Mechanism Study On Goat Follicle Related MRNA-miRNA Expression Profiles Basing On Transcriptome Sequencing

As a local superior species in Guangdong,Leizhou goat has the advantages of early maturity,coarse feeding resistance,good meat quality,and low odor.However,for the unscientific breeding system and low fertility,the reproductive performance of Leizhou goat was poor.Reproductive performance was closely related to follicular development.The microenvironment in which the oocytes live determined whether oocytes could develop and mature eventually,and oocytes will face different fates in different follicular microenvironments.Therefore,the comparative study of small follicles and mature follicles is helpful to understand the gene expression and regulation patterns of different follicles,enrich the molecular mechanism of follicle development,and provide a theoretical basis for improving the reproductive effectiveness of Leizhou goats in practice.In this study,5 healthy Leizhou goats with 2-3 years were selected and slaughtered after simultaneous oestrus treatment.The single follicles were derived from ovarian under stereoscopic microscope to obtain large follicle(diameter >8 mm)and small follicles(diameter < 3 mm).Each large follicle pool was composed of 1 large follicle of each goat,and each small follicle pool was composed of 5-10 small follicles from same goat.RNA-Seq and small RNA sequencing were performed to obtain the gene and miRNA expression profiles of Leizhou goats between large follicle and small follicle.The character of gene and miRNA were identified using differently expressed analysis,target genes prediction,function analysis.Mi RNA-mRNA interactions network was constructed to screen the key genes,miRNAs and related signaling pathways which may be associated with follicular development.The main results are as follows:(1)The follicle RNA was extracted to construct 10 RNA-seq libraries and a total of144.82 GB sequencing data were obtained by sequencing.3000 differentially expressed genes(DEGs)were identified in large follicles vs small follicles,of which 856 genes were up-regulated in large follicles.The DEGs significantly identified 332 GO functions(244biological processes,51 cellular components and 37 molecular functions)related to immune response and activity of transmembrane transporters.KEGG had significantly enriched 89 pathways including steroid synthesis,cAMP signaling pathway with downstream pathway and cellular immune pathway.The genes NDRG4,HEY2,TC2 N,CDO1,FDXR and GADD45 B may play important roles in follicular maturation.(2)10 small RNA sequencing libraries were constructed,434 known miRNAs were identified,and 1782 novel miRNAs were predicted.44 differentially expressed miRNAs(DEmiRNAs)were identified in large follicles vs small follicles,of which 24 miRNAs were up-regulated in large follicles.Mi R-202-5p,miR-128-3p and miR-450-5p may be highly correlated with follicular development.Function enrichment analysis of their target genes shown that miRNAs may be involved in regulation of follicular development by participating in steroid hormone biosynthesis,Th17 cell differentiation,Th1 and Th2 cell differentiation,TGF-signaling pathway,Hippo signaling pathway,MAPK signaling pathway and cell adhesion factor pathway through their target genes.(3)By constructing the miRNA-mRNA interaction network associated with follicular development in Leizhou goats,an interaction subset containing 14 DEmiRNAs and 25 DEGs was predicted.Among them,miR-380-5p-PIK3CG/GLI3,miR-494-PIK3CG/GLI3,miR-758-PDE4A/PTCH1 and miR-202-5p-PDE4A/PTCH1 were involved in the inhibition of cAMP signaling in large follicles,and miR-202-3p-GLI2/PTCH1,miR-296-3p-GLI2/PTCH1 were involved in the inhibition of follicle Hedgehog signaling.miR-411a-3p,miR-433 and miR-543-3p which down-regulated BCL2L11 and FGF10 were involved in the inhibition of PI3K-Akt signaling pathway in large follicles,and miR-128-3p,miR-144-3p and miR-411a-3p were involved in cellular immunosuppression in large follicles by down-regulating VAV1.(4)8 DEGs and 5 DEmiRNAs were randomly selected for RT-qPCR verification,and the quantitative results were basically consistent with the sequencing results,indicating a high reliability of the sequencing results.In conclusion,this study improved the gene and miRNA expression profiles of goat follicles,providing a data basis for the study on the regulatory mechanisms of gene and noncoding RNA in goat follicular development and laying a foundation for the breeding improvement of Leizhou goat.