| FoxO1(forkhead box O1)belongs to the Fox family and is also an important member of the FoxO subfamily.It involved in biological processes such as cell metabolism,cell cycle and apoptosis regulation,angiogenesis,and tumorigenesis.FoxO1,as a transcription factor,plays an important role in follicular development.Although FoxO1 has been widely studied,the effect of FoxO1 on the function of ovine granulosa cells has not been reported.This study first explored the expression pattern of FoxO1 in sheep ovarian follicles.Ovaries were collected and granulosa cells harvested from small follicle(diameter ≤ 3 mm),medium follicle(3 mm <diameter < 5 mm),big follicle(diameter ≥ 5 mm).The localization of FoxO1 in different diameter follicles was analyzed by immunohistochemistry.And the expression of FoxO1 in granulosa cells of small,medium and large follicle was detected by q RT-PCR and Western blotting.Next,the CDS sequence of sheep ovarian FoxO1 gene was amplified and carried out bioinformatics analysis.Finally,functional analysis of the FoxO1 gene was performed in sheep granulosa cells using RNA interference and lentivirus-mediated overexpression technology.The proliferation activity of granulosa cells after FoxO1 silencing was detected by CCK-8 assay,and the expression levels of apoptosis related genes,cell cycle related genes and steroid-related genes were detected by q RT-PCR after FoxO1 silencing and overexpression.The main results of this study were as follows:(1)The immunohistochemical analysis showed that FoxO1 was expressed in the granular layer of sheep follicles.QRT-PCR results suggested that the expression of FoxO1 m RNA in medium follicle was higher than that in small and big follicle(P < 0.01),and the expression of FoxO1 m RNA in small follicle was higher than that in big follicle(P < 0.01).Furthermore,Western blotting data showed that the expression of FoxO1 protein in big follicle was lower than that in small and medium follicle(P < 0.01),but there was no significant difference between small and medium follicles.(2)The total length of the sheep FoxO1 gene CDS sequence is 1827 bp and the predicted sheep FoxO1 protein is composed of 608 amino acids with a molecular weight of 64.60 k Da and an isoelectric point of 6.57.It is an unstable hydrophilic protein with no signal peptide and transmembrane region.It also has 3 potential N-glycosylation sites and 90 potential phosphorylation sites,with a typical FH superfamily domain.Multiple alignments suggested that the FoxO1 protein sequence was highly homologous to FoxO1 from other species.And the phylogenetic analysis showed that the sheep have the closest relationship with goat,and the furthest relationship with rat and mouse.In addition,using STRING software to predict the interaction of sheep FoxO1 protein,we found that sheep FoxO1 protein may interact with 10 proteins including SIRT1,Smad2,Smad3,AKT2 and AKT3.(3)CCK-8 assays suggested that sheep GCs proliferation increased with FoxO1 knockdown.FoxO1 overexpression in sheep GCs promoted P21 and P27 m RNA expression,whereas knockdown did the opposite.Moreover,the expression of apoptosis-related genes(caspase3,caspase8,caspase9,Bax/Bcl-2)was significantly increased by FoxO1 overexpression and decreased by FoxO1 knockdown in vitro(P <0.05).FoxO1 upregulation enhanced the expression of steroidgenic genes(CYP11A1,3β-HSD),while silencing of FoxO1 reduced steroidgenic genes(CYP11A1,STAR)expression.This study showed that FoxO1 was expressed in the granulosa cell layer of sheep follicles,and the proliferation of granulosa cells was closely related to the expression level of FoxO1.In addition,FoxO1 can regulate the transcription of genes related to cell cycle,apoptosis and steroidogenesis.The data in this study showed that FoxO1 played an important role in the growth and development of follicles,which laid a foundation for further study on the role of FoxO1 in GCs of sheep. |
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