FIGURE 5.

MET proto-oncogene is a target of miR-1. A, the 3′-UTR of MET harbors two miR-1 cognate sites. Luciferase activity regulated by 3′-UTR of MET is inhibited by ectopic expression of miR-1. A549 cells were co-transfected with firefly luciferase-3′-UTR (MET) and hsa-miR-1 or control RNA (60 nm) along with SV40-β-gal (as internal control). After 48 h, firefly luciferase and β-galactosidase activities were measured. MET-3′-UTR deleted of both or individual miR-1 complementary sites were transfected following the same protocol. The results are means of three independent experiments ± S.D. B, the levels of miR-1 targets are reduced in A549 cells expressing miR-1. Cell extracts were subjected to Western blot analysis with specific antibodies. C, quantification of Western blot data in B. A reproducible result was obtained in two independent experiments. D and E, real time RT-PCR analysis of MET and FoxP1 miR-1-expressing cells. Data were normalized to 18 S rRNA. The results are means of triplicate assays ± S.D. F, luciferase activity controlled by Pim-1–3′-UTR is inhibited by miR-1. Cells were co-transfected with the pIS0-Pim-1–3′-UTR-firefly luciferase reporter, Renilla luciferase, along with hsa-miR-1. After 48 h, luciferase activities were measured. G, Pim-1 expression in miR-1 expressing A549 clones was measured by real time RT-PCR.