Cellular stress induced alterations in microRNA let-7a and let-7b expression are dependent on p53.
Genotoxic stressors, such as radiation, induce cellular damage that activates pre-programmed repair pathways, some of which involve microRNAs (miRNA) that alter gene expression. The let-7 family of miRNA regulates multiple cellular processes including cell division and DNA repair pathways. However, the role and mechanism underlying regulation of let-7 genes in response to stress have yet to be elucidated. In this study we demonstrate that let-7a and let-7b expression decreases significantly following exposure to agents that induce stress including ionizing radiation. This decrease in expression is dependent on p53 and ATM in vitro and is not observed in a p53(-/-) colon cancer cell line (HCT116) or ATM(-/-) human fibroblasts. Chromatin Immunoprecipitation (ChIP) analysis showed p53 binding to a region upstream of the let-7 gene following radiation exposure. Luciferase transient transfections demonstrated that this p53 binding site is necessary for radiation-induced decreases in let-7 expression. A radiation-induced decrease in let-7a and let-7b expression is also observed in radiation-sensitive tissues in vivo and correlates with altered expression of proteins in p53-regulated pro-apoptotic signaling pathways. In contrast, this decreased expression is not observed in p53 knock-out mice suggesting that p53 directly repress let-7 expression. Exogenous expression of let-7a and let-7b increased radiation-induced cytotoxicity in HCT116 p53(+/+) cells but not HCT116 p53(-/-) cells. These results are the first demonstration of a mechanistic connection between the radiation-induced stress response and the regulation of miRNA and radiation-induced cytotoxicity and suggest that this process may be a molecular target for anticancer agents.
Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
Anthony D
AD
Saleh
Jason E
JE
Savage
Liu
L
Cao
Benjamin P
BP
Soule
David
D
Ly
William
W
DeGraff
Curtis C
CC
Harris
James B
JB
Mitchell
Nicole L
NL
Simone
D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000528:radiation effects; D045325:HCT116 Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D018384:Oxidative Stress / Q000235:genetics / Q000528:radiation effects; D010766:Phosphorylation / Q000528:radiation effects; D011485:Protein Binding / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D014158:Transcription, Genetic / Q000528:radiation effects; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D035683:MicroRNAs; C488588:PUMA protein, mouse; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D051028:bcl-2-Associated X Protein; C487931:mirnlet7 microRNA, human; C542687:mirnlet7 microRNA, mouse; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases