Over-expression of miR-100 is responsible for the low-expression of ATM in the human glioma cell line: M059J

Abstract

M059J and M059K cells were isolated from different portions of the same human malignant glioma. M059J cells are more radiosensitive than M059K cells due to the absence of DNA-PKcs and low-expression of ATM. The mechanism concerning the absence of DNA-PKcs in M059J is due to the frameshift mutation in PRKDC (DNA-PKcs gene); however, the reason for the low-expression of ATM in M059J cells remains unclear. We showed here that the main reason for the lower ATM level in M059J cells was not related to the transcriptional regulation or protein degradation but was related to post-transcriptional regulation. Based on database information, we found that the 3'-untranslational region (UTR) of ATM contains a miR-100 binding site. By using an RNase protection assay and qRT-PCR, we identified that miR-100 is highly-expressed in M059J cells. We further demonstrated that miR-100 bound to the 3'-UTR of ATM. Knocking down miR-100 promotes ATM expression in M059J cells. Up-regulating miR-100 in M059K cells and other cancer cells reduces ATM expression and sensitizes these cells to ionizing radiation. These results indicate that ATM is a target of miR-100, elucidating that the low-expression of ATM in M059J cells is mainly due to the high expression of miR-100. These results also suggest that miR-100 could be a useful tool to target ATM and sensitize tumor cells to ionizing radiation.

Fig. 1

miR-100 is over-expressed in M059J cells. (A) Different expression of miR-100 between M059J and M059K cells was detected by using qRT-PCR. RNU48 was used as an internal loading control. (B) Different expression of miR-100 between M059J and M059K cells was detected by using an RNase protection assay. RNU48 was used as an internal loading control.

Fig. 2

ATM is the target of miR-100. (A) Three putative regions of ATM 3’-UTR for miR-100 binding (b1, b2, b3) and corresponding mutant miR-100 binding sites (mb1, mb2, mb3) in which the seed sequences were mutated as indicated. (B) Effects of miR-100-binding sites on luciferase activity. 293T cells were co-transfected with the firefly luciferase reporter plasmid containing wild-type (b1, b2 or b3) or mutant ATM 3'-UTR (mb1, mb2 or mb3), with (b or mb) or without miR-100 (mock). Luciferase activity was assayed 48 h after transfection. The data represent mean ± SE of three independent experiments and normalized to their respective controls (mock) as 100%. **, P < 0.01, compared with cells treated with control RNA.

Fig. 3

Over-expressed miR-100 is responsible for the low-expression in M059J cells. (A) The effects of the miR-100 inhibitor or the Dicer siRNA on ATM expression in M059J cells. Ku70 was used as an internal loading control. (B) Up-regulating miR-100 in M059K cells. The images reflect GFP signals, which represent the infection efficiencies of the lentivirus vectors. The miR-100 level was measured by qRT-PCR. (C) The effects of up-regulation of miR-100 on ATM expression in M059K cells.

Fig. 4

The effects of up-regulation of miR-100 on the cell radiosensitivity. (A) The effects of up-regulation of miR-100 on M059K cell radiosensitivity. At 72 h after infection with the lentivirus encoding pri-miR-100, the cells were exposed to 4 Gy. The clonogenic assay was performed as described in METHODS. Data shown are the mean ± SE from three independent experiments; **, P < 0.01, compared with the cells infected with the lentiviral vector without miR-100. (B) The effects of the ATM siRNA on protein expression. The cells were treated with the ATM siRNA or a control RNA (100 nM) for 48 h. The cells were collected for Western blot assay. (C) The effects of the ATM siRNA on the M059K cell radiosensitivity. The cells were treated with the RNAs as described above and then the cells were collected for clonogenic assay. The data represent mean ± SE of three independent experiments, **: P < 0.01.