Effect of GEN1 interference on the chemosensitivity of the breast cancer MCF-7 and SKBR3 cell lines

Abstract

Chemotherapy is a notable method for the treatment of breast cancer. Numerous genes associated with the sensitivity of cancer to chemotherapy have been found. In recent years, evidence has suggested that a particular structure termed Holliday junction (HJ) plays a crucial role in cancer chemosensitivity. Targeting HJ resolvases, such as structure-specific endonuclease subunit SLX4 (Slx4) and MUS81 structure-specific endonuclease subunit (Mus81), significantly increases the chemosensitivity of tumor cells. Flap endonuclease GEN homolog 1 (GEN1) is a HJ resolvase that belongs to the Rad2/xeroderma pigmentosum complementation group G nuclease family. Whether GEN1 affects the chemosensitivity of tumor cells in a similar manner to Slx4 and Mus81 remains unknown. The aim of the present study was to determine the effect of GEN1 interference on the chemosensitivity of breast cancer cell lines. The investigation of the function of GEN1 was performed using MCF-7 and SKBR3 cells. Short hairpin RNA was used to suppress the expression of GEN1, and western blot analysis and reverse transcription-quantitative polymerase chain reaction were used to detect gene expression. In addition, a cell counting kit-8 assay was performed to detect the viability of cells and flow cytometry was performed to test apoptosis levels. Suppression of GEN1 in SKBR3 cells effectively increased the sensitivity to the chemotherapeutic drug 5-fluorouracil (5-FU), while MCF-7 cells showed no significant change in sensitivity following GEN1 suppression. However, when GEN1 was targeted in addition to Mus81, the MCF-7 cells also demonstrated a significantly increased sensitivity to 5-FU. In addition, when the level of Mus81 was low, GEN1 expression was increased under a low concentration of 5-FU. The present results suggest that GEN1 may play different roles in different breast cancer cell lines. The function of GEN1 may be affected by the level of Mus81 in the cell line. In addition, GEN1 interference may improve the sensitivity to chemotherapy induced by targeting Mus81 alone.

Keywords: 5-fluorouracil; GEN1; Mus81; breast cancer cells; chemosensitivity.

Figure 1.

Survival of SKBR3 and MCF-7 cells under various concentrations of 5-FU. Cell survival was determined using an CCK-8 assay. In total, 1×10⁴ SKBR3 or MCF-7 cells were planted into 96-well culture plates and various concentrations of 5-FU were added 24 h later. Subsequent to the exposure of cells to 5-FU for 48 h, 10 µl CCK-8 medium was added to each well and the optical density values were determined using a microplate reader 1 h later. 5-FU, 5-fluorouracil; CCK-8, cell counting kit-8.

Figure 2.

SKBR3 cells demonstrated increased sensitivity to 5-FU subsequent to suppression of the GEN1 gene. (A) The silencing of GEN1 in SKBR3 cells was confirmed by reverse transcription-quantitative polymerase chain reaction and western blot analysis. (B) Cells in the sh-Ctrl and sh-GEN1 groups were seeded onto 96-well culture plates at a density of 10,000 cells per well subesequent to 24 h of GEN1 suppression. The cell survival was determined using cell counting kit-8 subsequent to treatment of cells with 5-FU for 48 h. The results are expressed as the mean ± standard deviation of triplicate samples from three experiments. The sh-GEN1 group demonstrated significantly enhanced sensitivity to 5-FU conpared with the sh-Ctrl group (P<0.05). (C) The cells were treated with the half maximal inhibitory concentration of 5-FU (8.77 µg/ml) and the survival of the cells in the sh-GEN1 group was significantly decreased compared with the cells in the sh-Ctrl group (P<0.05). *P<0.05, sh-Ctrl vs. sh-GEN1 group. 5-FU, 5-fluorouracil; GEN1, flap endonuclease GEN homolog 1; sh-Ctrl; control short hairpin RNA; sh-GEN1, GEN1 short hairpin RNA.

Figure 3.

Silencing GEN1 in the MCF-7 cell line had no significant effect on the sensitivity to 5-FU. (A) The silencing of GEN1 in MCF-7 cells was confirmed by reverse transcription-quantitative polymerase chain reaction and western blot analysis. (B) Cells in the sh-Ctrl and sh-GEN1 groups were seeded onto 96-well culture plates at a density of 10,000 cells per well subsequent to 24 h of GEN1 suppression. The cell survival were determined using cell counting kit-8 subsequent to treatment of the cells with 5-FU for 48 h. The results are expressed as the mean ± standard deviation of triplicate samples from three experiments. Following GEN1 suppression, the MCF-7 cell line did exhibit a significant change in its sensitivity to 5-FU. (C) The cells were treated with the half maximal inhibitory concentration of 5-FU (7.73 µg/ml) and the survival rates of the two groups demonstrated no evident change. *P<0.05, sh-Ctrl vs. sh-GEN1 group. 5-FU, 5-fluorouracil; GEN1, flap endonuclease GEN homolog 1; sh-Ctrl; control short hairpin RNA; sh-GEN1, GEN1 short hairpin RNA.

Figure 4.

The effect of silencing GEN1 on apoptosis in SKBR3 and MCF-7 cells. (A) The apoptosis of SKBR3 cells was observed under a fluorescence microscope (magnification, ×200). (B) The apoptosis of SKBR3 cells was detected by flow cytometry. (C) The apoptosis of MCF-7 was observed under a fluorescence microscope (magnification, ×200). (D) The apoptosis of MCF-7 cells was detected by flow cytometry. GEN1, flap endonuclease GEN homolog 1; sh-Ctrl; control short hairpin RNA; sh-GEN1, GEN1 short hairpin RNA; PI, propidium iodide.

Figure 5.

The MCF-7 cell line demonstrated increased sensitivity to 5-FU subsequent to the simultaneous suppression of the GEN1 and Mus81 genes. Reverse transcription-quantitative polymerase chain reaction and western blot analysis confirmed the silencing of the GEN1 and Mus81 genes by assessing the (A) mRNA and (B) protein expression. (C) The sh-Ctrl-1, sh-Ctrl-2, sh-GEN1, sh-Mus81 and sh-GEN1+sh-Mus81 groups were transfected with 4 µg control plasmid, 8 µg control plasmid, 4 µg sh-GEN1 plasmid, 4 µg sh-Mus81 plasmid and 4 µg sh-GEN1 plasmid + 4 µg sh-Mus81 plasmid, respectively. No significant difference in cell survival was identified between the sh-Ctrl-1 and sh-Ctrl-2 groups and the sh-GEN1 group. In contrast to the suppression of GEN1 alone, MCF-7 cells demonstrated significantly enhanced sensitivity to 5-FU when the GEN1 and Mus81 genes were simultaneously suppressed (P<0.05). *P<0.05. GEN1, flap endonuclease GEN homolog 1; Mus81, MUS81 structure-specific endonuclease subunit; sh-Ctrl; control short hairpin RNA; sh-GEN1, GEN1 short hairpin RNA; sh-Mus81, Mus81 short hairpin RNA; 5-FU, 5-fluorouracil.

Figure 6.

Under a low concentration of 5-FU, the GEN1 expression level increased when the Mus81 level was low. (A) The level of GEN1 in the MCF-7 cell line increased when Mus81 was suppressed and a low concentration of 5-FU was administered. (B) The level of GEN1 in the MCF-7 cell line did not change significantly when cells were treated with a low concentration of 5-FU. The GEN1 level in the SKBR3 cell line, however, increased significantly under the same conditions. (C) The expression levels of Mus81 in MCF-7 and SKBR3 cells without treatment. *P<0.05. GEN1, flap endonuclease GEN homolog 1; Mus81, MUS81 structure-specific endonuclease subunit; 5-FU, 5-fluorouracil.