Fig. 1.

Functional screen identifies miR-584-5p as a new therapeutic adjuvant in MB. a Outline of the primary screen and list of drug-sensitizer, drug-desensitizer, and drug-neutral miRNAs. A total of 1902 miRNA mimics arrayed in 96-well plates were screened in triplicates. b Line graphs showing relative viability of DAOY cells transfected with miR-NC or indicated VCR-sensitizer miRNAs (miR-584-5p, miR-1266, miR-126-5p, miR-335-5p, miR-9-5p, miR-449b; obtained from primary screen) for 24 h followed by treatment with vehicle or increasing concentration of VCR for 72 h. Cell viability was assessed using alamarBlue® cell viability assay. p-value was calculated using one-way ANOVA followed by Dunnett’s multiple-comparisons test. Error bars represent the mean ± SEM of three independent experiments (performed in sixtuplicate for each experiment). c–g Dose–response curves showing VCR sensitivity in miRNA negative control (NC) (blue line) and miR-584-5p mimic-transfected D556Med, D458Med, D425Med, DAOY, and primary MB BT-28 cells. MB cells were transfected with miR-NC or miR-584 mimic followed by treatment with VCR or vehicle for 72 h. Cell viability was assessed using alamarBlue cell viability assay. The p-values were determined by the sum-of-squares F test. Error bars represent mean ± standard error of the mean (SEM) of three independent experiments (performed in sixtuplicate for each experiment). h Synergistic effect of miR-584-5p with VCR. D556Med cells were treated with increasing concentrations of miR-584-5p and VCR before being subjected to cell viability assay using alamarBlue cell viability assay. Compusyn software (http://www.combosyn.com/) was used to calculate combination indices (CIs). The p-values were determined by the sum-of-squares F test. Error bars represent mean ± SEM of three independent experiments (performed in sixtuplicate for each experiment)