Fig 1.
Predicted miR-17-5p guide strand binding sites in the 3'UTR of PDCD4 and PTEN mRNAs.
aThe potential oncomiR:mRNA binding sites were identified by rna22, Targetscan, or miRanda. bTop strand is mRNA (5' ➔ 3') and the bottom strand is oncomiR (3' ➔ 5'). cCalculated at http://mfold.rna.albany.edu/?q=mfold.
Fig 2.
Treatment with complementary DNA-LNA chimeras knocked down endogenous miRNAs in MDA-MB-231 TNBC cells.
A: qPCR of miR-17-5p 12 hr and 48 hr post-transfection with anti-miR-17-5p. B: qPCR of miR-21-5p 12 hr and 48 hr post-transfection with anti-miR-21-5p. Results represent absolute values of miRNA/internal control U6 normalized to mock transfected. Values are the average of three measurements ±.
Fig 3.
miR-17-3p is a potential regulator of PDCD4 protein level and competes with miR-17-5p for inhibition of PDCD4 and PTEN mRNAs.
miR-21-5p guide strand regulates PDCD4 protein level without competing with its passenger strand miR-21-3p. A: Mirbase search of miR-17-3p, forming the lower arm of the miR-17 pre-miRNA hairpin. B: Homologous sequences between miR-17-5p and miR-17-3p are highlighted in yellow. C: PDCD4 and PTEN protein Western blots at 48 hr post transfection with anti-miR-17-5p. D: PDCD4 and PTEN protein Western blots at 48 hr post transfection with anti-miR-17-3p. E: PDCD4 protein Western blot at 48 hr post transfection with anti-miR-21. β-actin was used as loading control. Values are the average of three blots ± s.d. after normalization to β-actin and to control/treatment group. Each blot was subjected to gamma setting adjustments.
Fig 4.
Predicted miR-17-3p passenger strand binding sites in the 3'UTR of PDCD4 mRNA.
aThe potential oncomiR:mRNA binding sites were identified by rna22, Targetscan, or miRanda. bTop strand is mRNA (5' ➔ 3') and the bottom strand is oncomiR (3' ➔ 5'). cCalculated at http://mfold.rna.albany.edu/?q=mfold.
Fig 5.
Predicted miR-17-3p passenger strand binding sites in the 3'UTR of PTEN mRNA.
aThe potential oncomiR:mRNA binding sites were identified by rna22, Targetscan, or miRanda. bTop strand is mRNA (5' ➔ 3') and the bottom strand is oncomiR (3' ➔ 5'). cCalculated at http://mfold.rna.albany.edu/?q=mfold.
Fig 6.
Predicted anti-miR-17-5p binding sites in the 3'UTR of PDCD4 mRNA as a mimic of miR-17-3p passenger strand.
aThe potential oncomiR:mRNA binding sites were identified by rna22, Targetscan, or miRanda. bTop strand is mRNA (5' ➔ 3') and the bottom strand is oncomiR (3' ➔ 5'). cCalculated at http://mfold.rna.albany.edu/?q=mfold.
Fig 7.
Predicted anti-miR-17-5p binding sites in the 3'UTR of PTEN mRNA as a mimic of miR-17-3p passenger strand.
aThe potential oncomiR:mRNA binding sites were identified by rna22, Targetscan, or miRanda. bTop strand is mRNA (5' ➔ 3') and the bottom strand is oncomiR (3' ➔ 5'). cCalculated at http://mfold.rna.albany.edu/?q=mfold.
Fig 8.
Predicted anti-miR-17-3p binding sites in the 3'UTR of PDCD4 and PTEN mRNAs as a mimic of miR-17-5p passenger strand.
aThe potential oncomiR:mRNA binding sites were identified by rna22, Targetscan, or miRanda. bTop strand is mRNA (5' ➔ 3') and the bottom strand is oncomiR (3' ➔ 5'). cCalculated at http://mfold.rna.albany.edu/?q=mfold.
Fig 9.
Minimum energy structure predicted with Amber 12 for miR-17-3p:PTEN mRNA duplex in explicit H2O with 100 mM NaCl, pH 7.0, at 300°K.
Fig 10.
Predicted miR-21-5p guide strand binding sites in the 3'UTR of PDCD4 mRNA.
aThe potential oncomiR:mRNA binding sites were identified by rna22, Targetscan, or miRanda. bTop strand is mRNA (5' ➔ 3') and the bottom strand is oncomiR (3' ➔ 5'). cCalculated at http://mfold.rna.albany.edu/?q=mfold.
Fig 11.
Effect of complementary DNA-LNA chimeras on the PDCD4 and PTEN mRNA levels in MDA-MB-231 cells.
A: qPCR of PDCD4 mRNA at 12 hr and 48 hr after transfection. B: qPCR of PTEN mRNA at 12 hr and 48 hr after transfection. C: Relative expression of PDCD4 mRNA from 12 hr to 48 hr after transfection with anti-miR-17-5p and anti-miR-17-3p. D: Relative expression of PTEN mRNA from 12 hr to 48 hr after transfection with anti-miR-17-5p and anti-miR-17-3p. Results represent absolute values of miRNA/internal control gene GAPDH normalized to mock transfected. Values are the average of three measurements ± s.d. * indicates p<0.05, ** indicates p<0.01.
Fig 12.
Both miR-17-5p and miR-17-3p can directly modulate the translation of PDCD4 and PTEN.
A: PDCD4 and PTEN protein Western blots at 48 hr post transfection with miR-17-5p mimic or miR-17-3p mimic. β-actin was used as loading control. Values are the average of three blots ± s.d. after normalization to β-actin and to control/treatment group. Each blot was subjected to gamma setting adjustments. B—E: Luciferase activity after co-transfecting MDA-MB-231 cells with indicated luciferase reporter constructs in the presence or absence of miRNA mimic. All luciferase signals from pMir-report firefly luciferase vectors are normalized to signals from pRL-TK Renilla luciferase vector. The ratio of normalized signal in the presence of mimic to signal in the absence of mimic for each construct is then calculated. The pMir-report luciferase vector is used as negative control. Results represent fold changes of the above ratio relative to vector control. Values are the average of at least three measurements ± s.e.m * indicates p<0.05, ** indicates p<0.01.
Fig 13.
Anti-miR-17-5p DNA-LNA can directly modulate the translation of PDCD4 and PTEN mRNAs through interactions with multiple binding sites from the 3’UTR.
Luciferase activity after co-transfecting MDA-MB-231 cells with indicated luciferase reporter constructs in the presence or absence of DNA-LNA inhibitor. A: PDCD4. B: PTEN. All luciferase signals from pMir-report are normalized to signals from pRL-TK Renilla luciferase vector. The ratio of normalized signal in the presence of DNA-LNA inhibitor to signal in the absence of inhibitor for each construct is then calculated. The pMir-report luciferase vector was used as a negative control. Results represent fold changes of the above ratio relative to vector control. Values are the average of at least three measurements ± s.e.m * indicates p<0.05, ** indicates p<0.01.
Fig 14.
Schematic view of competition between anti-miR-17-5p and miR-17-5p for inhibition of PDCD4 mRNA.