Bub1 is a critical component of the spindle assembly checkpoint (SAC) and closely linked to cell proliferation and differentiation. We previously found that spontaneous abortion embryos contained a low level of Bub1 protein but normal mRNA level, while the knockdown of Bub1 leads to abnormal numerical chromosomes in embryonic cells. Here, we investigated the mechanism through which governs the post-transcriptional regulation of Bub1 protein expression level. We first conducted bioinformatics analysis and identified eight putative miRNAs that may target Bub1. Luciferase reporter assay confirmed that miR-450a-3p can directly regulate Bub1 by binding to the 3′-untranslated region of Bub1 mRNA. We found that the overexpression of miR-450a-3p in mouse embryonic fibroblast (MEF) cells down-regulated Bub1 protein level, repressed cell proliferation, increased apoptosis and restricted most cells in G1 phase of the cell cycle. Furthermore, when the fertilized eggs were microinjected with miR-450a-3p mimics, the cleavage of zygotes was effectively suppressed. Our results strongly suggest that an abnormally decreased Bub1 level regulated by miRNAs may be implicated in the pathogenesis of spontaneous miscarriage. Therefore, the blockade of miR-450a-3p may be explored as a novel therapeutic strategy for preventing spontaneous miscarriages.
References
[1]
Draviam VM, Xie S, Sorger PK (2004) Chromosome segregation and genomic stability. Curr Opin Genet Dev 14: 120–125.
[2]
Kops GJ, Foltz DR, Cleveland DW (2004) Lethality to human cancer cells through massive chromosome loss by inhibition of the mitotic checkpoint. Proc Natl Acad Sci U S A 101: 8699–8704.
[3]
Musacchio A, Salmon ED (2007) The spindle-assembly checkpoint in space and time. Nat Rev Mol Cell Biol 8: 379–393.
[4]
Doak SH, Jenkins GJ, Parry EM, Griffiths AP, Baxter JN, et al. (2004) Differential expression of the MAD2, Bub1 and HSP27 genes in Barrett's oesophagus-their association with aneuploidy and neoplastic progression. Mutat Res 547: 133–144.
[5]
Steuerwald N, Cohen J, Herrera RJ, Sandalinas M, Brenner CA (2001) Association between spindle assembly checkpoint expression and maternal age in human oocytes. Mol Hum Reprod 7: 49–55.
[6]
Boyarchuk Y, Salic A, Dasso M, Arnaoutov A (2007) Bub1 is essential for assembly of the functional inner centromere. J Cell Biol 176: 919–928.
[7]
Niikura Y, Dixit A, Scott R, Perkins G, Kitagawa K (2007) Bub1 mediation of caspase-independent mitotic death determines cell fate. J Cell Biol 178: 283–296.
[8]
Perera D, Tilston V, Hopwood JA, Barchi M, Boot-Handford RP, et al. (2007) Bub1 maintains centromeric cohesion by activation of the spindle checkpoint. Dev Cell 13: 566–579.
[9]
Tilston V, Taylor SS, Perera D (2009) Inactivating the spindle checkpoint kinase Bub1 during embryonic development results in a global shutdown of proliferation. BMC Res Notes 2: 190.
[10]
Meraldi P, Sorger PK (2005) A dual role for Bub1 in the spindle checkpoint and chromosome congression. EMBO J 24: 1621–1633.
[11]
Li SS, Liu YH, Tseng CN, Chung TL, Lee TY, et al. (2006) Characterization and gene expression profiling of five new human embryonic stem cell lines derived in Taiwan. Stem Cells Dev 15: 532–555.
[12]
Wells D, Bermudez MG, Steuerwald N, Thornhill AR, Walker DL, et al. (2005) Expression of genes regulating chromosome segregation, the cell cycle and apoptosis during human preimplantation development. Hum Reprod 20: 1339–1348.
[13]
Shi Q, Hu M, Luo M, Liu Q, Jiang F, et al. (2011) Reduced expression of Mad2 and Bub1 proteins is associated with spontaneous miscarriages. Mol Hum Reprod 17: 14–21.
[14]
Ambros V (2001) microRNAs: tiny regulators with great potential. Cell 107: 823–826.
Kloosterman WP, Plasterk RH (2006) The diverse functions of microRNAs in animal development and disease. Dev Cell 11: 441–450.
[17]
He L, Hannon GJ (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5: 522–531.
[18]
Song L, Tuan RS (2006) MicroRNAs and cell differentiation in mammalian development. Birth Defects Res C Embryo Today 78: 140–149.
[19]
Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120: 15–20.
[20]
Bernstein E, Kim SY, Carmell MA, Murchison EP, Alcorn H, et al. (2003) Dicer is essential for mouse development. Nat Genet 35: 215–217.
[21]
Brown BD, Gentner B, Cantore A, Colleoni S, Amendola M, et al. (2007) Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state. Nat Biotechnol 25: 1457–1467.
[22]
Chan MC, Hilyard AC, Wu C, Davis BN, Hill NS, et al. (2010) Molecular basis for antagonism between PDGF and the TGFbeta family of signalling pathways by control of miR-24 expression. EMBO J 29: 559–573.
[23]
Taylor SS, McKeon F (1997) Kinetochore localization of murine Bub1 is required for normal mitotic timing and checkpoint response to spindle damage. Cell 89: 727–735.
[24]
Tang Z, Shu H, Oncel D, Chen S, Yu H (2004) Phosphorylation of Cdc20 by Bub1 provides a catalytic mechanism for APC/C inhibition by the spindle checkpoint. Mol Cell 16: 387–397.
[25]
Kuersten S, Goodwin EB (2003) The power of the 3′ UTR: translational control and development. Nat Rev Genet 4: 626–637.
[26]
Orom UA, Nielsen FC, Lund AH (2008) MicroRNA-10a binds the 5′UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell 30: 460–471.
[27]
Yu J, Ryan DG, Getsios S, Oliveira-Fernandes M, Fatima A, et al. (2008) MicroRNA-184 antagonizes microRNA-205 to maintain SHIP2 levels in epithelia. Proc Natl Acad Sci U S A 105: 19300–19305.
[28]
Gao F, Ponte JF, Papageorgis P, Levy M, Ozturk S, et al. (2009) hBub1 deficiency triggers a novel p53 mediated early apoptotic checkpoint pathway in mitotic spindle damaged cells. Cancer Biol Ther 8: 627–635.
[29]
Gjoerup OV, Wu J, Chandler-Militello D, Williams GL, Zhao J, et al. (2007) Surveillance mechanism linking Bub1 loss to the p53 pathway. Proc Natl Acad Sci U S A 104: 8334–8339.
[30]
Jeganathan K, Malureanu L, Baker DJ, Abraham SC, van Deursen JM (2007) Bub1 mediates cell death in response to chromosome missegregation and acts to suppress spontaneous tumorigenesis. J Cell Biol 179: 255–267.
[31]
Hernando E, Orlow I, Liberal V, Nohales G, Benezra R, et al. (2001) Molecular analyses of the mitotic checkpoint components hsMAD2, hBub1 and hBub3 in human cancer. Int J Cancer 95: 223–227.
[32]
Masuda A, Takahashi T (2002) Chromosome instability in human lung cancers: possible underlying mechanisms and potential consequences in the pathogenesis. Oncogene 21: 6884–6897.
