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Aberrant gene expression patterns in acute myeloid leukemia (AML) with balanced chromosomal translocations are often associated with dysregulation of epigenetic modifiers. The AML1/ETO (RUNX1/MTG8) fusion protein, caused by the translocation (8;21)(q22;q22), leads to the epigenetic repression of its target genes. We aimed in this work to identify critical epigenetic modifiers, on which AML1/ETO-positive AML cells depend on for proliferation and survival using shRNA library screens and global transcriptomics approaches. Using shRNA library screens, we identified 41 commonly depleted genes in two AML1/ETO-positive cell lines Kasumi-1 and SKNO-1. We validated, genetically and pharmacologically, DNMT1 and ATR using several AML1/ETO-positive and negative cell lines. We also demonstrated in vivo differentiation of myeloblasts after treatment with the DNMT1 inhibitor decitabine in a patient with an AML1/ETO-positive AML. Bioinformatic analysis of global transcriptomics after AML1/ETO induction in 9/14/18-U937 cells identified 973 differentially expressed genes (DEGs). Three genes (PARP2, PRKCD, and SMARCA4) were both downregulated after AML1/ETO induction, and identified in shRNA screens. In conclusion, using unbiased shRNA library screens and global transcriptomics, we have identified several driver epigenetic regulators for proliferation in AML1/ETO-positive AML. DNMT1 and ATR were validated and are susceptible to pharmacological inhibition by small molecules showing promising preclinical and clinical efficacy.
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Dysregulation of long noncoding RNAs (lncRNAs), such as maternally expressed gene 3 (MEG3) and long intergenic noncoding RNA regulator of reprogramming (linc-ROR), plays a crucial role in colorectal cancer progression. We aimed to assess linc-ROR silencing and MEG3 activation on the colorectal cancer cell proliferation simultaneously; and explore the underlying mechanisms in the TP53-associated Pathway. The MEG3 and linc-ROR shRNA were cloned under the bidirectional CEA promoter (UM1). Subsequently, additional vectors were constructed to express linc-ROR shRNA (UM2) and MEG3 (UM3). After transfecting colorectal cancer cell lines with these recombinant vectors, experiments on cell viability, apoptosis, and cell cycle analysis were conducted. Furthermore, TP53's transcriptional activity and associated genes were assessed using quantitative real-time polymerase chain reaction (qRT-PCR). Interestingly, UM1 significantly inhibited the proliferation of both cell lines than UM2 and UM3. In response to UM1, TP53 transcript remarkably increased in HCT116 cells (10.46) than SW480 cells (6.16); which resulted in up-regulation of TP53INP1, TP53I3, GDF15, CCKN1A and BAX, and down-regulation of G1 cyclins (D1, E1). The rate of apoptosis increased in HCT116 (36.35 %) and SW480 (16.64 %) cells than control. Moreover, UM1-transfected HCT116 cells exhibited a notable arrest in the G0/G1 phase, accompanied by a reduction in the G2/M cell population. Compared to unidirectional vectors, the concurrent targeting approach enhanced TP53 activation at the transcription level. The cell response to UM1 resulted in rapid upregulation of TP53, leading to inhibition of cell proliferation, increased apoptosis, and cell cycle arrest. These findings suggest that the synergistic effect of targeting both MEG3 and linc-ROR could serve as a promising therapeutic strategy for TP53-associated colon cancer.
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In this study, the nonclinical pharmacokinetics of OLX702A-075-16, an RNA interference (RNAi) therapeutic currently in development, were investigated. OLX702A-075-16 is a novel N-acetylgalactosamine conjugated asymmetric small-interfering RNA (GalNAc-asiRNA) used for the treatment of an undisclosed liver disease. Its unique 16/21-mer asymmetric structure reduces nonspecific off-target effects without compromising efficacy. We investigated the plasma concentration, tissue distribution, metabolism, and renal excretion of OLX702A-075-16 following a subcutaneous administration in mice and rats. For bioanalysis, high-performance liquid chromatography with fluorescence detection (HPLC-FD) was used. The results showed rapid clearance from plasma (0.5 to 1.5 h of half-life) and predominant distribution to the liver, and/or kidney. Less than 1% of the liver concentration of OLX702A-075-16 was detected in the other tissues. Metabolite profiling using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) revealed that the intact duplex OLX702A-075-16 was the major compound in plasma. The GalNAc moiety was predominantly metabolized from the sense strand in the liver, with the unconjugated sense strand of OLX702A-075-16 accounting for more than 95% of the total exposure in the rat liver. Meanwhile, the antisense strand was metabolized by the sequential loss of nucleotides from the 3'-terminus by exonuclease, with the rat liver samples yielding the most diverse truncated forms of metabolites. Urinary excretion over 96 h was less than 1% of the administered dose in rats. High plasma protein binding of OLX702A-075-16 likely inhibited its clearance through renal filtration. Significance Statement This study presents the first comprehensive characterization of the in vivo pharmacokinetics of GalNAc-asiRNA. The pharmacokinetic insights gained from this research will aid in understanding toxicology and efficacy, optimizing delivery platforms, and improving the predictive power of preclinical species data for human applications.
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Objective: This study aims to verify the protective effect of the Kelch-like ECH-associated protein1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways by studying the effect of plasmids containing Nrf2-small hairpin RNA (shRNA) interference down-regulation of Nrf2 on zearalenone (ZEA) -induced intestinal porcine epithelial cells (IPEC-J2) oxidative stress. Methods: We constructed an IPEC-J2 model that interferes with Nrf2 expression, set blank (Control), negative control group (Sh-control), positive control group (Sh-Nrf2), and added 10, 20, and 40 µmol/L ZEA experimental group (Sh-Nrf2+ZEA10, Sh-Nrf2+ZEA20, and Sh-Nrf2+ZEA40). Results: The study results showed that, compared with the Sh-Nrf2 group, ZEA significantly increased the apoptosis rate of IPEC-J2 in a time- and dose-dependent manner. Compared with the Sh-Nrf2 group, the activities of T-SOD and GSH-PX and relative expressions of Keap1 at mRNA and protein level in the Sh-Nrf2+ZEA20 and Sh-Nrf2+ZEA40 groups were significantly reduced, the MDA level, and the fluorescence intensity around and within the nucleus of ROS and Nrf2, and the relative expressions of Nrf2, Nqo1, and Ho1 at mRNA and protein level significantly increased. Conclusion: These results further prove that interfering with the expression of Nrf2 in IPEC-J2 cells affected the activation of the Keap1-Nrf2 signaling pathway and reduced the ability of cells to resist ZEA-induced oxidative stress. Therefore, the Keap1-Nrf2 signaling pathway had an important protective effect in ZEA-induced intestinal oxidative stress.
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We describe in this review the historical evidence leading up to the concept and design of Vigil and subsequent clinical applications including safety and efficacy in a randomized, controlled Phase IIB trial. Vigil (gemogenovatucel-T) is a unique triple function targeted immunotherapy that demonstrates preclinical and clinical systemic anticancer activity. Construction of Vigil involves harvest of autologous malignant tissue for neoantigen targeting (ideally containing clonal neoantigens) followed by a two-day process involving transfection with a plasmid to provide a permissive 'training environment' for the patient's immune system. Transfected plasmid components contain an expressive human GMCSF DNA segment to enhance anticancer immune functional response and a second component expressing bi-shRNAfurin which reduces TGFß isomers (TGFß1 and TGFß2) thereby reducing cancer inhibition of the targeted immune response. Results generated to date justify advancement to confirmatory clinical trials supporting product regulatory approval.
Vigil is an anticancer treatment that employs three methods of enhancing the body's immune system to identify and kill cancer cells. The construction of Vigil involves cancer cells from the same person being treated (personalized therapy) in combination with added anticancer genetic signals to enhance the number and function anti-anticancer immune cells and to guide the immune cells to the cancer and not to normal organs of the body. In this manner, an army of immune cells are created that can move to attacking the cancer using blood vessels to get to the cancer anywhere it tries to grow in the body. One study (Phase I) performed with this product to determine safety and dose range demonstrated an optimal dose and schedule. Another study (Phase IIA) showed initial clinical benefit. A third more complex study (Phase IIB) in patients treated with Vigil compared with standard of care without Vigil demonstrated the ability to prolong the patients life and time without their cancer getting worse without any significant side effects associated with the treatment in a unique subset of ovarian cancer patients, those with the ability to repair their DNA. Based on the composite of these results, Vigil is an attractive targeted immunotherapy justified for late-stage clinical testing.
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Pemphigus is an autoimmune disease that affects the skin and mucous membranes, induced by the deposition of pemphigus IgG, which mainly targets desmogleins 1 and 3 (Dsg1 and 3). This autoantibody causes steric interference between Dsg1 and 3 and the loss of cell adhesion, producing acantholysis. This molecule and its cellular effects are clinically reflected as intraepidermal blistering. Pemphigus vulgaris-IgG (PV-IgG) binding involves p38MAPK-signaling-dependent caspase-3 activation. The present work assessed the in vitro effect of PV-IgG on the adherence of HaCaT cells dependent on caspase-3. PV-IgG induced cell detachment and apoptotic changes, as demonstrated by annexin fluorescent assays. The effect of caspase-3 induced by PV-IgG was suppressed in cells pre-treated with caspase-3-shRNA, and normal IgG (N-IgG) as a control had no relevant effects on the aforementioned parameters. The results demonstrated that shRNA reduces caspase-3 expression, as measured via qRT-PCR and via Western blot and immunofluorescence, and increases cell adhesion. In conclusion, shRNA prevented in vitro cell detachment and the late effects of apoptosis induced by PV-IgG on HaCaT cells, furthering our understanding of the molecular role of caspase-3 cell adhesion dependence in pemphigus disease.
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Apoptosis , Autoanticuerpos , Caspasa 3 , Adhesión Celular , Pénfigo , ARN Interferente Pequeño , Humanos , Pénfigo/inmunología , Pénfigo/patología , Caspasa 3/metabolismo , Autoanticuerpos/inmunología , ARN Interferente Pequeño/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Línea Celular , Células HaCaT , Desmogleína 3/inmunología , Desmogleína 3/metabolismo , Desmogleína 3/genética , Queratinocitos/metabolismoRESUMEN
Adeno-associated virus (AAV) vectors have become the leading platform for gene delivery in both preclinical research and therapeutic applications, making the production of high-titer AAV preparations essential. To date, most AAV-based studies use constitutive promoters (e.g., CMV, CAG), which are also active in human embryonic kidney (HEK)-293 producer cells, thus leading to the expression of the transgene already during production. Depending on the transgene's function, this might negatively impact producer cell performance and result in decreased AAV vector yields. Here, we evaluated a panel of diverse microRNA (miRNA)-based shRNA designs to identify a highly potent artificial miRNA for the transient suppression of transgenes during AAV production. Our results demonstrate that insertion of miRNA target sites into the 3' UTR of the transgene and simultaneous expression of the corresponding miRNA from the 3' UTR of conventional AAV production plasmids (rep/cap, pHelper) enabled efficient silencing of toxic transgene expression, thereby increasing AAV vector yields up to 240-fold. This strategy not only allows to maintain the traditional triple-transfection protocol, but also represents a universally applicable approach to suppress toxic transgenes, thereby boosting vector yields with so far unprecedented efficiency.
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BACKGROUND: More and more evidence has shown the process of Parkinson's disease (PD). Probably, inflammation exerts a crucial role between them. Therefore, the aim of this study was to analyze the impact of interleukin-6 receptor (IL-6R) expression on the IL-6/signal transducer and activator of transcription 3 (STAT3)/hypoxia-inducible factor-1α (HIF-1α) inflammatory signaling pathway within a mouse model of PD with type 2 diabetes mellitus (T2DM) as co-morbidity. METHODS: We chose healthy wild-type C57BL/6J male mice at the age of 10 weeks to prepare a mouse model of PD with T2DM co-morbidity. Adeno-associated virus (AAV) overexpressing IL-6R or AAV IL-6R-shRNA genes were injected into the substantia nigra (SN) of the mice. The behavioral indices of the pole test were used for examining the motor function of the mice. Using immunofluorescence analysis, the impacts of IL-6R on the level of tyrosine hydroxylase (TH) and anti-ionized calcium-binding adaptor molecule 1 (IBA-1) on dopaminergic neurons and microglia were examined. Additionally, enzyme-linked immunosorbent assay (ELISA) was adopted for determining the expressions of HIF-1α and inflammatory cytokines like tumor necrosis factor-α (TNF-α), IL-1ß, IL-6, and IL-4 in the serum. In this study, the protein expression levels of TH, α-Synuclein (α-Syn), IBA-1, IL-6, IL-6R, phosphorylated and total signal transducer and activator of transcription 3 (p-STAT3 (Tyr705) and STAT3) and HIF-1α in the SN were tested via western blotting. To ascertain the mRNA expressions of TNF-α, IL-1ß, IL-6, IL-4, and HIF-1α, we used quantitative Real-Time Polymerase Chain Reaction (RT-qPCR). RESULTS: IL-6R-shRNA treatment could markedly shorten the total time of PD in the T2DM co-morbidity mouse model based on the pole test results, reverse the decrease in TH-positive neurons stimulated by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), and lower the activation of microglia (all p < 0.05). Further, IL-6R-shRNA treatment hindered the expression of IL-6, p-STAT3 (Tyr705), and HIF-1α in the SN, lowered the levels of TNF-α, IL-1ß, IL-6, IL-4, and HIF-1α in the serum, and mRNA expressions of TNF-α, IL-1ß, IL-6, and HIF-1α in the SN (all p < 0.05). In contrast, IL-6R overexpression reduced TH levels, upregulated the level of IBA-1, IL-6, p-STAT3 (Tyr705), and HIF-1α, increased the level of IL-1ß, TNF-α, IL-6, IL-4, and HIF-1α (all p < 0.05) in the serum and SN in the PD mouse model with T2DM as a co-morbidity. CONCLUSIONS: PD progression with T2DM as a co-morbidity can be boosted by AAV IL-6R-overexpression through upregulation of the IL-6/STAT3/HIF-1α axis. Conversely, AAV IL-6R-shRNA treatment suppressed the IL-6/STAT3/HIF-1α pathway and alleviated neuroinflammation, thus weakening the development of PD with T2DM as a co-morbidity.
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Diabetes Mellitus Tipo 2 , Modelos Animales de Enfermedad , Subunidad alfa del Factor 1 Inducible por Hipoxia , Interleucina-6 , Ratones Endogámicos C57BL , Enfermedad de Parkinson , Receptores de Interleucina-6 , Factor de Transcripción STAT3 , Transducción de Señal , Animales , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Interleucina-6/metabolismo , Masculino , Ratones , Receptores de Interleucina-6/metabolismo , Receptores de Interleucina-6/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/genéticaRESUMEN
Gain-of-function mutations in SCN8A cause developmental and epileptic encephalopathy (DEE), a disorder characterized by early-onset refractory seizures, deficits in motor and intellectual functions, and increased risk of sudden unexpected death in epilepsy. Altered activity of neurons in the corticohippocampal circuit has been reported in mouse models of DEE. We examined the effect of chronic seizures on gene expression in the hippocampus by single-nucleus RNA sequencing in mice expressing the patient mutation SCN8A-p.Asn1768Asp (N1768D). One hundred and eighty four differentially expressed genes were identified in dentate gyrus granule cells, many more than in other cell types. Electrophysiological recording from dentate gyrus granule cells demonstrated an elevated firing rate. Targeted reduction of Scn8a expression in the dentate gyrus by viral delivery of an shRNA resulted in doubling of median survival time from 4 months to 8 months, whereas delivery of shRNA to the CA1 and CA3 regions did not result in lengthened survival. These data indicate that granule cells of the dentate gyrus are a specific locus of pathology in SCN8A-DEE.
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Giro Dentado , Canal de Sodio Activado por Voltaje NAV1.6 , Neuronas , Animales , Canal de Sodio Activado por Voltaje NAV1.6/genética , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Giro Dentado/patología , Giro Dentado/metabolismo , Ratones , Neuronas/metabolismo , Neuronas/patología , Ratones Transgénicos , Masculino , MutaciónRESUMEN
Nonviral vectors, such as liposomes, offer potential for targeted gene delivery in cancer therapy. Liposomes, composed of phospholipid vesicles, have demonstrated efficacy as nanocarriers for genetic tools, addressing the limitations of off-targeting and degradation commonly associated with traditional gene therapy approaches. Due to their biocompatibility, stability, and tunable physicochemical properties, they offer potential in overcoming the challenges associated with gene therapy, such as low transfection efficiency and poor stability in biological fluids. Despite these advancements, there remains a gap in understanding the optimal utilization of nanoliposomes for enhanced gene delivery in cancer treatment. This review delves into the present state of nanoliposomes as carriers for genetic tools in cancer therapy, sheds light on their potential to safeguard genetic payloads and facilitate cell internalization alongside the evolution of smart nanocarriers for targeted delivery. The challenges linked to their biocompatibility and the factors that restrict their effectiveness in gene delivery are also discussed along with exploring the potential of nanoliposomes in cancer gene therapy strategies by analyzing recent advancements and offering future directions.
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Deregulated cyclin-dependent kinase 5 (Cdk5) activity closely correlates with hyperphosphorylated tau, a common pathology found in neurodegenerative diseases. Previous postmortem studies had revealed increased Cdk5 immunoreactivity in amyotrophic lateral sclerosis (ALS); hence, we investigated the effects of Cdk5 inhibition on ALS model mice and neurons in this study. For the in vitro study, motor neuron cell lines with wild-type superoxide dismutase 1 (SOD1) or SOD1G93A and primary neuronal cultures from SOD1G93A transgenic (TG) mice or non-TG mice were compared for the expression of proteins involved in tau pathology, neuroinflammation, apoptosis, and neuritic outgrowth by applying Cdk5-small interfering RNA or Cdk5-short hairpin RNA (shRNA). For the in vivo study, SOD1G93A mice and non-TG mice were intrathecally injected with adeno-associated virus 9 (AAV9)-scramble (SCR)-shRNA or AAV9-Cdk5-shRNA at the age of 5 weeks. Weight and motor function were measured three times per week from 60 days of age, longevity was evaluated, and the tissues were collected from 90-day-old or 120-day-old mice. Neurons with SOD1G93A showed increased phosphorylated tau, attenuated neuritic growth, mislocalization of SOD1, and enhanced neuroinflammation and apoptosis, all of which were reversed by Cdk5 inhibition. Weights did not show significant differences among non-TG and SOD1G93A mice with or without Cdk5 silencing. SOD1G93A mice treated with AAV9-Cdk5-shRNA showed significantly delayed disease onset, delayed rotarod failure, and prolonged survival compared with those treated with AAV9-SCR-shRNA. The brain and spinal cord of SOD1G93A mice intrathecally injected with AAV9-Cdk5-shRNA exhibited suppressed tau pathology, neuroinflammation, apoptosis, and an increased number of motor neurons compared to those of SOD1G93A mice injected with AAV9-SCR-shRNA. Cdk5 inhibition could be an important mechanism in the development of a new therapeutic strategy for ALS.
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Introduction: Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies. However, its efficacy in solid tumors is limited by the immunosuppressive tumor microenvironment that compromises CAR T cell antitumor function in clinical settings. To overcome this challenge, researchers have investigated the potential of inhibiting specific immune checkpoint receptors, including A2aR (Adenosine A2 Receptor) and Tim3 (T cell immunoglobulin and mucin domain-containing protein 3), to enhance CAR T cell function. In this study, we evaluated the impact of genetic targeting of Tim3 and A2a receptors on the antitumor function of human mesothelin-specific CAR T cells (MSLN-CAR) in vitro and in vivo. Methods: Second-generation anti-mesothelin CAR T cells were produced using standard cellular and molecular techniques. A2aR-knockdown and/or Tim3- knockdown anti-mesothelin-CAR T cells were generated using shRNA-mediated gene silencing. The antitumor function of CAR T cells was evaluated by measuring cytokine production, proliferation, and cytotoxicity in vitro through coculture with cervical cancer cells (HeLa cell line). To evaluate in vivo antitumor efficacy of manufactured CAR T cells, tumor growth and mouse survival were monitored in a human cervical cancer xenograft model. Results: In vitro experiments demonstrated that knockdown of A2aR alone or in combination with Tim3 significantly improved CAR T cell proliferation, cytokine production, and cytotoxicity in presence of tumor cells in an antigen-specific manner. Furthermore, in the humanized xenograft model, both double knockdown CAR T cells and control CAR T cells could effectively control tumor growth. However, single knockdown CAR T cells were associated with reduced survival in mice. Conclusion: These findings highlight the potential of concomitant genetic targeting of Tim3 and A2a receptors to augment the efficacy of CAR T cell therapy in solid tumors. Nevertheless, caution should be exercised in light of our observation of decreased survival in mice treated with single knockdown MSLN-CAR T cells, emphasizing the need for careful efficacy considerations.
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Receptor 2 Celular del Virus de la Hepatitis A , Inmunoterapia Adoptiva , Mesotelina , Receptores Quiméricos de Antígenos , Neoplasias del Cuello Uterino , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Animales , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/genética , Femenino , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/terapia , Neoplasias del Cuello Uterino/genética , Ratones , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Línea Celular Tumoral , Microambiente Tumoral/inmunología , Ratones SCIDRESUMEN
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.
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Neoplasias Pancreáticas , ARN Circular , Humanos , ARN Circular/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/metabolismo , Animales , Terapia Molecular Dirigida/métodosRESUMEN
Circular RNAs (circRNAs) are a type of noncoding RNA that are formed by back-splicing from eukaryotic protein-coding genes. The most frequently reported and well-characterized function of circRNAs is their ability to act as molecular decoys, most often as miRNA and protein sponges. However, the functions of most circRNAs still need to be better understood. To more fully understand the biological relevance of validated circRNAs, knockdown functional analyses can be performed using antisense oligonucleotides, RNA interference (RNAi) experiments (e.g., targeting back-splicing junction sites), the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)-9 system (e.g., generating circRNA-specific knockouts), and CRISPR-Cas13 technology to effectively target circRNAs without affecting host genes. In this review, I summarize the feasibility and effectiveness of circRNA knockdown through antisense strategies for investigating the biological roles of circRNAs in cultured cells and animal models.
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Oligonucleótidos Antisentido , ARN Circular , ARN Circular/genética , Animales , Humanos , Oligonucleótidos Antisentido/genética , Técnicas de Silenciamiento del Gen/métodos , Sistemas CRISPR-Cas/genética , Interferencia de ARNRESUMEN
Porcine reproductive and respiratory syndrome (PRRS) is one of the most detrimental contagious swine ailments worldwide. Currently, no effective drugs are available for its treatment. Targeting the structural and non-structural proteins (NSP) of the type 2 PRRS virus (PRRSV-2) with small interfering RNA (siRNA) is an effective approach to inhibit PRRSV replication. NSP4, which is highly conserved and possesses 3â¯C-like serine protease activity (3CLSP), can cleave PRRSV self-proteins, thereby contributing to viral replication. To investigate the mechanism by which NSP4 regulates PRRSV-2 replication and screen for effective siRNA inhibitors of PRRSV-2 replication, the recombinant plasmid pEGFP-C1-NSP4 was constructed, and a control siRNA pair and two siRNA pairs targeting the PRRSV-2 NSP4 gene (shRNA-ctr, shRNA-150, and shRNA-536) were synthesized and cloned into the pSilencer4.1-CMV vector. After 24â¯h of incubation, Marc-145 cells were transfected with recombinant plasmids, and subsequently infected with different PRRSV-2 (XH-GD, ZQ-GD, GDr180, and JXA1-R). Subsequently, the effects of NSP4 overexpression, shRNA on PRRSV-2 replication were evaluated by assessing cytopathic effects (CPE), TCID50, quantitative real-time PCR (qPCR), immunofluorescence assays (IFA), and Western blotting. The data from these CPE, TCID50, qPCR, and IFA experiments revealed that NSP4 overexpression significantly enhanced PRRSV-2 replication and shRNA targeting NSP4 can inhibit PRRSV-2 replication in Marc-145 cells, indicating that shRNA could serve as candidate molecules for fundamental research on PRRSV-2.
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Virus del Síndrome Respiratorio y Reproductivo Porcino , ARN Interferente Pequeño , Proteínas no Estructurales Virales , Replicación Viral , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Animales , Porcinos , Línea Celular , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virologíaRESUMEN
shRNA-mediated strategy of miRNA overexpression based on RNA Polymerase III (Pol III) expression cassettes is widely used for miRNA functional studies. For some miRNAs, e.g., encoded in the genome as a part of a polycistronic miRNA cluster, it is most likely the only way for their individual stable overexpression. Here we have revealed that expression of miRNAs longer than 19 nt (e.g. 23 nt in length hsa-miR-93-5p) using such approach could be accompanied by undesired predominant generation of 5' end miRNA isoforms (5'-isomiRs). Extra U residues (up to five) added by Pol III at the 3' end of the transcribed shRNA during transcription termination could cause a shift in the Dicer cleavage position of the shRNA. This results in the formation of 5'-isomiRs, which have a significantly altered seed region compared to the initially encoded canonical hsa-miR-93-5p. We demonstrated that the commonly used qPCR method is insensitive to the formation of 5'-isomiRs and cannot be used to confirm miRNA overexpression. However, the predominant expression of 5'-isomiRs without three or four first nucleotides instead of the canonical isoform could be disclosed based on miRNA-Seq analysis. Moreover, mRNA sequencing data showed that the 5'-isomiRs of hsa-miR-93-5p presumably regulate their own mRNA targets. Thus, omitting miRNA-Seq analysis may lead to erroneous conclusions regarding revealed mRNA targets and possible molecular mechanisms in which studied miRNA is involved. Overall, the presented results show that structures of shRNAs for stable overexpression of miRNAs requires careful design to avoid generation of undesired 5'-isomiRs.
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MicroARNs , ARN Interferente Pequeño , MicroARNs/genética , MicroARNs/metabolismo , Humanos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ribonucleasa III/metabolismo , Ribonucleasa III/genética , ARN Polimerasa III/metabolismo , ARN Polimerasa III/genética , Células HEK293 , Isoformas de ARN/genética , Isoformas de ARN/metabolismoRESUMEN
Elevated brain angiotensin II activity plays a key role in the development of neurogenic hypertension. While blood pressure (BP) control in neurogenic hypertension has been successfully demonstrated by regulating central angiotensin II activity, current techniques involving cerebrovascular injections of potential therapeutic agents are not suitable for clinical translation. To address this gap, we present the synthesis of dual-functionalized liposomes functionalized with targeting ligand and cell-penetrating peptide. Functionalized liposomes were synthesized using the thin film hydration technique and loaded with plasmid DNA encoding short hairpin RNA targeted toward angiotensin II receptors (PEAS), via the post-insertion method. The synthesized liposomes had a cationic surface charge, an average size of 150 nm, and effectively entrapped more than 89% of loaded PEAS. These liposomes loaded with PEAS demonstrated biocompatibility and efficient delivery to brain-derived cell lines, resulting in a remarkable reduction of more than 70% in receptor expression within 7 days. To assess the therapeutic potential, spontaneously hypertensive rats were administered intravenous injections of functionalized liposomes loaded with PEAS, and the changes in mean arterial pressure were monitored for 45 days. Remarkably, this treatment led to a significant (p < 0.001) decrease in BP of more than 30 mm Hg compared with saline-treated rats.
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To explore the role of YAP, a key effector of the Hippo pathway, in temporomandibular joint (TMJ) ankylosis. The temporal and spatial expression of YAP was detected via immunohistochemistry and multiplex immunohistochemistry on postoperative Days 1, 4, 7, 9, 11, 14 and 28 in a sheep model. Isolated mesenchymal stem cells (MSCs) from samples of the Day 14. The relative mRNA expression of YAP was examined before and after the osteogenic induction of MSCs. A YAP-silenced MSC model was constructed, and the effect of YAP knockdown on MSC function was examined. YAP is expressed in the nucleus of the key sites that determine the ankylosis formation, indicating that YAP is activated in a physiological state. The expression of YAP increased gradually over time. Moreover, the number of cells coexpressing of RUNX2 and YAP-with the osteogenic active zone labelled by RUNX2-tended to increase after Day 9. After the osteogenic induction of MSCs, the expression of YAP increased. After silencing YAP, the osteogenic, proliferative and migratory abilities of the MSCs were inhibited. YAP is involved in the early development of TMJ bony ankylosis. Inhibition of YAP using shRNA might be a promising way to prevent or treat TMJ ankylosis.
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Anquilosis , Células Madre Mesenquimatosas , Osteogénesis , Trastornos de la Articulación Temporomandibular , Animales , Células Madre Mesenquimatosas/metabolismo , Trastornos de la Articulación Temporomandibular/metabolismo , Trastornos de la Articulación Temporomandibular/patología , Trastornos de la Articulación Temporomandibular/genética , Anquilosis/metabolismo , Anquilosis/patología , Anquilosis/genética , Proteínas Señalizadoras YAP/metabolismo , Articulación Temporomandibular/metabolismo , Articulación Temporomandibular/patología , Ovinos , Proliferación Celular , Modelos Animales de Enfermedad , Diferenciación Celular , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Movimiento Celular , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
MHV-A59 is a beta-coronavirus that causes demyelinating encephalitis and hepatitis in mice. Recently, the mouse infection model of MHV-A59 has been used as an alternative animal infection model for SARS-CoV and SARS-CoV-2, aiding the development of new antiviral drugs. In this study, the MHV-A59 model was employed to investigate the potential of SARS-CoV-2 UTRs as new targets for antiviral drugs. Optimal targets within the MHV-A59 UTRs were identified using a shRNA and siRNA design tool, focusing on RNA secondary stem-loop (SL) structures in the UTRs. We then examined whether the designed RNAi constructs could inhibit MHV-A59 replication. In the 5'UTR, the stem-loop 1 (SL1) was identified as the most effective target, while in the 3'UTR, the minimal element for the initiation of negative-strand RNA synthesis (MIN) proved to be the most effective. Importantly, siRNAs targeting SL1 and MIN structures significantly reduced total RNA synthesis, negative-strand genomic RNA synthesis, subgenomic (sg) RNA synthesis, viral titer, and the plaque size of MHV-A59 compared to the control. Although not statistically significant, the combination of siSL1 and siMIN had a stronger effect on inhibiting MHV-A59 replication than either siRNA monotherapy. Interestingly, while the SL1 structure is present in both MHV and SARS-CoV-2, the MIN structure is unique to MHV. Thus, the SL1 of SARS-CoV-2 may represent a novel and promising target for RNAi-based antiviral drugs.
RESUMEN
Haemophilic arthropathy (HA), a common comorbidity in haemophilic patients leads to joint pain, deformity and reduced quality of life. We have recently demonstrated that a long non-coding RNA, Neat1 as a primary regulator of matrix metalloproteinase (MMP) 3 and MMP13 activity, and its induction in the target joint has a deteriorating effect on articular cartilage. In the present study, we administered an Adeno-associated virus (AAV) 5 vector carrying an short hairpin (sh)RNA to Neat1 via intra-articular injection alone or in conjunction with systemic administration of a capsid-modified AAV8 (K31Q) vector carrying F8 gene (F8-BDD-V3) to study its impact on HA. AAV8K31Q-F8 vector administration at low dose, led to an increase in FVIII activity (16%-28%) in treated mice. We further observed a significant knockdown of Neat1 (~40 fold vs. untreated injured joint, p = 0.005) in joint tissue of treated mice and a downregulation of chondrodegenerative enzymes, MMP3, MMP13 and the inflammatory mediator- cPLA2, in mice receiving combination therapy. These data demonstrate that AAV mediated Neat1 knockdown in combination with F8 gene augmentation can potentially impact mediators of haemophilic joint disease.