RESUMO
The design of RNA sequences with desired structural properties presents a challenging computational problem with promising applications in biotechnology and biomedicine. Most regulatory RNAs function by forming RNA-RNA interactions, e.g., in order to regulate mRNA expression. It is therefore natural to consider problems where a sequence is designed to form a desired RNA-RNA interaction and switch between structures upon binding. This contribution demonstrates the use of the Infrared framework to design interacting sequences. Specifically, we consider the regulation of the rpoS mRNA by the sRNA DsrA and design artificial 5 ' UTRs that place a downstream protein coding gene under control of DsrA. The design process is explained step by step in a Jupyter notebook, accompanied by Python code. The text discusses setting up design constraints for sampling sequences in Infrared, computing quality measures, constructing a suitable cost function, as well as the optimization procedure. We show that not only thermodynamic but also kinetic folding features can be relevant. Kinetics of interaction formation can be estimated efficiently using the RRIkinDP tool, and the chapter explains how to include kinetic folding features from RRIkinDP directly in the cost function. The protocol implemented in our Jupyter notebook can easily be extended to consider additional requirements or adapted to novel design scenarios.
Assuntos
Conformação de Ácido Nucleico , Termodinâmica , Biologia Computacional/métodos , Software , Cinética , RNA/genética , RNA/química , RNA/metabolismo , Regiões 5' não Traduzidas , RNA Mensageiro/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Algoritmos , Dobramento de RNARESUMO
Non-viral nanoparticles (NPs) have seen heightened interest as a delivery method for a variety of clinically relevant nucleic acid cargoes in recent years. While much of the focus has been on lipid NPs, non-lipid NPs, including polymeric NPs, have the possibility of improved efficacy, safety, and targeting, especially to non-liver organs following systemic administration. A safe and effective systemic approach for intracellular delivery to the lungs could overcome limitations to intratracheal/intranasal delivery of NPs and improve clinical benefit for a range of diseases including cystic fibrosis. Here, engineered biodegradable poly (beta-amino ester) (PBAE) NPs are shown to facilitate efficient delivery of mRNA to primary human airway epithelial cells from both healthy donors and individuals with cystic fibrosis. Optimized NP formulations made with differentially endcapped PBAEs and systemically administered in vivo lead to high expression of mRNA within the lungs in BALB/c and C57 B/L mice without requiring a complex targeting ligand. High levels of mRNA-based gene editing were achieved in an Ai9 mouse model across bronchial, epithelial, and endothelial cell populations. No toxicity was observed either acutely or over time, including after multiple systemic administrations of the NPs. The non-lipid biodegradable PBAE NPs demonstrate high levels of transfection in both primary human airway epithelial cells and in vivo editing of lung cell types that are targets for numerous life-limiting diseases particularly single gene disorders such as cystic fibrosis and surfactant deficiencies.
Assuntos
Pulmão , Camundongos Endogâmicos C57BL , Nanopartículas , Polímeros , RNA Mensageiro , Animais , Pulmão/metabolismo , Humanos , Nanopartículas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Polímeros/química , Camundongos Endogâmicos BALB C , Camundongos , Fibrose Cística , Feminino , Ligantes , Células Epiteliais/metabolismoRESUMO
QuantiGene™ 2.0 technique could be used to investigate the gene expression signature of the immune system senescence and thus to understand the molecular mechanism involved in the defects of the immune response during aging.QuantiGene™ 2.0 technique is a multiplex platform allowing the simultaneous analysis of several target RNA molecules (up to 80) present in a single sample. QuantiGene Assays use an accurate method for multiplexed or for single gene expression quantitation. QuantiGene 2.0 uses magnetic beads which are dyed internally with two fluorescence dyes, exhibiting a unique spectral signal and providing specificity and multiplexing capability of the technique. QuantiGene Assays incorporate branched-DNA technology for gene expression profiling.Branched-DNA system is responsible for the high sensitivity of the system. In fact, it permits to detect low levels of mRNA molecules. This branched-DNA system allows for the direct measurement of RNA transcripts by using signal amplification rather than target amplification. The assay protocol is spread over 2 days. First, immune cells are lysed to release the target RNA, which is incubated with oligonucleotide probe set targeted with beads capable to hybridize with the target RNA. Signal amplification is performed by sequential hybridization of the branched-DNA pre-amplifier, amplifier, and label probe molecules. The last step involves the incubation with Streptavidin-conjugated R-phycoerythrin. The fluorescent reporter generates a signal directly proportional to the levels of RNA molecules present in the cells. Luminex instrument evaluates the median intensity of fluorescence, which is proportional to the number of RNA target molecules present in the cells.
Assuntos
Perfilação da Expressão Gênica , Perfilação da Expressão Gênica/métodos , Humanos , RNA/genética , Hibridização de Ácido Nucleico/métodos , RNA Mensageiro/genéticaRESUMO
The aging immune system undergoes significant changes, leading to a state known as immunosenescence. Understanding the molecular mechanisms underlying immunosenescence is crucial for developing targeted interventions to enhance immune functions in older individuals. This bio-protocol review focuses on the application of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the mRNA quantification of cytokine-inducible SH2-containing protein (CISH), an immune regulator overexpressed in T-cell responses from older adults. We outline a comprehensive protocol for the quantitative assessment of CISH mRNA expression, providing a valuable tool for researchers investigating immunosenescence.
Assuntos
Imunossenescência , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Citocinas/metabolismo , Envelhecimento/imunologia , Envelhecimento/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
To function effectively as an integrated system, the transcriptional and post-transcriptional machineries must communicate through mechanisms that are still poorly understood. Here, we focus on the zinc-finger Sfp1, known to regulate transcription of proliferation-related genes. We show that Sfp1 can regulate transcription either by binding to promoters, like most known transcription activators, or by binding to the transcribed regions (gene bodies), probably via RNA polymerase II (Pol II). We further studied the first mode of Sfp1 activity and found that, following promoter binding, Sfp1 binds to gene bodies and affects Pol II configuration, manifested by dissociation or conformational change of its Rpb4 subunit and increased backtracking. Surprisingly, Sfp1 binds to a subset of mRNAs co-transcriptionally and stabilizes them. The interaction between Sfp1 and its client mRNAs is controlled by their respective promoters and coincides with Sfp1's dissociation from chromatin. Intriguingly, Sfp1 dissociation from the chromatin correlates with the extent of the backtracked Pol II. We propose that, following promoter recruitment, Sfp1 accompanies Pol II and regulates backtracking. The backtracked Pol II is more compatible with Sfp1's relocation to the nascent transcripts, whereupon Sfp1 accompanies these mRNAs to the cytoplasm and regulates their stability. Thus, Sfp1's co-transcriptional binding imprints the mRNA fate, serving as a paradigm for the cross-talk between the synthesis and decay of specific mRNAs, and a paradigm for the dual-role of some zinc-finger proteins. The interplay between Sfp1's two modes of transcription regulation remains to be examined.
The ability to fine-tune the production of proteins in a cell is essential for organisms to exist. An imbalance in protein levels can be the cause of various diseases. Messenger RNA molecules (mRNA) link the genetic information encoded in DNA and the produced proteins. Exactly how much protein is made mostly depends on the amount of mRNA in the cell's cytoplasm. This is controlled by two processes: the synthesis of mRNA (also known as transcription) and mRNA being actively degraded. Although much is known about mechanisms regulating transcription and degradation, how cells detect if they need to degrade mRNA based on the levels of its synthesis and vice versa is poorly understood. In 2013, researchers found that proteins known as 'RNA decay factors' responsible for mRNA degradation are actively moved from the cell's cytoplasm into its nucleus to instruct the transcription machinery to produce more mRNA. Kelbert, Jordán-Pla, de-Miguel-Jiménez et al. including some of the researchers involved in the 2013 work investigated how mRNA synthesis and degradation are coordinated to ensure a proper mRNA level. The researchers used advanced genome engineering methods to carefully manipulate and measure mRNA production and degradation in yeast cells. The experiments revealed that the protein Sfp1 a well-characterized transcription factor for stimulating the synthesis of a specific class of mRNAs inside the nucleus can also prevent the degradation of these mRNAs outside the nucleus. During transcription, Sfp1 bound directly to mRNA. The investigators could manipulate the co-transcriptional binding of Sfp1 to a certain mRNA, thereby changing the mRNA stability in the cytoplasm. This suggests that the ability of Sfp1 to regulate both the production and decay of mRNA is dependent on one another and that transcription can influence the fate of its transcripts. This combined activity can rapidly change mRNA levels in response to changes in the cell's environment. RNA plays a key role in ensuring correct levels of proteins. It can also function as an RNA molecule, independently of its coding capacity. Many cancers and developmental disorders are known to be caused by faulty interactions between transcription factors and nucleic acids. The finding that some transcription factors can directly regulate both mRNA synthesis and its destruction introduces new angles for studying and understanding these diseases.
Assuntos
RNA Polimerase II , RNA Mensageiro , Fatores de Transcrição , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Estabilidade de RNA , Regiões Promotoras Genéticas , Ligação Proteica , Dedos de Zinco , Transcrição Gênica , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Citoplasma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiaeRESUMO
The mechanisms underlying stimuli-induced dynamic structural remodeling of RNAs for the maintenance of cellular physiological function and survival remain unclear. Here, we showed that in MGMT promoter-methylated glioblastoma (GBM), the RNA helicase DEAD-box helicase 46 (DDX46) is phosphorylated by temozolomide (TMZ)-activated checkpoint kinase 1 (CHK1), resulting in a dense-to-loose conformational change and an increase in DDX46 helicase activity. DDX46-mediated tertiary structural remodeling of LINC01956 exposes the binding motifs of LINC01956 to the 3' untranslated region of O6-methylguanine DNA methyltransferase (MGMT). This accelerates recruitment of MGMT mRNA to the RNA export machinery and transportation of MGMT mRNA from the nucleus to the cytoplasm, leading to increased MGMT abundance and TMZ resistance. Using patient-derived xenograft (PDX) and tumor organoid models, we found that treatment with the CHK1 inhibitor SRA737abolishes TMZ-induced structural remodeling of LINC01956 and subsequent MGMT up-regulation, resensitizing TMZ-resistant MGMT promoter-methylated GBM to TMZ. In conclusion, these findings highlight a mechanism underlying temozolomide-induced RNA structural remodeling and may represent a potential therapeutic strategy for patients with TMZ-resistant MGMT promoter-methylated GBM.
Assuntos
RNA Helicases DEAD-box , Metilases de Modificação do DNA , Resistencia a Medicamentos Antineoplásicos , Glioblastoma , RNA Longo não Codificante , Temozolomida , Proteínas Supressoras de Tumor , Glioblastoma/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/metabolismo , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Humanos , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Metilases de Modificação do DNA/metabolismo , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Regiões Promotoras Genéticas/genética , Metilação de DNA/genética , Metilação de DNA/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/metabolismo , Quinase 1 do Ponto de Checagem/genética , Linhagem Celular Tumoral , Camundongos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Fosforilação/efeitos dos fármacosRESUMO
How eukaryotic ribosomes traverse messenger RNA (mRNA) leader sequences to search for protein-synthesis start sites remains one of the most mysterious aspects of translation and its regulation. While the search process is conventionally described by a linear "scanning" model, its exquisitely dynamic nature has restricted detailed mechanistic study. Here, we observed single Saccharomyces cerevisiae ribosomal scanning complexes in real time, finding that they scan diverse mRNA leaders at a rate of 10 to 20 nt s-1. We show that specific binding of a protein to its mRNA leader sequence substantially arrests scanning. Conversely, impairing scanning-complex guanosine 5'-triphosphate hydrolysis results in native start-site bypass. Our results illustrate an mRNA-centric, kinetically controlled regulatory model where the ribosomal pre-initiation complex amplifies a nuanced energetic landscape to regulate scanning and start-site selection fidelity.
Assuntos
RNA Mensageiro , Ribossomos , Saccharomyces cerevisiae , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Imagem Individual de Molécula/métodos , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Guanosina Trifosfato/metabolismoRESUMO
In mammalian females, the transition from dormancy in primordial follicles to follicular development is critical for maintaining ovarian function and reproductive longevity. In mice, the quiescent primary oocyte of the primordial follicle contains a Balbiani body (B-body), an organelle aggregate comprised of a spherical structure of Golgi complexes. Here we show that the structure of the B-body is maintained by microtubules and actin. The B-body stores mRNA-capping enzyme and 597 mRNAs associated with mRNA-decapping enzyme 1 A (DCP1A). Gene ontology analysis results indicate that proteins encoded by these mRNAs function in enzyme binding, cellular component organization and packing of telomere ends. Pharmacological depolymerization of microtubules or actin led to B-body disassociation and nascent protein synthesis around the dissociated B-bodies within three hours. An increased number of activated developing follicles were observed in ovaries with prolonged culture and the in vivo mouse model. Our results indicate that the mouse B-body is involved in the activation of dormant primordial follicles likely via translation of the B-body-associated RNAs in primary oocytes.
Assuntos
Oócitos , Folículo Ovariano , Animais , Oócitos/metabolismo , Camundongos , Feminino , Folículo Ovariano/metabolismo , Folículo Ovariano/citologia , RNA/metabolismo , RNA/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Microtúbulos/metabolismo , Actinas/metabolismo , Actinas/genética , Complexo de Golgi/metabolismoRESUMO
mRNA-based therapeutics increasingly demonstrate significant potential in treating various diseases, including infectious diseases, cancers, and genetic disorders. Effective delivery systems are crucial for advancing mRNA therapeutics. Lipid nanoparticles (LNPs) serve as an excellent carrier, widely validated for their safety and tolerability in commercially available mRNA vaccines. Standard LNPs typically consist of four components: ionizable lipids (ILs), helper lipids, cholesterol, and polyethylene glycol-lipids (PEG-lipids), with the structural design of ILs gradually becoming a focal point of research interest. The chemical structures and formulations of the other components also significantly affect the delivery efficiency, targeting specificity, and stability of LNPs. The complex formulations of LNPs may hinder the clinical transformation of mRNA therapeutics and have raised widespread concerns about their safety. This review aims to summarize the progress of LNPs-based mRNA therapeutics in clinical trials, focusing on adverse effects that occurred during these trials. It also discusses representative innovations in LNP components, highlighting challenges and potential ways in this research field. We firmly believe this review will promote further improvements and designs of LNP compositions to optimize mRNA therapeutics. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Biology-Inspired Nanomaterials > Lipid-Based Structures.
Assuntos
Lipídeos , Nanopartículas , RNA Mensageiro , Humanos , Nanopartículas/química , Lipídeos/química , Animais , LipossomosRESUMO
Background: Chemical modifications on RNA profoundly affect RNA function and regulation. m6A, the most abundant RNA modification in eukaryotes, plays a pivotal role in diverse cellular processes and disease mechanisms. However, its importance is understudied in human CKD samples regarding its influence on pathological mechanisms. Methods: Liquid chromatographytandem mass spectrometry and methylated RNA immunoprecipitation sequencing were used to examine alterations in m6A levels and patterns in CKD samples. Overexpression of the m6A writer METTL3 in cultured kidney tubular cells was performed to confirm the effect of m6A in tubular cells and explore the biological functions of m6A modification on target genes. In addition, tubule-specific deletion of Mettl3 (Ksp-Cre Mettl3f/f) mice and antisense oligonucleotides inhibiting Mettl3 expression were used to reduce m6A modification in an animal kidney disease model. Results: By examining 127 human CKD samples, we observed a significant increase in m6A modification and METTL3 expression in diseased kidneys. Epitranscriptomic analysis unveiled an enrichment of m6A modifications in transcripts associated with the activation of inflammatory signaling pathways, particularly the cyclic guanosine monophosphateAMP synthase (cGAS)-stimulator of IFN genes (STING) pathway. m6A hypermethylation increased mRNA stability in cGAS and STING1 as well as elevated the expression of key proteins within the cGAS-STING pathway. Both the tubule-specific deletion of Mettl3 and the use of antisense oligonucleotides to inhibit Mettl3 expression protected mice from inflammation, reduced cytokine expression, decreased immune cell recruitment, and attenuated kidney fibrosis. Conclusions: Our research revealed heightened METTL3-mediated m6A modification in fibrotic kidneys, particularly enriching the cGAS-STING pathway. This hypermethylation increased mRNA stability for cGAS and STING1, leading to sterile inflammation and fibrosis.
Assuntos
Adenosina , Fibrose , Proteínas de Membrana , Metiltransferases , Nucleotidiltransferases , RNA Mensageiro , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , RNA Mensageiro/metabolismo , Animais , Metiltransferases/metabolismo , Metiltransferases/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Humanos , Transdução de Sinais , Camundongos , Rim/patologia , Rim/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologiaRESUMO
Technological innovation yielded opportunities to obtain mRNA expression data for prostate cancer (PCa) patients even prior to biopsy, which can be used in a precision medicine approach to treatment decision-making. This can apply in particular to predict the risk of, and time to biochemical recurrence (BCR). Most mRNA-based models currently proposed to this end are designed for risk classification and post-operative prediction. Effective pre-operative prediction would facilitate early treatment decision-making, in particular by indicating more appropriate therapeutic pathways for patient profiles who would likely not benefit from a systematic prostatectomy regime. The aim of this study is to investigate the possibility to leverage mRNA information pre-operatively for BCR-free survival prediction. To do this, we considered time-to-event machine learning (ML) methodologies, rather than classification models at a specific survival horizon. We retrospectively analysed a cohort of 135 patients with clinical follow-up data and mRNA information comprising over 26,000 features (data accessible at NCBI GEO database, accession GSE21032). The performance of ML models including random survival forest, boosted and regularised Cox models were assessed, in terms of model discrimination, calibration, and predictive accuracy for overall, 3-year and 5-year survival, aligning with common clinical endpoints. Results showed that the inclusion of mRNA information could yield a gain in performance for pre-operative BCR prediction. ML-based time-to-event models significantly outperformed reference nomograms that used only routine clinical information with respect to all metrics considered. We believe this is the first study proposing pre-operative transcriptomics models for BCR prediction in PCa. External validation of these findings, including confirmation of the mRNA variables identified as potential key predictors in this study, could pave the way for pre-operative precision nomograms to facilitate timely personalised clinical decision-making.
Assuntos
Neoplasias da Próstata , RNA Mensageiro , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/cirurgia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/mortalidade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estudos Retrospectivos , Idoso , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Intervalo Livre de Doença , Aprendizado de Máquina , Prostatectomia , Prognóstico , Biomarcadores Tumorais/genéticaRESUMO
MAT2B works together with MAT2A to synthesize S-Adenosyl methionine (SAM) as the primary methyl donor. MAT2B, despite lacking catalytic activity, exerts regulatory control over the enzymatic activity of MAT2A. In addition to the enzymatic activity regulation, we find that, in an NADP+-dependent manner, MAT2B binds and stabilizes MAT2A. Disruption of the cellular NADP+ remodels the protein level of MAT2A. The pentose phosphatase pathway regulates the level of MAT2A protein through the interaction of NADP+ with MAT2B. Additionally, MAT2B-MAT2A interaction regulates the mRNA m6A modification and stability. In liver tumors, the Mat2a mRNA level is elevated but the protein level is decreased by the restricted NADP+. Blocking the interaction between MAT2B and MAT2A by the keto diet can suppress liver tumor growth. These findings reveal that MAT2B is essential for regulating the protein levels of MAT2A and connecting SAM synthesis to mRNA m6A.
Assuntos
Adenosina , Neoplasias Hepáticas , Metionina Adenosiltransferase , Metionina Adenosiltransferase/metabolismo , Metionina Adenosiltransferase/genética , Humanos , Adenosina/metabolismo , Adenosina/análogos & derivados , Animais , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , NADP/metabolismo , Camundongos , S-Adenosilmetionina/metabolismo , Linhagem Celular Tumoral , Ligação ProteicaRESUMO
Background: In the general population, primary human papillomavirus (HPV) testing is advocated for cervical cancer (CC) screening. HPV E6/E7 mRNA (Aptima HPV, AHPV) assays have garnered considerable traction due to their higher specificity when compared with HPV DNA assays. Here, we investigated age-specific primary AHPV screening assays and different triage strategies versus cytology to identify the best approach. Methods: Between April 2018 and December 2021, we recruited female participants from 34 communities across Liaoning province and Qingdao City, China. Primary cervical screening protocols included liquid-based cytology (LBC) and AHPV assays, with females positive for any assays undergoing colposcopy. Genotyping (AHPV-GT) was conducted on all HPV-positive samples. Our primary outcomes were the identification of age-specific detection rates, colposcopy referral rates, and sensitivity and specificity values for high-grade squamous intraepithelial lesions or worse (HSIL+). AHPV and different triage strategy performances were also examined across different age cohorts. Results: Our investigation included 9911 eligible females. Age-specific abnormal cytology rates were in the 6.1%-8.0% range, and were highest in 45-54-year olds. When compared with 35-44-or 45-54-year olds, HPV prevalence was highest in 55-64-year olds (12.2% or 11.6% vs.14.1%, P = 0.048 and P = 0.002, respectively). In 35-44-year olds, AHPV sensitivity for detecting HSIL+ was 96.6 (95% confidence interval [CI]: 89.7-100) - significantly higher than LBC sensitivity (65.5 [95% CI: 48.3-82.8], P < 0.001). When compared with LBC, HSIL+ detection rates by AHPV-GT using reflex LBC triage increased by 31.5% (9.6 vs. 7.3), and colposcopy referral rates decreased by 16.4% (5.1% vs. 6.1%). In 45-54-year olds, HSIL+ detection rates for AHPV-GT using reflex LBC triage were lower than LBC rates (6.2 vs. 6.6). In 55-64-year olds, AHPV sensitivity (97.2 [95% CI: 91.7-100.0]) was higher than LBC sensitivity (66.7 [95% CI: 50.0-80.6], P = 0.003). The area under the curve (AUC) value was not significantly different between AHPV-GT with reflex LBC triage and LBC (0.845 [95% CI: 0.771-0.920] vs. 0.812 [95% CI: 0.734-0.891], P = 0.236). Conclusions: Primary AHPV screening using different triage strategies were different across different age cohorts. Thus, AHPV may be an appropriate primary screening method for 35-44 and 55-64 year old females, while AHPV-GT with reflex LBC triage may be more apt for 35-44 year old females.
Assuntos
Detecção Precoce de Câncer , Infecções por Papillomavirus , Sensibilidade e Especificidade , Triagem , Neoplasias do Colo do Útero , Humanos , Feminino , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologia , Pessoa de Meia-Idade , China/epidemiologia , Adulto , Detecção Precoce de Câncer/métodos , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Triagem/métodos , Idoso , Fatores Etários , Colposcopia , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , RNA Mensageiro/genética , Proteínas Oncogênicas Virais/genética , Adulto Jovem , Genótipo , Programas de Rastreamento/métodos , Papillomavirus Humano , CitologiaRESUMO
Realizing the immense clinical potential of mRNA-based drugs will require continued development of methods to safely deliver the bioactive agents with high efficiency and without triggering side effects. In this regard, lipid nanoparticles have been successfully utilized to improve mRNA delivery and protect the cargo from extracellular degradation. Encapsulation in lipid nanoparticles was an essential factor in the successful clinical application of mRNA vaccines, which conclusively demonstrated the technology's potential to yield approved medicines. In this review, we begin by describing current advances in mRNA modifications, design of novel lipids and development of lipid nanoparticle components for mRNA-based drugs. Then, we summarize key points pertaining to preclinical and clinical development of mRNA therapeutics. Finally, we cover topics related to targeted delivery systems, including endosomal escape and targeting of immune cells, tumors and organs for use with mRNA vaccines and new treatment modalities for human diseases.
Assuntos
Sistemas de Liberação de Medicamentos , Nanopartículas , RNA Mensageiro , Humanos , RNA Mensageiro/genética , RNA Mensageiro/administração & dosagem , Nanopartículas/química , Sistemas de Liberação de Medicamentos/métodos , Vacinas de mRNA , Lipídeos/química , LipossomosRESUMO
Lipid nanoparticles (LNPs) are currently the most commonly used non-viral gene delivery system. Their physiochemical attributes, encompassing size, charge and surface modifications, significantly affect their behaviors both in vivo and in vitro. Nevertheless, the effects of these properties on the transfection and distribution of LNPs after intramuscular injection remain elusive. In this study, LNPs with varying sizes, lipid-based charges and PEGylated lipids were formulated to study their transfection and in vivo distribution. Luciferase mRNA (mLuc) was entraped in LNPs as a model nucleic acid molecule. Results indicated that smaller-sized LNPs and those with neutral potential presented superior transfection efficiency after intramuscular injection. Surprisingly, the sizes and charges did not exert a notable influence on the in vivo distribution of the LNPs. Furthermore, PEGylated lipids with shorter acyl chains contributed to enhanced transfection efficiency due to their superior cellular uptake and lysosomal escape capabilities. Notably, the mechanisms underlying cellular uptake differed among LNPs containing various types of PEGylated lipids, which was primarily attributed to the length of their acyl chain. Together, these insights underscore the pivotal role of nanoparticle characteristics and PEGylated lipids in the intramuscular route. This study not only fills crucial knowledge gaps but also provides significant directions for the effective delivery of mRNA via LNPs.
Assuntos
Lipídeos , Nanopartículas , Tamanho da Partícula , Polietilenoglicóis , RNA Mensageiro , Transfecção , Nanopartículas/química , Animais , Polietilenoglicóis/química , Injeções Intramusculares , Lipídeos/química , Transfecção/métodos , Camundongos , Técnicas de Transferência de Genes , Humanos , Luciferases/metabolismo , Luciferases/genética , Propriedades de Superfície , LipossomosRESUMO
BACKGROUND: Oral cancers, which include tumors of the oral cavity, salivary glands, and pharynx, are becoming increasingly prevalent worldwide. Squamous cell carcinoma accounts for over 90% of malignant oral lesions, with oral squamous cell carcinoma (OSCC) being notably common in the Indian subcontinent and other regions of Asia. This is especially true in South-Central Asia, including Sri Lanka, where it is particularly prevalent among men. This study aims to evaluate the levels of Vascular Endothelial Growth Factor-A (VEGF-A) and Cytokeratin-19 (CK-19) mRNAs in whole blood as a potential method for the early detection of OSCC. METHODS: The study included 40 patients (each from OSCC, Oral Submucous Fibrosis (OSF), Oral Leukoplakia (OLK), Oral Lichen Planus (OLP), and 10 healthy controls. The expression levels of VEGF-A and CK-19 mRNAs were measured from extracellular RNA extracted from whole blood samples using real-time reverse transcription polymerase chain reaction (RT-PCR) with sequence-specific primers. Receiver operating characteristic (ROC) curve analysis was used to evaluate the effectiveness of these biomarkers in detecting OSCC. RESULTS: The results demonstrated a significant increase in blood transcripts of the candidate mRNAs CK-19 and VEGF-A in patients with OSCC, OSF, OLK, and OLP. The Wilcoxon signed-rank test revealed a p-value of 0.002 for each specific comparison between diseased patients and healthy controls (i.e., OSCC vs. HC, OSF vs. HC, OLP vs. HC, OLK vs. HC) for both CK-19 and VEGF-A. When these two biomarkers were used together, they provided a 60% predictive probability for patients with OSCC (p = 0.023). CONCLUSION: This study highlights the efficacy of blood mRNA transcriptome diagnostics in detecting OSCC. This innovative clinical approach has the potential to be a robust, efficient, and reliable tool for early cancer detection. Blood-based transcriptomes could be further explored for their effectiveness in various health contexts and for routine health monitoring.
Assuntos
Biomarcadores Tumorais , Carcinoma de Células Escamosas , Queratina-19 , Leucoplasia Oral , Neoplasias Bucais , Fibrose Oral Submucosa , RNA Mensageiro , Fator A de Crescimento do Endotélio Vascular , Humanos , Fator A de Crescimento do Endotélio Vascular/sangue , Fator A de Crescimento do Endotélio Vascular/genética , Neoplasias Bucais/sangue , Neoplasias Bucais/genética , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/diagnóstico , Masculino , RNA Mensageiro/sangue , Fibrose Oral Submucosa/sangue , Fibrose Oral Submucosa/genética , Feminino , Leucoplasia Oral/sangue , Leucoplasia Oral/genética , Pessoa de Meia-Idade , Biomarcadores Tumorais/sangue , Queratina-19/sangue , Adulto , Líquen Plano Bucal/sangue , Líquen Plano Bucal/genética , Estudos de Casos e Controles , Lesões Pré-Cancerosas/sangue , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Detecção Precoce de Câncer/métodos , Idoso , Reação em Cadeia da Polimerase em Tempo Real , Curva ROCRESUMO
In our cells, a limited number of RNA binding proteins (RBPs) are responsible for all aspects of RNA metabolism across the entire transcriptome. To accomplish this, RBPs form regulatory units that act on specific target regulons. However, the landscape of RBP combinatorial interactions remains poorly explored. Here, we perform a systematic annotation of RBP combinatorial interactions via multimodal data integration. We build a large-scale map of RBP protein neighborhoods by generating in vivo proximity-dependent biotinylation datasets of 50 human RBPs. In parallel, we use CRISPR interference with single-cell readout to capture transcriptomic changes upon RBP knockdowns. By combining these physical and functional interaction readouts, along with the atlas of RBP mRNA targets from eCLIP assays, we generate an integrated map of functional RBP interactions. We then use this map to match RBPs to their context-specific functions and validate the predicted functions biochemically for four RBPs. This study provides a detailed map of RBP interactions and deconvolves them into distinct regulatory modules with annotated functions and target regulons. This multimodal and integrative framework provides a principled approach for studying post-transcriptional regulatory processes and enriches our understanding of their underlying mechanisms.
Assuntos
RNA Mensageiro , Proteínas de Ligação a RNA , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Transcriptoma , Processamento Pós-Transcricional do RNA , Regulação da Expressão Gênica , Células HEK293 , Análise de Célula Única , Redes Reguladoras de Genes , Regulon/genéticaRESUMO
Background: Inflammatory Bowel Diseases (IBDs), encompassing Ulcerative Colitis (UC) and Crohn's Disease (CD), are chronic, recurrent inflammatory conditions of the gastrointestinal tract. The microRNA (miRNA) -mRNA regulatory network is pivotal in the initiation and progression of IBDs. Although individual studies provide valuable insights into miRNA mechanisms in IBDs, they often have limited scope due to constraints in population diversity, sample size, sequencing platform variability, batch effects, and potential researcher bias. Our study aimed to construct comprehensive miRNA-mRNA regulatory networks and determine the cellular sources and functions of key miRNAs in IBD pathogenesis. Methods: To minimize potential bias from individual studies, we utilized a text mining-based approach on published scientific literature from PubMed and PMC databases to identify miRNAs and mRNAs associated with IBDs and their subtypes. We constructed miRNA-mRNA regulatory networks by integrating both predicted and experimentally validated results from DIANA, Targetscan, PicTar, Miranda, miRDB, and miRTarBase (all of which are databases for miRNA target annotation). The functions of miRNAs were determined through gene enrichment analysis of their target mRNAs. Additionally, we used two large-scale single-cell RNA sequencing datasets to identify the cellular sources of miRNAs and the association of their expression levels with clinical status, molecular and functional alternation in CD and UC. Results: Our analysis systematically summarized IBD-related genes using text-mining methodologies. We constructed three comprehensive miRNA-mRNA regulatory networks specific to IBD, CD, and UC. Through cross-analysis with two large-scale scRNA-seq datasets, we determined the cellular sources of the identified miRNAs. Despite originating from different cell types, hsa-miR-142, hsa-miR-145, and hsa-miR-146a were common to both CD and UC. Notably, hsa-miR-145 was identified as myofibroblast-specific in both CD and UC. Furthermore, we found that higher tissue repair and enhanced glucose and lipid metabolism were associated with hsa-miR-145 in myofibroblasts in both CD and UC contexts. Conclusion: This comprehensive approach revealed common and distinct miRNA-mRNA regulatory networks in CD and UC, identified cell-specific miRNA expressions (notably hsa-miR-145 in myofibroblasts), and linked miRNA expression to functional alterations in IBD. These findings not only enhance our understanding of IBD pathogenesis but also offer promising diagnostic biomarkers and therapeutic targets for clinical practice in managing IBDs.
Assuntos
Mineração de Dados , Redes Reguladoras de Genes , Doenças Inflamatórias Intestinais , MicroRNAs , RNA Mensageiro , Análise de Célula Única , Humanos , MicroRNAs/genética , RNA Mensageiro/genética , Doenças Inflamatórias Intestinais/genética , Análise de Célula Única/métodos , Biologia Computacional/métodos , Análise de Sequência de RNA/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Doença de Crohn/genéticaRESUMO
Omeprazole, a drug of choice for the management of gastric hyperacidity, influences serotonergic neurotransmission in brain regions and its long-term use is known to cause stress-related behavioral deficits including anxiety. Aim of the current study was to explore the effects of omeprazole treatment on immobilization-induced anxiety in rats, specifically on the role of serotonin (5-HT). In view of the role of serotonin-1A (5-HT1A) autoreceptor in the availability of 5-HT in brain regions, mRNA expression of this autoreceptor was performed in raphe nuclei. Similarly, because of the role of hippocampal 5-HT neurotransmission in anxiety-like disorders, expression of the 5-HT1A heteroreceptors was determined in this region. We found that the treatment with omeprazole reduces anxiety-like behavior in rats, increases the expression of 5-HT1A autoreceptor in the raphe and decreases the hippocampal expression of 5-HT1A heteroreceptor. This suggests a role of 5-HT1A receptor types in omeprazole-induced behavioral changes. It also indicates a potential role of omeprazole in the management of serotonergic disorders.
Assuntos
Ansiedade , Modelos Animais de Doenças , Hipocampo , Omeprazol , Receptor 5-HT1A de Serotonina , Estresse Psicológico , Animais , Receptor 5-HT1A de Serotonina/metabolismo , Receptor 5-HT1A de Serotonina/efeitos dos fármacos , Omeprazol/farmacologia , Masculino , Ratos , Ansiedade/tratamento farmacológico , Ansiedade/metabolismo , Estresse Psicológico/metabolismo , Estresse Psicológico/tratamento farmacológico , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Ratos Wistar , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Serotonina/metabolismo , Núcleos da Rafe/metabolismo , Núcleos da Rafe/efeitos dos fármacos , RNA Mensageiro/metabolismo , Restrição Física , ImobilizaçãoRESUMO
Messenger RNA (mRNA) therapy has been applied to the treatment of various human diseases including malignant tumors. Increasing evidences have shown that mRNA can enhance the efficacy of cancer immunotherapy by modulating the functions of immune cells and stimulating their activity. However, mRNA is a type of negatively charged biomacromolecules that are susceptible to serum nucleases and cannot readily cross the cell membrane. In the past few decades, various nanoparticles (NPs)-based delivery systems have been rationally designed and developed to facilitate the intracellular uptake and cytosolic delivery of mRNA. More importantly, by means of the specific recognition between the targeting ligands decorated on NP surface and receptors specifically expressed on immune cells, these mRNA delivery systems could be functionalized to target immune cells to further enhance the mRNA-based cancer immunotherapy. In this review, we briefly introduced the advancements of mRNA in cancer therapy, discussed the challenges faced by mRNA delivery, and systematically summarized the recent development in NPs-based mRNA delivery systems targeting various types of immune cells for cancer immunotherapy. The future development of NPs-mediated targeted mRNA delivery and their challenges in clinical translation are also discussed.