Construction Of Human NR4A1 Adenovirus Vector And Investigation Of It's Role On The Development Of Polycystic Ovary Syndrome

Objective:NR4A1(also known as NGFI-B,nur77 and TR3)is a member of the steroid/thyroid/retinoid receptor super-family that acts as a transcriptional factor to regulate gene expression.By differential cDNA microarray hybridization in our laboratory,we identify 290 gene differentially expressed between normal and PCOS ovaries,and among this group the most interesting gene is NR4A1,which is down-regulated in PCOS ovary.Numerial studies indicate NR4A1 are expressed in many tissues and involved in various biologic functions,such as cell apoptosis or proliferation,steroid hormone production,signal conduction and so on. Howerve,the role of NR4A1 in the development of PCOS have not been documented before.In this study,we cloned the full length cDNA of human NR4A1 and constructed its recombinant adenovirus vector.Then, NR4A1 recombinant adenovirus vector was transfected into mouse theca cells on the platform of mouse preantral follicle culture approach. Fundamentally investigate the effect of NR4A1 on testosterone production in mouse theca cells.The aim of this study was to explor the effects of NR4A1 on steroid hormone production in polycystic ovary syndrome and further demonstrate the role and mechanism of NR4A1 on the etiology and pathophysiology development of polycystic ovary syndrome at molecular level,which will provide strategies focusing on prevention and treatment of PCOS.Methods:(1)NR4A1 cDNA was amplified by RT-PCR from human ovary and was cloned into PGEM-T vector.NR4A1 cDNA was then subcloned into the shuttle vector pAdTrack-CMV for the construction of pAdtrack-NR4A1.The shuttle vector was linearized with PmeⅠand subsequently transformed into E.coli BJ-5183 cells with adenovirus backbone plasmid pAdEasy-1 to achieve homologous recombination. After digestion with PacⅠ,it was transfected into AD-293 cells for packaging and amplification.The appearance of recombinant adenovirus were determined by GFP expression.The expression of NR4A1 gene was confirmed by PCR and Westem blot analyses.(2)Mouse preantral follicles(diameter:200μm)isolated from ovaries of 20-day-old female mice were cultured in vitro.After 24 hours' culture,theca cells were infected with recombinant AdCMV-NR4A1 adenovirus.Overexpression of NR4A1 gene in mouse theca cells was demonstrated by RT-PCR and western blot.The culture media were collected to compare the difference of testosterone level between NR4A1-overexpression group and control group by high sensitive RIA.Results:(1)A 1797bp cDNA was amplified by RT-PCR from human ovary with correct sequence.Then,recombinant adenovirus vector AdCMV-NR4A1 was constructed successfully.After transfected it into AD-293 cells in which the recombinant adenovirus are packaged and amplified,the high titer recombinant adenoviruses were harvest.(2) Mouse preantral follicle culture approach was established successfully. NR4A1 recombinant adenovirus vector was transfected into mouse theca cells by this platform.Compared with control group,NR4A1 gene overexpressed in mouse theca cells infected with AdCMV-NR4A1.The culture media were collected to compare the difference of testosterone level between NR4A1-overexpression group and control group by high sensitive RIA.The result indicates that overexpression of NR4A1 in mouse theca cells significantly suppressed testosterone secretion (p＜0.05). Conclusion:(1)Human NR4A1 gene has been cloned successfully and its recombinant adenovirus vector constructed.This lay a foundation for further research on the role and mechanism of NR4A1 gene in PCOS. (2)The mouse preantral follicle culture approach can help to investigate the role and mechanism of some differentially expressed gene and proteins in pathophysiological development of PCOS.(3)Overexpression of NR4A1 in mouse theca cells significantly suppressed testosterone secretion,which suggested NR4A1 might has some effects on the testosterone production.The decreased expression of NR4A1 in PCOS may contribute to the development of hyperandrogenism.