Expression of miRNA-Targeted and Not-Targeted Reporter Genes Shows Mutual Influence and Intercellular Specificity
Abstract
:1. Introduction
2. Results
2.1. miR-21, miR-24 and Let-7 Influence the Expression Not Only of Targeted Renilla Reporter Genes but Also of Non-Targeted Firefly Luciferase Genes
2.2. Sucrose Gradient Centrifugation of Complexes Formed by Firefly and Renilla mRNAs
2.3. Influence of Anti-miR-21, Anti-miR-24, and Different Anti-Let-7 Oligonucleotides on Expression of Reporter Luciferases
2.4. Proteins Potentially Engaged in Regulation of Translation Are Differently Expressed in Me45 and HCT116 Cells
3. Discussion
3.1. Specific miRNA Effects in Cells of the Same Type and Differences between the Effects of the Same miRNA in Different Cell Types
3.2. Pitfalls in Normalization of Results for miRNA-Targeted Gene Expression to Those for a Non-Targeted Gene
4. Materials and Methods
4.1. Cell Lines
4.2. Plasmids
4.3. Anti-microRNA Oligonucleotides (Anti-miRs)
4.4. Transfection Protocol
4.5. Extraction and Assays of RNA
4.6. Luciferase Assays
4.7. Sucrose Gradient Centrifugation
4.8. Calculation of Cell Numbers Serving for Assays
4.9. Microarray Analyses
4.10. Statistical Tests
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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No Target | miR-21 | miR-24 | Let-7 | ||
---|---|---|---|---|---|
Me45 cells | Renilla | 5.56 ± 0.10 | 0.04 ± 0.01 | 56.82 ± 5.62 | 10.15 ± 1.15 |
firefly | 0.18 ± 0.01 | 0.16 ± 0.01 | 3.23 ± 0.46 | 1.75 ± 0.06 | |
HCT116 cells | Renilla | 7.82 ± 0.21 | 0.12 ± 0.02 | 14.88 ± 0.94 | 17.45 ± 1.40 |
firefly | 4.71 ± 0.25 | 1.01 ± 0.05 | 2.46 ± 0.42 | 2.39 ± 0.05 |
Gene | HCT116 Cells | Me45 Cells |
---|---|---|
ABCE1 | 18.68 ± 3.05 | 11.79 ± 6.36 |
CNOT6/CCR4 | 5.66 ± 0.78 | 4.43 ± 2.35 |
CNOT7/CAF1 | 34.72 ± 6.09 | 17.75 ± 7.97 |
CNOT8/CAF2 | 6.19 ± 0.81 | 4.99 ± 1.84 |
CSDE1 | 19.50 ± 2.20 | 19.16 ± 4.97 |
DDX17 | 4.05 ± 1.74 | 4.49 ± 1.45 |
DDX3X | 12.05 ± 1.73 | 10.60 ± 4.78 |
DDX3Y | 0.85 ± 0.80 | 6.23 ± 2.39 |
DDX5 | 62.49 ± 15.75 | 84.26 ± 21.25 |
DICER1 | 1.67 ± 0.41 | 2.16 ± 0.38 |
EEF2 | 59.93 ± 10.37 | 104.99 ± 36.10 |
EIF2C1/AGO1 | 3.81 ± 0.74 | 10.81 ± 4.05 |
EIF2C2/AGO2 | 2.87 ± 0.52 | 2.71 ± 0.71 |
HNRNPH1 | 22.61 ± 5.34 | 21.80 ± 2.97 |
HNRNPL | 20.70 ± 1.39 | 8.18 ± 2.19 |
HNRNPM | 26.78 ± 5.77 | 20.08 ± 11.52 |
HNRNPU | 32.20 ± 6.20 | 25.66 ± 7.63 |
HSP90AA1 | 149.67 ± 14.05 | 112.88 ± 20.58 |
HSP90AB1 | 186.57 ± 26.71 | 145.69 ± 33.39 |
HSPA1A | 20.82 ± 10.19 | 10.42 ± 4.24 |
HSPA5 | 89.45 ± 23.53 | 114.94 ± 28.48 |
HSPA8 | 156.33 ± 28.72 | 149.77 ± 81.01 |
IGF2BP2 | 8.73 ± 0.93 | 17.40 ± 3.16 |
IGF2BP3 | 9.22 ± 1.37 | 8.03 ± 4.71 |
MKNK2 | 10.94 ± 2.08 | 7.51 ± 2.32 |
PABPC1 | 127.80 ± 22.45 | 144.75 ± 20.27 |
PABPC3 | 42.33 ± 7.50 | 31.14 ± 5.15 |
PABPC4 | 15.94 ± 3.45 | 51.53 ± 8.44 |
RBM14 | 6.77 ± 1.31 | 13.66 ± 3.41 |
SRP14 | 52.32 ± 12.92 | 78.13 ± 19.86 |
SYNCRIP | 16.92 ± 2.65 | 8.68 ± 3.94 |
TNRC6B | 4.38 ± 0.85 | 5.49 ± 0.89 |
Gene Name | Forward Primer | Reverse Primer |
---|---|---|
Renilla Luciferase | ACAAGTACCTCACCGCTTGG | GACACTCTCAGCATGGACGA |
Firefly Luciferase | GCTAAGAGCACCCTGATCG | CCTCTGGGGTAATCAGAATGG |
GAPDH | TTTGGCTACAGCAACAGGGTG | TTCCTCTTGTGCTCTTGCTGG |
RPL41 | TCCTGCGTTGGGATTCCGTG | ACGGTGCAACAAGCTAGCGG |
hsa-Let-7i-5p | GTGAGGTAGTAGTTTGTGCTGTT | Universal from miRNA 1st-Strand cDNA Synthesis kit |
hsa-miR-24-3p | TGGCTCAGTTCAGCAGGAACA | Universal from miRNA 1st-Strand cDNA Synthesis kit |
U75 | AGCCTGTGATGCTTTAAGAGTAG | Universal from miRNA 1st-Strand cDNA Synthesis kit |
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Hudy, D.; Rzeszowska-Wolny, J. Expression of miRNA-Targeted and Not-Targeted Reporter Genes Shows Mutual Influence and Intercellular Specificity. Int. J. Mol. Sci. 2022, 23, 15059. https://doi.org/10.3390/ijms232315059
Hudy D, Rzeszowska-Wolny J. Expression of miRNA-Targeted and Not-Targeted Reporter Genes Shows Mutual Influence and Intercellular Specificity. International Journal of Molecular Sciences. 2022; 23(23):15059. https://doi.org/10.3390/ijms232315059
Chicago/Turabian StyleHudy, Dorota, and Joanna Rzeszowska-Wolny. 2022. "Expression of miRNA-Targeted and Not-Targeted Reporter Genes Shows Mutual Influence and Intercellular Specificity" International Journal of Molecular Sciences 23, no. 23: 15059. https://doi.org/10.3390/ijms232315059