Abstract
The miR-17–92 cluster encodes 7 miRNAs inside a single polycistronic transcript, and is known as a group of oncogenic miRNAs that contribute to tumorigenesis in several cancers. However, their direct targets remain unclear, and it has been suggested that a single miRNA is capable of reducing the production of hundreds of proteins. The majority of reports on the identification of miRNA targets are based on computational approaches or the detection of altered mRNA levels, despite the fact that most miRNAs are thought to regulate their targets primarily by translational inhibition in higher organisms. In this study, we examined the target profiles of miR-19a, miR-20a and miR-92-1 in MCF-7 breast cancer cells by a quantitative proteomic strategy to identify their direct targets. A total of 123 proteins were significantly increased after the endogenous miR-19a, miR-20a and miR-92-1 were knocked down, and were identified as potential targets by two-dimensional electrophoresis and a mass spectrometric analysis. Among the upregulated proteins, four (PPP2R2A, ARHGAP1, IMPDH1 and NPEPL1) were shown to have miR-19a or miR-20a binding sites on their mRNAs. The luciferase activity of the plasmids with each binding site was observed to decrease, and an increased luciferase activity was observed in the presence of the specific anti-miRNA-LNA. A Western blot analysis showed the expression levels of IMPDH1 and NPEPL1 to increase after treatment with anti-miR-19a, while the expression levels of PPP2R2A and ARHGAP1 did not change. The expression levels of IMPDH1 and NPEPL1 did not significantly change by anti-miR-19a-LNA at the mRNA level. These results suggest that the IMPDH1 and NPEPL1 genes are direct targets of miR-19a in breast cancer, while the exogenous expression of these genes is not associated with the growth suppression of MCF-7 cells. Furthermore, our proteomic approaches were shown to be valuable for identifying direct miRNA targets.
Highlights
MicroRNAs are endogenous small non-coding singlestranded RNAs, 19 to 23 in length [1,2]
We found that all of these miRNAs, except miR-18 family (miR-18a), were expressed at a significantly highly level in MCF-7 breast cancer cells compared to the other cell lines (Fig. 1B)
Since the miR-17-92 cluster is categorized into four groups by alignment of their nucleotide sequences; the miR-17 family, the miR-18 family, the miR-19 family and the miR-92 family [21], in this study, we focused on three representative miRNAs, miR-19a, miR-20a, and miR-92-1
Summary
MicroRNAs (miRNAs) are endogenous small non-coding singlestranded RNAs, 19 to 23 in length [1,2]. MiRNAs have been suggested to have oncogenic or tumor suppressive functions through their negative post-transcriptional regulation of proteincoding genes [3,4]. Many miRNAs exhibit binding activity to the 39 untranslated region (39UTR) of target mRNAs as a result of sequence complementarity. It has been estimated that the miRNAs in a whole cell regulate approximately 30% of all protein-coding genes. By targeting multiple transcripts and affecting the expression of numerous proteins, miRNAs play key roles in cellular development, differentiation, proliferation and apoptosis [6,7,8,9]. Several studies have demonstrated that more than 50% of miRNAs are located in cancer-associated genomic regions [10], suggesting that miRNAs may play an important role in cancer
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