Ionizing radiation-induced oxidative stress alters miRNA expression

Abstract

Background: MicroRNAs (miRNAs) are small, highly conserved, non-coding RNA that alter protein expression and regulate multiple intracellular processes, including those involved in the response to cellular stress. Alterations in miRNA expression may occur following exposure to several stress-inducing anticancer agents including ionizing radiation, etoposide, and hydrogen peroxide (H(2)O(2)).

Methodology/principal findings: Normal human fibroblasts were exposed to radiation, H(2)O(2), or etoposide at doses determined by clonogenic cell survival curves. Total RNA was extracted and miRNA expression was determined by microarray. Time course and radiation dose responses were determined using RT-PCR for individual miRNA species. Changes in miRNA expression were observed for 17 miRNA species following exposure to radiation, 23 after H(2)O(2) treatment, and 45 after etoposide treatment. Substantial overlap between the miRNA expression changes between agents was observed suggesting a signature miRNA response to cell stress. Changes in the expression of selected miRNA species varied in response to radiation dose and time. Finally, production of reactive oxygen species (ROS) increased with increasing doses of radiation and pre-treatment with the thiol antioxidant cysteine decreased both ROS production and the miRNA response to radiation.

Conclusions: These results demonstrate a common miRNA expression signature in response to exogenous genotoxic agents including radiation, H(2)O(2), and etoposide. Additionally, pre-treatment with cysteine prevented radiation-induced alterations in miRNA expression which suggests that miRNAs are responsive to oxidative stress. Taken together, these results imply that miRNAs play a role in cellular defense against exogenous stress and are involved in the generalized cellular response to genotoxic oxidative stress.

Figure 1. miRNA Micorarray changes following exposure to radiation, H₂O₂, and etoposide.

1522 cells were collected one hour after exposure to 10Gy of radiation, etoposide, or H₂O₂. The microarray (A) was performed using RNA isolated from treated cells and compared to RNA from untreated controls. (B) Scatter plot of miRNA expression in control versus radiation, etoposide, or H₂O₂ samples.

Figure 2. Venn Diagram showing miRNA species altered by stressors.

1522 cells were collected one hour after exposure to 10Gy of radiation, etoposide, or H₂O₂. miRNAs with expression that is significantly different from control as determined by microarray are shown illustrating the overlapping signature of exogenous genotoxic stresses.

Figure 3. Response of miRNAs to genotoxic stress.

1522 cells were collected one hour after exposure to 10Gy of radiation, etoposide, or H₂O₂ and evaluated by RT-PCR for expression of (A) let-7a and (B) let-7b. A decrease in let-7a expression is noted after treatment with all three agents. A decrease in let-7b expression is noted for radiation and etoposide while an increase in expression is noted after H₂O₂ treatment. All results represent the average of three separate experiments all done in triplicate. Error bars represent one standard deviation about the arithmetic mean and * denotes p<0.05.

Figure 4. Response of miRNAs to radiation dose and time after radiation.

1522 cells were irradiated to doses ranging from 0.25Gy to 10Gy. Samples were collected one hour after irradiation and evaluated by RT-PCR for expression of (A) let-7a and (B) let-7b. A dose-dependent, linear decrease in expression from 0.25Gy to 1Gy was noted with no further response seen at higher doses. In addition, RT-PCR was performed on samples collected at several time points ranging from 30 min to 48 hrs after irradiation (10Gy). A decrease in (C) let-7a and (D) let-7b expression was noted at 30 min with a return to baseline expression by 24 hrs after irradiation. All results represent the average of three separate experiments all done in triplicate. Error bars represent one standard deviation about the arithmetic mean and * denotes p<0.05.

Figure 5. The Effect of the Free Radical Scavenger Cysteine on miRNA expression.

(A) 1522 cells were treated with cysteine (0.2, 0.5, 1 or 2 µM) for one hour, then DCF (dichhlorofluroisceine) for 15 min, then irradiated to 5, 10 or 20Gy. ROS expression, measured for 15 min after irradiation, increased with increasing radiation dose and decreased with increasing doses of cysteine. ROS expression is reported as area under the curve (AUC) for plot of luminescence intensity over time. Subsequently, 1522 cells were collected one hour after treatment with cysteine (1 µM) alone, radiation (10Gy) alone, or cysteine added 1 hour prior to irradiation. Cysteine alone did not alter miRNA expression, however pre-treatment with cysteine prevented a radiation-induced decrease in (B) let-7b and (C) let-7a expression. All results represent the average of three separate experiments all done in triplicate. Error bars represent one standard deviation about the arithmetic mean and * denotes p<0.05.