45A non-coding RNA (ncRNA) overexpression induces modifications of neuroblastoma (NB) cell cytoskeleton leading to a cascade of reactions that interferes with proliferation control, cell migration, tumorigenic potential, and cell adhesion properties. In detail, 45A-overexpressing cells show non-specific metastatic engraftment whereas the silencing of its expression drives to the production of liver-specific metastasis.
We investigated by Real-time RT-PCR, western blot, and immunofluorescence analysis the different expression and/or localization of GTSE1, MCAK, Aurora B, p53, EGFR, and the altered organisation of tubulin in different neuroblastoma cell (SKNBE2) models stably overexpressing or downregulating 45A ncRNA. Indeed, the proper regulation of these proteins\u2019 expression and function is fundamental for cytoskeleton organization as their impairment leads to the particularly dangerous condition of chromosomal instability (CIN).
We demonstrate that 45A ncRNA not only directly regulates the expression of aforementioned proteins but can even affect GTSE1 subcellular localization: in 45A-overexpressing cells, the protein is accumulated in nuclei, while 45A-downregulation leads to a significant GTSE1 cytoplasm relocation, together with simultaneous cytoplasmatic sequestration of p53, driven by GTSE1/p53 interaction. This shuttling of the oncosopressor decreases the apoptotic potential of 45A-downregulating cells, justifying the resistance to toxoids that we observed.
Furthermore, 45A-overexpression leads to an increased number of abnormal spindles, thus promoting CIN, and possibly explaining the increased tumorigenic potential exhibited by 45A-overexpressing cells.
Lastly, we also observed EGFR nucleolar sequestration in 45A overexpressing cells, a mechanism observed in several kind of tumors and correlated with malignancy.
These data highlight the role of 45A ncRNA in the functional regulation of several proteins expression involved in microtubules dynamics, causing different drugs responses and suggesting its possible relevance in prognosis