Trehalose Promotes Autophagy In Porcine Ovarian Granulosa Cells Via MiR-221-5p＿R-4/NRBF2

During the development of mammalian follicles,most follicles have follicular atresia and fail to ovulate,resulting in a waste of genetic resources.Studies have shown that autophagy in ovarian granulosa cells is one of the key factors causing follicular atresia,but the factors that induce autophagy in granulosa cells are still unclear.As an autophagy inducer,trehalose exerts a protective effect by inducing autophagy in various types of cells,yet its mechanism during follicle development has not been fully clarified.In this study,porcine ovarian granulosa cells(PGCs)were used as the experimental object to study the effect and mechanism of trehalose on PGCs.The main findings are as follows:1.The effect of trehalose on the function of PGCs in vitro(1).PGCs were treated with 0.01,0.1,1,10 and 100 mM trehalose for 8 h,16 h and 24 h,respectively.There was no statistical difference in cell viability among the treatment groups.(2).When PGCs were treated with trehalose for 16 h,0.1 mM trehalose significantly increased the content of estradiol(E2)in cell culture medium while the content of E2 in the 1mM trehalose treatment group was significantly higher than that in the 100 mM treatment group.Besides,the content of progesterone(P4)in the trehalose treatment group was significantly up-regulated.And the P4 content of cells treated with 1 mM trehalose for 24 h was also significantly up-regulated.When treated for 16 h,compared with the effect of 100 mM trehalose,1 mM trehalose significantly up-regulated the mRNA levels of StAR,FSHR,and PGR.When treated for 24 h,compared with the control group,the transcription levels of StAR,FSHR,and PGR in PGCs were significantly increased,and the transcription level of StAR in the 16 h treatment group was also significantly higher.(3).1 mM trehalose treatment for 16 h promoted the expression of autophagy genes ATG3,ATG7,BECN,LC3 and LC3-Ⅱ proteins in PGCs,down-regulated the expression of p62 protein,and increased the number of autophagosomes in cells.From the above results,it can be seen that trehalose can regulate the hormone secretion of PGCs and can also promote autophagy.2.Transcriptomic study of trehalose effects on PGCs(1).Whole transcriptome sequencing of trehalose-treated PGCs identified a total of 279 differentially expressed genes(DEGs),including 89 up-regulated and 190 down-regulated genes.Through GO analysis and KEGG signaling pathway analysis,CYP1A1,PTGS2,NRBF2,DDIT4,BNIP3,ATG9 B,NOS3 and FOS were enriched in steroid hormone biosynthesis and autophagy pathways.(2).Screening the data of non-coding RNA,6 differentially expressed miRNAs were found,namely hsa-miR-98-3p＿1ss22CT,ssc-miR-221-5p＿R-4,ssc-miR-184,ssc-miR-362＿R-1,ssc-miR-660 and PC-3p-68235＿30.(3).The randomly selected DEGs and miRNAs were verified by qRT-PCR,and the PCR results were consistent with the sequencing results.Based on the significance of differential expression,it was decided to conduct in-depth research on the regulation of autophagy by miR-221-5p-R-4 in PGCs.3.MiR-221-5p＿R-4 mediates autophagy via NRBF2 in PGCs(1).In PGCs,miR-221-5p＿R-4 mimic significantly up-regulated the expression of autophagy protein LC3-Ⅱ and inhibited the expression of p62.LC3-Ⅱ expression was inhibited after miR-221-5p＿R-4 inhibition,while p62 protein level increased,indicating that miR-221-5p＿R-4 expression promotes autophagy.(2).Using Target Scan(v5.0),miRanda(v3.3a)software and dual luciferase reporter assay,it was proved that miR-221-5p＿R-4 targets and binds to the autophagy-related gene NRBF2.MiR-221-5p＿R in PGCs-4 negatively regulates the expression of NRBF2 protein.(3).When NRBF2 was silenced in PGCs,the mRNA level of autophagy-related gene Vps34 was decreased,the expression of autophagy protein LC3-Ⅱ was up-regulated,and Beclin1 protein was down-regulated,indicating that silencing NRBF2 would affect autophagy.If NRBF2 was silenced while suppressing miR-221-5p＿R-4 expression,NRBF2 silencing would reverse the regulation of miR-221-5p＿R-4 on autophagy.In conclusion,in PGCs,1 mM trehalose treatment for 16 h can significantly promote the function of PGCs and promote autophagy.Trehalose regulates autophagy in PGCs by inhibiting the level of NRBF2 protein through promoting the expression of miR-221-5p＿R-4.The findings provide theoretical support for the study of the mechanism of trehalose in porcine follicle development.