P65 Regulates Transcription Of FGFR1 And Affects The Function Of Granulosa Cells In Porcine Ovary

Fibroblast growth factor receptor 1(FGFR1)is a transmembrane protein that binds to its ligand FGFs and activates signaling pathways such as PLCγ,MAPK and PI3 KAKT,and plays an important role in cell proliferation,differentiation,survival and migration.Excessive apoptosis of ovarian granulosa cells may induce follicular atresia and impede follicular development.The study found that FGFR1 and p65 are involved in the regulation of proliferation and apoptosis of ovarian granulosa cells,affecting the growth and development of follicles.In the early stage of the study,there were six p65 binding sites on the FGFR1 promoter,so it is speculated that p65 may affect the follicular development by regulating the transcriptional expression of FGFR1.To explore the function of FGFR1 and p65 genes in the development of porcine ovarian follicles,q RT-PCR method was used to detect the expression of FGFR1 and p65 m RNA in porcine ovarian immature and mature ovarian tissue,as well as large and small follicular granulosa cells;Construction of FGFR1 and p65 eukaryotic expression vectors and si RNA to study the effects of FGFR1 and p65 on granulosa cells proliferation,apoptosis and estrogen secretion;To construct recombinant vector of FGFR1 promoter reporter gene with deletion of p65 binding site and detect the activity change of FGFR1.The main results of this study are as follows:(1)The expression level of FGFR1/p65 m RNA in mature ovarian tissue and large follicle(≥ 5 mm)granulosa cells were significantly higher than that in immature ovarian tissue and small follicle(≤ 3 mm)granulosa cells.(2)The results of EdU,ELISA and Annexin V-FITC/PI double staining assay showed that FGFR1 gene promoted granulosa cells proliferation and estrogen secretion,inhibited granulosa cells apoptosis.(3)There are potential binding sites of transcription factors p65 in the FGFR1 promoter regions-1673/-1663,-1283/1273,-1056/-1046,-746/-736,-534/-524,-348/-338,Double fluorescence activity assay and Ch IP assay showed that transcription factors p65 bind to the-348/-338 region to positively regulate the transcription of FGFR1 gene.(4)The results of EdU,ELISA and Annexin V-FITC/PI double staining assay showed that transcription factor p65 promoted granulosa cell proliferation and estrogen secretion,inhibited granulosa cell apoptosis.Conclusion: The transcription factor p65 may binds to the-348/-338 region of FGFR1 promoter to positively regulate the expression of FGFR1,promote granulosa cells proliferation and estrogen secretion,inhibit the apoptosis of granulosa cells.