A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective small interfering RNA (siRNA) therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle (LNP) delivery system. Multiple siRNAs targeting highly conserved regions of the SARS-CoV-2 virus were screened, and three candidate siRNAs emerged that effectively inhibit the virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel LNP formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.

Dual VP28 and VP37 dsRNA encapsulation in IHHNV virus-like particles enhances shrimp protection against white spot syndrome virus.

Accumulative evidence of using double stranded (ds) RNA encapsulated into virus like particle (VLP) nanocarrier has open feasibility to fight against shrimp viral infection in aquaculture field. In this study, we co-encapsulated VP37 and VP28 dsRNA into hypodermal and hematopoietic necrosis virus (IHHNV) like particle and investigated its protection against white spot syndrome virus (WSSV). Five micrograms of each dsRNA were used as starting materials to load into VLP, while the loading efficiency was slightly different, i.e, VP37 dsRNA had somewhat a better load into VLP's cavity. It was apparent that co-encapsulation of dual dsRNA showed a superior WSSV silencing ability than the single dsRNA counterpart as evidence by the lower WSSV gene expression and its copy number in the gill tissues. Besides, we also demonstrated that co-encapsulated dual dsRNA into IHHNV-VLP stimulated the increased number of hemocytes and the corresponding PO activity as well as up-regulated proPO gene expression in hemocytes to resist viral invasion after an acute stage of WSSV infection. This synergistic action of dual dsRNA encapsulated into IHHNV-VLPs could thus act to delay time of shrimp death and reduced shrimp cumulative mortality greater than the single, naked dsRNA treatment and positive control groups. The obtaining results would encourage the feasibility to use it as a new weapon to fight WSSV infection in shrimp aquaculture.

Structurally modulated codelivery of siRNA and Argonaute 2 for enhanced RNA interference.

Small interfering RNA (siRNA) represents a promising class of inhibitors in both fundamental research and the clinic. Numerous delivery vehicles have been developed to facilitate siRNA delivery. Nevertheless, achieving highly potent RNA interference (RNAi) toward clinical translation requires efficient formation of RNA-induced gene-silencing complex (RISC) in the cytoplasm. Here we coencapsulate siRNA and the central RNAi effector protein Argonaute 2 (Ago2) via different delivery carriers as a platform to augment RNAi. The physical clustering between siRNA and Ago2 is found to be indispensable for enhanced RNAi. Moreover, by utilizing polyamines bearing the same backbone but distinct cationic side-group arrangements of ethylene diamine repeats as the delivery vehicles, we find that the molecular structure of these polyamines modulates the degree of siRNA/Ago2-mediated improvement of RNAi. We apply this strategy to silence the oncogene STAT3 and significantly prolong survival in mice challenged with melanoma. Our findings suggest a paradigm for RNAi via the synergistic coassembly of RNA with helper proteins.

Impact of Phosphorothioate Chirality on Double-Stranded siRNAs: A Systematic Evaluation of Stereopure siRNA Designs.

Oligonucleotides are important therapeutic approaches, as evidenced by recent clinical successes with antisense oligonucleotides (ASOs) and double-stranded short interfering RNAs (siRNAs). Phosphorothioate (PS) modifications are a standard feature in the current generation of oligonucleotide therapeutics, but generate isomeric mixtures, leading to 2n isomers. All currently marketed therapeutic oligonucleotides (ASOs and siRNAs) are complex isomeric mixtures. Recent chemical methodologies for stereopure PS insertions have resulted in preliminary rules for ASOs, with multiple stereopure ASOs moving into clinical development. Although siRNAs have comparatively fewer PSs, the field has yet to embrace the idea of stereopure siRNAs. Herein, it has been investigated whether the individual isomers contribute equally to the in vivo activity of a representative siRNA. The results of a systematic evaluation of stereopure PS incorporation into antithrombin-3 (AT3) siRNA are reported and demonstrate that individual PS isomers dramatically affect in vivo activity. A standard siRNA design with six PS insertions was investigated and it was found that only about 10 % of the 64 possible isomers were as efficacious as the stereorandom control. Based on this data, it can be concluded that G1R stereochemistry is critical, G2R is important, G21S is preferable, and G22 and P1/P2 tolerate both isomers. Surprisingly, the disproportionate loss of efficacy for most isomers does not translate into significant gain for the productive isomers, and thus, warrants further mechanistic studies.

Functional divergence of white genes in Henosepilachna vigintioctopunctata revealed by RNA interference.

Henosepilachna vigintioctopunctata is a serious pest of Solanaceae and Cucurbitaceae in many Asian countries. RNA interference (RNAi) can effectively reduce transcript abundance in this beetle, offering opportunities to explore the biological function of specific genes. The white gene encodes a half-type ATP-binding cassette transporter that plays an essential role in tryptophan, guanine and uric acid transport across membranes. Mutations that disrupt the function of white are known to cause eye pigmentation phenotypes in many insect species. Here, we found evidence for five white gene paralogues present in H. vigintioctopunctata transcriptome datasets sequenced from a range of developmental stages. We individually knocked down each of the five white genes through the injection of corresponding double-stranded RNAs (dsRNAs) to the fourth-instar larvae to determine whether functional divergence has occurred. We found that injecting 1 µg dswhite3 caused compound eye colour of pupae and adults to develop as red/brown and brown, respectively, compared with black eyes in control beetles. Injection of 2 µg dswhite3 increased RNAi efficacy and produced a clearer eye colour phenotype. At both doses, the ocular diaphragm (a ring of black pigment surrounding each eye) did not change in the white3 RNAi hypomorphs. Moreover, our data revealed that injection of dswhite2 at the fourth-instar larval stage impaired the climbing ability of both male and female adults. Our results confirmed, for the first time, functional divergence of duplicated white genes in an insect species.

Lipids help double-stranded RNA in endosomal escape and improve RNA interference in the fall armyworm, Spodoptera frugiperda.

RNA interference (RNAi) is a valuable method for understanding the gene function and holds great potential for insect pest management. While RNAi is efficient and systemic in coleopteran insects, RNAi is inefficient in lepidopteran insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda cells by formulating dsRNA with Cellfectin II (CFII) transfection reagent. The CFII formulated dsRNA was protected from degradation by endonucleases present in Sf9 cells conditioned medium, hemolymph and midgut lumen contents collected from the FAW larvae. Lipid formulated dsRNA also showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing Sf9 cells and tissues to CFII formulated dsRNA caused a significant knockdown of endogenous genes. CFII formulated dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation and mortality. Processing of dsRNA into siRNA was detected in Sf9 cells and Spodoptera frugiperda larvae treated with CFII conjugated 32 P-UTP labeled dsGFP. Overall, the present study concluded that delivering dsRNA formulated with CFII transfection reagent helps dsRNA escapes from the endosomal accumulation and improved RNAi efficiency in the FAW cells and tissues.

Chitosan nanoparticles help double-stranded RNA escape from endosomes and improve RNA interference in the fall armyworm, Spodoptera frugiperda.

RNA interference (RNAi) is a promising technology for the development of next-generation insect pest control products. Though RNAi is efficient and systemic in coleopteran insects, it is inefficient and variable in lepidopteron insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda by conjugating double-stranded RNA (dsRNA) with biodegradable chitosan (Chi). dsRNA conjugated with chitosan was protected from degradation by endonucleases present in Sf9 cell-conditioned medium, hemolymph, and midgut lumen contents collected from the FAW larvae. Chi-dsRNA complexes showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing chitosan formulated dsRNA in Sf9 cells and the tissues induced a significant knockdown of endogenous genes. Chi-dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation, and mortality. Processing of dsRNA into small interfering RNA was detected with chitosan-conjugated 32 P-UTP-labeled ds green fluorescent protein in Sf9 cells and FAW larval tissues. Overall, these data suggest that dsRNA conjugated with chitosan helps dsRNA escape from the endosomes and improves RNAi efficiency in FAW cells and tissues.

Girardia dorotocephala transcriptome sequence, assembly, and validation through characterization of piwi homologs and stem cell progeny markers.

Planarian flatworms are popular models for the study of regeneration and stem cell biology in vivo. Technical advances and increased availability of genetic information have fueled the discovery of molecules responsible for stem cell pluripotency and regeneration in flatworms. Unfortunately, most of the planarian research performed worldwide utilizes species that are not natural habitants of North America, which limits their availability to newcomer laboratories and impedes their distribution for educational activities. In order to circumvent these limitations and increase the genetic information available for comparative studies, we sequenced the transcriptome of Girardia dorotocephala, a planarian species pandemic and commercially available in North America. A total of 254,802,670 paired sequence reads were obtained from RNA extracted from intact individuals, regenerating fragments, as well as freshly excised auricles of a clonal line of G. dorotocephala (MA-C2), and used for de novo assembly of its transcriptome. The resulting transcriptome draft was validated through functional analysis of genetic markers of stem cells and their progeny in G. dorotocephala. Akin to orthologs in other planarian species, G. dorotocephala Piwi1 (GdPiwi1) was found to be a robust marker of the planarian stem cell population and GdPiwi2 an essential component for stem cell-driven regeneration. Identification of G. dorotocephala homologs of the early stem cell descendent marker PROG-1 revealed a family of lysine-rich proteins expressed during epithelial cell differentiation. Sequences from the MA-C2 transcriptome were found to be 98-99% identical to nucleotide sequences from G. dorotocephala populations with different chromosomal number, demonstrating strong conservation regardless of karyotype evolution. Altogether, this work establishes G. dorotocephala as a viable and accessible option for analysis of gene function in North America.

Effects of FABP knockdown on flight performance of the desert locust, Schistocerca gregaria.

During migratory flight, desert locusts rely on fatty acids as their predominant source of energy. Lipids mobilized in the fat body are transported to the flight muscles and enter the muscle cells as free fatty acids. It has been postulated that muscle fatty acid binding protein (FABP) is needed for the efficient translocation of fatty acids through the aqueous cytosol towards mitochondrial ß-oxidation. To assess whether FABP is required for this process, dsRNA was injected into freshly emerged adult males to knock down the expression of FABP. Three weeks after injection, FABP and its mRNA were undetectable in flight muscle, indicating efficient silencing of FABP expression. At rest, control and treated animals exhibited no morphological or behavioral differences. In tethered flight experiments, both control and treated insects were able to fly continually in the initial, carbohydrate-fueled phase of flight, and in both groups, lipids were mobilized and released into the hemolymph. Flight periods exceeding 30 min, however, when fatty acids become the main energy source, were rarely possible for FABP-depleted animals, while control insects continued to fly for more than 2 h. These results demonstrate that FABP is an essential element of skeletal muscle energy metabolism in vivo.

A Plant-Derived Nucleic Acid Reconciles Type I IFN and a Pyroptotic-like Event in Immunity against Respiratory Viruses.

Nucleic acids carrying pathogen-associated molecular patterns trigger innate immune responses and are used to activate host immunity. Although synthetic nucleic acids have been used for that purpose, they have shown limitations for in vivo and clinical applications. To address this issue, we tested a naturally occurring dsRNA extracted from rice bran (rb-dsRNA) and characterized it as a potent ligand of TLR3 and MDA5. In this study, intranasal administration of rb-dsRNA induced production of type I IFNs by alveolar macrophages and protected mice from morbidity and mortality resulting from respiratory virus infection, such as influenza A virus. This protection was completely absent in mice lacking both TRIF and MDA5, indicating the essential role of TLR3- and MDA5-dependent pathways. Interestingly, IFNAR1-deficient mice retained residual antiviral protection, which was abolished by pharmacological inhibition of caspase 1, but not IL-1ß signaling. In fact, rb-dsRNA activated caspase 1 via TRIF, resulting in the release of IL-1ß and LDH. In addition to the direct antiviral activity, rb-dsRNA modulated the immune cell population in the lungs by repopulating virus-depleted alveolar macrophages. Our data demonstrate that rb-dsRNA orchestrates IFN-dependent and -independent direct antiviral protection and that it is a potent immune stimulator modulating antiviral immunity in the lungs. These findings open doors to a range of precise immune-modulating studies and therapeutic options.

Impairment of pupation by RNA interference-aided knockdown of Broad-Complex gene in Leptinotarsa decemlineata (Say).

Dietary delivery of bacterially expressed double-stranded RNA (dsRNA) has a great potential for management of Leptinotarsa decemlineata. An important first step is to discover possible RNA-interference (RNAi)-target genes effective against larvae, especially the old larvae. In the present paper, five putative Broad-Complex (BrC) cDNAs (Z1-Z4, and Z6) were identified in L. decemlineata. The expression of the five LdBrC isoforms was suppressed by juvenile hormone signaling, whereas the transcription was upregulated by 20-hydroxyecdysone signaling at the fourth (final) instar larval stage. Feeding of bacterially expressed dsBrC (derived from a common fragment of the five LdBrC variants) in the third- and fourth-instar larvae successfully knocked down the target mRNAs. For the fourth-instar LdBrC RNAi hypomorphs, they had a higher larval mortality compared with the controls. Moreover, most dsBrC-fed beetles did not pupate normally. After removal of the apolysed larval cuticle, a miniature adult was found. The adult head, compound eyes, prothorax, mesothorax, metathorax were found on the dorsal view. Distinct adult cuticle pigmentation was seen on the prothorax. The mouthparts, forelegs, midlegs, and hindlegs could be observed on the ventral view of the miniature adults. For the third-instar LdBrC RNAi specimens, around 20% moribund beetles remained as prepupae and finally died. Therefore, LdBrC is among the most attractive candidate genes for RNAi to control the fourth-instar larvae in L. decemlineata.

Extracellular RNA is transported from one generation to the next in Caenorhabditis elegans.

Experiences during the lifetime of an animal have been proposed to have consequences for subsequent generations. Although it is unclear how such intergenerational transfer of information occurs, RNAs found extracellularly in animals are candidate molecules that can transfer gene-specific regulatory information from one generation to the next because they can enter cells and regulate gene expression. In support of this idea, when double-stranded RNA (dsRNA) is introduced into some animals, the dsRNA can silence genes of matching sequence and the silencing can persist in progeny. Such persistent gene silencing is thought to result from sequence-specific interaction of the RNA within parents to generate chromatin modifications, DNA methylation, and/or secondary RNAs, which are then inherited by progeny. Here, we show that dsRNA can be directly transferred between generations in the worm Caenorhabditis elegans Intergenerational transfer of dsRNA occurs even in animals that lack any DNA of matching sequence, and dsRNA that reaches progeny can spread between cells to cause gene silencing. Surprisingly, extracellular dsRNA can also reach progeny without entry into the cytosol, presumably within intracellular vesicles. Fluorescently labeled dsRNA is imported from extracellular space into oocytes along with yolk and accumulates in punctate structures within embryos. Subsequent entry into the cytosol of early embryos causes gene silencing in progeny. These results demonstrate the transport of extracellular RNA from one generation to the next to regulate gene expression in an animal and thus suggest a mechanism for the transmission of experience-dependent effects between generations.

Metabolically stabilized double-stranded mRNA polyplexes.

The metabolic instability of mRNA currently limits its utility for gene therapy. Compared to plasmid DNA, mRNA is significantly more susceptible to digestion by RNase in the circulation following systemic dosing. To increase mRNA metabolic stability, we hybridized a complementary reverse mRNA with forward mRNA to generate double-stranded mRNA (dsmRNA). RNase A digestion of dsmRNA established a 3000-fold improved metabolic stability compared to single-stranded mRNA (ssmRNA). Formulation of a dsmRNA polyplex using a PEG-peptide further improved the stability by 3000-fold. Hydrodynamic dosing and quantitative bioluminescence imaging of luciferase expression in the liver of mice established the potent transfection efficiency of dsmRNA and dsmRNA polyplexes. However, hybridization of the reverse mRNA against the 5' and 3' UTR of forward mRNA resulted in UTR denaturation and a tenfold loss in expression. Repeat dosing of dsmRNA polyplexes produced an equivalent transient expression, suggesting the lack of an immune response in mice. Co-administration of excess uncapped dsmRNA with a dsmRNA polyplex failed to knock down expression, suggesting that dsmRNA is not a Dicer substrate. Maximal circulatory stability was achieved using a fully complementary dsmRNA polyplex. The results established dsmRNA as a novel metabolically stable and transfection-competent form of mRNA.

An RNAi-Based Control of Fusarium graminearum Infections Through Spraying of Long dsRNAs Involves a Plant Passage and Is Controlled by the Fungal Silencing Machinery.

Meeting the increasing food and energy demands of a growing population will require the development of ground-breaking strategies that promote sustainable plant production. Host-induced gene silencing has shown great potential for controlling pest and diseases in crop plants. However, while delivery of inhibitory noncoding double-stranded (ds)RNA by transgenic expression is a promising concept, it requires the generation of transgenic crop plants which may cause substantial delay for application strategies depending on the transformability and genetic stability of the crop plant species. Using the agronomically important barley-Fusarium graminearum pathosystem, we alternatively demonstrate that a spray application of a long noncoding dsRNA (791 nt CYP3-dsRNA), which targets the three fungal cytochrome P450 lanosterol C-14α-demethylases, required for biosynthesis of fungal ergosterol, inhibits fungal growth in the directly sprayed (local) as well as the non-sprayed (distal) parts of detached leaves. Unexpectedly, efficient spray-induced control of fungal infections in the distal tissue involved passage of CYP3-dsRNA via the plant vascular system and processing into small interfering (si)RNAs by fungal DICER-LIKE 1 (FgDCL-1) after uptake by the pathogen. We discuss important consequences of this new finding on future RNA-based disease control strategies. Given the ease of design, high specificity, and applicability to diverse pathogens, the use of target-specific dsRNA as an anti-fungal agent offers unprecedented potential as a new plant protection strategy.

Nosemosis control in European honey bees, Apis mellifera, by silencing the gene encoding Nosema ceranae polar tube protein 3.

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene and is conserved in a wide range of eukaryotic organisms. The RNAi mechanism has provided unique opportunities for combating honey bee diseases caused by various parasites and pathogens. Nosema ceranae is a microsporidian parasite of European honey bees, Apis mellifera, and has been associated with honey bee colony losses in some regions of the world. Here we explored the possibility of silencing the expression of a N. ceranae putative virulence factor encoding polar tube protein 3 (ptp3) which is involved in host cell invasion as a therapeutic strategy for controlling Nosema parasites in honey bees. Our studies showed that the oral ingestion of a dsRNA corresponding to the sequences of N. ceranae ptp3 could effectively suppress the expression of the ptp3 gene in N. ceranae-infected bees and reduce Nosema load. In addition to the knockdown of ptp3 gene expression, ingestion of ptp3-dsRNA also led to improved innate immunity in bees infected with N. ceranae along with an improvement in physiological performance and lifespan compared with untreated control bees. These results strongly suggest that RNAi-based therapeutics hold real promise for the effective treatment of honey bee diseases in the future, and warrant further investigation.

Morphological changes in different caste adult ant specificities of Polyrhachis vicina Roger (Hymenoptera, Formicidae) caused in estrogen-related receptor.

The estrogen-related receptor (ERR) gene is a member of the nuclear receptor subfamily. Previous studies have indicated that ERR plays important roles in regulating insect growth and development. How ERR is associated with ant caste specificities remains unclear. In this study, we attempted to identify the role of ERR in the regulation of different adult caste specificities of Polyrhachis vicina Roger. Significant variations were detected in the ants including PvERR expressions, some physiological indexes and morphological traits including survival rate, body weight, body length, head width and abdominal appearance by different techniques. The results revealed that when PvERR expressions is up-regulated, boundaries of the abdominal segments were indistinct on the ventral side of the abdomen in males. Down-regulation of PvERR expressions caused abdominal swelling in males and a distended ventral abdomen in females and workers. Variation in PvERR expressions led to a remarkable decline in ant survival rates, particularly for males. These results indicated that different caste adults appeared to have different degrees of sensitivity in physiological response and morphological changes caused by variation in PvERR expressions. Thus, our data demonstrate that PvERR plays an important role in regulating the different adult caste specificities of P. vicina.

Rapid and persistent RNAi response in western corn rootworm adults.

RNA interference (RNAi) has proven effective for controlling pest insects such as western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). Previous studies have shown that WCR adults display a robust RNAi response to orally-administered double-stranded RNA (dsRNA). However, it is unclear how quickly the response occurs after ingestion or how long RNAi effect lasts after WCR stop ingesting diet containing dsRNA. In the current study, WCR adult females were provided with diet treated with dsRNAs of Laccase 2 and Argonaute 2, two nonessential genes, for 8 days. RNAi response in WCR females commenced as early as 10 h after exposure to dsRNA and lasted up to 40 days after exposure to dsRNA ended. Our results show that dsRNA-mediated RNAi response in WCR females is rapid and long-lasting. These findings suggest that even a short-term ingestion of transgenic plants expressing dsRNA by WCR may have a sustained impact on this insect.

Evaluation of some potential target genes and methods for RNAi-mediated pest control of the corn earworm Helicoverpa zea.

In this study, we explored the efficacy of knockdown four genes required for proper nervous system function by RNAi, in the corn earworm Helicoverpa zea (Boddie). Three of these genes encode components of validated insecticide target sites. We synthesized cDNA sequences orthologous to the Drosophila melanogaster genes Para (paralyticts), TipE (temperature-induced paralysis locus E), GluCl (glutamate-gated chloride channel), and Notch, and used these fragments to synthesize double-stranded RNAs (dsRNAs). We then performed experiments in an attempt to induce RNAi-mediated effects on gene expression and viability using three modes of delivery of the dsRNAs: microinjection of eggs, soaking of eggs and feeding of larvae. Microinjection of dsRNAs into eggs induced reduced hatch rates and knockdown of target gene expression for GluCl, para and TipE, but not for Notch. However, neither feeding nor soaking eggs in dsRNA solutions resulted in discernable RNAi effects. These results demonstrated the susceptibility to RNAi effects of the expression of H. zea genes encoding insecticide target sites, which suggests future avenues of research toward practical applications.

RNA Interference of the Muscle Actin Gene in Bed Bugs: Exploring Injection Versus Topical Application for dsRNA Delivery.

Bed bugs are one the most troublesome household pests that feed primarily on human blood. RNA interference (RNAi) is currently being pursued as a potential tool for insect population management and has shown efficacy against some phytophagous insects. We evaluated the different techniques to deliver dsRNA specific to bed bug muscle actin (dsactin) into bed bugs. Initially, stability of dsRNA in human blood was studied to evaluate the feasibility of feeding method. Adult bed bugs were injected with dsRNA between last thoracic segment and first abdominal segment on the ventral side, with a dose of 0.2 µg dsactin per insect. In addition to injection, dsactin was mixed in acetone and treated topically in the abdomens of fifth stage nymphs. We found the quick degradation of dsRNA in blood. Injection of dsactin caused significant depletion of actin transcripts and substantial reduction in oviposition and lethality in female adults. Topically treated dsRNA in fifth stage nymphs had no effect on actin mRNA expression and survival. Our results demonstrated that injection is a reliable method of dsRNA delivery into bed bugs while topical treatment was not successful. This research provides an understanding on effective delivery methods of dsRNA into bed bugs for functional genomics research and feasibility of the RNAi based molecules for pest management purposes.

Pro-apoptotic signaling induced by Retinoic acid and dsRNA is under the control of Interferon Regulatory Factor-3 in breast cancer cells.

Breast cancer is one of the most lethal malignancies for women. Retinoic acid (RA) and double-stranded RNA (dsRNA) are considered signaling molecules with potential anticancer activity. RA, co-administered with the dsRNA mimic polyinosinic-polycytidylic acid (poly(I:C)), synergizes to induce a TRAIL (Tumor-Necrosis-Factor Related Apoptosis-Inducing Ligand)- dependent apoptotic program in breast cancer cells. Here, we report that RA/poly(I:C) co-treatment, synergically, induce the activation of Interferon Regulatory Factor-3 (IRF3) in breast cancer cells. IRF3 activation is mediated by a member of the pathogen recognition receptors, Toll-like receptor-3 (TLR3), since its depletion abrogates IRF3 activation by RA/poly(I:C) co-treatment. Besides induction of TRAIL, apoptosis induced by RA/poly(I:C) correlates with the increased expression of pro-apoptotic TRAIL receptors, TRAIL-R1/2, and the inhibition of the antagonistic receptors TRAIL-R3/4. IRF3 plays an important role in RA/poly(I:C)-induced apoptosis since IRF3 depletion suppresses caspase-8 and caspase-3 activation, TRAIL expression upregulation and apoptosis. Interestingly, RA/poly(I:C) combination synergizes to induce a bioactive autocrine/paracrine loop of type-I Interferons (IFNs) which is ultimately responsible for TRAIL and TRAIL-R1/2 expression upregulation, while inhibition of TRAIL-R3/4 expression is type-I IFN-independent. Our results highlight the importance of IRF3 and type-I IFNs signaling for the pro-apoptotic effects induced by RA and synthetic dsRNA in breast cancer cells.