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Transforming growth factor-β (TGF-β) signaling and microRNAs (miRNAs) are important gene regulatory components in cancer. Usually in advanced malignant stages, TGF-β signaling is elevated but global miRNA expression is suppressed. Such a gene expression signature is well illustrated in a fibrosis (or mesenchymal) subtype of ovarian cancer (OC) that is of poor prognosis. However, the interplay between the two pathways in the OC subtype has not yet been elucidated. nc886 is a recently identified non-coding RNA implicated in several malignancies. The high expression of nc886 is associated with poor prognosis in 285 OC patients. Herein, we find that in OC nc886 expression is induced by TGF-β and that nc886 binds to Dicer to inhibit miRNA maturation. By preventing the miRNA pathway, nc886 emulates TGF-β in gene expression patterns and potentiates cell adhesion, migration, invasion, and drug resistance. Here we report nc886 to be a molecular link between the TGF-β and miRNA pathways. Ovarian cancers often display elevated TGF-β signaling but repressed miRNA expression. Here the authors identify that non-coding RNA nc886 expression is induced by TGF-β, which then binds to DICER and impairs miRNA maturation.

Without a glucocorticoid (GC) ligand, the transcription factor glucocorticoid receptor (GR) is largely cytoplasmic, with its GC-binding domain held in high affinity conformation by a cluster of chaperones. Binding a GC causes serial dis- and re-associations with chaperones, translocation of the GR to the nucleus, where it binds to DNA sites and associates with coregulatory proteins and basic transcription complexes. Herein, we describe the effects of a potent protective osmolyte, trimethylamine N-oxide (TMAO), on a conditions-dependent \"activation-labile\" mutant GR (GRact/l), which under GR-activating conditions cannot bind GCs in cells or in cell cytosols. In both cells and cytosols, TMAO restores binding to GRact/l by stabilizing it in complex with chaperones. Cells bathed in much lower concentrations of TMAO than those required in vitro show restoration of GC binding, presumably due to intracellular molecular crowding effects.

In the original version of the Supplementary Information file associated with this Article, Supplementary Fig. 18 panel b was inadvertently replaced with a duplicate of panel a. The error has now been fixed and the corrected version of the Supplementary Information PDF is available to download from the HTML version of the Article.

Background Microvascular endothelial barrier dysfunction is the central enigma in spotted fever group (SFG) rickettsioses. Angiogenin (ANG) is one of the earliest identified angiogenic factors, of which some are relevant to the phosphorylation of VE-cadherins that serve as endothelial adherens proteins. Although exogenous ANG is known to translocate into the nucleus of growing endothelial cells (ECs) where it plays a functional role, nuclear ANG is not detected in quiescent ECs. Besides its nuclear role, ANG is thought to play a cytoplasmic role, owing to its RNase activity that cleaves tRNA to produce small RNAs. Recently, such tRNA-derived RNA fragments (tRFs) have been shown to be induced under stress conditions. All these observations raise an intriguing hypothesis about a novel cytoplasmic role of ANG, which is induced upon infection with Rickettsia and generates tRFs that may play roles in SFG rickettsioses. Methods C3H/HeN mice were infected intravenously with a sublethal dose of R. conorii . At days 1, 3, and 5 post infection (p.i.), liver, lung and brain were collected for immunofluorescence (IF) studies of R. conorii and angiogenin (ANG). Human umbilical vein endothelial cells (HUVECs) were infected with R. conorii for 24, 48, and 72 hrs before incubation with 1μg/ml recombinant human ANG (rANG) in normal medium for 2 hrs. HUVEC samples were subjected to IF, exogenous ANG translocation, endothelial permeability, and immunoprecipitation phosphorylation assays. To identify small non-coding RNAs (sncRNAs) upon rickettsial infection, RNAs from pulverized mouse lung tissues and HUVECs were subjected to library preparation and deep sequencing analysis using an Illumina 2000 instrument. Identified sncRNAs were confirmed by Northern hybridization, and their target mRNAs were predicted in silico using BLAST and RNA hybrid programs. Results In the present study, we have demonstrated endothelial up-regulation of ANG, co-localized with SFG rickettsial infection in vivo . We also have provided direct evidence that rickettsial infection sensitizes human ECs to the translocation of exogenous ANG in a compartmentalized pattern at different times post-infection. Typically, exogenous ANG translocates into the nucleus at 24 hrs and to the cytoplasm at 72 hrs post-infection. The ANG cytoplasmic translocation enhances phosphorylation and destabilization of VE-cadherin and attenuates endothelial barrier function. Of note, deep sequencing analysis detected tRFs, mostly derived from the 5'-halves of host tRNAs, that are induced by ANG. Northern hybridization validates the two most abundantly cloned tRFs derived from tRNA-ValGTG and tRNA-GlyGCC, in both mouse tissues and human cells. Bioinformatics analysis predicted that these tRFs may interact with transcripts associated with the endothelial barrier, the host cell inflammatory response, and autophagy. Conclusions Our data provide new insight into the role of compartmentalized ANG during SFG rickettsioses, and highlight its possible mediation through tRFs.

DNA-reactive compounds are harnessed for cancer chemotherapy. Their genotoxic effects are considered to be the main mechanism for the cytotoxicity to date. Because this mechanism preferentially affects actively proliferating cells, it is postulated that the cytotoxicity is specific to cancer cells. Nonetheless, they do harm normal quiescent cells, suggesting that there are other cytotoxic mechanisms to be uncovered. By employing doxorubicin as a representative DNA-reactive compound, we have discovered a cytotoxic mechanism that involves a cellular noncoding RNA (ncRNA) nc886 and protein kinase R (PKR) that is a proapoptotic protein. nc886 is transcribed by RNA polymerase III (Pol III), binds to PKR, and prevents it from aberrant activation in most normal cells. We have shown here that doxorubicin evicts Pol III from DNA and, thereby, shuts down nc886 transcription. Consequently, the instantaneous depletion of nc886 provokes PKR and leads to apoptosis. In a short-pulse treatment of doxorubicin, these events are the main cause of cytotoxicity preceding the DNA damage response in a 3D culture system as well as the monolayer cultures. By identifying nc886 as a molecular signal for PKR to sense doxorubicin, we have provided an explanation for the conundrum why DNA-damaging drugs can be cytotoxic to quiescent cells that have the competent nc886/PKR pathway.

nc886 (= pre-miR-886 or vtRNA2-1) is a non-coding RNA that has been recently identified as a natural repressor for the activity of PKR (Protein Kinase R). The suppression of nc886 activates PKR and thereby provokes a cell death pathway. When combined with the fact that nc886 is suppressed in a wide range of cancer cells, the nc886-PKR relationship suggests a tumor surveillance model. When neoplastic cells develop and nc886 decreases therein, PKR is released from nc886 and becomes the active phosphorylated form, which initiates an apoptotic cascade to eliminate those cells. The nc886-PKR pathway is distinct from conventional mechanisms, such as the immune surveillance hypothesis or intrinsic mechanisms that check/proofread the genomic integrity, and thus represents a novel example of tumor surveillance.

We have recently identified nc886 (pre-miR-886 or vtRNA2-1) as a novel type of non-coding RNA that inhibits activation of protein kinase R (PKR). PKR’s pro-apoptotic role through eukaryotic initiation factor 2 α (eIF2α) phosphorylation is well established in the host defense against viral infection. Paradoxically, some cancer patients have elevated PKR activity; however, its cause and consequence are not understood. Initially, we evaluated the expression of nc886, PKR and eIF2α in non-malignant cholangiocyte and cholangiocarcinoma (CCA) cells. nc886 is repressed in CCA cells and this repression is the cause of PKR’s activation therein. nc886 alone is necessary and sufficient for suppression of PKR via direct physical interaction. Consistently, artificial suppression of nc886 in cholangiocyte cells activates the canonical PKR/eIF2α cell death pathway, suggesting a potential significance of the nc886 suppression and the consequent PKR activation in eliminating pre-malignant cells during tumorigenesis. In comparison, active PKR in CCA cells does not induce phospho-eIF2α nor apoptosis, but promotes the pro-survival nuclear factor-κB pathway. Thus, PKR has a dual life or death role during tumorigenesis. Similarly to the CCA cell lines, nc886 tends to be decreased but PKR tends to be activated in our clinical samples from CCA patients. Collectively from our data, we propose a tumor surveillance model for nc886’s role in the PKR pathway during tumorigenesis.

Gene therapy promises to revolutionize medicine by providing a method to treat diseases at their core level-the living cell-with the ultimate drug-the DNA. However, its success relies on designing transfection systems that can overcome the different delivery barriers present in living organisms. Viral vectors are the most efficient, but they are hampered by safety issues related to their immunogenicity and sometimes fatal toxicity. Synthetic non-viral vectors-cationic lipids, polymers and related gemini surfactants and oligomeric polycations provide promising alternatives to the use of viruses for delivering genes therapeutically, but their efficiency must be increased 1 Fig 1.Heterocyclic cationic lipids in particular were shown to display a good transfection profile and low cytotoxic side effects. Our strategy2 for generating new synthetic cationic vectors for gene delivery bearing pyridinium polar heads is based on the use of highly reactive pyrylium salts, which can react with a primary amine to generate the polar head in single high-yield step. Switching from a monoamine to a di- or polyamine one can readily access the classes of cationic lipids, gemini surfactants or lipophilic polycations. We present a structure-activity relationship study which identified several lead compounds such as 1-7 that display better transfection profiles than commercial transfection systems in vitro and in vivo. The study offers insights on the influence of the structural elements on the physico-chemical properties and transfection efficiency of these classes on non-viral transfection systems, as well as the optimum formulations found to be the most efficient on various tumor cell lines, as well as for in vivo studies. The advantages of the present technology to generate fluorescent molecular markers will also be discussed.

Three oxidized analogs of cholesterol have been characterized for their ability to cause apoptotic cell death in CEM‐C7‐14 human leukemic cells. In addition to testing 15‐ketocholestenol (K15), 15‐ketocholestenol hydroxyethyl ether (CK15), and 7‐ketocholesterol hydroxyethyl ether (CK7), an oxysterol of known apoptotic response, 25‐hydroxycholesterol (25OHC), served as a standard for comparison. Growth studies based on dye exclusion by viable cells while using a sublethal concentration of oxysterols ranked their potency for cell kill as 25OHC>K15>CK15>CK7. Both the TUNEL assay (terminal deoxynucleotidyl transferase‐mediated dUTP‐X nick end labeling), which quantifies the amount of DNA nicks caused by a toxic agent, and the MTT assay, which measures cell metabolism and thus reflects cell viability, substantiated the same rank order. An ELISA assay for evaluating release of DNA fragments into the cytosol after treatment gave a similar potency order. The oncogene c‐myc mRNA was suppressed by all three oxysterols. with 25OHC and K15 being the most potent suppressors. Hoechst and Annexin V staining documented that these oxysterols kill cells by an apoptotic pathway as evidenced by condensation of nuclear chromatin and plasma membrane inversion, respectively. From these in vitro studies, we believe that 25OHC, K15, and possibly CK15 have the potential to be chemotherapeutic agents.

A new approach for generating cationic lipids for gene delivery bearing pyridinium polar heads was described recently 1 . It is based on the reaction of pyrylium salts with primary amines thus generating the polar head and the linker of the non-viral transfection vector in a single, high yield step. The versatility of the synthesis allows the choice of a large variety of aliphatic or aromatic linkers, yielding cationic lipids such as 1-4, some of which act better than commercial transfection systems. The method also allows the easy access to the class of gemini surfactants such as 5 and congeners, and the facile choice of the counterion of the final positively charged species. Additionally, the use of styryl pyrylium salts can yield fluorescent transfection systems and membrane markers such as 6. Detailed insights into their chemical synthesis and transfection properties on different tumor cell lines are presented, together with a complete structure - activity relationship study for each class of lipophilic pyridinium compounds Figure 1.