Mechanism Of MiR-26b-5p Regulating The Proliferation Of Follicular Granulosa Cells In Goose

Follicular development is one of the important factors affecting egg production in goose,and it is determined by the number and states of follicular granulosa cells.The proliferation and differentiation of follicular granulosa cells in pre-hierarchical follicles can ensure the normal development and the maturation of follicles and enter hierarchical stage.The researched have revealed that less than 1%of the pre-hierarchical follicles can be selected as the dominant follicles,and most follicles become atresia due to the apoptosis of follicular granulosa cells.Therefore,it was necessary to carry out the research about regulatory mechanism of the proliferation of follicular granulosa cells in goose,which contributed to promote normal follicular development,increase the number of dominant follicles and improve the quantity of eggs.miRNAs can regulate the transcription of target genes to affect cell proliferation and follicular development by partial or complete complementary paired with the 3'UTR region of the target gene.Our previous research revealed the expression of miR-26b-5p was significantly different between laying geese and broody geese,and the target gene of different miRNA was enriched in the TGF-?/SMADs signaling pathway.So the hypothesis on miR-26b-5p involvement in the proliferation of follicular granulosa cells through TGF-?/SMADs signaling pathway was proposed.In present study,the target gene of miR-26b-5p was investigated,and the mechanism of miR-26b-5p regulating the cell proliferation was explored.The main results of this experiment were as follows:1.miR-26b-5? participated in the proliferation of follicular granulosa cells and promoted the secretion of E2 in goose.In order to reveal the effect of miR-26b-5p on the proliferation of follicular granulosa cells in goose,the miR-26b-5p mimics and inhibitor were transfected into cells,and the cell proliferation and apoptosis were detected by CCK-8 kit and flow cytometry,respectively.The result showed that the proliferation rate of follicular granulosa cells increased significantly by 81.2%(P<0.05)after transfection with miR-26b-5p mimics,while cell proliferation was inhibited after transfection with miR-26b-5? inhibitor(P<0.01).Whereas the apoptosis rate of follicular granulosa cells increased by 28%after transfection with miR-26b-5p inhibitor(P<0.01).Furthermore,the expression of apoptosis marker genes was determined by qRT-PCR,the follicular granulosa cells transfected with miR-26b-5p mimics resulted in significantly up-regulation of anti-apoptotic gene BCL-2 and significantly down-regulation of pro-apoptotic gene Caspase-3(P<0.01),while those transfected with miR-26b-5p inhibitor in an opposite trend(P<0.01).In addition,transfection with miR-26b-5p mimic could significantly induce the secretion of E2 and reduce the secretion of P4,while transfection with miR-26b-5p inhibitor also showed an opposite trend(P<0.05).The above results confirmed that miR-26b-5p could promote the proliferation of follicular granulosa cells,increase the secretion of E2 and reduce the secretion of P4 in goose.2.miR-26b-5p targeted INHBB to promote the proliferation of follicular granulosa cells.In order to indentify the action mode of miR-26b-5p promoting the proliferation of follicular granulosa cells,the INHBB and SMAD4 were predicted as potential target genes of miR-26b-5p,and furtherly identified by Dual Luciferase Reporter Assay.The results suggested that miR-26b-5p could bind to the 3'UTR of INHBB and significantly inhibit luciferase activity(P<0.01),but it couldn't inhibit the luciferase activity of SMAD4 Additionally,the miR-26b-5p could significantly reduce the INHBB expression and INHB secretion(P<0.01).The above results demonstrated that miR-26b-5p involved in regulating the proliferation of follicular granulosa cells by targeting INHBB in goose.3.Transcription factor SMAD4 regulated the transcription of miR-26b-5p to participate in the proliferation of follicular granulosa cells in goose.To elucidate the molecular mechanism of miR-26b-5p participating in the proliferation of follicular granulosa cells in goose,the squence of pre-miR-26b-5p was obtained,and the three SMADs-binding sites on the promoter region of goose miR-26b-5p(SBE1:-827--815,SBE2:-796--784,SBE3:-298--286)were pridicted and verified.The results showed the 293T cell transfected SBE3-containing fragments was significantly increased fluorescence activity,so SMAD4 enhanced the transcription activity of miR-26b-5p as a transcription factor.In order to further investigate the role of SMAD4 in the proliferation,the upstream specific blocker of the TGF-?/SMADs signaling pathway in which SMAD4 is located was selected,and the expreesion of miR-26b-5p,cell apoptosis and hormone secretion were detected.The specific blocker not only inhibited significantly the expression of SMAD4 and miR-26b-5p(P<0.05),but also promoted INHBB expression(P<0.01).Moreover,the specific blocker also accelerated significantly cell apoptosis.The secretion of E2 was also significantly reduced,at the same time,the secretion of P4 and INHB were increased significantly(P<0.05).The above results fully demonstrated that SMAD4 could be used as a transcription factor to initiate miR-26b-5p transcription and participated in the proliferation of follicular granulosa cells through the TGF-?/SMADs signaling pathway.In summary,miR-26b-5p was transcriptionally regulated by the transcription factor SMAD4 and participates in the proliferation of follicular granulosa cells targeting INHBB,which provided some theoretical basis for increasing the number of follicular granulosa cells and improving the egg production in goose.