Role of miR-100 in the radioresistance of colorectal cancer cells

Abstract

The prognosis of radioresistant colorectal cancer (CRC) is generally poor. Abnormal expression of microRNAs (miRNAs) is involved in the radiosensitivity of various tumor cells as these RNAs regulate biological signaling pathways. However, radioresistance-associated miRNAs in CRC have not yet been identified. In this study, we filtered out HCT116 and CCL-244 from seven CRC cell lines that showed the highest difference in radiosensitivity in a clonogenic assay. MiRNA sequencing identified 33 differentially expressed miRNAs (13 up-regulated and 20 down-regulated) in CCL-244 and 37 in HCT116 (20 up-regulated and 17 down-regulated) cells. MiR-100 was significantly down-regulated in CCL-244 cells after X-ray irradiation but not in HCT116 cells. Quantitative real-time PCR showed that the expression of miR-100 in CRC tissues was significantly lower than that in normal tissues. Thus, miR-100 seems to be involved in the radioresistance of CCL-244 cells. MiR-100 up-regulation sensitized CCL-244 cells to X-ray irradiation, which probably led to apoptosis and DNA double-strand breaks in these. In conclusion, to our knowledge, this is the first study to show that miR-100 may play an important role in regulating the radiosensitivity of CRC, and it may act as a new clinical target for CRC radiotherapy.

Keywords: Colorectal cancer; miR-100; miRNA profiling; radioresistance; radiosensitivity.

Figure 1

Comparison of the radiosensitivities of the seven CRC cell lines to X-ray irradiation. A. Cells were exposed to X-rays at different irradiation doses: 0, 2, 4, 6, and 8 Gy. After 14 days, the surviving fraction was calculated as a ratio of the number of colonies formed divided by the total number of cells plated times the plating efficiency. B. D₀ values for each type of colorectal cancer cell line.

Figure 2

miRNA sequencing results of colon cancer cells before and after X-ray irradiation. A. miRNA expression profiles of CCL-244 and HCT116 cells before and after X-ray irradiation determined by miRNA sequencing; the heatmap was generated with miRNAs whose expressions were down- or up-regulated more than 2-fold compared to controls. B. Ten miRNAs were differentially expressed in CCL-244 or HCT116 cells after exposure to 8-Gy X-ray irradiation compared with nonirradiated control cells. The irradiated/nonirradiated expression ratio was expressed as Log2. The bars indicate the means for this experiment. C. Expression of miR-100 in CCL-244 cells before and after exposure to 8-Gy X-ray irradiation (**P < 0.01); expression of miR-100 in HCT116 cells before and after exposure to 8-Gy X-ray irradiation (P > 0.05). D. Quantitative real-time PCR to confirm the expression of miR-100 after transfection and to examine consistency with sequencing results (*P < 0.05).

Figure 3

Different expression of miR-100 in CRC tissues compared with matched noncancerous colorectal tissues. Quantitative real-time PCR was performed to compare the expression of miR-100 between 30 CRC tissues and matched noncancerous colorectal tissues. U6 RNA was used as an internal control. The mean ΔCt of miR-100 in the 30 CRC tissues was compared with that in the matched noncancerous colorectal tissues (**P < 0.01). The mean and standard deviation of expression levels relative to U6 expression levels are shown and normalized to the expression in the normal tissue of each matched pair.

Figure 4

miR-100 regulated the radiosensitivity of CRC cells CCL-244. A. Quantitative real-time PCR was used to confirm the transfection efficiency. U6 RNA was used as an internal control. The mean ΔCt of miR-100 in CCL-244 cells treated with miR-100 mimics and negative control miR-100 mimics (*P < 0.05). B. CCL-244 cells treated with miR-100 mimics and negative control miR-100 mimics. After 24 h, the cells were subjected to 8-Gy irradiation. After 48 h, CCK-8 was used to detect cell viabilities (miR-100 mimics compared with miR-100 negative control, *P < 0.05 or untransfected, **P < 0.01). These results are representative of at least three separate experiments. C. CCL-244 cells treated with miR-100 mimics and negative control miR-100 mimics. After 24 h, the cells were subjected to 0, 2, 4, 6 and 8 Gy of irradiation. After 14 days, the colony formation rate in different treatment groups was examined.

Figure 5

miR-100 promoted X-ray-induced apoptosis of CCL-244 cells. A. Flow cytometry assay to determine the apoptosis of CCL-244 cells transfected with miR-100 mimics or negative control miR-100 mimics and non-transfected cells. Percentage of apoptotic cells in non-transfected and transfected cells subjected to 0 and 8-Gy irradiation. Data are means ± SEM of three independent experiments. miR-100 significantly increased the X-ray-induced apoptosis of CCL-244 cells compared with non-transfected (**P < 0.01) and negative control miR-100 mimics (*P < 0.05) as well as the apoptosis of nonirradiated CCL-244 cells compared with non-transfected (*P < 0.05) and negative control miR-100 mimics (*P < 0.05). B. Western blot analysis of apoptotic marker proteins p53, Caspase-3, Bcl-2 or NF-κB in CCL-244 cells with miR-100 in transfected and non-transfected cells before and after exposure to 8-Gy X-ray irradiation. β-Actin was used as an internal control.

Figure 6

miR-100 increased γ-H2AX foci caused by X-ray irradiation and retarded DNA double strand breaks repair. A. Representative images of γ-H2AX foci for miR-100 mimics, miR-100 negative control, and non-transfected treated groups exposed to 4-Gy X-ray irradiation at different time points. B. Representative images of γ-H2AX foci in different groups of cells after 4-Gy X-ray irradiation. CCL-244 cells were stained for γ-H2AX at the indicated times and the mean number of γ-H2AX foci per cell (foci/cell) were then counted (**P < 0.01).