Construction of shRNA interference vector for GABRA1 gene in laying hens and its molecular regulatory effects on prehierarchical follicle granulosa cells
CHEN Xiaoxia
YANG Hui
ZOU Xianqun
YU Jing
LIU Yongjie
Abstract：The study aimed to investigate the regulatory role of the γ-aminobutyric acid type A receptor subunit α1(GABRA1)gene in prehierarchical follicle granulosa cells of laying hens.Using targeted short hairpin RNA(shRNA)interference technology,an interference expression vector was constructed.The experiment was divided into three groups:Blank control(BC group),negative control(NC group),and shRNA GABRA1 interference group(shRNA GABRA1 group).The vectors were transfected into prehierarchical follicle granulosa cells(4～8 mm)of laying hens.After 48 hours of transfection,real-time quantitative PCR(RT-qPCR)and Western blot were used to analyze the mRNA and protein expression levels of genes related to cell proliferation(CCND1),cell differentiation,steroid synthesis genes,steroid synthesis rapid regulatory protein(StAR)cytochrome P450 family 11 subfamily A member 1(CYP11A1),apoptosis genes caspase-3(Caspase-3),and B-cell lymphoma/leukemia-2(Bcl-2).Additionally,EDU assay and flow cytometry were employed to assess cell proliferation and apoptosis.The results showed that,compared to the BC and NC groups,the mRNA and protein expression levels of CCND1,StAR,CYP11A1,and Bcl-2 were extremely upregulated in the shRNA GABRA1 group(P＜0.01),while the expression of Caspase-3 was extremely downregulated(P＜0.01).The proliferation index of granulosa cells significantly increased(P＜0.05),and the total apoptosis rate significantly decreased(P＜0.05).The study shows that GABRA1 can inhibit the proliferation and differentiation of early follicular granulosa cells,promote apoptosis,and then affect the growth and development of follicles.
Keywords：shRNA interferenceGABRA1granulosa cellsproliferationapoptosis
Publication Date：2025-03-28
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：7( 71-77 )