RNA Interference Pathway Research Articles

The microgravity enhanced polymer-mediated siRNA gene silence by improving cellular uptake

Graphical abstractMicrogravity (MG) effect is a weightlessness phenomenon caused by the distance from the ground or low gravity of other planets outside the earth’s atmosphere. The various effects of MG have been corroborated in human and animal studies and modeled in cell-based analogs. However, the impact of MG on siRNA performance remains to be elucidated, which is crucial for aerospace medicine. In this study, we prepared nucleic acid nanomicelles (EAASc/siRNA) by using tri-block copolymer of PEG45-PAMA40-P(C7A36-DBA37) (EAASc) and siRNA and explored its working mechanism under simulated microgravity (SMG) condition generated by a random positioning machine (RPM). The binding ability of EAASc to siRNA and silence activity were firstly confirmed in normal gravity (NG) environment. Evaluation of PLK1 mRNA expression revealed that gene inhibition efficiencies were increased by 28.7% (HepG2) and 28.9% (A549) under SMG condition, compared with those under NG condition. In addition, mechanism exploration indicated that morphology and migration capability of cancer cells were significantly changed, the internalization of EAASc/siRNA by cells was magnified when the cells were incubated with RPM. No significant difference was observed regarding the expression profiles of genes involved in RNA interference (RNAi) pathway, including Ago2, Dicer, TRBP, and so on. Taken together, siRNA activity was elevated under SMG condition owning to increased cellular internalization. This study, for the first time to our knowledge, provides valuable theory for development and application of siRNA therapeutic in space in the future.

RNAi Screening to Identify Factors That Control Circular RNA Localization.

Thousands of eukaryotic protein-coding genes are noncanonically spliced to generate circular RNAs that have covalently linked ends. These transcripts are resistant to degradation by exonucleases, which enables some to accumulate to higher levels than the associated linear mRNA. In general, exonic circular RNAs accumulate in the cytoplasm, but functions for most of these transcripts remain unknown. It has been proposed that some may modulate the activity of microRNAs or RNA-binding proteins, be translated to yield protein products, or regulate innate immune responses. Recent work has revealed that circular RNAs are exported from the nucleus in a length-dependent manner and that the subcellular localization of these transcripts can be controlled by the DExH/D-box helicase Hel25E in Drosophila. Here, we describe how RNAi screening combined with subcellular fractionation and quantitative reverse transcription PCR (RT-qPCR) can be used to identify regulators of circular RNA localization in Drosophila cells. Long double-stranded RNAs (dsRNAs) that activate the RNA interference (RNAi) pathway are used to deplete factors of interest followed by biochemical fractionation to separate nuclear and cytoplasmic RNAs. RT-qPCR primers that amplify across the backsplicing junction of specific circular RNAs are then used to quantify the relative amounts of these transcripts in the nuclear and cytoplasmic compartments. In total, this approach can be broadly used to characterize circular RNA nuclear export and localization mechanisms, including to identify novel regulatory factors and their breadth of circular RNA targets.

Germline AGO2 mutations impair RNA interference and human neurological development

ARGONAUTE-2 and associated miRNAs form the RNA-induced silencing complex (RISC), which targets mRNAs for translational silencing and degradation as part of the RNA interference pathway. Despite the essential nature of this process for cellular function, there is little information on the role of RISC components in human development and organ function. We identify 13 heterozygous mutations in AGO2 in 21 patients affected by disturbances in neurological development. Each of the identified single amino acid mutations result in impaired shRNA-mediated silencing. We observe either impaired RISC formation or increased binding of AGO2 to mRNA targets as mutation specific functional consequences. The latter is supported by decreased phosphorylation of a C-terminal serine cluster involved in mRNA target release, increased formation of dendritic P-bodies in neurons and global transcriptome alterations in patient-derived primary fibroblasts. Our data emphasize the importance of gene expression regulation through the dynamic AGO2-RNA association for human neuronal development.

A Novel Anphevirus in Aedes albopictus Mosquitoes Is Distributed Worldwide and Interacts with the Host RNA Interference Pathway.

The Asian tiger mosquito Aedes albopictus is a competent vector for several human arboviruses including dengue, chikungunya and Zika viruses. Mosquitoes also harbor insect-specific viruses (ISVs) that may modulate host physiology and potentially affect the transmission of viruses that are pathogenic to vertebrates, thus representing a potential tool for vector control strategies. In Ae. albopictus we identified a novel anphevirus (family Xinmoviridae; order Mononegavirales) provisionally designated here as Aedes albopictus anphevirus (AealbAV). AealbAV contains a ~12.4 kb genome that is highly divergent from currently known viruses but displays gene content and genomic organization typical of known anpheviruses. We identified AealbAV in several publicly available RNA-Seq datasets from different geographical regions both in laboratory colonies and field collected mosquitoes. Coding-complete genomes of AealbAV strains are highly similar worldwide (>96% nucleotide identity) and cluster according to the geographical origin of their hosts. AealbAV appears to be present in various body compartments and mosquito life stages, including eggs. We further detected AealbAV-derived vsiRNAs and vpiRNAs in publicly available miRNA-Seq libraries of Ae. albopictus and in samples experimentally coinfected with chikungunya virus. This suggests that AealbAV is targeted by the host RNA interference (RNAi) response, consistent with persistent virus replication. The discovery and characterization of AealbAV in Ae. albopictus will now allow us to identify its infection in mosquito populations and laboratory strains, and to assess its potential impact on Ae. albopictus physiology and ability to transmit arboviruses.

Zika Virus Replication in Myeloid Cells during Acute Infection Is Vital to Viral Dissemination and Pathogenesis in a Mouse Model.

Zika virus (ZIKV) can establish infection in immune privileged sites such as the testes, eye, and placenta. Whether ZIKV infection of white blood cells is required for dissemination of the virus to immune privileged sites has not been definitively shown. To assess whether initial ZIKV replication in myeloid cell populations is critical for dissemination during acute infection, recombinant ZIKVs were generated that could not replicate in these specific cells. ZIKV was cell restricted by insertion of a complementary sequence to a myeloid-specific microRNA in the 3' untranslated region. Following inoculation of a highly sensitive immunodeficient mouse model, crucial immune parameters, such as quantification of leukocyte cell subsets, cytokine and chemokine secretion, and viremia, were assessed. Decreased neutrophil numbers in the spleen were observed during acute infection with myeloid-restricted ZIKV that precluded the generation of viremia and viral dissemination to peripheral organs. Mice inoculated with a nontarget microRNA control ZIKV demonstrated increased expression of key cytokines and chemokines critical for neutrophil and monocyte recruitment and increased neutrophil influx in the spleen. In addition, ZIKV-infected Ly6Chi monocytes were identified in vivo in the spleen. Mice inoculated with myeloid-restricted ZIKV had a decrease in Ly6Chi ZIKV RNA-positive monocytes and a lack of inflammatory cytokine production compared to mice inoculated with control ZIKV.IMPORTANCE Myeloid cells, including monocytes, play a crucial role in immune responses to pathogens. Monocytes have also been implicated as "Trojan horses" during viral infections, carrying infectious virus particles to immune privileged sites and/or to sites protected by physical blood-tissue barriers, such as the blood-testis barrier and the blood-brain barrier. In this study, we found that myeloid cells are crucial to Zika virus (ZIKV) pathogenesis. By engineering ZIKV clones to encode myeloid-specific microRNA target sequences, viral replication was inhibited in myeloid cells by harnessing the RNA interference pathway. Severely immunodeficient mice inoculated with myeloid-restricted ZIKV did not demonstrate clinical signs of disease and survived infection. Furthermore, viral dissemination to peripheral organs was not observed in these mice. Lastly, we identified Ly6Cmid/hi murine monocytes as the major myeloid cell population that disseminates ZIKV.

Cancer biology functional genomics: From small RNAs to big dreams.

The year 2021 marks the 20th anniversary of the first publications reporting the discovery of the gene silencing mechanism, RNA interference (RNAi) in mammalian cells. Along with the many studies that delineated the proteins and substrates that form the RNAi pathway, this finding changed our understanding of the posttranscriptional regulation of mammalian gene expression. Furthermore, the development of methods that exploited the RNAi pathway began the technological revolution that eventually enabled the interrogation of mammalian gene function—from a single gene to the whole genome—in only a few days. The needs of the cancer research community have driven much of this progress. In this perspective, we highlight milestones in the development and application of RNAi‐based methods to study carcinogenesis. We discuss how RNAi‐based functional genetic analysis of exemplar tumor suppressors and oncogenes furthered our understanding of cancer initiation and progression and explore how such studies formed the basis of genome‐wide scale efforts to identify cancer or cancer‐type specific vulnerabilities, including studies conducted in vivo. Furthermore, we examine how RNAi technologies have revealed new cancer‐relevant molecular targets and the implications for cancer of the first RNAi‐based drugs. Finally, we discuss the future of functional genetic analysis, highlighting the increasing availability of complementary approaches to analyze cancer gene function.

Overcoming Barriers for siRNA Therapeutics: From Bench to Bedside.

The RNA interference (RNAi) pathway possesses immense potential in silencing any gene in human cells. Small interfering RNA (siRNA) can efficiently trigger RNAi silencing of specific genes. FDA Approval of siRNA therapeutics in recent years garnered a new hope in siRNA therapeutics. However, their therapeutic use is limited by several challenges. siRNAs, being negatively charged, are membrane-impermeable and highly unstable in the systemic circulation. In this review, we have comprehensively discussed the extracellular barriers, including enzymatic degradation of siRNAs by serum endonucleases and RNAases, rapid renal clearance, membrane impermeability, and activation of the immune system. Besides, we have thoroughly described the intracellular barriers such as endosomal trap and off-target effects of siRNAs. Moreover, we have reported most of the strategies and techniques in overcoming these barriers, followed by critical comments in translating these molecules from bench to bedside.

Transcriptome analysis of the innate immune system of Hyalomma asiaticum

Ticks are considered to be the second most important vectors of human infectious diseases. The innate immune system is the key factor that affects its vector competence. Hyalomma asiaticum is the primary vector of Crimean–Congo hemorrhagic fever virus (CCHFV). However, the immune system of H. asiaticum remains virtually unknown. Here, a high throughput full-length mRNA sequencing method was adopted to define the immunotranscriptome of H. asiaticum infected with the fungal pathogen Beauveria bassiana and gram-negative bacterium Enterobacter cloacae. The analysis yielded 22,300 isoforms with an average length of 3233 bps. In total, 68 potential immunity-related genes were identified based on similarity to the homologs known to be involved in immunity. These included most members of the Toll and JAK/STAT signaling pathways, but not the IMD signaling pathway. Moreover, two copies of Dicer-2 and five copies of Argonaute-2 were detected. These genes are postulated to be involved in the RNA interference (RNAi) pathway, which is an important defense against RNA viruses. Overall, this study provides the foundation for understanding the immune response of H. asiaticum to CCHFV.

Lyophilization and nebulization of pulmonary surfactant-coated nanogels for siRNA inhalation therapy

RNA interference (RNAi) enables highly specific silencing of potential target genes for treatment of pulmonary pathologies. The intracellular RNAi pathway can be activated by cytosolic delivery of small interfering RNA (siRNA), inducing sequence-specific gene knockdown on the post-transcriptional level. Although siRNA drugs hold many advantages over currently applied therapies, their clinical translation is hampered by inefficient delivery across cellular membranes. We previously developed hybrid nanoparticles consisting of an siRNA-loaded nanosized hydrogel core (nanogel) coated with Curosurf®, a clinically used pulmonary surfactant (PS). The latter enhances both particle stability as well as intracellular siRNA delivery, which was shown to be governed by the PS-associated surfactant protein B (SP-B). Despite having a proven in vitro and in vivo siRNA delivery potential when prepared ex novo, clinical translation of this liquid nanoparticle suspension requires the identification of a long-term preservation strategy that maintains nanoparticle stability and potency. In addition, to achieve optimal pulmonary deposition of the nanocomposite, its compatibility with state-of-the-art pulmonary administration techniques should be evaluated. Here, we demonstrate that PS-coated nanogels can be lyophilized, reconstituted and subsequently nebulized via a vibrating mesh nebulizer. The particles retain their physicochemical integrity and their ability to deliver siRNA in a human lung epithelial cell line. The latter result suggests that the functional integrity of SP-B in the PS coat towards siRNA delivery might be preserved as well. Of note, successful lyophilization was achieved without the need for stabilizing lyo- or cryoprotectants. Our results demonstrate that PS-coated siRNA-loaded nanogels can be lyophilized, which offers the prospect of long-term storage. In addition, the formulation was demonstrated to be suitable for local administration with a state-of-the-art nebulizer for human use upon reconstitution. Hence, the data presented in this study represent an important step towards clinical application of such nanocomposites for treatment of pulmonary disease.

Characterization of natural antisense transcripts arisen from the locus encoding Toxoplasma gondii ubiquitin-like protease

Natural antisense transcripts (NATs) are non-protein coding RNAs that could play an important role in regulating the expression of their counterpart protein encoding sense transcript. Although NATs are widespread in most eukaryotic genomes, very little is known about their functions. This study focuses on gaining a better understanding of the function of NATs in Toxoplasma gondii, a pathogenic unicellular eukaryote. Previously, we characterized the gene encoding the first committed enzyme in sumoylation, named ubiquitin-like protease 1 (TgUlp1), and showed that the expression of TgUlp1 is vital to the life cycle of T. gondii. Interestingly, the locus of TgUlp1 also transcribes a NAT species. Using a dual luciferase assay, we identified the promoter of TgUlp1 NAT to be located within the 3’-region of its counterpart coding sequence. While TgUlp1 mRNA level was detected at a lower level throughout the life cycle of T. gondii, its NAT level was upregulated when the parasite converts from actively replicating tachyzoite form to slowly growing bradyzoite form. To investigate the effect of TgUlp1 NAT on the expression of its counterpart mRNA, we used a reporter system bearing TgUlp1 mRNA sequences and showed that the single-stranded TgUlp1 NAT and its in vitro RNase III processed products have the ability to lower the expression of the reporter system. Using a transgenic Dicer-knockout (TgDicer-KO) strain, we showed that TgDicer is required for the function of TgUlp1 NAT in vivo. The findings strongly suggest that the RNA interference pathway is necessary for the function of TgUlp1 NAT.

Diverse Defenses: A Perspective Comparing Dipteran Piwi-piRNA Pathways.

Animals face the dual threat of virus infections hijacking cellular function and transposons proliferating in germline genomes. For insects, the deeply conserved RNA interference (RNAi) pathways and other chromatin regulators provide an important line of defense against both viruses and transposons. For example, this innate immune system displays adaptiveness to new invasions by generating cognate small RNAs for targeting gene silencing measures against the viral and genomic intruders. However, within the Dipteran clade of insects, Drosophilid fruit flies and Culicids mosquitoes have evolved several unique mechanistic aspects of their RNAi defenses to combat invading transposons and viruses, with the Piwi-piRNA arm of the RNAi pathways showing the greatest degree of novel evolution. Whereas central features of Piwi-piRNA pathways are conserved between Drosophilids and Culicids, multiple lineage-specific innovations have arisen that may reflect distinct genome composition differences and specific ecological and physiological features dividing these two branches of Dipterans. This perspective review focuses on the most recent findings illuminating the Piwi/piRNA pathway distinctions between fruit flies and mosquitoes, and raises open questions that need to be addressed in order to ameliorate human diseases caused by pathogenic viruses that mosquitoes transmit as vectors.

High-Temperature-Responsive Poplar lncRNAs Modulate Target Gene Expression via RNA Interference and Act as RNA Scaffolds to Enhance Heat Tolerance.

High-temperature stress is a threat to plant development and survival. Long noncoding RNAs (lncRNAs) participate in plant stress responses, but their functions in the complex stress response network remain unknown. Poplar contributes to terrestrial ecological stability. In this study, we identified 204 high-temperature-responsive lncRNAs in an abiotic stress-tolerant poplar (Populus simonii) species using strand-specific RNA sequencing (ssRNA-seq). Mimicking overexpressed and repressed candidate lncRNAs in poplar was used to illuminate their regulation pattern on targets using nano sheet mediation. These lncRNAs were predicted to target 185 genes, of which 100 were cis genes and 119 were trans genes. Gene Ontology enrichment analysis showed that anatomical structure morphogenesis and response to stress and signaling were significantly enriched. Among heat-responsive LncRNAs, TCONS_00202587 binds to upstream sequences via its secondary structure and interferes with target gene transcription. TCONS_00260893 enhances calcium influx in response to high-temperature treatment by interfering with a specific variant/isoform of the target gene. Heterogeneous expression of these two lncRNA targets promoted photosynthetic protection and recovery, inhibited membrane peroxidation, and suppressed DNA damage in Arabidopsis under heat stress. These results showed that lncRNAs can regulate their target genes by acting as potential RNA scaffolds or through the RNA interference pathway.

Double-stranded RNA in exosomes: Potential systemic RNA interference pathway in the Colorado potato beetle, Leptinotarsa decemlineata

Despite extensive research during the past decade elucidating the mechanism of RNA interference (RNAi) in insects, it is not clear how ingested or injected double-stranded RNA (dsRNA) triggers RNAi response in the whole body or even its progeny, which is referred to as systemic RNAi. In the present study, we aim to understand how the dsRNA delivered into cells causes systemic RNAi using Colorado potato beetle cells (Lepd-SL1). We first tested if dsRNA treatment induces systemic RNAi in Lepd-SL1 cells. Exposure of a new batch of Lepd-SL1 cells to the conditioned medium where Lepd-SL1 cells treated with dsRNA targeting inhibitor of apoptosis were grown for 6 h induced apoptosis in these new batch of cells. We hypothesized the exosomes in the conditioned medium are responsible for RNAi-inducing effect. To test this hypothesis, we isolated exosomes from the conditioned medium from Lepd-SL1 cells that had been treated with dsGFP (dsRNA targeting gene coding for green fluorescent protein) or dsLuc (dsRNA targeting gene coding for the luciferase) were grown. RNA present in the purified exosomes was analyzed to check if long dsRNA or siRNA is accumulated in them. The results from the electrophoretic mobility shift assay clearly showed that the long dsRNAs are present in the exosomes. By knockdown of candidate genes involved in endosome recycling and generation pathways, we found that Rab4 and Rab35 are involved in exosome production and transport.

Multiple Argonaute family genes contribute to the siRNA-mediated RNAi pathway in Locusta migratoria

Argonautes (Ago) are important core proteins in RNA interference (RNAi) pathways of eukaryotic cells. Generally, it is thought that Ago1, Ago2 and Ago3 are involved in the miRNA (microRNA), siRNA (small interfering RNA) and piRNA (Piwi-interacting RNA)-mediated RNAi pathways, respectively. As a main component of the RNA-induced silencing complex (RISC), Ago2 plays an indispensable role in using siRNA to recognize and cut target messenger RNAs resulting in suppression of transcript levels, but the contributions of Ago1 and Ago3 to the siRNA-mediated RNAi pathway remain to be explored in many insect species. In this study, we investigated the contributions of four Ago genes (named LmAgo1, LmAgo2a and LmAgo2b and LmAgo3) to RNAi efficiency in Locusta migratoria by using both in vivo and in vitro experiments. Our results showed that suppression of each of the Ago genes significantly impaired RNAi efficiency when targeting Lmβ-tubulin transcripts, resulting in recovery of 48, 43.3, 61.4 or 26% of Lmβ-tubulin transcripts following RNAi-mediated suppression of LmAgo1, LmAgo2a, LmAgo2b, and LmAgo3, respectively. Furthermore, overexpression of LmAgo1, LmAgo2a, LmAgo2b, or LmAgo3 in a PAc5.1-V5/HisB vector and co-transfection with psicheck2 fluorescence vector in S2 cells reduced luciferase fluorescence by 38.3, 58.9, 53.3 or 55.6%, respectively. Taken together, our results showed that LmAgo1, LmAgo2a, LmAgo2b, and LmAgo3 each make significant contributions to RNAi efficiency in L. migratoria and suggest that the involvement of all four enzymes could be one of the major factors supporting robust RNAi responses observed in this species.

C.elegans interprets bacterial non-coding RNAs to learn pathogenic avoidance.

Summary:C. elegans must distinguish pathogenic from nutritious bacterial food sources among the many bacteria it is exposed to in its environment1. Here we show that a single exposure to purified small RNAs isolated from pathogenic Pseudomonas aeruginosa (PA14) is sufficient to induce pathogen avoidance, both in the treated animals and in four subsequent generations of progeny. The RNA interference and piRNA pathways, the germline, and the ASI neuron are required for bacterial small RNA-induced avoidance behavior and transgenerational inheritance. A single P. aeruginosa non-coding RNA, P11, is both necessary and sufficient to convey learned avoidance of PA14, and its C. elegans target, maco-1, is required for avoidance. Our results suggest that this ncRNA-dependent mechanism evolved to survey the worm’s microbial environment, use this information to make appropriate behavioral decisions, and pass this information on to its progeny.

Small RNA Isolation and Library Construction for Expression Profiling of Small RNAs from Neurospora crassa and Fusarium oxysporum and Analysis of Small RNAs in Fusarium oxysporum-Infected Plant Root Tissue.

Due to crucial roles in gene regulation, noncoding small RNAs (sRNAs) of 20-30 nucleotides (nt) have been intensively studied in mammals and plants and are implicated in significant diseases and metabolic disorders. Elucidation of biogenesis mechanisms and functional characterization of sRNAs is often achieved using tools such as separation of small-sized RNA and deep sequencing. Although RNA interference pathways, such as quelling and meiotic silencing, have been well-described in Neurospora crassa, knowledge of sRNAs in other filamentous fungi is still limited compared to other eukaryotes. As a prerequisite for study, isolation and sequence analysis of sRNAs is necessary. We developed a protocol for isolation and library construction of sRNAs of 20-30nt for deep sequencing in two filamentous fungi, N. crassa and Fusarium oxysporum f.sp. lycopersici. Using 200-300μg total RNA, sRNA was isolated by size-fractionation and ligated with adapters and amplified by RT-PCR for deep sequencing. Sequence analysis of several cDNA clones showed that the cloned sRNAs were not tRNAs and rRNAs and were fungal genome-specific. In order to validate fungal miRNAs that were imported into the host cell, we developed a straightforward method to isolate protoplasts from tomato roots infected by Fusarium oxysporum f.sp. lycopersici using enzymatic digestion.

Mechanism and Function of Antiviral RNA Interference in Mice.

Distinct mammalian RNA viruses trigger Dicer-mediated production of virus-derived small-interfering RNAs (vsiRNA) and encode unrelated proteins to suppress vsiRNA biogenesis. However, the mechanism and function of the mammalian RNA interference (RNAi) response are poorly understood. Here, we characterized antiviral RNAi in a mouse model of infection with Nodamura virus (NoV), a mosquito-transmissible positive-strand RNA virus encoding a known double-stranded RNA (dsRNA)-binding viral suppressor of RNAi (VSR), the B2 protein. We show that inhibition of NoV RNA replication by antiviral RNAi in mouse embryonic fibroblasts (MEFs) requires Dicer-dependent vsiRNA biogenesis and Argonaute-2 slicer activity. We found that VSR-B2 of NoV enhances viral RNA replication in wild-type but not RNAi-defective MEFs such as Argonaute-2 catalytic-dead MEFs and Dicer or Argonaute-2 knockout MEFs, indicating that VSR-B2 acts mainly by suppressing antiviral RNAi in the differentiated murine cells. Consistently, VSR-B2 expression in MEFs has no detectable effect on the induction of interferon-stimulated genes or the activation of global RNA cleavages by RNase L. Moreover, we demonstrate that NoV infection of adult mice induces production of abundant vsiRNA active to guide RNA slicing by Argonaute-2. Notably, VSR-B2 suppresses the biogenesis of both vsiRNA and the slicing-competent vsiRNA-Argonaute-2 complex without detectable inhibition of Argonaute-2 slicing guided by endogenous microRNA, which dramatically enhances viral load and promotes lethal NoV infection in adult mice either intact or defective in the signaling by type I, II, and III interferons. Together, our findings suggest that the mouse RNAi response confers essential protective antiviral immunity in both the presence and absence of the interferon response.IMPORTANCE Innate immune sensing of viral nucleic acids in mammals triggers potent antiviral responses regulated by interferons known to antagonize the induction of RNA interference (RNAi) by synthetic long double-stranded RNA (dsRNA). Here, we show that Nodamura virus (NoV) infection in adult mice activates processing of the viral dsRNA replicative intermediates into small interfering RNAs (siRNAs) active to guide RNA slicing by Argonaute-2. Genetic studies demonstrate that NoV RNA replication in mouse embryonic fibroblasts is inhibited by the RNAi pathway and enhanced by the B2 viral RNAi suppressor only in RNAi-competent cells. When B2 is rendered nonexpressing or nonfunctional, the resulting mutant viruses become nonpathogenic and are cleared in adult mice either intact or defective in the signaling by type I, II, and III interferons. Our findings suggest that mouse antiviral RNAi is active and necessary for the in vivo defense against viral infection in both the presence and absence of the interferon response.

Advances with RNAi-Based Therapy for Hepatitis B Virus Infection.

Infection with hepatitis B virus (HBV) remains a global health challenge. Approximately 292 million people worldwide are chronically infected with HBV and the annual mortality from the infection is approaching 900,000. Despite the availability of an effective prophylactic vaccine, millions of individuals are at risk of potentially fatal complicating cirrhosis and hepatocellular carcinoma. Current drug treatments can suppress viral replication, slow the progression of liver fibrosis, and reduce infectivity, but can rarely clear the viral covalently closed circular DNA (cccDNA) that is responsible for HBV persistence. Alternative therapeutic strategies, including those based on viral gene silencing by harnessing the RNA interference (RNAi) pathway, effectively suppress HBV replication and thus hold promise. RNAi-based silencing of certain viral genes may even lead to disabling of cccDNA during chronic infection. This review summarizes different RNAi activators that have been tested against HBV, the advances with vectors used to deliver artificial potentially therapeutic RNAi sequences to the liver, and the current status of preclinical and clinical investigation.

LncRNA TTN-AS1 promotes the progression of cholangiocarcinoma via the miR-320a/neuropilin-1 axis

Neuropilin-1 regulated by miR-320a participates in the progression of cholangiocarcinoma by serving as a co-receptor that activates multiple signaling pathways. The present study sought to investigate upstream lncRNAs that control the expression of miR-320a/neuropilin-1 axis and dissect some of the underlying mechanisms. Here we report lncRNA TTN-AS1 (titin-antisense RNA1) acts as a sponging ceRNA to downregulate miR-320a and is highly expressed in human cholangiocarcinoma tissues and cells. The expression of the above three molecules is correlated with the clinicopathologic parameters of cholangiocarcinoma patients. In this study, multiple bioinformatics tools and databases were employed to seek potential lncRNAs that have binding sites with miR-320a and TTN-AS1 was identified because it exhibited the largest folds of alteration between cholangiocarcinoma and normal bile duct epithelial cells. The regulatory role of TTN-AS1 on miR-320a was further evaluated by luciferase reporter and RNA pulldown assays, coupled with in situ hybridization and RNA immunoprecipitation analyses, which showed that TTN-AS1 bound to miR-320a through an argonaute2-dependent RNA interference pathway in the cytoplasm of cholangiocarcinoma cells. Knockdown and overexpression assays showed that the regulatory effect between TTN-AS1 and miR-320 was in a one-way manner. TTN-AS1 promoted the proliferation and migration of cholangiocarcinoma cells via the miR-320a/ neuropilin-1 axis. The function of TTN-AS1 on tumor growth and its interaction with miR-320a were confirmed in animal models. Further mechanistic studies revealed that TTA-AS1, through downregulating miR-320a, promoted cell cycle progression, epithelial–mesenchymal transition, and tumor angiogenesis by upregulating neuropilin-1, which co-interacted with the hepatocyte growth factor/c-Met and transforming growth factor (TGF)-β/TGF-β receptor I pathways. In conclusion, the present results demonstrate that lncRNA TTA-AS1 is a sponging ceRNA for miR-320a, which in turn downregulates neuropilin-1 in cholangiocarcinoma cells, indicating these three molecules represent potential biomarkers and therapeutic targets in the management of cholangiocarcinoma.

Design and Evaluation of AgoshRNAs with 3'-Terminal HDV Ribozymes to Enhance the Silencing Activity.

Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNA (shRNA) molecules for targeted gene silencing has become a benchmark technology. Plasmid and viral vector systems can be used to express shRNA precursor transcripts that are processed by the cellular RNAi pathway to trigger sequence-specific gene knockdown. Intensive RNAi investigations documented that only a small percentage of computationally predicted target sequences can be used for efficient gene silencing, in part because not all shRNA designs are active. Many factors influence the shRNA activity and guidelines for optimal shRNA design have been proposed. We recently described an alternatively processed shRNA molecule termed AgoshRNA with a ~18base pairs (bp) stem and a 3-5 nucleotides(nt) loop. This molecule is alternatively processed by the Argonaute (Ago) protein into a single guide RNA strand that efficiently induces the RNAi mechanism. The design rules proposed for regular shRNAs do not apply to AgoshRNA molecules and therefore new rules had to be defined. We optimized the AgoshRNA design and managed to create a set of active AgoshRNAs targeted against the human immunodeficiency virus (HIV). In an attempt to enhance the silencing activity of the AgoshRNA molecules, we included the hepatitis delta virus (HDV) ribozyme at the 3' terminus, which generates a uniform 3' end instead of a 3' U-tail of variable length. We evaluated the impact of this 3'-end modification on AgoshRNA processing and its gene silencing activity and we demonstrate that this novel AgoshRNA-HDV design exhibits enhanced antiviral activity.