High Throughput CircRNAs Sequencing Profile Of Follicle Fluid Of Polycystic Ovary Syndrome Patients

ObjectBy detecting the circular RNA(circRNA)in the follicular fluid of patients with polycystic ovary syndrome(PCOS)and non-PCOS patients,the overall expression level of circRNA in follicular fluid was detected.According to the circRNA of PCOS group Differences in expression in the control group explored circRNAs that may be involved in the development of PCOS disease and the functional pathways involved.MethodsThe follicular fluid of female patients who received adjuvant reproductive technology treatment at the Reproductive Center of Subei People’s Hospital of Yangzhou City,Jiangsu Province from July 2017 to September 2017 was collected.The clinical data of all patients and the results of basic hormones and gynecological ultrasound were analyzed.According to the Rotterdam diagnostic rule,3 cases of PCOS group were selected,and 3 cases of follicular fluid of female patients treated with male factors alone were used as control group.The exosomes in the follicular fluid samples were isolated by multi-step differential centrifugation.The total RNA of the exosomes was linearly purified and randomly broken into short fragments.The preliminary sequencing library was constructed by these fragments and finally amplified by PCR.The library was sequenced,library sequencing was performed using Illumina Hiseq4000,and the amount of expression was calculated.The bwa software was used to compare the sequencing results with the reference genome of the human gene pool,and the candidate circRNA was identified by CIRCexplorer software to select potential circRNAs.Finally,the characteristic part of the exon in the candidate circRNA is compared with the known gene and annotated into the functional category of the gene.The bioinformatics analysis mainly uses the two databases of GO and KEGG to enrich the parental genes of the identified circRNA.The circRNA with different expression between the PCOS group and the control group was searched,and the gene function of the parental gene was GO annotated and enriched,and the functional pathway of the parental gene was enriched by KEGG.Results1.The total number of effective fragments obtained from the follicular fluid of PCOS patients was about 60.8 million,and the control group was about 64.6 million.About 88%of the effective sequences could be successfully aligned to the human GRCH38 genome,and there was no significant difference between the samples.However,the trans-splicing fragment of the PCOS group accounted for approximately 2.68%,while the control group accounted for approximately 1.11%.The number of reverse splicing fragments in the effective fragments obtained by the PCOS group was significantly higher than that of the control group.The trans-splicing process is more active in the PCOS group.2.A total of 16771 circRNAs and 5308 parental genes were identified in six follicular fluid samples.Among them,12907 circRNAs and 4311 parental genes were identified in 3 samples of PCOS group,and 5972 circRNAs and 3092 parental genes were identified in non-PCOS group.The identified circRNAs were not expressed in each group,and the distribution between groups was not the same.3.The expression of circRNA in the two groups was mainly between 10-1000 TPM,and the expression showed a skewed distribution.The expression level was expressed by quartiles.The expression of circRNA in the PCOS group at 75%was within 650 TPM,while that in the control group at 75%was within 200 TPM.The length of CRC in the two groups was mainly distributed in the range of 200-1000 bp.Most of the circRNAs contained only one exon,and the content of exons in circRNA increased,and the expression was significantly reduced.4.According to the differential p1RNA expression difference and p value significance of the two groups,412 differentially expressed circRNAs were screened,among which 167 were up-regulated and 245 were down-regulated.Upregulated:circRNA-15918_GREB1L,circRNA-2702 ANKH,circRNA-7788 HTT,circRNA-5762 SPHKAP,circRNA-8717 FANCL,etc.,downregulated:circRNA-5172_NBPF20,ciRNA-14485 MBOAT,ciRNA-15481_ATP6V0D1,ciRNA-6172_LINC-PINT,and ciRNA-6485 LYRM4 and so on.5.The GO function enrichment of the parental genes of differential circRNA is highly enriched in terms of cell stress response,programmed cell death,autophagy regulation,ATPase activity,and oxidative phosphorylation activity.In addition,there is a high degree of enrichment in terms of virion binding and anchoring junction.6.The KEGG function enrichment of the parental gene of the differential circRNA is highly enriched in the functions of oxidative stress,glutathione metabolism and autophagy regulation.In addition,it is highly enriched in the functional pathways of Vibrio cholerae infection,Shigella,Salmonella infection,pathogenic E.coli infection,and bacterial invasion of epithelial cells.At the same time,there are more enrichment in the functional pathways of neurological diseases such as Huntington’s disease and Alzheimer’s disease.7.Select four differentially expressed circRNAs,namely ciRNA-7323 TIAM1,ciRNA-2003_BECN1,ciRNA-1880_RHOA,and ciRNA-10775 RUVBL1,and predict that the four circRNAs may interact as miRNA sponges with the discovered miRNAs.Among them,ciRNA-7323 TIAM1 may bind to 128 miRNAs,and the potential site of miR-1304 can bind to the methylenetetrahydrofolate reductase gene translation region leading to premature birth,and can also induce granulosa cell apoptosis.Conclusions1.The trans-splicing process of PCOS patients is more active in vivo,and the expression of circRNA is more diverse and abundant.2.Various kinds of circRNA are abnormally expressed in follicular fluid of patients with polycystic ovary syndrome.Differential circRNA may cause PCOS diseases through pathological processes such as bacterial infection,oxidative stress,autophagy and neuropathy.3.CircRNA-7323_TIAM1,ciRNA-2003_BECN1,ciRNA-1880_RHOA and ciRNA-10775_RUVBL1 differentially expressed circRNA may serve as targets for further study of the pathogenesis of PCOS.