Investigation of the mechanism of p27 regulation by Fli-3 mir-17–92. (A) Expression analysis of the proteins associated with p27 ubiquitination (Skp2, p-Akt, cyclin D1, and CDK2) in NIH-3T3 cells infected with Fli-3-mir-17–92 and the control groups. The intensity of NIH3T3 band was used as control (1.0). Mean fold changes are shown at the bottom. (B) Posttranscriptional regulation of p27 by mir-17–92 cluster through a specific binding to the p27 3′UTR was examined using luciferase assay. The 3′UTR of p27 was cloned into pGL3 vector and designated pGL3-p27-UTR. NIH-3T3-mir-17–92 cells were transfected with pGL3-p27-UTR (experimental group, Ex) or pGL3 control vector (control group, Clt) and subjected to luciferase assay. 293T cells were cotransfected with either pGL3-p27-UTR (Ex) or vector alone (Clt) and either Fli-3 mir-17–92 or vector alone and used to determine the luciferase activity. (C) KH16 cells were electroporated with the same amount of pGL3-p27-UTR (Ex) or pGL3 vector (Clt) and subjected to luciferase assay. [β]-Galactosidase plasmid was also included in the transfections and firefly luciferase activity was normalized to [β]-galactosidase expression. Error bars are SD.