Figure 1.
Design and generation of plasmids for the overexpression of microRNA* species.
(A) Design of the stem-loop miRNA precursor. The mature sequences of miRNA* species were incorporated into the 3′ arm of the precursor, whereas the complementary sequences of the miRNA* species were incorporated into the 5′ arm. Note that the complementary sequences could either be completely complementary to the miRNA* species or could be modified by mutating several bases in its seed region, which were not complementary to the miRNA* species. (B) The designed hsa-miR-146b-3p stem-loop precursor. The red characters in the 3′ arm correspond with the sequences of hsa-miR-146b-3p. The 5′ arm sequences are the modified complementary sequences of hsa-miR-146b-3p, and the sequences above are hsa-miR-146b-5p, with blue characters denoting seed sequences and solid lines between them indicating the similarity of the two sequences. The dotted lines show the modified sites in the 5′ arm sequences and their corresponding nucleotides in hsa-miR-146b-3p sequences. (C) The uppermost oligo is the DNA insert of hsa-miR-146b-3p precursor obtained by annealing two single stranded DNA oligos with BamH1 and EcoR1 sticky ends. The DNA insert was incorporated into plvx-shRNA2 between the recognition sites for the restriction enzymes BamH1 and EcoR1. The representation of the vector was modified from an illustration in the instructions provided with the product.
Figure 2.
Successful overexpression of hsa-miR-146b-3p by plvx-hs-146b-3p with no detectable increase of hsa-miR-146b-5p.
HeLa cells were transfected with the indicated plasmids (400 ng per well) and RNA was collected and extracted 24 h later. The expression of hsa-miR-146b-3p (A) and hsa-miR-146b-5p (B) was detected using qRT–PCR. The same experiments were done in Hep G2 cells (C) for hsa-miR-146b-3p; (D) for hsa-miR-146b-5p and HEK 293T cells (E) for hsa-miR-146b-3p; (F) for hsa-miR-146b-5p. Each graph shows the mean of three independent experiments that measured the relative expression levels (2−deltaCT) of the two miRNAs to the reference gene RNU48. Error bars represent SEMs. * means p value ≤0.05; ** means p value ≤0.01; *** means p value ≤0.001; ns means no significance.
Figure 3.
plvx-hs-146b-3p overexpressed functional hsa-miR-146b-3p with no detectable activity change of hsa-miR-146b-5p.
(A) Transcription products from plvx-hs-146b-3p had hsa-miR-146b-3p activities, without changing the activity of hsa-miR-146b-5p. HeLa cells were transfected with psicheck-ctrl (20 ng per well), psicheck-146b-3psensor (20 ng per well), or psicheck-146b-5psensor (20 ng per well), in combination with either plvx-ctrl (100 ng per well) or plvx-hs-146b-3p (100 ng per well), as indicated in the graph. (B) The repression of psicheck-146b-3psensor activity could be rescued using hsa-miR-146b-3p inhibitors. HeLa cells were transfected with psicheck-146b-3psensor (20 ng per well) in combination of plvx-ctrl (100 ng per well) or plvx-hs-146b-3p (100 ng per well) under the presentation of hsa-miR-146b-3p inhibitors (100 nM) or inhibitor negative controls (100 nM), as indicated in the graph. (C) Designed modified complementary sequences (designed anti-hsa-miR-146b-3p) could not repress psicheck-146b-3psensor activity as effectively as hsa-miR-146b-3p. HeLa cells were transfected with psicheck-146b-3psensor (20 ng per well) in combination with single stranded RNA mimics of negative control sequences (100 nM), designed anti-hsa-miR-146b-3p (100 nM), hsa-miR-146b-3p (100 nM), and hsa-miR-410 (100 nM), as indicated in the graph. The miRNA hsa-miR-410, which had no ability to repress psicheck-146b-3psensor activity, was used as a negative control miRNA. (D) The miRNA hsa-miR-146b-3p overexpressed by plvx-hs-146b-3p could repress the activity of bulged sensor of hsa-miR-146b-3p (pMIR-hs-146b-3p) and this repression was abolished by mutation of the seed sequences of the bulged sensor. HeLa cells were transfected with pMIR-hs-146b-3p (2 ng per well) or pMIR-hs-146b-3p-mut (2 ng per well), together with plvx-ctrl (400 ng per well) or plvx-hs-146b-3p (400 ng per well), as indicated in the graph. Renilla luciferase vector (5 ng per well) was delivered simultaneously as a transfection control. For all assays, protein was collected 24 h after transfection. Luciferase activity was quantified and expressed as relative luciferase activity. The data represent one of at least three independent experiments, each of which involved four replicates. Error bars represent SEMs. ** means p value ≤0.01; *** means p value ≤0.001; ns means no significance.
Figure 4.
Successful overexpression of hsa-miR-127-3p and hsa-miR-142-3p.
HeLa cells were transfected with the indicated plasmids (400 ng per well) and RNA was collected and extracted 24 h later. Levels of expression of hsa-miR-127-3p (A) and hsa-miR-142-3p (B) were detected using qRT–PCR. Each graph shows the representative result of at least three independent experiments. Relative expression levels (2−deltaCT), to the reference gene RNU48, were plotted. Error bars represent SEMs. ** means p value ≤0.01.