Abstract
The miR-17-92 cluster is involved in animal development and homeostasis, and its dysregulation leads to human diseases such as cancer. In the present study, we investigated the functional link between miR-17-92 cluster and Wnt/β-catenin signaling pathway in ICP2 and DF1 cells. We demonstrated that ectopic expression of either LEF1 or β-catenin increased the promoter activity of the miR-17-92 cluster host gene (MIR17HG) and combined ectopic expression of LEF1 and β-catenin further enhanced the promoter activity; while knockdown of either LEF1 or β-catenin reduced the MIR17HG promoter activity. Both LEF1 and β-catenin could directly bind to the MIR17HG promoter. Furthermore, we demonstrated that low doses of lithium chloride (LiCl), an activator of Wnt/β-catenin signaling pathway, increased MIR17HG promoter activity and the endogenous expression of the miR-17-92 cluster, while high doses of LiCl had the opposite effects. Treatment with XAV-939, an inactivator of the Wnt/β-catenin pathway, reduced the endogenous expression of miR-17-92 cluster. Finally, we found that low doses of LiCl promoted the proliferation of ICP2 and DF1 cells, while high doses of LiCl inhibited the proliferation of ICP2 and DF1 cells. Taken together, our results reveal that MIR17HG is a target of LEF1 and the Wnt/β-catenin pathway and suggest that the miR-17-92 cluster may, at least in part, mediate the proliferation-promoting effect of the Wnt/β-catenin pathway in cell proliferation.
Highlights
MicroRNAs are single-stranded RNA molecules of 19–22 nucleotides in length, which regulate gene expression post-transcriptionally (Bartel, 2004; Eulalio et al, 2008)
The results showed that compared to the empty vector pEASY-Blunt-M2, at low doses (0.1–0.2 μg), pEASY-Blunt-M2-LEF1 increased the miR-17-92 cluster host gene (MIR17HG) promoter activity by over 1.4-fold in ICP2 and DF1 cells (P < 0.05), whereas at high doses (0.5–0.7 μg), pEASY-Blunt-M2LEF1 decreased the promoter activity in ICP2 cells (P < 0.05) (Figure 1)
To determine whether the potential LEF1 binding site is required for the regulation of MIR17HG transcription by LEF1, the consensus LEF1 binding site CTTTGTT was substituted by CGAATTC in the MIR17HG promoter reporter construct, and the resulting LEF1 binding site-mutated reporter construct was designated pGL3-MT-MIR17HG-Luc
Summary
MicroRNAs (miRNAs) are single-stranded RNA molecules of 19–22 nucleotides in length, which regulate gene expression post-transcriptionally (Bartel, 2004; Eulalio et al, 2008). They typically bind to the 3 -untranslated region (3 UTR) of target mRNAs, resulting in translational repression or mRNA degradation (Bartel, 2009). To date, accumulating evidence has demonstrated that miRNAs exert crucial roles in cell proliferation, differentiation, apoptosis, development, and oncogenesis. MicroRNAs genes are scattered across chromosomes either individually or in clusters, in which two or more miRNAs are transcribed from adjacent miRNA genes within a short distance. The polycistronic miRNA cluster, miR-17-92, is one of the best-studied miRNA clusters.
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