The Role Of FTO And Nicotinamide In Steroid Hormone Abnormality Of Polycystic Ovary Syndrome

Chapter I The Role of FTO in Steroid Hormone Abnormality of Polycystic Ovary SyndromeObjectivePolycystic ovary syndrome(PCOS)is a highly complicated reproductive endocrine disease,which is characterized by hyperandrogenism,abnormal menstruation and polycystic ovary.About 50%of PCOS women accompanied with metabolic abnormalities such as obesity and insulin resistance.The etiology of PCOS is complex and many single nucleotide polymorphisms(SNPs)are related to PCOS.FTO is the first susceptibility gene of BMI and obesity found in genome-wide association analysis.In subsequent studies,multiple SNPs of FTO were found significantly correlated with the risk of PCOS.The FTO rs9939609 A/T polymorphism showed significant correlation with PCOS in Chinese women independent from BMI.It is suggested that FTO might play an important role in reproductive endocriminal disease,except metabolic disorder and obesity.In this study,we detected the expression of FTO in granulosa cells of PCOS patients and control population,analyzed the correlation between the expression of FTO in granulosa cells and clinical subtypes and further investigated the expression characteristics of FTO in different PCOS subtypes.Based on the results,the function of FTO in steroid anabolism was further explored by constructing FTO knockout and over expression human granulosa cell lines and conditional Fto knockout mice.MethodsIn this study,a total of 131 Chinese Han women(67 PCOS cases and 64 control cases)were collected.The diagnosis of PCOS was according to the Rotterdam criteria.The control cases were infertile women underwent IVF-ET or ICSI-ET due to male factors.The expression of was detected by real-time quantitative PCR and the difference of expression between PCOS group and control group was analyzed.The correlations between FTO expression level and clinical characteristics were analyzed by Spearman correlation.The expression of FTO in different PCOS subtypes was further analyzed.FTO knockout and overexpression human granulosa cell lines were constructed by CRISPR-Cas9 and lentivirus transfection respectively.Knockout and overexpression efficiency were detected by genomic DNA PCR,real-time quantitative PCR and Western blot.CCK-8 experiment was used to analyze the effect of FTO on the activity of granulosa cells;flow cytometry was performed to detect the role of FTO in the cell cycle and apoptosis of granulosa cells;real-time quantitative PCR and Western blot were performed to detect the changes of differentiation marker genes of granulosa cells.Based on the characteristics of steroids synthesis in granulosa cell lines,the level of estrogen in the supernatant of granulosa cells was detected by electrochemiluminescence.The expression of Sulfotransferase Family IE Member 1(SULT1E1),the key enzyme of estrogen metabolism,was detected by real-time quantitative PCR and Western blot.In addition,the rescue experiment was performed via RNA interference to knock down the expression of SULT1E1 in granulosa cells,which further confirmed that FTO affected the metabolism of estrogen by regulating the expression of SULT1E1 The regulatory factors of SULT1E1 were predicted according to database.The real-time quantitative PCR and Western blot were used to detect the expression of transcription factor C/EBP?,which could potentially regulate the expression of SULT1E1.Chromatin immunoprecipitation was performed to further confirm that transcription factor C/EBP? could bind and regulate the promoter region of SULT1E1.Moreover,the alternative splicing variants of androgen receptor(AR)was performed by real-time quantitative PCR to explore the potential role of epigenetic effects of FTO in steroid hormone regulation.Fto conditioned knockout(Foxl2-Cre)mice were also constructed.Hematoxylin-Eosin(HE)staining was performed to observe the changes of ovarian morphology and follicles.The serum was collected to detect the levels of estradiol,testosterone and progesterone via radioimmunoassay.The roles of Fto on steroid hormones in mice granulosa cells were investigated.ResultsIn the first section of this chapter,through detecting the expression of FTO in the granulosa cells of PCOS cases and control,it showed that the expression of FTO has no significant change in PCOS granulosa cells compared with controls.The expression level of FTO of control group showed significantly positive correlation with BMI and metabolic characteristics,which was consistent with the existing researches.The analysis in PCOS subtypes showed that the expression of FTO decreased significantly in the PCOS with hyperandrogenism,suggesting that FTO may be involved in the steroid hormone anabolism.In the second section,it showed that FTO had no effect on the cell activity,cell cycle and apoptosis of granulosa cells,but affected the expression of granulosa cell differentiation marker genes and related transcription factors(CYP19A1,C/EBP?,FOXL2,GATA4,etc.).Further detection of the estrogen level in the cellular supernatant showed that FTO knockout could significantly reduce the estrogen level in the cellular supernatant.The mechanism study showed that FTO knockout could promote the expression of transcription factor C/EBP?,and then regulated the expression of SULT1E1,which enhanced the sulfide metabolism ability of estrogen,and decreased the active estrogen level.In addition,FTO knockout may affect alternative splicing of AR through epigenetic effect and regulated the anabolism of steroid hormones.In the third section,by constructing Fto conditional knockout(Foxl2-Cre)mice,it showed that Fto was knockout in ovary,pituitary and hypothalamus of mice.The Fto knockout mice also showed smaller ovary,lower serum estradiol and higher testosterone after PMSG injection 44 hours.It preliminarily demonstrated that Fto may affect the anabolism of steroid hormones in mice.ConclusionThe expression of FTO in granulosa cells of PCOS patients was significantly decreased.The function study showed that FTO knockout increased transcription factor C/EBPP,and then increased the target genes SULT1E1 expression,which affected the estrogen sulfated metabolism of granulosa cells.In the meanwhile,FTO may affected alternative splicing of AR and further had an influence on the anabolism of steroid hormones.In conclusion,FTO might be involved in the reproductive endocrine by regulating several critical genes of steroidogenesis and metabolism.Chapter II The Role of Nicotinamide in Steroid HormoneSynthesisObjectivePolycystic ovary syndrome(PCOS)is a disorder with highly clinical heterogeneity,which is characterized by reproductive dysfunction and metabolic disorders.Acne is one of the common clinical manifestations of hyperandrogenism in PCOS.And the occurrence of hyperandrogenemia with PCOS in type 1 diabetic women reaches 24%.Nicotinamide is a derivative of vitamin B3,which exists in many foods.At present,it is widely used in the adjuvant treatment of acne vulgaris and the control of type 1 diabetes development,and it has low side effects.However,there is no report on the role of nicotinamide in PCOS.This study designed to explore the effect of nicotinamide on androgen anabolism through NCI-H295R cell model in starved medium.Combined with the transcriptome analysis,the changes and regulatory networks of genes and signaling pathways related to steroid anabolism after nicotinamide treatment were investigated to explore the possible role of nicotinamide in steroids abnormality of PCOS.MethodsIn this study,NCI-H295R,an ideal model for studying androgen synthesis and metabolism,was used to treat the cells with nicotinamide at different doses(1mM,5mM and 25mM).CCK-8 assay was used to analysis the effect of nicotinamide on cell activity.The cellular supernatant was collected to detect the level of testosterone,which was normalized by the cellular protein content.The potential molecular targets of nicotinamide in androgen regulation were analyzed by transcriptome sequencing.Real time quantitative PCR was used to verify differential gene expression.Cluster analysis and principal component analysis were used for the differentially expressed genes(DEGs,fold change>2,FDR<0.01)cluster and dimension reduction.Gene set enrichment analysis(GSEA)analysis was used to analyze the gene set enrichment of all regulatory genes of nicotinamide treated NCI-H295R cells.KEGG and GO analysis were performed to analyze the pathway enrichment of the DEGs after nicotinamide treatment.The String database was used to construct the protein-protein interaction network of DEGs in the crucial pathways.ResultsIt showed that 25 mM nicotinamide could inhibit NCI-H295R cell activity.Nicotinamide also inhibited the production of testosterone in NCI-H295R cells in a dose-dependent manner.Transcriptome analysis showed that there were 2835 DEGs(fold change>2,FDR<0.01)after 25mM nicotinamide treatment,among which 1631 genes were significantly up-regulated,and 1204 genes were significantly down-regulated.Many genes related to steroid anabolism(CYP11A1,CYP17A1,HSD3?2,SULT2A1,AKR1C3,IGF1,etc.)were significantly decreased.GSEA analysis showed that various gene sets were significantly changed,including E2F targets gene set,TGF?signaling gene set,androgen response gene set,estrogen response gene set,targets of IGF1 and IGF2 gene set,steroid hormone biosynthesis gene set,cell cycle gene set,mitotic related pathway gene set,steroid dehydrogenase activity gene set and ovulation cycle gene set.KEGG analysis showed that the DEGs were significantly enriched in TGF? signaling pathway,cytokine-cytokine receptor interaction,and ovarian steroidogenesis pathway.Go analysis of DEGs revealed that the main biological processes were cell proliferation and differentiation,SMAD protein signal transduction and cellular hormone metabolic process.In terms of the molecular function,it was enriched in RNA binding and cellular components as protein-containing complex.The results of protein-protein interaction analysis showed that there were many close interactions among proteins encoded by the DEGs in steroid hormone synthesis,TGF?signaling pathway and cell proliferation.ConclusionIn summary,nicotinamide inhibited androgen synthesis in a dose-dependent manner.On the one hand,many key enzymes in androgen biosynthesis pathway significantly changed,such as CYP11A1,CYP17A1,HSD3?2 and AKR1C3;on the other hand,cell cycle,cell proliferation and hormone synthesis pathways were significantly enriched in DEGs,indicating that nicotinamide might regulate androgen anabolism through these pathways.These findings can not only explain the mechanism of nicotinamide in the treatment of acne,but also provide theoretical evidence for nicotinamide as a complementary and alternative medicines for hyperandrogenism of PCOS therapy.