Figure 4.

AsiRNAs have less or no inhibition of the activities of other siRNAs or miRNAs. (a) siRNAs that target CREB3 mRNA [siCREB3 (19+2)] and competitor siRNA variants (shown on the x-axis) were co-transfected into HeLa cells at the indicated concentrations. CREB3 mRNA levels were analyzed by quantitative real-time reverse transcription–PCR 24 hours after transfection. The CREB3 mRNA levels plotted on the y-axis in the figure were calculated as the CREB3 mRNA level divided by the GAPDH (control) mRNA level. All data in the graph represent mean ± SD values of three independent experiments. The black bars labeled 0 nmol/l and siCREB3(19+2) 1 nmol/l represent transfections of Lipofectamine 2000 only and 1 nmol/l of siCREB3(19+2) only, respectively. For experiments represented by the gray bars, 10 nmol/l competitor siRNAs (siTIG3 19+2, siTIG3 17+2A, siTIG3 16+3A, or siTIG3 15+4A) were transfected with 1 nmol/l siCREB3(19+2). (b) HeLa cells were transfected with 1 nmol/l siCREB3(19+2) with or without 10 nmol/l Survivin siRNAs (Survivin 19+2, Survivin 17+2A, or Survivin 16+3A) into HeLa cells. See the legend of a for details. (c) Changes in the gene-silencing activity of miR-21 by transfection of exogenous siRNAs into HeLa cells. HeLa cells were transfected with a pMIR-luc–based firefly luciferase reporter plasmid (200 ng/ml) that contained a miR-21 binding site, the control pRL-SV40 Renilla luciferase expression vector (2 ng/ml), and one of several siTIG3 variants at a concentration of 10 nmol/l. The firefly luciferase activity was normalized by dividing it by the Renilla luciferase activity. Luciferase activity of the control luciferase reporter was set as one. All data in the graph represent means ± SD values of two independent experiments. Mock, mock-transfected control HeLa cells; anti-miR-19 and anti-miR-21, HeLa cells treated with an antagomir against miR-19 or miR-21, respectively.