Small hairpin RNA

About: Small hairpin RNA is a research topic. Over the lifetime, 9279 publications have been published within this topic receiving 285471 citations.

Tetraspanin protein CD9 interacts with metalloprotease CD10 and enhances its release via exosomes.

Abstract: Tetraspanins interact with a wide variety of transmembrane and intracellular proteins called molecular partners, and modulate their function. In this article, we describe a new partner of tetraspanin web, membrane metalloprotease CD10, which is selectively associated with CD9. By constructing chimeras between tetraspanins CD9 and CD82 (the latter does not interact with CD10) or by using site-directed mutagenesis, we determined that a portion of the large extracellular loop from the CCG motif to transmembrane domain 4, as well as the C-terminal tail of CD9, are involved in the interaction with CD10. The stable expression of wild-type CD9 in K562 CD10-positive cells enhanced the level of CD10 released with exosomes five-fold. In contrast, the expression of chimeric CD9, which contained the cytoplasmic C-terminal domain from CD82, had little effect on CD10 release. Short hairpin RNA knockdown of CD9 expression in Nalm-6 pre-B cells resulted in a two-fold reduction in the amount of endogenous CD10 released with microvesicles. The peptidase activity of CD10 measured either on cells or on exosomes correlated with the level of CD10 expression, and was not significantly modulated by CD9 expression as such. Our data suggest that the interaction of CD10 with tetraspanin CD9 can play an important role in the redistribution of peptidase activity from the cell surface to outer microenvironments. In bone marrow, where CD10 presumably contributes to the maturation of pre-B cells and migration of B cells to the blood circulation, release of CD10 peptidase activity with exosomes may effectively regulate extracellular matrix microenvironments. Structured digital abstract CD9 physically interacts with CD179b, Tetraspanin-9, CD63, CD81, HLA class II, Uncharacterized protein C14orf166B, Tetraspanin-14, HLA class I, Galactokinase, Protein disulfide-isomerase A6, CD98, CD19, CD316, CD146, CD92, Annexin A6, CD156c, CD71, CD10, CD29, CD315, DAAM1 and CD49f by anti bait coimmunoprecipitation (View interaction) CD10 physically interacts with CD9 by anti bait coimmunoprecipitation (View Interaction: 1, 2, 3, 4) CD10 physically interacts with CD179b, Protein FAM207A, CD9, CD81, Uncharacterized protein C22orf13, HLA class II, Uncharacterized protein C3orf26, HLA class I, Protein C14orf21, Annexin A6 and DAAM1 by anti bait coimmunoprecipitation (View interaction)

Heat Shock Protein-90 Inhibitors Increase MHC Class I-Related Chain A and B Ligand Expression on Multiple Myeloma Cells and Their Ability to Trigger NK Cell Degranulation

Abstract: Modulation of the host immune system represents a promising therapeutic approach against cancer, including multiple myeloma. Recent findings indicate that the NK group 2D (NKG2D)- and DNAX accessory molecule-1 (DNAM-1)-activating receptors play a prominent role in tumor recognition and elimination by cytotoxic lymphocytes, suggesting that the levels of NKG2D and DNAM-1 ligand expression on tumor cells may be a critical factor to improve the immune response against cancer. In this study, we tested the effect of 17-allylaminogeldanamycin and radicicol, drugs targeting the heat shock protein-90 (HSP-90) chaperone protein and displaying antimyeloma activity, on the expression of NKG2D and DNAM-1 ligands in human myeloma cell lines. We demonstrate that HSP-90 inhibitors are able to up-regulate both MHC class I chain-related (MIC) A and MICB protein surface and mRNA expression in human myeloma cell lines, without any significant effect on the basal expression of the DNAM-1 ligand poliovirus receptor CD155, or induction of nectin-2 and UL16-binding proteins. Activation of the transcription factor heat shock factor-1 by HSP-90 inhibitors is essential for the up-regulation of MICA/MICB expression and knockdown of heat shock factor-1 using small hairpin RNA interference blocks this effect. Moreover, in vitro and in vivo binding of heat shock factor-1 to MICA and MICB promoters indicates that it may enhance NKG2D ligand expression at the transcriptional level. Finally, exposure to HSP-90 inhibitors renders myeloma cells more efficient to activate NK cell degranulation and a blocking Ab specific for NKG2D significantly reduces this effect. Thus, these results provide evidence that targeting NKG2D ligands expression may be an additional mechanism supporting the antimyeloma activity of HSP-90 inhibitors and suggest their possible immunotherapeutic value.

A Novel Function of YWHAZ/β-Catenin Axis in Promoting Epithelial–Mesenchymal Transition and Lung Cancer Metastasis

Abstract: YWHAZ, also known as 14-3-3zeta, has been reportedly elevated in many human tumors, including non-small cell lung carcinoma (NSCLC) but little is known about its specific contribution to lung cancer malignancy. Through a combined array-based comparative genomic hybridization and expression microarray analysis, we identified YWHAZ as a potential metastasis enhancer in lung cancer. Ectopic expression of YWHAZ on low invasive cancer cells showed enhanced cell invasion, migration in vitro, and both the tumorigenic and metastatic potentials in vivo. Gene array analysis has indicated these changes associated with an elevation of pathways relevant to epithelial-mesenchymal transition (EMT), with an increase of cell protrusions and branchings. Conversely, knockdown of YWHAZ levels with siRNA or short hairpin RNA (shRNA) in invasive cancer cells led to a reversal of EMT. We observed that high levels of YWHAZ protein are capable of activating β-catenin-mediated transcription by facilitating the accumulation of β-catenin in cytosol and nucleus. Coimmunoprecipitation assays showed a decrease of ubiquitinated β-catenin in presence of the interaction between YWHAZ and β-catenin. This interaction resulted in disassociating β-catenin from the binding of β-TrCP leading to increase β-catenin stability. Using enforced expression of dominant-negative and -positive β-catenin mutants, we confirmed that S552 phosphorylation of β-catenin increases the β-catenin/YWHAZ complex formation, which is important in promoting cell invasiveness and the suppression of ubiquitnated β-catenin. This is the first demonstration showing YWHAZ through its complex with β-catenin in mediating lung cancer malignancy and β-catenin protein stability.

Brief Report Efficient Lentiviral Vectors for Short Hairpin RNA Delivery into Human Cells

Abstract: RNA interference is an evolutionarily conserved process of gene silencing that in plants serves as a natural defense mechanism against exogenous viral agents. RNA interference is becoming an important tool for the study of biological processes through reverse genetics and has potential for therapeutic applications in humans; however, effective delivery is still a major issue. Small interfering RNA (siRNA) and short hairpin RNA (shRNA) have been introduced into cells by transfection of chemically synthesized and RNA expression via plasmid cassettes utilizing RNA polymerase III transcription. The employment of siRNA/shRNA for gene knockout requires an efficient stable transfection or transduction process. Here, we report the successful construction of lentiviral vectors to express shRNA stably in human cells. We demonstrate that lentiviral vectors expressing siRNA directed to the reporter gene luciferase, when stably transduced into human cells without drug selection, are capable of protecting the cells from infection by a lentiviral vector encoding humanized firefly luciferase as a reporter gene. We observed 16- to 43-fold reduction of gene expression in infected cells transduced with shRNA vectors relative to cells transduced with control vectors. This model system demonstrates the utility of lentiviral vectors to stably express shRNA as both a cellular gene knockout tool and as a means to inhibit exogenous infectious agents such as viruses in human cells.

RNA-binding protein Musashi1 modulates glioma cell growth through the post-transcriptional regulation of Notch and PI3 kinase/Akt signaling pathways.

Abstract: Musashi1 (MSI1) is an RNA-binding protein that plays critical roles in nervous-system development and stem-cell self-renewal. Here, we examined its role in the progression of glioma. Short hairpin RNA (shRNA)-based MSI1-knock down (KD) in glioblastoma and medulloblastoma cells resulted in a significantly lower number of self renewing colony on day 30 (a 65% reduction), compared with non-silencing shRNA-treated control cells, indicative of an inhibitory effect of MSI1-KD on tumor cell growth and survival. Immunocytochemical staining of the MSI1-KD glioblastoma cells indicated that they ectopically expressed metaphase markers. In addition, a 2.2-fold increase in the number of MSI1-KD cells in the G2/M phase was observed. Thus, MSI1-KD caused the prolongation of mitosis and reduced the cell survival, although the expression of activated Caspase-3 was unaltered. We further showed that MSI1-KD glioblastoma cells xenografted into the brains of NOD/SCID mice formed tumors that were 96.6% smaller, as measured by a bioluminescence imaging system (BLI), than non-KD cells, and the host survival was longer (49.3±6.1 days vs. 33.6±3.6 days; P<0.01). These findings and other cell biological analyses suggested that the reduction of MSI1 in glioma cells prolonged the cell cycle by inducing the accumulation of Cyclin B1. Furthermore, MSI1-KD reduced the activities of the Notch and PI3 kinase-Akt signaling pathways, through the up-regulation of Numb and PTEN, respectively. Exposure of glioma cells to chemical inhibitors of these pathways reduced the number of spheres and living cells, as did MSI1-KD. These results suggest that MSI1 increases the growth and/or survival of certain types of glioma cells by promoting the activation of both Notch and PI3 kinase/Akt signaling.