RNAi-mediated Gene Knockdown Research Articles

Liprin-\u03b11 modulates cancer cell signaling by transmembrane protein CD82 in adhesive membrane domains linked to cytoskeleton

BackgroundPPFIA1 is located at the 11q13 region commonly amplified in cancer. The protein liprin-α1 encoded by PPF1A1 contributes to the adhesive and invasive structures of cytoskeletal elements and is located at the invadosomes in cancer cells. However, the precise mechanism of liprin-α1 function in cancer progression has remained elusive.MethodsInvasion regulating activity of liprin-α1 was examined by analyzing the functions of squamous cell carcinoma of head and neck (HNSCC) cell lines in three-dimensional collagen I after RNAi mediated gene knockdown. Transcriptome profiling and Gene Set Enrichment Analysis from HNSCC and breast cancer cells were used to identify expression changes relevant to specific cellular localizations, biological processes and signaling pathways after PPFIA1 knockdown. The significance of the results was assessed by relevant statistical methods (Wald and Benjamini-Hochberg). Localization of proteins associated to liprin-α1 was studied by immunofluorescence in 2D and 3D conditions. The association of PPFIA1 amplification to HNSCC patient survival was explored using The Cancer Genome Atlas data.ResultsIn this study, we show that liprin-α1 regulates biological processes related to membrane microdomains in breast carcinoma, as well as protein trafficking, cell-cell and cell-substrate contacts in HNSCC cell lines cultured in three-dimensional matrix. Importantly, we show that in all these cancer cells liprin-α1 knockdown leads to the upregulation of transmembrane protein CD82, which is a suppressor of metastasis in several solid tumors.ConclusionsOur results provide novel information regarding the function of liprin-α1 in biological processes essential in cancer progression. The results reveal liprin-α1 as a novel regulator of CD82, linking liprin-α1 to the cancer cell invasion and metastasis pathways.

Development of a Gateway-compatible pCAMBIA binary vector for RNAi-mediated gene knockdown in plants

RNA interference (RNAi), based on hairpin RNA (hpRNA) expression, plays an important role in functional analysis of plant genes. Traditional methods for making RNAi constructs usually involve multiple time-consuming cloning steps. We have developed a Gateway-compatible binary vector for RNAi-mediated gene knockdown in plants from pCAMBIA2301 and pHANNIBAL vectors. The new plant RNAi binary vector, named pCAMBIA2301-GW-RNAi, has two inverted repeated Gateway cassettes driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter. This enables site-specific recombination at two sites by one Gateway LR reaction without restriction enzymes and ligases. The pCAMBIA2301-GW-RNAi vector's effectiveness was evaluated by Agrobacterium-mediated transient co-expression assays of overexpression and silencing constructs of HvCEBiP in Nicotiana benthamiana followed by western blot analysis. Obtained results show that the developed RNAi vector successfully knocked down 35S-driven expression of HvCEBiP, as expression levels of the encoded HvCEBiP protein were significantly reduced.

Neuronal IL-4Rα modulates neuronal apoptosis and cell viability during the acute phases of cerebral ischemia.

Ischemic stroke caused by an embolus or local thrombosis results in neural tissue damage (an infarct) in the territory of the occluded cerebral artery. Decades of studies have increased our understanding of the molecular events during cerebral infarction; however, translation of these discoveries to druggable targets for ischemic stroke treatment has been largely disappointing. Interleukin-4 (IL-4) is a multifunctional cytokine that exerts its cellular activities via the interleukin-4 receptor α (IL-4Rα). This cytokine receptor complex is associated with diverse immune and inflammatory responses. Recent studies have suggested a role of the cytokine IL-4 in long-term ischemic stroke recovery, involving immune cell activity. In contrast, the role of the receptor, IL-4Rα especially in the acute phase of infarction is unclear. In this study, we determined that IL-4Rα is expressed on neurons and that during the early phases of cerebral infarction (24 h) levels of this receptor are increased to regulate cellular apoptosis factors through activation of STAT6. In this context, we show a neuroprotective role for IL-4Rα in an in vivo surgical model of cerebral ischemia and in ex vivo brain slice explants, using both genetic knockout of this receptor and RNAi-mediated gene knockdown. IL-4Rα may therefore represent a novel target and pathway for therapeutic development in ischemic stroke.

A novel gene-family involved in spermatophore generation in the economically important salmon louse Lepeophtheirus salmonis.

The salmon louse (Lepeophtheirus salmonis) is an important parasite of Atlantic salmon (Salmo salar). It is widely spread in aquaculture facilities and leads to economic losses every year. As it has developed resistances against many common treatments, new control methods must be established. Here we characterize a novel gene family of the salmon louse, consisting of two genes, which has not been described in other species before. We analyzed temporal expression patterns of both genes, the localization of mRNA and protein. An RNAi mediated gene knockdown lead to information about the function of the protein. Overall, these two genes are expressed only in sperm ducts of male sea lice. The mucin-like proteins can additionally be found in the wall of spermatophores, which are responsible for sperm transfer to females. Knockdown showed that both genes are essential for successful fertilization of females. Overall, all results indicate that the two analyzed genes are necessary for reproduction in sea lice as they are essential for the formation of a wall surrounding the spermatophores, which is needed for fertilization. Therefore, we name them Mucin-like spermatophore wall protein 1 & 2 (MLSWP1 & MLSWP2). Analysis of sequence data from other copepod species suggests that MLSWPs are present in many copepod species and may also play a similar role in reproduction in those species.

Improved oil quality in transgenic soybean seeds by RNAi-mediated knockdown of GmFAD2-1B.

Soybean oil contains approximately 20% oleic acid and 63% polyunsaturated fatty acids, which limits its uses in food products and industrial applications because of its poor oxidative stability. Increasing the oleic acid content in soybean seeds provides improved oxidative stability and is also beneficial to human health. Endoplasmic reticulum-associated delta-12 fatty acid desaturase 2 (FAD2) is the key enzyme responsible for converting oleic acid (18:1) precursors to linoleic acid (18:2) in the lipid biosynthetic pathway. In this study, a 390-bp conserved sequence of GmFAD2-1B was used to trigger a fragment of RNAi-mediated gene knockdown, and a seed-specific promoter of the β-conglycinin alpha subunit gene was employed to downregulate the expression of this gene in soybean seeds to increase the oleic acid content. PCR and Southern blot analysis showed that the T-DNA had inserted into the soybean genome and was stably inherited by the progeny. In addition, the expression analysis indicated that GmFAD2-1B was significantly downregulated in the seeds by RNAi-mediated post-transcription gene knockdown driven by the seed-specific promoter. The oleic acid content significantly increased from 20 to ~80% in the transgenic seeds, and the linoleic and linolenic acid content decreased concomitantly in the transgenic lines compared with that in the wild types. The fatty acid profiles also exhibited steady changes in three consecutive generations. However, the total proteinand oil contents and agronomic traits of the transgenic lines did not show a significant difference compared with the wild types.

Individual co-variation between viral RNA load and gene expression reveals novel host factors during early dengue virus infection of the Aedes aegypti midgut.

Dengue virus (DENV) causes more human infections than any other mosquito-borne virus. The current lack of antiviral strategies has prompted genome-wide screens for host genes that are required for DENV infectivity. Earlier transcriptomic studies that identified DENV host factors in the primary vector Aedes aegypti used inbred laboratory colonies and/or pools of mosquitoes that erase individual variation. Here, we performed transcriptome sequencing on individual midguts in a field-derived Ae. aegypti population to identify new candidate host factors modulating DENV replication. We analyzed the transcriptomic data using an approach that accounts for individual co-variation between viral RNA load and gene expression. This approach generates a prediction about the agonist or antagonist effect of candidate genes on DENV replication based on the sign of the correlation between gene expression and viral RNA load. Using this method, we identified 39 candidate genes that went undetected by conventional pairwise comparison of gene expression levels between DENV-infected midguts and uninfected controls. Only four candidate genes were detected by both methods, emphasizing their complementarity. We demonstrated the value of our approach by functional validation of a candidate agonist gene encoding a sterol regulatory element-binding protein (SREBP), which was identified by correlation analysis but not by pairwise comparison. We confirmed that SREBP promotes DENV infection in the midgut by RNAi-mediated gene knockdown in vivo. We suggest that our approach for transcriptomic analysis can empower genome-wide screens for potential agonist or antagonist factors by leveraging inter-individual variation in gene expression. More generally, this method is applicable to a wide range of phenotypic traits displaying inter-individual variation.

Luciferase shRNA Presents off-Target Effects on Voltage-Gated Ion Channels in Mouse Hippocampal Pyramidal Neurons.

RNA interference (RNAi) is a straightforward approach to study gene function from the in vitro cellular level to in vivo animal behavior. Although RNAi-mediated gene knockdown has become essentially routine in neuroscience over the past ten years, off-target effects of short hairpin RNAs (shRNAs) should be considered as the proper choice of control shRNA is critical in order to perform meaningful experiments. Luciferase shRNA (shLuc), targeting firefly luciferase, and scrambled shRNAs (shScrs) have been widely used as controls for vertebrate cell research. However, thorough validation of control shRNAs has not been made to date. Here, we performed thorough physiological and morphological studies against control shRNAs in mouse hippocampal CA1 pyramidal neurons. As expected, all control shRNAs exhibited normal basal synaptic transmission and dendritic morphology. However, to our surprise, shLuc exerted severe off-target effects on voltage-gated ion channel function, while the shScr had no detectable changes. These results indicate that thorough validation of shRNA is imperative and, in the absence of such validation, that shScr is the best available negative control for gene knockdown studies.

Methylmercury Uptake into BeWo Cells Depends on LAT2-4F2hc, a System L Amino Acid Transporter.

The organic mercury compound methylmercury (MeHg) is able to target the fetal brain. However, the uptake of the toxicant into placental cells is incompletely understood. MeHg strongly binds to thiol-S containing molecules such as cysteine. This MeHg-l-cysteine exhibits some structural similarity to methionine. System L plays a crucial role in placental transport of essential amino acids such as leucine and methionine and thus has been assumed to also transport MeHg-l-cysteine across the placenta. The uptake of methylmercury and tritiated leucine and methionine into the choriocarcinoma cell line BeWo was examined using transwell assay and small interfering (si)RNA mediated gene knockdown. Upon the downregulation of large neutral amino acids transporter (LAT)2 and 4F2 cell-surface antigen heavy chain (4F2hc), respectively, the levels of [3H]leucine in BeWo cells are significantly reduced compared to controls treated with non-targeting siRNA (p < 0.05). The uptake of [3H]methionine was reduced upon LAT2 down-regulation as well as methylmercury uptake after 4F2hc silencing (p < 0.05, respectively). These findings suggest an important role of system L in the placental uptake of the metal. Comparing the cellular accumulation of mercury, leucine, and methionine, it can be assumed that (1) MeHg is transported through system L amino acid transporters and (2) system L is responsible for the uptake of amino acids and MeHg primarily at the apical membrane of the trophoblast. The findings together can explain why mercury in contrast to other heavy metals such as lead or cadmium is efficiently transported to fetal blood.

In vitro stimulation of vitellogenin expression by insulin in the mud crab, Scylla paramamosain, mediated through PI3K/Akt/TOR pathway

Vitellogenin (vtg) synthesis, known as vitellogenesis, is one of most important processes in the ovarian development of oviparous animals. Recently, multiple insulin-like peptides (ILPs) have been reported in crustacean species due to the application of transcriptome sequencing. In this context, the present study reports that the addition of an exogenous ILP, bovine insulin, stimulates vtg (termed Sp-vtg) expression in hepatopancreatic explants from the mud crab, Scylla paramamosain, by in vitro experiments. Homologous genes of key factors in ILP signaling, Sp-PI3K, Sp-Akt, Sp-Rheb and Sp-TOR, have been isolated in S. paramamosain based on a transcriptome database. Further experiments reveal that the RNAi-mediated Sp-Akt gene knockdown and the inhibitors of Sp-PI3K and Sp-TOR block the stimulation of Sp-vtg expression by insulin. The combined results implicate the endogenous ILP and its corresponding signaling in the regulation of Sp-vtg synthesis in S. paramamosain.

Activin A-induced signalling controls hair follicle neogenesis.

Acquisition of potent human dermal papilla (DP) cells that can induce hair follicle neogenesis is an overarching concern, and various approaches have been tried. In an attempt to solve the problem, we previously introduced the three-dimensional (3D) culture of human DP cells and observed de novo formation of hair follicles when conducting a patch hair reconstitution assay using 3D cultured DP spheres with mouse epidermal cells. In this study, we have subsequently focused our attention on activin A, one of the notably upregulated proteins in DP spheres compared with 2D cultured DP cells. We then adopted a small interfering RNA-mediated gene knock-down approach and hair reconstitution assay to investigate the role of activin A. We observed that human DP spheres with activin A knock-down are severely impaired in hair follicle neogenesis when combined with mouse epidermal cells. In addition, activin receptor 2B (ActvR2B) knock-down mouse epidermal cells showed severe impairment of hair follicle neogenesis when combined with human DP spheres. Moreover, recombinant activin A treatment of mouse epidermal cells increased the expression of downstream genes of the activin pathway. Taken together, our data strongly suggest that activin A-induced signalling plays a critical role in hair follicle neogenesis, which has not been previously reported.

Lipopolysaccharide Attenuates Induction of Proallergic Cytokines, Thymic Stromal Lymphopoietin, and Interleukin 33 in Respiratory Epithelial Cells Stimulated with PolyI:C and Human Parechovirus.

Epidemiological studies based on the “hygiene hypothesis” declare that the level of childhood exposure to environmental microbial products is inversely related to the incidence of allergic diseases in later life. Multiple types of immune cell-mediated immune regulation networks support the hygiene hypothesis. Epithelial cells are the first line of response to microbial products in the environment and bridge the innate and adaptive immune systems; however, their role in the hygiene hypothesis is unknown. To demonstrate the hygiene hypothesis in airway epithelial cells, we examined the effect of lipopolysaccharide (LPS; toll-like receptor 4 ligand) on the expression of the proallergic cytokines thymic stromal lymphopoietin (TSLP) and interleukin 33 (IL33) in H292 cells (pulmonary mucoepidermoid carcinoma cells). Stimulation with the TLR ligand polyI:C and human parechovirus type 1 (HPeV1) but not LPS-induced TSLP and IL33 through interferon regulatory factor 3 (IRF3) and NF-κB activity, which was further validated by using inhibitors (dexamethasone and Bay 11-7082) and short hairpin RNA-mediated gene knockdown. Importantly, polyI:C and HPeV1-stimulated TSLP and IL33 induction was reduced by LPS treatment by attenuating TANK-binding kinase 1, IRF3, and NF-κB activation. Interestingly, the basal mRNA levels of TLR signaling proteins were downregulated with long-term LPS treatment of H292 cells, which suggests that such long-term exposure modulates the expression of innate immunity signaling molecules in airway epithelial cells to mitigate the allergic response. In contrast to the effects of LPS treatment, the alarmin high-mobility group protein B1 acts in synergy with polyI:C to promote TSLP and IL33 expression. Our data support part of the hygiene hypothesis in airway epithelia cells in vitro. In addition to therapeutic targeting of TSLP and IL33, local application of non-pathogenic LPS may be a rational strategy to prevent allergies.

AMP deaminase suppression increases biomass, cold tolerance and oil content in green algae

The green algae, Chlamydomonas reinhardtii, is currently one of the most targeted organisms for renewable biofuel production, yet its growth depends strictly on warm (subtropical) conditions. This requirement makes C. reinhardtii not only susceptible to cold stress, but also unsuitable for culturing large scale algae year round in temperate regions. Using RNAi mediated gene knockdown, we targeted the AMP degradation enzyme, AMP deaminase (AMPD), to reflect psychrophilic-like purine metabolism (purine metabolism in cold adapted organisms) in C. reinhardtii. The engineered C. reinhardtii strains elevated intracellular steady-state ATP levels by more than twofold across their viable temperature range, displayed ~3-fold higher growth rate and biomass, and ~25% higher lipid/oil accumulation compared to wild-type under both normal and cold growth conditions. Our findings can be extrapolated to engineer plants that grow under cold stress conditions for sustainable agriculture and plant-based biofuel production.

RNAi-mediated gene knockdown by microinjection in the model entomopathogenic nematode Heterorhabditis bacteriophora.

BackgroundParasitic nematodes threaten the health of humans and livestock and cause a major financial and socioeconomic burden to modern society. Given the widespread distribution of diseases caused by parasitic nematodes there is an urgent need to develop tools that will elucidate the genetic complexity of host-parasite interactions. Heterorhabditis bacteriophora is a parasitic nematode that allows simultaneous monitoring of nematode infection processes and host immune function, and offers potential as a tractable model for parasitic nematode infections. However, molecular tools to investigate these processes are required prior to its widespread acceptance as a robust model organism. In this paper we describe microinjection in adult H. bacteriophora as a suitable means of dsRNA delivery to knockdown gene transcripts.MethodsRNA interference was used to knockdown four genes by injecting dsRNA directly into the gonad of adult hermaphrodite nematodes. RNAi phenotypes were scored in the F1 progeny on the fifth day post-injection, and knockdown of gene-specific transcripts was quantified with real-time quantitative RT-PCR (qRT-PCR).ResultsRNAi injection in adult hermaphrodites significantly decreased the level of target transcripts to varying degrees when compared with controls. The genes targeted by RNAi via injection included cct-2, nol-5, dpy-7, and dpy-13. In each case, RNAi knockdown was confirmed phenotypically by examining the progeny of injected animals, and also confirmed at the transcriptional level by real-time qRT-PCR.ConclusionsHere we describe for the first time the successful use of microinjection to knockdown gene transcripts in H. bacteriophora. This technique can be used widely to study the molecular basis of parasitism.

Disruption of NF-&amp;kappa;B signaling by fluoxetine attenuates MGMT expression in glioma cells

BackgroundResistance to temozolomide (TMZ) in glioma is modulated by the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). This study aimed to examine the effects of fluoxetine (FLT) on MGMT expression in glioma cells and to investigate its underlying mechanisms.Materials and methodsExpression of MGMT, GluR1, or IκB kinase β (IKKβ) was attenuated using short hairpin RNA-mediated gene knockdown. The 3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the growth inhibition induced by FLT or TMZ. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) was conducted to detect apoptotic cells. Western blotting was conducted to analyze the protein expression of MGMT, IKKβ, and NF-κB/p65 following FLT treatment. The murine subcutaneous xenograft model was used to evaluate the combinational effect of TMZ and FLT.ResultsFLT markedly reduced MGMT expression in glioma cells, which was independent of GluR1 receptor function. Further, FLT disrupted NF-κB/p65 signaling in glioma cells and consequently attenuated NF-κB/p65 activity in regulating MGMT expression. Importantly, FLT sensitized MGMT-expressing glioma cells to TMZ, as FLT enhanced TMZ’s ability to impair the in vitro tumorigenic potential and to induce apoptosis in glioma cells. Knockdown of MGMT or IKKβ expression abolished the synergistic effect of FLT with TMZ in glioma cells, which suggested that FLT might sensitize glioma cells to TMZ through down-regulation of MGMT expression. Consistently, TMZ combined with FLT markedly attenuated NF-κB/p65 activity, reduced MGMT expression, and suppressed in vivo tumor growth in the murine subcutaneous xenograft model.ConclusionOur findings demonstrated that FLT attenuated MGMT expression by disrupting NF-κB signaling and sensitized glioma cells to TMZ, which may warrant further investigation toward possible clinical application in MGMT-expressing glioma.

FOXM1 is a downstream target of LPA and YAP oncogenic signaling pathways in high grade serous ovarian cancer.

Lysophosphatidic acid (LPA), a prototypical ligand for G protein coupled receptors, and Forkhead box protein M1 (FOXM1), a transcription factor that regulates expression of a wide array of genes involved in cancer initiation and progression, are two important oncogenic signaling molecules in human epithelial ovarian cancers (EOC). We conducted in vitro mechanistic studies using pharmacological inhibitors, genetic forms of the signaling molecules, and RNAi-mediated gene knock-down to uncover the molecular mechanisms of how these two molecules interact in EOC cells. Additionally, in vivo mouse studies were performed to confirm the functional involvement of FOXM1 in EOC tumor formation and progression. We show for the first time that LPA up-regulates expression of active FOXM1 splice variants in a time- and dose-dependent manner in the human EOC cell lines OVCA433, CAOV3, and OVCAR5. Gi-PI3K-AKT and G12/13-Rho-YAP signaling pathways were both involved in the LPA receptor (LPA1-3) mediated up-regulation of FOXM1 at the transcriptional level. In addition, down-regulation of FOXM1 in CAOV3 xenografts significantly reduced tumor and ascites formation, metastasis, and expression of FOXM1 target genes involved in cell proliferation, migration, or invasion. Collectively, our data link the oncolipid LPA, the oncogene YAP, and the central regulator of cell proliferation/mutagenesis FOXM1 in EOC cells. Moreover, these results provide further support for the importance of these pathways as potential therapeutic targets in EOC.

Cell-type phylogenetics and the origin of endometrial stromal cells.

A challenge of genome annotation is the identification of genes performing specific biological functions. Here, we propose a phylogenetic approach that utilizes RNA-seq data to infer the historical relationships among cell types and to trace the pattern of gene-expression changes on the tree. The hypothesis is that gene-expression changes coincidental with the origin of a cell type will be important for the function of the derived cell type. We apply this approach to the endometrial stromal cells (ESCs), which are critical for the initiation and maintenance of pregnancy. Our approach identified well-known regulators of ESCs, PGR and FOXO1, as well as genes not yet implicated in female fertility, including GATA2 and TFAP2C. Knockdown analysis confirmed that they are essential for ESC differentiation. We conclude that phylogenetic analysis of cell transcriptomes is a powerful tool for discovery of genes performing cell-type-specific functions.

Identification of protective linear B-cell epitopes on the subolesin/akirin orthologues of Ornithodoros spp. soft ticks

Subolesin/akirin is a protective antigen that is highly conserved across hematophagous vector species and is therefore potentially useful for the development of a universal vaccine for vector control, including soft ticks. Recent results have shown that in Ornithodoros erraticus and O. moubata soft ticks, RNAi-mediated subolesin gene knockdown inhibits tick oviposition and fertility by more than 90%; however, vaccination with recombinant subolesins resulted in remarkably low protective efficacies (5–24.5% reduction in oviposition). Here we report that vaccination with subolesin recombinants induces non-protective antibodies mainly directed against immunodominant linear B-cell epitopes located on highly structured regions of the subolesin protein, probably unrelated to its biological activity, while leaving the unstructured/disordered regions unrecognized. Accordingly, for a new vaccine trial we designed four synthetic peptides (OE1, OE2, OM1 and OM2) from the unrecognized/disordered regions of the Ornithodoros subolesin sequences and coupled them to keyhole limpet haemocyanin (KLH). These KLH-peptide conjugates induced the synthesis of antibodies that recognized linear B-cell epitopes located on the unstructured loops of the subolesin protein and provided up to 70.1% and 83.1% vaccine efficacies in O. erraticus and O. moubata, respectively. These results show that the protective effect of subolesin-based vaccines is highly dependent on the particular epitope recognized by antibodies on the subolesin sequence and strongly suggest that the biological activity of subolesin is exerted through its unstructured regions. The results reported here contribute to our understanding of the mechanism of protection of subolesin-based vaccines and reveal novel protective peptides that could be included among the array of candidate antigens useful for developing anti-vector vaccines based on subolesin/akirin.

Reverse genetics in the tide pool: knock-down of target gene expression via RNA interference in the copepod Tigriopus californicus.

Reverse genetic tools are essential for characterizing phenotypes of novel genes and testing functional hypotheses generated from next-generation sequencing studies. RNA interference (RNAi) has been a widely used technique for describing or quantifying physiological, developmental or behavioural roles of target genes by suppressing their expression. The marine intertidal copepod Tigriopus californicus has become an emerging model for evolutionary and physiological studies, but this species is not amenable to most genetic manipulation approaches. As crustaceans are susceptible to RNAi-mediated gene knock-down, we developed a simple method for delivery of gene-specific double-stranded RNA that results in significant suppression of target gene transcription levels. The protocol was examined on five genes of interest, and for each, at least 50% knock-down in expression was achieved. While knock-down levels did not reach 100% in any trial, a well-controlled experiment with one heat-shock gene showed unambiguously that such partial gene suppression may cause dramatic changes in phenotype. Copepods with suppressed expression of heat-shock protein beta 1 (hspb1) exhibited dramatically decreased tolerance to high temperatures, validating the importance of this gene during thermal stress, as proposed by a previous study. The application of this RNAi protocol in T.californicus will be invaluable for examining the role of genes putatively involved in reproductive isolation, mitochondrial function and local adaptation.

S100A8 is a novel therapeutic target for anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is one of the most deadly human malignancies. It is 99% lethal, and patients have a median survival of only 6 months after diagnosis. Despite these grim statistics, the mechanism underlying the tumorigenic capability of ATC cells is unclear. S100A8 and S100A9 proteins have emerged as critical mediators in cancer. The aim was to investigate the expression and function of S100A8 and S100A9 in ATC and the mechanisms involved. We determined the expression of S100A8 and S100A9 in human ATC by gene array analysis and immunohistochemistry. Using RNAi-mediated stable gene knockdown in human ATC cell lines and bioluminescent imaging of orthotopic and lung metastasis mouse models of human ATC, we investigated the effects of S100A8 and S100A9 on tumorigenesis and metastasis. We demonstrated that S100A8 and S100A9 were overexpressed in ATC but not in other types of thyroid carcinomas. In vivo analysis in mice using ATC cells that had S100A8 knocked down revealed reduced tumor growth and lung metastasis, as well as significantly prolonged animal survival. Mechanistic investigations showed that S100A8 promotes ATC cell proliferation through an interaction with RAGE, which activates the p38, ERK1/2 and JNK signaling pathways in the tumor cells. These findings establish a novel role for S100A8 in the promoting and enhancing of ATC progression. They further suggest that the inhibition of S100A8 could represent a relevant therapeutic target, with the potential of enabling a more effective treatment path for this deadly disease.

Requirement for CRIF1 in RNA interference and Dicer-2 stability

RNA interference (RNAi) is a eukaryotic gene-silencing system. Although the biochemistry of RNAi is relatively well defined, how this pathway is regulated remains incompletely understood. To identify genes involved in regulating the RNAi pathway, we screened for genetic mutations in Drosophila that alter the efficiency of RNAi. We identified the Drosophila homolog of the mammalian CR6-interacting factor 1 (CRIF1), also known as growth arrest and DNA-damage-inducible 45-gamma interacting protein (Gadd45GIP1), as a potential new regulator of the RNAi pathway. Loss-of-function mutants of Drosophila CRIF1 (dCRIF) are deficient in RNAi-mediated target gene knock-down, in the biogenesis of small interfering RNA (siRNA) molecules, and in antiviral immunity. Moreover, we show that dCRIF may function by interacting with, and stabilizing, the RNase III enzyme Dicer-2. Our results suggest that dCRIF may play an important role in regulating the RNAi pathway.