Abstract #3976: The response of the microRNA let-7b to ionizing radiation may involve p53

Introduction: The cellular response to ionizing radiation (IR) involves large-scale changes in expression patterns and cellular function. Our research has shown that microRNAs are among genes that change with IR exposure, however the mechanistic role of specific miRNAs in the cellular stress response is yet to be elucidated. IR causes an increase in intracellular free radicals which is a main cause of IR-induced cell death. A better understanding of the role of microRNAs in the stress response of cells could offer insight into these pathways. We have previously shown that expression of the miRNA let-7b changes in response to IR. In addition, p53 is also induced by IR-induced DNA damage and we identified two p53 binding sites in the promoter region of the gene encoding let-7b. In this study we aim to determine the mechanistic role of the miRNA species let-7b in the IR response, specifically whether p53 expression is associated with changes in let-7b expression following IR exposure. Methods: Genomatix software was used to determine potential binding sites for the let-7b transcription factor. Wild type and p53 knock out HCT116 cells, a human colon cancer cell line, were grown in culture. Cells were collected in log-phase growth to generate whole cell lysates. Western blots were run using anti-p53 antibody. RNA was collected from cells in log-growth phase following treatment with radiation (2Gy or 10Gy), the DNA-damaging agent etoposide, or H2O2 to induce free radical stress, using the trizol extraction method. RT-PCR was then performed to evaluate let-7b expression. Results: Using the Genomatix software, two p53 binding sites were identified 700-1000 base pairs upstream of let-7b in the target region for transcription factors. Western blots confirmed that p53 protein was present in HCT116 wild type cells and absent in p53 knock out cells. In wild type cells, let-7b expression decreased by 94% following treatment with 2Gy radiation, 60% after 10Gy, 77% after treatment with etoposide, and 63% after treatment with H2O2, which is similar to our prior findings in other cell types. Conversely, in p53 knock out cells let-7b increased by 41% after 2Gy radiation, 10% after 10Gy, 20% after treatment with etoposide, and 33% after treatment with H2O2. Studies employing chromatin immunoprecipitation to confirm that p53 is regulating the let-7b response are on going. Conclusions: Expression of let-7b decreases after IR exposure in response to both free radical stress and DNA damage. This response occurs in cells that express p53 but not in p53 knock out cells suggesting that p53 may regulate let-7b expression. These results suggest that let-7b may be important in the regulation and fine tuning of the p53 cell stress response. We further hypothesize that increased levels of let-7b could alter cellular stress response and repair processes after IR exposure and that therapeutics aimed at inducing overexpression of let-7b may be developed for use as radiation sensitizers. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3976.