RESUMO
Cancer is a multifactorial and deadly disease. Despite major advancements in cancer therapy in the last two decades, cancer incidence is on the rise and disease prognosis still remains poor. Furthermore, molecular mechanisms of cancer invasiveness, metastasis, and drug resistance remain largely elusive. Targeted cancer therapy involving the silencing of specific cancer-enriched proteins by small interfering RNA (siRNA) offers a powerful tool. However, its application in clinic is limited by the short half-life of siRNA and warrants the development of efficient and stable siRNA delivery systems. Oncolytic adenovirus-mediated therapy offers an attractive alternative to the chemical drugs that often suffer from innate and acquired drug resistance. In continuation to our reports on the development of oncolytic adenovirus-mediated delivery of shRNA, we report here the replication-incompetent (dAd/shErbB3) and replication-competent (oAd/shErbB3) oncolytic adenovirus systems that caused efficient and persistent targeting of ErbB3. We demonstrate that the E1A coded by oAd/shErbB, in contrast to dAd/shErbB, caused downregulation of ErbB2 and ErbB3, yielding stronger downregulation of the ErbB3-oncogenic signaling axis in in vitro models of lung and breast cancer. These results were validated by in vivo antitumor efficacy of dAd/shErbB3 and oAd/shErbB3.
Assuntos
Neoplasias da Mama , Terapia Viral Oncolítica , Vírus Oncolíticos , Adenoviridae/fisiologia , Apoptose/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Vetores Genéticos , Humanos , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/fisiologia , RNA Interferente Pequeno/genética , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Following its evoked release, dopamine (DA) signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DA transporter (DAT). DAT surface availability is dynamically regulated by endocytic trafficking, and direct protein kinase C (PKC) activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that PKC-stimulated DAT endocytosis requires both Ack1 inactivation, which releases a DAT-specific endocytic brake, and the neuronal GTPase, Rit2, which binds DAT. However, it is unknown whether Rit2 is required for PKC-stimulated DAT endocytosis in DAergic terminals or whether there are region- and/or sex-dependent differences in PKC-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls PKC-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that PKC activation acutely decreased DAT surface expression selectively in ventral, but not dorsal, striatum. AAV-mediated, conditional Rit2 knockdown in DAergic neurons impacted baseline DAT surface:intracellular distribution in DAergic terminals from female ventral, but not dorsal, striatum. Further, Rit2 was required for PKC-stimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that PKC activation drives DAT-Rit2 surface dissociation and that the DAT N terminus is required for both PKC-mediated DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate PKC-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate PKC-regulated DAT internalization and reveal unexpected region-specific differences in PKC-stimulated DAT trafficking in bona fide DAergic terminals.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Endocitose , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Corpo Estriado/citologia , Corpo Estriado/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Monoméricas de Ligação ao GTP/genética , Ligação Proteica , Proteína Quinase C/metabolismoRESUMO
Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome caused by mutations in the NF1 tumor suppressor gene. NF1 encodes neurofibromin, a GTPase-activating protein for RAS proto-oncogene GTPase (RAS). Plexiform neurofibromas are a hallmark of NF1 and result from loss of heterozygosity of NF1 in Schwann cells, leading to constitutively activated p21RAS. Given the inability to target p21RAS directly, here we performed an shRNA library screen of all human kinases and Rho-GTPases in a patient-derived NF1-/- Schwann cell line to identify novel therapeutic targets to disrupt PN formation and progression. Rho family members, including Rac family small GTPase 1 (RAC1), were identified as candidates. Corroborating these findings, we observed that shRNA-mediated knockdown of RAC1 reduces cell proliferation and phosphorylation of extracellular signal-regulated kinase (ERK) in NF1-/- Schwann cells. Genetically engineered Nf1flox/flox;PostnCre+ mice, which develop multiple PNs, also exhibited increased RAC1-GTP and phospho-ERK levels compared with Nf1flox/flox;PostnCre- littermates. Notably, mice in which both Nf1 and Rac1 loci were disrupted (Nf1flox/floxRac1flox/flox;PostnCre+) were completely free of tumors and had normal phospho-ERK activity compared with Nf1flox/flox ;PostnCre+ mice. We conclude that the RAC1-GTPase is a key downstream node of RAS and that genetic disruption of the Rac1 allele completely prevents PN tumor formation in vivo in mice.
Assuntos
Técnicas de Silenciamento de Genes , Segunda Neoplasia Primária , Neurofibroma Plexiforme , Neurofibromatose 1 , Neuropeptídeos/deficiência , Proteínas rac1 de Ligação ao GTP/deficiência , Animais , Camundongos , Camundongos Knockout , Segunda Neoplasia Primária/enzimologia , Segunda Neoplasia Primária/genética , Segunda Neoplasia Primária/patologia , Segunda Neoplasia Primária/prevenção & controle , Neurofibroma Plexiforme/enzimologia , Neurofibroma Plexiforme/genética , Neurofibroma Plexiforme/prevenção & controle , Neurofibromatose 1/enzimologia , Neurofibromatose 1/genética , Neurofibromatose 1/patologia , Neurofibromina 1/deficiência , Neurofibromina 1/metabolismo , Neuropeptídeos/metabolismo , Proto-Oncogene Mas , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Dysregulation of the ErbB family of receptor tyrosine kinases is involved in the progression of many cancers. Antibodies targeting the dimerization domains of family members EGFR and HER2 are approved cancer therapeutics, but efficacy is restricted to a subset of tumors and resistance often develops in response to treatment. A third family member, HER3, heterodimerizes with both EGFR and HER2 and has also been implicated in cancer. Consequently, there is strong interest in developing antibodies that target HER3, but to date, no therapeutics have been approved. To aid the development of anti-HER3 antibodies as cancer therapeutics, we combined antibody engineering and functional genomics screens to identify putative mechanisms of resistance or synthetic lethality with antibody-mediated anti-proliferative effects. We developed a synthetic antibody called IgG 95, which binds to HER3 and promotes ubiquitination, internalization, and receptor down-regulation. Using an shRNA library targeting enzymes in the ubiquitin proteasome system, we screened for genes that effect response to IgG 95 and uncovered the E3 ubiquitin ligase RNF41 as a driver of IgG 95 anti-proliferative activity. RNF41 has been shown previously to regulate HER3 levels under normal conditions and we now show that it is also responsible for down-regulation of HER3 upon treatment with IgG 95. Moreover, our findings suggest that down-regulation of RNF41 itself may be a mechanism for acquired resistance to treatment with IgG 95 and perhaps other anti-HER3 antibodies. Our work deepens our understanding of HER3 signaling by uncovering the mechanistic basis for the anti-proliferative effects of potential anti-HER3 antibody therapeutics.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias da Mama/prevenção & controle , Proliferação de Células , Neoplasias Pancreáticas/prevenção & controle , Receptor ErbB-3/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Camundongos , Camundongos SCID , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Receptor ErbB-3/antagonistas & inibidores , Homologia de Sequência , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RNA-based therapeutics are considered as novel treatments for human diseases. Our previous study demonstrated that treatment with short-hairpin RNA against Ido1 (IDO shRNA) suppresses tumor growth, detects Th1-bias immune responses, and elevates expression of tryptophan transfer RNA (tRNATrp) in total splenocytes. In addition, depletion of Ly6g+ neutrophils attenuates the effect of IDO shRNA. The aim of this study was to investigate the regulatory network and the expression profile of tRNAs and other non-coding RNAs in IDO shRNA-treated spleens. The total splenocytes and magnetic bead-enriched splenic neutrophils were collected from the lung tumor bearing mice, which were treated with IDO shRNA or scramble IDO shRNA, and the collected cells were subsequently subjected to RNA sequencing. The gene ontology analysis revealed the different enrichment pathways in total splenocytes and splenic neutrophils. Furthermore, the expression of tRNA genes was identified and validated. Six isoacceptors of tRNA, with different expression patterns between total splenocytes and splenic neutrophils, were observed. In summary, our findings not only revealed novel biological processes in IDO shRNA-treated total splenocytes and splenic neutrophils, but the identified tRNAs and other non-coding RNAs may contribute to developing a novel biomarker gene set for evaluating the clinical efficiency of RNA-based cancer immunotherapies.
Assuntos
Antineoplásicos/administração & dosagem , Regulação da Expressão Gênica/genética , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Neutrófilos/fisiologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA de Transferência/genética , Baço/fisiologia , Animais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Indolamina-Pirrol 2,3,-Dioxigenase/administração & dosagem , Neoplasias Pulmonares/genética , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , RNA Interferente Pequeno/administração & dosagem , Baço/efeitos dos fármacosRESUMO
The amygdala plays a key role in emotional-affective aspects of pain and in pain modulation. The central nucleus (CeA) serves major amygdala output functions related to emotional-affective behaviors and pain modulation. Our previous studies implicated the corticotropin-releasing factor (CRF) system in amygdala plasticity and pain behaviors in an arthritis model. We also showed that serotonin (5-HT) receptor subtype 5-HT2CR in the basolateral amygdala (BLA) contributes to increased CeA output and neuropathic pain-like behaviors. Here, we tested the novel hypothesis that 5-HT2CR in the BLA drives CRF1 receptor activation to increase CeA neuronal activity in neuropathic pain. Extracellular single-unit recordings of CeA neurons in anesthetized adult male rats detected increased activity in neuropathic rats (spinal nerve ligation model) compared to sham controls. Increased CeA activity was blocked by local knockdown or pharmacological blockade of 5-HT2CR in the BLA, using stereotaxic administration of 5-HT2CR short hairpin RNA (shRNA) viral vector or a 5-HT2CR antagonist (SB242084), respectively. Stereotaxic administration of a CRF1 receptor antagonist (NBI27914) into the BLA also decreased CeA activity in neuropathic rats and blocked the facilitatory effects of a 5-HT2CR agonist (WAY161503) administered stereotaxically into the BLA. Conversely, local (BLA) knockdown of 5-HT2CR eliminated the inhibitory effect of NBI27914 and the facilitatory effect of WAY161503 in neuropathic rats. The data suggest that 5-HT2CR activation in the BLA contributes to neuropathic pain-related amygdala (CeA) activity by engaging CRF1 receptor signaling.
Assuntos
Tonsila do Cerebelo/metabolismo , Neuralgia/etiologia , Neuralgia/metabolismo , Neurônios/metabolismo , Receptor 5-HT2C de Serotonina/genética , Receptores de Hormônio Liberador da Corticotropina/genética , Tonsila do Cerebelo/fisiopatologia , Animais , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Masculino , Neuralgia/fisiopatologia , Ratos , Receptor 5-HT2C de Serotonina/metabolismo , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Antagonistas do Receptor 5-HT2 de Serotonina/farmacologiaRESUMO
The mechanisms behind the induction of malignancy and chemoresistance in breast cancer cells are still not completely understood. Inflammation is associated with the induction of malignancy in different types of cancer and is highlighted as an important factor for chemoresistance. In previous work, we demonstrated that the inflammatory cytokine interleukin 1ß (IL-1ß)-induced upregulation of genes was associated with chemoresistance in breast cancer cells. Here, we evaluated the participation and the expression profile of TP63 in the induction of resistance to cisplatin. By loss-of-function assays, we identified that IL-1ß particularly upregulates the expression of the tumor protein 63 (TP63) isoform ΔNP63α, through the activation of the IL-1ß/IL-1RI/ß-catenin signaling pathway. Upregulation of ΔNP63α leads to an increase in the expression of the cell survival factors epidermal growth factor receptor (EGFR) and phosphatase 1D (Wip1), and a decrease in the DNA damage sensor, ataxia-telangiectasia mutated (ATM). The participation of these processes in the increase of resistance to cisplatin was confirmed by silencing TP63 expression or by inhibition of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) activity in the IL-1ß/IL-1RI/ß-catenin signaling pathway. These data reinforced the importance of an inflammatory environment in the induction of drug resistance in cancer cells and uncovered a molecular mechanism where the IL-1ß signaling pathway potentiates the acquisition of cisplatin resistance in breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos , Interleucina-1beta/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Neoplasias da Mama/imunologia , Cisplatino , Receptores ErbB , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Tipo I de Interleucina-1/metabolismo , Regulação para Cima , beta Catenina/metabolismoRESUMO
KRAS is the most frequently mutated oncogene in human cancer, but its therapeutic targeting remains challenging. Here, we report a synthetic lethal screen with a library of deubiquitinases and identify USP39, which encodes an essential splicing factor, as a critical gene for the viability of KRAS-dependent cells. We show that splicing fidelity inhibitors decrease preferentially the proliferation rate of KRAS-active cells. Moreover, depletion of DHX38, encoding an USP39-interacting splicing factor, also reduces the viability of these cells. In agreement with these results, USP39 depletion caused a significant reduction in pre-mRNA splicing efficiency, as demonstrated through RNA-seq experiments. Furthermore, we show that USP39 is up-regulated in lung and colon carcinomas and its expression correlates with KRAS levels and poor clinical outcome. Accordingly, our work provides critical information for the development of splicing-directed antitumor treatments and supports the potential of USP39-targeting strategies as the basis of new anticancer therapies.
Assuntos
Neoplasias do Colo/patologia , Neoplasias Pulmonares/patologia , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteases Específicas de Ubiquitina/metabolismo , Animais , Apoptose , Western Blotting , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Células Tumorais Cultivadas , Proteases Específicas de Ubiquitina/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Calcium (Cav1 and Cav2) and sodium channels possess homologous CaM-binding motifs, known as IQ motifs in their C termini, which associate with calmodulin (CaM), a universal calcium sensor. Cav3 T-type channels, which serve as pacemakers of the mammalian brain and heart, lack a C-terminal IQ motif. We illustrate that T-type channels associate with CaM using co-immunoprecipitation experiments and single particle cryo-electron microscopy. We demonstrate that protostome invertebrate (LCav3) and human Cav3.1, Cav3.2, and Cav3.3 T-type channels specifically associate with CaM at helix 2 of the gating brake in the I-II linker of the channels. Isothermal titration calorimetry results revealed that the gating brake and CaM bind each other with high-nanomolar affinity. We show that the gating brake assumes a helical conformation upon binding CaM, with associated conformational changes to both CaM lobes as indicated by amide chemical shifts of the amino acids of CaM in 1H-15N HSQC NMR spectra. Intact Ca2+-binding sites on CaM and an intact gating brake sequence (first 39 amino acids of the I-II linker) were required in Cav3.2 channels to prevent the runaway gating phenotype, a hyperpolarizing shift in voltage sensitivities and faster gating kinetics. We conclude that the presence of high-nanomolar affinity binding sites for CaM at its universal gating brake and its unique form of regulation via the tuning of the voltage range of activity could influence the participation of Cav3 T-type channels in heart and brain rhythms. Our findings may have implications for arrhythmia disorders arising from mutations in the gating brake or CaM.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Calmodulina/fisiologia , Caveolina 3/metabolismo , Ativação do Canal Iônico , Animais , Sítios de Ligação , Encéfalo/fisiologia , Cálcio/metabolismo , Calmodulina/metabolismo , Coração/fisiologia , Humanos , Invertebrados , PeriodicidadeRESUMO
Persistent or chronic infection with the hepatitis B virus (HBV) represents one of the most common viral diseases in humans. The hepatitis B virus deploys the hepatitis B virus X protein (HBx) as a suppressor of host defenses consisting of RNAi-based silencing of viral genes. Because of its critical role in countering host defenses, HBx represents an attractive target for antiviral drugs. Here, we developed and optimized a loss-of-function screening procedure, which identified a potential pharmacophore that abrogated HBx RNAi suppression activity. In a survey of 14,400 compounds in the Maybridge Screening Collection, we prioritized candidate compounds via high-throughput screening based on reversal of green fluorescent protein (GFP)-reported, RNAi-mediated silencing in a HepG2/GFP-shRNA RNAi sensor line. The screening yielded a pharmacologically active compound, N-(2,4-difluorophenyl)-N'-[3-(1H-imidazol-1-yl) propyl] thiourea (IR415), which blocked HBx-mediated RNAi suppression indicated by the GFP reporter assay. We also found that IR415 reversed the inhibitory effect of HBx protein on activity of the Dicer endoribonuclease. We further confirmed the results of the primary screen in IR415-treated, HBV-infected HepG2 cells, which exhibited a marked depletion of HBV core protein synthesis and down-regulation of pre-genomic HBV RNA. Using a molecular interaction analysis system, we confirmed that IR415 selectively targets HBx in a concentration-dependent manner. The screening assay presented here allows rapid and improved detection of small-molecule inhibitors of HBx and related viral proteins. The assay may therefore potentiate the development of next-generation RNAi pathway-based therapeutics and promises to accelerate our search for novel and effective drugs in antiviral research.
Assuntos
Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/crescimento & desenvolvimento , Ensaios de Triagem em Larga Escala , Interferência de RNA , Bibliotecas de Moléculas Pequenas/farmacologia , Replicação Viral/efeitos dos fármacos , Células Hep G2 , Humanos , Modelos Moleculares , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Brain-derived neurotrophic factor (BDNF) is an active neurotrophin abundantly expressed throughout the nervous system. It plays an important role in synaptic transmission, plasticity, neuronal proliferation, differentiation, survival, and death. The Bdnf gene in rodents has eight non-coding exons and only a single coding exon (IX). Despite its recognized regulation by neuronal activity, relatively little is known about its transcriptional regulation, and even less about the transcription factor candidates that may play such a role. The goal of the present study was to probe for such a candidate that may regulate exon IX in the rat Bdnf gene. Our in silico analysis revealed tandem binding sites for nuclear respiratory factor 2 (NRF-2) on the promoter of exon IX. NRF-2 is of special significance because it co-regulates the expressions of mediators of energy metabolism (cytochrome c oxidase) and mediators of neuronal activity (glutamatergic receptors). To test our hypothesis that NRF-2 also regulates the Bdnf gene, we performed electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), promoter cloning, and site-directed mutagenesis, real-time quantitative PCR (RT-qPCR), and Western blotting analysis. Results indicate that NRF-2 functionally regulates exon IX of the rat Bdnf gene. The binding sites of NRF-2 are conserved between rats and mice. Overexpressing NRF-2 up-regulated the expression of Bdnf exon IX, whereas knocking down NRF-2 down-regulated such expression. These findings are consistent with our hypothesis that NRF-2, in addition to regulating the coupling between neuronal activity and energy metabolism, also regulates the expression of BDNF, which is intimately associated with energy-demanding neuronal activity.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Éxons/fisiologia , Regulação da Expressão Gênica/fisiologia , Fator 2 Relacionado a NF-E2/metabolismo , Elementos de Resposta/fisiologia , Transcrição Gênica/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Metabolismo Energético/fisiologia , Técnicas de Silenciamento de Genes , Camundongos , Fator 2 Relacionado a NF-E2/genética , Neurônios/metabolismo , RatosRESUMO
The mechanism of LDL receptor (LDLR) degradation mediated by the proprotein convertase subtilisin/kexin type 9 (PCSK9) has been extensively studied; however, many steps within this process remain unclear and still require characterization. Recent studies have shown that PCSK9 lacking its Cys/His-rich domain can still promote LDLR internalization, but the complex does not reach the lysosome suggesting the presence of an additional interaction partner(s). In this study we carried out an unbiased screening approach to identify PCSK9-interacting proteins in the HepG2 cells' secretome using co-immunoprecipitation combined with mass spectrometry analyses. Several interacting proteins were identified, including glypican-3 (GPC3), phospholipid transfer protein, matrilin-3, tissue factor pathway inhibitor, fibrinogen-like 1, and plasminogen activator inhibitor-1. We then validated these interactions by co-immunoprecipitation and Western blotting. Furthermore, functional validation was examined by silencing each candidate protein in HepG2 cells using short hairpin RNAs to determine their effect on LDL uptake and LDLR levels. Only GPC3 and phospholipid transfer protein silencing in HepG2 cells significantly increased LDL uptake in these cells and displayed higher total LDLR protein levels compared with control cells. Moreover, our study provides the first evidence that GPC3 can modulate the PCSK9 extracellular activity as a competitive binding partner to the LDLR in HepG2 cells.
Assuntos
Carcinoma Hepatocelular/metabolismo , Glipicanas/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Pró-Proteína Convertase 9/metabolismo , Receptores de LDL/metabolismo , Carcinoma Hepatocelular/genética , Glipicanas/genética , Células Hep G2 , Humanos , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Neoplasias Hepáticas/genética , Proteínas Matrilinas/genética , Proteínas Matrilinas/metabolismo , Proteínas de Neoplasias/genética , Pró-Proteína Convertase 9/genética , Ligação Proteica , Receptores de LDL/genéticaRESUMO
Targeted therapies in personalized medicine require the knowledge about the molecular changes within the patient that cause the disease. With the beginning of the new century, a plethora of new technologies became available to detect these changes and use this information as starting point for drug development. Next-generation genome sequencing and sophisticated genome-wide functional genomics' methods have led to a significant increase in the identification of novel drug target candidates and understanding of the relevance of these genomic and molecular changes for the diseases. As functional genomic tool for target identification, high-throughput gene silencing through RNA interference screening has become the established method. RNAi is discussed with its advantages and challenges in this chapter. Furthermore the potential of CRISPR/Cas9, a gene-editing method that has recently been adapted for use as functional screening tool, will be briefly reviewed.
Assuntos
Descoberta de Drogas , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Interferência de RNARESUMO
RNA interference (RNAi) is a potent and specific post-transcriptional gene silencing process. Since its discovery, tremendous efforts have been made to translate RNAi technology into therapeutic applications for the treatment of different human diseases including respiratory diseases, by manipulating the expression of disease-associated gene(s). Similar to other nucleic acid-based therapeutics, the major hurdle of RNAi therapy is delivery. Pulmonary delivery is a promising approach of delivering RNAi therapeutics directly to the airways for treating local conditions and minimizing systemic side effects. It is a non-invasive route of administration that is generally well accepted by patients. However, pulmonary drug delivery is a challenge as the lungs pose a series of anatomical, physiological and immunological barriers to drug delivery. Understanding these barriers is essential for the development an effective RNA delivery system. In this review, the different barriers to pulmonary drug delivery are introduced. The potential of RNAi molecules as new class of therapeutics, and the latest preclinical and clinical studies of using RNAi therapeutics in different respiratory conditions are discussed in details. We hope this review can provide some useful insights for moving inhaled RNAi therapeutics from bench to bedside.
RESUMO
Annexin A2 (AnxA2) was reported to be an extracellular endogenous inhibitor of proprotein convertase subtilisin kexin type 9 (PCSK9) activity on cell-surface LDL receptor degradation. In this study, we investigated the effect of silencing the expression of AnxA2 and PCSK9 in HepG2 and Huh7 cells to better define the role of AnxA2 in PCSK9 regulation. AnxA2 knockdown in Huh7 cells significantly increased PCSK9 protein levels as opposed to AnxA2 knockdown in HepG2 cells. However, HepG2 cells overexpressing AnxA2 had lower levels of PCSK9 protein. Overall, our data revealed a plausible new role of AnxA2 in the reduction of PCSK9 protein levels via a translational mechanism. Moreover, the C-terminal Cys/His-rich domain of PCSK9 is crucial in the regulation of PCSK9 activity, and we demonstrated by far-Western blot assay that the M1 and M2 domains are necessary for the specific interaction of PCSK9's C-terminal Cys/His-rich domain and AnxA2. Finally, we produced and purified recombinant PCSK9 from humans and mice, which was characterized and used to perform 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate LDL cell-based assays on the stable knockdown HepG2 and Huh7 cells. We also demonstrated for the first time the equipotency of human and mouse PCSK9 R218S on human cells.
Assuntos
Anexina A2/metabolismo , Pró-Proteína Convertases/biossíntese , Biossíntese de Proteínas/fisiologia , Serina Endopeptidases/biossíntese , Animais , Anexina A2/química , Anexina A2/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Células Hep G2 , Humanos , Camundongos , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/química , Pró-Proteína Convertases/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Serina Endopeptidases/química , Serina Endopeptidases/genéticaRESUMO
Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3'-untranslated region (UTR) of endogenous dhfr. Thus, shRNAs were designed to target the 3'-UTR of endogenous dhfr, and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80% in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity (q EPO) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr. This study reveals that an expression vector including shRNA that targets the 3'-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector.
Assuntos
Regiões 3' não Traduzidas , Eritropoetina/biossíntese , Técnicas de Silenciamento de Genes/métodos , RNA Interferente Pequeno/genética , Tetra-Hidrofolato Desidrogenase/genética , Transgenes , Animais , Células CHO , Cricetulus , Eritropoetina/genética , Vetores Genéticos , Humanos , Metotrexato/metabolismo , Plasmídeos , RNA Interferente Pequeno/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Ativação Transcricional/efeitos dos fármacos , Regulação para CimaRESUMO
We have reviewed the literature for circular RNAs (circRNAs) with efficacy in preclinical pancreatic-cancer related in vivo models. The identified circRNAs target chemoresistance mechanisms (n=5), secreted proteins and transmembrane receptors (n=15), transcription factors (n=9), components of the signaling- (n=11), ubiquitination- (n=2), autophagy-system (n=2), and others (n=9). In addition to identifying targets for therapeutic intervention, circRNAs are potential new entities for treatment of pancreatic cancer. Up-regulated circRNAs can be inhibited by antisense oligonucleotides (ASO), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs) or clustered regularly interspaced short-palindromic repeats-CRISPR associated protein (CRISPR-CAS)-based intervention. The function of down-regulated circRNAs can be reconstituted by replacement therapy using plasmids or virus-based vector systems. Target validation experiments and the development of improved delivery systems for corresponding agents were examined.
Assuntos
Neoplasias Pancreáticas , RNA Circular , Humanos , RNA Circular/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/metabolismo , Animais , Terapia de Alvo Molecular/métodosRESUMO
Short hairpin RNAs (shRNAs) have emerged as a powerful tool for gene knockdown in various cellular systems, including chimeric antigen receptor (CAR) T cells. However, the elements of shRNAs that are crucial for their efficacy in developing shRNA-containing CAR T cells remain unclear. In this study, we evaluated the impact of different shRNA elements, including promoter strength, orientation, multiple shRNAs, self-targeting, and sense and antisense sequence composition on the knockdown efficiency of the target gene in CAR T cells. Our findings highlight the importance of considering multiple shRNAs and their orientation to achieve effective knockdown. Moreover, we demonstrate that using a strong promoter and avoiding self-targeting can enhance CAR T cell functionality. These results provide a framework for the rational design of CAR T cells with shRNA-mediated knockdown capabilities, which could improve the therapeutic efficacy of CAR T cell-based immunotherapy.
RESUMO
BACKGROUND/AIM: We evaluated the impact of FosL1, a member of the activated protein-1 family, on the pathways leading to regional metastasis of head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: We examined the influence of small interfering RNA (siRNA) and short heparin RNA (shRNA) mediated knockdown of FosL1 on cell migration, invasion, and proliferation in vitro as well as on regional metastasis in vivo. The prognostic significance of FosL1 was also analyzed using the Kaplan- Meier plotter using data from an HNSCC patient database. RESULTS: Down-regulation of FosL1 inhibited cell migration, invasion, and proliferation in vitro, decreased the incidence of regional metastases, and prolonged the survival of mice in vivo. We also determined that HNSCC patients with higher expression levels of FosL1 had a significantly shorter survival time than those with low expression of FosL1. CONCLUSION: FosL1 plays a crucial role in promoting cell migration, invasion, and proliferation in HNSCC.
Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Invasividade Neoplásica/genética , Proteínas Proto-Oncogênicas c-fos/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Animais , Linhagem Celular Tumoral , Regulação para Baixo/genética , Humanos , Metástase Linfática/genética , Metástase Linfática/patologia , Camundongos , Camundongos Nus , Invasividade Neoplásica/patologia , Prognóstico , RNA Interferente Pequeno/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologiaRESUMO
Knockdown assays are widely used to study the functions of a gene of interest. RNA interference (RNAi) describes a set of well-known methods used to reduce the expression of a target gene by degrading its mRNA with short hairpin RNAs (shRNAs) or short interfering RNAs (siRNAs). Knockdown of chimeric RNAs present different challenges than standard RNAi targeting for regular genes. Most specifically, sequence homology restricts the targeting region to the chimeric junction and can result in off-target effects on the parental genes. In this chapter, we provide guidelines and procedures for RNAi design of chimeric RNAs, knockdown of chimeric RNAs, downstream experiments for chimeric RNA functional studies and necessary controls to accompany each set of experiments.