Construction Of Mouse NR4A1 Adenovirus-delivered SiRNA Vector And Mouse NR4A1 Adenovirus Vector And Investigation Of It's Role In The Hormone Synthesis In Theca Cells

Objective: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder affecting women of reproductive age, the pathophysiology have yet to be determined. 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 and proliferation, steroid hormone production, insulin resistance and so on. However, the role of NR4A1 in the development of PCOS has not been documented before. In this study, we construct mouse NR4A1 recombinant adenovirus-delivered siRNA vector and then the theca cells mouse preantral follicles cultured in vitro infected with recombinant adenovirus. Fundamentally investigate the effect of NR4A1 on androgen producing enzymes in mouse theca cells. The aim of this study was to explore the effect of NR4A1 steroid hormone production in polycystic ovary syndrome and further demonstrate the role and mechanism of NR4A1 on the pathophysiology development of polycystic ovary syndrome at molecular level.Methods: (1) The synthetical oligonueleotide sequence, which could produce RNA interference to mouse NR4A1 was ligated into a shuttle vector pShuttle-H1, the shuttle vector was linearized with PmeI and homogenous recombination was conducted in the E.coli BJ-5l83 cells containing an adenoviral backbone plasmid pAdeasy-1.After linearized with PacI, the recon was transfected into AD-293 cells mediated by liposome and packaged to be infective adenovirus, which was repeatly infected and amplified. The expression of the NR4A1 protein in the infected MLTC-1 cells was detected by Western blot.(2) Mouse NR4A1 cDNA was amplified by PCR and was cloned into the shuttle vector pAdTrack-CMV,the shuttle vector was linearized with PmeI and homogenous recombination was conducted in the E.coli BJ-5l83 cells containing an adenoviral backbone plasmid pAdeasy-1.After linearized with PacI, the recon was transfected into AD-293 cells mediated by liposome and packaged to be infective adenovirus, which was repeatly infected and amplified. The expression of the NR4A1 protein in the infected AD-293 cells was detected by Western blot.(3) Individual preantral follicles of 200μm in diameter were isolated by manual dissection from ovaries of 18~20-day-old female mice were cultured in 100μl culture medium in vitro. Follicle diameter was measured by using a precalibrated ocular micrometer and culture medium was replaced with fresh medium every day. Follicle cultures were maintained for 3 days at 37°C under an atmosphere of 5% CO2。(4) After 24 hours of culture, follicles which exhibit an intact basement membrane, a dense complement of granulosa cells, a centrally located oocyte and attached theca cells were selected and infected with recombinant adenovirus Ad-H1-siRNA/NR4A1 or Ad-CMV-NR4A1. After an additional 48 hours incubation period, the follicles were collected and the knockdown of NR4A1 gene in mouse theca cells of follicles was demonstrated by western blot, and after 24 hours of infection, the effect of NR4A1 on androgen producing enzymes(StAR,CYP11A1,CYP17A1,HSD3B2) in mouse theca cells of follicles was analysed by Realtime PCR. (5) Mouse follicles were treated with Forskolin for 1, 2, 4, 6 hours separately, the expression of NR4A1 and androgen producing enzymes was detected by Realtime PCR.Results: (1) It was confirmed by restriction endonuclease analysis and sequencing that oligonueleotide sequence was inserted into pShuttle-H1 successfully. Recombinants were selected by kanamycin resistance and confirmed by PacI.The restriction endonuclease analysis confirmed that correct recombinant adenovirus plasmid was constructed (30 kb and 4.5 kb or 3.0 kb fragments were obtained after PacI digestion). The NR4A1 protein expression in MLTC-1 cells dramatically decreased. (2) It was confirmed by restriction endonuclease analysis and sequencing that mouse NR4A1 gene sequence was inserted into pAdTrack-CMV correctly. The NR4A1 protein expression in AD-293 cells dramatically increased.(3) During 3 days culture period, the follicles grown from 200μm to 320μm~400μm in diameter at last and the secretion of estradiol was also increased. (4) The expression of NR4A1 protein in the mouse theca cells of follicles infected with recombinant adenovirus Ad-H1-siRNA/NR4A1 was significantly decreased compared with the control groups, and the expression of NR4A1 protein in the mouse theca cells of follicles infected with Ad-CMV-NR4A1 was significantly increased. The mRNA expression of androgen producing enzymes (StAR, CYP11A1, CYP17A1, HSD3B2) in mouse theca cells of follicles after Ad-H1-siRNA/NR4A1 infection was significantly decreased compared with the control. The mRNA expression of androgen producing enzymes in mouse theca cells of follicles after Ad-CMV-NR4A1 infection was significantly increased compared with the control. (5) NR4A1 was rapidly induced with FSK, with an induction peak being reached after 1h of treatment. The expression of androgen producing enzymes were relative slowly induced with FSK,with an induction peak being reached after 2h or later of treatment.Conclusion:(1) The recombinant adenovirus exerting RNA interference to NR4A1 gene and mouse NR4A1 recombinant adenovirus have been constructed successfully, which offers a basis for the research into the pathogenesis of PCOS. (2) The mouse preantral follicle culture approach can help us investigate the role and mechanism of some differentially expressed gene and proteins in pathophysiological development of PCOS. (3) Knockdown of NR4A1 gene in mouse theca cells of follicles in vitro significantly suppressed mRNA transcription of androgen producing enzymes (StAR, CYP11A1, CYP17A1, HSD3B2) and overexpress of NR4A1 gene in mouse theca cells of follicles in vitro significantly stimulated mRNA transcription of androgen producing enzymes,which suggested NR4A1 might has some effects on the androgen production. The decreased expression of NR4A1 in PCOS might be related to the pathophysiological development of PCOS. (4) NR4A1 might be involved in theca cell androgen synthesis regulating by cAMP/PKA signaling pathway.