Figure 7. Arhgap1 is a miR-34a target gene.
(A) 3′-UTRs analyzed. Shown are size (right) and location of predicted miR-34a binding sites. Poorly conserved sites were defined on the basis of criteria established by TargetScan. mut1 and mut2, mutations generated in 2 putative miR-34a binding sites in the Arhgap1 3′-UTR. Flt1 3′-UTR is a negative control. (B and C) miR-34a–induced repression of 3′-UTR reporters. Luciferase assays on 344SQ cells transiently cotransfected with the indicated 3′-UTR reporters and control or miR-34a precursors. Data are mean ± SD (n = 3). *P < 0.001; #P < 0.01. (D) Correlation of miR-34a levels in KP cell lines (from Figure 2A), with densitometric analysis of ARHGAP1 protein expression by Western analysis. ACTIN was used as a loading control. Correlation (R and P, 1-tailed Spearman rank correlation test) is indicated. (E) miR-34a downregulated ARHGAP1. Western blot analysis of ARHGAP1 in 344SQ_vector and 344SQ_miR-34a cells. Densitometric analysis (numbers below ARHGAP1 gel) is shown relative to 344SQ_vector cells (set at 1.0). (F) Q-PCR analysis of ARHGAP1 mRNA levels in MDA-MB-231 cells 72 hours after transient transfection with pre-miR negative control or pre–miR-34a precursors. Data are mean ± SD (n = 3). P values were determined by 2-tailed Student’s t test. See complete unedited blots in the supplemental material.