CircWRNIP1 Regulates Proliferation And Progesterone Synthesis Of Chicken Follicular Granulosa Cells Through MiR-10a-5p/MAPRE1/CDK2 Pathway

Egg production of chickens depends mainly on the level of follicle development.As a new type of endogenous non-coding RNA,circRNA has a more stable ring structure than linear RNA and can participate in a variety of biological processes in the body by targeting and regulating downstream gene expression through molecular sponge mechanism.Previous studies have shown that circRNA is widely involved in the physiological processes of oocyte and surrounding somatic cells proliferation,apoptosis and steroid synthesis in animal ovaries,thus regulating follicle development.The previous sequencing of normal developing follicles in laying hens and atretic follicles in broody chickens by my research group found that the expression level of circWRNIP1 in normal follicles was significantly higher than that in atretic follicles,suggesting that circWRNIP1 might be involved in regulating the growth and development of chicken follicles.In this study,chicken primary granulosa cells were cultured in vitro.The mechanism of circWRNIP1 in the development of chicken granulosa cells was detected by real-time quantitative PCR(RT-qPCR),western blotting,flow cytometry,cell counting kit-8(CCK-8),enzyme linked immunosorbent assay(ELISA),dual-luciferase reporter gene and co-immunoprecipitation(Co-IP).The main research results are as follows:1.The expression level of novel＿circ＿0008863 in follicles(SWF,LWF,SYF,LYF)of laying hens was significantly higher than that of nesting hens(P<0.05 or P<0.01).The cyclic structure of novel＿circ＿0008863 was analyzed and identified as circWRNIP1,which was formed by reverse splicing of exon 2 and exon 3 of WRN helicase interacting protein 1(WRNIP1)gene on chicken chromosome 2.2.Overexpression of circWRNIP1 in chicken primary granulosa cells in vitro promoted mRNA expression levels of proliferation-related genes PCNA,CCND1 and CDK2(P<0.05),the opposite result was obtained after interfering circWRNIP1 expression.The results of CCK-8 and flow cytometry showed that overexpression of circWRNIP1 could significantly increase the number of proliferating cells.In conclusion,circWRNIP1 promoted the proliferation of chicken granulosa cells.3.Overexpression of circWRNIP1 in chicken primary granulosa cells significantly increased the mRNA and protein expressions of progesterone synthesis-related genes CYP11A1 and STAR(P<0.05 or P<0.01),the effect was opposite after circWRNIP1 interference.ELISA results showed that overexpression of circWRNIP1 could increase the secretion of progesterone in granulosa cells.These results indicated that circWRNIP1 promoted the synthesis of progesterone in granulosa cells.4.The prediction through RNAhybrid website showed that miR-10a-5p may be the target miRNA of circWRNIP1.Dual-luciferase reporter assay results indicated that miR-10a-5p could be targeted to circWRNIP1 3’UTR sequence.Overexpression or suppression of circWRNIP1 in cultured granulosa cells can decrease or increase the mRNA level of miR-10a-5p(P<0.01).These results indicate that circWRNIP1 can target miR-10a-5p.5.mRNA levels of PCNA,CCND1,CDK2,CYP11A1 and STAR were significantly decreased after overexpression of miR-10a-5p in cultured granulosa cells in vitro(P<0.05 or P<0.01),and the opposite result was found after interference with miR-10a-5p.CCK-8and flow cytometry showed that overexpression of miR-10a-5p inhibited cell proliferation.Further ELISA showed that overexpression of miR-10a-5p resulted in decreased secretion of progesterone in granulosa cells.These results indicated that miR-10a-5p could inhibit granulosa cell proliferation and progesterone synthesis.6.Bioinformatics predicted that microtubule-associated protein RP/EB family member1(MAPRE1)might be the target gene of miR-10a-5p.Dual-luciferase assay found that miR-10a-5p binds to the 3’UTR sequence of the MAPRE1 gene.mRNA and protein expression levels of MAPRE1 were decreased or increased after overexpression or interference of miR-10a-5p in chicken granulosa cells in vitro(P<0.05 or P<0.01),indicating that miR-10a-5p could inhibit the expression of MAPRE1.7.Functional verification of MAPRE1 gene showed that mRNA levels of PCNA,CCND1,CDK2,CYP11A1 and STAR were significantly increased after MAPRE1 was overexpressed in chicken granulosa cells in vitro(P<0.05 or P<0.01),the results were opposite after interference of MAPRE1 expression.In addition,the results of CCK-8,flow cytometry,Western Blot and ELISA showed that MAPRE1 promoted granulosa cell proliferation and progesterone synthesis.Rescue tests showed that circWRNIP1,after targeting miR-10a-5p,could alleviate the inhibition of miR-10a-5p on the expression level of MAPRE1,and could relieve the inhibition of miR-10a-5p on granulosa cell proliferation and progesterone synthesis.8.Co-IP and Western Blot detection showed that MAPRE1 could combine with CDK2 to form a complex,and the CDK2 protein level increased or decreased after overexpression or interference of MAPRE1 in granulosa cells(P<0.05).These results indicate that MAPRE1 binds to CDK2 and promotes the expression of CDK2,thus playing a role in granulosa cell proliferation and progesterone synthesis.In conclusion,circWRNIP1 regulates the expression of downstream target gene MAPRE1 by targeting miR-10a-5p through the molecular sponge mechanism,regulates the proliferation of chicken follicular granulosa cells and progesterone synthesis,thus affecting follicular development.The results can provide reference for studying the molecular mechanism of circRNA regulating follicle development in hens,and provide theoretical basis for molecular breeding to improve laying performance of laying hens.