The MiRNA Selection And Target Gene Function Verification In Different Season Of Estrous Sheep

In China,most of sheep breeds are seasonal estrous,which limits the production efficiency of sheep industry.In the actual production of sheep,affected by the external factors,some sheep in the non-breeding season also appeared estrus traits,but to make it stable perennial estrus,we must understand this non-breeding season in the performance of the molecular regulation mechanism.Mammalian reproductive activity is mainly depended on the regulation of the hypothalamic-pituitary-gonadal axis,while researchers on ncRNA can find out much more genes that involved in the regulation from a number of ways.In this study,Kazakh sheep was used as the research object,we used the high-throughput sequencing technique to construct the hypothalamus-pituitary-ovarian tissue library of non-breeding season and breeding season.The miRNAs and their target genes were screened by seasonal factors,with a view to providing a reference for changing the seasonal estrus of sheep.The experimental results are as follows:1.In this study,high-throughput sequencing technique was used to construct the small RNA library of hypothalamus(H)-pituitary(P)-ovary(O)tissue with non-breeding season estrus(EN)as the tester group and breeding season estrus(EB)as the control group,analyzed by the bioinformation methods.Parts of miRNAs were choosen to test the quality of miRNA libraries by q RT-PCR.While the predicted target genes were subjected to GO enrichment and KEGG enrichment analysis.The results showed that:(1)? Compared the two hypothalamus(HEN vs HEB)libaries,15 known and 126 novel miRNAs were differently expressed;? Compared the two pituitary(PEN vs PEB)libaries,the 12 known and 114 novel miRNAs were differently expressed;? Compared the two ovary(OEN vs OEB)libaries,the 48 known and135 novel miRNAs were differently expressed;(4)oar-miR-136 and oar-miR-148 a are common differentially expressed miRNAs in hypothalamus-pituitary-ovarian tissue(2)6 differentially expressed miRNAs(4 known and 2 newly discovered)were selected randomly to confirm differential expression by Quantitative RT-PCR,and the results were consistent with the high-throughput sequencing data;(3)The GO analysis suggested that,the target genes function were the cell process,the development process,the growth process,the metabolism process,biology regulation process,propagation process and enzyme regulation activity process,et al.KEGG analysis of its regulatory pathway revealed mammalian circadian rhythm pathway,GnRH signaling pathway,Progesterone-mediated oocyte maturation pathway,Insulin signaling pathway,Steroid hormone biosynthesis and Melanogenesis and other important pathway were significantly enriched.2.The target genes of oar-miR-136 and oar-miR-148 a were screened by bioinformatics and KEGG pathway,oar-miR-136 obtained two potential target genes for PLCB1 and GNAQ.oar-miR-148 a obtained two potential target genes for PLCB1 and NRAS.Construction of wild-type and mutant-type dual-luciferase vector,transfection of Hela cells,comparison of luciferase activity.The results showed that oar-miR-136 only inhibited the luciferase activity of wild-type GNAQ recombinant plasmid(P <0.01),indicating that there was target relationship between oar-miR-136 and GNAQ,and oar-miR-148 a only inhibited the luciferase activity of wild-type PLCB1 recombinant plasmid(P <0.05),indicating that the existence target relationship between oar-miR-148 a and PLCB1.3.MiR-136 minic and miR-148 a minic and control group were transfected into hypothalamic neurons cells,the related estrus genes(KiSS-1,GPR54,TSHB,RFRP,DIO2,DIO3 and GnRH)were selected to detect by Q-PCR.MiR-136 minic and miR-148 a minic and control group were transfected into ovarian granulosa cells,the related hormone genes(ER?,PR and INHBA)were selected to detect by Q-PCR.Theresults showed that:(1)After transfection of hypothalamic neurons cells,compared with the control group,for miR-136 minics,GNAQ gene was significantly down-regulated,KiSS-1,DIO2,RFRP and GnRH genes were significantly up-regulated(P <0.05),TSHB gene was significantly decreased(P<0.05),GPR54 and DIO3 genes were not significant.For miR-148 a minics,PLCB1 gene was down-regulated(P<0.05),KISS1 and GPR54 genes were significantly increased(P<0.05),other genes were not significant.(2)After transfection of ovarian granulosa cells,compared with the control group,for miR-136 minics,GNAQ gene expression increased(P<0.05),and the expression of ER? gene was significantly down-regulated(P <0.05),and the PR and INHBA genes were not significant(P<0.01).For miR-148 a minics,PLCB1 gene was down-regulated(P<0.05),the hormone related genes ER?,PR and INHBA were significantly increased.It was revealed that miR-136 regulates the expression of the estrus gene by inhibiting the expression of target gene,the change of genes was similar to that of breeding season estrus,miR-148 a regulated the expression of the hormone related gene by inhibiting the expression of target gene,the final regulation of the development of follicles,and then played a role in the estrus.In summary,the miRNAs of the hypothalamus-pituitary-ovary small RNA of non-breeding season and breeding season were successfully constructed by high-throughput sequencing technique.The differentially expressed miRNAs were screened and the target gene was predicted by KEGG pathway and prediction software.At the same time,the candidate target genes of specific miRNAs were verified,and finally the function of target gene was verified at the cellular level.The results provide a basis for deepening the role of miRNAs in seasonal estrus,so as to provide reference for sheep to achieve estrus during non-breeding season.