RNAi Effect Research Articles

Study on the inhibition of non-small cell lung cancer mediated by chitosan-based gene carrier delivering STAT3-shRNA

Systemic chemotherapy and radiotherapy often yield poor effect in the postoperative treatment of non-small cell lung cancer (NSCLC) and induce drug resistance. Herein, we proposed a targeted therapeutic approach utilizing gene carrier-mediated specific shRNA method. Firstly, the targeted short hairpin shRNA sequence, designed based on the STAT3 gene sequence, was inserted into the eukaryotic expression vector pGPU6/GFP/Neo to form the recombinant plasmid STAT3-shRNA. Next, a novel gene carrier, Vitamin E Succinate-Chitosan-Histidine (VES-CTS-His, VCH), was synthesized through an acylation reaction. The VCH was combined with pGPU6/GFP/Neo STAT3-shRNA recombinant plasmid by electrostatic interactions to form stable particles. VCH/pDNA, with typical nanoscale dimensions, could accumulate in tumor tissues through the EPR effect and enter tumor cells via endocytosis. VCH exhibited good pH responsiveness and could dissociate in the acidic microenvironment of tumors, thereby releasing the plasmids. Subsequently, the plasmids could downregulate STAT3 expression through RNAi effect. Inhibiting or blocking the expression of the STAT3 gene could significantly enhance the apoptotic induction and growth inhibition effects on NSCLC cells through the PI3K and mTOR signaling pathways, thereby achieving the goal of tumor treatment. This study provides a novel method for the construction of novel non-viral gene carriers and clinical gene-targeted therapy for NSCLC.

CYP303A1 regulates molting and metamorphosis through 20E signaling in Nilaparvata lugens Stål (Hemiptera: Delphacidae)

Cytochrome P450s play a crucial role in the breakdown of external substances and perform important activities in the hormone system of insects. It has been understood that P450s were essential in the metabolism of ecdysteroids. CYP303A1 is a highly conserved CYP in most insects, but its specific physiological functions remain poorly understood in Nilaparvata lugens Stål. In this study, NlCYP303A1 was identified and highly expressed in the pre-molt stages, predominantly in the cuticle-producing tissues. Silencing of NlCYP303A1 caused a lethal phenotype with a molting defect. Moreover, the 20E titers, the expression levels of Halloween genes, and critical genes associated with the 20E signaling pathway in N. lugens nymphs were significantly decreased with the silencing NlCYP303A1. We further performed additional backfilling of 20E to rescue the RNAi effects on NlCYP303A1. The gene expression levels that were previously reduced caused by silencing NlCYP303A1 were significantly elevated. However, the molting defects of nymphs were not effectively improved. The results demonstrated NlCYP303A1 plays a crucial role in the molting and metamorphosis of N. lugens by regulating the 20E signaling pathway and cuticular formation, enhances the understanding of the functional role of CYP 2 clans, and identifies candidate gene for RNAi-based control of N. lugens.

Establishment of RNAi-Mediated Pest Control Method for Red Imported Fire Ant, Solenopsis invicta.

RNAi plays a crucial role in insect gene function research and pest control field. Nonetheless, the variable efficiency of RNAi across diverse insects and off-target effects also limited its further application. In this study, we cloned six essential housekeeping genes from Solenopsis invicta and conducted RNAi experiments by orally administering dsRNA. Then, we found that mixing with liposomes significantly enhanced the RNAi efficiency by targeting for SiV-ATPaseE. Additionally, we observed a certain lethal effect of this dsRNA on queens by our established RNAi system. Furthermore, no strict sequence-related off-target effects were detected. Finally, the RNAi effect of large-scale bacteria expressing dsRNA was successfully confirmed for controlling S. invicta. In summary, this study established an RNAi system for S. invicta and provided a research template for the future development of nucleic acid drugs based on RNAi.

The development of an egg-soaking method for delivering dsRNAs into spider mites

Recently, the first sprayable RNAi biopesticide, Ledprona, against the Colorado potato beetle, Leptinotarsa decemlineata, has been registered at the United States Environmental Protection Agency. Spider mites (Acari: Tetranychidae), a group of destructive agricultural and horticultural pests, are notorious for rapid development of insecticide/acaricide resistance. The management options, on the other hand, are extremely limited. RNAi-based biopesticides offer a promising control alternative to address this emerging issue. In this study, we i) developed an egg-soaking dsRNA delivery method; ii) evaluated the factors influencing RNAi efficiency, and finally iii) investigated the potential mode of entry of this newly developed egg-soaking RNAi method. In comparison to other dsRNA delivery methods, egg-soaking method was the most efficient, convenient/practical, and cost-effective method for delivering dsRNAs into spider mites. RNAi efficiency of this RNAi method was affected by target genes, dsRNA concentration, developmental stages, and mite species. In general, the hawthorn spider mite, Amphitetranychus viennensis, is more sensitive to RNAi than the two-spotted spider mite, Tetranychus urticae, and both of them have dose-dependent RNAi effect. For different life stages, egg and larvae are the most sensitive life stages to dsRNAs. For different target genes, there is no apparent association between the suppression level and the resultant phenotype. Finally, we demonstrated that this egg-soaking RNAi method acts as both stomach and contact toxicity. Our combined results demonstrate the effectiveness of a topically applied dsRNA delivery method, and the potential of a spray induced gene silencing (SIGS) method as a control alternative for spider mites.

A nematode-inducible promoter can effectively drives RNAi construct to confer Meloidogyne incognita resistance in tomato.

Heterologous expression of a nematode-responsive promoter in tomato successfully driven the RNAi constructs to impart root-knot nematode resistance. The root-knot nematode Meloidogyne incognita seriously afflicts the global productivity of tomatoes. Nematode management options are extremely reliant on chemical methods, however, only a handful of nematicides are commercially available. Additionally, nematodes have developed resistance-breaking phenotypes against the commercially available Mi gene-expressing tomatoes. Nematode resistance in crop plants can be enhanced using the bio-safe RNAi technology, in which plants are genetically modified to express nematode gene-specific dsRNA/siRNA molecules. However, the majority of the RNAi crops conferring nematode tolerance have used constitutive promoters, which have many limitations. In the present study, using promoter-GUS fusion, we functionally validated two nematode-inducible root-specific promoters (pAt1g74770 and pAt2g18140, identified from Arabidopsis thaliana) in the Solanum lycopersicum-M. incognita pathosystem. pAt2g18140 was found to be nematode-responsive during 10-21days post-inoculation (dpi) and became non-responsive during the late infection stage (28 dpi). In contrast, pAt1g74770 remained nematode-responsive for a longer duration (10-28 dpi). Next, a number of transgenic lines were developed that expressed RNAi constructs (independently targeting the M. incognita integrase and splicing factor genes) driven by the pAt1g74770 promoter. M. incognita parasitic success (measured by multiplication factor ratio) in pAt1g74770:integrase and pAt1g74770:splicing factor RNAi lines were significantly reduced by 60.83-74.93% and 69.34-75.31%, respectively, compared to the control. These data were comparable with the RNAi lines having CaMV35S as the promoter. Further, a long-term RNAi effect was evident, because females extracted from transgenic lines were of deformed shape with depleted transcripts of integrase and splicing factor genes. We conclude that pAt1g74770 can be an attractive alternative to drive localized expression of RNAi constructs rather than using a constitutive promoter. The pAt1g74770-driven gene silencing system can be expanded into different plant-nematode interaction models.

Identification, structural modeling, gene expression analysis and RNAi effect of putative phospholipase A2 in the lone star tick Amblyomma americanum

Amblyomma americanum, also known as the lone star tick, is a small arachnid that feeds on blood and can spread disease to humans and other animals. Despite the overlapped ecological niche, geographic distribution, and host selection, there is no proof that A. americanum transmits the pathogen Borrelia burgdorferi that causes Lyme disease. Studies have shown that phospholipase A2 (PLA2) may act as a tool to eliminate B. burgdorferi, but particular PLA2 genes in A. americanum have not been identified and functionally characterized. Using the de novo sequencing method, we identified 42 putative A. americanum PLA2 (pAaPLA2) homologs in the present study, of which three pAaPLA2 had calcium binding sites and canonical histidine catalytic sites. Then, we determined phylogenetic relationships, sequence alignments, and conserved protein motifs of these pAaPLA2s. Protein structural analysis demonstrated that pAaPLA2s primarily consisted of α-helices, β-sheets, and random coils. These genes were predicted to be engaged in the phospholipid metabolic process, arachidonic acid secretion, and PLA2 activity by functional annotation analysis. A transcriptional factor (Bgb) was discovered that interacted with pAaPLA2 proteins that may have unrecognized roles in regulating neuronal development. Based on the RNA-seq data, we surveyed expression profiles of key pAaPLA2-related genes to reveal putative modulatory networks of these genes. RNAi knockdown of pAaPLA2_1, a dominant isoform in A. americanum, led to decreased bacterial inhibition ability, suggesting pAaPLA2 may play an important role in mediating immune responses. Collectively, this study provides essential evidence of the identification, gene structure, phylogeny, and expression analysis of pAaPLA2 genes in A. americanum, and offers a deeper understanding of the putative borreliacidal roles in the lone star tick.

Nicotinamide-N-methyltransferase regulates lipid metabolism via SAM and 1-methylnicotinamide in the AML12 hepatocyte cell line.

Nicotinamide-N-methyltransferase (NNMT) is an enzyme that consumes S-adenosyl-methionine (SAM) and nicotinamide (NAM) to produce S-adenosyl-homocysteine (SAH) and 1-methylnicotinamide (MNAM). How much NNMT contributes to the quantity regulation of these four metabolites depends on whether NNMT is a major consumer or producer of these metabolites, which varies among various cellular contexts. Yet, whether NNMT critically regulates these metabolites in the AML12 hepatocyte cell line has been unexplored. To address this, we knockdown Nnmt in AML12 cells and investigate the effects of Nnmt RNAi on metabolism and gene expression. We find that Nnmt RNAi accumulates SAM and SAH, whereas it reduces MNAM with NAM being unaltered. These results indicate that NNMT is a significant consumer of SAM and critical for MNAM production in this cell line. Moreover, transcriptome analyses reveal that altered SAM and MNAM homeostasis is accompanied by various detrimental molecular phenotypes, as exemplified by the down-regulations of lipogenic genes, such as Srebf1. Consistent with this, oil-red O-staining experiments demonstrate the decrease of total neutral lipids upon Nnmt RNAi. Treating Nnmt RNAi AML12 cells with cycloleucine, an inhibitor of SAM biogenesis suppresses SAM accumulation and rescues the decrease of neutral lipids. MNAM also shows activity to elevate neutral lipids. These results suggest that NNMT contributes to lipid metabolism by maintaining proper SAM and MNAM homeostasis. This study provides an additional example where NNMT plays a critical role in regulating SAM and MNAM metabolism.

Silencing of insect dsRNase genes enhances the plastid-mediated RNAi effect on the Colorado potato beetle

Silencing of insect dsRNase genes enhances the plastid-mediated RNAi effect on the Colorado potato beetle

Analyses of ecdysteroid transporters in the fat body of Tribolium castaneum.

The control of insect moulting and metamorphosis involves ecdysteroids that orchestrate the execution of developmental genetic programs by binding to dimeric hormone receptors consisting of the ecdysone receptor (EcR) and ultraspiracle (USP). In insects, the main ecdysteroids comprise ecdysone (E), which is synthesized in the prothoracic gland and secreted into the haemolymph, and 20-hydroxyecdysone (20E), which is considered the active form by binding to the nuclear receptor of the target cell. While biosynthesis of ecdysteroids has been studied in detail in different insects, the transport systems involved in guiding these steroid hormones across cellular membranes have just recently begun to be studied. By analysing RNAi phenotypes in the red flour beetle, Tribolium castaneum, we have identified three transporter genes, TcABCG-8A, TcABCG-4D and TcOATP4-C1, whose silencing results in phenotypes similar to that observed when the ecdysone receptor gene TcEcRA is silenced, that is, abortive moulting and abnormal development of adult compound eyes during the larval stage. The genes of all three transporters are expressed at higher levels in the larval fat body of T. castaneum. We analysed potential functions of these transporters by combining RNAi and mass spectrometry. However, the analysis of gene functions is challenged by mutual RNAi effects indicating interdependent gene regulation. Based on our findings, we propose that TcABCG-8A, TcABCG-4D and TcOATP4-C1 participate in the ecdysteroid transport in fat body cells, which are involved in E → 20E conversion catalysed by the P450 enzyme TcShade.

Compartmentalized PGRP expression along the dipteran Bactrocera dorsalis gut forms a zone of protection for symbiotic bacteria.

All metazoan guts are subject to opposing pressures wherein the immune system must eliminate pathogens while tolerating the presence of symbiotic microbiota. The Imd pathway is an essential defense against invading pathogens in insect guts, but tolerance mechanisms are less understood. Here, we find PGRP-LB and PGRP-SB express mainly in the anterior and middle midgut in a similar pattern to symbiotic Enterobacteriaceae bacteria along the Bactrocera dorsalis gut. Knockdown of PGRP-LB and PGRP-SB enhances the expression of antimicrobial peptide genes and reduces Enterobacteriaceae numbers while increasing abundance of opportunistic pathogens. Microbiota numbers recover to normal levels after the RNAi effect subsided. In contrast, high expression of PGRP-LC in the foregut allows increased antibacterial peptide production to efficiently filter the entry of pathogens, protecting the symbiotic bacteria. Our study describes a mechanism by which regional expression of PGRPs construct a protective zone for symbiotic microbiota while maintaining the ability to fight pathogens.

Identification and functional analysis of two potential RNAi targets for chitin degradation in Holotrichia parallela Motschulsky (Insecta Coleoptera)

Chitin metabolism enzymes are safe and desirable targets for pest management. β-N-acetylglucosaminidase (NAG) and N-acetylglucosamine kinase (NAGK) are involved in chitin degradation. NAG is the main glycosidase that works synergistically with chitinases. NAGK is a key enzyme for the generation of UDP-Nacetylglucosamine (UDP-GlcNAc) and for the conversion of GlcNAc into GlcNAc 6-phosphate (GlcNAc-6-P). In this study, NAG and NAGK genes were identified from Holotrichia parallela, a polyphagous soil pest that causes serious damage to crops. The spatiotemporal expression investigated by RT-qPCR indicated that the two genes are expressed in all larval developmental stages. HpNAG is highly expressed in the integument and HpNAGK overexpressed in the midgut. After injection of dsHpNAG and dsHpNAGK, a significant RNAi effect was found after 72 h and larvae stopped growing. The survival rates of larvae were 13.3% and 16.7%, respectively. RNAi of HpNAG and HpNAGK regulated the expression levels of chitin metabolism-related genes, indicating that these two genes could be critical in the chitin metabolism. Furthermore, silencing HpNAG and HpNAGK reduced the thickness of the cuticle, and decreased its content of chitin. The study will lay a foundation for further clarifying the mechanism of chitin metabolism and provide potential targets for the biological control of H. parallela larvae.

Multifunctional Therapeutic Cyclodextrin-Appended Dendrimer Complex for Treatment of Systemic and Localized Amyloidosis.

Amyloidosis pathologically proceeds via production of amyloidogenic proteins by organs, formation of protein aggregates through structural changes, and their deposition on tissues. A growing body of evidence demonstrates that amyloidosis generally develops through three critical pathological steps: (1) production of amyloid precursor proteins, (2) amyloid formation, and (3) amyloid deposition. However, no clinically effective therapy that is capable of targeting each pathological step of amyloidosis independently is currently available. Here, we combined therapeutic effects and developed a short hairpin RNA expression vector (shRNA) complex with a cyclodextrin-appended cationic dendrimer (CDE) as a novel multitarget therapeutic drug that is capable of simultaneously suppressing these three steps. We evaluated its therapeutic effects on systemic transthyretin (ATTR) amyloidosis and Alzheimer's disease (AD) as localized amyloidosis, by targeting TTR and amyloid β, respectively. CDE/shRNA exhibited RNAi effects to suppress amyloid protein production and also achieved both inhibition of amyloid formation and disruption of existing amyloid fibrils. The multitarget therapeutic effects of CDE/shRNA were confirmed by evaluating TTR deposition reduction in early- and late-onset human ATTR amyloidosis model rats and amyloid β deposition reduction in AppNL-G-F/NL-G-F AD model mice. Thus, the CDE/shRNA complex exhibits multifunctional therapeutic efficacy and may reveal novel strategies for establishing curative treatments for both systemic and localized amyloidosis.

Theranostic Ruthenium Polypyridine Nanoparticles for Targeted Chimera Delivery into Ovarian Cancer Cells

Efficient in vivo delivery of small interfering RNAs (siRNAs) to target cells is challenging in clinical applications. Ruthenium (II) polypyridyl complexes have been discovered as imaging theranostic and anticancer agents due to their photophysical and biological properties. However, the clinical implementation of ruthenium complexes is limited by cancer cell selectivity. This study presents a novel siRNA delivery nanoplatform by ruthenium polypyridine complex nanoparticles (RPNs). The EGFR RNA aptamer and Notch3 siRNA chimera-loaded RPNs showed superior RNAi effects against Notch3 gene compared to Lipofectamine. Also, RPN-chimera complexes exhibited significant in vivo antitumor effects against ovarian cancer, which exhibited much potential in future cancer imaging guided gene therapy.

Localized efficacy of environmental RNAi in Tetranychus urticae

Environmental RNAi has been developed as a tool for reverse genetics studies and is an emerging pest control strategy. The ability of environmental RNAi to efficiently down-regulate the expression of endogenous gene targets assumes efficient uptake of dsRNA and its processing. In addition, its efficiency can be augmented by the systemic spread of RNAi signals. Environmental RNAi is now a well-established tool for the manipulation of gene expression in the chelicerate acari, including the two-spotted spider mite, Tetranychus urticae. Here, we focused on eight single and ubiquitously-expressed genes encoding proteins with essential cellular functions. Application of dsRNAs that specifically target these genes led to whole mite body phenotypes—dark or spotless. These phenotypes were associated with a significant reduction of target gene expression, ranging from 20 to 50%, when assessed at the whole mite level. Histological analysis of mites treated with orally-delivered dsRNAs was used to investigate the spatial range of the effectiveness of environmental RNAi. Although macroscopic changes led to two groups of body phenotypes, silencing of target genes was associated with the distinct cellular phenotypes. We show that regardless of the target gene tested, cells that displayed histological changes were those that are in direct contact with the dsRNA-containing gut lumen, suggesting that the greatest efficiency of the orally-delivered dsRNAs is localized to gut tissues in T. urticae.

Immunomodulatory effect and safety of TNF-α RNAi mediated by oral yeast microcapsules in rheumatoid arthritis therapy.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that requires long-term treatment and monitoring. Inhibition of inflammatory gene expression by gene therapy is a significant breakthrough in RA treatment, but the lack of a safe and effective gene delivery system hinders its application. Since oral administration can significantly reduce wound infection caused by parenteral administration, it also has the advantages of high patient compliance and convenience. Therefore, oral administration may be the best option for the treatment of this chronic disease. In this study, we developed a novel oral drug system by delivering tumor necrosis factor-α (TNF-α) short hairpin RNA (shRNA) mediated by non-pathogenic yeast to evaluate its regulation of systemic immune inflammation and safety in RA. Non-pathogenic yeast can resist the destruction of the gastrointestinal acid-base environment and can be recognized by the intestinal macrophages and act on systemic inflammatory lesions. Oral administration of yeast-mediated TNF-α shRNA significantly reduced the expression of TNF-α predominant pro-inflammatory factors in intestinal macrophages and joint synovium, and up-regulated the expression of anti-inflammatory cytokine IL-10 and M2 macrophages, systematically regulating the inflammatory response. This yeast-mediated oral gene delivery system can not only significantly inhibit knee joint synovial inflammation, but also has no toxic effects on peripheral blood and major organs. Therefore, yeast-mediated oral delivery of TNF-α shRNA may be used as a novel gene therapy strategy to treat RA through immunomodulating the mononuclear phagocyte system from the intestine to the joint synovium, and ultimately regulating systemic and local immune inflammation, providing new ideas for the clinical treatment of RA.

Investigating Variant Surface Glycoprotein Dynamics under Inhibition of Fatty Acid Synthesis

Trypanosoma brucei is a hemoflagellate that causes African Sleeping Sickness in humans and nagana in cattle. The T. brucei surface is coated with ten million identical copies of single Variant Surface Glycoprotein (VSG), one of ~2500 VSGs encoded in the genome. VSG helps T. brucei evade host immune response by two mechanisms: antigenic variation (changing the surface coat) and endocytosis (internalizing immune complexes, recycling the VSG while degrading the immune components). For both strategies, the VSG glycosylphosphatidylinositol (GPI) membrane anchor and its two myristates are thought to play a key role in VSG mobility and trafficking. The role of fatty acid synthesis (FAS) in VSG function is unknown, but it is likely needed to maintain VSG GPI‐anchor myristoylation. To reduce FAS, we use RNAi‐mediated depletion of Acetyl‐CoA Carboxylase (ACC), the first enzyme in the FAS pathway. We found that ACC RNAi resulted in a significant 30‐40% reduction in both receptor‐mediated and fluid‐phase endocytosis. To determine the effect of ACC RNAi on VSG dynamics, we assessed VSG half‐life by surface biotinylation and streptavidin blotting. We found that ACC RNAi resulted in ~40% decrease in surface VSG half‐life (p<0.05). Currently, we are examining two possible mechanisms leading to increased VSG turnover upon ACC RNAi: shedding in the media or intracellular degradation. Our preliminary data rules out degradation by proteasome or lysosome and reveals that ACC RNAi altered shedding of multiple surface proteins, including VSG. For the future, we will use proteomics to identify and quantify the proteins shed in the media, and we will investigate the mechanisms of their release.

Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine-rich preference for virus- and non-virus-based expression vectors in plants.

SummaryArtificial microRNA (amiRNA) technology has allowed researchers to direct efficient silencing of specific transcripts using as few as 21 nucleotides (nt). However, not all the artificially designed amiRNA constructs result in selection of the intended ~21‐nt guide strand amiRNA. Selection of the miRNA guide strand from the mature miRNA duplex has been studied in detail in human and insect systems, but not so much for plants. Here, we compared a nuclear‐replicating DNA viral vector (tomato mottle virus, ToMoV, based), a cytoplasmic‐replicating RNA viral vector (tobacco mosaic virus, TMV, based), and a non‐viral binary vector to express amiRNAs in plants. We then used deep sequencing and mutational analysis and show that when the structural factors caused by base mismatches in the mature amiRNA duplex were excluded, the nucleotide composition of the mature amiRNA region determined the guide strand selection. We found that the strand with excess purines was preferentially selected as the guide strand and the artificial miRNAs that had no mismatches in the amiRNA duplex were predominantly loaded into AGO2 instead of loading into AGO1 like the majority of the plant endogenous miRNAs. By performing assays for target effects, we also showed that only when the intended strand was selected as the guide strand and showed AGO loading, the amiRNA could provide the expected RNAi effects. Thus, by removing mismatches in the mature amiRNA duplex and designing the intended guide strand to contain excess purines provide better control of the guide strand selection of amiRNAs for functional RNAi effects.

Drosophila ZIP13 over-expression or transferrin1 RNAi influences the muscle degeneration of Pink1 RNAi by elevating iron levels in mitochondria.

Disruption of iron homeostasis in the brain of Parkinson's disease (PD) patients has been reported for many years, but the underlying mechanisms remain unclear. To investigate iron metabolism genes related to PTEN-induced kinase 1 (Pink1) and parkin (E3 ubiquitin ligase), two PD-associated proteins that function to coordinate mitochondrial turnover via induction of selective mitophagy, we conducted a genetic screen in Drosophila and found that altered expression of genes involved in iron metabolism, such as Drosophila ZIP13 (dZIP13) or transferrin1 (Tsf1), significantly influences the disease progression related to Pink1 but not parkin. Several phenotypes of Pink1 mutant and Pink1 RNAi but not parkin mutant were significantly rescued by over-expression (OE) of dZIP13 (dZIP13 OE) or silencing of Tsf1 (Tsf1 RNAi) in the flight muscles. The rescue effects of dZIP13 OE or Tsf1 RNAi were not exerted through mitochondrial disruption or mitophagy; instead, the iron levels in mitochondira were significantly increased, resulting in enhanced activities of enzymes participating in respiration and increased ATP synthesis. Consistently, the rescue effects of dZIP13 OE or Tsf1 RNAi on Pink1 RNAi can be inhibited by decreasing the iron levels in mitochondria through mitoferrin (dmfrn) RNAi. This study suggests that dZIP13, Tsf1, and dmfrn might act independently of parkin in a parallel pathway downstream of Pink1 by modulating respiration and indicates that manipulation of iron levels in mitochondria may provide a novel therapeutic strategy for PD associated with Pink1.

Cloning and Functional Characterization of a Double-Stranded RNA-Degrading Nuclease in the Tawny Crazy Ant (Nylanderia fulva).

RNA interference is a powerful tool that post-transcriptionally silences target genes. However, silencing efficacy varies greatly among different insect species. Recently, we attempted to knock down some housekeeping genes in the tawny crazy ant (Nylanderia fulva), a relatively new invasive species in the southern United States, but only achieved relatively low silencing efficiency when dsRNA was orally administered. Here, we detected divalent cation-dependent, dsRNA-degrading activity in the midgut fluid of worker ants in ex vivo assays. To determine whether dsRNA degradation could contribute to low effectiveness of oral RNAi in N. fulva, we cloned its sole dsRNase gene (NfdsRNase). The deduced amino acid sequence contained a signal peptide and an endonuclease domain. Sequence alignment indicated a high degree of similarity with well-characterized dsRNases, particularly the six key residues at active sites. We also identified dsRNase homologs from five other ant species and found a tight phylogenetic relationship among ant dsRNases. NfdsRNase is expressed predominantly in the abdomen of worker ants. Oral delivery of dsRNA of NfdsRNase significantly reduced the expression of NfdsRNase transcripts, and substantially suppressed dsRNA-degrading activity of worker ants’ midgut fluids as well. Our data suggest that dsRNA stability in the alimentary tract is an important factor for gene silencing efficiency in N. fulva, and that blocking NfdsRNase in gut lumen could potentially improve RNAi, a novel pest management tactic in control of N. fulva and other ant species.

Heparanase-1 is downregulated in chemoradiotherapy orbital rhabdomyosarcoma and relates with tumor growth as well as angiogenesis.

To determine the role of heparanase-1 (HPSE-1) in orbital rhabdomyosarcoma (RMS), and to investigate the feasibility of HPSE-1 targeted therapy for RMS. Immunohistochemistry was performed to analyze HPSE-1 expression in 51 cases of orbital RMS patients (including 28 cases of embryonal RMS and 23 cases of alveolar RMS), among whom there were 27 treated and 24 untreated with preoperative chemoradiotherapy. In vitro, studies were conducted to examine the effect of HPSE-1 silencing on RMS cell proliferation and tube formation of human umbilical vein endothelial cells (HUVECs). RD cells (an RMS cell line) and HUVECs were infected with HPSE-1 shRNA lentivirus at a multiplicity of infection (MOI) of 10 and 30 separately. Real-time PCR and Western blot were applied to detect the mRNA and protein expression levels of HPSE-1. Cell viability of treated or control RD cells was evaluated by cell counting kit-8 (CCK-8) assay. Matrigel tube formation assay was used to evaluate the effect of HPSE-1 RNAi on the tube formation of HUVECs. Immunohistochemistry showed that the expression rate of HPSE-1 protein was 92.9% in orbital embryonal RMS and 91.3% in orbital alveolar RMS. Tissue from alveolar orbital RMS did not show relatively stronger staining than that from the embryonal orbital RMS. However, despite the types of RMS, comparing the cases treated chemoradiotherapy with those untreated, we have observed that chemoradiotherapy resulted in weaker staining in patients' tissues. The expression levels of HPSE-1 declined significantly in both the mRNA and protein levels in HPSE-1 shRNA transfected RD cells. The CCK-8 assay showed that lentivirus-mediated HPSE-1 silencing resulted in significantly reduced RD cells viability in vitro. Silencing HPSE-1 expression also inhibited VEGF-induced tube formation of HUVECs in Matrigel. HPSE-1 silencing may be a promising therapy for the inhibition of orbital RMS progression.