Abstract
The DNA damage response (DDR) triggers widespread changes in gene expression, mediated partly by alterations in micro(mi) RNA levels, whose nature and significance remain uncertain. Here, we report that miR-34a, which is upregulated during the DDR, modulates the expression of protein phosphatase 1γ (PP1γ) to regulate cellular tolerance to DNA damage. Multiple bio-informatic algorithms predict that miR-34a targets the PP1CCC gene encoding PP1γ protein. Ionising radiation (IR) decreases cellular expression of PP1γ in a dose-dependent manner. An miR-34a-mimic reduces cellular PP1γ protein. Conversely, an miR-34a inhibitor antagonizes IR-induced decreases in PP1γ protein expression. A wild-type (but not mutant) miR-34a seed match sequence from the 3' untranslated region (UTR) of PP1CCC when transplanted to a luciferase reporter gene makes it responsive to an miR-34a-mimic. Thus, miR-34a upregulation during the DDR targets the 3' UTR of PP1CCC to decrease PP1γ protein expression. PP1γ is known to antagonize DDR signaling via the ataxia-telangiectasia-mutated (ATM) kinase. Interestingly, we find that cells exposed to DNA damage become more sensitive - in an miR-34a-dependent manner - to a second challenge with damage. Increased sensitivity to the second challenge is marked by enhanced phosphorylation of ATM and p53, increased γH2AX formation, and increased cell death. Increased sensitivity can be partly recapitulated by a miR-34a-mimic, or antagonized by an miR-34a-inhibitor. Thus, our findings suggest a model in which damage-induced miR-34a induction reduces PP1γ expression and enhances ATM signaling to decrease tolerance to repeated genotoxic challenges. This mechanism has implications for tumor suppression and the response of cancers to therapeutic radiation.