CRED9: A differentially expressed elncRNA regulates expression of transcription factor CEBPA.

Enhancer RNAs (eRNA) are non-coding transcripts produced from active enhancers and have potential gene regulatory function. CCAAT enhancer-binding protein alpha (CEBPA) is a transcription factor generally involved in metabolism, cell cycle inhibition, hematopoiesis, adipogenesis, hepatogenesis, and is associated with tumorigenesis. In this study, we demonstrate that an enhancer-associated long non-coding RNA (elncRNA), transcribed from an enhancer located 9kb downstream from the transcriptional start site (TSS) of CEBPA, positively regulates the expression of CEBPA. As a result, we named this elncRNA 'CEBPA regulatory elncRNA downstream 9kb' or 'CRED9'. CRED9 expression level positively correlates with CEBPA mRNA expression across multiple cell lines as detected by RT droplet digital PCR. Knockdown of CRED9 resulted in a reduction of CEBPA mRNA expression in Hep3B cells. Additionally, CRED9 knockdown in Hep3B and HepG2 cells resulted in lower CEBPA protein expression. We also found that knockdown of CRED9 in Hep3B cells caused a 57.8% reduction in H3K27ac levels at the +9kb CEBPA enhancer. H3K27ac has previously been described as a marker of active enhancers. Taken together, the evidence presented here supports a previously proposed model whereby, in some contexts, eRNA transcripts are necessary to amplify and maintain H3K27ac levels at a given enhancer. Ultimately, this study adds to the growing body of evidence that elncRNA transcripts have important roles in enhancer function and gene regulation.

Dual Mechanisms of Action of Self-Delivering, Anti-HIV-1 FANA Oligonucleotides as a Potential New Approach to HIV Therapy.

Currently, the most effective and durable therapeutic option for HIV-1 infection is combination antiretroviral therapy (cART). Although cART is powerful and can delay viral evolution of drug resistance for decades, it is associated with limitations, including an inability to eradicate the virus and a potential for adverse effects. Therefore, it is imperative to discover new HIV therapeutic modalities. In this study, we designed, characterized, and evaluated the in vitro potency of 2'-deoxy-2'-fluoroarabinonucleotide (FANA) modified antisense oligonucleotides (ASOs) targeting highly conserved regions in the HIV-1 genome. Carrier-free cellular internalization of FANA ASOs resulted in strong suppression of HIV-1 replication in HIV-1-infected human primary cells. In vitro mechanistic studies suggested that the inhibitory effect of FANA ASOs can be attributed to RNase H1 activation and steric hindrance of dimerization. Using 5'-RACE PCR and sequencing analysis, we confirmed the presence of human RNase H1-mediated target RNA cleavage products in cells treated with FANA ASOs. We observed no overt cytotoxicity or immune responses upon FANA ASO treatment. Together, our results strongly suggest that FANA ASOs hold great promise for antiretroviral therapy. The dual ability of FANA ASOs to target RNA by recruiting RNase H1 and/or sterically blocking RNA dimerization further enhances their therapeutic potential.

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry.

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious cycle further requires the formation of viral/host protein complexes with HIV-1 RNA to regulate the splicing and enable nucleocytoplasmic transport. The HIV-1 Rev protein accomplishes the nuclear export of HIV-1 mRNAs through multimerization with intronic cis-acting targets - the Rev response element (RRE). A nucleolar localization signal (NoLS) exists within the COOH-terminus of the Rev arginine-rich motif (ARM), allowing the accumulation of Rev/RRE complexes in the nucleolus. Nucleolar factors are speculated to support the HIV-1 infectious cycle through various other functions in addition to mediating mRNA-independent nuclear export and splicing. We describe an immunoprecipitation method of wild-type (WT) Rev in comparison to Rev nucleolar mutations (deletion and single-point Rev-NoLS mutations) in the presence of HIV-1 replication for mass spectrometry. Nucleolar factors implicated in the nucleocytoplasmic transport (nucleophosmin B23 and nucleolin C23), as well as cellular splicing factors, lose interaction with Rev in the presence of Rev-NoLS mutations. Various other nucleolar factors, such as snoRNA C/D box 58, are identified to lose interaction with Rev mutations, yet their function in the HIV-1 replication cycle remain unknown. The results presented here demonstrate the use of this approach for the identification of viral/host nucleolar factors that maintain the HIV-1 infectious cycle. The concepts used in this approach are applicable to other viral and disease models requiring the characterization of understudied pathways.

Multi-center phase II trial of bortezomib and rituximab maintenance combination therapy in patients with mantle cell lymphoma after consolidative autologous stem cell transplantation.

BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive and incurable lymphoma. Standard of care for younger patients with MCL is induction chemotherapy followed by autologous stem cell transplantation (auto-HCT). Rituximab maintenance after auto-HCT has been shown to improve progression-free survival (PFS) and overall survival (OS) in MCL. Bortezomib maintenance therapy has also been shown to be tolerable and feasible in this setting. However, the combination of bortezomib and rituximab as maintenance therapy post-auto-HCT has not been studied. METHODS: We conducted a multicenter, phase II trial of bortezomib given in combination with rituximab as maintenance in MCL patients after consolidative auto-HCT. Enrolled patients (n = 23) received bortezomib 1.3 mg/m2 subcutaneously weekly for 4 weeks every 3 months (up to 24 months) and rituximab 375 mg/m2 intravenously weekly for 4 weeks every 6 months (up to 24 months) for a total duration of 2 years. The primary study endpoint was disease-free survival (DFS). RESULTS: With a median follow-up of 35.9 months, the 2-year DFS probability was 90.2% (95% CI 66-97), and 2-year OS was 94.7% (95% CI 68-99). The most frequent grade 3/4 toxic events were neutropenia (in 74% of patients) and lymphopenia (in 35%). The incidence of peripheral neuropathy was 48% for grade 1, 9% for grade 2, and 0% for grade 3/4. We also examined the role of quantitative cyclin D1 (CCND1) mRNA in monitoring minimal residual disease. CONCLUSION: Combined bortezomib and rituximab as maintenance therapy in MCL patients following auto-HCT is an active and well-tolerated regimen. TRIAL REGISTRATION: ClinicalTrials.gov NCT01267812 , registered Dec 29, 2010.

High throughput sequencing analysis of RNA libraries reveals the influences of initial library and PCR methods on SELEX efficiency.

The systemic evolution of ligands by exponential enrichment (SELEX) technique is a powerful and effective aptamer-selection procedure. However, modifications to the process can dramatically improve selection efficiency and aptamer performance. For example, droplet digital PCR (ddPCR) has been recently incorporated into SELEX selection protocols to putatively reduce the propagation of byproducts and avoid selection bias that result from differences in PCR efficiency of sequences within the random library. However, a detailed, parallel comparison of the efficacy of conventional solution PCR versus the ddPCR modification in the RNA aptamer-selection process is needed to understand effects on overall SELEX performance. In the present study, we took advantage of powerful high throughput sequencing technology and bioinformatics analysis coupled with SELEX (HT-SELEX) to thoroughly investigate the effects of initial library and PCR methods in the RNA aptamer identification. Our analysis revealed that distinct "biased sequences" and nucleotide composition existed in the initial, unselected libraries purchased from two different manufacturers and that the fate of the "biased sequences" was target-dependent during selection. Our comparison of solution PCR- and ddPCR-driven HT-SELEX demonstrated that PCR method affected not only the nucleotide composition of the enriched sequences, but also the overall SELEX efficiency and aptamer efficacy.

Dual functional BAFF receptor aptamers inhibit ligand-induced proliferation and deliver siRNAs to NHL cells.

The B-cell-activating factor (BAFF)-receptor (BAFF-R) is restrictedly expressed on B-cells and is often overexpressed in B-cell malignancies, such as non-Hodgkin's lymphoma. On binding to its ligand BAFF, proliferation and cell survival are increased, enabling cancer cells to proliferate faster than normal B-cells. Nucleic acid aptamers can bind to target ligands with high specificity and affinity and may offer therapeutic advantages over antibody-based approaches. In this study, we isolated several 2'-F-modified RNA aptamers targeting the B-cell-specific BAFF-R with nanomolar affinity using in vitro SELEX technology. The aptamers efficiently bound to BAFF-R on the surface of B-cells, blocked BAFF-mediated B-cell proliferation and were internalized into B-cells. Furthermore, chimeric molecules between the BAFF-R aptamer and small interfering RNAs (siRNAs) were specifically delivered to BAFF-R expressing cells with a similar efficiency as the aptamer alone. We demonstrate that a signal transducer and activator of transcription 3 (STAT3) siRNA delivered by the BAFF-R aptamer was processed by Dicer and efficiently reduced levels of target mRNA and protein in Jeko-1 and Z138 human B-cell lines. Collectively, our results demonstrate that the dual-functional BAFF-R aptamer-siRNA conjugates are able to deliver siRNAs and block ligand mediated processes, suggesting it might be a promising combinatorial therapeutic agent for B-cell malignancies.

Interplay between HIV-1 infection and host microRNAs.

Using microRNA array analyses of in vitro HIV-1-infected CD4(+) cells, we find that several host microRNAs are significantly up- or downregulated around the time HIV-1 infection peaks in vitro. While microRNA-223 levels were significantly enriched in HIV-1-infected CD4(+)CD8(-) PBMCs, microRNA-29a/b, microRNA-155 and microRNA-21 levels were significantly reduced. Based on the potential for microRNA binding sites in a conserved sequence of the Nef-3'-LTR, several host microRNAs potentially could affect HIV-1 gene expression. Among those microRNAs, the microRNA-29 family has seed complementarity in the HIV-1 3'-UTR, but the potential suppressive effect of microRNA-29 on HIV-1 is severely blocked by the secondary structure of the target region. Our data support a possible regulatory circuit at the peak of HIV-1 replication which involves downregulation of microRNA-29, expression of Nef, the apoptosis of host CD4 cells and upregulation of microRNA-223.

Small interfering RNAs targeting cyclin D1 and cyclin D2 enhance the cytotoxicity of chemotherapeutic agents in mantle cell lymphoma cell lines.

Cyclin D1 (CCND1) is a known cell cycle regulator whose overexpression is a hallmark of mantle cell lymphoma (MCL). Although molecular techniques have unified the diagnostic approach to MCL, no therapeutic advances have been made to target this particular pathway. The significance of CCND1 in the pathogenesis and treatment of MCL has yet to be defined. We have taken advantage of RNA interference (RNAi) to down-regulate CCND1 expression in two MCL cell lines (Granta-519 and Jeko-1) to investigate the cytotoxic effect of combining RNAi with conventional chemotherapeutic agents. We designed four small interfering RNAs (siRNAs) specific to CCND1, one specific to CCND2, and one dual-targeting siRNA that simultaneously down-regulates CCND1 and CCND2. Etoposide and doxorubicin were used as chemotherapeutics in combination with the siRNAs. The transfected siRNAs in MCL cell lines triggered 40-60% reduction in target mRNA and protein levels. Importantly, the siRNA-mediated reduction in cyclins resulted in decreased IC(50) (50% inhibitory concentration) values for both doxorubicin and etoposide. The combination of siRNA-mediated inhibition of the cyclins along with chemotherapeutic agents could potentially be used to lower the effective doses of the chemotherapeutic agents and reduce drug-related toxicities.

Silencing of gene expression in cultured cells using small interfering RNAs.

The discovery of RNA interference (RNAi) and related small RNA-mediated regulatory pathways has significantly altered the understanding of gene regulation in eukaryotic cells. In the RNAi pathway, small interfering RNAs (siRNAs) approximately 21 to 23 nucleotides in length serve as the regulatory molecules that guide and induce sequence-specific gene silencing. The use of siRNA-mediated silencing as a tool for investigating gene function is well established in cultured mammalian cells. This unit provides basic approaches to explore the field of RNAi, and hopes to address the importance of optimizing transfection conditions after empirical determinations in order to understand various degrees of silencing efficiency.

A role for the Dicer helicase domain in the processing of thermodynamically unstable hairpin RNAs.

In humans a single species of the RNAseIII enzyme Dicer processes both microRNA precursors into miRNAs and long double-stranded RNAs into small interfering RNAs (siRNAs). An interesting but poorly understood domain of the mammalian Dicer protein is the N-terminal helicase-like domain that possesses a signature DExH motif. Cummins et al. created a human Dicer mutant cell line by inserting an AAV targeting cassette into the helicase domain of both Dicer alleles in HCT116 cells generating an in-frame 43-amino-acid insertion immediately adjacent to the DExH box. This insertion creates a Dicer mutant protein with defects in the processing of most, but not all, endogenous pre-miRNAs into mature miRNA. Using both biochemical and computational approaches, we provide evidence that the Dicer helicase mutant is sensitive to the thermodynamic properties of the stems in microRNAs and short-hairpin RNAs, with thermodynamically unstable stems resulting in poor processing and a reduction in the levels of functional mi/siRNAs. Paradoxically, this mutant exhibits enhanced processing efficiency and concomitant RNA interference when thermodynamically stable, long-hairpin RNAs are used. These results suggest an important function for the Dicer helicase domain in the processing of thermodynamically unstable hairpin structures.

Rice grassy stunt tenuivirus nonstructural protein p5 interacts with itself to form oligomeric complexes in vitro and in vivo.

We investigated the interaction of Rice grassy stunt tenuivirus (RGSV) nonstructural protein p5, a protein of 22 kDa encoded on vRNA 5, with all 12 RGSV proteins by using a GAL4 transcription activator-based yeast two-hybrid system. The p5 protein interacted only with itself and not with any other viral protein; the interacting domains were localized within the N-terminal 96 amino acids of p5. The p5-p5 interaction was reproduced in an Sos recruitment-mediated yeast two-hybrid system as well in by far-Western blots. Native p5 protein extracted from RGSV-infected rice tissue was detected in a large complex with a molecular mass of approximately 260 kDa composed of 12 molecules of p5 or a p5 oligomer with an unidentified host factor(s).