Differential Inhibition of Target Gene Expression by Human microRNAs
Abstract
:1. Introduction
2. Material and Methods
2.1. Target Selection
2.2. Cell Cultures
2.3. Molecular Cloning
2.4. Transfection and Reporter Assay
2.5. Bioinformatics and Statistical Analyses
2.6. miRNA Overexpression and RNA-seq
3. Results
3.1. Construction of Target Reporter Gene Libraries
3.2. Reporter Assays to Measure Target Repression by miRNAs
3.3. Parameters that Correlated with Target Repression by miRNAs
3.4. Relationships between Reporter Inhibition and Endogenous Gene Expression
3.5. Differential Reduction of Target mRNA Expression by miRNAs
4. Discussion
Supplementary Material
Author Contributions
Funding
Conflicts of Interest
References
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miR-1 | miR-122 | miR-124 | |||||||
---|---|---|---|---|---|---|---|---|---|
Selected | Confirmed | Ratio | Selected | Confirmed | Ratio | Selected | Confirmed | Ratio | |
TargetScan | 165 | 140 | 84.8% | 149 | 124 | 83.2% | 174 | 128 | 73.6% |
miRanda | 159 | 129 | 81.1% | 162 | 134 | 82.7% | 151 | 109 | 72.2% |
PicTar | 84 | 70 | 83.3% | 57 | 47 | 82.5% | 104 | 76 | 73.1% |
TargetScan only | 19 | 17 | 89.5% | 19 | 17 | 89.5% | 13 | 11 | 84.6% |
miRanda only | 28 | 19 | 67.9% | 42 | 34 | 84.0% | 9 | 8 | 88.9% |
PicTar only | 1 | 1 | 100% | 2 | 2 | 100% | 8 | 6 | 75.0% |
All three | 64 | 52 | 81.3% | 43 | 33 | 76.7% | 67 | 48 | 71.6% |
miRTarBase | 10 | 10 | 100% | 14 | 13 | 92.9% | 5 | 5 | 100% |
Single MRE | 171 | 140 | 81.9% | 181 | 152 | 84.0% | 166 | 117 | 70.5% |
Multiple MREs | 25 | 22 | 88.0% | 13 | 11 | 84.6% | 30 | 28 | 93.3% |
Total | 196 | 162 | 82.7% | 194 | 163 | 84.0% | 196 | 145 | 74.0% |
mRNA Properties | miR-1 | miR-122 | miR-124 | |||||||
---|---|---|---|---|---|---|---|---|---|---|
n | r | p | n | r | p | n | r | p | ||
AU% | 50 nt | 131 | −0.25 | 0.004 | 143 | −0.32 | <0.001 | 116 | 0.00 | 0.991 |
100 nt | 130 | −0.22 | 0.013 | 136 | −0.24 | 0.006 | 115 | 0.03 | 0.716 | |
200 nt | 110 | −0.23 | 0.017 | 125 | −0.25 | 0.005 | 99 | −0.07 | 0.502 | |
300 nt | 84 | −0.17 | 0.120 | 98 | −0.24 | 0.020 | 77 | −0.04 | 0.713 | |
whole | 162 | −0.17 | 0.032 | 163 | −0.15 | 0.061 | 145 | 0.05 | 0.583 | |
ΔG of predicted secondary structure | 50 nt | 131 | −0.11 | 0.198 | 143 | −0.22 | 0.009 | 116 | −0.01 | 0.899 |
100 nt | 130 | −0.18 | 0.046 | 136 | −0.23 | 0.008 | 115 | 0.02 | 0.843 | |
200 nt | 110 | −0.20 | 0.033 | 125 | −0.27 | 0.003 | 99 | −0.03 | 0.751 | |
300 nt | 84 | −0.13 | 0.250 | 98 | −0.27 | 0.008 | 77 | −0.04 | 0.711 | |
whole | 162 | −0.18 | 0.024 | 163 | −0.03 | 0.685 | 145 | −0.09 | 0.276 | |
ΔG of miRNA and MRE hybrid | 140 | 0.23 | 0.005 | 152 | −0.01 | 0.926 | 117 | 0.03 | 0.722 | |
Local AU content | 129 | −0.14 | 0.109 | 140 | −0.32 | <0.001 | 107 | −0.12 | 0.219 |
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Li, P.; Chen, Y.; Juma, C.A.; Yang, C.; Huang, J.; Zhang, X.; Zeng, Y. Differential Inhibition of Target Gene Expression by Human microRNAs. Cells 2019, 8, 791. https://doi.org/10.3390/cells8080791
Li P, Chen Y, Juma CA, Yang C, Huang J, Zhang X, Zeng Y. Differential Inhibition of Target Gene Expression by Human microRNAs. Cells. 2019; 8(8):791. https://doi.org/10.3390/cells8080791
Chicago/Turabian StyleLi, Peng, Yi Chen, Conslata Awino Juma, Chengyong Yang, Jinfeng Huang, Xiaoxiao Zhang, and Yan Zeng. 2019. "Differential Inhibition of Target Gene Expression by Human microRNAs" Cells 8, no. 8: 791. https://doi.org/10.3390/cells8080791