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1.
Nat Commun ; 12(1): 3460, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103491

RESUMO

Lack or dysfunction of the lymphatics leads to secondary lymphedema formation that seriously reduces the function of the affected organs and results in degradation of quality of life. Currently, there is no definitive treatment option for lymphedema. Here, we utilized nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) encoding murine Vascular Endothelial Growth Factor C (VEGFC) to stimulate lymphatic growth and function and reduce experimental lymphedema in mouse models. We demonstrated that administration of a single low-dose of VEGFC mRNA-LNPs induced durable, organ-specific lymphatic growth and formation of a functional lymphatic network. Importantly, VEGFC mRNA-LNP treatment reversed experimental lymphedema by restoring lymphatic function without inducing any obvious adverse events. Collectively, we present a novel application of the nucleoside-modified mRNA-LNP platform, describe a model for identifying the organ-specific physiological and pathophysiological roles of the lymphatics, and propose an efficient and safe treatment option that may serve as a novel therapeutic tool to reduce lymphedema.


Assuntos
Linfangiogênese/genética , Vasos Linfáticos/patologia , Linfedema/patologia , Nucleosídeos/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Animais , Vasos Sanguíneos/patologia , Proliferação de Células/efeitos dos fármacos , Toxina Diftérica/farmacologia , Modelos Animais de Doenças , Células HEK293 , Humanos , Imunidade/efeitos dos fármacos , Injeções Intradérmicas , Lipídeos/administração & dosagem , Lipídeos/química , Vasos Linfáticos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , Nanopartículas/química , Especificidade de Órgãos , Poli C/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tamoxifeno/farmacologia , Fator C de Crescimento do Endotélio Vascular/administração & dosagem , Fator C de Crescimento do Endotélio Vascular/metabolismo
2.
Nat Commun ; 12(1): 3426, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103516

RESUMO

Adaptive plasticity in stress responses is a key element of plant survival strategies. For instance, moderate heat stress (HS) primes a plant to acquire thermotolerance, which allows subsequent survival of more severe HS conditions. Acquired thermotolerance is actively maintained over several days (HS memory) and involves the sustained induction of memory-related genes. Here we show that FORGETTER3/ HEAT SHOCK TRANSCRIPTION FACTOR A3 (FGT3/HSFA3) is specifically required for physiological HS memory and maintaining high memory-gene expression during the days following a HS exposure. HSFA3 mediates HS memory by direct transcriptional activation of memory-related genes after return to normal growth temperatures. HSFA3 binds HSFA2, and in vivo both proteins form heteromeric complexes with additional HSFs. Our results indicate that only complexes containing both HSFA2 and HSFA3 efficiently promote transcriptional memory by positively influencing histone H3 lysine 4 (H3K4) hyper-methylation. In summary, our work defines the major HSF complex controlling transcriptional memory and elucidates the in vivo dynamics of HSF complexes during somatic stress memory.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , Complexos Multiproteicos/metabolismo , Transcrição Genética , Proteínas de Arabidopsis/genética , Epistasia Genética , Genes de Plantas , Loci Gênicos , Fatores de Transcrição de Choque Térmico/genética , Histonas/metabolismo , Cinética , Lisina/metabolismo , Metilação , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
3.
BMC Bioinformatics ; 22(Suppl 10): 271, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34058988

RESUMO

BACKGROUND: Translational regulation is one important aspect of gene expression regulation. Dysregulation of translation results in abnormal cell physiology and leads to diseases. Ribosome profiling (RP), also called ribo-seq, is a powerful experimental technique to study translational regulation. It can capture a snapshot of translation by deep sequencing of ribosome-protected mRNA fragments. Many ribosome profiling data processing tools have been developed. However, almost all tools analyze ribosome profiling data at the gene level. Since different isoforms of a gene may produce different proteins with distinct biological functions, it is advantageous to analyze ribosome profiling data at the isoform level. To meet this need, previously we developed a pipeline to analyze 610 public human ribosome profiling data at the isoform level and constructed HRPDviewer database. RESULTS: To allow other researchers to use our pipeline as well, here we implement our pipeline as an easy-to-use software tool called RPiso. Compared to Ribomap (a widely used tool which provides isoform-level ribosome profiling analyses), our RPiso (1) estimates isoform abundance more accurately, (2) supports analyses on more species, and (3) provides a web-based viewer for interactively visualizing ribosome profiling data on the selected mRNA isoforms. CONCLUSIONS: In this study, we developed RPiso software tool ( http://cosbi7.ee.ncku.edu.tw/RPiso/ ) to provide isoform-level ribosome profiling analyses. RPiso is very easy to install and execute. RPiso also provides a web-based viewer for interactively visualizing ribosome profiling data on the selected mRNA isoforms. We believe that RPiso is a useful tool for researchers to analyze and visualize their own ribosome profiling data at the isoform level.


Assuntos
Biossíntese de Proteínas , Ribossomos , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Software
4.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068231

RESUMO

Stress granules (SGs) are membrane-less assemblies arising upon various stresses in eukaryotic cells. They sequester mRNAs and proteins from stressful conditions and modulate gene expression to enable cells to resume translation and growth after stress relief. SGs containing the translation initiation factor eIF3a/Rpg1 arise in yeast cells upon robust heat shock (HS) at 46 °C only. We demonstrate that the destabilization of Rpg1 within the PCI domain in the Rpg1-3 variant leads to SGs assembly already at moderate HS at 42 °C. These are bona fide SGs arising upon translation arrest containing mRNAs, which are components of the translation machinery, and associating with P-bodies. HS SGs associate with endoplasmatic reticulum and mitochondria and their contact sites ERMES. Although Rpg1-3-labeled SGs arise at a lower temperature, their disassembly is delayed after HS at 46 °C. Remarkably, the delayed disassembly of HS SGs after the robust HS is reversed by TDP-43, which is a human protein connected with amyotrophic lateral sclerosis. TDP-43 colocalizes with HS SGs in yeast cells and facilitates cell regrowth after the stress relief. Based on our results, we propose yeast HS SGs labeled by Rpg1 and its variants as a novel model system to study functions of TDP-43 in stress granules disassembly.


Assuntos
Grânulos Citoplasmáticos/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fator de Iniciação 3 em Eucariotos/química , Resposta ao Choque Térmico , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/genética , Retículo Endoplasmático/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Humanos , Mitocôndrias/metabolismo , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
5.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068434

RESUMO

(1) Background: Understanding the function of circular RNAs (circRNAs), a class of noncoding RNA, in psoriatic skin can provide important insights into the complex regulation of genes contributing to the pathogenesis of psoriasis. (2) Methods: A novel method was applied to RNA-seq datasets from 93 skin biopsy samples to comprehensively identify circRNAs of all types, i.e., canonical circRNAs from the intron-exon junctions of mRNAs and interior circRNAs (i-circRNAs) from the interior regions of exons, introns, and intergenic regions. Selected circRNAs were experimentally validated by qRT-PCR and Sanger sequencing. CircRNAs with abundant and differential expression were identified and their putative function as competing endogenous RNAs (ceRNAs) was analyzed by an integrated analysis of circRNAs, microRNAs, and mRNAs. (3) Results: With a comprehensive search using no information of splicing signals, we systematically identified 179 highly abundant circRNAs in psoriatic skin. Many of these were reported for the first time and many were differentially expressed in involved versus normal or uninvolved skin. Validation based on three additional RNA-seq datasets confirmed most of the identified circRNAs in psoriatic skin. Experimental analyses confirmed the expression of the well-known circRNA CDR1as, a canonical circRNA, and a novel i-circRNA in psoriasis. We also identified many circRNAs that may act as ceRNAs to regulate the expression of mRNA genes in psoriasis-related signaling pathways in psoriasis. (4) Conclusions: The result of the study suggested that circRNAs are abundant in psoriatic skin, have distinct characteristics, and contribute to psoriatic pathogenesis.


Assuntos
Regulação da Expressão Gênica , MicroRNAs/metabolismo , Psoríase/genética , RNA Circular/genética , RNA Mensageiro/metabolismo , Pele/metabolismo , Estudos de Casos e Controles , Humanos , MicroRNAs/genética , Psoríase/patologia , RNA Circular/metabolismo , RNA Mensageiro/genética , RNA-Seq
6.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069987

RESUMO

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the accumulation and translation of their target mRNAs through sequence complementarity. miRNAs have emerged as crucial regulators during maize somatic embryogenesis (SE) and plant regeneration. A monocot-specific miRNA, mainly accumulated during maize SE, is zma-miR528. While several targets have been described for this miRNA, the regulation has not been experimentally confirmed for the SE process. Here, we explored the accumulation of zma-miR528 and several predicted targets during embryogenic callus induction, proliferation, and plantlet regeneration using the maize cultivar VS-535. We confirmed the cleavage site for all tested zma-miR528 targets; however, PLC1 showed very low levels of processing. The abundance of zma-miR528 slightly decreased in one month-induced callus compared to the immature embryo (IE) explant tissue. However, it displayed a significant increase in four-month sub-cultured callus, coincident with proliferation establishment. In callus-regenerated plantlets, zma-miR528 greatly decreased to levels below those observed in the initial explant. Three of the target transcripts (MATE, bHLH, and SOD1a) showed an inverse correlation with the miRNA abundance in total RNA samples at all stages. Using polysome fractionation, zma-miR528 was detected in the polysome fraction and exhibited an inverse distribution with the PLC1 target, which was not observed at total RNA. Accordingly, we conclude that zma-miR528 regulates multiple target mRNAs during the SE process by promoting their degradation, translation inhibition or both.


Assuntos
Zea mays/embriologia , Zea mays/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Desenvolvimento Vegetal/genética , Polirribossomos/genética , Polirribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Regeneração/genética , Zea mays/metabolismo
7.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070066

RESUMO

Megalin has been proposed as an endocytic receptor for aminoglycosides as well as estrogen and androgen. We aimed to investigate the otoprotective effects of antiandrogens (flutamide, FM) on kanamycin (KM)-induced hearing loss in rats. Rats were divided into four groups. The KM group was administered KM (20 mg/kg/day) for 5 days, while the FM group received FM (15 mg/kg/day) for 10 days. In the KM + FM group, KM and FM (15 mg/kg/day) were simultaneously injected for 5 days and then FM was injected for 5 days. Auditory brainstem responses were measured. Western blotting and/or quantitative reverse transcriptase-polymerase chain reaction were performed for megalin, cytochrome P450 1A1 (Cyp1a1), Cyp1b1, metallothionein 1A (MT1A), MT2A, tumor necrosis factor (TNF)-α, caspase 3, and cleaved caspase 3. The FM + KM group showed attenuated auditory thresholds when compared with the KM group at 4, 8, 16, and 32 kHz (all p < 0.05). The KM + FM group showed lower megalin and Cyp1b1 levels than the KM group (all p < 0.05). The KM + FM group revealed lower MT1A, TNFα, and caspase 3 protein levels, compared with those in the KM group (all p < 0.05). Androgen receptor inhibition protects against cochlear injuries in KM-induced hearing loss rats by attenuating megalin expression, revealing anti-inflammatory and anti-apoptotic effects.


Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Perda Auditiva Neurossensorial/prevenção & controle , Animais , Antibacterianos/toxicidade , Limiar Auditivo/efeitos dos fármacos , Cóclea/efeitos dos fármacos , Cóclea/patologia , Cóclea/fisiopatologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Flutamida/farmacologia , Expressão Gênica/efeitos dos fármacos , Perda Auditiva Neurossensorial/induzido quimicamente , Perda Auditiva Neurossensorial/fisiopatologia , Canamicina/toxicidade , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Metalotioneína/genética , Metalotioneína/metabolismo , Substâncias Protetoras/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo
8.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070162

RESUMO

During mRNA transcription, diverse RNA-binding proteins (RBPs) are recruited to RNA polymerase II (RNAP II) transcription machinery. These RBPs bind to distinct sites of nascent RNA to co-transcriptionally operate mRNA processing. Recent studies have revealed a close relationship between transcription and co-transcriptional RNA processing, where one affects the other's activity, indicating an essential role of protein-RNA interactions for the fine-tuning of mRNA production. Owing to their limited amount in cells, the detection of protein-RNA interactions specifically assembled on the transcribing RNAP II machinery still remains challenging. Currently, cross-linking and immunoprecipitation (CLIP) has become a standard method to detect in vivo protein-RNA interactions, although it requires a large amount of input materials. Several improved methods, such as infrared-CLIP (irCLIP), enhanced CLIP (eCLIP), and target RNA immunoprecipitation (tRIP), have shown remarkable enhancements in the detection efficiency. Furthermore, the utilization of an RNA editing mechanism or proximity labeling strategy has achieved the detection of faint protein-RNA interactions in cells without depending on crosslinking. This review aims to explore various methods being developed to detect endogenous protein-RNA interaction sites and discusses how they may be applied to the analysis of co-transcriptional RNA processing.


Assuntos
Imunoprecipitação/métodos , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação , Perfilação da Expressão Gênica , Humanos , Imunoprecipitação/tendências , Ligação Proteica , RNA Polimerase II/metabolismo , Transcrição Genética , Transcriptoma
9.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070163

RESUMO

Preeclampsia is associated with an increased cardiovascular morbidity of mother and offspring, thus contributing to a substantial burden in women and children's health. It has been proven that endothelial progenitor cell (EPC) numbers and functional characteristics are impaired in cardiovascular disease and preeclampsia, although causative factors for the latter have remained elusive. MicroRNA (miRNA) modifications are a potential mechanism through which exposure to an altered environment translates into the development of chronic disease. In this study, we examined whether development of preeclampsia corresponds to alterations of miRNAs in maternal- and cord-blood-derived EPC. To test this end, we analyzed maternal and neonatal miRNAs via RNA sequencing from endothelial cells of preeclamptic and healthy controls in different cell culture passages. We were able to demonstrate differentially represented miRNAs in all groups. Hsa-miR-1270 showed significantly different levels in cord blood EPC from preeclampsia versus control and was negatively correlated with mRNA levels of its predicted targets ANGPTL7 and TFRC. Transfection with an hsa-miR-1270 inhibitor decreased the tube formation capacity and chemotactic motility but did not change proliferation in vitro. Target predictions and gene set enrichment analyses identified alternative splicing as a significantly enriched pathway for hsa-miR-1270. The top miRNAs in three other groups were predicted to target transcriptional and developmental pathways. Here, we showed for the first time significantly different levels of miRNAs and differently represented mRNA levels of predicted target genes in EPC derived from preeclampsia. Understanding the effects of preeclampsia on the epigenetic mechanisms of EPC will be crucial and may provide initial insights for further evaluation of the benefits of therapies targeting this cell population.


Assuntos
Células Progenitoras Endoteliais/metabolismo , MicroRNAs/genética , Pré-Eclâmpsia/genética , Adulto , Proteínas Semelhantes a Angiopoietina/genética , Antígenos CD/genética , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Quimiotaxia , Feminino , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Perfilação da Expressão Gênica , Humanos , Recém-Nascido , Masculino , MicroRNAs/sangue , MicroRNAs/metabolismo , Neovascularização Patológica/genética , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/metabolismo , Gravidez , RNA Mensageiro/sangue , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores da Transferrina/genética , Adulto Jovem
10.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070203

RESUMO

Alternative transcript cleavage and polyadenylation is linked to cancer cell transformation, proliferation and outcome. This has led researchers to develop methods to detect and bioinformatically analyse alternative polyadenylation as potential cancer biomarkers. If incorporated into standard prognostic measures such as gene expression and clinical parameters, these could advance cancer prognostic testing and possibly guide therapy. In this review, we focus on the existing methodologies, both experimental and computational, that have been applied to support the use of alternative polyadenylation as cancer biomarkers.


Assuntos
Regiões 3' não Traduzidas , Biomarcadores Tumorais/genética , Processamento Alternativo , Biomarcadores Tumorais/metabolismo , Biologia Computacional , Bases de Dados de Ácidos Nucleicos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/metabolismo , Poliadenilação , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA-Seq , Análise de Célula Única
11.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070217

RESUMO

Rhes is one of the most interesting genes regulated by thyroid hormones that, through the inhibition of the striatal cAMP/PKA pathway, acts as a modulator of dopamine neurotransmission. Rhes mRNA is expressed at high levels in the dorsal striatum, with a medial-to-lateral expression gradient reflecting that of both dopamine D2 and adenosine A2A receptors. Rhes transcript is also present in the hippocampus, cerebral cortex, olfactory tubercle and bulb, substantia nigra pars compacta (SNc) and ventral tegmental area of the rodent brain. In line with Rhes-dependent regulation of dopaminergic transmission, data showed that lack of Rhes enhanced cocaine- and amphetamine-induced motor stimulation in mice. Previous studies showed that pharmacological depletion of dopamine significantly reduces Rhes mRNA levels in rodents, non-human primates and Parkinson's disease (PD) patients, suggesting a link between dopaminergic innervation and physiological Rhes mRNA expression. Rhes protein binds to and activates striatal mTORC1, and modulates L-DOPA-induced dyskinesia in PD rodent models. Finally, Rhes is involved in the survival of mouse midbrain dopaminergic neurons of SNc, thus pointing towards a Rhes-dependent modulation of autophagy and mitophagy processes, and encouraging further investigations about mechanisms underlying dysfunctions of the nigrostriatal system.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Doença de Parkinson/metabolismo , Animais , Autofagia , Encéfalo/metabolismo , Encéfalo/patologia , Corpo Estriado/metabolismo , Corpo Estriado/patologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação ao GTP/deficiência , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Humanos , Levodopa/metabolismo , Camundongos , Camundongos Knockout , Mitofagia , Modelos Neurológicos , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Transmissão Sináptica
12.
Nat Commun ; 12(1): 3285, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078899

RESUMO

In peripheral nerves, Schwann cells form myelin and provide trophic support to axons. We previously showed that the mitochondrial protein prohibitin 2 can localize to the axon-Schwann-cell interface and is required for developmental myelination. Whether the homologous protein prohibitin 1 has a similar role, and whether prohibitins also play important roles in Schwann cell mitochondria is unknown. Here, we show that deletion of prohibitin 1 in Schwann cells minimally perturbs development, but later triggers a severe demyelinating peripheral neuropathy. Moreover, mitochondria are heavily affected by ablation of prohibitin 1 and demyelination occurs preferentially in cells with apparent mitochondrial loss. Furthermore, in response to mitochondrial damage, Schwann cells trigger the integrated stress response, but, contrary to what was previously suggested, this response is not detrimental in this context. These results identify a role for prohibitin 1 in myelin integrity and advance our understanding about the Schwann cell response to mitochondrial damage.


Assuntos
Nervo Femoral/metabolismo , Mitocôndrias/metabolismo , Proteínas Repressoras/genética , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Nervo Tibial/metabolismo , Animais , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/metabolismo , Axônios/metabolismo , Axônios/ultraestrutura , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Nervo Femoral/patologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/patologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/deficiência , Células de Schwann/patologia , Nervo Isquiático/patologia , Estresse Fisiológico , Nervo Tibial/patologia , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo , gama-Glutamilciclotransferase/genética , gama-Glutamilciclotransferase/metabolismo
13.
Nat Commun ; 12(1): 3308, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083519

RESUMO

The spatial partitioning of the transcriptome in the cell is an important form of gene-expression regulation. Here, we address how intron retention influences the spatio-temporal dynamics of transcripts from two clinically relevant genes: TERT (Telomerase Reverse Transcriptase) pre-mRNA and TUG1 (Taurine-Upregulated Gene 1) lncRNA. Single molecule RNA FISH reveals that nuclear TERT transcripts uniformly and robustly retain specific introns. Our data suggest that the splicing of TERT retained introns occurs during mitosis. In contrast, TUG1 has a bimodal distribution of fully spliced cytoplasmic and intron-retained nuclear transcripts. We further test the functionality of intron-retention events using RNA-targeting thiomorpholino antisense oligonucleotides to block intron excision. We show that intron retention is the driving force for the nuclear compartmentalization of these RNAs. For both RNAs, altering this splicing-driven subcellular distribution has significant effects on cell viability. Together, these findings show that stable retention of specific introns can orchestrate spatial compartmentalization of these RNAs within the cell. This process reveals that modulating RNA localization via targeted intron retention can be utilized for RNA-based therapies.


Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Telomerase/genética , Animais , Compartimento Celular , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Células HeLa , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Íntrons , Camundongos , Mitose , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Estabilidade de RNA , Especificidade da Espécie
14.
Nat Commun ; 12(1): 3318, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083536

RESUMO

Dormancy, a reversible quiescent cellular state characterized by greatly reduced metabolic activity, protects from genetic damage, prolongs survival and is crucial for tissue homeostasis and cellular response to injury or transplantation. Dormant cells have been characterized in many tissues, but their identification, isolation and characterization irrespective of tissue of origin remains elusive. Here, we develop a live cell ratiometric fluorescent Optical Stem Cell Activity Reporter (OSCAR) based on the observation that phosphorylation of RNA Polymerase II (RNApII), a hallmark of active mRNA transcription elongation, is largely absent in dormant stem cells from multiple lineages. Using the small intestinal crypt as a model, OSCAR reveals in real time the dynamics of dormancy induction and cellular differentiation in vitro, and allows the identification and isolation of several populations of transcriptionally diverse OSCARhigh and OSCARlow intestinal epithelial cell states in vivo. In particular, this reporter is able to identify a dormant OSCARhigh cell population in the small intestine. OSCAR therefore provides a tool for a better understanding of dormant stem cell biology.


Assuntos
RNA Polimerase II/metabolismo , Fase de Repouso do Ciclo Celular/fisiologia , Animais , Separação Celular , Quinase 9 Dependente de Ciclina/metabolismo , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Transcrição Genética
15.
Int J Nanomedicine ; 16: 3241-3254, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34007176

RESUMO

Purpose: Immune checkpoint inhibitors (ICIs) and sonodynamic therapy (SDT) are types of immunotherapy. In order to combine soluble programmed cell death protein 1 (sPD-1)-mediated immune checkpoint therapy and chlorin e6 (Ce6)-assisted SDT, nanobubbles (NBs) were generated to simultaneously load sPD-1 and Ce6. Materials and Methods: The sPD-1/Ce6-NBs, which were prepared by thin-film hydration and mechanical oscillation, had a stable physical condition, and delivered sPD-1 and Ce6 in a targeted manner. NBs could strengthen tumor suppression by increasing tumor-targeting accumulation of Ce6 and sPD-1, and by inducing ultrasound-targeted NB destruction. A mouse H22 cell hepatoma xenograft model was used to evaluate the synergetic immunotherapeutic effect and mechanism of sPD-1/Ce6-NBs. Results: By observing the tumor inhibition rate, tissue and cell apoptosis, apoptosis-related genes and protein expression, the best immunotherapeutic effect was exhibited by the sPD-1/Ce6-NBs group. The immunotherapeutic mechanism initially demonstrated that when tumor cells were transfected by sPD-1 delivered by NBs, which downregulated the expression of programmed death-ligand 1 (PD-L1) in tumor cells, and blocked the PD-1/PD-L1 signaling pathway, which improved T-cell-mediated tumor inhibition. Furthermore, ICIs combined with SDT induced immunogenic cell death by translocating calreticulin to the cell surface and then synergistically enhancing antitumor immune responses. Conclusion: In conclusion, sPD-1/Ce6-NBs were successfully designed. Ultrasound-mediated sPD-1/Ce6-NBs are potentially effective delivery systems for combination immunotherapy of hepatocellular carcinoma.


Assuntos
Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/terapia , Imunoterapia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Nanopartículas/química , Porfirinas/uso terapêutico , Receptor de Morte Celular Programada 1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos BALB C , Nanopartículas/ultraestrutura , Porfirinas/farmacocinética , Porfirinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Carga Tumoral
16.
Nat Commun ; 12(1): 3074, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031373

RESUMO

Single-cell RNA sequencing combined with spatial information on landmark genes enables reconstruction of spatially-resolved tissue cell atlases. However, such approaches are challenging for rare cell types, since their mRNA contents are diluted in the spatial transcriptomics bulk measurements used for landmark gene detection. In the small intestine, enterocytes, the most common cell type, exhibit zonated expression programs along the crypt-villus axis, but zonation patterns of rare cell types such as goblet and tuft cells remain uncharacterized. Here, we present ClumpSeq, an approach for sequencing small clumps of attached cells. By inferring the crypt-villus location of each clump from enterocyte landmark genes, we establish spatial atlases for all epithelial cell types in the small intestine. We identify elevated expression of immune-modulatory genes in villus tip goblet and tuft cells and heterogeneous migration patterns of enteroendocrine cells. ClumpSeq can be applied for reconstructing spatial atlases of rare cell types in other tissues and tumors.


Assuntos
Transporte Biológico/genética , Transporte Biológico/fisiologia , Biologia Computacional/métodos , Intestinos/fisiologia , Animais , Diferenciação Celular , Enterócitos/metabolismo , Células Enteroendócrinas/metabolismo , Células Epiteliais/metabolismo , Epitélio , Expressão Gênica , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Análise de Sequência de RNA
17.
Nat Commun ; 12(1): 2940, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011995

RESUMO

Resistance to endocrine treatment occurs in ~30% of ER+ breast cancer patients resulting in ~40,000 deaths/year in the USA. Preclinical studies strongly implicate activation of growth factor receptor, HER2 in endocrine treatment resistance. However, clinical trials of pan-HER inhibitors in ER+/HER2- patients have disappointed, likely due to a lack of predictive biomarkers. Here we demonstrate that loss of mismatch repair activates HER2 after endocrine treatment in ER+/HER2- breast cancer cells by protecting HER2 from protein trafficking. Additionally, HER2 activation is indispensable for endocrine treatment resistance in MutL- cells. Consequently, inhibiting HER2 restores sensitivity to endocrine treatment. Patient data from multiple clinical datasets supports an association between MutL loss, HER2 upregulation, and sensitivity to HER inhibitors in ER+/HER2- patients. These results provide strong rationale for MutL loss as a first-in-class predictive marker of sensitivity to combinatorial treatment with endocrine intervention and HER inhibitors in endocrine treatment-resistant ER+/HER2- breast cancer patients.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Reparo de Erro de Pareamento de DNA , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Camundongos SCID , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Proteínas MutL/genética , Proteínas MutL/metabolismo , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-2/genética , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Nat Commun ; 12(1): 2939, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011960

RESUMO

Elucidation of non-canonical protein functions can identify novel tissue homeostasis pathways. Herein, we describe a role for the Bcl-2 family member BAD in postnatal mammary gland morphogenesis. In Bad3SA knock-in mice, where BAD cannot undergo phosphorylation at 3 key serine residues, pubertal gland development is delayed due to aberrant tubulogenesis of the ductal epithelium. Proteomic and RPPA analyses identify that BAD regulates focal adhesions and the mRNA translation repressor, 4E-BP1. These results suggest that BAD modulates localized translation that drives focal adhesion maturation and cell motility. Consistent with this, cells within Bad3SA organoids contain unstable protrusions with decreased compartmentalized mRNA translation and focal adhesions, and exhibit reduced cell migration and tubulogenesis. Critically, protrusion stability is rescued by 4E-BP1 depletion. Together our results confirm an unexpected role of BAD in controlling localized translation and cell migration during mammary gland development.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Glândulas Mamárias Humanas/metabolismo , Proteína de Morte Celular Associada a bcl/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Movimento Celular/genética , Feminino , Técnicas de Introdução de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Morfogênese , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Fosforilação , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina/química , Proteína de Morte Celular Associada a bcl/deficiência , Proteína de Morte Celular Associada a bcl/genética
19.
Biomed Res Int ; 2021: 5541780, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33937393

RESUMO

Objective: This study is aimed at investigating the enriched functions of polymeric immunoglobulin receptor (PIGR) and its correlations with liver fibrosis stage. Methods: PIGR mRNA expression in normal liver, liver fibrosis, hepatic stellate cells (HSCs), and hepatitis virus infection samples was calculated in Gene Expression Omnibus (GEO) and Oncomine databases. Enrichment analysis of PIGR-related genes was conducted in Metascape and Gene Set Enrichment Analysis (GSEA). Logistic model and ROC curve were performed to evaluate the correlations between pIgR and liver fibrosis. Results: PIGR mRNA was upregulated in advanced liver fibrosis, cirrhosis compared to normal liver (all p < 0.05). PIGR mRNA was also overexpressed in activated HSCs compared to senescent HSCs, liver stem/progenitor cells, and reverted HSCs (all p < 0.05). Enrichment analysis revealed that PIGR-related genes involved in the defense response to virus and interferon (IFN) signaling. In GEO series, PIGR mRNA was also upregulated by hepatitis virus B, C, D, and E infection (all p < 0.05). After adjusting age and gender, multivariate logistic regression models revealed that high PIGR in the liver was a risk factor for liver fibrosis (OR = 82.2, p < 0.001). The area under curve (AUC), positive predictive value (PPV), negative predictive value (NPV), sensitivity, and specificity of PIGR for liver fibrosis stage >2 were 0.84, 0.86, 0.7, 0.61, and 0.90. Conclusion: PIGR was correlated with liver fibrosis and might involve in hepatitis virus infection and HSC transdifferentiation.


Assuntos
Biologia Computacional , Progressão da Doença , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Receptores de Imunoglobulina Polimérica/metabolismo , Regulação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatite B/patologia , Hepatite B/virologia , Vírus da Hepatite B/genética , Humanos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Modelos Logísticos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Imunoglobulina Polimérica/genética
20.
Biomed Res Int ; 2021: 6699910, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33937412

RESUMO

Cartilage injury of the knee joint is very common. Due to the limited self-healing ability of articular cartilage, osteoarthritis is very likely to occur if left untreated. Bone marrow mesenchymal stem cells (BMMSCs) are widely used in the study of cartilage injury due to their low immunity and good amplification ability, but they still have disadvantages, such as heterogeneous undifferentiated cells. MicroRNAs can regulate the chondrogenic differentiation ability of MSCs by inhibiting or promoting mRNA translation and degradation. In this research, we primarily investigated the effect of microRNA-210-3p (miR-210-3p) on chondrogenic and adipogenic differentiation of BMMSCs in vitro. Our results demonstrate that miR-210-3p promoted chondrogenic differentiation and inhibited adipogenic differentiation of rat BMMSCs, which was related to the HIF-3α signalling pathway. Additionally, miR-210-3p promotes mRNA and protein levels of the chondrogenic expression genes COLII and SOX9 and inhibits mRNA and protein levels of the adipogenic expression genes PPARγ and LPL. Thus, miR-210-3p combined with BMMSCs is a candidate for future clinical applications in cartilage regeneration and could represent a promising new therapeutic target for OA.


Assuntos
Adipogenia/genética , Condrogênese/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Masculino , MicroRNAs/genética , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Fatores de Transcrição/genética
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