Regulation of miR-18a on the 3′-UTR of ATM.

<p>(A) The miR-18a alignment site (underlined) within ATM 3′UTR of different species is conserved. (B) Mature human miR-18a sequence and region of 3′-UTR of human ATM containing the recognition site, which was cloned into a construct with <i>Renilla</i> luciferase. The mutant 3′-UTR contains a seed region with 5 mutated nucleotides (underlined). (C) Expression of miR-18a in HCT-116 cells transfected with miRNA precursor control (pre-miR-ctrl) or miR-18a precursor (pre-miR-18a) at 15 nM. (D) Luciferase activity in HCT-116 cells co-transfected with the <i>Renilla</i> luciferase construct (containing wildtype or mutant miR-18a seed region), <i>Firefly</i> luciferase construct and pre-miR-ctrl or pre-miR-18a at 15 nM. Level of activity was calculated by normalizing <i>Renilla</i> luciferase to <i>Firefly</i> luciferase. NS denotes no statistical significance. p value was determined by student t-test. Mean and standard deviation (SD) was calculated from three independent experiments. (E) Immunoblot of endogenous ATM expression in HCT-116 cells 48 hours after transfection of pre-miR-ctrl and pre-miR-18a. (F) Immunoblot of the phosphorylated form of CHK-2 protein, a direct downstream target of ATM, in HCT-116 cells, after transfection of pre-miR-ctrl or pre-miR-18a under the treatment with DMSO or 2 µM etoposide for 1 hour.