cynoglossus semilaevis.GO function and KEGG pathway analysis were performed on all detected circRNAs,The significant enrichment GO terms is cellular process(GO:0009987),single-organism(GO:0044699),metabolic process(GO:0008152),cell part(GO:0044464),cell(GO:0005623),binding(GO:0005488),catalytic activity(GO:0003824).And the significant enrichment pathway of differentially expressed genes is Erb B signaling pathway(ko04012),Adherens junction(ko04520),Gn RH signaling pathway(ko04912),Cell adhesion molecules,CAMs(ko04514),Phosphatidylinositol signaling system(ko04070),Oocyte meiosis(ko04114).The RNAhybrid+svm_light,Miranda and Targetscan software were used to predict the circ RNA-mi RNA targeting relationship for all detected circRNAs.And the results showed that the detected circ RNA contained abundant binding sites of mi RNA,and a total of 11238 circRNAs were detected to target 3186 mi RNAs,generating 1841040 targeting relationships.(2)We select the source genes from pathways related to ovarian development,and the m RNAs related to ovarian development for the prediction of the targeting relationship and differential screening.Then the RNAhybrid(v2.1.2)+svm_light(v6.01),Miranda(v3.3a)and Target Scan(Version: 7.0)were used for target gene prediction.And the predicted target gene intersection was taken as the prediction result of mi RNA-m RNA.The results of network analysis showed that circ RNA-mi RNA-m RNA in the tissues of Cynoglossus semilaevis may have a complex regulatory network.Based on the targeted prediction results of circ RNA-mi RNA,cytoscape was used to construct the regulation network of circ RNA-mi RNA-m RNA.Base on network interaction,GO function and KEGG pathway analysis,the selected circ RNA are fouse on the pathway of Gn RH signaling pathway,Oocyte meiosis,progesterone-mediated oocyte maturation,steroid hormone biosynthesis,TGF-beta signaling pathway and PPAR signaling pathway.(3)The results of RNA-seq were verified by q RT-PCR of circ-ADCY6,circ-ESR1,circ-TGFBR2,circ-CPT1 A,circ-PLD1,circ-VTG?-1(novel_circ₀00969),circ-VTG?-2(novel_circ₀00968),circ-cgbb,circ-LHCGR,circ-CYP21A2,which include the screened and different expression between different comparison groups.The results showed that the expression trend of RNA-seq and q RT-PCR was highly consistent.It was also found that circRNAs have different expression patterns in different developmental stages and thus perform different functions in ovarian development and maturation.Sanger sequencing was performed on the common PCR products of convergent prmers and divergent primers to verify the complete sequence of the predicted circRNAs in the high-throughput sequencing results,so as to farther prove the accuracy of the RNA-seq results.RNase R digestion experiment and sanger sequencing results were used to verify that the circ RNA was ring forming and had RNase R tolerance.And the structure stability of circRNAs was further demonstrated by such experiments.In this study,the expression profile of circRNAs in Cynoglossus semilaevis was described for the first time.And a large number of circRNAs were found in the growth and reproduction axes of Cynoglossus semilaevis.The mechanism of circ RNA regulating ovarian development and maturation of Cynoglossus semilaevis was preliminarily revealed to provide a theoretical basis for improving the reproductive performance of Cynoglossus semilaevis.