Aneuploid Abortion Positively Correlate With MAD1 Overexpression and mir125b Depression

Background: Aneuploid is the most frequent cause of early embryo abortion, and any defect in chromosome segregation would fail to satisfy spindle assembly checkpoint (SAC) during mitosis, which could lead to the halted metaphase and aneuploid occurrence. Mitotic checkpoint complex(MCC), a complex compound of MAD1 (cid:0) MAD2 (cid:0) Cdc20 (cid:0) BUBR1 and BUB3, plays an important role in SAC activation. Studies have conrmed that the overexpression of MAD2 and BUBR1 can facilitate the correct chromosome segregation and embryo stability. Research identications also proved that miR-125b negatively regulated MAD1 expression by binding to its 3’UTR. However, the expression of mir125b, MAD1 and BUB3 genes in aneuploidy embryos of spontaneous abortion has not been reported. Methods: In this study, embryonic villi from miscarriage pregnant women were collected and divided into two groups (aneuploidy and euploidy) by HLPA and FISH analysis. The RNA levels of mir125b, MAD1 and BUB3 were detected through QRT-PCR, while Western blot was further used to analyze the protein levels of MAD1 and BUB3. Results: SPSS 17.0 statistical analysis(P<0.05) showed that mir125b and BUB3 were signicantly down-regulated in aneuploidy group compared to the control group, MAD1 was signicantly up-regulated in RNA level; Additionally, MAD1 protein level was also signicantly higher while BUB3 was mildly increased in aneuploidy abortion villus. Correlation analysis revealed that the expression of MAD1 was negatively correlated with Mir125b. Conclusion: these results suggested that aneuploid abortion was positively correlated with MAD1 overexpression which might be caused by insucient mir125b. the expression of mir125b, MAD1 and BUB3 genes in aneuploidy embryos of spontaneous abortion has not been reported. This study was designed to investigate the expression of mir125b, MAD1 and BUB3 in embryonic villi cells from aneuploidy abortion embryos, and to further explore the mechanism of spontaneous abortion.


Preface
Aneuploidy is a common and natural occurrence in early human embryos, which would cause disordered cell-cycle and retarded embryonic growth, even miscarriage 1 . A systematic review and meta-analysis associated with human embryos chromosome in 2011 reported that 73% of all human embryos contain aneuploid cells 2,3 . Chromosomal instability (CIN) plays a key role in aneuploidy and tumorigenesis 4 . In this context, spindle assembly checkpoint (SAC) is crucial to ensure delity of chromosome segregation during mitosis. Aneuploidy is known as a frequent factor from a defective mitotic checkpoint 5 . Any defect in kinetochore-spindle attachment between sister chromatids would fail to satisfy SAC, which could lead to the halted metaphase until the defect is corrected 6 .
Obviously, the process of chromosome segregation during mitosis is extremely complicated, which contains a series of cascade reactions, and multiple proteins are involved. Once all chromatids are bioriented at metaphase, E3 ubiquitin-ligase anaphase-promoting complex/cyclosome (APC/C) catalyses the proteasomal degradation of securin, an inhibitory chaperone of separase. Activated separase then cleaves the cohesion complex required for the physical linkage of sister chromatids. Owing to loss of cohesion, separated chromatids move to opposite spindle poles and cells enter anaphase 7,8 . Furthermore, APC/C activity depends on an adaptor-protein Cdc20 (cell division cycle 20) 9 . When SAC is 'on', APC/C remains inactive due to sequestered Cdc20 by Mad2 and BubR1/Bub3 in the form of mitotic checkpoint complex (MCC). Premature anaphase progression is thus prevented by the 'wait-anaphase' signal generated from the diffusible MCC 10 .
MCC, a complex compound of MAD1 MAD2 Cdc20 BUB1 and BUB3, plays an important role in SAC activation. One of the rst events in SAC activation is the recruitment of an adaptor protein Mad1 to the kinetochore. Mad1 is crucial both in Mad2-transportation from cytosol to nucleus and its kinetochore localization 6 . According to 'template' model, Mad2 exists in two states: open/free (O-Mad2),and closed (bound to either Mad1 or Cdc20 (C-Mad2)).Studies have con rmed that reduced expression of Mad2 and Bub1 proteins is associated with spontaneous miscarriages, whereas MAD2 overexpression can facilitate the correct chromosome segregation and embryo stability 11 .
Many mitotic cell cycle regulators often have modi ed functions in meiosis important for the meiotic chromosome segregation program.A noteworthy example of a regulatory pathway with increased roles in meiosis is the spindle checkpoint.
Research shows that he spindle checkpoint protein Mad2 Bub1 and Bub3 have a more important role in meiosis than in mitosis .Mad2 cells have enhanced chromosome mis-segregation in meiosis I and premature anaphase I onset.Loss of BUB1 or BUB3 in meiosis,causes massive chromosome missegregation in meiosis II.though there were fewer cells with massive chromosome missegregation in meiosis I, aneuploidy still occurred 12 .
Research from S Bhattacharjya et.al showed that miR-125b negatively regulates MAD1 expression by binding to its 3'UTR 13 . To date, the expression of mir125b, MAD1 and BUB3 genes in aneuploidy embryos of spontaneous abortion has not been reported. This study was designed to investigate the expression of mir125b, MAD1 and BUB3 in embryonic villi cells from aneuploidy abortion embryos, and to further explore the mechanism of spontaneous abortion. detection, 100 embryonic villi samples were divided into two groups: 50 cases with chromosomal abnormalities (abnormal group), and 50 cases without chromosomal abnormalities (normal group). This trial was approved by the Ethics Committee.

Aneuploidy detection by HLPA
The chromosomal abnormalities of 24 chromosomes were detected by a method of modi ed MLPA (Multiplex ligation-dependent probe ampli cation) assay named HLPA (High-throughput ligationdependent probe ampli cation), which was carried out using CNVplex detection kit (Genesky Technologies (Suzhou) Inc.). The main principle of this method: Using the highly speci city of ligase to perform a set of hybridization and ligation reaction on the target regions to distinguish the ploidy of the target regions. At the step of ligation, sequence tags within different length and different uorescein (PET, VIC, NED, and FAM) were introduced to the ends of the probes and then ligated to the target regions to get ligated products of different lengths. Then, universal primers labeled with uorescent markers were used to amplify the concatenate products by PCR. After ampli cation, the PCR products were separated and detected by uorescence capillary electrophoresis, then the copy number of target regions were calculated by analyzing the peak height of electrophoretogram, the detailed work ow refer to the study Chen, S et al reported 14 . There were totally 170 (for Chr1-12 and Chr16-17, there 8 probes for each chromosome; for Chr13-15, Chr21-22 and ChrY, there 5 probes for each chromosome; for Chr18-Chr20 and ChrX, there 7 probes for each chromosome) pairs of probes targeting 24 chromosomes were designed for the aneuploidy detection. The experimental steps were as follows: First, 2 μL gDNA (30ng/μL) mixed with 1μL probe mix(10uM), 1.25 μL 4× DNA lysis buffer and 5.75 μL DNA diluent were denatured for 2 min at 98 °C and then put on ice immediately. Then, 2μL 10× ligation buffer, 0.5μL ligase and 7.5 μL doubledistilled water were added to the rst step products to start the ligation under the following the program: 5 cycles of 94°C for 1 min and 60°C for 3 h, then 94 °C for 2 min, and 72 °C for 10 min. Reactions were stopped by adding 20 μL of 20 mM EDTA. After ligation, 1 μL of ligation products, 10 μL 2× PCR Master Mix, 1 μL primer mix and 8 μL double-distilled water were mixed evenly to perform the multiplex PCR ampli cation. The PCR program was as follows: 95°C for 2 min;5 cycles of 94°C for 20 s, 62°C for 40s decreasing 1°C per cycle, and 72°C for 1.5 min; then 27 cycles of 94°C for 20 s, 57°C for 40 s, and 72°C for 1.5 min; then 68°C for 1h; Finally ,cooling the system to 4°C to stop the reaction. The last step was capillary electrophoresis and data analysis: PCR products were diluted 5-fold. 1μL diluted products mixed with 0.1μL LIZ 500 size standard (Applied Biosystems, Foster City, CA, USA) and 8.9 μL Hi-DiTM formamide (Applied Biosystems) were denatured at 95 °C for 5 min and uorescently labeled products were separated by on an ABI3130XL genetic analyzer (Applied Biosystems). Data were analyzed with GeneMapper software v4.1 (Applied Biosystems).
Then the slides were dehydrated in ethanol series (70%, 85% and 100%) for 2 min each, and dried at room temperature in a slanted position. Denaturing When dry, slides were placed in 5-nmol/L dithiothreitol (DDT, Sigma) and 1% Triton X-100 solution (Sigma) at 37°C for 13 min. After that, the slides were denatured in 70% Formamide (Sigma) and 2×SSC solution for 5 min at 71°C. The slides were washed again in 2× SSC and dehydrated in ethanol series. Before hybridization, the DNA probes mixed with hybridization buffer were immersed in a water-bath for 5 minutes at 72°C to denature. Hybridization The denatured probe was then applied to each glass slide containing the fixed villi cells and was hybridized overnight at 37°C. Then, slides were washed in 0.7× SSC at 71°C for 2 minutes, dried at room temperature, and counterstained with DAPI in antifade and covered with glass coverslips for analysis.

Quantitative real-time PCR.
Total RNA was isolated using TRIZOL (Invitrogen) according to manufacturer's protocol. 5ug isolated RNA was treated with DNase (Promega, Madison, WI, USA) and 1ug of the DNase-treated RNA was used for cDNA preparation using random hexamer (Invitrogen) and MMLV-RT (Promega). For miRNAs, 200ng isolated RNA was used for cDNA preparation in a master mix containing stem-loop primers speci c for the desired miRNAs (Sigma), dNTPs (Invitrogen) and MMLV-RT (Promega). Real-time PCR was performed in the 7500 Fast Real-Time PCR System (Applied Biosystems) using power SYBR Green PCR Master Mix (Applied Biosystems). The comparative threshold cycle method (△△Ct) was used to quantify relative amounts of product transcripts with GAPDH (for mRNAs) and U6 (for miRNAs) as endogenous reference controls. Primer sets for MAD1, BUB3 and GAPDH are listed in Supplementary Table 1. Fold activation values were calculated as mean of independent experiments.

Karyotype analysis
Villi tissues were obtained after operation of uterine cleanup. The chromosomal aneuploidy was detected by HLPA.50 cases with chromosomal abnormalities were listed in Table2. To con rm the aneuploidy detection results by HLPA, we further carried out FISH assay. The results were exhibited in Figure 1. The relative expression of miR125b and BUB3 were signi cantly down-regulated in the abnormal group compared to normal group(P<0.05), whereas MAD1 was signi cantly increased at RNA level (p <0.05) Fig.2 . Figure2:miR125b was signi cantly down-regulated in the abnormal group compared to normal group(P<0.05) MAD1 gene in abnormal group was signi cantly up-regulated than that in normal group, and the relative expression of BUB3 gene in abnormal group was lower than that in normal group (p <0.05).
Western blotting analysis of MAD1 and BUB3 proteins The relative expression of BUB3 protein in abnormal group was slightly up-regulated, while the relative expression of MAD1 protein in abnormal group was signi cantly up-regulated (Figure3). BUB3 protein in abnormal group was slightly up-regulated, while the relative expression of MAD1 protein in abnormal group was signi cantly up-regulated (P<0.05).

Discussion
Aneuploid is the most frequent etiology of miscarriage, and elucidating the molecular mechanism of aneuploid is important for genetic consultation from aborted women. SAC is vital to ensure delity of chromosome segregation during mitosis, and aneuploidy could generate a defective SAC. It is well known that MCC exert an important in uence on the whole SAC course. MAD1, MAD2, Cdc20, BUBR1 and BUB3 have been con rmed to be important components for MCC. Across these proteins, MAD2 and BUBR1 overexpression can facilitate the correct chromosome segregation and embryo stability, but the expression levels of MAD1 and BUB3 in aneuploid abortion still been unknown.
Literature retrieval discovered that MiR-125b is a highly conserved miRNA that functions as a tumor suppressor or an oncogene depending upon cellular contexts. S Bhattacharjya et.al reported that miR-125b negatively regulates MAD1 expression by binding to its 3'UTR, miR-125b up-or down regulation could give rise to CIN or lead to accelerated proliferation and cell death12. Indeed, it was earlier reported that Mad1 expression was heightened in cancer cells and its high expression was correlated with cellular proliferation 16 . At the molecular level, Mad1-bound and free-Mad2 are both essential to maintain SAC.
Moreover, excess MAD1 was superior to CDC20 in binding with free-MAD2, whereas CDC20 could bind to and activate APC/C. All these lead to rapid mitotic exit and SAC abolishment 17,18 .
Two crucial regulators of cell cycle, cohesin and the SAC share common regulators to coordinate faithful chromosome segregation and orchestrate meiotic progression, Research show that cohesin release factor Wapl interacts with Bub3 to mediate the SAC function in oocyte meiosis I,Nevertheless,Wapl regulates Bub3 through an unknown way that requires further investigation 19 .Other research shows that ,in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1 and concomitant massive chromosome missegregation caused by incorrect chromosome-spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1mediated phosphorylation. they propose a new role for the Bub1-Bub3 pathway in maintaining the balance between kinetochore localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset 12 .
In this study,the relative expression of BUB3 gene in abnormal group was lower than that in normal group,but the relative expression of BUB3 protein in abnormal group was slightly up-regulated,The relationship between BUB3 gene expression regulatory pathway and aneuploidy requires further investigation.
In this study, Mir125b was signi cantly decreased while the mRNA and protein levels of MAD1 were higher in abnormal group compared to normal group; Moreover, the MAD1 expression was negatively correlated with Mir125b. Taken together, we hypothesize that insu cient miR-125b increased MAD1 expression levels, which resulted in decreased free-Mad2, actived APC/C. All these dysregulation lead to SAC abolishment and accumulation of aneuploid cells in embryo, which nally cause miscarriage through child-to-mother communication mechanism.
This will help us to further explore the elaborate regulatory mechanism between miR-125b and MAD1 in aneuploid-induced spontaneous abortion. The relative expression of miR125b, MAD1 and BUB3 Figure 3