Ionizing radiation results in the generation of reactive oxygen species that damage cellular macromolecules including the induction of double strand breaks in DNA which can be cytotoxic. We hypothesized that the observed reductions in let-7a and b could be expression a direct response to oxidative stress or due to DNA damage based signaling pathways. Since both oxidative stress and DNA damage stabilize p53 protein levels, p53 could be involved in the signaling of the observed let-7 decrease. p53 wild type and knock out cells were used and we found that in the p53 wt cells both let7a and let 7b expression decreased significantly even at the lowest treatment dose. However, in the p53-/- cells let-7a and b expression did not decrease, and at 2.0 Gy a significant increase in expression was observed. We also found that the genes for let7-a3 and let-7b are clustered within approximately 900 bps on chromosome 22 and both precursor miRNAs overlap with novel vega gene OTTHUMG00000030111 (referred to as let-7b gene) which has been reported to be the host transcript for these miRNAs. Although let-7a is also expressed from other locations in the genome, because of the presence of let-7b we selected this locus to probe for a p53 interaction. Scanning of the DNA sequence upstream of the let-7b gene with Genomatix MatInspector software revealed a possible p53 binding site -450 to -438 from the transcription start site (Figure 3A). ChIP was performed with p53 antibody and PCR primers bracketing this site to probe for an interaction with p53. RT-PCR of IP samples indicated an interaction with p53 only following exposure to 2.0 Gy. These data suggest that following radiation, p53 interacts with DNA upstream of the let-7b gene and maybe a mechanism for repression of let-7a and let-7b. Given these observations, we sought to determine if let-7 expression is repressed in several tissues in vivo, and if the degree of repression correlates with overall radiation sensitivity. C57BL/6J mice, wild type and p53 -/- were treated with 2.0 Gy radiation. One hour after radiation, mice were sacrificed and several tissues were collected including, radiation sensitive tissues (small intestine, lungs, and bone marrow) and radiation resistant tissues (brain, muscle, and skin) Total RNA was extracted and let-7a and let-7b was assessed. In radiation sensitive tissues, let-7 a and b expression was significantly reduced. In comparison, in radiation resistant tissues, the only significant decrease in let-7a or let-7b expression observed was a 30% decrease in let-7b in muscle tissue. Otherwise, let-7 expression remains either unchanged or increased. In bone marrow, lung and brain tissues, this increase was quite dramatic. These findings in vivo confirm our previous in citro results and support the hypotheses that p53 plays an important role in radiation-induced changes in let-7a and let-7b expression and therefore, in microRNA-induced changes in radiation sensitivity.
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