Study Of Circular RNA Expression Profile And Mechanisms In PCOS Ovarian Granulosa Cells

Polycystic ovary syndrome?Polycystic ovary syndrome,PCOS?is a common endocrine metabolic disorder in women and the main cause of infertility in women of childbearing age.It has a high risk of developing endometrial cancer,type 2 diabetes,cardiovascular disease and non-alcoholic fatty liver throughout a woman's lifetime.It affects 5%-10% women of childbearing age,and there is a lack of medical cure and preventive measures,so it is a hot and difficult area in the field of reproductive research.With the development of high-throughput sequencing and chip technology,circular RNA?circular RNA,circ RNA?has become a research hot spot in tumors,neurological diseases and other fields because of its stable expression and wide range of functions,and there is limited researches in reproductive area.Bidirectional somatic cell-oocyte signaling regulates follicle growth via nutritive substance and steroidogenesis.Granulosa cell is a good non-invasive research method in the field of female reproduction.In the past year,there are few reports focus on the expression of circ RNA in granulosa cells women with PCOS,but the function and mechanism of circ RNA in granulosa cells in PCOS is still blank.To explore the roles of circ RNAs in the development of PCOS,the following three parts of the study are carried out.The first part is to study the differential expression of circ RNA and m RNA in granulosa cells of non-PCOS and PCOS patients,to construct a Ce RNA network,and to do matrix analysis of clinical indicators;the second part is to study the expression of target circular RNA in granulosa cells of PCOS patients in luteinized granulosa cells.To study the localization and stability of target circ RNA and its function in granulosa cell proliferation / apoptosis in KGN cell line.The third part is to study the molecular mechanism of target circular RNA in granulosa cell proliferation/apoptosis in KGN cell line.The purpose of this study is to understand the differential expression of circ RNA in patients with PCOS,and to provide a new theoretical basis for clarifying the pathogenic mechanism of target circ RNA in PCOS.Part I.Study on the expression profile of circular RNA and m RNA of granulosa cells in patients with PCOSObjective:The expression pattern of circ RNA and m RNA in luteinized granulosa cells of patients with or without PCOS was explored using microarray.Methods:The circ RNA spectra of granulosa cells were collected from 3 PCOS patients and 3 non-PCOS patients.The differential circ RNA was analyzed by GO analysis and KEGG analysis,and the ce RNA network was constructed.13 differentially expressed circ RNAs were verified by q RT-PCR and their correlation with biochemical indexes were analyzed.Results:1.Circ RNA expression profiles in granulosa cells from PCOS The results showed a total of 86205 circ RNAs were detected in PCOS luteinized granulosa cells.Among them,595 circ RNAs were significantly differentially expressed with a citeria of fold change>2 and P < 0.05.Circ RNAs was distributed on different chromosomes.There were significant differences in the expression of 13 TOP circ RNAs and 3 maternal m RNA?EML5,FASD1,FASD2?.Both androgen and AMH's level were positively correlated with hsa_circ₀032873,hsa_circ₀032868,hsa_circ₀032872,hsa_circ₀032883,hsa_circ₀032875,hsa_circ₀032871.2.KEGG analysis of circ RNA differential expression in granulosa cells of patients with or without PCOS focused in several signaling pathways that may be involved in the occurrence and development of PCOS,such as hormone synthesis,salt / lipid absorption pathway and so on.Conclusion:There are a large number of differentially expressed circ RNAs in granulosa cells of patients with or without PCOS,and significant differences in circ RNAs involve in multiple metabolic pathways.Part two: the biological function of candidate circ RNA on the proliferation and apoptosis of granulosa cells.Objective:To identify the structural characteristics of candidate circ RNA and to investigate the biological functions of circ RNA in KGN cells.Methods:1.The expression of candidate circ RNA was analyzed by RT-PCR with divergent or convergent primers in c DNA and g DNA of KGN.The expression of circ RNA and maternal m RNA was detected by q RT-PCR with Rnase R treatment,to test circ RNA's stablity.QRT-PCR was used to detect the expression of circ RNAs in another 20 pairs of primary luteinized granulosa cells.2.To detect the biological function of candidate circ RNA si RNA was transfectin into the granulosa cell line?KGN?.CCK8,flow cytometry and western blot were detected on the proliferation and apoptosis of KGN cells.And the candidate circ RNA overexpression plasmid was further transfected into the KGN cells.The proliferation and apoptosis of granulosa cells were detected as above.Results:1.Ring structure and stability analysis of candidate circ RNA Circ EML5-1?has_circ₀032875?arose from exons 2-10 of the EML5 gene and formed the mature back-spliced sequence.Using c DNA and g DNA from KGN cells as templates,the convergent primers could amplify circ EML5-1.However,the divergent primers could amplify only circ EML5-1 using c DNA templates,but not g DNA templates.RNase R digestion experiments show that circ EML5-1 is more stable than the corresponding linear RNA.The expression of circ EML5-1 was higher in cells with PCOS compared with cells with non-PCOS.2.Cell viability was decreased by circ EML5-1 knockdown.Circ EML5-1knockdown also led to an increased apoptotic rate.Consistently,western blot analysis showed that circ EML5-1 knockdown could lead to increased expression of Bax,cleaved-caspase3?proteins of apoptosis?.The reverse result was obtained after overexpression of circ EML5-1 using cyclization vector.Conclusion:Circ EML5-1 exists in the cytoplasm of granulosa cells and forms a ring independently.It is more stable than the maternal gene and has the function of affecting the proliferation and apoptosis of granulosa cells.Part three: the molecular mechanism of circ EML5-1 involved in the regulation of PCOSMethods:Bioinformatics network analysis predicts the downstream mi RNA and downstream m RNA of circ EML5-1.QRT-PCR technique was used to verify the changes of mi RNA content after overexpression circ RNA.QRT-PCR was used to detect the expression of mi R-494 in another 20 pairs of primary luteinized granulosa cells.Flow cytometry and western blot techniques were used to verify the biological function of mi R-494 in KGN cell line.RIP,Pull down and luciferace reporter system were used to explore the biological interaction between circ EML5-1 and downstream mi RNA.The interaction between downstream mi RNA and downstream m RNA was explored by using luciferace reporting system.Finally,the recovery experiment was used to verify the effects of circ EML5-1 and downstream mi RNA on downstream m RNA expression and cell function.Results:Mi R-494 and IGF1-R were selected as the possible target genes and signal pathways of circ EML5-1 by bioinformatics analysis.RIP,Pull down and double luciferase report system experiments all proved that circ EML5-1 could sponge mi R-494.The expression of mi R-494 was lower in cells with PCOS compared with cells with non-PCOS.Mi R-494 can inhibit the proliferation of granulosa cells and promote apoptosis,while circ EML5-1 can significantly reduce the expression of mi R-494 in KGN cells and negatively regulate its apoptosis-promoting effect.Double luciferase reporting system shows that mi R-494 and IGF1-R can be combined.The results of q PCR showed that mi R-494 could significantly reduce the expression of IGF1-R m RNA in KGN cells,while the down-regulation of mi R-494 could partially reverse the inhibition of IGF1-R protein expression after circ EML5-1 silencing and its effect on the proliferation and apoptosis of granulosa cells.Conclusion:Increased circ EML5-1 expression efficiently promoted growth and decreased apoptosis of granulosa cell through circ EML5-1/mi R-494/IGF1-R regulatory network.Circ EML5-1 might play a role in PCOS.