Identification and validation of KIT-targeting miR-26a chimera that inhibits human breast cancer growth in vitro and in vivo. (A) Overall survival of patients with basal-like breast cancer with higher or lower expression of miR-26a-2 based on expression scores compared with the mean value in TCGA cohort. (B) The binding of anti-KIT antibody and KIT DNA aptamer to MDA-MB-231 cell line. Other cell-targeting aptamer is specific for Ramos cells. (C) The secondary structure of KIT aptamer–miR-26a chimera. KIT DNA aptamer was linked with C3 linker to complementary RNA passenger sequence that was bound to miR-26a mimic sequence. Another RNA passenger sequence binding to the 5′ of miR-26 mimic was conjugated with triethylene glycol (TEG)–cholesterol. (D) Specific miR-26a delivery by the miR-26a chimera. Two days after incubation with the miR-26a chimera, significant increase of miR-26a expression in the MDA-MB-231 cells compared with control (ctrl) chimera treatment was detected by quantitative polymerase chain reaction. Data (mean ± standard deviation [SD]) were pooled from 3 experiments. (E) miR-26a chimera suppressed the growth and induced apoptosis of MDA-MB-231 cells in a dose-dependent manner. After 3 days of culture with miR-26a chimera or ctrl chimera, MDA-MB-231 cell numbers (left) were counted using hemocytometer, and percentage of annexin V⁺ cells (right) was determined by flow cytometry. Data (mean ± SD) were pooled from 2 experiments. (F) Significant suppression of EZH2 protein in miR-26a chimera–treated MDA-MB-231 cells detected by immunoblot. (G) Relative expression of miR-26a in tumor harvested from NSG mice bearing human breast tumor with MDA-MB-231 cells. Significant increase of miR-26a was observed in KIT⁺ tumor cells 3 days after IV injection with 670 pmol per 20 g of miR-26a chimera. Data (mean ± SD) were pooled from 2 experiments involving a total of 6 mice per group. (H) Therapeutic effect of miR-26a chimera. The tumor-bearing mice were treated daily with miR-26a chimera (670 pmol per 20 g) for 5 or 10 days (first injection defined as day 0). Data (mean ± SD) were pooled from 2 experiments involving a total of 5 mice per group. Tumor volume over time (left). Significant difference between miR-26 chimera (×5) versus miR26 chimera (×10; 2-way repeated-measures analysis of variance followed by Bonferroni post-test for day 0 to day 18 detected the significant difference (interaction P = .0012). Kaplan-Myer survival curve (right). Log-rank test detected significant differences between ctrl chimera group and miR-26a chimera groups (ctrl chimera vs miR-26 chimera [×5], P = .002; ctrl chimera vs miR-26 chimera [×10], P = .002). The difference in survival between miR-26 chimera (×5) and miR26 chimera (×10) did not reach statistical significance (P = .077). *P < .05, **P < .01. Error bars indicate SD.