IGF-1调控RBM3表达抑制低温应激诱导牦牛卵丘细胞凋亡

RNA-Binding Motif Protein 3(RBM3)Expression is Regulated by Insulin-Like Growth Factor(IGF-1)for Protecting Yak(Bos grunniens)Cumulus Cells from Apoptosis During Hypothermia Stress

【目的】探索动物机体遭受低温应激及细胞冷冻过程中胰岛素样生长因子1(insulin-like growth factor,IGF-1)与RNA结合基序蛋白3(RNA-binding motif protein 3,RBM3)之间的作用关系,及IGF-1参与哺乳动物细胞抑制低温损伤的机制。【方法】体外培养牦牛卵丘细胞,采用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)、蛋白免疫印迹(Western blot,WB)和免疫荧光技术检测不同浓度(0、50、100、200 ng·mL^-1)IGF-1和低温应激(30℃、25℃)对RBM3表达影响。卵丘细胞经最佳浓度IGF-1(100 ng·mL^-1)和对照组(0 IGF-1)作用30 h后,低温(30℃、25℃)应激8 h,比较RBM3表达水平。评估在低温应激组、100 ng·mL^-1IGF-1处理组和100 ng·mL^-1IGF-1+RBM3抑制剂处理组,卵丘细胞25℃应激8 h后凋亡水平差异,并从基因和蛋白水平检测3组卵丘细胞中凋亡相关基因Bax和Bcl-2的表达水平。【结果】(1)IGF-1作用卵丘细胞后RBM3基因和蛋白的表达水平显著上升(P<0.05),其在100 ng·mL^-1IGF-1处理组最高,免疫荧光检测显示100和200 ng·mL^-1IGF-1处理组卵丘细胞胞核和细胞质均可检测到RBM3,而0和50 ng·mL^-1处理组,RBM3仅定位在细胞质。(2)卵丘细胞经受低温应激时,RBM3的表达水平显著增加,但其在30℃和25℃应激处理组差异不显著;100 ng·mL^-1IGF-1作用卵丘细胞30 h后,其再次接受25℃、8 h低温应激,卵丘细胞中RBM3的表达水平显著高于低温应激前未经IGF-1处理卵丘细胞中RBM3的表达水平,且该处理组卵丘细胞胞质和胞核均可检测到RBM3。(3)25℃应激后,100 ng·mL^-1IGF-1处理组卵丘细胞的凋亡率为(15.94±2.03)%,显著低于其在未经IGF-1处理组和100 ng·mL^-1IGF-1+RBM3抑制剂处理组中卵丘细胞的凋亡率,两者分别为(25.86±1.09)%和(20.14±2.65)%,在100 ng·mL^-1IGF-1处理组Bcl-2的表达水平显著高于其余两个处理组(P<0.05),而Bax的表达水平显著低于其余两个处理组(P<0.05)。【结论】RBM3参与牦牛卵丘细胞低温应激调控,IGF-1可调控其在低温应激中的表达水平,从而降低低温诱导的卵丘细胞凋亡。本研究为揭示IGF-1和RBM3参与动物机体或细胞免受低温损伤的分子机制提供了关键信息,为体细胞和生殖细胞冷冻技术的提高提供了理论依据。

【Objective】The aim of this study was to verify the relationship between insulin-like growth factor 1(insulin-like growth factor,IGF-1)and RNA binding motif protein 3(RBM3)during hypothermia stress and cell freezing in animals,and to investigate the mechanism of IGF-1 involved in inhibiting hypothermia injury of mammalian cells.【Method】Yak(Bos grunniens)cumulus cells were cultured in vitro,the effects of different concentrations(0,50,100 and 200 ng·mL^-1)IGF-1 and hypothermia stress(25℃and 30℃)on RBM3 expression in yak cumulus cells were detected by the methods of real-time fluorescence quantitative PCR(qRT-PCR),Western blot(WB)and immunofluorescence.The expression of RBM3 was compared when the cumulus cells treated with 100 ng·mL^-1and 0 IGF-1 for 30 h and then stressed at low temperature(25℃and 30℃)for 8 h.The difference of apoptosis level of cumulus cells was evaluated from the groups that treated with 0 ng·mL^-1IGF-1,100 ng·mL^-1IGF-1,100 ng·mL^-1IGF-1+RBM3 inhibitor for 30 h and then stressed at 25℃for 8 h.The expression of apoptosis-related genes(Bax and Bcl-2)in cumulus cells from three groups was also detected at mRNA and protein levels.【Result】(1)The expression level of RBM3 in cumulus cells treated with IGF-1 was significantly higher than that in control group(0 ng·mL^-1),which was the highest in cumulus cells from 100 ng·mL^-1IGF-1 group.The RBM3 protein could be detected in nucleus and cytoplasm of cumulus cells,which only expressed in cytoplasm under 0 and 50 ng·mL^-1treatment groups.(2)The levels of RBM3 could be increased by hypothermia stress,and which also could be enhanced when 100 ng·mL^-1IGF-1 was added before cold stress.The RBM3 protein could be detected in nucleus and cytoplasm of cumulus cells after hypothermia stress and treated with IGF-1.(3)After stress at 25℃for 8 h,the apoptosis rate of cumulus cells treated with 100 ng·mL^-1IGF-1 was(15.94±2.03)%,which was significantly lower than those of cumulus cells without IGF-1 treatment(25.86±1.09)%or with 100 ng·mL^-1IGF-1+RBM3 inhibitor(20.14±2.65)%.The expression level of Bcl-2 was significantly higher than those in the control and IGF-1+RBM3 inhibitor groups(P<0.05),while the expression level of Bax was significantly lower than control and IGF-1+RBM3 inhibitor groups(P<0.05).【Conclusion】RBM3 was involved in the regulation of hypothermia stress in yak cumulus cells,and IGF-1 could regulate its expression level in hypothermia stress,which helped to reducing the apoptosis of cumulus cells induced by low temperature.The results in this study provided the key information for revealing the molecular mechanism of IGF-1 and RBM3 involved in the protection of animal bodies or cells from hypothermia damage,and provided theoretical basis for the improvement of somatic and germ cell freezing techniques.