The Mechanism Of TRIM28 In Polycystic Ovary Syndrome

OBJECTIVE: The objective of the study was to optimize the identification system of AR splicing variant and explore the expression pattern of TRIM28 in granulosa cells(GCs)of polycystic ovary syndrome(PCOS)women.DESIGN: Case-control study.MATERIALS AND METHODS: 139 PCOS women and 120 women with tubal blockage undergoing IVF-ET in our center were recruited.GCs and follicular fluids were collected during oocyte retrieval.Nested PCR was used to optimize the identification system.The expression pattern of TRIM28 in granulosa cells of PCOS women and oocytes were determined by RT-PCR,immunofluorescence(IF).RIA and ECL were used to test the hormone level of serum and follicular fluid.RESULTS:(1)The optimized system could quickly identify the PCOS patients with AR splicing variant.The sensitivity and accuracy were significantly improved,compared with the previous study;(2)There have been different results between ECL and RIA in testing testosterone and estradiol of follicular fluid,and the RIA results were more in line with the pathophysiological changes of the hyperandrogenism;(3)We found the TRIM28 m RNA and protein level in granulosa cells of PCOS women was significantly up-regulated compared with the control group(P<0.05);(4)TRIM28 is highly expressed in human oocytes.CONCLUSION: Through the optimized identification system,we can quickly and accurately classify PCOS patients so that they can be better targeted therapy.Our finding indicates that TRIM28 is highly expressed in GCs of PCOS patients,which suggesting that it plays a role the pathogenesis of PCOS.OBJECTIVE: The objective of the study was to investigate the regulation of TRIM28 on the steroidhormone synthesis and involved molecular mechanism based on stably transfecting cell models.DESIGN: Basic research.MATERIALS AND METHODS: A human granulosa-like tumor cell line(KGN)was identified by STR analysis,stably transfected by lentivirus and used for in vitro test.We performed RT-PCR,real-time PCR,lentivirus construction,stable transfection,RNA and Ch IP sequencing,ELISA,Co-IP,Western blotting,PKA activity assay,luciferase reporter assay and bisulfite sequencing.RESULTS:(1)In the transcriptome study,940 differentially expressed genes were selected for further analysis from KGN with TRIM28 gene overexpression or empty vector,and 2517 were selected from interference group,with a total of 259 repeated differentially expressed genes.And a series of genes were identified as significantly enriched in ovarian steroidogenesis pathway.(2)The combination analysis of RNA sequence and Ch IP sequence revealed that PDE10 A could be regulated by TRIM28 and the m RNA expression of PDE10 A was significantly up-regulated in both GCs of PCOS women and KGN with TRIM28 overexpression compared with the control group(P<0.05).(3)Since PDE10 A hydrolyzes cyclic adenosine monophosphate(c AMP),the c AMP and PKA activation of TRIM28 overexpression KGN was inhibited under the FSH stimulated compared to control(P<0.05),therefore leading to a decrease in the phosphorylation of ERKs.(4)Inhibitor of PDE10 A could partially reverse changes inhibition for ERK pathway induced by TRIM28.(5)TRIM28 could bind to the PDE10 A intron region as an enhancer regulating its transcription;(6)TRIM28 could also interact with TP53 to enhance PDE10 A transcription.CONCLUSION: Our finding strongly indicates the elevated TRIM28 level reduces ERK phosphorylation by up-regulating the expression of PDE10 A,leading to ovarian steroidogenesis dysfunction in GCs,resulting in polycystic ovary syndrome especially ovarian hyperandrogenism.OBJECTIVE: The objective of the study was to identify the effect of Trim28 overexpression on reproduction and endocrinology based on transgenic mice models.DESIGN: Animal experiment.MATERIALS AND METHODS: The ovarian specific overexpression of Trim28 transgenicm mice constructed by CRIAPR/Casp9 and Cre/lox P techniques were used as animal models,and the phenotypic observation lasted for 20 weeks to clarify the role of Trim28 in the pathophysiological changes of PCOS and explore the molecular mechanism involved.The research process involved RT-PCR,real-time PCR,qualitative and semi-quantitative detection of PKA activity,glucose and insulin tolerance tests,body composition analysis and fat imaging based on MRI,tissue HE staining,vaginal smear,etc.RESULTS:(1)Compared with the control group,Trim28 ovarian specific overexpression mice exhibited increased body weight,fat content began from 12 weeks and a bi-modal body-weight distribution.The m RNA levels of the obesity related genes Pcsk1,Npc1 and Fto were significantly up-regulated compared with the control group.(2)The Trim28 female mice showed impaired insulin tolerance at 12 weeks compared with the control group.And the m RNA level of Irs1 gene was down-regulated and Glut4 up-regulated.(3)The ovarian HE staining results of Trim28 mice indicated that immature follicles increased,vaginal smears showed prolonged estrus and cycle disorder.Elisa showed that testosterone level in peripheral serum of Trim28 female mice was increased.The m RNA levels of Hsd3b1/2,Hsd11b1,Cyp11a1,Cyp17a1,Srd5a2 were significantly up-regulated and meanwhile the expression levels of Pde2 a,and Pde4 a also showed the same changes.CONCLUSION: The above phenotypes were highly similar to the clinical manifestations of PCOS women and echoed with the molecular mechanism of Part II,which further confirmed the key role of TRIM28 in the pathophysiological changes of PCOS at the in vivo level.