CircRALGPS2 Regulates Chicken Follicle Granulosa Cell Apoptosis And Autophagy Via Encoding A Novel Protein And Sponging MiRNA

Chicken egg production is a low heritability trait,which is difficult to improve through breeding of this breed.Although cross breeding can improve egg production quickly,it is easy to cause chicken quality decline.Therefore,it is urgent to improve the laying ability of local chickens by molecular breeding technology on the basis of maintaining the excellent meat quality.The laying rate of chicken mainly depends on the development level of germ cells and follicles in ovary.Chicken follicle development is a very complex biological process,which is controlled by endocrine hormones as well as a large number of functional genes and transcription factors.In recent years,more and more studies have shown that non-coding Rnas play a key role in follicle development in animals.Circular RNA(circRNA),as an endogenous non-coding RNA,has a more stable transcriptional regulatory function than linear RNA due to its special closed circular structure.Therefore,the excavation of circRNAs regulating follicle growth and development can provide important theoretical support for the development of molecular breeding programs to improve the laying performance of local chickens.Tianfu broiler is a high-quality chicken supporting line.In this study,the normal follicles and atretic follicles of Tianfu broiler breeders were sequenced by circRNA RNA-seq and circRNA RNC-seq,and a circRALGPS2 that may regulate follicle development and atresia was screened.Then the primary granulosa cells of chickens were cultured in vitro.We investigated the function and molecular mechanism of circRALGPS2 in regulating chick follicle development and atresia through molecular biological methods.The main results were as follows:1.Physiological and morphological examination showed that serum PRL hormone levels increased,LH and FSH hormone levels decreased.The pregraded follicles also showed atrophy and atresia.The process of follicle atresia is accompanied by thinning of granulosa layer and enhanced apoptosis and autophagy of granulosa cells.2.A total of 311 differentially expressed circRNAs were identified by circRNA RNA-seq,and 297 differentially expressed circRNAs were identified by circRNA RNC-seq.There were38 circRNAs with common differences,including circRALGPS2,circ THADA,circ FAF1,circ VAT1 L and circRAB2 A,the source genes were mainly enriched in TGF-β,Fox O and Gn RH signaling pathways.3.It was identified that circRALGPS2 is a circular RNA,formed by the cyclization of the second to the eighth exon of RALGPS2.circRALGPS2 is more highly expressed in atretic follicles,mainly in the cytoplasm of granulosa cells,and highly expressed in the uterus and ovary.4.CircRALGPS2 has IRES activity and can encode a 212aa-sized peptide,named circRALGPS2-212 aa.In primary chicken granulosa cells,circRALGPS2-212 aa can promote apoptosis and autophagy.In addition,the results of co-immunoprecipitation and Western Bolt showed that circRALGPS2-212 aa could directly act on PARP1 and PARP1 directly act on HMGB1.PARP1 could promote apoptosis of chicken granulosa cells,while HMGB1 promoted autophagy of chicken granulosa cells5.Combined with mi RNA transcriptome sequencing of normal and atretic follicles of Tianfu broiler chickens,the circRALGPS2/mi R-200a-3p/TGFβ2 regulatory network was constructed by biogenic prediction and dual luciferase reporter.circRALGPS2 can release the expression of TGFβ2 by adsorbing mi R-200a-3p,and activate the downstream TGFβ/SMAD pathway through the release of TGFβ2,thus promoting the apoptosis and autophagy of chicken granulosa cells.In this study,our results indicated that circRALGPS2 regulates follicle development and atresia by promoting apoptosis and autophagy in chicken granulosa cells.circRALGPS2 regulates apoptosis and autophagy via the circRALGPS2-212aa/PARP1/HMGB1 axis and the circRALGPS2/mi R-200a-3p/TGFβ2/SMAD axis,which refines the molecular mechanisms underlying follicular development,atresia and its disorders in chicken.Chicken is an important model for the study of human ovarian diseases,therefore,this results also provide new strategies for the diagnosis and treatment of ovarian dysfunction.