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. 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. 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. 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.

中文翻译：

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非整倍体流产与MAD1过表达和miR-125b下调呈正相关

非整倍体是早期胚胎流产的最常见原因。染色体分离中的任何缺陷都将无法在有丝分裂期间满足纺锤体装配检查点（SAC），停止中期并导致非整倍性。包含MAD1，MAD2，Cdc20，BUBR1和BUB3的有丝分裂检查点复合物（MCC）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通过与其3'UTR结合而负调控MAD1表达。但是，迄今为止尚未报道自发流产的非整倍体胚胎中的miR-125b，Mad1和Bub3基因表达。在这项研究中，根据高通量连接依赖探针扩增（HLPA）和荧光原位杂交（FISH）分析，收集流产孕妇的胚胎绒毛并将其分为两组（非整倍体和整倍体）。通过实时定量PCR（qRT-PCR）检测miR-125b，MAD1和BUB3的RNA水平；通过蛋白质印迹分析MAD1和BUB3的蛋白水平。统计分析（p <0.05）显示，与对照组相比，非整倍体组miR-125b和BUB3显着下调，而MAD1显着上调。此外，在非整倍体流产绒毛中，MAD1蛋白水平显着较高，而BUB3蛋白仅轻度升高。相关分析表明，MAD1的表达与miR-125b负相关。这些结果表明非整倍体流产与MAD1过表达呈正相关，这可能是由于miR-125b水平不足引起的。综上所述，我们的发现首先证实了MAD1和miR-125b之间的负调控模式，为进一步研究非整倍体流产提供了基础。