Rama Ana R, Perazzoli Gloria, Cabeza Laura, Mesas Cristina, Quiñonero Francisco, García-Pinel Beatriz, Vélez Celia
Institute of Biopathology and Regenerative Medicine (IBIMER), Center of Biomedical Research (CIBM), University of Granada, Granada, Spain.
Institute of Biosanitary Research from Granada (ibs. GRANADA), Granada, Spain.
Nucleic Acid Ther. 2020 Oct;30(5):325-334. doi: 10.1089/nat.2020.0861. Epub 2020 May 19.
MicroRNA (miRNA) sponges allow the selective blockade of a complete family of associated miRNAs, which induce post-transcriptional gene silencing in their target through binding to 3'UTR mRNA. miRNA-365 and miRNA-145 are downregulated in colorectal cancer (CRC) but not in healthy tissues. Based on this, we constructed two vectors by inserting miRNA sponges (one for miRNA-365 and other for miRNA-145), and used enhanced green fluorescent protein () as a 3'UTR reporter gene to analyze the ability of each sponge to catch its respective miRNA. Quantitative polymerase chain reaction (qPCR) results corroborated that the expression levels of both miRNAs were lower in CRC cell lines than in normal colon cell lines. 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 in 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.
微小RNA（miRNA）海绵能够选择性地阻断与之相关的整个miRNA家族，这些miRNA通过与3'UTR mRNA结合在其靶标中诱导转录后基因沉默。miRNA - 365和miRNA - 145在结直肠癌（CRC）中表达下调，但在健康组织中并非如此。基于此，我们通过插入miRNA海绵构建了两个载体（一个针对miRNA - 365，另一个针对miRNA - 145），并使用增强型绿色荧光蛋白（EGFP）作为3'UTR报告基因来分析每个海绵捕获其相应miRNA的能力。定量聚合酶链反应（qPCR）结果证实，与正常结肠细胞系相比，两种miRNA在CRC细胞系中的表达水平较低。流式细胞术分析显示，在转染了两种海绵的细胞系中，EGFP表达水平降低，在正常细胞系中较高，而CRC细胞系中下降最小。此外，这种下降与通过qPCR获得的两种miRNA的表达水平成反比。荧光显微镜证实了这些结果，显示荧光有类似的下降。我们提出了一种新的载体系统，以特定方式将基因表达导入CRC细胞而不影响健康细胞，防止对健康组织造成损害。
