| The ovary is an important reproductive organ of female animals and an important organ for follicular development and atresia.Follicles are the structural and functional units of the ovary.The formation of follicles,the recruitment of follicles,the selection of dominant follicles and ovulation play an important role in the reproductive performance of sows.The growth process of follicles is accompanied by the development and differentiation of Granulosa cells(GCs).The proliferation and apoptosis of GCs are closely related to follicular development and atresia.MicroRNA(miRNA)are short non-coding RNA.Numerous studies have shown that miRNA and their target genes in GCs affect the molecular mechanism of GCs,which in turn affects the growth and development of follicles.Based on previous studies,this study combined the miRNA sequencing and pre-experimental results of the ovarian follicle and luteal phase of the sow in the previous study group to screen for miR-21-5p which was significantly expressed in sows and speculated that it participated.The reproductive process of pigs.The position of miR-21-5p in porcine ovarian tissue and GCs was firstly investigated by fluorescence in situ hybridization(FISH).The function of miR-21-5p on GCs was detected by flow cytometry,MTT colorimetry and ELISA.The effect of bioinformatics,construction of dual luciferase reporter gene,qRT-PCR and Western blot to verify the negative regulation of miR-21-5p on Smad7;followed by immunofluorescence technique to explore Smad7 in pig ovarian tissue and GCs The location was determined by flow cytometry,MTT colorimetry and ELISA to detect the effect of Smad7 on GCs.The results of the study have deepened the understanding of the molecular mechanism of pig GCs and have important significance for improving the reproductive performance of sows.(1)Fluorescence in situ hybridization showed that miR-21-5p was widely expressed in the cytoplasm of porcine follicular membrane and GCs.Immunofluorescence showed that Smad7 was widely expressed in porcine follicular membrane and GCs nucleus.(2)The results of flow cytometry showed that miR-21-5p or Smad7-siRNA could inhibit the apoptosis of GCs,and significantly increase S-phase cells and proliferation index.MTT method was used to demonstrate that miRNA-21-5p or Smad7-siRNA could promote the proliferation of GCs.(3)The results of ELISA showed that miR-21-5p mimics or Smad7-siRNA could increase the level of estradiol and reduce the level of progesterone in GCs.(4)Bioinformatics predicted that the key gene Smad7 in the transforming growth factor-β(TGF-β)signaling pathway is targeted by miR-21-5p.Further,it was confirmed by the dual luciferase reporter that miR-21-5p binds to the Smad7 3’UTR and inhibits luciferase activity.(5)The qRT-PCR and Western blotting showed that miR-21-5p could negatively regulate Smad7 expression at transcriptional level and translation level.These results revealed that miR-21-5p can inhibit the apoptosis of GCs and promote the proliferation of GCs through Smad7,providing a reference for understanding miRNA in regulating ovarian follicle development,and laying the foundation for further research on Smad family in GCs. |
