背景:非整倍体是早期胚胎流产的最常见原因。染色体分离的任何缺陷都无法满足有丝分裂过程中的纺锤体组装检查点(SAC),停止中期并引起非整倍性。有丝分裂检查点复合体(MCC),MAD1、MAD2、Cdc20、BUBR1和BUB3在SAC激活中起着至关重要的作用。研究证实,MAD2和BUBR1的过表达可以促进正确的染色体分离和胚胎稳定性。研究还证明miR-125b通过结合其3'UTR负调节MAD1表达。然而,miR-125b,Mad1和Bub3基因在自然流产的非整倍体胚胎中的表达迄今尚未见报道。
方法:在本研究中,收集来自流产妊娠的胚胎绒毛,并根据高通量连接依赖性探针扩增(HLPA)和荧光原位杂交(FISH)分析将其分为两组(非整倍性和整倍性)。miR-125b的RNA水平,通过定量实时PCR(qRT-PCR)检测MAD1和BUB3;通过蛋白质印迹分析MAD1和BUB3的蛋白质水平。
结果:统计学分析(p<0.05)显示,与对照组相比,非整倍体组中miR-125b和BUB3显著下调,MAD1显著上调。此外,非整倍体流产绒毛中的MAD1蛋白水平明显升高,但BUB3蛋白仅轻度增加。相关性分析显示MAD1与miR-125b的表达呈负相关。
结论:这些结果表明,非整倍体流产与MAD1过表达呈正相关,这可能是由miR-125b水平不足引起的。一起来看,我们的发现首先证实了MAD1和miR-125b之间的负调控模式,为非整倍体流产机制的进一步研究提供依据。
BACKGROUND: Aneuploidy is the most frequent cause of early-embryo abortion. Any defect in chromosome segregation would fail to satisfy the spindle assembly checkpoint (SAC) during mitosis, halting metaphase and causing aneuploidy. The mitotic checkpoint complex (MCC), comprising MAD1, MAD2, Cdc20, BUBR1 and
BUB3, plays a vital role in SAC activation. Studies have confirmed that overexpression of MAD2 and BUBR1 can facilitate correct chromosome segregation and embryo stability. Research also proves that miR-125b negatively regulates MAD1 expression by binding to its 3\'UTR. However, miR-125b, Mad1 and
Bub3 gene expression in aneuploid embryos of spontaneous abortion has not been reported to date.
METHODS: In this study, embryonic villi from miscarried pregnancies were collected and divided into two groups (aneuploidy and euploidy) based on High-throughput ligation-dependent probe amplification (HLPA) and Fluorescence in situ hybridization (FISH) analyses. RNA levels of miR-125b, MAD1 and
BUB3 were detected by Quantitative real-time PCR (qRT-PCR); protein levels of MAD1 and
BUB3 were analysed by Western blotting.
RESULTS: statistical analysis (p < 0.05) showed that miR-125b and
BUB3 were significantly down-regulated in the aneuploidy group compared to the control group and that MAD1 was significantly up-regulated. Additionally, the MAD1 protein level was significantly higher in aneuploidy abortion villus, but BUB3 protein was only mildly increased. Correlation analysis revealed that expression of MAD1 correlated negatively with miR-125b.
CONCLUSIONS: These results suggest that aneuploid abortion correlates positively with MAD1 overexpression, which might be caused by insufficient levels of miR-125b. Taken together, our findings first confirmed the negative regulatory mode between MAD1 and miR-125b, providing a basis for further mechanism researches in aneuploid abortion.