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. 2020 Mar;97(3):159–170. doi: 10.1124/mol.119.118315

Fig. 3.

Fig. 3.

miR-9-3p and miR-9-5p directly interact with the TOP2α/170 3′-UTR. (A) K562 and K/VP.5 cells were transfected with the psiTOP2α/170/UTR luciferase reporter construct (1 µg). Forty-eight hours later, luciferase activities were measured. Renilla luciferase activity was normalized to firefly luciferase activity. Results shown are the mean ± S.D. from 17 experiments performed on separate days; *P < 0.001, K/VP.5 luciferase vs. K562 luciferase levels. (B) K562 cells were cotransfected with the psiTOP2α/170/UTR luciferase reporter construct (1 µg) and either control, miR-9-3p, or miR-9-5p mimics (25 nM). Forty-eight hours later, total RNA was isolated and reverse transcribed. qPCR experiments were performed utilizing K562 cDNAs and TaqMan hydrolysis assays specific for miR-9-3p and miR-9-5p. Results shown are the mean ± S.D. from five experiments performed on separate days; *P < 0.001, miR-9-3p levels in miR-9-3p mimic–transfected K562 vs. nontransfected K562 cells, and miR-9-5p levels in miR-9-5p mimic–transfected K562 vs. nontransfected K562 cells, respectively. mir-9-5p was not significantly increased in mir-9-3p mimic–transfected cells (P = 0.340) nor was mir-9-3p significantly increased in mir-9-5p mimic–transfected cells (P = 0.223). Calculated 2−ΔCt values from qPCR assays were log transformed to assure distribution normality prior to data analysis using a two-tailed paired Student’s t test. (C) K562 cells were cotransfected with the psiTOP2α/170/UTR luciferase reporter construct (1 µg) and either control, miR-9-3p, or miR-9-5p mimics (25 nM). Forty-eight hours later, luciferase activities were measured. Results shown are the mean ± S.D. from nine experiments performed on separate days; P < 0.05 for miR-9-3p mimic and miR-9-5p mimic vs. control mimic–transfected K562 cells. (D) K562 cells were cotransfected with luciferase constructs, wherein the miR-9-3p and miR-9-5p putative binding sites were mutated (shown schematically) and either control, miR-9-3p, or miR-9-5p mimics (25 nM). Forty-eight hours later, luciferase activities were measured. Results shown are the mean ± S.D. from three to five experiments performed on separate days. N.S., not significant.