The Expression Profile Of High-throughput LncRNA Sequencing In Exosomes From The Follicular Fluid Of Patients With Polycystic Ovary Syndrome

Objective:In this study,high-throughput sequencing of follicular fluid exosomes in patients with polycystic ovary syndrome(PCOS)was performed to verify that the long noncoding RNA(lncRNA)were involved in normal follicular development and abnormal follicular development in PCOS patients.To investigate the molecular functions of differentially expressed lncRNAs,metabolic pathways and functional signaling pathways which will be the foundation of diagnosing biomarkers or potential therapeutic approaches.Methods:Selected infertility patients who received in vitro fertilization/intrafollicular sperm injection-embryo transfer(IVF/ICSI-ET)assisted at the Reproductive Center of Northern Jiangsu People’s Hospital,Yangzhou City,Jiangsu Province from January 2018 to December 2018.Total of 56 subjects were included this research,28 PCOS and 28 control with fallopian tube factors.We compared the basic clinical data between control and PCOS group included basic endocrine hormones,BMI,and antral follicle count.Follicle fluid exosomes were extracted by ultracentrifugation,and observed and detected by transmission electron microscopy(TEM).Nanoparticle Tracking Analysis(NTA)was performed to measure the size distribution and concentration of isolated particles of follicular fluid.And to identify the isolated exosomes,western blotting analysis was conducted to detect the expression of CD9.3 PCOS exosomes and 3 controls exosomes were selected for sequencing and database construction,and performed genes cluster analysis,GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes).Six significant differentially expressed lncRNAs were selected to verify the accuracy of sequencing results by RT-qPCR.Screening selected differentially expressed lncRNA interacting was mapped by the GeneMANIA website(http://genemania.org).Results:1.Compared with control group,PCOS group were no statistically significant differences in age,progesterone(P),estrogen(E2),follicle stimulating hormone(FSH),number of retrieved oocytes,number of transferable embryos,fasting blood-glucose and prolactin(PRL)between two groups(P>0.05).However,there were statistically significant differences in luteinizing hormone(LH),body mass index(BMI),and antral follicle count in the two groups(P<0.05).2.Successfully isolated and identified exosomes from follicular fluid.The morphology of exosome observed under a transmission electron microscope was a circular vesicle with a bilayer membrane structure.The nanoparticle tracking system(NTA)measured the concentration and size of exosomeswhich showedthe particle concentration was 4.9×106 particles/mL,and the peak diameter of exosome was 127 nm.Western blot results showed that exosomes from follicular fluid were positive for the exosomes’ marker CD9.3.Good quality RNA integrity number(RIN value)in exosomes.The Norgen kit was extracted exosomes RNA,and the NanoDrop ND-2000(ThermoScientific)was employed for quantification.The results were consistent with the results of most exosomal RNA studies.The A260/280 ratio of exosomal RNA was between 1.1-1.6.Additionally,the Agilent Bioanalyzer 2100(Agilent Technologies)was employed to detect RNA quality,which showed obvious RNA peaks,which indicated good RNA integrity.In that,it showed that exosomal RNA in this study was qualified and can be performed for further library sequencing.4.The lncRNAs in the follicular fluid exosomes of the PCOS group were compared with the control group.The volcano plot showed the lncRNA was differentially expressed.There were 1253 up-regulated lncRNAs and 613 down-regulated lncRNAs.5.GO and KEGG and cluster analysis,three major aspects were included in GO analysis,including cellular component,biological processes and molecular functions.In cellular component,they were mainly concentrated intracellular.Biological processes are mainly concentrated in cellular component organization or biogenesis,cellular component organization.And molecular functions are mainly enriched in such as RNA binding and protein binding.KEGG results showed that differential lncRNAs are in oocyte meiosis,insulin signaling pathway,insulin resistance,insulin secretion,type Ⅰdiabetes mellitus,Type Ⅱ diabetes mellitus,and estrogen signaling pathway.6.RT-qPCR results were consistent with the sequencing results.Six lncRNAs were selected for verification,the expression levels of DNMT1,DNMT2,DNMT3A and H19 in the exosomes of the polycystic ovary syndrome group were statistically significantly lower compared with the control group(P<0.05),while the expression of SGK1 was statistically significant increased compared with the control group(P<0.05),indicated that the sequencing results were accurate and reliable.7.Gene interaction mapping of differentially expressed lncRNAs showed that these genes are closely related and interacted with each other,especially in DNA methylation and regulation of gene expression.Conclutions:1.This study is the first time to determine the entire genome IncRNA expression pattern of follicular fluid exosomes in PCOS patients by sequencing.Compared with the control group,different expression lncRNAs may participate in the development and pathogenesis of PCOS.2.DNMT1,DNMT2,DNMT3A,H19 and SGK1 are DNA methylation,epigenetics genes,which affect the family heredity of PCOS patients,and may essential in the pathogenesis of PCOS.