The miR-183, miR-96, and miR-182 are involved in FOXO1 repression in cHL cell lines. (A) Expression of miR-183, miR-96, and miR-182. The total RNA was extracted from CD19⁺ tonsillar cells obtained from 5 different patients. cHL and NHL cell lines and expression of miRNAs were measured by quantitative RT-PCR. U6 mRNA was used as reference. The data were analyzed with the comparative C_t method and represent mean ± SD. (B) miRNA regulation of FOXO1 3′-UTR. miRNA activity in L428 cells was measured by electroporation of psi-CHECK-2 vector bearing either a specific wild-type target sequence (WT) common for miR-96 and miR-182, or for miR-183 sequence. The relevant mutated sequences (MUT) with mutated nucleotides in the seed region were used as control. Activity is expressed as mean ± SD of ratio of Renilla to firefly luciferase activity normalized to the mutated control. (C) Inhibition of miRNA expression by specific anti-miRNAs. L428 cells were transfected with anti-miRNAs or with the negative control RNAs. Twenty-four hours later, miRNA expression was measured by quantitative RT-PCR and analyzed and presented as described for panel A. (D) Effect of miRNA overexpression on FOXO1 levels. L428 cell line was transfected with anti-miRs or with the negative control RNAs. Forty-eight hours after transfection, the cells were harvested and FOXO1 expression was analyzed by immunoblot. ACTB was used as a loading control. The protein expression was quantified with the help of ImageJ 64 software (http://rsbweb.nih.gov/ij). All experiments were done in triplicate.