LncRNA366.2/miR-1576/WNT6 Regulates Endometrial Epithelial Cell Functions In Hu Sheep

Fecundity is one of the important factors for the development and economic benefits of animal industry.Most of sheep breeds in China are seasonal estrus and generally have a low fertility.With the development of standardized indoor feeding sheep industry in our country,higher requirements have been put forward for the fertility of sheep.Therefore,breeding new prolificacy sheep and rapidly expanding its population size are the focus of current work.Hu sheep,originated in the Taihu Lake area,is well-known for many characters,such as early sexual maturity,high lambing rate,strong adaptability and easy indoor feeding.As an excellent local variety in China,Hu sheep has been widely introduced to across the country.Hu sheep has become the most popular maternal parents for cross breeding,and is also the ideal animals to study the regulatory mechanism of high prolificacy sheep.The mechanism of mammalian fertility is very complex.As we all known,ovulation and uterine receptivity are the two main controlling factors for mammalian fertility.Ewes carrying the marker gene FecB of high ovulation rate still reveal low prolificacy.Therefore,in addition to ovulation performance,the development and receptivity of uterus are closely related to reproductive efficiency of Hu sheep.Presently,there are many reports on the regulation of ovulation performance,but few systematic research in the effects of uterine development on the fertility.Studies have shown that IncRNAs are involved in mammalian reproductive activities such as testicular development,oocyte maturation and embryo implantation,which provide a new idea for research the mechanism of Hu sheep prolificacy.In the present study,RNA sequencing(RNA-seq)was applied to screen and analyze the lncRNA/miRNA/mRNA regulatory network involved in endometrial function in Hu sheep with different fecundity and FecB genotypes.The function and mechanism of candidate genes were investigated in vitro.This study includes the following four parts:1.Analysis of uterine morphology and related gene expression in Hu sheep with different prolificacyThe objective of this research was to evaluate the relationship between the difference of uterine phenotype and prolificacy.Based on the lambing records and genotype analysis,a total of 9 multiparous Hu sheep of similar age and body condition were divided into three groups including a high prolificacy group of Hu sheep carrying the FecBB genotype(HBB,n=3,litter size=3),a low prolificacy group carrying the FecBB genotype(LBB,n=3,litter size=1),and a low prolificacy groups carrying FecBB+ genotype(LB+,n=3,litter size=1).After synchronous estrus,blood samples and uterine tissue were collected at the second estrus(natural estrus).Results of IHC showed the density of uterine glands(UGs)and microvessel density(MVD)were higher in the HBB group of sheep than that in the LB+and LBB groups(P<0.05).The uterine coefficients and numbers of ductal gland invaginations in the HBB group were higher than that of the LB+group(P<0.05).Results of ELISA analysis showed serum progesterone(P4)concentration in the high prolificacy Hu sheep was lower than that of the LBB group(P<0.05).Interestingly,serum placental growth factor(PLGF)concentration appeared to be higher in the high prolificacy group than in the two low prolificacy groups(P<0.05).The results of RT-qPCR showed that the expression levels of growth factors in HBB group were significantly higher than those in LBB and LB+groups(P<0.05)such as,HOXA10,HOXA11,IGF-1,VEGFA and LGR5 which related to uterine gland development of Hu sheep.In a word,the significant differences in uterus microenvironment and endocrine level of Hu sheep different prolificacy.2.Analysis of DE mRNA,lncRNA and miRNA in endometrium of Hu sheep with different prolificacyThe purpose of this study was to analyze the expression patterns of mRNA,lncRNA,and miRNA in the endometrium of Hu sheep by RNA-Seq,and to screen candidate DE lncRNAs,DE miRNAs,and DE mRNA related to the fecundity of Hu sheep.Results of endometrial mRNA sequencing showed 743 and 874 DE mRNAs were screened in HBB vs.LB+and HBB vs.LBB groups,respectively.KEGG pathway analysis revealed that the endometrial DE mRNAs in Hu sheep were significantly enriched in the signaling pathways involved in uterine reproduction including NF-Kappa B signaling pathway,TGF-βsignaling pathway,Wnt signaling pathway,TNF signaling pathway,ECM receptor signaling pathway,and Hippo signaling pathway;GO pathway analysis revealed that the endometrial DE mRNAs in Hu sheep were significantly enriched in growth factor activity signaling pathway(P<0.05).Results of IncRNA sequencing showed 261 and 347 DE lncRNAs were screened in HBB vs.LB+and HBB vs.LBB groups,respectively.DE lncRNAs targets were enriched in some signaling pathways involved in endometrial functions,such as the oxytocin signaling pathway,estrogen signaling pathway,NF-Kappa B signaling pathway and WNT signaling pathway.Results of miRNA sequencing showed 55 and 58 DE miRNAs were screened in HBB vs.LB+and HBB vs.LBB groups,respectively.The ceRNA networks related to fertility were constructed by conjoint analysis of lncRNA/miRNA/mRNA.A candidate lncRNA/miRNA/WNT6 regulatory network was selected from the endometrium of Hu sheep with different prolificacy.In conclusion,the DE lncRNAs,DE miRNAs,and DE mRNA related to the fecundity of Hu sheep were screened,and ceRNA networks were also constructed,which provide a new theoretical reference for the identification of candidate genes related to prolificacy.3.WNT6 regulates endometrial epithelial cells(EECs)proliferation and migration in Hu sheepThis study was conducted to investigate the effects of WNT6 on Hu sheep EECs and UGs development in vitro.In endometrial tissue,WNT6 was highly expressed in the HBB group compared to the two low prolificacy groups(P<0.05).Results of tissue expression profile showed that a highly expressed level of WNT6 was detected in the endometrium,ovary and oviduct.Furthermore,results of immunofluorescent staining revealed WNT6 localized to EECs.Functionaly,WNT6 knockdown significantly inhibited the expression ofβ-catenin in EECs.However,overexpression of WNT6 significantly increased the expression of β-catenin.In immunoprecipitation test,WNT6 was detected to affect the activation of WNT/β-catenin signaling pathway by regulating the activity of AXIN1-centered degradation complex.Results also discovered that WNT6 promoted the proliferation,migration and cell cycle progression of EECs(P<0.05).We found that WNT6 can promote endometrial organogenesis by enhancing the expression of organogenesis related genes.Taken together,WNT6 is a crucial regulator for EECs proliferation and migration,which enhances the development of sheep UGs.4.LncRNA366.2/miR-1576/WNT6 regulates EECs proliferation and migration in Hu sheepThis study aims to investigate the roles of lncRNA366.2/miR-1576/WNT6 on Hu sheep EECs and UGs development.Firstly,the targeting relationship between miR-1576 and WNT6 predicted by RNA-seq bioinformatics analysis was verified by dual luciferase reporter assay.Then,functional assay was performed by supression/overexpression on WNT6 in EECs.The results showed that the Inhibitor of miR-1576 significantly enhanced the expression of WNT6,and then activated the proliferation,cell cycle and migration process of EECs(P<0.05).In contrast,the addition of miR-1576 Mimics significantly inhibited WNT6 expression,thus interfering with EECs proliferation,cell cycle and migration(P<0.05).The targeting relationship between miR-1576 and candidate lncRNAs was verified by RT-qPCR and dual luciferase reporter assay.Then,roles of lncRNA366.2 was investigated in EECs.The results indicated that WNT/β-catenin activation was significantly inhibited by the interference of lncRNA366.2,and then interfering with EECs proliferation,cell cycle,migration,and organogenesis of sheep(P<0.05);The results of co-transfection of miR-1576 Inhibitor and si-lncRNA366.2 showed that lncRNA366.2 competitively inhibited the binding of miR-1576 to WNT6,and regulating the proliferation,migration,and organogenesis of sheep via the Wnt/β-catenin pathway.This study illustrated the effects of lncRNA366.2/miR-1576/WNT6 network on the development of UGs in Hu sheep,and provides a new theoretical reference for the discovery of candidate genes related to fecundity.