Supplementary Figures 1-12 - PDF file 897K, Relative expression of miR-520d-3p (miR-520d) and miR-520d-5p (miR-520d*) in ovarian cancer cell lines (Figure S1), Kaplan-Meier curves representing the percent progression-free survival in patients with ovarian cancer based on miR-520d-3p median expression levels (Figure S2), miR-520d-5p does not target EphA2 (Figure S3), miR-520d-3p and EphA2 expression levels in miR-520d-3p overexpressing stable clones (Figure S4), Effect of miR-520d-3p expression on cell proliferation (Figure S5), miR-520d-3p and EphA2 levels in xenograft models of miR-520d-3p overexpressing HeyA8 and SKOV3ip1 models (Figure S6), Efficiency of EphA2 targeting siRNAs in HeyA8 cells (Figure S7), Combination of miR-520d-3p and siRNA-EphA2 treatment shows enhanced EphA2 inhibition in vitro (Figure S8), Dose-response curve of miR-520d-3p, si-EphA2-1 and combined miR-520d-3p + si-EphA2-1 in SKOV3ip1 and HeyA8 cells (Figure S9), Treatment with anti-miR-520d-3p can restore EphA2 protein levels in a dose-dependent manner in SKOV3ip1 cells (Figure S10), Effect of combined miR-520d-3p+siEphA2-1 treatment on angiogenesis, proliferation and apoptosis in HeyA8 cells (Figure S11), and Differentially expressed coding genes in miR-520d-3p overexpressing stable clones (Figure S12)
ARTICLE ABSTRACT
Development of improved RNA interference–based strategies is of utmost clinical importance. Although siRNA-mediated silencing of EphA2, an ovarian cancer oncogene, results in reduction of tumor growth, we present evidence that additional inhibition of EphA2 by a microRNA (miRNA) further “boosts” its antitumor effects. We identified miR-520d-3p as a tumor suppressor upstream of EphA2, whose expression correlated with favorable outcomes in two independent patient cohorts comprising 647 patients. Restoration of miR-520d-3p prominently decreased EphA2 protein levels, and suppressed tumor growth and migration/invasion both in vitro and in vivo. Dual inhibition of EphA2 in vivo using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) nanoliposomes loaded with miR-520d-3p and EphA2 siRNA showed synergistic antitumor efficiency and greater therapeutic efficacy than either monotherapy alone. This synergy is at least in part due to miR-520d-3p targeting EphB2, another Eph receptor. Our data emphasize the feasibility of combined miRNA–siRNA therapy, and will have broad implications for innovative gene silencing therapies for cancer and other diseases.Significance: This study addresses a new concept of RNA inhibition therapy by combining miRNA and siRNA in nanoliposomal particles to target oncogenic pathways altered in ovarian cancer. Combined targeting of the Eph pathway using EphA2-targeting siRNA and the tumor suppressor miR-520d-3p exhibits remarkable therapeutic synergy and enhanced tumor suppression in vitro and in vivo compared with either monotherapy alone. Cancer Discov; 3(11); 1302–15. ©2013 AACR.See related commentary by Kasinski and Slack, p. 1220This article is highlighted in the In This Issue feature, p. 1207
