Fig 2: Aurka controls breast cancer stem cells through regulation of Wnt/ß-catenin.(A) On the left, relative quantification of AurkA and wnt3a in MCF-AurkA+ cells (AurkA). MCF-7 carrying empty vector (empty) were considered as control. On the right, (a) Cytofluorimetric panels show distribution of MCF-AurkA or MCF-empty cells depending on CD44-CD24 staining; (b,c) Distribution of CD44+ or CD24+ subpopulation, respectively, in MCF-empty (top panels) and MCF-AurkA cells (bottom panels). Black arrow in (b) highlights the increase of CD44+ cells in AurkA overexpressing cells (AurkA+). Black bar in (c) highlights the decrease of CD24 expression cells in AurkA overexpressing. (B) On the left, relative quantification of AurkA and wnt3a after AurkA silencing at low (sh8, shMin) or high (sh5, shMax) efficiency in MDA-MB-231 cells. MDA-MB-231 cells carrying empty vector (empty) were considered as control. (a) On the right, cytofluorimetric panels showing distribution of MDA-shAurkA versus MDA-empty cells depending on ALDH-expression (bottom panels). +DEAB empty or shAurkA were negative controls (top panel). (b) ALDH positive cells were scored as reported in the right graph. (C) MFE (Mammosphere Forming Efficiency) valued in AurkA+ MCF-7 or in shAurkA MDA-MB-231. Control cells are indicated as empty. Representative images for each sample were showed at 200X magnification (Scale bar: 800 µm). Data are representative of biological triplicates.

Fig 3: MiR-26b inhibits hepatocellular carcinoma cell proliferation, invasion and migration in vitro by targeting Zinc ribbon domain-containing 1 (ZNRD1). SMMC-7721 or Hep 3B cells were transfected with NC, miR-26b mimics or miR-26b mimics & ZNRD1 overexpression plasmid. A, The expression levels of ZNRD1, Wnt3a, ß-catenin, APC, and cyclinD1 were analyzed by western blot 48 h later. B, Cell proliferation was determined by CCK-8 cell proliferation assays. C, Colonies of SMMC-7721 or Hep 3B in different groups were determined by colony formation assay. D, Cell invasion capability of SMMC-7721 or Hep 3B in different groups was analyzed by transwell assay. E, Cell migration capability of SMMC-7721 or Hep 3B cells in different groups was analyzed by wound-healing assay. *P < .05, **P < .01

Fig 4: Zinc ribbon domain-containing 1 (ZNRD1) activates Wnt/ß-catenin signaling pathway in hepatocellular carcinoma (HCC). A, GSVA analysis showed that Wnt/ß-catenin pathway in tumor compared with adjacent nontumor were identified as the top three activated regulated pathway in TCGA HCC cancer cohort. B, The Gene Set Enrichment Analysis of the correlation between ZNRD1 expression and gene signatures of Wnt/ß-catenin signaling pathway in HCC. C and D, SMMC-7721 or Hep 3B cells were transfected with Ctrl or sh-CLK3 and the expression of Wnt3a, ß-catenin, APC, and cyclinD1 was analyzed by western blot. Relative quantification analysis was based on grayscale values. *P < .05, **P < .01

Fig 5: Snail mediates repression of miR-128 in response to Aurka overexpression.(A) AurkA overexpression does not promote Snail protein increase as found in MCF-AurkA (AurkA+) versus control cells (empty) Evaluation of Snail protein levels in MCF-7 and MCF-AurkA. In contrast, AurkA knock-down severely impaired Snail protein levels in MDA-shAurkA (sh5, sh7, sh8) and KBr2-shAurkA (shAurkA) in comparison with control cells (empty). (B) AurkA antibody pulls down Snail in MCF7 and MDA-MB-231 cells after a co-immunoprecipitation assay (IP anti-AurkA). As control, protein lysates from both cell lines were tested for Snail protein levels (control). A marker was loaded in the last lane as a control of molecular weight. (C) AurkA overexpression in MCF-7 cells induced nuclear translocation of Snail (MCF, AurkA+). Control cells show a moderate cytoplasmic staining (MCF, empty). Nuclear accumulation of Snail in MDA-MB-231 cells (MDA, empty) was inhibited by AurkA knock-down (MDA, shAurkA). Nuclei were counterstained with DAPI. Collectively our findings supports a signaling pathway showing that AurkA may promote nuclear translocation of Snail to repress miR-128 gene transcription. As results, wnt3a mRNA accumulates increasing Wnt3a protein levels. (D) On the left, Q-PCR confirmed association between AurkA and miR-128 levels in 32 human tissues from breast cancer patients. Asterisk highlights samples were association is missing. On the right, Graphic representation of of samples (percentage, %) showing a strong correlation AurkA-mir-128 (AurkA > 1.5/miR-128 < 1.5 and AurkA < 1.5/miR-128 > 1.5), or where AurkA-miR-128 correlation is missing (AurkA > 1.5/miR-128 > 1.5 and AurkA < 1.5/miR-128 < 1.5).