The Study Of CircRAB11A Targeting MiR-24-5p Regulated The Development Of Chicken Follicular Granulosa Cells Through ERK1/2 And PI3K/AKT Signaling Pathways

The egg production rate of chickens depends mainly on the developmental status of follicles.The proliferation of follicular granulosa cells is crucial for normal,growth of primordial follicles,mediating the developmental process during the follicular growth phase,while apoptosis of granulosa cells mediates the atresia process during the late follicular growth phase,so granulosa cells play an important regulatory role in follicular development process.As a class of endogenous non-coding RNAs,circular RNAs(circ RNAs),which possess a special closed-loop structure and can be protected from degradation by exonucleases,are more stable than linear RNAs and can perform regulatory functions more effectively,and have been shown to be widely involved in regulating follicle development in animals.In my group,we sequenced circ RNAs from normal and atretic follicles in chickens and found that the expression of circ RAB11 A in normal follicles was significantly higher than that in atretic follicles.Therefore,it was hypothesized that circ RAB11 A may be involved in regulating the development of chicken follicle.In this study,chicken follicular granulosa cells cultured in vitro were used as experimental materials to interfere with or overexpress circ RAB11 A in cells using si RNA and overexpression vectors.circ RAB11 A was amplified by PCR,sequenced by Sanger,analyzed by real-time quantitative fluorescence PCR(q PCR),CCK-8 kit,Ed U staining,TUNEL staining,protein immunoblotting(Western Blot),dual luciferase reporter analysis and other experimental methods to detect the effect of circ RAB11 A on proliferation and apoptosis of chicken granulosa cells and its mechanism of action,the main results are as follows:1.The ring formation validation experiments show that circ RAB11 A is a circular RNA formed by reverse splicing of exons 2 to 4 of the RAB11 A gene.circ RAB11 A was relatively highly expressed in the reproductive organs of hens and was more consistently expressed in the hierarchical follicles.circ RAB11 A overexpression is followed by a significant increase in the m RNA expression of cell proliferation-related genes(CCND1,CCND2,CDK2 and PCNA)and m RNA expression of the antiapoptotic gene BCL2 was significantly increased(P<0.01),while m RNA expression of cell pro-apoptosis-related genes(Caspase3,Caspase8,Caspase9)was significantly decreased(P<0.05),as well as Caspase9 protein expression was significantly decreased(P<0.05).m RNA expression of CCK-8,Ed U and TUNEL assays showed that overexpression of circ RAB11 A could effectively promote proliferation and inhibit apoptosis of chicken follicular granulosa cells.This shows that circ RAB11 A has the function of promoting granulosa cell proliferation and inhibiting their apoptosis.2.The results of dual luciferase experiments indicated that circ RAB11 A can act as a molecular sponge for miR-24-5p and regulate the expression of miR-24-5p.Compared with the control group,interference with miR-24-5p significantly upregulated the m RNA expression of CCND1,CDK2,PCNA,and BCL2(P<0.05).In addition,the m RNA levels of pro-apoptotic genes(Caspase3,Caspase8)significantly decreased under the action of miR-24-5p inhibitors(P<0.05).Meanwhile,CCK-8,Ed U,TUNEL,and Western Blot tests have demonstrated that interfering with miR-24-5p in vitro cells can promote the proliferation of granulosa cells and inhibit their apoptosis.The above results suggest that circ RAB11 A regulates granulosa cell proliferation and apoptosis through sponge adsorption of miR-24-5p.3.According to the target relationship verification,miR-24-5p can target the3’UTR of EGFR.At the same time,after knockdown of EGFR,the m RNA expression of proliferation related genes(CCND1,CDK2,and PCNA)decreased(P<0.05),the m RNA expression of apoptosis related genes(Caspase3,Caspase8)increased(P<0.05),the protein expression of Caspase9 increased(P<0.05),cell activity,and the number of Ed U positive cells decreased(P<0.05),and TUNEL positive cells increased(P<0.05).At the same time,Western Blot experiments have found that EGFR may regulate the proliferation and apoptosis of granulosa cells through ERK1/2 and PI3K/AKT signaling pathways.4.The results of the rescue assay showed that miR-24-5p mimic could counteract part of the inhibitory effect on miR-24-5p after overexpression of circ RAB11 A,and also abrogate the promotional effect of overexpression of circ RAB11 A on cell proliferation-related genes and the inhibitory effect of apoptosis-related genes.Specifically,expression of circ RAB11 A activated ERK1/2 and PI3K/AKT signaling pathways through circ RAB11A/miR-24-5p axis,promoted proliferation of chicken follicular granulosa cells and inhibited apoptosis of granulosa cells.Comprehensive analysis showed that circ RAB11 A could activate ERK1/2 and PI3K/AKT signaling pathways through circ RAB11A/miR-24-5p/EGFR axis to promote proliferation and inhibit apoptosis of chicken follicle granulosa cells,thus regulating chicken follicle growth and development.The results can provide a reference for studying the important role of circ RNA in the growth and development of chicken follicles and provide theoretical support for molecular breeding in local chickens to improve egg production ability.