miR-10 family expression in relation to in vitro and in vivo chemotherapy response in AML. (A) Baseline miR-10a-5p expression in untreated AML samples according to chemotherapy response (no-CR vs CR). (B) Baseline miR-10b-5p expression in untreated AML samples according to chemotherapy response (no-CR vs CR). Measures of expression were obtained after normalization and background subtraction of miRNA microarray data from 54 newly diagnosed AML patients. (C) Baseline miR-10a-5p levels in AML cell lines detected by quantitative real-time polymerase chain reaction. Results are shown after normalization with U44. (D) Cell growth curves of KG-1a and Kasumi-1 cells (left panel) or K562 and OCI-AML3 cells (right panel) transfected/infected in vitro with 100 nM of synthetic miR-10a-5p, miR-10a-5p overexpressing lentivirus (pMIR-10a), or anti–miR-10a-5p (anti–10a-5p) oligonucleotides and their respective controls (scrambled oligonucleotides or empty vector [pMIR-EV]). The rationale for using a lentivirus expressing miR-10a-5p to infect Kasumi-1 cells was based on the difficulty in achieving successful overexpression of synthetic miR-10a-5p using nucleoporation methods. The efficiency of infection of the Kasumi-1 cell line is 50% using a multiplicity of infection of 5. The efficiency of nucleoporation of the KG-1a cell line is about 80% to 90%.⁹  Cells were harvested and counted at 24-hour intervals using a ViCell counter (Beckman Coulter). Each sample was run in triplicate. (E) Annexin V/propidium iodide (PI) assays in KG-1a and Kasumi-1 (left panel) and K562 and OCI-AML3 (right panel) cells after 48 hours of transfection/infection with synthetic miR-10a-5p/lentivirus pMIR-10a, anti–10a-5p, or controls (scrambled oligonucleotides or empty vector) in the presence or absence of 5 μM cytarabine (ARA-C) or control (phosphate-buffered saline [PBS]). Experiments were repeated in triplicate. (F) Annexin V/PI assays in 2 primary CN-AML patient samples with NPM1-FLT3 wild-type and low level of miR-10a-5p. About 3 × 10⁶ cells were cultured with StemSpan SFEM supplemented with 20% fetal bovine serum and StemSpan CC100 cytokine cocktail (STEMCELL Technologies) along with transferrin-conjugated nanoparticles (NPs) encapsulated with 0.4 µM synthetic miR-10a-5p or scrambled controls. Nanoparticle-conjugated miRNAs was used to improve cell delivery as described in detail.¹⁰  In addition, ARA-C (5 µM) or control (PBS) was added to the culture media. The miR-10a-5p expression in primary AML blasts after nanoparticle-conjugated synthetic miRNA coculture was assessed by quantitative real-time polymerase chain reaction (supplemental Figure 2). (G) Annexin V/PI assays in 2 primary CN-AML patient samples with FLT3 wild-type and NPM1-mutated/high level of miR-10a-5p. About 3 × 10⁶ cells were cultured with ARA-C (5 µM) or control (PBS) media as described in (F) along with transferrin-conjugated NPs encapsulated with 0.4 µM synthetic anti–10a-5p or scrambled controls (see supplemental Figure 2 for miR-10-5p expression after nanoparticle-miRNA treatment).