Mir-361-5p Regulates Follicular Granulosa Cells Apoptosis And Follicular Atresia In Pigs Via VRGFA

Follicular atresia is the main factor limiting the reproductive potential of female animals,and its regulation mechanism is one of the focuse of livestock and poultry reproduction research.Follicular fate(ovulation or atresia)and the state of granulosa cells(proliferation or apoptosis)are inseparable from the development of ovarian vascular network.Previous studies had shown that the degeneration of ovarian vascular network during porcine follicular atresis.Our laboratory research had shown that the transcription level of VEGFA is significantly decreased in pig during follicle atresia.while it is not clear the effect of VEGFA in follicular atresia and how VEGFA is regulated is related to granulosa cell apoptosis in prcine ovarian follicles.Bioinformatics analysis indicated that miR-361-5p which was increased during porcine follicular atresia has potential to bind to the 3’ UTR of VEGFA.From this we hypothesized that miR-361-5p regulates porcine granulosa cell apoptosis and follicular atresia by regulating VEGFA.In this study,we focus on VEGFA gene in porcine follicular granulosa cells to analyse its role in regulating GCs apoptosis and follicular atresia,and the mechanisms of VEGFA regulation by miR-361-5p.It has theoretical and practical value in revealing the molecular mechanism of high fertility of pigs,screening molecular markers of high fertility,and providing new ideas for related diseases interventions and treatments.Results of this research are listed as followings:1 VEGFA expression during porcine ovarian follicle development and atresiaWe first use immunohistochemical staining(IHC)technology to explore the expression of VEGFA protein levels during porcine ovarian follicular development and atresia.The results showed that the epithelial cells of primordial follicle showed weak VEGFA expression,and the expression of VEGFA in granulosa cells was gradually increased with the growth of follicles,especially in granulosa cells surrounding the antrum and cumulus oocytes;the expression of VEGFA in theca was observed in the secondary and tertiary follicles.During atresia of secondary and tertiary follicle,VEGFA expression decreased with degree of atresia.qRT-PCR and Western blot were used to quantitatively detect the expression of VEGFA mRNA and protein in healthy follicles and atresia follicles.The results showed that VEGFA was mainly produced by granulosa cells and secreted into follicular fluid and follicular cells to exert its function.During the follicles atresia,VEGFA expression in the whole follicle,theca,antral granulosa cells,follicular fluid was significantly decreased,while the expression in the thecal granulosa cells was increased.VEGFA level in follicular fluid of healthy follicles and atresia was detected by enzyme-linked immunosorbent assay(ELISA).The results showed that the VEGFA concentration in follicular fluid of healthy follicles was significantly higher than that of atresia follicles.In addition,granulosa cells cultured in vitro were treated with VEGFA siRNA.Western blot and flow cytometry(FACS)were used to detect active caspase 3 expression and apoptosis of granulosa cells.The results showed that the decrease of VEGFA significantly increased the active caspase-3 protein expression and increased apoptosis rate of granulosa cells.These results indicated that VEGFA has a positive effect in maintaining follicular development,inhibiting granulosa cell apoptosis and follicular atresia.2 Expression of miR-361-5p during follicular atresiaThe expression of miR-361-5p in follicles was detected by qRT-PCR.The results showed that miR-361-5p was mainly distributed in GCs of healthy follicles and in theca of atresia follicles.With follicular atresia the expression of miR-361-5p in the whole follicle,granulosa cells,thecal granulosa cells and follicular fluid was significantly increased.In situ hybridization(FISH)was used to detect the expression of miR-361-5p in follicles.The results showed that miR-361-5p was significantly higher in atresia follicles than in healthy follicles.Treatment with miR-361-5p mimics or inhibitor of granulosa cells revealed that miR-361-5p mimics caused a significant increased active caspase-3 expression and granulosa cell apoptosis rate,whereas miR-361-5p inhibitor resulted active caspase-3 expression and apoptosis rate significantly reduced.3 miR-361-5p regulates granulosa cell apoptosis by targeting VEGFABioinformatics analysis revealed that miR-3 61-5p and the VEGFA 3’untranslated region(UTR)have base-complementing sequences.The results of the dual luciferase reporter system assay demonstrated that miR-361-5p targeted the 3’ UTR of VEGFA.After miR-361-5p mimic or inhibitor treatment,qRT-PCR and Western Blot were used to detect the expression level of VEGFA,which confirmed the negative regulation of miR-361-5p on VEGFA in granulosa cells.The results of miR-361-5p inhibitor and VEGFA siRNA co-transduction experiments confirmed that interference with VEGFA reversed the inhibitory effect of miR-361-5p inhibitor on granulosa cell apoptosis.These results demonstrate that VEGFA is necessary in maintain cell survival and porcine follicular development,and miR-361-5p regulates granulosa cell apoptosis by targeting VEGFA 3’UTR and downregulating its mRNA levels.