The Effects Of MiR-458b-5p Regulatory Gene CTNNB1 On The Proliferation Of Follicular Granulosa Cells In Chickens

In poultry,the egg production performance is affected by many factors,but the key factor to affect egg production traits of poultry is the follicle development process.Early studies in our laboratory found that high-throughput sequencing results showed differential expression of miRNA-458b-5p in the ovaries of parturient and non-parturient hens.In order to study the role of miRNA-458b-5p in follicular development of chickens,the expression of miRNA-458b-5p in follicles was detected by qRT-PCR.The candidate target genes were identified according to high-throughput results,and then to validate the targeting relationship.Finally,we studied the effects of miRNA-458b-5p on the proliferation of chicken grade follicular granulosa cells by targeting the target genes.The results were as follows:(1)The results of RT-qRT-PCR showed that chicken miRNA-458b-5p was highly expressed in the theca cells and low expressed in granulosa cells of grade follicles,and there was a significant difference in the expression of miRNA-458b-5p between theca cells and granulosa cells(P<0.05).The target gene CTNNB1 was highly expressed in granulosa cells and low expressed in theca cells of grade follicles.The expression of CTNNB1 between theca cells and granulosa cells was significantly different(P<0.05).In grade follicles,the expression of CTNNB1 gene decreased with the increase of follicles,and the trend was similar in different breeds of chicken.The results of RT-qRT-PCR showed that the expression of miRNA-458b-5p and CTNNB1 gene showed a negative correlation.(2)The target gene of miR-458b-5p was predicted to be the gene CTNNB1 by the Targetscan.The results of double luciferase reporter gene activity assay showed that transfection of the miR-458b-5p mimics resulted in a significant decrease in the activity ratio of the wild-type vector dual luciferase reporter gene(P<0.05).It was confirmed by Western Blotting that miR-458b-5p can inhibit the synthesis of ?-catenin protein.These results indicate that miR458b-5p has a targeting relationship with gene CTNNB1,and miR458b-5p can inhibit the translation of gene CTNNB1 by binding to the 3'UTR of gene CTNNB1.(3)According to the results of granulosa cells by CCK-8 reagent,miR-458b-5p can inhibit the proliferation of chicken grade follicular granulosa cells cultured in vitro.According to the results of flow cytometry detection of granulosa cell apoptosis,it was confirmed that miR-458b-5p could not cause apoptosis of chicken grade follicular granulosa cells.(4)After transfecting miR-458b-5p mimics into chicken grade follicular granulosa cells,RT-qPCR results showed that it could cause the change in the expression of target gene CTNNB1,which then affected the expression level of the related genes changed in Wnt/?-catenin signaling pathway.The results indicate that miR-458b-5p does have some effects on the Wnt/?-catenin signaling pathway.Transfecting miR-458b-5p mimics into chicken grade follicles granulosa cells,the expression of LH receptors mRNA changed significantly,which suggested that luteinizing hormone was associated with the synthesis of ?-catenin protein.In conclusion,through a series of experimental results,it can be proved that ggamiRNA-458b-5p targeting regulatory target gene CTNNB1,overexpression of gga-miRNA-458b-5p mimics can inhibit the synthesis of ?-catenin protein in chicken grade follicular granulosa cells and the proliferation of chicken grade follicular granulosa cells,and to affect the expression of some related genes in Wnt/?-catenin signaling pathway.