Effect of the tumor suppressor gene ING4 on the proliferation of MCF-7 human breast cancer cells

Abstract

Inhibitor of growth 4 (ING4) is a member of the ING family and acts as a tumor suppressor protein. To investigate the impact of ING4 on breast cancer proliferation, the present study examined the antitumor effects caused by upregulation in the expression of ING4 and its possible mechanism of effect in MCF-7 cells. A plasmid-based expression system encoding the ING4 gene was used to construct a stable cell line and overexpress ING4 in MCF-7 cells. Real-time PCR and western blot analysis were used to detect the mRNA and protein expression levels of ING4, respectively. Cell growth was examined by methylthiazolyltetrazolium (MTT) assay. Cell cycle distribution and cell apoptosis were measured by flow cytometry. The expression of p21, p53 and bax genes were tested by real-time PCR and western blot analysis. The stably transfected cell lines pcDNA3.1(+)/ING4 (with the ING4 gene) and pcDNA3.1(+) (an empty vector) were established. The expression levels of ING4 mRNA and protein in the stable cell line expressing pcDNA3.1(+)/ING4 were significantly higher than those of the two control cell lines. The cell proliferation of stably transfected cells was inhibited, and the inhibitory rate was 62.58±2.93%. Based on the changes in cell cycle distribution in stably transfected cells compared with two control cell lines, a number of cells were blocked in the G0/G1 phase 67.82±3.78% (P<0.05). In addition, the apoptotic rate was significantly higher, at 31.51±3.02% (P<0.05). Real-time PCR revealed that p21 and bax mRNA expression were increased (P<0.01), but the expression of p53 did not change significantly (P>0.05) in the stably transfected cell lines. Western blot analysis results of p21, bax and p53 were in accordance with real-time PCR results. ING4 upregulation may inhibit breast cancer cell proliferation and accelerate the process of apoptosis. It is suggested that ING4 plays a significant role in the suppression of breast cancer progression.

Figure 1

ING4 expression in MCF-7 cells. (A) mRNA expression of ING4 detected by real-time PCR. Total RNA from the three types of cells were reverse transcribed and amplified by the primers from ING4 and β-actin. ING4 mRNA expression in the stable MCF-7 cells was increased significantly in comparison with the negative group and the blank group (P<0.01). Data values and error bars show the means and SD, respectively. (B) Protein expression of ING4 was detected by western blot analysis. Total cell lysates were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with an antibody against ING4. ING4 expression levels were normalized against β-actin expression. ING4, inhibitor of growth 4.

Figure 2

Proliferation curves were determined by MTT assay. Cell growth curves and the results of inhibitory rates of cell growth were applied to absorbance at 570 nm on a microreader (Bio-Rad, USA). The stably transfected cell proliferation was inhibited significantly in a time-dependent manner, and the highest inhibitory rate was 62.58±2.93% (P<0.05) on day 4. All data represent the mean of three independent experiments. ING4, inhibitor of growth 4.

Figure 3

The results of cell cycle distribution analysis by flow cytometry. The graphs show cell cycle profiles in the stably transfected cells and control cells. The percentages of cell populations of different cell cycle phases are shown in the upper-right corner. Many stably transfected cells were blocked in the G0/G1 phase (67.82±3.78%; P<0.05), and there was a reduction in the S phase (24.88±3.09%; P<0.05). ING4, inhibitor of growth 4.

Figure 4

The results of flow cytometry in the stably transfected group and controls. Cells were double-stained with FITC and PI, then screened by flow cytometry. The presented data shows that the apoptotic rate of the pcDNA3.1(+)/ING4 group was higher than that of the control group (P<0.05), whereas no apparent differences were observed between the two control groups. The means and SD are shown. ING4, inhibitor of growth 4.

Figure 5

ING4-induced expression changes in some of the genes involved in the cell cycle and apoptosis. (A) Real-time PCR analysis showed that compared with control group cells, the transcriptional expressions of p21 and bax were markedly upregulated in the stably transfected ING4 cells (P<0.05), while the transcriptional expression of p53 was not significantly different to control groups. All data represent the mean of three independent experiments. (B) The expression of p21, bax and p53 proteins in MCF-7 cells. The protein lysates were analyzed by western blot analysis with anti-p21, anti-bax and anti-p53 antibodies. The mRNA and protein expression levels were similar in all groups of cells. P21, bax and p53 levels were normalized against β-actin expression.