Comparative Analysis Circular RNA Expression Profiles Of Mouse Ovaries At Different Stage Of Development And The Function And Molecular Mechanism Of CircEGFR And CircGAS5 In Granulosa Cells

The ovary is an important part of the reproductive system of female animals.The main function of the ovaries is producing mature oocytes and secreting hormones.The sex hormones secreted by the ovaries are mainly estrogen,progesterone,etc.The sex hormone that ovarian secrete basically has estrogen,progesterone Estrogen and progesterone are the mainly sex hormones secreted by ovary,especially,secrete from granulosa cells(GCs)in the ovary.The abnormal secretion of hormones can cause female infertility,premature ovarian failure and other diseases.Circular RNA(circ RNA)is a type of non-coding RNA with a closed loop structure that may regulate gene expression.Accumulating evidence illustrates that circ RNA play important role in cell proliferation and development.However,there are few studies on the involvement of circ RNA in regulating ovarian development.Therefore,exploring the function of circ RNA in ovary and GCs is important for the diagnosis and treatment diseases which caused by abnormal hormone secretion.GCs in adult ovary showed obvious proliferation and differentiation compared with the GCs in neonatal ovary.We selected these two periods mouse ovaries,digested linear RNA with RNA exonuclease(RNase R),and then analyzed and compared circ RNAs expression profiles by high-throughput sequencing(RNA-Seq).327 circ RNA were different expression in adult and neonatal ovaries.In these circ RNAs,169 circ RNAs were up regulated and 158 circ RNAs were down regulated.The GO(Gene Ontology)analysis of differentially expressed circ RNAs showed that these circ RNAs were involved in ovulation cycle,cell proliferation regulation,developmental growth and other biological processes.The result of KEGG(Kyotoencyclopedia of genes and genomes)pathway analysis showed that these circ RNAs were involved in estrogen secretion,cell junction and vascular endothelial growth factor signaling pathways.There were 5 differentially expressed circ RNAs in the estrogen signaling pathway,including circ EGFR.circ EGFR is a circular transcript of the epidermal growth factor receptor(EGFR)gene,and it has been reported that EGFR is involved in the regulation of cell proliferation,differentiation,cell cycle and other processes.We examined the expression level of circ EGFR in the ovaries of two stages and found that the expression of circ EGFR in the adult mice ovaries was significantly higher than neonatal ovaries.And we found that circ EGFR was located in the cytoplasm of GCs in the ovary by RNA in situ hybridization.We found that overexpression of circ EGFR increased estrogen secretion,but had no effect on progesterone secretion in GCs,while circ EGFR overexpression promoted in GCs proliferation.Whereas knockdown of circ EGFR enhanced progesterone production,inhibited(E2)secretion by GCs and increased the apoptosis rate of GCs.The ce RNA net of circ EGFR showed that circ EGFR may sponge mi R-125a-3p,mi R-183-5p and mi R-1934-5p.The binding energy of circ EGFR and mi R-125a-3p is the least among these mi RNAs(-30.5 kcal/mol).Subsequently,we studied the effects of mi R-125a-3p on proliferation by CCK-8 and Ed U experiments in GCs.It was found that overexpression of mi R-125a-3p in GCs inhibited cell proliferation,while inhibition of endogenous mi R-125a-3p promoted GCs proliferation.Next,we verified the interaction between circ EGFR and mi R-125a-3p by dual luciferase reporter assay and RNA immunoprecipitation assay.We tested the expression level of Fyn after circ EGFR overexpression and interference by q PCR.The results showed that overexpression of circ EGFR increased Fyn expression,whereas interference with circ EGFR reduced Fyn expression.In granulosa cells,circ EGFR acts as a mi RNA molecule to adsorb mi R-125a-3p,which inhibits the expression of its target gene Fyn,and promotes granulosa cell proliferation.Knockdown of circ EGFR could release mi R-125a-3p,and the expression of Fyn was inhibited,resulting in inhibition of cell proliferation.It was shown that circ EGFR as a mi RNA sponge adsorbed mi R-125a-3p.circ EGFR could sponge mi R-125a-3p,and increased the expression of Fyn,and circ EGFR could promote the proliferation of GCs.Knockdown of circ EGFR increased mi R-125a-3p expression,and mi R-125a-3p inhibited the expression of Fyn,Knockdown of circ EGFR could inhibit the cell proliferation.We found circ GAS5 which is one of circular transcript of GAS5(Growth arrest-specific5)in circ RNAs that were higher expression in neonatal ovaries than adult ovaries.GAS5 is involved in the regulation of female germline stem cells proliferation in the ovary.While,the study of circ GAS5 and GAS5 in GCs has not been reported.In this study,Through CCK-8and Ed U experiments,we found that overexpression of circ GAS5 inhibited the proliferation of GCs,and interference with circ GAS5 had no significant effect on cell proliferation.Overexpression of GAS5 promoted cell proliferation,while knockdown of GAS5 inhibit the proliferation of GCs.Overexpression of GAS5 could increase estrogen secretion in GCs.In addition,GAS5 would relieve the inhibitory effect of circ GAS5 on cell proliferation.The above results indicate that circ GAS5 inhibits GCs proliferation,while GAS5 can promote cell proliferation.Meanwhile,it means that circ GAS5 and GAS5 plays antagonistic function in GCs.Taken together,our findings reveal the circ RNA profiles and explore the function of ovarian development,and comparative analysis the different circ RNAs,and we constructed the ce RNA networks which related estrogen secretion.Meanwhile,we uncovered the function of circ EGFR and circ GAS5 in cell proliferation and hormone secretion in ovary GCs.These findings provide a basis for research of circ RNA regulation ovarian development and hormone,thus providing diagnosis and treatment of female infertility,premature ovarian failure and other diseases.