Abstract #3967: Altered microRNA expression after irradiation may regulate the radiation induced toxicity of fibrosis
Authors: David Ly, Jason Savage, Anastasia Sowers, Benjamin Soule, Anthony Saleh, James Mitchell, Nicole Simone
Journal: Cancer Research (2009)

Introduction: Radiotherapy can be complicated by debilitating normal tissue toxicity such as radiation-induced fibrosis. We have previously shown that mir-21 and let-7b expression is altered by radiation  in vitro . We hypothesize that these miRNA species may target proteins involved in radiation-induced fibrosis and therefore may serve as clinical targets to prevent morbidity. TGF\#946; is associated with induction of fibrosis, but has been reported to be down-regulated in long-term fibrosis. The aim of this study is to demonstrate that radiation alters the expression of miRNA species  in vivo  that target fibrosis-associated proteins such as TGF\#946;. Methods: The right hind leg of 9 week old CH3 mice was irradiated to 35Gy in a single fraction with the left leg serving as an unirradiated control. Mice were euthanized at 2hrs, 7 days, 14 days, 30 days or 150 days after irradiation. Fibrosis in the irradiated leg was determined by the amount of leg shortening compared to the unirradiated leg. Leg tissue was homogenized and total RNA was extracted using a phenol-chloroform method. Expression of mir-21 and let-7b was determined using RT-PCR. Targetscan was used to determine potential downstream targets of both species that could contribute to long term fibrosis. Protein lysates were prepared from leg tissue and analyzed using western blots to evaluate expression of TGF\#946;-1. Results: Irradiated mouse legs were inspected at each time point. Let-7b expression decreased by 69% at two hours and 11% at 7 days while mir-21 decreased by 60% at two hours and by 70% at 7 days. Erythema and edema were first noted in the treatment field 14 days after irradiation. By 30 days, alopecia was noted with an average leg contraction of 13% and the mean miRNA expression increased above control for both let-7b (84%) and mir-21 (292%). By 150 days, the irradiated leg developed telangiectasias and decreased range of motion with an average leg contraction of 70% compared to the unirradiated leg. At that time, the mean expression of let-7b decreased by 26% while miR-21 expression increased by 7%. These findings confirm  in vivo  the results of our prior  in vitro  analysis. Targetscan revealed that multiple fibrosis-associated proteins are potential targets of mir-21 including RhoB, TGF\#946;1 and TGF\#946;R2. Western blots revealed that RhoB was unchanged but TGF\#946;1 was decreased in the irradiated leg compared to the irradiated leg at 150 days. Conclusions: Since miRNAs alter the expression of many genes, it is logical to suggest that some species may modify pathways involved in fibrosis. One potential target of mir-21 is TGF\#946;1. We demonstrate increased mir-21 expression associated with a decrease in TGF\#946;1 expression at 150 days in an  in vivo  model of long term radiation-induced fibrosis. These finding suggest that mir-21 may regulate TGF\#946;1 expression and, therefore, may be exploited to prevent normal tissue toxicity due to radiation.   Citation Information:  In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3967.