Abstract
BackgroundMicroRNAs are non-coding RNAs involved in the regulation of gene expression including DNA damage responses. Low doses of low energy X-ray radiation, similar to those used in mammographic exams, has been described to be genotoxic. In the present work we investigated the expression of miR-34a; a well described p53-regulated miRNA implicated in cell responses to X-ray irradiation at low doses.MethodsNon-cancerous breast cell line MCF-10A and cancerous T-47D and MCF-7 cell lines were submitted to a low-energy X-ray irradiation (ranging from 28–30 Kv) using a dose of 5 Gy. The expression level of miR-34a, let-7a and miR-21 was assessed by qRT-PCR at 4 and 24 hours post-irradiation. DNA damage was then measured by comet assay and micronuclei estimation in MCF-10A and MCF-7 cell lines, where an increase of miR-34a levels could be observed after irradiation. The rate of apoptotic cells was estimated by nuclear staining and fluorescence microscopy. These experiments were also performed at low doses (3; 12 and 48 mGy) in MCF-10A and MCF-7 cell lines.ResultsWe have observed an increase in miR-34a expression 4 hours post-irradiation at 5 Gy in MCF-10A and MCF-7 cell lines while its level did not change in T-47D, a breast cancer cell line bearing non-functional p53. At low doses, miR-34a was up-regulated in non-tumoral MCF-10A to a higher extent as compared to MCF-7. MiR-34a levels decreased 24 hours post-irradiation. We have also observed DNA damage and apoptosis at low-energy X-ray irradiation at low doses and the high dose in MCF-10A and MCF-7 4 and 24 hours post-irradiation relative to the mock control.ConclusionLow energy X-ray is able to promote DNA strand breaks and miR-34a might be involved in cell responses to low energy X-ray DNA damage. MiR-34a expression correlates with X-ray dose, time after irradiation and cell type. The present study reinforces the need of investigating consequences of low dose X-ray irradiation of breast cells.
Highlights
MicroRNAs are non-coding RNAs involved in the regulation of gene expression including DNA damage responses
To estimate DNA damage in different X-ray doses we first performed the comet assay with irradiated noncancerous breast cells MCF-10A and cancerous MCF-7 cells, using low-energy (28/30 kV) X-ray irradiation at low doses (3; 12 and 48 mGy) and at a high dose (5 Gy)
At 24 hours, miR-34a levels lowered, relative to the expression observed at 4 hours, showing an approximately 4 times fold-change when compared to the nontreated control, in both cell lines
Summary
MicroRNAs are non-coding RNAs involved in the regulation of gene expression including DNA damage responses. The global action of miRNAs is to repress gene expression and, by this way, they are involved in important cellular processes including DNA damage response (DDR) [6,7] Ionizing radiation such as X-rays can harm cells by direct DNA breakage or indirectly through the creation of free radicals which will contribute to increase and prolong DNA damage [8]. The ATM-mediated DDR activates the tumor suppressor protein p53, a transcription factor critical for genomic stability, regulating cell cycle progression and DNA repair, as well as apoptosis In this same sense, it is expected that miRNAs are involved in mechanisms such those regulated by p53 [9,10]. Our results show an overexpression of miR34a in the non-cancerous MCF-10A cells in response to DNA damage caused by low-doses of X-ray radiation
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