Figure 7.

miR-34 blocks cell migration and EMT-associated invasion by silencing Snail1. (A) Immunoblot analysis and Snail1 3′ UTR reporter activities 48 h after transduction of an miR-34a expression vector into HPV-E6–expressing A549 or MCF-7 cells. Snail1 3′ UTR reporter activity (normalized to a cotransfected XV-40 promoter renilla luciferase construct) was determined after HPV-E6 expression alone in A549 or MCF-7 cells, or alternatively, after cotransfection with HPV-E6 in tandem with a control (control) or miR-34a (miR-34a) synthetic RNA oligonucleotide. Results are expressed as the mean ± 1 SD of three or more experiments (*, P ≤ 0.01). (B) Reporter activities of a wt E-cadherin promoter construct (blue) or its E-box mutant (red) were determined in HPV-E6–expressing A549 or MCF-7 cells 48 h after transfection with a control or miR-34a expression vector. E-cadherin promoter activity was determined as described in Fig. 2 E (mean ± 1 SD; n = 3; *, P ≤ 0.01). (C) The migratory activity of HPV-E6–expressing A549 cells relative to cells transfected with a control vector (Mock) alone or cells transfected with HPV-E6 and either a Snail1 siRNA alone, miR-34a oligonucleotide alone, or miR-34a oligonucleotide and a Snail1 expression vector in tandem was determined after a 2-d culture period. Inhibition of endogenous miR-34a in wt A549 cells with a miR-34a antisense oligonucleotide (anti–miR-34a) increased cell migration after a 4-d culture period via a process reversed by Snail1 siRNA (anti–miR-34a/siSnail1) or a Snail1 shRNA (not depicted). Results are expressed as the mean ± 1 SD (n = 5; *, P ≤ 0.01). (D) HCT116-p53^−/− cells were transfected with a control RNA oligonucleotide (control) alone, miR-34a oligonucleotide alone, or miR-34a in combination with mock (miR-34a/Mock) or Snail1 expression vectors (miR-34a/Snail1). After a 24-h culture period, the cells were labeled with fluorescent nanobeads and cultured atop the chorioallantoic membrane of live chick embryos for 3 d. Tissues were then removed, H&E-stained cross sections were prepared for light microscopy, or unstained frozen sections were examined by fluorescence microscopy. The boxed areas in the H&E-stained sections (the first and third rows in the panel) were magnified and examined by fluorescence microscopy to visualize directly the invasive activity of the green-colored cancer cells. The upper face of the chorioallantoic membrane is indicated by the broken lines in the fluorescent images. Bar, 50 µm. (E) HCT116-p53^−/− cells were transfected with control or miR-34a oligonucleotides, and Snail1 or E-cadherin protein expression levels were monitored by immunoblot analysis after a 24-h culture period. (F) After transfection with an siRNA control (Scr) or one of two Snail1-specific siRNAs (si1Snail1 and si2Snail1), HCT116-p53^−/− cells were cultured for 24 h in vitro before being inoculated on the chick chorioallantoic membrane. Relative invasion depth was monitored after a 3-d assay period as described in D. Alternatively, HCT116-p53^−/− cells were transfected with control or miR-34a oligonucleotide alone, or with a miR-34a oligonucleotide in tandem with either a control expression vector (miR-34a/Mock) or a Snail1 expression vector (mi-R34a/Snail1), and cultured for 24 h in vitro before being placed atop the chorioallantoic membrane. Results are expressed as the mean ± 1 SD (error bars; n = 3; *, P ≤ 0.01).