RESUMO
Trans-acting hammerhead ribozyme inherits the advantages of being the smallest and best-characterized RNA-cleaving ribozyme, offering high modularity and the ability to cleave any desired sequence without the aid of any protein, as long as the target sequence contains a cleavage site. However, achieving precise control over the trans-acting hammerhead ribozyme would enable safer and more accurate regulation of gene expression. Herein, we described an intracellular selection of hammerhead aptazyme that contains a theophylline aptamer on stem II based on toxin protein IbsC. Based on the intracellular selection, we obtained three new cis-acting hammerhead aptazymes. Moreover, the corresponding trans-acting aptazymes could be efficiently induced by theophylline to knock down different targeted genes in eukaryotic cells. Notably, the best one, T195, exhibited a ligand-dependent and dose-dependent response to theophylline, and the cleavage efficiency could be enhanced by incorporating multiplex aptazymes.
RESUMO
As a promising therapeutic approach for the correction of pathogenic mutations, the RNA editing process is reversible and tunable without permanently altering the genome. RNA editing mediated by human ADAR proteins offers distinct advantages, including high specificity and low propensity to cause immunogenicity. Herein, we describe a small molecule-inducible RNA editing strategy by incorporating aptazymes into the guide RNA of ADAR-based RNA editing technology. Once small molecules are added or removed, aptazymes trigger self-cleavage to release the guide RNA, achieving small molecule-controlled RNA editing. To satisfy different RNA editing applications, both turn-on and turn-off A-to-I RNA editing of target mRNA have been realized by using on/off-switch aptazymes. Theoretically speaking, this strategy can be applied to various ADAR-based editing systems, which could improve the safety and potential clinical applications of RNA editing technology.
Assuntos
Edição de RNA , RNA , Humanos , Edição de RNA/genética , RNA Mensageiro/genéticaRESUMO
Organelles play specific and indispensable roles in cellular processes, and their dysfunction causes a series of diseases. Monitoring their variations is crucial to understanding their roles in physiopathology. Unlike most organelle imaging tools based on chemical probes or proteins, we developed a novel molecular tool for cell organelle imaging based on nucleic acids. Real-time imaging of mitochondria and nucleus in living cells was realized easily through using RNA-based tools, which are composed of Organelle-targeted RNA Sequence and Fluorogen-activating RNA aptamer. This genetically encoded Organelle-targeted RNA imaging system provides a powerful platform for measuring the properties of organelles and intracellular structures in living cells, due to its nontoxicity, low fluorescence background, ease of construction, and modular properties.
Assuntos
Aptâmeros de Nucleotídeos , Aptâmeros de Nucleotídeos/química , Fluorescência , Corantes Fluorescentes/química , Organelas/química , RNA/análiseRESUMO
Trans-cleaving techniques have been most enthusiastically embraced in the development of therapy for genetic diseases, particularly in the correction of monogenic recessive mutations at the messenger RNA level. However, easy degradation and poor catalytic activity in vivo remain significant obstacles to trans-cleaving of the hammerhead ribozyme. Herein, we found a novel scaffold RNA that stabilizes the ribozyme structure in trans-cleaving and promotes the knockdown efficiency of the hammerhead ribozyme in specific regions of living cells. We can give the trans-cleaving hammerhead ribozyme the ability to knock down specific genes in specific cell regions by changing different scaffolds. Therefore, our study proves the potential usefulness of the RNA knockdown strategy with high-specific trans-cleaving hammerhead ribozyme as a therapeutic approach in gene therapy.
RESUMO
Hyperglycemia and hyperlipidemia (glycolipotoxicity)-triggered islet ß-cell dysfunction is known to drive the progression of obesity-related type 2 diabetes, however the underlying mechanisms have not been clearly elucidated. The current study aimed to investigate the role of mitogen-activated protein kinase phosphatase 5 (MKP-5) in islet cells under glucolipotoxic conditions. Using gene overexpression and knockdown approaches, we demonstrated that MKP-5 could alleviate glucolipotoxicity-induced apoptosis via the endoplasmic reticulum (ER) stress and mitochondrial apoptosis pathways owing to the altered regulation of caspase family members and ER stress-related molecules in MIN6 and primary islet cells. Overexpression of MKP-5 reversed the glucose and palmitic acid (GP)-induced impairment of insulin secretion as well as the abnormal decreases in the expression of islet functional genes, thereby maintaining the normal insulin secretory functionality, whereas the absence of MKP-5 aggravated islet cell dysfunction. In parallel, the production of ROS and increased inflammation-associated genes in response to GP were also reduced upon MKP-5 overexpression. Further, inhibition of JNK or P38 MAPK pathways resisted to glucolipotoxicity observed in MKP-5 knockdown MIN6 cells. These findings indicate that MKP-5 is an important mediator for glucolipotoxicity-induced islet cell dysfunction and apoptosis, with JNK and P38 as the critical downstream pathways.
Assuntos
Apoptose/fisiologia , Fosfatases de Especificidade Dupla/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Glucose/toxicidade , Ilhotas Pancreáticas/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Fosfatases da Proteína Quinase Ativada por Mitógeno/fisiologia , Palmitatos/toxicidade , Animais , Linhagem Celular Tumoral , Dieta Hiperlipídica/efeitos adversos , Fosfatases de Especificidade Dupla/genética , Técnicas de Silenciamento de Genes , Humanos , Insulina/metabolismo , Insulinoma/patologia , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Neoplasias Pancreáticas/patologia , Proteínas Recombinantes/metabolismo , Regulação para CimaRESUMO
STAT3 is persistently activated in a wide variety of human tumours, and aberrant STAT3 activity promotes tumour growth, invasion and metastasis. To explore STAT3 down-regulation in human oesophageal cancer cells, cell proliferation, apoptosis and mitochondrial mechanisms were explored in oesophageal carcinoma TE1 cell cultures. We demonstrate for the first time that STAT3 down-regulation by RNAi is sufficient to inhibit oesophageal cancer cell proliferation inducing cell apoptosis. Further, we demonstrate that mitochondrial transmembrane potential is impaired thereby leading to collapsed mitochondrial membrane potential, abnormal mitochondrial membrane depolarization, nuclear DNA fragmentation and cell cycle G2/M arrest under the conditions of STAT3 down-regulation. Thus, our results suggest that STAT3 inhibition is a valid approach to induce oesophageal carcinoma cell mitochondrial-dependent apoptosis in therapeutic strategies against oesophageal cancers.