Figure 1.

Flt1 is a miR-200 target gene. A, Kaplan–Meier analysis of 2 cohorts of lung denocarcinoma patients on the basis of VEGFR1 expression (top quartile vs. lower 3 quartiles) in resected primary tumor specimens. The number of patients analyzed (n; top right corner) and the results of statistical tests (P; bottom left corner) are indicated for each cohort. B, miR-200 targets sequences in the Flt1 gene 3′-UTR. Reporter constructs containing wild-type and mutant Flt1 3′UTR are illustrated diagrammatically. 344SQ cells were transiently cotransfected with pre-miRs (10 nmol/L) and reporter plasmids (500 ng) that express luciferase alone (control) or are linked to the full-length 3′-UTR of Zeb1 (Zeb1) or Flt1 that is wild-type (Flt1) or mutated at the 5′ (mut1) or 3′ (mut2) putative miR-200 binding site. Results were normalized on the basis of renilla luciferase and expressed as the mean values of triplicate wells. *, P < 0.05. C, miR-200 suppresses VEGFR1 expression. Western blot analysis (left) of 344SQ cells and murine lung endothelial cells (MEC) as a positive control. Quantitative RT-PCR analysis (bar graphs) of 344SQ cells stably transfected with miR-200 (200b) or empty (control) lentiviral vectors normalized on the basis of L32 ribosomal protein mRNA levels and expressed as mean values of triplicate cultures relative to control transfectants, which were set at 1.0. Western blot analysis (right) of VEGFR1 in the same transfectants, which was quantified densitometrically, normalized on the basis of actin, and expressed relative to control, which was set at 1.0.