Function And Mechanism Of Plasma Exosome MicroRNAs In Polycystic Ovary Syndrome

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Difference expression of plasma exosome microRNAs in polycystic ovary syndrome and control womenBackground:Polycystic ovary syndrome(PCOS)is a highly prevalent endocrine-metabolic disorder,affecting 6-10%of women.It is the first common cause of anovulatory infertility,characterized by ovulatory dysfunction,hyperandrogenism,polycystic ovary morphology,and metabolic disturbances such as insulin resistance,obesity,and dyslipidemia,which confers to increased risk of type 2 diabetes mellitus(T2DM),cardiovascular disease,etc.Nowadays,the pathogenesis of PCOS has not been well elucidated.Genetic variations are widely believed to serve as a causal contributor.Several candidate genes have been revealed through genome wide association studies(GWAS)and pedigree studies.However,despite their apparent association with PCOS,none were identified to have a direct causal effect on the occurrence of the syndrome due to the genetic variations.MicroRNA(miRNA)is a kind of single-stranded noncoding RNA composed of 20-24 nucleotides,which can diversely regulate gene expression post-transcriptionally through cleavage or translational repression of specific target messenger RNAs(mRNAs).Circulating miRNAs exist in both the supernatant(combined with proteins or lipoproteins)and extracellular vesicles(EVs).EVs are heterogeneous membranous vesicles originating from different cells.The smallest subpopulation of them with a diameter of 30-150nm is named exosome.It is considered to be secreted depending on the fusion of multivesicular bodies with the plasma membrane.Exosomes can carry and release the contents into the target cells,acting as an intercellular mediator.Previous studies reported that miRNAs were more stable in exosome than in the supernatant and might have bigger weight in gene regulation.As a systemic disease,PCOS compromises multi-organs rather than the ovary alone.It is assumed that circulating exosome have different miRNA expression in PCOS,which may transport aberrant regulating information to the target organs and result in the functional abnormality of related genes and pathways.Objective:The aim of the study was to identify the difference of exosome between PCOS and control women,and to clarify the plasma exosome miRNA expression profile of PCOS women.Method:Plasma exosomes were isolated from PCOS women and age-matched normal controls by ExoQuick Precipitation methods.MiRNA sequencing was conducted among 15 PCOS women and 15 control women,and the expression profile of exosomal miRNAs was further determined by real time PCR in a larger sample size of 75 PCOS and 75 control women.Gene ontology(GO)and Kyoto Encyclopedia of Genomes pathway(KEGG)enrichment analyses were performed.Correlation analysis and receiver operating characteristic(ROC)curve analysis were used to test the association between PCOS phenotypes and miRNA expression.Result:PCOS subjects displayed a significantly elevated concentration of smaller exosomes,while totol exosome concentration is similar between groups.MiRNA sequencing showed 34 exosomal miRNAs differentially expressed between PCOS women and controls.Five miRNAs were validated differentially expressed by qRT-PCR,of which miR-18a-3p,miR-20b-5p and miR-106a-5p were down-regulated in PCOS,while miR-126-3p and miR-146a-5p were up-regulated in PCOS.GO and KEGG analysis revealed that the predicted target functions of these differentially expressed miRNAs mainly involved in axon guidance,MAPK signaling,endocytosis,circadian rhythm and cancer pathways.The expression of these 5 miRNAs mainly correlated with menstrual cycle,antral follicle counting(AFC),anti mullerian hormone(AMH),testosterone and luteinizing hormone(LH)levels.Conclusion:The diameter distribution of plasma exosome was smaller in PCOS women compared of control women.The expression profile of plasma miRNA in PCOS women was different from that in normal controls,and the miRNA expression was associated to PCOS reproductive phenotypes.Exosomes can act as the vector of pathogenic miRNAs which may lead to PCOS.Chapter ? Pathogenic effect of plasma exosomes in polycystic ovary syndromeBackground:Exosomes can carry and release contents into target cells to act as intercellular mediators.Their lipid bilayer structure can protect the contents from degradation or modification,to ensures the stability of the cargos.In recent years,the function of exosome in female reproductive diseases has been gradually revealed.Exosomes can act as carriers of signaling molecules,and play an important role in reprodective process such as follicular development and embryo implantation,as well as in ovarian tumor,PCOS,premature ovarian insufficiency(POI)and other reproductive diseases.PCOS is a complex and heterogeneous endocrine disorder.However,the etiology of PCOS is still poorly understood.In the previous chapter we found the plasma exosome of PCOS women and the exosome derived miRNAs are different from those of normal women.It is suggested that the plasma exosome of PCOS women may contain some PCOS pathogenic factors,which may lead to abnormal reproductive phenotypes.Objective:To explore the effect of PCOS plasma exosome on reproductive phenotypes of mice,by injecting PCOS plasma exosome into wild type mice via tail vein.Method:The plasma exosome of PCOS and normal women were isolated and injected separately into 8-week-old ICR female mice via tail vein.The changes in estrous cycle,serum hormone levels and ovarian morphology were observed.Result:ICR female mice were injected with plasma exosome of PCOS women,control women,or 150?l PBS respectively,and to observe the estrous cycle of mice for 4 weeks.The results showed that most of the mice injected with PCOS exosomes had markedly disordered estrous cycles,presented as irregularly prolonged cycles and a significant decrease in the number of intact estrous cycles in 2 weeks after injection.However,after 2 weeks of injection,the estrous cycle gradually tended to be normal,and the number of intact cycles was no longer significantly different.The serum hormones and ovarian morphology of diestrus mice were detected after exosome injection for 48 hours.It was found that the ovarian morphology of female mice did not change after injection of PCOS exosomes,and the levels of luteinizing hormone(LH)and follicle stimulating hormone(FSH)increased,but with no significant difference.Other mice were injected with 5 IU pregnant horse serum gonadotropin(PMSG)intraperitoneally after exosome injection for 48 hours.Forty four hours after PMSG treatment,the ovarian morphology was not different between mice injected with PCOS exosome or control exosome.Serum estradiol,testosterone,LH and FSH levels were slightly increased in PCOS group,with no significant difference.Conclusion:PCOS plasma exosome can make wild type mice appear PCOS-like reproductive phenotypes,including irregular estrous cycle,and slightly increasing of estradiol,testosterone and LH levels.Chapter ? The etiological mechanism of plasma exosome microRNA in polycystic ovary syndromeBackground:PCOS is a complex condition with reproductive,metabolic and psychological features.As a common reproductive endocrine disorder in reproductive-age women,PCOS is characterised by hyperandrogenism,irregular ovulation,,and polycystic ovaries,whose effects mainly occur in follicles.The follicle development process of PCOS women is abnormal,presented as accumulation of excessive pre-antral and small antral follicles,resulting in the failure in dominant follicle selection.Granulosa cells are important somatic cells that surround oocytes.Normal proliferation and differentiation of granulosa cells are critical for the development of ovarian follicles.Additionally,granulosa cells are essential for steroid hormone production.In PCOS women,abnormal follicular development may partly result from dysfunction of granulosa cells.MiRNA is a type of single-stranded noncoding RNA that can diversely regulate target gene expression.MiRNA plays an important role in post-transcriptional regulation in gene expression,and a single miRNA can regulate hundreds of target genes.In the ovary,miRNA can affect the growth and development of follicles by regulating the proliferation,differentiation,apoptosis and steroid hormone synthesis of granulosa cells.Recent studies reported that the differentially expressed miRNA in granulosa cells would be involved in the pathogenesis of PCOS.MiRNA is more stable in exosomes than in the supernatant.Therefore,the exosomal miRNAs may be essential in the regulation of granulosa cells,and further affect the development of follicles.In previous study,we have confirmed that PCOS plasma exosome can make wild type mice appear PCOS-like reproductive phenotypes to a certain extent.The previous sequencing results showed the expression of miRNA in PCOS plasma exosome was different from that in control women.Therefore,we speculate that the the pathogenic effect of PCOS exosome is caused by miRNAs in exosome.Objective:The aim of the study was to identify the function of differentially expressed exosomal miRNAs in PCOS pathogenisis,and to partly explain etiological mechanisms of PCOS via epigenetic regulation.Method:In the previous study,we identified five differentially expressed exosomal miRNAs in PCOS plasma by high-throughput sequencing and following qRT-PCR validation,named as miR-18a-3p,miR-20b-5p,mir-106a-5p,miR-126-3p and miR-146a-5p.KGN cells were cultured for detecting the proliferation,apoptosis and steroid hormone synthesis,after enhancing or repressing the miRNAs expression.Of all these five miRNAs,the most obvious phenotypes were observed with miR-126-3p.PDGFRB is one of target genes of miR-126-3p.The function of KGN cells were identified after overexpressing miR-126-3p or repressing the target gene PDGFRB.The downstream pathways of PDGFRB were also detected.Result:The exosomes were stained and co-cultured with granulosa cells for 24 hours.The stained exosomes were observed in KGN cells,indicated that granulosa cells might be the target cells of plasma exosomes,and the miRNAs in exosomes might be released into granulosa cells,therefore affected the target gene expression.Overexpression or inhibition of miR-18a-3p,miR-20b-5p,miR-106a-5p,miR-126-3p or miR-146a-5p affected the proliferation,apoptosis and steroid hormone synthesis of KGN cells,among which miR-126-3p has the strongest effect.Overexpression of miR-126-3p increased estradiol synthesis and decreased progesterone synthesis,and significantly repressed the proliferation of KGN cells.PDGFRB was found to be the downstream target gene of miR-126-3p.Immunohistochemical detection showed that PDGFRB was highly expressed in ovarian granulosa cells.Suppressing endogenous PDGFRB expression in KGN cells showed similar result as overexpression of miR-126-3p.Moreover,overexpression of miR-126-3p or inhibition of PDGFRB can both reduce the expression of phosphorylated-Akt,indicating that miR-126-3p may restrain proliferation of granulosa cells by supressing the expression of PDGFRB and its downstream PI3K-Akt pathway.Conclusion:The differentially expressed plasma exosomal miRNAs in PCOS women negatively regulate the biological function of granulosa cells,which may affect the PCOS progression.MiR-126-3p may affect the proliferation of granulosa cells through target gene PDGFRB and its downstream PI3K-Akt pathway.