Study On The Regulation And Cell Function Of STAT3 Gene In Porcine Ovary Granulosa Cells

The ovary is one of the main reproductive organs of the sow,and is the site of follicular development and ovulation.The development of mammalian follicles is a complex and continuous physiological process that includes the recruitment of primordial follicles,the development of preantral follicles,the selective growth of antral follicles,and the maturation and atresia.The granulosa cells play a crucial role in the development of follicles as well as atresia.The signal transducer and activator of transcription 3(STAT3)is a member of the signal transduction and activator of transcription protein family and plays an important role in cell proliferation,differentiation,and apoptosis.Studies have shown that STAT3 gene is expressed in the ovary and participates in the growth and development of the follicle as well as the atresia process.In this experiment,the 5'regulatory region sequence of STAT3 gene was obtained by PCR amplification,and a dual luciferase reporter gene recombinant containing a 5'regulatory region deletion fragment was constructed,and then transfected into ovarian granulosa cells to analyze the promoter activity and identify the core regulatory regions in the promoter region.Bioinformatics website predicts and analyzes the potential transcription factor C/EBP? binding site in the core regulatory region of the STAT3 gene promoter,and then the chromatin immunoprecipitation(ChIP)and binding site mutation experiments were used to verify the binding of transcription factor C/EBP? to STAT3 core regulatory regions.The transcription factor C/EBP? overexpression vector and small interference RNA were constructed and transfected into porcine ovarian granulosa cells,then we used qRT-PCR,flow cytometry,Edu staining and Western Blot and other experimental techniques to analyze the effect of transcription factors on the regulation of STAT3 gene expression at the level of gene transcription and translation and the effect on the apoptosis and proliferation of granulosa cells.The STAT3 overexpression vector and the small interfering RNA were constructed and transfected into porcine ovarian granulosa cells,then Flow cytometry and Edu staining were used to study the function of STAT3 in apoptosis and proliferation of granulosa cells.Finally,the effect of transcription factors on the function of STAT3 in granulosa cells was verified by subgroup co-transfection.The main findings are as follows:(1)The STAT3 promoter-2199/+375 region was amplified by PCR and a promoterdeleted fragment recombinant vector was constructed.The activity of P3(-1034/+375)was significantly lower than that of P2(-1532/+375)with P4(-587/+375)by double fluorescence activity analysis,it was determined that the-1532/-1034 fragment contained a binding site for positive regulatory elements.(2)Transcriptional factors in the-1532/-1034 region were predicted by some bioinformatics websites such as TFBIND,JASPAR,and Bimas.ChIP demonstrated that C/EBP? recognizes and binds the-1397/-1387 region of STAT3 promoter.(3)The transcription factor C/EBP? expression vector was constructed and transfected into porcine ovarian granulosa cells.The results showed that C/EBP? promoted the transcriptional activity,mRNA production and protein synthesis of STAT3 core regulatory region;Interfered with C/EBP? inhibited transcriptional activity,mRNA production and protein synthesis of STAT3 core regulatory region,indicating that transcription factor C/EBP? promotes STAT3 gene transcription.(4)The target gene STAT3 expression vector was constructed.Through flow cytometry and Edu staining,it was found that STAT3 and C/EBP? can inhibit apoptosis of porcine ovarian granulosa cells and promote cell proliferation.C/EBP? enhanced the anti-apoptotic and pro-proliferative effects of STAT3 in ovarian granulosa cells.Taken together,the main conclusion of this study was that C/EBP? binds to the STAT3 gene promoter-1397/-1387 region and promotes its transcription and translation,and can enhance the anti-apoptotic and pro-proliferative effects of STAT3 in porcine ovarian granulosa cells.