Fig 1.
Expression of RARB mRNA levels in PTC tumors relative to paired control tissue (n = 48), normalized to the reference gene GAPDH.
SQ-PCR reaction for each sample was performed in triplicate.
Fig 2.
Expression of miR-146a-5p (A) and miR-146b-5p (B) in PTC tumors and matched control tissue from the same patient (n = 48), normalized by the reference gene RNU6B. The data are medians +/- SD (95% CI). Statistical analysis was performed using Wilcoxon paired test (***P <0,001).
Fig 3.
Positions of putative miRNA binding sites in RARB 3′UTR identified by TargetScan and RNAHybrid.
miRNAs binding to 3′UTRs requires only partial complementarity to the target. Nucleotides of perfect complementarity are shown as seed match.
Fig 4.
Luciferase activity in transfected HeLa cells.
HeLa cells transfected with pGL3-3'UTR-RARB and pcDNA3-miR-146a-5p (A) or pcDNA3-miR-146b-5p (B). Luciferase activity is showed as a percentage relative to the control (cells transfected with pcDNA3-miR-421). The results are normalized by Renilla luciferase and derive from three experiments, each performed in triplicates. The graph shows the mean, along with deviations from mean (SEM). Statistical analysis was performed using an unpaired t test (*** P<0.001).
Fig 5.
Reduction of endogenous RARB mRNA is shown by real-time PCR Taqman assay in the A549 cell line transfected with pcDNA3-miR-146a-5p (A) or pcDNA3-miR-146b-5p (B) or control plasmid. Data are expressed as mean values +/- SEM. Statistical analysis was performed using an unpaired t test (*** p <0.001). Western blotting for RARβ and β-actin (loading control) in the A549 cells transfected with pcDNA3-miR-146a-5p (C), pcDNA3-miR-146b-5p (D) or control plasmid has shown no significant reduction of protein level. Cells were harvested 72 h after transfection; the relative density of bands was quantified by densitometry.
Fig 6.
Induction of RARB is shown by a real-time PCR Taqman assay in the K1 cell line after inhibition of endogenous miR-146a-5p (C) or miR-146b-5p (D) by means of transfection with sponge plasmids.
Data are expressed as mean values +/- SEM. Statistical analysis was performed using an unpaired t test (** p< 0.01).
Fig 7.
Effect of inhibition of miR-146a-5p and miR-146b-5p on the intensity of cell division.
Proliferation of K1 cells transfected with pGL3-sponge-miR-146a-5p (dark gray) or pGL3-sponge-miR-146b-5p (light gray) was analyzed by the xCELLigence system, operating on the basis of electrical resistance measurement. Resistance was measured every 30 minutes for 80 hours. The final cell index values show the difference between the resistance generated by the cells in each time point and the resistance of the medium without cells. The graph indicates proliferation index at each time point and SD for each measurement