Follicular fluid HD-sevs-mir-128-3p is a key molecule in regulating bovine granulosa cells autophagy

Follicular fluid (FF) is rich in extracellular vesicles (EVs). EVs carries a variety of miRNA involved in regulating follicular development, the function of cells in follicles, primordial follicular formation, follicular recruitment and selection, follicular atresia, oocyte communication, granulosa cells (GCs) function and luteinization and other biological processes of follicular development. Previous studies in our laboratory have shown that bovine follicular fluid (bFF) high density-small extracellular vesicles (HD-sEVs)-miRNA was enriched in autophagy-related pathways. However, the mechanism of bFF EVs carrying miRNA regulating GCs autophagy is not clear. Thus, this study carried out a series of studies on the previous HD-sEVs sequencing data and miR-128-3p contained in bFF HD-sEVs. A total of 38 differentially expressed genes were detected by RNA-Seq after overexpression of miR-128-3p in bovine GCs (bGCs). Through cell transfection, Western blot (WB) and Immunofluorescence (IF), it was proved that overexpression of miR-128-3p could promote the expression of LC3 (microtubule-associated protein I light chain 3), inhibit p62, promote the number of autophagosome, promote the formation of autophagy lysosome and autophagy flow, and activate bGCs autophagy. MiR-128-3p inhibitor significantly inhibited the expression of LC3 and monodansylcadaverine (MDC) in bGCs, and promoted the expression of autophagy substrate p62, indicating that HD-sEVs-miR-128-3p could activate bGCs autophagy. In addition, through double luciferase assay, bioinformatics analysis, WB and RT-qPCR, it was concluded that bFF HD-sEVs-miR-128-3p could target TFEB (transcription factor EB) and FoxO4 (Forkhead box O4) and activate GCs autophagy.