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.

Lipid nanoparticle siRNA systems for silencing the androgen receptor in human prostate cancer in vivo.

Abstract: The androgen receptor (AR) plays a critical role in the progression of prostate cancer. Silencing this protein using short-hairpin RNA (shRNA) has been correlated with tumor growth inhibition and decreases in serum prostate specific antigen (PSA). In our study, we have investigated the ability of lipid nanoparticle (LNP) formulations of small-interfering RNA (siRNA) to silence AR in human prostate tumor cell lines in vitro and in LNCaP xenograft tumors following intravenous (i.v.) injection. In vitro screening studies using a panel of cationic lipids showed that LNPs containing the ionizable cationic lipid 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) exhibited the most potent AR silencing effects in LNCaP cells. This is attributed to an optimized ability of DLin-KC2-DMA-containing LNP to be taken up into cells and to release the siRNA into the cell cytoplasm following endocytotic uptake. DLin-KC2-DMA LNPs were also effective in silencing the AR in a wild-type AR expressing cell line, LAPC-4, and a variant AR expressing cell line, CWR22Rv1. Importantly, it is demonstrated that LNP AR-siRNA systems containing DLin-KC2-DMA can silence AR gene expression in distal LNCaP xenograft tumors and decrease serum PSA levels following i.v. injection. To our knowledge, this is the first report demonstrating the feasibility of LNP delivery of siRNA for silencing AR gene expression in vivo.

A Hairpin Structure in the R Region of the Human Immunodeficiency Virus Type 1 RNA Genome Is Instrumental in Polyadenylation Site Selection

Abstract: Reverse transcription of a retroviral genome produces a double-stranded DNA copy that is longer than the RNA template at both the 5′ and 3′ ends (40). This additional genetic information is generated in an intricate reverse transcription mechanism that includes strand transfers onto redundant sequences. All retroviral RNA genomes therefore contain a repeat (R) region that constitutes the extreme 5′ and 3′ ends of the viral transcript. The length of this R region varies significantly among retroviruses. It can be as short as 16 nucleotides in the mouse mammary tumor virus and as long as 228 nucleotides in the human T-cell leukemia virus type 1 (HTLV-1). At the 3′ end of the viral RNA, the AAUAAA polyadenylation signal is recognized by cellular enzymes that produce a polyadenylated RNA. Some retroviruses with an extended R region encode the polyadenylation signal within the R region such that it is present at both the 5′ and 3′ ends of the viral transcript. This necessitates differential regulation either to repress recognition of the 5′ polyadenylation signal or to enhance usage of the 3′ signal. The human immunodeficiency virus type 1 (HIV-1) has been reported to have both regulatory features. Usage of the 3′ polyadenylation site is promoted by an upstream enhancer motif that is uniquely present at the 3′ end of viral transcripts (13, 20, 23, 41, 42). This upstream sequence element (USE) appears to stabilize binding of the cleavage polyadenylation specificity factor (CPSF) to the AAUAAA hexamer motif (24). Repression of the 5′ polyadenylation site is mediated by two mechanisms. First, the 5′ polyadenylation site becomes active when moved further downstream in the transcript (14, 44). Thus, the 5′ HIV-1 polyadenylation site is repressed because it is positioned too close to the transcription initiation site. A possible mechanistic explanation for this effect was recently provided by the observation that polyadenylation factors gain access to the nascent transcript through the RNA polymerase II complex (34). It is possible that the transcription complex engaged in synthesis of the HIV-1 leader transcript is not yet competent for polyadenylation. A second possible repression mechanism is that the 5′ polyadenylation site is negatively influenced by the major splice donor signal (SD) that is uniquely present downstream of the 5′ polyadenylation site (3, 4). Mutational inactivation of the SD site in full-length HIV-1 transcripts triggered usage of the 5′ polyadenylation site (3). This repression is mediated by binding of the U1 snRNP to the splice donor site, but it is currently unknown how this affects 5′ polyadenylation site usage (4). This example may be one of a growing number of cases in which the splicing machinery influences the process of polyadenylation (16). Previous studies with reporter gene constructs indicated that the HIV-1 sequence represents an inherently efficient polyadenylation signal. Transfection studies with mutant HIV-1 polyadenylation signals indicated that the efficiency of this polyadenylation site can be down-modulated by stable RNA structure (30). When in vitro evolution techniques were used to select for functional variants of the HIV-1 polyadenylation site, sequences lacking stable secondary structure were obtained (27, 28). Notwithstanding the potential repressive effect of RNA structure, we previously reported that the natural HIV-1 polyadenylation site is embedded within a hairpin structure of intermediate stability, which we have termed the polyA hairpin (Fig. ​(Fig.11 and ​and2),2), which flanks the well-known trans-acting responsive (TAR) element (17). Although the sequence of this part of the viral genome varies significantly among different HIVs and simian immunodeficiency viruses (SIVs), all isolates can fold a similar polyA hairpin structure of comparable thermodynamic stability (9). The phylogenetic conservation suggested a critical role for this structured RNA motif in virus replication, and this was confirmed in studies with mutant viruses (18). Opening of the hairpin structure in both the 5′R and 3′R regions of the HIV-1 genome did severely affect virus replication. Through prolonged culturing of these mutant viruses, revertant viruses with improved replication capacity were obtained. Analysis of such phenotypic revertants revealed that additional mutations had been introduced into the sequences encoding the polyA hairpin. Although different mutations were observed in individual revertants, all nucleotide changes restored the hairpin conformation (10). These results demonstrate an absolute requirement for this structured RNA motif in virus replication, but they do not reveal the function of the 5′ or 3′ element. Previous studies indicated that the 5′ polyA hairpin contributes to packaging of the RNA genome into virus particles (18, 33), but the hairpin may play additional roles in the replication cycle. FIG. 1 A tandem hairpin motif is present at both ends of HIV-1 genomic RNA. (A) A schematic of HIV-1 proviral DNA is shown at top with the LTRs subdivided into the U3, R, and U5 domains. The terminal R and U5 elements (R region is boxed) are also present in ... FIG. 2 The wild-type and mutant polyA hairpins. The wild-type polyA hairpin conformation was established by several lines of evidence: RNA structure probing, phylogenetic comparison, and the analysis of mutants (reviewed in reference 7). The hairpin was mutated ... In this study, we set out to test whether the HIV-1 polyA hairpin is involved in differential regulation of polyadenylation. For instance, the stem-loop structure may either stimulate polyadenylation in the 3′R context or inhibit polyadenylation in the 5′R context. We therefore analyzed the viral transcripts generated by HIV-1 genomes with mutant or revertant hairpin motifs at either the 5′ or 3′ end. We report that destabilization of the 5′ polyA hairpin triggers premature polyadenylation, suggesting that the wild-type structure is involved in occlusion of the 5′ polyadenylation site. In the 3′ context with the USE enhancer, the wild-type polyA hairpin does not interfere with efficient polyadenylation. However, 3′ polyadenylation can be efficiently inhibited by further stabilization of the hairpin. These results suggest that the role of the polyA hairpin is to create a regulatable polyadenylation site, which can be either repressed in the presence of silencers (5′ situation) or activated in the presence of an enhancer (3′ situation). The thermodynamic stability of the polyA hairpin needs to be fine-tuned to allow the on-off switching of polyadenylation.

Connexin43 enhances glioma invasion by a mechanism involving the carboxy terminus.

Abstract: Gliomas are particularly difficult to cure owing largely to their invasive nature. The neoplastic changes of astrocytes which give rise to these tumors frequently include a reduction of connexin43 (Cx43), the most abundant connexin isoform expressed in astrocytes. Cx43 is a subunit of gap junctions (GJ), intercellular channels which directly link the cytosol of adjacent cells and allow the regulated passage of ions and small molecules. To examine the role of Cx43 in glioma motility, we identified two variant C6 cell lines which endogenously express high (C6-H) or low (C6-L) levels of Cx43. In wound healing and transwell assays, C6-H cells were more motile than C6-L cells. To deduce whether Cx43 mediated these differences, assays were conducted on C6-H cells retrovirally transduced with Cx43 shRNA. Coincident with the stable knockdown of endogenous Cx43, a decrease in motility and invasion was observed. Gap junctional intercellular communication was also decreased, however motility assays conducted in the presence of GJ inhibitors did not reveal significant differences in cell motility. C6 cells transfected with full length or C-terminal truncated Cx43 (Cx43DeltaCT) were subjected to the aforementioned motility assays to expose alternate mechanisms of Cx43-mediated motility. Cells expressing full length Cx43 exhibited increased motility while cells expressing Cx43DeltaCT did not. This report, the first in which RNAi has been employed to reduce Cx43 expression in gliomas, indicates that the downregulation of Cx43 decreases motility of C6 cells. Furthermore, it is the first report to suggest that the Cx43 CT plays an important role in glioma motility.

Transient Receptor Potential Melastatin 2 Is Required for Lipopolysaccharide-Induced Cytokine Production in Human Monocytes

Abstract: Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable nonselective cation channel that is stimulated by oxidative stress and specifically activated by intracellular ADP-ribose. Because TRPM2 is highly expressed in immunocytes, a role of this channel in inflammation processes has been proposed. The aim of the current study was to determine the function of TRPM2 in LPS-induced cytokine production of human monocytes. Incubation of human primary monocytes with LPS resulted in an upregulation of TRPM2 mRNA, protein, and of ADP-ribose-induced membrane currents. By using short hairpin RNA to downregulate TRPM2 expression in THP-1 monocytes, we demonstrate that TRPM2 is required for the LPS-induced production of IL-6, IL-8, IL-10, and TNF-alpha. Application of LPS led to a time-dependent increase in intracellular Ca(2+) concentrations in THP-1 cells that was clearly reduced by downregulation of TRPM2. Omission of extracellular Ca(2+) strongly decreased TNF-alpha production in TRPM2-expressing cells. Thus, TRPM2-mediated Ca(2+) entry is a central mechanism for LPS-induced cytokine production in monocytic cells. The identification of TRPM2 as a major player in this LPS-dependent process makes it a promising tool in modulating monocyte functions.

Down-Regulation of WAVE3, a Metastasis Promoter Gene, Inhibits Invasion and Metastasis of Breast Cancer Cells

Abstract: The expression of WAVE3, an actin-cytoskeleton and reorganization protein, is elevated in malignant human breast cancer, yet the role of WAVE3 in promoting tumor progression remains undefined. We have recently shown that knockdown of WAVE3 expression in human breast adenocarcinoma MDA-MB-231 cells using small interfering RNA resulted in a significant reduction of cell motility, migration, and invasion, which correlated with a reduction in the levels of active p38 mitogen-activated protein kinase. Here, we investigated the effect of stable suppression of WAVE3 by short hairpin RNA on tumor growth and metastasis in xenograft models. Breast cancer MDA-MB-231 cells expressing short hairpin RNA to WAVE3 (shWAVE3) showed a significant reduction in Matrigel invasion and formation of lung colonies after tail-vein injection in SCID mice. In the orthotopic model, we observed a reduction in growth rate of the primary tumors, as well as in the metastases to the lungs. We also show that suppression of p38 mitogen-activated protein kinase activity by dominant-negative p38 results in comparable phenotypes to the knockdown of WAVE3. These studies provide direct evidence that the WAVE3-p38 pathway contributes to breast cancer progression and metastasis.