miR-18a impairs DNA damage response through downregulation of ataxia telangiectasia mutated (ATM) kinase

Abstract

The DNA damage response (DDR) encompasses multi-step processes by which cells evolve to sense DNA damage, transduce the signal and initiate the repair of damaged DNA. Ataxia Telangiectasia Mutated (ATM) Kinase, which functions as the primary sensor and transducer of DNA damage signal, has been demonstrated to play an important role in the DDR and cancer prevention. Hence, understanding the molecular mechanisms underlying the regulation of ATM has received much attention. Here, we found that miR-18a was upregulated in both cell lines and patients' tissue samples of breast cancer. Furthermore, we demonstrated that ectopically expressing miR-18a downregulated ATM expression by directly targeting the ATM-3'-UTR and abrogated the IR-induced cell cycle arrest. Similar to the effect of ATM siRNA, overexpressing miR-18a in breast cancer cells reduced the DNA damage repair ability and the efficiency of homologous recombination-based DNA repair (HRR) and sensitized cells to γ-irradiation (IR) treatment. However, inhibition of miR-18a led to augmentation of DNA damage repair, increase of HRR efficiency and reduced cellular radiosensitivity. Moreover, we showed that the phorsphorylation level and nuclear foci formation of H2AX and 53BP1, the downstream substrates of ATM kinase, were significantly deceased in miR-18a overexpressing cells. Taken together, our results uncover a new regulatory mechanism of ATM expression and suggest that miR-18a might be a novel therapeutic target.

Figure 1. Upregulation of miR-18a abrogated IR-induced cell cycle arrest.

A, Real-time PCR analysis of miR-18a expression in normal breast epithelial cells (NBEC) and breast cancer cell lines, including ZR-75-1, ZR-75-30, SKBR3, T47D, MDA-MB-231, MDA-MB-435, MDA-MB-453, BT474 and BT-549. B, The expression of miR-18a was examined in 10 paired breast tumor tissues (tumor) and their adjacent normal tissues (normal). The average miR-18a expression was normalized by U6 expression. Each bar represents the mean of three independent experiments. C, Flow cytometric analysis of indicated breast cancer cells transduced with miR-18a mimic or NC, treated with or without 2.0 Gy IR. Statistic analysis revealed the proportion change of cells at each phase of cell cycle from three independent experiments under different miR-18a expression levels with or without IR treatment, using the algorithm (S^+IR−S^-IR)/S^-IR×100%. D, Representative micrographs (left) and quantification analysis (right) of BrdUrd incorporating-cells in indicated cells with or without 2.0 Gy IR.

Figure 2. miR-18a downregulated ATM expression via directly targeting the ATM 3′-UTR.

A, Predicted miR-18a target sequence in 3′UTR of ATM (ATM-3′-UTR) and a mutant that contained two mutated nucleotides in the ATM 3′-UTR (ATM-3′-UTR -MUT). B, Western blotting analysis of ATM expression in indicated cells transfected with miR-18a mimic- or miR-18a inhibitor-oligonucleotides. C, Western blot analysis that showed GFP expression in indicated cells. α-Tubulin was used as the loading control. D, Luciferase assay on indicated cells that were transduced with the pGL3-ATM-3′UTR reporter with increasing amounts (10, 50 nM) of miR-18a mimic- or miR-18a inhibitor-oligonucleotides. E, Luciferase assay on indicated cells transduced with pGL3-ATM-3′UTR or pGL3-ATM-3′UTR-MUT reporter with increasing amounts (10, 50 nM) of miR-18a mimic-oligonucleotides. F, The expression (left) and correlation (right) of miR-18a and ATM in nine freshly prepared human breast cancer tissues. Error bars represent the mean ± SD from three independent experiments. * P<0.05.

Figure 3. miR-18a impaired the ATM signaling pathway.

A, Western blotting analysis of ATM expression, phosphorylated CHK2 (p-CHK2) and total CHK2 protein in indicated cells treated with or without IR. α-Tubulin was used as the loading control. B, Western blotting analysis of the expression levels of γ-H2AX, phosphorylated 53BP1 (p-53BP1) and total 53BP1 protein in indicated cells treated with IR (2.0 Gy). α-Tubulin was used as the loading control. C and D, The representative pictures (left panel) and quantification (right panel) of IR (2.0 Gy)-induced γ-H2AX (C) and 53BP1 (D) foci were analyzed in indicated breast cancer cells transfected with NC, miR-18a or scramble ATM-siRNA. The foci numbers of at least 300 cells were counted. Error bars represent the mean ± SD from three independent experiments. * P<0.05.

Figure 4. miR-18a reduces cell HRR frequency and enhances cell radiation sensitivity.

A, DSB-induced HRR assay indicated that the overexpression of miR-18a decreased the spontaneous HR frequency in HT1080-1885 cells. Western blotting analysis of the expression levels of ATM and HA-tagged I-SceI endonuclease. α-Tubulin was used as the loading control. B, Overexpressing miR-18a or silencing ATM expression enhanced the sensitivity of breast cancer cells to IR treatment. But further overexpressing miR-18a mimic had no additional sensitivity to IR in the ATM-silenced cells. The viabilities of the indicated cells were assayed after indicated doses of γ-radiation by the clonogenic cell survival assay. Error bars represent the mean ± SD from three independent experiments. * P<0.05.

Figure 5. Inhibition of miR-18a increased cell HRR frequency and reduced radiosensitivity.

A, Western blotting analysis of the expression of ATM, γ-H2AX, phosphorylated 53BP1 (p-53BP1) and total 53BP1 protein in indicated cells treated with IR (2.0 Gy). α-Tubulin was used as the loading control. B, DSB-induced HRR assay indicated that inhibition of miR-18a increased the spontaneous HR frequency in HT1080–1885 cells. Western blotting analysis of expression levels of ATM and HA-tagged I-SceI endonuclease. α-Tubulin was used as the loading control. C, Inhibition of miR-18a reduced the sensitivity of breast cancer cells to IR treatment. The viabilities of the indicated cells were assayed after indicated doses of γ-radiation by the clonogenic cell survival assay. D and E, The representative pictures (left panel) and quantification (right panel) of IR (2.0 Gy)-induced γ-H2AX (D) and 53BP1 (E) foci were analyzed in indicated breast cancer cells transfected with either the NC or miR-18a inhibitor. The foci numbers of at least 300 cells were counted.