Figure 7.
Validation of antitumor mi/siRNAs for ovarian and breast cancer
(A) 206/EphA2, a mi/siRNA designed to target EphA2 with miR-1/206 activity based on the AGO CLIP results of ovarian cancer cell A2780;16 AGO-bound regions in EphA2 (gray line, upper panel) with indication of miR-1/206 seed site (red line) are shown. Sequences of miR-206, 206/EphA2, and its target site in EphA2 (pink shading) are indicated in the lower panel. (B) Efficiency of on-target repression mediated by 206/EphA2; IC50 from luciferase reporter assays as performed in Figure 3C. (C) Repressive activity of 206/EphA2 on EphA2 (on-target) and ADAR1 (miR-206 target), measured by qRT-PCR. (D) Effect of 206/EphA2 on migration activity of A2780; wound-healing assays (left panel) and quantitative results (right panel) as measured in Figure 2D. More images are shown in Figure S7A. (E) Cell death assays of 206/EphA2-transfected A2780; flow cytometry (left panel) and its quantitative results (right panel), as conducted in Figures 1E and 1F. Relatively low responses from 206/EphA2 could be explained by low expression of EphA2 in A2780. (F and G) The same wound-healing and cell death assays as performed in (D) and (E) except that 206/EphA2-6Ø was introduced into another ovarian cancer cell line, SK-OV-3. More images are shown in Figure S7B. (H) 206/Her2, a mi/siRNA designed to target Her2 (ERBB2) with miR-1/206 activity, derived from the AGO CLIP results of the breast cancer cell line, MCF7. (I) On-target repressive efficacy of 206/Her2 as estimated in (B). (J) Repression of Her2 (on-target) and ADAR1 (miR-206 target), quantitated by qRT-PCR in SKBR3. The same experiment performed in another breast cancer cell line, BT474, is shown in Figure S7C. (K and L) Wound-healing (K) and cell death assay (L) as performed in (D) and (E) except with 206/Her2 and 206/Her2-6Ø in MCF7. More wound-healing results are shown in Figure S7D. All p values are from two-sided t tests. ∗p < 0.05, relative to NT; n ≥ 3; repeated with biologically independent samples. Graphs show mean and error bars show SD unless otherwise indicated.