MiR-128-3p Regulates Chicken Granulosa Cell Function Via 14-3-3β/FoxO And PPAR-γ/LPL Signalling Pathways

MicroRNAs(miRNAs)are a class of non-coding RNAs with a length of about 22 nt,which inhibit the translation process of mRNA by binding to the 3’-UTR of target genes.A lot of researches showed that miRNA play an important role in regulating the growth and development of animal ovaries,fallopian tubes,uterus and other tissues and organs related to reproduction.The function of chicken follicle granulosa cells(Granulosa cells,GC)directly affects the development and ovulation of follicles,thereby affecting the egg production process of chickens.Therefore,the study of the functions of chicken follicle granulosa cells provides breeding targets for improving chicken egg production.In this study,we found via miRNA sequencing that a large number of miRNAs,including miR-128-3p,were differentially expressed in granulosa cells of F1 and F5 grade follicles in chickens.Next,in this study,miR-128-3p was overexpressed and interfered with,and its target genes were predicted and verified by RT-qPCR,Western Blot,CCK-8,EdU,flow cytometry,and Oil Red O staining.The effects of miR-128-3p on the proliferation and apoptosis of granulosa cells,hormone synthesis and lipid droplet deposition were preliminarily proved by methods such as ELISA and ELISA.The specific results are as follows:(1)miRNA sequencing revealed 25 miRNAs that were differentially expressed in F1 and F5 grade follicles,among which miR-128-3p was upregulated in F1 follicle grade granulosa cells and downregulated in F5 grade granulosa cells.(2)The results of bioinformatics prediction,dual luciferase reporter system,RT-qPCR,and Western Blot showed that YWHAB and PPAR-γ were the target genes of miR-128-3p,and miR-128-3p was expressed at the mRNA and protein levels Both can inhibit the expression of YWHAB(14-3-3β)and PPAR-γ.(3)The results of RT-qPCR and Western Blot showed that miR-128-3p regulates proliferation and apoptosis-related genes and proteins(Cyclin D1,CDK2,PCNA,Bcl-2,Caspase3,Caspase9),steroid hormone secretion-related genes and proteins(CYP11A1,CYP19A1,3β-HSD,StAR)lipid metabolism-related genes and proteins(FASN,LPL,SREBP1,PPAR-α,PPAR-β)and 14-3-3β/FoxO signaling pathway-related proteins(14-3-3β,p-FoxO1,p-FoxO3,p-FoxO4)expression levels.The results of CCK-8 and EdU showed that miR-128-3p inhibited the proliferation of granulosa cells;the results of flow cytometry showed that miR-128-3p promoted the apoptosis of granulosa cells.After overexpression and interference treatment of YWHAB gene on granulosa cells,it was found that YWHAB can promote the proliferation of granulosa cells and inhibit apoptosis through the 14-3-3β/FoxO pathway.(4)After overexpression and interference of miR-128-3p,it was found by Oil Red O staining and ELISA that miR-128-3p inhibited the formation of lipid droplets in granulosa cells and the secretion of progesterone and estrogen.Next,the Western Blot test proved that miR-128-3p inhibited lipid synthesis,estrogen and progesterone synthesis by inhibiting the PPAR-γ/LPL pathway.In summary,miR-128-3p regulates the 14-3-3β/FoxO and PPAR-γ/LPL signaling pathways by targeting YWHAB and PPAR-γ genes,thereby inhibiting the proliferation of chicken granulosa cells and promoting the apoptosis of granulosa cells.And inhibit the production of estrogen and progesterone in granulosa cells and the process of lipid droplet deposition.