Untranslated Region Luciferase Assay Research Articles

MicroRNA-200a suppresses the Wnt/β-catenin signaling pathway by interacting with β-catenin.

The Wnt/β-catenin signaling pathway is crucial for human organ development and is involved in tumor progression of many cancers. Accumulating evidence suggests that the expression of β-catenin is, in part, regulated by specific microRNAs (miRNAs). The purpose of this study was to determine the expression of a recently identified epithelial to mesenchymal transition (EMT)-associated tumor suppressor microRNA (miR)-200a, in cancer cells. We also aimed to identify specific miR-200a target genes and to investigate the antitumor effects of miR-200a on the Wnt/β-catenin signaling pathway. We employed TOP/FOP flash luciferase assays to identify the effect of miR-200a on the Wnt/β-catenin pathway and we confirmed our observations using fluorescence microscopy. To determine target genes of miR-200a, a 3′ untranslated region (3′ UTR) luciferase assay was performed. Cell viability, invasion and wound healing assays were carried out for functional analysis after miRNA transfection. We further investigated the role of miR-200a in EMT by Western blot analysis. We found fluctuation in the expression of miR-200a that was accompanied by changes in the expression of members of the Wnt/β-catenin signaling pathway. We also determined that miR-200a can directly interact with the 3′ UTR of CTNNB1 (the gene that encodes β-catenin) to suppress Wnt/β-catenin signaling. MiR-200a could also influence the biological activities of SGC790 and U251 cells. Our results demonstrate that miR-200a is a new tumor suppressor that can regulate the activity of the Wnt/β-catenin signaling pathway via two mechanisms. MiR-200a is a candidate target for tumor treatment via its regulation of the Wnt/β-catenin signaling pathway.

MiR-382 targeting of kallikrein 5 contributes to renal inner medullary interstitial fibrosis

Previously we have shown that microRNA miR-382 can facilitate loss of renal epithelial characteristics in cultured cells. This study examined the in vivo role of miR-382 in the development of renal interstitial fibrosis in a mouse model. Unilateral ureteral obstruction was used to induce renal interstitial fibrosis in mice. With 3 days of unilateral ureteral obstruction, expression of miR-382 in the obstructed kidney was increased severalfold compared with sham-operated controls. Intravenous delivery of locked nucleic acid-modified anti-miR-382 blocked the increase in miR-382 expression and significantly reduced inner medullary fibrosis. Expression of predicted miR-382 target kallikrein 5, a proteolytic enzyme capable of degrading several extracellular matrix proteins, was reduced with unilateral ureteral obstruction. Anti-miR-382 treatment prevented the reduction of kallikrein 5 in the inner medulla. Furthermore, the protective effect of the anti-miR-382 treatment against fibrosis was abolished by renal knockdown of kallikrein 5. Targeting of kallikrein 5 by miR-382 was confirmed by 3'-untranslated region luciferase assay. These data support a completely novel mechanism in which miR-382 targets kallikrein 5 and contributes to the development of renal inner medullary interstitial fibrosis. The study provided the first demonstration of an in vivo functional role of miR-382 in any species and any organ system.

MicroRNA-1826 directly targets beta-catenin (CTNNB1) and MEK1 (MAP2K1) in VHL-inactivated renal cancer

The aim of this project is to identify new therapeutic microRNAs (miRNAs) for von Hippel-Lindau (VHL)-inactivated renal cancer cells. We initially identified several potential miRNAs targeting CTNNB1 and MEK1 using several targets scan algorithms. Only miR-1826 was found to target CTNNB1 and MEK1. Therefore, we focused on miRNA-1826 and performed 3' untranslated region (UTR) luciferase assay, functional analyses and association study between miR-1826 expression and renal cancer patient outcomes. miR-1826 expression was significantly lower in renal cancer tissues compared with non-neoplastic areas and lower expression was significantly associated with overall shorter survival and earlier recurrence after radical nephrectomy. Following miR-1826 transfection, 3' UTR luciferase activity and protein expression of beta-catenin and MEK1 were significantly downregulated in renal cancer cells. Introduction of miR-1826 also inhibited renal cancer cell proliferation, invasion and migration. Additionally, miR-1826 promoted apoptosis and G(1) arrest in VHL-inactivated renal cancer cells. Knockdowns of CTNNB1 and MEK1 by small interfering RNAs reproduced the tumor-suppressive effect of miR-1826. Our data suggest that the miR-1826 plays an important role as a tumor suppressor by downregulating beta-catenin and MEK1 in VHL-inactivated renal cancers.

Axotomy-induced miR-21 promotes axon growth in adult dorsal root ganglion neurons.

Following injury, dorsal root ganglion (DRG) neurons undergo transcriptional changes so as to adopt phenotypic changes that promote cell survival and axonal regeneration. Here we used a microarray approach to profile changes in a population of small noncoding RNAs known as microRNAs (miRNAs) in the L4 and L5 DRG following sciatic nerve transection. Results showed that 20 miRNA transcripts displayed a significant change in expression levels, with 8 miRNAs transcripts being altered by more than 1.5-fold. Using quantitative reverse transcription PCR, we demonstrated that one of these miRNAs, miR-21, was upregulated by 7-fold in the DRG at 7 days post-axotomy. In dissociated adult rat DRG neurons lentiviral vector-mediated overexpression of miR-21 promoted neurite outgrowth on a reduced laminin substrate. miR-21 directly downregulated expression of Sprouty2 protein, as confirmed by Western blot analysis and 3′ untranslated region (UTR) luciferase assays. Our data show that miR-21 is an axotomy-induced miRNA that enhances axon growth, and suggest that miRNAs are important players in regulating growth pathways following peripheral nerve injury.

MicroRNA-21 Regulates Vascular Smooth Muscle Cell Function via Targeting Tropomyosin 1 in Arteriosclerosis Obliterans of Lower Extremities

The goal of this study was to determine the expression signature and the potential role of microRNAs in human arteries with arteriosclerosis obliterans (ASO). The expression profiles of microRNAs in human arteries with ASO and in normal control arteries were determined by quantitative reverse transcription-polymerase chain reaction array. Among the 617 detected microRNAs, multiple microRNAs were aberrantly expressed in arteries with ASO. Some of these dysregulated microRNAs were further verified by quantitative reverse transcription-polymerase chain reaction. Among them, microRNA-21 (miR-21) was mainly located in arterial smooth muscle cells (ASMCs) and was increased by more than 7-fold in ASO that was related to hypoxia inducible factor 1-α. In cultured human ASMCs, cell proliferation and migration were significantly decreased by inhibition of miR-21. 3'-Untranslated region luciferase assay confirmed that tropomyosin 1 was a target of miR-21 that was involved in miR-21-mediated cellular effects, such as cell shape modulation. The results suggest that miR-21 is able to regulate ASMC function by targeting tropomyosin 1. The hypoxia inducible factor-1 α/miR-21/tropomyosin 1 pathway may play a critical role in the pathogenesis of ASO. These findings might provide a new therapeutic target for human ASO.

A Comprehensive Analysis of MicroRNA Expression During Human Keratinocyte Differentiation In Vitro and In Vivo

Here, we report a comprehensive investigation of changes in microRNA (miRNA) expression profiles on human keratinocyte (HK) differentiation in vitro and in vivo. We have monitored expression patterns of 377 miRNAs during calcium-induced differentiation of primary HKs, and have compared these patterns with miRNA expression profiles of epidermal stem cells, transient amplifying cells, and terminally differentiated HKs from human skin. Apart from the previously described miR-203, we found an additional nine miRNAs (miR-23b, miR-95, miR-210, miR-224, miR-26a, miR-200a, miR-27b, miR-328, and miR-376a) that are associated with HK differentiation in vitro and in vivo. In situ hybridization experiments confirmed miR-23b as a marker of HK differentiation in vivo. Additionally, gene ontology analysis and functional validation of predicted miRNA targets using 3'-untranslated region-luciferase assays suggest that multiple miRNAs that are upregulated on HK differentiation cooperate to regulate gene expression during skin development. Our results thus provide the basis for further analysis of miRNA functions during epidermal differentiation.