CircEML1 Modulates Steroid Synthesis And Secretion Of Hen’s Follicular Granulosa Cells Via Targeting The Gga-miR-449a/IGF2BP3 Axis

In recent years,numerous researches have reported that non-coding RNAs（ncRNA）including their competing endogenous RNA（ceRNA）networks play a crucial role in the regulation of the female reproductive processes in mammals,such as oocyte growth,follicular proliferation,differentiation and apoptosis,steroid synthesis and hormones production,the ovulation and atresia of mature follicle and so on.However,unlike mammals,chicken has a unique reproduction system with numbers of various sizes of follicles in the mature ovary to maintain continuous ovulation,and involves a series of dynamic processes of follicle recruitment,selection,dominance and ovulation,which directly impact the laying performance of hens.Currently,there remains little knowledge about the regulatory role of non-coding RNAs and their ceRNA networks in the reproductive mechanism of hens.Therefore,in the present study,we analyzed the whole transcriptome data of ovarian tissues of hens at different ages（15weeks,20weeks,30weeks and 68weeks）and explored the potential circular RNA（circRNA）-micro RNA（miRNA）-mRNA associated ceRNA regulatory networks,to research their effects on the development of follicular granulosa cells（GCs）and steroid synthesis through the potential related signal pathways of laying hens,and will provide to the theoretical reference for improving the ncRNAs regulatory mechanism in the reproduction of chicken.The results are as follows:Trial 1.Screening potential circRNA-miRNA-mRNA associated ceRNA network regulated follicular steroid synthesis based on the whole transcriptome resequencingThe ovarian tissues of hens at different physiological periods（15W,20W,30W and 68W）were selected for the whole RNA-seq and the potential ceRNA networks were screened using the bioinformatics analysis and granulosa cell trial.In the results,a total of 1090 differentially expressed（DE）mRNAs,209 DE miRNAs and 403 DE mRNAs were found in the RNA-seq dataset and it demonstrated that ceRNA networks played crucial roles in ovarian development,biological metabolism,amino acid biosynthesis,angiogenesis and steroid synthesis of hens by the analysis of circRNAmiRNA-mRNA interaction analysis and functional enrichment.Additionally,it has been found that gga-miR-449a,gga-miR-449b-5p and gga-miR-449c-5p of gga-miR449 family with the same seed sequence targeted multiple genes related steroid synthesis after further evaluating and filtering.Then we preliminarily valid and obtained their common potential target gene IGF2BP3 and potential circRNA novel_circ₀010840 through follicular GCs trial and the prediction of RNAhybird software.Furthermore,according to the expression profiles of granulosa and theca layer of different follicles,we found that gga-miR-449a and gga-miR-449b-5p were highly expressed in follicular granulosa layer,whereas the high expression of IGF2BP3 was found in follicular theca layer,as well as circEML1 had higher expression in granulosa layer than that in theca layer.Taken together,we preliminarily built three potential ceRNA networks:novel_circ₀010840/gga-miR-449a/IGF2BP3,novel_circ₀010840/gga-miR-449b-5p/IGF2BP3 and novel_circ₀010840/gga-miR-449c-5p/IGF2BP3,which provided the basis for the following study of the regulation of ceRNA network on ovarian function.Trial 2.The regulatory role of circEML1 in the development and steroid synthesis of follicular GCsThe PCR amplification,RNase R treatment,the nuclear mass separation analysis and cell trials were performed to verify the structure characteristic,distribution and function of circEML1（novel_circ₀010840）.The results showed that circEML1 originated from back-spliced exon 2,3 and 4 circularization of echinoderm microtubule-associated protein（EMAP）-like 1（EML1）gene and had a higher abundance in cytoplasmic of GCs.In the cell trial,it was demonstrated that overexpression of circEML1 had no significant effect on the viability and proliferation of follicular GCs,however,it significantly promoted the expression of CCND1 and CCND2 and inhibit CDK2 expression（p<0.01）,which are key genes related cell proliferation.Meanwhile,overexpression of circEML1 could notably upregulate the expressions of the key steroidogenesis genes-CYP19A1 and StAR,as well as promote the secretions of estrogen（E2）and progesterone（P4）in GCs（p<0.05 or p<0.01）,which indicated that circEML1 played an important regulatory role in the steroid synthesis and hormones’ secretions of follicular GCs.Furthermore,we further verified the target relationship of ceRNA network and found that circEML1 could serve as a sponge to bind gga-miR-449a in cytoplasmic by the dual luciferase reporter system and FISH assays,as well as reduced the expression of gga-miR-449a and promoted the mRNA and protein expression of the potential target gene IGF2BP3 in GCs.Trial 3.The regulation of gga-miR-449a in the development and steroid synthesis of follicular GCsThe assays of CCK8,qRT-PCR,EdU,flow cytometry,ELISA and dual-luciferase reporter were performed to detect the effect of gga-miR-449a on follicular GCs’proliferation and steroid synthesis of hens,as well as its target relationship with IGF2BP3.In the results,it was showed that there was no significant change in the proliferation of GCs between overexpression of gga-miR-449a and NC group,whereas gga-miR449a mimic significantly upregulated the expressions of CDK2 and CDK6 and decreased the expressions of CCND1 and CCND2（p<0.05 or p<0.01）,which were generally opposite of those in the treatment of overexpression of circEML1.Furthermore,gga-miR-449a mimic could remarkably inhibit the expressions of CYP19A1 and StAR,and depressed the secretion of E2 and P4（p<0.05 or p<0.01）.Conversely,gga-miR-449a inhibitor promoted the expression of CYP19A1 and StAR and the production of E2 and P4（p<0.05 or p<0.01）,which were consistent with the results of overexpression of circEML1.These findings indicated that circEML1 could regulate steroid synthesis and E2/P4 level in follicular GCs by sponging gga-miR-449a.Furthermore,it was demonstrated that gga-miR-449a could suppress the mRNA and protein expression of IGF2BP3,and reduced the enzymatic activity of luciferase through binding to the 3’UTR region of IGF2BP3,which further improve the mechanism of ceRNA network（e.g.circEML1 could release the repression of gga-miR449a on IGF2BP3 expression via competitively binding gga-miR-449a）.Trial 4.The regulatory role of IGF2BP3 and its related signaling pathways on the development and steroid synthesis of follicular GCsThe overexpression plasmid and interference fragment of IGF2BP3 were constructed and the assays of CCK8,qRT-PCR,EdU,flow cytometry and ELISA were performed to valid the regulation of IGF2BP3 in the proliferation and steroid synthesis of follicular GCs.The results demonstrated that IGF2BP3 had no remarkable effect on the proliferation of granulosa cells.However,it significantly promoted the expressions of CCND1 and CCND2,whereas decreased the expressions of CDK2 and CDK6 after transfected with overexpression of IGF2BP3 in GCs（p<0.05 or p<0.01）,which were generally consistent with those in the treatments of overexpression of circEMLl and inhibition of gga-miR-449a.Furthermore,IGF2BP3 facilitated the expression of CYP19A1 and StAR,and promoted the releases of E2 and P4 in GCs（p<0.05 or p<0.01）,which were also consistent with the results of overexpression of circEMLl and inhibition of gga-miR-449a.These findings highlighted that circEML1 could promote the steroid synthesis and secretion of follicular GCs by sponging gga-miR449a and regulating IGF2BP3 expression.Moreover,we investigated five possible signaling pathways inhibitors（AKT-I,PI3K-I,mTOR-I,p38MAPK-I and IGF-I）to search the regulatory mechanism of IGF2BP3 related pathways on the steroid synthesis and hormones in GCs.It was suggested that IGF2BP3 probably facilitate steroid synthesis and E2/P4 secretion through activating mTOR/p38MAPK signaling pathways in follicular GCs.Trial 5.The rescue experiment-to verify the regulatory role of the ceRNA network on steroid synthesis and secretion of layer granulosa cellsIn the functional rescue experiment,the assays of qRT-PCR and ELISA were conducted to valid the interaction between circEMLl and gga-miR-449a on target gene and steroid synthesis by co-transfecting overexpression of circEML1 with NC mimic or gga-miR-449a.In the results,it was revealed that gga-miR-449a mimic could reverse the suppression of circEMLl in gga-miR-449a expression and the promotion in the mRNA and protein expression of IGF2BP3（p<0.05 or p<0.01）.Meanwhile,it also eliminated the facilitation of circEML1 in the expression of CYP19A1 and StAR,as well as the levels of E2 and P4（p<0.05 or p<0.01）.All of these results demonstrated that the ceRNA network could modulate the steroid synthesis and the secretions of E2/P4 in follicular GCs.In summary,the ceRNA network（circEML1/gga-miR-449a/IGF2BP3）played a major role in the regulation of the ovarian function of hens,e.g.circEML1 could regulate steroid synthesis and the secretions of E2/P4 in follicular GCs by sponging gga-miR-449a to enhance the expression of IGF2BP3.In addition,the further study suggested that the mTOR/p38MAPK signaling pathway played an important role in the regulation of ceRNA network.