ATM–dependent downregulation of miR-335 post-IR.

Abstract

<p>(A) RT-qPCR indicates downregulation of miR-335 expression 2 hours after 10 Gy in WT but not A-T LCLs. (B) MiR-335 downregulation following IR- or doxorubicin-induced DNA damage by RT-qPCR in MCF7 (ATM +/+) cells. (C) Inhibition of ATM kinase activity in MCF7 cells with KU-55933 for 2 hours prior to IR exposure abolishes the downregulation of miR-335 expression 2 hours after IR. (D) MiR-335 is located in the second intron of <i>MEST</i>. The arrows indicate two predicted CREB binding sites in the <i>MEST</i> promoter (−938 to −949, −214 to −216 related to translational start site, respectively). (E) RT-qPCR shows that transcription levels of <i>MEST</i> mimic those of miR-335 in response to IR, supporting their anticipated co-regulation. (F) Suppression of CREB by siRNA reduced endogenous miR-335 levels and abolished the post-IR down-regulation of miR-335 observed in siCTL transfected HeLa cells. (G) Results of CREB ChIP assay 2 hours post-IR in HeLa cells indicated the binding of CREB to the <i>MEST</i> promoter before IR and release from the promoter after IR.</p