Aguilera, A., Perazzoli, G., Cabeza, L., Mesas, C., Quiñonero, F., García-Pinel, B., Velez, C., & Rama, A.R. (2019). Novel miRNA Sponges to Specifically Modulate Gene Expression in Colon Cancer Cells. Preprints. https://doi.org/10.20944/preprints201911.0014.v1
Aguilera, A., Celia Velez and Ana Rosa Rama. 2019 "Novel miRNA Sponges to Specifically Modulate Gene Expression in Colon Cancer Cells" Preprints. https://doi.org/10.20944/preprints201911.0014.v1
miRNA sponges allow the selective blockade of a complete family of associated miRNAs which induce posttranscriptional gene silencing in its target through binding to 3´UTR mRNA. MiRNA-365 and miRNA-145 are down-regulated in colorectal cancer (CRC), but not in health tissues. Based on this, we constructed two vectors by inserting miRNA sponge (one for miRNA-365 and other for miRNA-145), and used EGFP (enhanced green fluorescent protein) as a 3′ UTR reporter gene to analyse the ability of each sponge to catch its respective miRNA. qPCR results corroborated that the expression levels of both miRNAs were lower in CRC cell lines than in normal colon cell line. Flow cytometry analysis revealed a decrease of the EGFP expression levels in the cell lines transfected with both sponges, being higher on the normal cell line while CRC cell lines presented a minimal decline. Also, this decrease was inversely proportional to the levels of expression of both miRNAs obtained by qPCR. These results were corroborated by fluorescence microscopy, showing a similar decrease fluorescence. We propose a new vector system to carry in a specific way the expression of genes to CRC cells without affecting healthy cells, preventing damage to healthy tissues.
miRNA; Sponges; miRNA-365; miRNA-145; CRC
Biology and Life Sciences, Biochemistry and Molecular Biology
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