Identification Of Human Ovary-derived CircRNAs And Their Potential Roles In Ovarian Aging

Part 1 Identification and characterization of human ovary-derived circular RNAs[Purpose]Circular RNAs(circRNAs)were recently shown to exert effects on multiple pathological processes by acting as miRNA sponges.However,the roles of circRNAs in ovarian senescence are largely unknown.The objective of this study was to identify the circRNAs involved in ovarian aging and predict their potential biological functions.[Methods]We collected ovarian cortex samples from women undergoing laparoscopic surgeries in the Department of Obstetric and Gynecology of our hospital due to benign gynaecological diseases between February 2017 and October 2017.RNA-seq was performed on those specimens which met the requirements to identify the expression profile of ovary-derived circRNAs.The presence of ovarian-derived circRNA and the reliability of the RNA-seq results were further validated in a large cohort of samples by qRT-PCR and Sanger sequencing.Molecular biology experiments were performed to validate the general biological properties of the ovary-derived circRNAs.We also carried out bioinformatics analysis on the differentially expressed circRNAs,predicting their potential biological functions,and constructing a circRNA-miRNA-mRNA interaction network.[Results]A total of 78 ovarian cortex samples were collected in this study,among which six specimens(three for the young group(age between 20 to 30)and three for the aging group(age between 40 to 50))were enrolled for RNA-seq.As a reslut,48,220 circRNAs were identified,of which 194 circRNAs were significantly up-regulated and 207 circRNAs were down-regulated(fold change>2,P<0.05).qRT-PCR and Sanger sequencing both validated the existence of these circRNAs.And eight randomly selected circRNAs were further validated in a larger cohort of ovary samples(n=44).Bioinformatics analysis demonstrated that the metabolic process,regulated secretory pathway,oxidation-reduction process,steroid hormone biosynthesis,and insulin secretion pathways,which may be associated with ovarian aging,were significantly enriched(P<0.05).The biological characteristics of ovary-derived circRNA,such as back-splicing,RNase R resistance,stability,and alternative splicing,were further validated.Bioinformatics predicted that most of the circRNAs harboured miRNA binding sites,of which circDDX10-miR-1301-3p/miR-4660-SIRT3 axis may be involved in the regulation of ovarian function[Conclusions]Our study indicates that circRNAs are aberrantly expressed in the aging ovary and may play potential roles in the development of ovarian senescencePart 2 Expression of human ovary-derived circular RNA in granulosa cells of follicular fluid and its relationship with clinical outcomes of assisted reproductive techniques[Purpose]This study aims to analyze the correlation between the levels of circRNAs in granulosa cells(GCs)of human follicular fluid and female age,BMI,ovarian reserve function,and assisted reproductive technology(ART).[Methods]239 samples of human follicular fluid were collected from infertile women undergoing ART treatment in the center of reproductive medicine of our hospital from October 2017 to July 2018.The GCs were isolated by Percoll density gradient centrifugation and stored at room temperature for 0 h,6 h,12 h,and 24 h.qRT-PCR was used to detect the expression of three circRNAs and their corresponding mRNA validated from Part 1 in granulosa cells.The expression levels of circDDX10 in GCs of human follicular fluid were detected and further validated,and the correlation between ovarian reserve function and clinical reproductive outcome of ART were studied.[Results]In this study,239 human follicular fluid samples were collected and 210 GCs samples were finally isolated and obtained.The expression levels of circRNAs in GCs did not change markedly within 24 h(P>0.05),while the corresponding mRNAs degraded rapidly.Further analysis of the three validated circRNAs showed that the expression levels of circDDX10 in GCs of human follicular fluid decreased steadily with aging and statistically significance(P<0.01).But this did not happen with BMI(P>0.05).Moreover,the expression levels of circDDX10 in human follicular GCs were positively correlated with AMH(r=0.45,P<0.01)and AFC(r=0.32,P<0.01).However,there was no obvious correlation with FSH and E2(P>0.05).A subgroup analysis was performed according to the quartiles of circDDX10 expression levels(Q1,Q2,Q3,Q4).The results showed obvious differences among the subgroups regarding age,AMH,AFC and duration of infertility(P<0.01).However,there were no significant differences concerning BMI,FSH and E2(P>0.05).The number of oocytes retrieved in the Q3 and Q4 groups was remarkedly higher than thoset in Q1 and Q2 groups(P<0.01).No significant differences among groups regarding normal fertilization rates were found(P>0.05).In terms of good quality embryo rates,the differences between groups were statistically significant(P<0.05 or P<0.01),of which Q4 group was significantly higher than Q1 group(P<0.05).No significant differences were observed among groups with respect to βhCG positive rate or clinical pregnancy rate(P>0.05),although there remained a positive correlation with the levels of circDDX10.[Conclusions]Human ovary-derived circRNAs can be stably expressed in human follicular GCs.The expression levels of circDDX10 are closely related to ovarian reserve function,oocytes retrieved,and good quality embryo rate,which is expected to be a valuable predictor for ART outcomes.Part 3 Roles and preliminary mechanisms of human ovary-derived circular RNA in ovarian aging[Purpose]To further elucidate the effect of ovarian tissue-derived circRNA,circDDX10,on ovarian function,and preliminarily explore the role of circDDX10 involved in ovarian granulosa cell senescence and the specific mechanisms[Methods]Molecular biology experiments were performed to validate the general biological properties of ovary-derived circRNAs,circDDX10.We constructed specific circDDX10 silencing sequences(si-circDDX10 group)and overexpression vector(over-circDDX10 group,OV group)to transfect human granulosa cell lines(COV434)Null interference sequences(Negative Control,NC group)and plasmid empty vector(EV group)were used as controls,respectively.The expression levels of apoptosis-related genes/proteins,including BAX,BCL-2,CASPASE-3 and CASPASE-9 were detected by qRT-PCR and Western blot,respectively.The apoptosis of granulosa cells was further detected by TUNEL.Cell proliferation activities of granulosa cells at 0 h,24 h,48 h,72 h,and 96 h were determined by CCK-8 kit.The expression levels of steroid hormone synthesis-related genes(CYP11A1,CYP17A1,CYP19A1,StAR and HSD17B1)in each group were detected by qRT-PCR.And the levels of estradiol(E2)and progesterone(P)in the supernatant of each group were further determined by kits(chemiluminescence immunoassay,CLIA)[Results]circDDX10,back-spliced by exon 7 to exon 10 of DDX10 gene,was mainly located in the nucleus of granulosa cells.Consistent with other circRNAs,circDDX10 was also produced by back-splicing,without polyA tail,RNase R resistant,stable and difficult to be degraded.Silencing circDDX10 resulted in significant up-regulation of apoptosis-related genes BAX,CASPASE-3 and CASPASE-9 in granulosa cells(P<0.05),but had no significant effect on the expression of BCL-2(P>0.05)Overexpression of circDDX10 distinctly inhibited the expression of CASPASE-3 and CASPASE-9(P<0.05),but did not influence the expression levels of BAX and BCL-2(P>0.05).Similar to the change on the transcriptional level,silencing circDDX10 in granulosa cells promoted the expression of apoptosis-related proteins BAX,CASPASE-3 and CASPASE-9,while inhibiting the expression of BCL-2 protein Overexpression of circDDX10 played an opposite role.Results of TUNEL further supported the above findings.The CCK-8 results showed that silencing circDDX10 resulted in decreased granulosa cell proliferation activity,whereas overexpression of circDDX10 increased the proliferation activity.Silencing circDDX10 significantly inhibited the expression levels of steroid hormone synthesis genes,CYP11A1 and CYP19A1(P<0.05),but had no distinct inhibitory effect on CYP17A1,StAR and HSD17B1 expression(P>0.05).Overexpression of circDDX10 remarkedly promoted the expression of CYP19A and HSD17B1(P<0.05),but not the expressions of CYP11A,CYP17A1 and StAR(P>0.05).The concentration of E2 in the si-circDDX10 group was markedly down-regulated(P<0.01),while no significant changes were observed on the concentration of P(P>0.05).Conversely,over-expression of circDDX10 significantly up-regulate the levels of E2<0.01),while no obvious changes were monitored on the levels of P concentration(P<0.05)[Conclusions]Silencing circDDX10 can promote granulosa cell apoptosis and inhibit granulosa cell proliferation,while overexpression of circDDX10 can play an opposite role.Silencing of circDDX10 inhibits the expression of genes involved in steroid hormone synthesis,while overexpression of circDDX10 promotes steroid hormone synthesis.circDDX10 may be involved in the regulation of ovarian granulosa cell function by affecting granulosa cell proliferation and steroid hormone synthesis.