【摘要】 [目的]本研究旨在分析促卵泡激素(FSH)对鸡颗粒细胞(GC)中miR-130a表达的影响，并探究miR-130a的靶基因及其在颗粒细胞增殖凋亡过程中的效果。[方法]体外分离培养鸡颗粒细胞，并使用不同浓度FSH处理24 h,检测miR-130a和PIK3CB基因的表达；通过双荧光素酶试验检测miR-130a的靶基因；转染miR-130a模拟物(mimics)和抑制剂后用试剂盒检测细胞活力以及细胞培养基中雌二醇(E2)和孕酮(P4)含量，荧光定量PCR和Western blot检测凋亡基因BCL2和BAX的表达。[结果]miR-130a和PIK3CB基因在FSH处理下的表达趋势相反。与对照组相比，共转染miR-130a模拟物和鸡源PIK3CB野生型过表达质粒(gga-PIK3CB-WT)后荧光素酶活性下降约40%(P<0.05),miR-130a过表达导致PIK3CB蛋白水平极显著降低(P<0.01),而抑制miR-130a能极显著增加PIK3CB蛋白水平(P<0.01)。miR-130a过表达降低了雌二醇(E2)的分泌(P<0.05),抑制了BCL2基因和BCL2蛋白的表达(P<0.05)及细胞增殖(P<0.05),促进了BAX基因(P<0.05)和BAX蛋白的表达(P<0.01);抑制miR-130a的表达能促进雌二醇(E2)分泌(P<0.05),并促进BCL2蛋白表达(P<0.01)和细胞增殖(P<0.01),抑制BAX基因(P<0.05)和BAX蛋白表达(P<0.01)。[结论]miR-130a通过靶向PIK3CB减少类固醇激素合成并抑制鸡颗粒细胞凋亡。更多还原

【Abstract】 [Objectives]The purpose of this study was to analyze the effect of follicle stimulating hormone(FSH)on the expression of miR-130a in chicken granular cells(GC),and explore the target gene of miR-130a and the effect of miR-130a on the proliferation and apoptosis of GC. [Methods]Chicken granular cells were isolated and cultured, treated with different concentrations of FSH for 24 hours, and the expression of miR-130a and PIK3CB genes were detected; the target gene of miR-130a was detected by double luciferase assay; after transfection of miR-130a mimics and inhibitor, the cell viability and the content of estradiol(E2)and progesterone(P4)in the cell culture medium were detected by the kit, and the expression of apoptotic genes BCL2 and BAX were detected by fluorescence quantitative PCR and Western blot. [Results]The expression trend of miR-130a and PIK3CB genes was opposite under FSH treatment. Compared with the control group, the luciferase activity decreased by about 40%(P<0.05)after co-transfection of miR-130a mimics and gga-PIK3CB-WT. The overexpression of miR-130a resulted in a significant decrease in PIK3CB protein level(P<0.01),while the inhibition of miR-130a significantly increased the PIK3CB protein level(P<0.01). Overexpression of miR-130a decreased the secretion of estrogen(E2)(P<0.05);the inhibition of miR-130a increased the secretion of estrogen(E2)(P<0.05),promoted the expression of BCL2 protein(P<0.01)and cell proliferation(P<0.01),and inhibited the expression of BAX gene(P<0.05)and BAX protein(P<0.01). [Conclusions]miR-130a reduces steroid hormone synthesis and inhibits apoptosis of chicken granular cells by targeting PIK3CB.更多还原