Regulation Of MicroRNA On Ovarian Maturation Of Cynoglossus Semilaevis

With the rapid development of the field of life science,mi RNA as a post-transcriptional regulator,has gradually entered the field of vision of researchers,and it is widely involved in the life activities of organisms through the regulation of genes.The Cynoglossus semilaevis is an important mariculture fish in northern of China.Its large-scale breeding of its seedlings promotes the development of aquaculture industry.Therefore,the research on reproductive endocrine regulation mechanism of ovarian development and maturation of Cynoglossus semilaevis has been paid more and more attention.In this paper,we used high-throughput sequencing technology to construct small RNA library of brain,pituitary,ovary and liver during IV,V and VI stages of ovarian development in Cynoglossus semilaevis.The data filtering and quality evaluation were used to identify mi RNA.Through mi RNA expression profile,principal component analysis,thermogram analysis,differential mi RNA and target gene enrichment analysis,as well as fluorescent quantitative q RT-PCR and double luciferase gene verification experiments,we obtained the following important results:1.By high-throughput sequencing of the brain,pituitary,ovary and liver tissue samples of three ovarian development stage of Cynoglossus semilaevis.36 small RNA libraries were obtained.And the 10892895-15687459 clean tags were filtered and screened.According to mi RNA target gene prediction and target gene enrichment analysis,3224 mi RNAs were identified.The number of predicted mi RNA target genes was 32743,and the number of target gene binding sites was 3246193.This shows the characteristics of mi RNA multi-target effect,and the relationship between mi RNA and target gene is multi to multi.According to mi RNA expression analysis,the number of down-regulated mi RNAs reduced from stage IV to stage VI in all tissues was higher than the number of up-regulated mi RNA.And the difference between ovary stage IVvs VI was most significant,with a total of 608 mi RNAs expressedsignificantly differently,of which 237 and 371 were up-regulated and down-regulated mi RNAs,respectively.2.To screen the mi RNA whose expression TPM is at least more than 50 and has negative correlation with the target gene,we constructed the m RNA mi RNA regulatory network map of each tissue during the ovarian development and maturation of Cynoglossus semilaevis,and identified some important mi RNAs(mir-34-x,mir-186-x,mir-2188-x,mir-153-x,mir-204-x,mir-211-x,mir-2188-y,mir-223-y,mir-724-x,mir-144-y,n Ovel-m0050-3p,novel-m0017-5p)at the core position.Those mi RNAs simultaneously regulate several m RNA,and reflect different biological functions at different ovarian stages.Through the target gene relationship,they screened out the important m RNA related to reproduction(IGF1R,hsd3 b,IGF2R,PGR,GHR,SSTR2,CYP19A1,LHCGR,AR,CYP17A1,sst2,GPR54,PGRMC1,ESR1,paqr7b).Through KEGG pathway analysis,we found that the following signaling pathways were enriched: Steroid hormone biosynthesis pathway,Oocyte meiosis pathway,Progesterone-mediated oocyte maturation pathway,Focal adhesion pathway,Endocytosis,Neuroactive ligand-receptor interaction pathway,Cytokine-cytokine receptor interaction pathway,Jak-STAT signaling pathway.These pathways play an important role in the process of ovarian development and maturation,and provide a reference for later biological experiments.3.We selected 9 mi RNA(mir-186-x,mir-153-x,mir-211-x,mir-223-y,mir-2188-x,mir-2188-y,mir-724-x,mir-221-x,mir-21-y)in 4 tissues with significant differences during the maturation of ovaries.And the differentially expressed genes in the maturation of ovaries,including igf2 r,esr1 and cyp19a1.Those mi RNA and genes were validated by q RT-PCR.The results were consistent with the expression trend of sequencing data.The results indicated the accuracy of sequencing data and the reliability of mi RNA-m RNA regulation network.4.By means of biological prediction,dual-luciferase reporter gene experiment and thedifferential expression of igf2 r and mir-186-x during the process of ovarian development and maturation,it is directly and indirectly proved that igf2 r is the target gene of mir-186-x.It is suggested that mir-186-x plays an important role in the regulation of reproductive endocrine system and ovarian maturation of Cynoglossus semilaevis.