Figure 1.
Expression profiles of selected mitotic spindle checkpoint genes in HCC microarray datasets.
Heat maps of relative fold change (A) and frequency of overexpression (B) of gene expression (rows) in three HCC microarray datasets (columns). Number of normal liver and HCC tumor samples per data set are shown below. (C) As the heat map shown, 13 mitotic spindle checkpoint genes differentially expressed between HCC and adjacent non-tumorous livers in 4 independent HCC microarray datasets. Missing data are denoted in gray. Transcriptional signal intensity is normalized in these datasets analyzed. The significance of differential expression between HCC and adjacent non-tumorous liver tissue was evaluated using a two-tailed t test, and P values are provided.
Table 1.
The 13 markedly overexpressed mitotic spindle checkpoint genes in HCC samples.
Figure 2.
Expression analysis of the mitotic checkpoint genes in HCC cell lines and established sorafenib-resistant sublines.
(A) The cytotoxic effects of sorafenib on sorafenib-naive and sorafenib-resistant cells of HCC cell lines Huh7 (upper panel) and HepG2 (lower panel). These cells were exposed to sorafenib at the indicated concentration for 24 h, and the cell viability was presented by the absorbance value at OD450 nm using CCK-8. *, P<0.05, **, P<0.01. (B) Scatter plots of fluorescence-activated cell sorting analysis with annexinV-FITC/PI staining in the sorafenib-naive and sorafenib-resistant cells of Huh7 and HepG2 exposed to 10 µM sorafenib (left panel). The histogram (right panel) shows the average percentages of annexin-positive cell population at three independent experiments ± standard deviation. (C) Expression analysis of the 13 mitotic spindle checkpoint genes in the sorafenib-naive and sorafenib-resistant cells of Huh7 and HepG2 using real-time PCR. Columns, mean (n = 3); bars, mean ± S.D. *, P<0.05, **, P<0.01.
Figure 3.
Expression profile of TTK in HCC specimens and cell lines.
(A) Real-time RT-PCR analysis of TTK was performed on 53 paired HCCs and adjacent non-tumor livers. The relative mRNA level of TTK was normalized based on that of β-actin, and presented by box-whisker Plot. The line within each box represents the median −ΔCt value; the upper and lower edges of each box represent the 75th and 25th percentile, respectively; the upper and lower bars indicate the highest and lowest values, respectively. ***, P<0.001. (B) Representative results of semi-quantitative RT-PCR of TTK from 16 pairs of HCC tumor (T) and corresponding non-tumor livers (N). (C) Western blot assay of 4 pairs of HCC specimens and adjacent non-tumor tissues with anti-TTK antibody, where β-actin was used as a loading control. (D) TTK protein expression was analyzed in Huh7, HepG2 cells and their sorafenib-resistant sublines by a western blotting assay.
Figure 4.
TTK overexpression promotes cell proliferation and resistance to sorafenib in HCC cells.
(A) Cell growth curves were described according to cell viability for five days (right panel), when recombinant pcDNA3.1-TTK and empty vector were transiently transfected into Huh7 and HepG2 cells respectively, confirmed by western blotting assay, where β-actin was used as an internal reference (left panel). (B) Representative dishes of anchor-dependent colony formation of Huh7 and HepG2 cells were shown, where these cells were transfected with recombinant pcDNA3.1-TTK and empty vector (left panel). The numbers of colonies were counted and statistically analyzed using a two-tailed t test (right panel). (C) The cytotoxic effects of sorafenib at different concentrations (0.5, 1, 2, 5, 10 µM) on cell viability was shown, where pcDNA3.1-TTK and empty vector were transfected into Huh7 (upper panel) and HepG2 (lower panel) cells, respectively. (D) As shown were the microscopic fields of anchorage-independent colony formation assay of Huh7 and HepG2 cells treated with 1 µM sorafenib, upon TTK overexpression (left panel). The numbers of colonies were counted and statistically analyzed using a two-tailed t test (right panel). *, P<0.05; **, P<0.01.
Figure 5.
Tumorigenicity assay in nude mice of HCC cell subcolonies with TTK overexpression upon sorefanib administration.
(A) Western blot analysis showed expression profile of exogenous FLAG-tagged TTK in those Huh7 cell subcolonies. (B) The relative cell viability or cytotoxicity assays of those offspring subcolonies of Huh7 cells with different TTK expression levels were performed, upon vehicle control DMSO, sorafenib or 5-Fluorouracil (5-FU) treatment, where the relative cell viability to DMSO control is shown in the histograms and statistically analyzed using a two-tailed t-test. *, P<0.05; **, P<0.01. (C) 2×106 offspring cells of C7 subcolony were subcutaneously inoculated into the flanks of nude mice, whereas the same amount of cells of C5 subcolony were inoculated into the opposite flank of the same mice (n = 5). These xenograft tumors were removed from the mice and photographed. (D) Tumor size was estimated by serial calibration, where mean tumor volume (± sd) (left panel) and tumor weights were statistically analyzed using two-tailed t-test. **, P<0.01 (right panel). (E) Representative pictures showed that immunohistochemistry staining assays of TTK and Ki-67 on xenograft tumours removed from the sorafenib-treated mice. All sections were counterstained with haematoxylin.
Figure 6.
TTK knockdown suppresses cell proliferation and enhances cytotoxic effects of sorafenib on HCC cells.
(A) TTK expression was obviously knocked down by the two siRNAs (si1-TTK, si2-TTK) in Huh7R and HepG2R (left panel), shown by western blotting assay and cell growth curves were shown according to cell viability for five days (right panel). (B) The microscopic fields shown were the anchorage-independent colony formation assay of Huh7R (upper panel) and HepG2R (lower panel) cells, where TTK was silenced by the two siRNAs (left panel). Colonies numbers were counted and statistically analyzed using a two-tailed t test. **, P<0.01 (right panel). (C) Effects of TTK knockdown on cell viability of sorafenib-naive and sorafenib-resistant cells of Huh7 (left panel), HepG2 (right panel). Cell viability was presented by the absorbance value at OD450 nm using CCK-8 kit. Columns, mean (n = 3); bars, mean ± S.D. **, P<0.01. (D) Cytotoxic effects of sorafenib at different concentrations (0.5, 1, 2, 5, 10 µM) on cell viability were shown, upon TTK knockdown in Huh7R (left panel) and HepG2R (right panel) cells, respectively.
Figure 7.
Tumorigenicity assay in nude mice of HCC subcolony cells with various TTK silence, upon sorefanib treatment.
(A) Western blot analysis displayed expression profile of endogenous TTK in those Huh7R derivative subcolonies. (B) The relative cell viability or cytotoxicity assays of those Huh7R offspring cell subcolonies with varying TTK silence were performed, when these cells were treated with vehicle control DMSO, sorafenib or 5-Fluorouracil (5-FU) respectively. The relative cell viability to DMSO is shown in the histograms and statistically analyzed using a two-tailed t-test. **, P<0.01. (C) 2×106 cells of the two Huh7R offspring subcolonies (p8 and p4) were subcutaneously inoculated into the two flanks of nude mice, respectively (n = 5). These xenograft tumors were removed from the mice and photograph. (D) Tumor size was estimated by serial calibration, where mean tumor volume (± sd) (left panel) and tumor weights were statistically analyzed using two-tailed t-test. **, P<0.01 (right panel). (E) Representative pictures showed that immunohistochemistry staining assays of TTK and Ki-67 on xenograft tumours removed from the sorafenib-treated mice. All sections were counterstained with haematoxylin.