RESUMEN
Transposable elements (TEs) provide a prime example of genetic conflict because they can proliferate in genomes and populations even if they harm the host. However, numerous studies have shown that TEs, though typically harmful, can also provide fuel for adaptation. This is because they code functional sequences that can be useful for the host in which they reside. In this review, I summarize the "how" and "why" of adaptation enabled by the genetic conflict between TEs and hosts. In addition, focusing on mechanisms of TE control by small piwi-interacting RNAs (piRNAs), I highlight an indirect form of adaptation enabled by conflict. In this case, mechanisms of host defense that regulate TEs have been redeployed for endogenous gene regulation. I propose that the genetic conflict released by meiosis in early eukaryotes may have been important because, among other reasons, it spurred evolutionary innovation on multiple interwoven trajectories - on the part of hosts and also embedded genetic parasites. This form of evolution may function as a complexity generating engine that was a critical player in eukaryotic evolution.
Asunto(s)
Elementos Transponibles de ADN , ARN Interferente Pequeño , Elementos Transponibles de ADN/genética , Animales , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Evolución Molecular , ARN de Interacción con PiwiRESUMEN
BACKGROUND: The Hippo pathway is a conserved tumour suppressor signalling pathway, and its dysregulation is often associated with abnormal cell growth and tumorigenesis. We previously revealed that the transcriptional coactivator Yes-associated protein (YAP), the key effector of the Hippo pathway, is a molecular target for glioblastoma (GBM), the most common malignant brain tumour. Inhibiting YAP with small interfering RNA (siYAP) or the specific inhibitor verteporfin (VP) can diminish GBM growth to a certain degree. RESULTS: In this study, to enhance the anti-GBM effect of siYAP and VP, we designed stepwise-targeting and hypoxia-responsive liposomes (AMVY@NPs), which encapsulate hypoxia-responsive polymetronidazole-coated VP and DOTAP adsorbed siYAP, with angiopep-2 (A2) modification on the surface. AMVY@NPs exhibited excellent bloodâbrain barrier crossing, GBM targeting, and hypoxia-responsive and efficient siYAP and VP release properties. By inhibiting the expression and function of YAP, AMVY@NPs synergistically inhibited both the growth and stemness of GBM in vitro. Moreover, AMVY@NPs strongly inhibited the growth of orthotopic U87 xenografts and improved the survival of tumour-bearing mice without adverse effects. CONCLUSION: Specific targeting of YAP with stepwise-targeting and hypoxia-responsive liposome AMVY@NPs carrying siYAP and VP efficiently inhibited GBM progression. This study provides a valuable drug delivery platform and creative insights for molecular targeted treatment of GBM in the future.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Liposomas , Ratones Desnudos , ARN Interferente Pequeño , Verteporfina , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Liposomas/química , Verteporfina/farmacología , Verteporfina/uso terapéutico , Animales , Humanos , Línea Celular Tumoral , Ratones , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Proteínas Señalizadoras YAP , Nanopartículas/química , Ratones Endogámicos BALB C , Factores de Transcripción/metabolismo , Angiomotinas , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , PéptidosRESUMEN
RNA interference (RNAi) is a powerful tool for sequence-specific gene knockdown in therapeutic and research applications. However, spatiotemporal control of RNAi is required to decrease nonspecific targeting, potential toxicity, and allow targeting of essential genes. Herein we describe a class of de-novo-designed RNA switches that enable sequence-specific regulation of RNAi in mammalian cells. Using cis-repressing RNA elements, we engineer RNA devices that only initiate microRNA biogenesis when binding with cognate trigger RNAs. We demonstrate that this conditional RNAi system, termed Orthogonal RNA Interference induced by Trigger RNA (ORIENTR), provides up to 14-fold increases in artificial miRNA biogenesis upon activation in orthogonal libraries. We show that integration of ORIENTR triggers with dCas13d enhances dynamic range to up to 31-fold. We further demonstrate that ORIENTR can be applied to detect endogenous RNA signals and to conditionally knockdown endogenous genes, thus enabling regulatory possibilities including cell-type-specific RNAi and rewiring of transcriptional networks via RNA profile.
Asunto(s)
MicroARNs , Interferencia de ARN , Activación Transcripcional , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Células HEK293 , Animales , Técnicas de Silenciamiento del Gen , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN/metabolismo , ARN/genéticaRESUMEN
Small interfering RNA (siRNA) highlights the immense therapeutic potential for cancer treatment. The major challenge in siRNA therapy is the effective RNA nanodrug delivery system, which is facilitated by the ligand and the carrier. In this study, we analyzed the binding specificity of linear RGD and circular RGD to αVß3 integrins by mapping the morphology using super-resolution direct stochastic optical reconstruction microscopy. Meanwhile, the binding dynamics was investigated using single-molecule force spectroscopy. Then, the effects of the ligand and carrier on RNA nanodrug cell entry dynamic parameters were evaluated at the single particle level by the force tracing technique. Furthermore, the delivery efficiency of RNA nanodrugs was assessed using AFM-based nanoindentation at the single cell level. This report will provide valuable insights for rational design strategies aiming to achieve improved efficiency for nanodrug delivery systems.
Asunto(s)
ARN Interferente Pequeño , Ligandos , Humanos , ARN Interferente Pequeño/química , ARN Interferente Pequeño/farmacología , Integrina alfaVbeta3/metabolismo , Integrina alfaVbeta3/química , Oligopéptidos/química , Sistemas de Liberación de Medicamentos , Portadores de Fármacos/química , Microscopía de Fuerza Atómica , Nanopartículas/químicaRESUMEN
Non-coding RNA expression has shown to have cell type-specificity. The regulatory characteristics of these molecules are impacted by changes in their expression levels. We performed next-generation sequencing and examined small RNA-seq data obtained from 6 different types of blood cells separated by fluorescence-activated cell sorting of severe COVID-19 patients and healthy control donors. In addition to examining the behavior of piRNA in the blood cells of severe SARS-CoV-2 infected patients, our aim was to present a distinct piRNA differential expression portrait for each separate cell type. We observed that depending on the type of cell, different sorted control cells (erythrocytes, monocytes, lymphocytes, eosinophils, basophils, and neutrophils) have altering piRNA expression patterns. After analyzing the expression of piRNAs in each set of sorted cells from patients with severe COVID-19, we observed 3 significantly elevated piRNAs - piR-33,123, piR-34,765, piR-43,768 and 9 downregulated piRNAs in erythrocytes. In lymphocytes, all 19 piRNAs were upregulated. Monocytes were presented with a larger amount of statistically significant piRNA, 5 upregulated (piR-49039 piR-31623, piR-37213, piR-44721, piR-44720) and 35 downregulated. It has been previously shown that piR-31,623 has been associated with respiratory syncytial virus infection, and taking in account the major role of piRNA in transposon silencing, we presume that the differential expression patterns which we observed could be a signal of indirect antiviral activity or a specific antiviral cell state. Additionally, in lymphocytes, all 19 piRNAs were upregulated.
Asunto(s)
COVID-19 , Citometría de Flujo , ARN Interferente Pequeño , SARS-CoV-2 , Humanos , COVID-19/genética , COVID-19/virología , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/genética , SARS-CoV-2/genética , Masculino , Femenino , Persona de Mediana Edad , Monocitos/metabolismo , Adulto , Células Sanguíneas/metabolismo , ARN de Interacción con PiwiRESUMEN
Malignant pleural effusions (MPEs) are hard to treat, and their onset usually signals terminal cancer. Immunotherapies hold promise but must overcome the immunosuppressive MPE microenvironment. Herein, we treat MPEs via synergistically combining two emerging cancer therapy modalities: enzyme-dynamic therapy (EDT) and metalloimmunotherapy. To do so, a nanoplatform termed "A-R-SOME" was developed which comprises MPE-targeted M1 type extracellular vesicles (EVs) loaded with (1) a manganese-based superoxide dismutase (SOD) enzyme, (2) stimulator of interferon genes (STING) agonist diABZI-2, and (3) signal transducer and an activator of transcription 3 (STAT3) small interfering RNA. Endogenous reactive oxygen species within tumors induced immunogenic cell death by EDT, along with STING activation by both Mn and diABZI-2, and suppression of the STAT3 pathway. Systemically administered A-R-SOME alleviated the MPE immunosuppressive microenvironment, triggered antitumor systemic immunity, and long-term immune memory, leading to the complete eradication of MPE and pleural tumors with 100% survival rate in an aggressive murine model. A-R-SOME-induced immune effects were also observed in human patient-derived MPE, pointing toward the translation potential of A-R-SOME as an experimental malignancy treatment.
Asunto(s)
Vesículas Extracelulares , Inmunoterapia , Derrame Pleural Maligno , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Animales , Humanos , Ratones , Superóxido Dismutasa/metabolismo , Microambiente Tumoral/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/química , ARN Interferente Pequeño/genética , Femenino , Factor de Transcripción STAT3/metabolismo , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismo , Línea Celular TumoralRESUMEN
A series of successes in RNA interference (RNAi) therapies for liver diseases using lipid nanoparticles and N-acetylgalactosamine have heralded a current era of RNA therapeutics. However, alternative delivery strategies are required to take RNAi out of the comfort zone of hepatocytes. Here we report SIRPα IgV/anti-CD47 siRNA (vS-siCD47) conjugates that selectively and persistently disrupt the antiphagocytic CD47/SIRPα axis in solid tumors. Conjugation of the SIRPα IgV domain protein to siRNAs enables tumor dash through CD47-mediated erythrocyte piggyback, primarily blocking the physical interaction between CD47 on cancer cells and SIRPα on phagocytes. After internalization of the vS-siCD47 conjugates within cancer cells, the detached free-standing anti-CD47 siRNAs subsequently attack CD47 through the RNAi mechanism. The dual-action approach of the vS-siCD47 conjugate effectively overcomes the "don't eat me" barrier and stimulates phagocyte-mediated tumor destruction, demonstrating a highly selective and potent CD47-blocking immunotherapy. This delivery strategy, employing IgV domain protein-siRNA conjugates with a dual mode of target suppression, holds promise for expanding RNAi applications beyond hepatocytes and advancing RNAi-based cancer immunotherapies for solid tumors.
Asunto(s)
Antígeno CD47 , ARN Interferente Pequeño , Receptores Inmunológicos , Antígeno CD47/metabolismo , Antígeno CD47/química , Humanos , ARN Interferente Pequeño/química , Animales , Ratones , Receptores Inmunológicos/metabolismo , Neoplasias/terapia , Neoplasias/genética , Neoplasias/patología , Antígenos de Diferenciación , Línea Celular TumoralRESUMEN
Endometrial receptivity is essential for successful embryo implantation and pregnancy initiation and is regulated via various signaling pathways. Adiponectin, an important adipokine, may be a potential regulator of reproductive system functions. The aim of the present study was to elucidate the regulatory role of adiponectin receptor 1 (ADIPOR1) in endometrial receptivity. The endometrial receptivity between RL952 and AN3CA cell lines was confirmed using an in vitro JAr spheroid attachment model. 293T cells were transfected with control or short hairpin (sh)ADIPOR1 vectors and RL952 cells were transduced with lentiviral particles targeting ADIPOR1. Reverse transcriptionquantitative PCR and immunoblot assays were also performed. ADIPOR1 was consistently upregulated in the endometrium during the midsecretory phase compared with that in the proliferative phase and in receptive RL952 cells compared with that in nonreceptive AN3CA cells. Stable cell lines with diminished ADIPOR1 expression caused by shRNA showed reduced Ecadherin expression and attenuated in vitro endometrial receptivity. ADIPOR1 regulated AMPactivated protein kinase (AMPK) activity in endometrial epithelial cells. Regulation of AMPK activity via dorsomorphin and 5aminoimidazole4carboxamide ribonucleotide affected Ecadherin expression and in vitro endometrial receptivity. The ADIPOR1/AMPK/Ecadherin axis is vital to endometrial receptivity. These findings can help improve fertility treatments and outcomes.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Cadherinas , Endometrio , Receptores de Adiponectina , Transducción de Señal , Receptores de Adiponectina/metabolismo , Receptores de Adiponectina/genética , Humanos , Femenino , Endometrio/metabolismo , Cadherinas/metabolismo , Cadherinas/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Línea Celular , Implantación del Embrión , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/genética , Adulto , Aminoimidazol Carboxamida/análogos & derivados , RibonucleótidosRESUMEN
piRNAs are crucial for transposon silencing, germ cell maturation, and fertility in male mice. Here, we report on the genetic landscape of piRNA dysfunction in humans and present 39 infertile men carrying biallelic variants in 14 different piRNA pathway genes, including PIWIL1, GTSF1, GPAT2, MAEL, TDRD1, and DDX4. In some affected men, the testicular phenotypes differ from those of the respective knockout mice and range from complete germ cell loss to the production of a few morphologically abnormal sperm. A reduced number of pachytene piRNAs was detected in the testicular tissue of variant carriers, demonstrating impaired piRNA biogenesis. Furthermore, LINE1 expression in spermatogonia links impaired piRNA biogenesis to transposon de-silencing and serves to classify variants as functionally relevant. These results establish the disrupted piRNA pathway as a major cause of human spermatogenic failure and provide insights into transposon silencing in human male germ cells.
Asunto(s)
Elementos Transponibles de ADN , Infertilidad Masculina , ARN Interferente Pequeño , Espermatogénesis , Testículo , Masculino , Humanos , Espermatogénesis/genética , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/genética , Elementos Transponibles de ADN/genética , Animales , Testículo/metabolismo , Ratones , Adulto , Silenciador del Gen , Ratones Noqueados , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Elementos de Nucleótido Esparcido Largo/genética , Espermatogonias/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ARN de Interacción con PiwiRESUMEN
Targeting non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), has recently emerged as a promising strategy for treating malignancies and other diseases. In recent years, the development of ncRNA-based therapeutics for targeting protein-coding and non-coding genes has also gained momentum. This review systematically examines ongoing and completed clinical trials to provide a comprehensive overview of the emerging landscape of ncRNA-based therapeutics. Significant efforts have been made to advance ncRNA therapeutics to early clinical studies. The most advanced trials have been conducted with small interfering RNAs (siRNAs), miRNA replacement using nanovector-entrapped miRNA mimics, or miRNA silencing by antisense oligonucleotides. While siRNA-based therapeutics have already received FDA approval, miRNA mimics, inhibitors, and lncRNA-based therapeutics are still under evaluation in preclinical and early clinical studies. We critically discuss the rationale and methodologies of ncRNA targeting strategies to illustrate this rapidly evolving field.
Asunto(s)
Ensayos Clínicos como Asunto , Neoplasias , ARN no Traducido , Humanos , Neoplasias/genética , Neoplasias/terapia , ARN no Traducido/genética , ARN no Traducido/uso terapéutico , MicroARNs/genética , MicroARNs/uso terapéutico , MicroARNs/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Animales , ARN Interferente Pequeño/uso terapéuticoRESUMEN
Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease, while there is a lack of pharmaceutical interventions to halt AAA progression presently. To address the multifaceted pathology of AAA, this work develops a novel multifunctional gene delivery system to simultaneously deliver two siRNAs targeting MMP-2 and MMP-9. The system (TPNs-siRNA), formed through the oxidative polymerization and self-assembly of epigallocatechin gallate (EGCG), efficiently encapsulates siRNAs during self-assembly. TPNs-siRNA safeguards siRNAs from biological degradation, facilitates intracellular siRNA transfection, promotes lysosomal escape, and releases siRNAs to silence MMP-2 and MMP-9. Additionally, TPNs, serving as a multi-bioactive material, mitigates oxidative stress and inflammation, fosters M1-to-M2 repolarization of macrophages, and inhibits cell calcification and apoptosis. In experiments with AAA mice, TPNs-siRNA accumulated and persisted in aneurysmal tissue after intravenous delivery, demonstrating that TPNs-siRNA can be significantly distributed in macrophages and VSMCs relevant to AAA pathogenesis. Leveraging the carrier's intrinsic multi-bioactive properties, the targeted siRNA delivery by TPNs exhibits a synergistic effect for enhanced AAA therapy. Furthermore, TPNs-siRNA is gradually metabolized and excreted from the body, resulting in excellent biocompatibility. Consequently, TPNs emerges as a promising multi-bioactive nanotherapy and a targeted delivery nanocarrier for effective AAA therapy.
Asunto(s)
Aneurisma de la Aorta Abdominal , Metaloproteinasa 9 de la Matriz , Ratones Endogámicos C57BL , Nanopartículas , ARN Interferente Pequeño , Aneurisma de la Aorta Abdominal/tratamiento farmacológico , Animales , Ratones , Nanopartículas/química , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Polifenoles/química , Polifenoles/farmacología , Catequina/análogos & derivados , Catequina/química , Catequina/farmacología , Té/química , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Humanos , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Técnicas de Transferencia de Gen , Estrés Oxidativo/efectos de los fármacos , Células RAW 264.7 , Apoptosis/efectos de los fármacosRESUMEN
Macrophages show high plasticity and play a vital role in the progression of metabolic dysfunction-associated steatohepatitis (MASH). X-box binding protein 1 (XBP1), a key sensor of the unfolded protein response, can modulate macrophage-mediated pro-inflammatory responses in the pathogenesis of MASH. However, how XBP1 influences macrophage plasticity and promotes MASH progression remains unclear. Herein, we formulated an Xbp1 siRNA delivery system based on folic acid modified D-α-tocopheryl polyethylene glycol 1000 succinate nanoparticles (FT@XBP1) to explore the precise role of macrophage-specific Xbp1 deficiency in the progression of MASH. FT@XBP1 was specifically internalized into hepatic macrophages and subsequently inhibited the expression of spliced XBP1 both in vitro and in vivo. It promoted M1-phenotype macrophage repolarization to M2 macrophages, reduced the release of pro-inflammatory factors, and alleviated hepatic steatosis, liver injury, and fibrosis in mice with fat-, fructose- and cholesterol-rich diet-induced MASH. Mechanistically, FT@XBP1 promoted macrophage polarization toward the M2 phenotype and enhanced the release of exosomes that could inhibit the activation of hepatic stellate cells. A promising macrophage-targeted siRNA delivery system was revealed to pave a promising strategy in the treatment of MASH.
Asunto(s)
Ácido Fólico , Macrófagos , ARN Interferente Pequeño , Proteína 1 de Unión a la X-Box , Animales , Masculino , Ratones , Estrés del Retículo Endoplásmico/efectos de los fármacos , Hígado Graso/metabolismo , Ácido Fólico/química , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Ratones Endogámicos C57BL , Nanopartículas/química , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
Introduction: Dysregulated calcium homeostasis and consequentially aberrant Ca2+ signalling could enhance survival, proliferation and metastasis in various cancers. Despite rapid development in exploring the ion channel functions in relation to cancer, most of the mechanisms accounting for the impact of ion channel modulators have yet to be fully clarified. Although harnessing small interfering RNA (siRNA) to specifically silence gene expression has the potential to be a pivotal approach, its success in therapeutic intervention is dependent on an efficient delivery system. Nanoparticles have the capacity to strongly bind siRNAs. They remain in the circulation and eventually deliver the siRNA payload to the target organ. Afterward, they interact with the cell surface and enter the cell via endocytosis. Finally, they help escape the endo-lysosomal degradation system prior to unload the siRNAs into cytosol. Carbonate apatite (CA) nanocrystals primarily is composed of Ca2+, carbonate and phosphate. CA possesses both anion and cation binding domains to target negatively charged siRNA molecules. Methods: Hybrid CA was synthesized by complexing CA NPs with a hydrophilic polysaccharide - hyaluronic acid (HA). The average diameter of the composite particles was determined using Zetasizer and FE-SEM and their zeta potential values were also measured. Results and Discussion: The stronger binding affinity and cellular uptake of a fluorescent siRNA were observed for HA-CA NPs as compared to plain CA NPs. Hybrid CA was electrostatically bound individually and combined with three different siRNAs to silence expression of calcium ion channel and transporter genes, TRPC6, TRPM8 and SLC41A1 in a human breast cancer cell line (MCF-7) and evaluate their potential for treating breast cancer. Hybrid NPs carrying TRPC6, TRPM8 and SLC41A1 siRNAs could significantly enhance cytotoxicity both in vitro and in vivo. The resultant composite CA influenced biodistribution of the delivered siRNA, facilitating reduced off target distribution and enhanced breast tumor targetability.
Asunto(s)
Apatitas , Neoplasias de la Mama , Ácido Hialurónico , Nanopartículas , ARN Interferente Pequeño , Humanos , Apatitas/química , Apatitas/farmacología , ARN Interferente Pequeño/química , ARN Interferente Pequeño/farmacocinética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/farmacología , ARN Interferente Pequeño/genética , Ácido Hialurónico/química , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Nanopartículas/química , Femenino , Animales , Supervivencia Celular/efectos de los fármacos , Línea Celular Tumoral , Células MCF-7 , Proliferación Celular/efectos de los fármacos , RatonesRESUMEN
The prognosis after heart transplantation continues to improve. Therefore, the prevention of chronic post-transplant sequelae, such as chronic kidney disease, allograft vasculopathy, and malignancies is becoming increasingly important. Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), is increasingly used for immunosuppression after heart transplantation. However, everolimus may cause a characteristic complex of adverse effects, including dyslipidemia. Currently there are no guidelines for the long-term screening and treatment of dyslipidemia in heart transplant recipients treated with everolimus. This article presents a clinical case of hypercholesterolemia that developed after the start of the everolimus treatment in a heart recipient. The patient was a 39-year-old man who underwent orthotopic heart transplantation for ischemic cardiomyopathy in 2012 (at the age of 27). In 2019, the patient's immunosuppressive therapy was converted from mycophenolate mofetil to everolimus due to the development of cardiac allograft vasculopathy. The change in the immunosuppressive therapy was associated with increases in total cholesterol and low-density lipoprotein cholesterol, which were not reversed with a combined lipid-lowering therapy (maximum doses of rosuvastatin, ezetimibe, fenofibrate). A decrease in lipid levels was achieved with a blocker of hepatic proprotein convertase subtilisin/kexin type 9 synthesis at the level of microribonucleic acid (inclisiran). This case demonstrates the difficulties in correcting dyslipidemia in patients with cardiac allograft, since the treatment with the immunosuppressant everolimus worsens existing dyslipidemia. However, the combination lipid-lowering therapy, that affects various elements of the pathogenesis (specifically, the combined inhibition of hydroxymethylglutaryl-CoA reductase with a statin, cholesterol absorption from the small intestine with ezetimibe, and PCSK9 messenger RNA with inclisiran), provides an effective control of blood lipids and minimizing the adverse effects of immunosuppressive therapy, such as cardiac allograft vasculopathy.
Asunto(s)
Everolimus , Trasplante de Corazón , Humanos , Masculino , Adulto , Everolimus/uso terapéutico , Inmunosupresores/uso terapéutico , Inmunosupresores/efectos adversos , Hiperlipidemias/tratamiento farmacológico , Inhibidores de PCSK9 , Complicaciones Posoperatorias/tratamiento farmacológico , Resultado del Tratamiento , ARN Interferente PequeñoRESUMEN
Nitric oxide (NO) regulates vascular homeostasis and plays a key role in revascularization and angiogenesis. The endothelial nitric oxide synthase (eNOS) enzyme catalyzes NO production in endothelial cells. Overexpression of the eNOS gene has been implicated in pathologies with dysfunctional angiogenic processes, such as cancer. Therefore, modulating eNOS gene expression using small interfering RNAs (siRNAs) represents a viable strategy for antitumor therapy. siRNAs are highly specific to the target gene, thus reducing off-target effects. Given the widespread distribution of endothelium and the crucial physiological role of eNOS, localized delivery of nucleic acid to the affected area is essential. Therefore, the development of an efficient eNOS-siRNA delivery carrier capable of controlled release is imperative for targeting specific vascular regions, particularly those associated with tumor vascular growth. Thus, this study aims to utilize ultrasound-mediated microbubble destruction (UMMD) technology with cationic microbubbles loaded with eNOS-siRNA to enhance transfection efficiency and improve siRNA delivery, thereby preventing sprouting angiogenesis. The efficiency of eNOS-siRNA transfection facilitated by UMMD was assessed using bEnd.3 cells. Synthesis of nitric oxide and eNOS protein expression were also evaluated. The silencing of eNOS gene in a model of angiogenesis was assayed using the rat aortic ring assay. The results showed that from 6 to 24 h, the transfection of fluorescent siRNA with UMMD was twice as high as that of lipofection. Moreover, transfection of eNOS-siRNA with UMMD enhanced the knockdown level (65.40 ± 4.50%) compared to lipofectamine (40 ± 1.70%). Silencing of eNOS gene with UMMD required less amount of eNOS-siRNA (42 ng) to decrease the level of eNOS protein expression (52.30 ± 0.08%) to the same extent as 79 ng of eNOS-siRNA using lipofectamine (56.30 ± 0.10%). NO production assisted by UMMD was reduced by 81% compared to 67% reduction transfecting with lipofectamine. This diminished NO production led to higher attenuation of aortic ring outgrowth. Three-fold reduction compared to lipofectamine transfection. In conclusion, we propose the combination of eNOS-siRNA and UMMD as an efficient, safe, non-viral nucleic acid transfection strategy for inhibition of tumor progression.
Asunto(s)
Aorta , Microburbujas , Óxido Nítrico Sintasa de Tipo III , Óxido Nítrico , ARN Interferente Pequeño , Transfección , Animales , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Transfección/métodos , Aorta/metabolismo , Óxido Nítrico/metabolismo , Ratones , Masculino , Línea Celular , Neovascularización Fisiológica/genéticaRESUMEN
Hepatocellular carcinoma (HCC) is the most common and deadliest subtype of liver cancer worldwide and, therefore, poses an enormous threat to global health. Understanding the molecular mechanisms underlying the development and progression of HCC is central to improving our clinical approaches. PIWI-interacting RNAs (piRNAs) are a class of small non-coding RNAs that bind to PIWI family proteins to regulate gene expression at transcriptional and post-transcriptional levels. A growing body of work shows that the dysregulation of piRNAs plays a crucial role in the progression of various human cancers. In this editorial, we report on the current knowledge of HCC-associated piRNAs and their potential clinical utility. Based on the editorial by Papadopoulos and Trifylli, on the role and clinical evaluation of exosomal circular RNAs in HCC, we highlight this other emerging class of non-coding RNAs.
Asunto(s)
Carcinoma Hepatocelular , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas , ARN Interferente Pequeño , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , ARN Interferente Pequeño/metabolismo , Exosomas/metabolismo , Exosomas/genética , ARN Circular/metabolismo , ARN Circular/genética , Progresión de la Enfermedad , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismoRESUMEN
RNA interference (RNAi) serves as an indispensable tool for gene function studies and has been substantiated through extensive research for its practical applications in the baculovirus expression vector system (BEVS). This chapter expands the RNAi toolkit in insect cell culture by including small interfering RNA (siRNA) in the protocol, in addition to the conventional use of double-stranded RNA (dsRNA). This chapter also brings attention to key design and reporting considerations, based on Minimum Information About an RNAi Experiment (MIARE) guidelines. Recommendations regarding online tools for dsRNA and siRNA design are provided, along with guidance on choosing suitable methods for measuring silencing outcomes.
Asunto(s)
Baculoviridae , Vectores Genéticos , Interferencia de ARN , ARN Bicatenario , ARN Interferente Pequeño , Animales , Baculoviridae/genética , ARN Bicatenario/genética , ARN Interferente Pequeño/genética , Vectores Genéticos/genética , Insectos/genética , Línea Celular , Células Sf9RESUMEN
BACKGROUND: Differentiation of pluripotent stem cells into desired lineages is the key aspect of regenerative medicine and cell-based therapy. Although RNA interference (RNAi) technology is exploited extensively for this, methods for long term silencing of the target genes leading to differentiation remain a challenge. Sustained knockdown of the target gene by RNAi is often inefficient as a result of low delivery efficiencies, protocol induced toxicity and safety concerns related to viral vectors. Earlier, we established octa-arginine functionalized hydroxyapatite nano vehicles (R8HNPs) for delivery of small interfering RNA (siRNA) against a pluripotency marker gene in mouse embryonic stem cells. Although we demonstrated excellent knockdown efficiency of the target gene, sustained gene silencing leading to differentiation was yet to be achieved. METHODS: To establish a sustained non-viral gene silencing protocol using R8HNP, we investigated various methods of siRNA delivery: double delivery of adherent cells (Adh-D), suspension delivery followed by adherent delivery (Susp + Adh), single delivery in suspension (Susp-S) and multiple deliveries in suspension (Susp-R). Sustained knockdown of a pluripotent marker gene followed by differentiation was analysed by reverse transcriptase-PCR, fluoresence-activated cell sorting and immunofluorescence techniques. Impact on cell viability as a result of repeated exposure of the R8HNP was also tested. RESULTS: Amongst the protocols tested, the most efficient knockdown of the target gene for a prolonged period of time was obtained by repeated suspension delivery of the R8HNP-siRNA conjugate. The long-term silencing of a pluripotency marker gene resulted in differentiation of R1 ESCs predominantly towards the extra embryonic and ectodermal lineages. Cells displayed excellent tolerance to repeated exposures of R8HNPs. CONCLUSIONS: The results demonstrate that R8HNPs are promising, biocompatible, non-viral alternatives for prolonged gene silencing and obtaining differentiated cells for therapeutics.
Asunto(s)
Diferenciación Celular , Durapatita , Células Madre Embrionarias de Ratones , ARN Interferente Pequeño , Animales , Ratones , Durapatita/química , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/efectos de los fármacos , ARN Interferente Pequeño/genética , Silenciador del Gen , Materiales Biocompatibles/química , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Nanopartículas/química , Transducción Genética , Interferencia de ARN , Técnicas de Silenciamiento del GenRESUMEN
BACKGROUND/AIMS: Tactile perception relies on mechanoreceptors and nerve fibers, including c-fibers, Aß-fibers and Aδ-fibers. Schwann cells (SCs) play a crucial role in supporting nerve fibers, with non-myelinating SCs enwrapping c-fibers and myelinating SCs ensheathing Aß and Aδ fibers. Recent research has unveiled new functions for cutaneous sensory SCs, highlighting the involvement of nociceptive SCs in pain perception and Meissner corpuscle SCs in tactile sensation. Furthermore, Piezo2, previously associated with Merkel cell tactile sensitivity, has been identified in SCs. The goal of this study was to investigate the channels implicated in SC mechanosensitivity and the release process of neurotrophic factor secretion. METHODS: Immortalized IFRS1 SCs and human primary SCs generated two distinct subtypes of SCs: undifferentiated and differentiated SCs. Quantitative PCR was employed to evaluate the expression of differentiation markers and mechanosensitive channels, including TRP channels (TRPV4, TRPM7 and TRPA1) and Piezo channels (Piezo1 and Piezo2). To validate the functionality of specific mechanosensitive channels, Ca2+ imaging and electronic cell sizing experiments were conducted under hypotonic conditions, and inhibitors and siRNAs were used. Protein expression was assessed by Western blotting and immunostaining. Additionally, secretome analysis was performed to evaluate the release of neurotrophic factors in response to hypotonic stimulation, with BDNF, a representative trophic factor, quantified using ELISA. RESULTS: Induction of differentiation increased Piezo2 mRNA expression levels both in IFRS1 and in human primary SCs. Both cell types were responsive to hypotonic solutions, with differentiated SCs displaying a more pronounced response. Gd3+ and FM1-43 effectively inhibited hypotonicity-induced Ca2+ transients in differentiated SCs, implicating Piezo2 channels. Conversely, inhibitors of Piezo1 and TRPM7 (Dooku1 and NS8593, respectively) had no discernible impact. Moreover, Piezo2 in differentiated SCs appeared to participate in regulatory volume decreases (RVD) after cell swelling induced by hypotonic stimulation. A Piezo2 deficiency correlated with reduced RVD and prolonged cell swelling, leading to heightened release of the neurotrophic factor BDNF by upregulating the function of endogenously expressed Ca2+-permeable TRPV4. CONCLUSION: Our study unveils the mechanosensitivity of SCs and implicates Piezo2 channels in the release of neurotrophic factors from SCs. These results suggest that Piezo2 may contribute to RVD, thereby maintaining cellular homeostasis, and may also serve as a negative regulator of neurotrophic factor release. These findings underscore the need for further investigation into the role of Piezo2 in SC function and neurotrophic regulation.