Study On The Mechanism Of DNA Methylation Modification And Fatty Acid Binding Protein 3 In Ovarian Insufficiency

Background Primary ovarian insufficient(POI)is one of the common endocrine diseases in gynecology.The fertility reduction,menopausal syndrome and other symptoms are serious problems for women's health.A large number of studies have confirmed that POI is related to genetic factors and environmental factors.But the pathogenesis of POI is not clear.Epigenetics is an important bridge to link environmental factors and genetic factors.DNA methylation as an important means of epigenetic modification has been the focus.Most of the previous POI studies on DNA methylation have mainly focused on individual genes.With the rapid development of science and technology genome-wide DNA methylation changes may be analyzed.In our study,we have performed whole-genome DNA methylation profiling in POI and a candidate gene associated with POI was determined,that is,fatty acid binding protein 3 gene(FABP3).FABP3 is a member of the family of fatty acid binding proteins(FABPs),which are a kind of small molecule protein in the cell.They usually were binding reversible hydrophobic ligands.FABP3 participates in the uptake,transport and intracellular metabolism of fatty acids.Therefore,FABP3 plays an important role in lipid metabolism.Abnormal lipid metabolism is closely related to POI.So it is speculated that FABP3 may be associated with POI.Objective To analyze DNA methylation of granulosa cells in POI and explore the function of different methylation genes associated with POI Method Follicular fluid were collected from two patients with POI and three women with healthy ovaries.Ovarian granulosa cells was isolate from follicular fluid.Granulosa cells was extracted genome DNA by using the kit,and then DNA was prepared for library preparation.The library preparations were sequenced on an Illumina Hiseq 2500 by Whole Genome Bisulfite Sequencing.Gene expression and DNA methylation of different methylation genes were analyzed.FABP3 gene became a candidate different methylation gene for POI.Crispr-cas9 genome editing technology was used to knock out FABP3 gene in human granulosa tumor-like cell line(KGN).Subsequent detection was performed on this cell line.First,the level of estradiol and cell proliferation were measured.The second step was to test CYP19A1 gene encoding aromatase enzyme that catalyses the final rate limiting step in E2 biosynthesis.The third step was to use DNA pull down technology to find the proteins binding with CYP19A1 gene.At last,the free fatty acids in FABP3 gene knockout KGN cells were analyzed.Results Genomic DNA methylation patterns in ovarian granulosa cells were compared between two groups of women.There was no significant difference between the POI patients and normal controls on a genome-wide scale.We extracted upstream?transcriptional start sites(TSS)?first exon,first intron?internal exon?internal intron?last exon and downstream from UCSC Genome Browsers and analyzed CpG methylation level in different regions.GO and KEGG analysis were carried out on the difference methylation region and differential methylation gene.The difference methylation gene was found to be enriched in 210 GO subclasses and 56 KEGG pathways.Through the functional analysis of the differential methylation gene,a candidate gene associated with POI was determined,that is,FABP3 gene.In FABP3 gene knock out KGN cells POI-like phenotype appeared:the level of estradiol level was low and the cell proliferation decreased.Gene expression and protein level of CYP19A1 was down-regulated in FABP3 gene knock out KGN cells.Nuclear factor ?B(NF-?B)that bind with CYP19A1 gene was found.Gene expression and protein level of inhibitor ?B alpha(I?B?)increased.Free fatty acids(FFA)significantly increased in FABP3 gene knock out KGN cells.POI-like phenotype reappeared in overexpression Peroxisome proliferator-activated receptors(PPARa)KGN cells.Conclusion DNA methylation of ovarian granulosa cells in primary ovarian failure were clarified.FABP3 regulated the production of estradiol in KGN cells.In FABP3 gene knock out KGN cells free fatty acid increased.FFA increased activity of PPARa.PPARa up-regulated I?B? and supressed activity of NF-?B.CYP19A1 was down-regulated and the production of estradiol decreased.FABP3 might be a new targeted gene associated with POI.