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. 2016 Jun 24;6:28436. doi: 10.1038/srep28436

Figure 5. Aurka promote stabilization of wnt3a through repression of miR-128.

Figure 5

(A) Q-PCR showed that AurkA knock-down in MDA-MB-231 cells promoted an increase of mir128 expression while reduced levels of mir15 and mir16. Effectively, there was a mechanism of miR128 inhibition by AurkA as revealed by Q-PCR after AurkA overexpression in MCF-7 (MCF-AurkA+) cells of knock-down in MDA-MB-231 and KBr2 cells (MDA-shAurkA and KBr2-shAurka). (B) miR-128 controls wnt3a. miR-128 inhibitors promoted Wnt3a protein stabilization in MCF-7 (lane 2), conversely specific miR-128 mimics (lane 4) repressed Wnt3a in MDA-MB-231 cells, untreated MCF-7 or MDA-MB-231 cells were considered as control (lane 1 and 3, respectively). (C) miR128 bind to wnt3a 3′UTR. Luciferase activity increased when HEK-293T cells were co-transfected with pMIR-wt-wnt3a and miR128 inhibitors (3rd bar), decreased in presence of pMIR-wt-wnt3a and mimics (mimicking miR128 activity, 2nd bar). In contrast, it was not affected in control cells co-transfected with pMIR-wt-wnt3a and miR128 scramble (1st bar) or co-transfected with pMIR-mut-wnt3a (carrying mutated 3′UTR of wnt3a) and miR128 scramble, mimics or inhibitor (respectively 4th, 5th, 6th bars). At the bottom a schematic view of pMIR-wt-wnt3a and pMIR-mut-wnt3a. Data are representative of biological triplicates. (D) AurkA expression affected Snail transcription. Snail cDNA increased after AurkA overexpression in MCF-7 (MCF-AurkA+) cells and increased after AurkA knock-down in MDA-MB-231 and KBr2 cells (MDA-shAurkA and KBr2-shAurka). (E) Increased luciferase activity was found when 293T cells were co-transfected with a vector carrying snail and pGL3-wt-Ebox1–2 (2nd gray bar). Conversely, luciferase activity was unaffected when 293T were co-transfected with a snail-vector and pGL3-mut-Ebox1–2 (carrying mutated Ebox1, Ebox2 or both, respectively 3th, 4th and 5th gray bars). Control cells, co-transfected with pGL3-wt-Ebox1–2 or pGL3-mut-Ebox1–2 with pGL3 (missing Snail gene) showed basal luciferase expression.