Fig 2: Inhibition of AURKB kinase activity represses the expression of CCND1 and decreases gastric cancer growth in vivo. (A) Photograph of xenograft tumors excised from mice treated with AZD1152 (25 mg/kg). (B) Tumor volumes were examined on the indicated days. Data are presented as the means ± SDs; **, P < 0.01. (C) Tumor mass of xenograft tumors excised from mice treated with AZD1152 (25 mg/kg). (D) Western blot analysis of AURKB, CCND1 and H3S10ph expression in xenograft tumors treated with AZD1152 (25 mg/kg). HSP70 and histone H3 were used as the endogenous controls. (E) Hematoxylin and eosin (H&E) staining and IHC staining of AURKB, CCND1, H3S10ph and Ki-67 in xenograft tumors excised from mice treated with AZD1152 (25 mg/kg) and control.

Fig 3: AURKB knockdown inhibits gastric cancer cell proliferation. (A) Quantitative real-time PCR analysis of the effect of AURKB knockdown by siRNA on the mRNA levels of AURKB in SGC7901 and BGC823 cells. The results shown are the means ± SDs of three independent experiments; **, P < 0.01 compared with the scrambled negative control (NC). (B) Western blot analysis of AURKB and H3S10ph expression in siRNA- and NC-transfected SGC7901 and BGC823 cells. HSP70 and histone H3 were used as loading controls. (C) CCK-8 assays showing that AURKB knockdown by siRNA significantly inhibited the proliferation of SGC7901 and BGC823 cells. The results shown are the means ± SDs of three independent experiments; **, P < 0.01 compared with the negative control. (D) Effects of AURKB knockdown by siRNA on the colony formation ability of SGC7901 and BGC823 cells. Left panel, representative images from colony formation assays. Right panel, the number of colonies formed by the indicated cells was quantified. Data are presented as the means ± SDs; **, P<0.01 compared with the negative control. (E) Flow cytometry analysis showing the cell cycle distribution of SGC7901 and BGC823 cells transfected with negative control (NC) or AURKB siRNA. Bar graphs showing the percentages of SGC7901 and BGC823 cells in the G0/G1, S and G2/M phases when treated with negative control (NC) or AURKB siRNAs (right panel). Each histogram bar represents the mean ± SD of three independent experiments.

Fig 4: Expression of AURKB and CCND1 is upregulated in gastric cancer tissues, and these expression levels are associated with poor prognosis in gastric cancer patients. (A) Representative images of immunohistochemical (IHC) staining of AURKB and CCND1 in human gastric cancer tissues and matched normal tissues (n = 80). (B) Total IHC score for AURKB and CCND1 in human gastric cancer tissues (Tumor) and matched normal tissues (Normal). **P < 0.01 compared with the matched normal tissue control. (C) The correlation between AURKB and CCND1 expression was evaluated using Pearson correlation analysis (n = 80; r = 0.5764; P < 0.01). (D) Analysis of data from the Kaplan-Meier plotter database suggested that high expression levels of AURKB (left panel; P = 0.0012) and CCND1 (right panel; P = 0.0026) are negatively correlated with 5-year overall survival in gastric cancer patients.

Fig 5: AZD1152 suppresses cell proliferation and represses the expression of CCND1 by inhibiting the enzymatic activity of AURKB in gastric cancer cells. (A) CCK-8 assays showing the effect of different concentrations of AZD1152 (0.5 μM, 2 μM and 10 μM; DMSO as the control) on the proliferation of SGC7901 and BGC823 cells. The results shown are the means ± SDs of three independent experiments; **, P < 0.01 compared with the DMSO control. (B) Colony formation assays showing the effects of different concentrations of AZD1152 (0.5 μM, 2 μM and 10 μM; DMSO as the control) on the colony formation ability of SGC7901 and BGC823 cells. Left panel, representative images from colony formation assays. Right panel, the number of colonies formed by the indicated cells was quantified. Data are presented as the means ± SDs; **, P<0.01 compared with the DMSO control. (C) Flow cytometry analysis showing the effect of different concentrations of AZD1152 (0.5 μM, 2 μM and 10 μM; DMSO as the control) on the cell cycle distribution. Bar graphs showing the percentages of SGC7901 and BGC823 cells in the G0/G1, S and G2/M phases when treated with different concentrations of AZD1152 (right panel). Each histogram bar represents the mean ± SD of three independent experiments. (D) Western blot analysis of the protein levels of AURKB, CCND1 and H3S10ph in SGC7901 and BGC823 cells treated with different concentrations of AZD1152 (0.5 μM, 2 μM and 10 μM; DMSO as the control). HSP70 and histone H3 were used as the endogenous loading controls. (E) Quantitative real-time PCR analysis of AURKB and CCND1 expression in SGC7901 and BGC823 cells treated with different concentrations of AZD1152 (0.5 μM, 2 μM and 10 μM; DMSO as the control); *, P < 0.05; **, P < 0.01. The results shown are the means ± SDs of three independent experiments. GAPDH was used as the endogenous control for mRNA expression analysis.