miR-151a induces partial EMT by regulating E-cadherin in NSCLC cells.

miR-151a and its host gene, focal adhesion kinase, FAK, are located in a region of chromosome 8q that is frequently amplified in solid tumors, including lung cancer. Lung cancer is the leading cause of cancer deaths worldwide and metastasis remains the major challenge in battling lung cancer mortality. Here, we demonstrate that miR-151a is overexpressed in non-small cell lung cancer (NSCLC) patient specimens, as compared to healthy lung. In addition, miR-151a overexpression promotes proliferation, epithelial-to-mesenchymal transition (EMT) and induces tumor cell migration and invasion of NSCLC cells. Blocking miR-151a expression using anti-miR-151a approaches significantly reduced NCSLC cell proliferative and motility potential. Furthermore, we determined that miR-151a significantly regulates E-cadherin expression. Finally, functional rescue experiments determined that overexpression of E-cadherin in miR-151a NSCLC cell lines potently repressed miR-151a-induced partial EMT and cell migration of NSCLC cells. In conclusion, our findings suggest that miR-151a functions as an oncomiR in NSCLC by targeting E-cadherin mRNA and inducing proliferation, migration and partial EMT.

Activation of the Wnt/ß-catenin signaling cascade after traumatic nerve injury.

Recent data have shown that preservation of the neuromuscular junction (NMJ) after traumatic nerve injury helps to improve functional recovery with surgical repair via matrix metalloproteinase-3 (MMP3) blockade. As such, we sought to explore additional pathways that may augment this response. Wnt3a has been shown to inhibit acetylcholine receptor (AChR) clustering via ß-catenin-dependent signaling in the development of the NMJ. Therefore, we hypothesized that Wnt3a and ß-catenin are associated with NMJ destabilization following traumatic denervation. A critical size nerve defect was created by excising a 10-mm segment of the sciatic nerve in mice. Denervated muscles were then harvested at multiple time points for immunofluorescence staining, quantitative real-time PCR, and western blot analysis for Wnt3a and ß-catenin levels. Moreover, a novel Wnt/ß-catenin transgenic reporter mouse line was utilized to support our hypothesis of Wnt activation after traumatic nerve injury. The expression of Wnt3a mRNA was significantly increased by 2 weeks post-injury and remained upregulated for 2 months. Additionally, ß-catenin was activated at 2 months post-injury relative to controls. Correspondingly, immunohistochemical analysis of denervated transgenic mouse line TCF/Lef:H2B-GFP muscles demonstrated that the number of GFP-positive cells was increased at the motor endplate band. These collective data support that post-synaptic AChRs destabilize after denervation by a process that involves the Wnt/ß-catenin pathway. As such, this pathway serves as a potential therapeutic target to prevent the motor endplate degeneration that occurs following traumatic nerve injury.

Position-independent expression of transgenes in zebrafish.

The variability in expression patterns of transgenes, caused by the influence of neighboring chromatin, is called 'position effect'. Border elements are DNA sequences, which have the ability to alleviate position effects. The abilities of two types of border elements, scs/scs' from the D. melanogaster 87A7 heat shock locus and the A-element from the chicken lysozyme gene, to protect transgenes from position effects were quantified in developing zebrafish embryos. The transgenic construct used was FV3CAT, which consists of the carp beta-actin transcriptional regulatory region, the chloramphenicol acetyltransferase (CAT) gene and the 3'-untranslated region from the Chinook salmon growth hormone gene. FV3CAT constructs flanked by either scs/scs'-elements or A-elements were introduced into zebrafish chromosomes and the spatial and temporal expression patterns of the transgenes were quantified in multiple generations of transgenic zebrafish. Levels of transgene expression were uniform in the pre-differentiated and fully differentiated populations of cells present during embryonic development. Levels of transgene expression were proportional to the numbers of integrated transgenes. Expression of transgenes per cell varied less than two-fold in different transgenic lines. Both types of border elements were able to prevent the influences of neighboring chromatin on transgene expression through three generations of fish. The results are consistent with the ability of border elements to function with equal efficiencies in the many cell types found in vertebrates. Thus, inclusion of border elements in genetic constructs can provide reliable and reproducible levels of gene expression in multiple lines of fish.