| The transcription factors RUNX1 and RUNX2 belong to the Runt-related(RUNX)gene family,and play critical roles in mammalian reproduction processes,such as follicle development and ovulation.However,the exact function of RUNX1 and RUNX2 in goat ovarian follicle is not clear.In the present study,the research objects were granulosa cells of Guanzhong dairy goat.In order to investigate the mechanism of hCG,mi R-181 b and mi R-222 regulated RUNX1 and RUNX2 expression,and the effects of RUNX1,RUNX2,hCG,mi R-181 b and mi R-222 on the proliferation,apoptosis and hormone secretion of granulosa cells,we used immunohistochemistry,immunofluorescence,real-time PCR,western blot,ELISA,CCK-8 and Ed U assay,flow cytometry and other methods.The main results are as follows:1.The expression of RUNX1 and RUNX2 in Guanzhong dairy goat ovaryReal-time PCR results showed that RUNX1 and RUNX2 mRNA were expressed in heart,liver,spleen,lung,kidney,muscle,fat,mammary gland,uterus,oviduct and ovary.And the expression of RUNX1 and RUNX2 m RNA were highest in mammary gland,lowest in muscle.RUNX1 and RUNX2 protein were detected in the oocytes and granulosa cells of preantral follicles,oocytes,granulosa cells and theca cells of antral follicles,and corpus luteum by immunohistochemistry.2.The effects of hCG on RUNX1 and RUNX2 expression in goat granulosa cellsThe granulosa cells were treated with 1 IU/m L hCG for different durations(0,2,4,8,12,16,24 h),the results showed that hCG-induced RUNX1 and RUNX2 m RNA expression peaked at 2 h,and hCG induced RUNX1 and RUNX2 protein expression in a time-dependent manner.The granulosa cells were treated with activators or inhibitor of hCG-induced intracellular signalling pathways,the results suggested that at 2 h,hCG induced RUNX1 m RNA expression by activating PKC and PI3 K,and hCG induced RUNX2 m RNA expression by activating adenylate cyclase,PKC and PI3 K.3.The effects of mi R-181 b and mi R-222 on hCG-induced RUNX1 and RUNX2 expression in goat granulosa cellsThere was a mi R-181 b binding site in RUNX1 3’UTR,and a mi R-221/222 binding site(mi R-221 and mi R-222 have the same seed sequences)in RUNX2 3’UTR according to the bioinformatics analysis results.The wild type or mutant dual-luciferase reporter vectors were constructed,and co-transfected into the 293 T cells and goat granulosa cells with the NC,mi R-181 b,mi R-221 or mi R-222 mimics for 24 h,separately.Mi R-181 b decreased the relative luciferase activity by binding to RUNX1 3’UTR,and mi R-222 decreased the relative luciferase activity by binding to RUNX2 3’UTR.Real-time PCR and western blot results showed that mi R-181 b reduced RUNX1 m RNA and protein expression,and mi R-222 reduced RUNX2 m RNA and protein expression,indicated that RUNX1 was a target gene of mi R-181 b,RUNX2 was a target gene of mi R-222.The granulosa cells were treated with 1 IU/m L hCG for different durations(0,2,4,8,12,16,24 h),the results suggested that hCG reduced mi R-181 b level at 2 h and maintained at a lower level.In contrast,hCG induced mi R-222 level at 2,4 and 12 h,with the highest level at 4 h and no change at other time.Then the granulosa cells were treated with activators or inhibitor of hCG-induced intracellular signalling pathways,the results showed that at 4 h,hCG decreased mi R-181 b expression by activating adenylate cyclase,PKA and PKC,however hCG increased mi R-222 expression by activating PKC and PI3 K.The granulosa cells were transfected with dual-luciferase reporter vector for 24 h,then the cells were treated with hCG,FSK or PMA for 4 h.The results revealed that hCG and PMA increased the relative uciferase activity of RUNX1-3’UTR,however,hCG decreased the relative uciferase activity of RUNX2-3’UTR.These results suggested that mi R-181 b and mi R-222 were involved in hCG-induced RUNX1 and RUNX2 expression,respectively.4.The effects of RUNX1 and RUNX2 on the proliferation,apoptosis and hormone secretion in goat granulosa cellsAfter the granulosa cells were treated with 1 IU/mL hCG for different durations(0,2,4,8,12,16,24 h),we found that hCG increased estradiol production at 12 h and 16 h.RUNX1 and RUNX2 expression were significantly decreased by si RNA.Without hCG treatment,knockdown of RUNX1 decreased the estradiol production,and did not effect the progesterone production.And knockdown of RUNX2 did not influence the estradiol and progesterone production.With hCG treatment,knockdown of RUNX1 and RUNX2 decreased the hCG-induced progesterone production,had no impact on the hCG-induced estradiol production.Knockdown of RUNX1 and RUNX2 significantly inhibited the natural and hCG-induced St AR,HSD3 B and CYP11A1 m RNA expression,but did not influence the natural and hCG-induced CYP19A1 m RNA expression.There were many RUNX1 and RUNX2 binding sites in the promotors of St AR,HSD3 B and CYP11A1.Futhermore,RUNX1 promoted CYP11A1,HSD3 B and St AR transcription by binding to-266~-256 and-252~-242 of CYP11A1 promotor,-1805~-1795,-1681~-1671 and-1327~-1317 of HSD3 B promotor,-1180~-1170,-1084~-1074 and-258~-248 of St AR promoter.And RUNX1 promoted CYP11A1,HSD3 B and St AR transcription by binding to-255~-241 of CYP11A1 promotor,-1808~-1794,-1684~-1670 and-1330~-1316 of HSD3 B promotor,-1087~-1073 of St AR promotor.Knockdown of RUNX1 and RUNX2 significantly induced granulosa cell proliferation at 24 h by CCK-8 and Ed U assay.At 48 h,knockdown of RUNX2 significantly reduced cell apoptosis,overexpression of RUNX2 induced granulosa cell apoptosis.However,RUNX1 had no effect on cell apoptosis.5.The effects of mi R-181 b and mi R-222 on the proliferation,apoptosis and hormone secretion in goat granulosa cellsThe effects of mi R-181 b and mi R-222 on hormone secretion of granulosa cells were detected by ELISA.Mi R-181 b and mi R-222 did not affect progesterone concentration with or without hCG treatment,mi R-222 significantly increased estradiol production in granulosa cells.We further found that mi R-181 b and mi R-222 increased granulosa cell proliferation at 48 h.At 48 h,mi R-181 b inhibited granulosa cell apoptosis.However,mi R-222 had no effect on cell apoptosis at 48 h.In summary,mi R-181 b and mi R-222 which were regulated by hCG,were involved in hCG-induced RUNX1 and RUNX2 expression,respectively.RUNX1 and RUNX2 binded to the promotors of St AR,HSD3 B and CYP11A1,induced transcription of these genes and stimulated hCG-induced progesterone production.In addition,RUNX1,RUNX2,mi R-181 b and mi R-222 regulated the proliferation and apoptosis of granulosa cells.The results of the present study provided the experimental evidence for the mechanism of RUNX1 and RUNX2 regulating follicle development and ovary function in goat. |
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