In two of the three experiments, Western blotting showed an increase in BUB1 protein, despite the steadily high miR-155 expression ( Figure 1D and Figure S1A,B), suggesting a possible mechanism of escape from miR-155 targeting. Considering these results, we measured BUB1 protein expression during the multistep immortalization of HDF LT/hTERT cells (from P14 to P24). In summary, the most adaptive clones expand during these passages, keeping normal metaphase plates. The number of such clones and related sub-clones evolves from P16 to P24, whereas single cells with aberrant karyotypes decrease ( Figure 1C Table S1). To better understand this apparent discrepancy, i.e., a higher number of aberrant karyotypes with normal metaphase plates, we grouped the identified karyotypes by separating cells with unique karyotypes from cells sharing a common karyotype or related karyotypes (considered sub-clonal). We found an initially high rate of abnormal metaphase (P16) followed by a reduction at P20 and P24 ( Figure 1B). We next evaluated the morphology of the metaphase plates of the same cells by immunofluorescence, considering abnormal those metaphases that showed chromosomes with distinct spindle-positioning defects and incomplete congression. To explore to what extent miR-155 affects aneuploidy in more advanced stages of transformation, we analyzed chromosomes at different passages during the immortalization process of normal HDF cells (HDF LT/hTERT cells at passages P16, P20 and P24) and found a similar number of karyotype abnormalities at P16 and P20, but a higher number at P24 ( Figure 1A). Our data reflect the critical implications of CIN in tumor initiation, highlighting miR-155 as a new player during tumor onset. Indeed, BUB1 deregulation is strongly associated to aneuploidy in mammalian cells. BUB1, the most studied of the three, is a serine/threonine kinase localized at the outer plate of the kinetochore whose main function is to ensure the correct chromosome alignment of the metaphase plate. We demonstrated that miR-155 targets BUB1, CENPF and ZW10, proteins implicated in preventing anaphase onset in case of unattached kinetochores. We recently shed light on its involvement in the spindle assembly checkpoint (SAC) and chromosome instability (CIN) during the early stages of the in vitro transformation of human normal dermal fibroblasts (HDF) by hTERT and SV40 large T antigen-enforced expression. Hsa-miR-155-5p ( miR-155) contributes to genomic instability through downregulation of proteins implicated in DNA mismatch repair, DNA damage repair, and maintenance of telomere integrity.

Our findings have revealed, at least in part, the molecular mechanisms behind the chromosomal stabilization of cell lines and the concept that, to survive, tumor cells cannot continuously change their genetic heritage but need to stabilize the most suitable karyotype. This mechanism occurs at advanced passages of cell transformation and allows the expansion of more favorable clones. Here we show that the heterogeneous nuclear ribonucleoprotein L (HNRNPL) binds to the polymorphic marker D2S1888 at the 3′UTR of BUB1 gene, impairs the miR-155 targeting, and restores BUB1 expression in chronic lymphocytic leukemia. During in vitro transformation of normal human fibroblast, upregulation of miR-155 downregulates spindle checkpoint proteins as the mitotic checkpoint serine/threonine kinase budding uninhibited by benzimidazoles 1 (BUB1), the centromere protein F (CENPF) and the zw10 kinetochore protein (ZW10), compromising the chromosome alignment at the metaphase plate and leading to aneuploidy in daughter cells. We recently demonstrated that miR-155 sustains aneuploidy at early stages of in vitro cellular transformation. Aneuploidy and overexpression of hsa-miR-155-5p ( miR-155) characterize most solid and hematological malignancies.

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