Downregulation of inhibitor of growth 3 is correlated with tumorigenesis and progression of hepatocellular carcinoma

Abstract

ING3, a member of the inhibitor of growth (ING) family, has been reported to be involved in transcription modulation, cell cycle control and the induction of apoptosis. Previous studies have demonstrated that the expression of ING3 decreased in melanoma and head and neck squamous cell carcinoma (HNSCC). The aim of this study was to investigate the role of ING3 in hepatocellular carcinoma (HCC) tumorigenesis and progression. The correlation between ING3 expression and clinicopathological variables of HCC was analyzed. Using the real-time reverse transcription-polymerase chain reaction (RT-PCR), it was found that ING3 was downregulated in HCC tissues compared with adjacent non-cancerous tissues (p<0.05). The immunohistochemical staining of tissue microarray data indicated a significant reduction of ING3 expression in 57.14% of HCC cases (64/112). In addition, the downregulation of ING3 was associated with the tumor differentiation stage. Most HCC samples of Edmondson-Steiner grades II to III exhibited inhibition of ING3 expression. The overexpression of ING3 in HCC cells was found to suppress cell proliferation, colony formation and cell migration, suggesting that ING3 acts as a tumor suppressor in HCC cells. Taken together, the data revealed that ING3 may serve as a suppression factor during tumorigenesis and progression of HCC.

Figure 1

Expression pattern of ING3 in HCC samples and cell lines. (A) The transcript level of ING3 was measured in 49 paired HCC and adjacent non-cancerous livers by quantitative RT-PCR, where GAPDH was used as an internal reference. The line within each box is the median -ΔCt value; the upper and lower edges of each box show the 75th and 25th percentile, respectively. The upper and lower bars are the highest and lowest values. P-value was calculated by paired sample t-test (p<0.001). (B) Representative semi-quantitative RT-PCR of ING3 of 3 pairs of HCC samples. C, cancer tissue; N, adjacent noncancerous liver. (C) Relative mRNA level of ING3 was evaluated in HCC cell lines and fetal liver by semiquantitative RT-PCR. HCC, hepatocellular carcinoma; ING3, inhibitor of growth 3; RT-PCR, reverse transcription-polymerase chain reaction.

Figure 2

Representative immunohistochemical detection of the ING3 protein expression in HCC and surrounding noncancerous tissues. HCC, hepatocellular carcinoma; ING3, inhibitor of growth 3.

Figure 3

ING3 overexpression inhibits cell proliferation and colony formation. (A) Exogenous ING3 was expressed in Hep3B and Huh7 cells by transient transfection with pcDNA3.0-ING3, confirmed by western blot analysis. Cells transfected with an empty vector were used as the control. (B) The growth curve of Hep3B cells with the exogenous ING3. Cells transfected with the empty vector pcDNA3.0 served as controls. The experiments were repeated three times and the spots represent the average values of triplicate wells, with SD included for each mean value. (C) ING3 suppressed the ability of Hep3B cells to form colonies. The relative numbers of colonies of Hep3B cells transfected with pcDNA3.0-ING3 and a control vector were calculated from three independent experiments (^***p<0.01). The lower panel showed the representative dishes of the experiments. (D) ING3 inhibited the colony formation of Huh7 cells (^*p<0.05 as compared with the control vectors). ING3, inhibitor of growth 3.

Figure 4

ING3 inhibits the migration of hepatoma cells. (A and B) ING3 overexpression reduced the migration of (A) Hep3B and (B) Huh7 cells. The two cell types were scraped with micropipette tips following transfection with pcDNA3.0-ING3 or pcDNA3.0. Images were captured at 0, 24 and 48 h following wounding. Cells transfected with an empty vector were used as controls. (C and D) The wound healing rates at 24 and 48 h following wounding of (C) Hep3B and (D) Huh7 cells. Values are the mean ± SD. ^*p<0.05. ING3, inhibitor of growth 3.