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1.
Stem Cell Res Ther ; 15(1): 208, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38992782

RESUMO

BACKGROUND: Mesenchymal stromal cells (MSCs) tropism for tumours allows their use as carriers of antitumoural factors and in vitro transcribed mRNA (IVT mRNA) is a promising tool for effective transient expression without insertional mutagenesis risk. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with antitumor properties by stimulating the specific immune response. The aim of this work was to generate modified MSCs by IVT mRNA transfection to overexpress GM-CSF and determine their therapeutic effect alone or in combination with doxorubicin (Dox) in a murine model of hepatocellular carcinoma (HCC). METHODS: DsRed or GM-CSF IVT mRNAs were generated from a cDNA template designed with specific primers followed by reverse transcription. Lipofectamine was used to transfect MSCs with DsRed (MSC/DsRed) or GM-CSF IVT mRNA (MSC/GM-CSF). Gene expression and cell surface markers were determined by flow cytometry. GM-CSF secretion was determined by ELISA. For in vitro experiments, the J774 macrophage line and bone marrow monocytes from mice were used to test GM-CSF function. An HCC model was developed by subcutaneous inoculation (s.c.) of Hepa129 cells into C3H/HeN mice. After s.c. injection of MSC/GM-CSF, Dox, or their combination, tumour size and mouse survival were evaluated. Tumour samples were collected for mRNA analysis and flow cytometry. RESULTS: DsRed expression by MSCs was observed from 2 h to 15 days after IVT mRNA transfection. Tumour growth remained unaltered after the administration of DsRed-expressing MSCs in a murine model of HCC and MSCs expressing GM-CSF maintained their phenotypic characteristic and migration capability. GM-CSF secreted by modified MSCs induced the differentiation of murine monocytes to dendritic cells and promoted a proinflammatory phenotype in the J774 macrophage cell line. In vivo, MSC/GM-CSF in combination with Dox strongly reduced HCC tumour growth in C3H/HeN mice and extended mouse survival in comparison with individual treatments. In addition, the tumours in the MSC/GM-CSF + Dox treated group exhibited elevated expression of proinflammatory genes and increased infiltration of CD8 + T cells and macrophages. CONCLUSIONS: Our results showed that IVT mRNA transfection is a suitable strategy for obtaining modified MSCs for therapeutic purposes. MSC/GM-CSF in combination with low doses of Dox led to a synergistic effect by increasing the proinflammatory tumour microenvironment, enhancing the antitumoural response in HCC.


Assuntos
Carcinoma Hepatocelular , Doxorrubicina , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Neoplasias Hepáticas , Células-Tronco Mesenquimais , RNA Mensageiro , Animais , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Linhagem Celular Tumoral , Transplante de Células-Tronco Mesenquimais/métodos , Humanos , Camundongos Endogâmicos C3H , Transfecção
2.
Elife ; 122024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-39010741

RESUMO

Multicellular organisms are composed of specialized cell types with distinct proteomes. While recent advances in single-cell transcriptome analyses have revealed differential expression of mRNAs, cellular diversity in translational profiles remains underinvestigated. By performing RNA-seq and Ribo-seq in genetically defined cells in the Drosophila brain, we here revealed substantial post-transcriptional regulations that augment the cell-type distinctions at the level of protein expression. Specifically, we found that translational efficiency of proteins fundamental to neuronal functions, such as ion channels and neurotransmitter receptors, was maintained low in glia, leading to their preferential translation in neurons. Notably, distribution of ribosome footprints on these mRNAs exhibited a remarkable bias toward the 5' leaders in glia. Using transgenic reporter strains, we provide evidence that the small upstream open-reading frames in the 5' leader confer selective translational suppression in glia. Overall, these findings underscore the profound impact of translational regulation in shaping the proteomics for cell-type distinction and provide new insights into the molecular mechanisms driving cell-type diversity.


Assuntos
Neuroglia , Biossíntese de Proteínas , Animais , Neuroglia/metabolismo , Neurônios/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Regulação da Expressão Gênica , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Encéfalo/metabolismo , Encéfalo/citologia , Ribossomos/metabolismo , Drosophila/genética
3.
Front Immunol ; 15: 1344995, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39011034

RESUMO

Background: Allergic rhinitis (AR) is a complex disease in which gene-environment interactions contribute to its pathogenesis. Epigenetic modifications, such as N6-methyladenosine (m6A) modification of mRNA, play important roles in regulating gene expression in multiple physiological and pathological processes. However, the function of m6A modification in AR and the inflammatory response is poorly understood. Methods: We used the ovalbumin (OVA) and aluminum hydroxide to induce an AR mouse model. Nasal symptoms, histopathology, and serum cytokines were examined. We performed combined m6A and RNA sequencing to analyze changes in m6A modification profiles. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and methylated RNA immunoprecipitation sequencing qPCR (MeRIP-qPCR) were used to verify differential methylation of mRNAs and the m6A methylation level. Knockdown or inhibition of Alkbh5 in nasal mucosa of mice was mediated by lentiviral infection or IOX1 treatment. Results: We showed that m6A was enriched in a group of genes involved in MAPK signaling pathway. Moreover, we identified a MAPK pathway involving Map3k8, Erk2, and Nfκb1 that may play a role in the disrupted inflammatory response associated with nasal inflammation. The m6A eraser, Alkbh5, was highly expressed in the nasal mucosa of AR model mice. Furthermore, knockdown of Alkbh5 expression by lentiviral infection resulted in high MAPK pathway activity and a significant nasal mucosa inflammatory response. Our findings indicate that ALKBH5-mediated m6A dysregulation likely contributes to a nasal inflammatory response via the MAPK pathway. Conclusion: Together, our data show that m6A dysregulation mediated by ALKBH5, is likely to contribute to inflammation of the nasal mucosa via the MAPK signaling pathway, suggesting that ALKBH5 is a potential biomarker for AR treatment.


Assuntos
Adenosina , Homólogo AlkB 5 da RNA Desmetilase , Modelos Animais de Doenças , Sistema de Sinalização das MAP Quinases , Mucosa Nasal , RNA Mensageiro , Rinite Alérgica , Animais , Mucosa Nasal/imunologia , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , Rinite Alérgica/imunologia , Rinite Alérgica/metabolismo , Rinite Alérgica/genética , Camundongos , Adenosina/análogos & derivados , Adenosina/metabolismo , Metilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Feminino , Camundongos Endogâmicos BALB C , Inflamação/genética , Inflamação/imunologia , Citocinas/metabolismo
4.
Life Sci Alliance ; 7(9)2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38986569

RESUMO

Maps of the RNA modification 5-methylcytosine (m5C) often diverge markedly not only because of differences in detection methods, data depand analysis pipelines but also biological factors. We re-analysed bisulfite RNA sequencing datasets from five human cell lines and seven tissues using a coherent m5C site calling pipeline. With the resulting union list of 6,393 m5C sites, we studied site distribution, enzymology, interaction with RNA-binding proteins and molecular function. We confirmed tRNA:m5C methyltransferases NSUN2 and NSUN6 as the main mRNA m5C "writers," but further showed that the rRNA:m5C methyltransferase NSUN5 can also modify mRNA. Each enzyme recognises mRNA features that strongly resemble their canonical substrates. By analysing proximity between mRNA m5C sites and footprints of RNA-binding proteins, we identified new candidates for functional interactions, including the RNA helicases DDX3X, involved in mRNA translation, and UPF1, an mRNA decay factor. We found that lack of NSUN2 in HeLa cells affected both steady-state levels of, and UPF1-binding to, target mRNAs. Our studies emphasise the emerging diversity of m5C writers and readers and their effect on mRNA function.


Assuntos
5-Metilcitosina , Metiltransferases , Biossíntese de Proteínas , RNA Mensageiro , Humanos , 5-Metilcitosina/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Células HeLa , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Especificidade por Substrato , Metilação , Estabilidade de RNA/genética , tRNA Metiltransferases
5.
Proc Natl Acad Sci U S A ; 121(29): e2403188121, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-38990950

RESUMO

The kinetoplastid parasite, Trypanosoma brucei, undergoes a complex life cycle entailing slender and stumpy bloodstream forms in mammals and procyclic and metacyclic forms (MFs) in tsetse fly hosts. The numerous gene regulatory events that underlie T. brucei differentiation between hosts, as well as between active and quiescent stages within each host, take place in the near absence of transcriptional control. Rather, differentiation is controlled by RNA-binding proteins (RBPs) that associate with mRNA 3' untranslated regions (3'UTRs) to impact RNA stability and translational efficiency. DRBD18 is a multifunctional T. brucei RBP, shown to impact mRNA stability, translation, export, and processing. Here, we use single-cell RNAseq to characterize transcriptomic changes in cell populations that arise upon DRBD18 depletion, as well as to visualize transcriptome-wide alterations to 3'UTR length. We show that in procyclic insect stages, DRBD18 represses expression of stumpy bloodstream form and MF transcripts. Additionally, DRBD18 regulates the 3'UTR lengths of over 1,500 transcripts, typically promoting the use of distal polyadenylation sites, and thus the inclusion of 3'UTR regulatory elements. Remarkably, comparison of polyadenylation patterns in DRBD18 knockdowns with polyadenylation patterns in stumpy bloodstream forms shows numerous similarities, revealing a role for poly(A) site selection in developmental gene regulation, and indicating that DRBD18 controls this process for a set of transcripts. RNA immunoprecipitation supports a direct role for DRBD18 in poly(A) site selection. This report highlights the importance of alternative polyadenylation in T. brucei developmental control and identifies a critical RBP in this process.


Assuntos
Regiões 3' não Traduzidas , Estágios do Ciclo de Vida , Proteínas de Protozoários , Proteínas de Ligação a RNA , Trypanosoma brucei brucei , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Estágios do Ciclo de Vida/genética , Regiões 3' não Traduzidas/genética , Animais , Transcriptoma , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Poli A/metabolismo , Poli A/genética , Poliadenilação
6.
Endocrinology ; 165(8)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38946397

RESUMO

Uterine leiomyoma or fibroids are prevalent noncancerous tumors of the uterine muscle layer, yet their origin and development remain poorly understood. We analyzed RNA expression profiles of 15 epigenetic mediators in uterine fibroids compared to myometrium using publicly available RNA sequencing (RNA-seq) data. To validate our findings, we performed RT-qPCR on a separate cohort of uterine fibroids targeting these modifiers confirming our RNA-seq data. We then examined protein profiles of key N6-methyladenosine (m6A) modifiers in fibroids and their matched myometrium, showing no significant differences in concordance with our RNA expression profiles. To determine RNA modification abundance, mRNA and small RNA from fibroids and matched myometrium were analyzed by ultra-high performance liquid chromatography-mass spectrometry identifying prevalent m6A and 11 other known modifiers. However, no aberrant expression in fibroids was detected. We then mined a previously published dataset and identified differential expression of m6A modifiers that were specific to fibroid genetic subtype. Our analysis also identified m6A consensus motifs on genes previously identified to be dysregulated in uterine fibroids. Overall, using state-of-the-art mass spectrometry, RNA expression, and protein profiles, we characterized and identified differentially expressed m6A modifiers in relation to driver mutations. Despite the use of several different approaches, we identified limited differential expression of RNA modifiers and associated modifications in uterine fibroids. However, considering the highly heterogenous genomic and cellular nature of fibroids, and the possible contribution of single molecule m6A modifications to fibroid pathology, there is a need for greater in-depth characterization of m6A marks and modifiers in a larger and diverse patient cohort.


Assuntos
Adenosina , Leiomioma , Neoplasias Uterinas , Leiomioma/genética , Leiomioma/metabolismo , Humanos , Feminino , Adenosina/análogos & derivados , Adenosina/metabolismo , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologia , Miométrio/metabolismo , Miométrio/patologia , Pessoa de Meia-Idade , Adulto , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Epigênese Genética
7.
Int J Mol Sci ; 25(13)2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-39000510

RESUMO

Poplar coma, the fluff-like appendages of seeds originating from the differentiated surface cells of the placenta and funicle, aids in the long-distance dispersal of seeds in the spring. However, it also poses hazards to human safety and causes pollution in the surrounding environment. Unraveling the regulatory mechanisms governing the initiation and development of coma is essential for addressing this issue comprehensively. In this study, strand-specific RNA-seq was conducted at three distinct stages of coma development, revealing 1888 lncRNAs and 52,810 mRNAs. The expression profiles of lncRNAs and mRNAs during coma development were analyzed. Subsequently, potential target genes of lncRNAs were predicted through co-localization and co-expression analyses. Integrating various types of sequencing data, lncRNA-miRNA-TF regulatory networks related to the initiation of coma were constructed. Utilizing identified differentially expressed genes encoding kinesin and actin, lncRNA-miRNA-mRNA regulatory networks associated with the construction and arrangement of the coma cytoskeleton were established. Additionally, relying on differentially expressed genes encoding cellulose synthase, sucrose synthase, and expansin, lncRNA-miRNA-mRNA regulatory networks related to coma cell wall synthesis and remodeling were developed. This study not only enhances the comprehension of lncRNA but also provides novel insights into the molecular mechanisms governing the initiation and development of poplar coma.


Assuntos
Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs , Populus , RNA Longo não Codificante , RNA Mensageiro , Populus/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , MicroRNAs/genética , Perfilação da Expressão Gênica/métodos , Sementes/genética , Sementes/crescimento & desenvolvimento
8.
Int J Mol Sci ; 25(13)2024 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-39000532

RESUMO

We hypothesized and investigated whether prenatal exposure to preeclampsia (PE) would simultaneously affect perinatal cardiovascular features and angiotensin system expressions. This prospective study was composed of mother-neonate dyads with (n = 49) and without maternal preeclampsia (n = 48) in a single tertiary medical center. The neonates exposed to PE had significantly larger relative sizes for the left and right coronary arteries and a higher cord plasma level of aminopeptidase-N, which positively correlated with the maternal diastolic blood pressures and determined the relative sizes of the left and right coronary arteries, whereas the encoding aminopeptidase-N (ANPEP) mRNA level in the PE cord blood leukocytes was significantly decreased, positively correlated with the neonatal systolic blood pressures (SBPs), and negatively correlated with the cord plasma-induced endothelial vascular cell adhesion molecule-1 mRNA levels. The PE cord plasma significantly induced higher endothelial mRNA levels of angiotensin II type 1 receptor (AT1R) and AT4R, whereas in the umbilical arteries, the protein expressions of AT2R and AT4R were significantly decreased in the PE group. The endothelial AT1R mRNA level positively determined the maternal SBPs, and the AT4R mRNA level positively determined the neonatal chamber size and cardiac output. In conclusion, PE may influence perinatal angiotensin system and cardiovascular manifestations of neonates across placentae. Intriguing correlations between these two warrant further mechanistic investigation.


Assuntos
Pré-Eclâmpsia , Humanos , Feminino , Gravidez , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/genética , Adulto , Recém-Nascido , Sangue Fetal/metabolismo , Pressão Sanguínea , Estudos Prospectivos , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Sistema Cardiovascular/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
9.
Ren Fail ; 46(2): 2371059, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38946402

RESUMO

BACKGROUND: Circular RNAs (circRNAs) have been shown to play critical roles in the initiation and progression of chronic glomerulonephritis (CGN), while their role from mesangial cells in contributing to the pathogenesis of CGN is rarely understood. Our study aims to explore the potential functions of mesangial cell-derived circRNAs using RNA sequencing (RNA-seq) and bioinformatics analysis. METHODS: Mouse mesangial cells (MMCs) were stimulated by lipopolysaccharide (LPS) to establish an in vitro model of CGN. Pro-inflammatory cytokines and cell cycle stages were detected by Enzyme-linked immunosorbent assay (ELISA) and Flow Cytometry experiment, respectively. Subsequently, differentially expressed circRNAs (DE-circRNAs) were identified by RNA-seq. GEO microarrays were used to identify differentially expressed mRNAs (DE-mRNAs) between CGN and healthy populations. Weighted co-expression network analysis (WGCNA) was utilized to explore clinically significant modules of CGN. CircRNA-associated CeRNA networks were constructed by bioinformatics analysis. The hub mRNAs from CeRNA network were identified using LASSO algorithms. Furthermore, utilizing protein-protein interaction (PPI), gene ontology (GO), pathway enrichment (KEGG), and GSEA analyses to explore the potential biological function of target genes from CeRNA network. In addition, we investigated the relationships between immune cells and hub mRNAs from CeRNA network using CIBERSORT. RESULTS: The expression of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α was drastically increased in LPS-induced MMCs. The number of cells decreased significantly in the G1 phase but increased significantly in the S/G2 phase. A total of 6 DE-mRNAs were determined by RNA-seq, including 4 up-regulated circRNAs and 2 down-regulated circRNAs. WGCNA analysis identified 1747 DE-mRNAs of the turquoise module from CGN people in the GEO database. Then, the CeRNA networks, including 6 circRNAs, 38 miRNAs, and 80 mRNAs, were successfully constructed. The results of GO and KEGG analyses revealed that the target mRNAs were mainly enriched in immune, infection, and inflammation-related pathways. Furthermore, three hub mRNAs (BOC, MLST8, and HMGCS2) from the CeRNA network were screened using LASSO algorithms. GSEA analysis revealed that hub mRNAs were implicated in a great deal of immune system responses and inflammatory pathways, including IL-5 production, MAPK signaling pathway, and JAK-STAT signaling pathway. Moreover, according to an evaluation of immune infiltration, hub mRNAs have statistical correlations with neutrophils, plasma cells, monocytes, and follicular helper T cells. CONCLUSIONS: Our findings provide fundamental and novel insights for further investigations into the role of mesangial cell-derived circRNAs in CGN pathogenesis.


Assuntos
Biologia Computacional , Glomerulonefrite , Células Mesangiais , RNA Circular , RNA Circular/genética , RNA Circular/metabolismo , Animais , Camundongos , Células Mesangiais/metabolismo , Glomerulonefrite/genética , Glomerulonefrite/metabolismo , Análise de Sequência de RNA , Redes Reguladoras de Genes , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Mapas de Interação de Proteínas/genética , Doença Crônica , Citocinas/metabolismo , Lipopolissacarídeos/farmacologia , Perfilação da Expressão Gênica , Modelos Animais de Doenças
10.
Oncoimmunology ; 13(1): 2373526, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38948931

RESUMO

Prostate cancer (PCa) is characterized as a "cold tumor" with limited immune responses, rendering the tumor resistant to immune checkpoint inhibitors (ICI). Therapeutic messenger RNA (mRNA) vaccines have emerged as a promising strategy to overcome this challenge by enhancing immune reactivity and significantly boosting anti-tumor efficacy. In our study, we synthesized Tetra, an mRNA vaccine mixed with multiple tumor-associated antigens, and ImmunER, an immune-enhancing adjuvant, aiming to induce potent anti-tumor immunity. ImmunER exhibited the capacity to promote dendritic cells (DCs) maturation, enhance DCs migration, and improve antigen presentation at both cellular and animal levels. Moreover, Tetra, in combination with ImmunER, induced a transformation of bone marrow-derived dendritic cells (BMDCs) to cDC1-CCL22 and up-regulated the JAK-STAT1 pathway, promoting the release of IL-12, TNF-α, and other cytokines. This cascade led to enhanced proliferation and activation of T cells, resulting in effective killing of tumor cells. In vivo experiments further revealed that Tetra + ImmunER increased CD8+T cell infiltration and activation in RM-1-PSMA tumor tissues. In summary, our findings underscore the promising potential of the integrated Tetra and ImmunER mRNA-LNP therapy for robust anti-tumor immunity in PCa.


Assuntos
Adjuvantes Imunológicos , Antígenos de Neoplasias , Vacinas Anticâncer , Células Dendríticas , Neoplasias da Próstata , RNA Mensageiro , Animais , Masculino , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/terapia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/tratamento farmacológico , Antígenos de Neoplasias/imunologia , Camundongos , Células Dendríticas/imunologia , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Humanos , Camundongos Endogâmicos C57BL , Linhagem Celular Tumoral , Vacinas de mRNA , Linfócitos T CD8-Positivos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Imunoterapia/métodos , Ativação Linfocitária/efeitos dos fármacos
11.
PLoS One ; 19(7): e0306323, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38976680

RESUMO

Left atrial strain (LAS) measured by two-dimensional speckle tracking echocardiography (2DSTE) is considered to be a marker of LA structural remodeling, but it remains unsettled. We investigated the potential usefulness and clinical relevance of LAS to detect atrial remodeling including fibrosis by analyzing gene expression in cardiovascular surgery patients. Preoperative 2DSTE was performed in 131 patients (92 patients with sinus rhythm [SR] patients including paroxysmal AF [PAF], 39 atrial fibrillation [AF]) undergoing cardiovascular surgery. Atrial samples were obtained from the left atrial appendages, and mRNA expression level was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) in 59 cases (24 PAF, 35 AF). Mean value of left atrial reservoir strain (mLASr) correlated with left atrial volume index (LAVI), and left atrial conduit strain (mLAScd). mLASr also correlated with left atrial contractile strain (mLASct) in SR patients including PAF. mLASr was significantly lower, and LAVI was higher, in the AF group, compared with SR patients including PAF. The expression of COL1A1 mRNA encoding collagen type I α1 significantly increased in AF patients (p = 0.031). mLASr negatively correlated with COL1A1 expression level, and multivariate regression analysis showed that mLASr was an independent predictor of atrial COL1A1 expression level, even after adjusting for age, sex, and BMI. But, neither mLAScd / mLASct nor LAVI (bp) correlated with COL1A1 gene expression. The expression level of COL1A1 mRNA strongly correlated with ECM-related genes (COL3A1, FN1). It also correlated ECM degradation-related genes (MMP2, TIMP1, and TIMP2), pro-fibrogenic cytokines (TGFB1 encoding TGFß1, END1, PDGFD, CTGF), oxidant stress-related genes (NOX2, NOX4), ACE, inflammation-related genes (NLRP, IL1B, MCP-1), and apoptosis (BAX). Among the fibrosis-related genes examined, univariable regression analysis showed that log (COL1A1) was associated with log (TGFB1) (adjusted R2 = 0.685, p<0.001), log (NOX4) (adjusted R2 = 0.622, p<0.001), log (NOX2) (adjusted R2 = 0.611, p<0.001), suggesting that TGFB1 and NOX4 was the potent independent determinants of COL1A1 expression level. mLASr negatively correlated with the ECM-related genes, and fibrosis-related gene expression level including TGFB1, NOX2, and NLRP3 in PAF patients. PAF patients with low mLASr had higher expression of the fibrosis-related gene expression, compared with those with high mLASr. These results suggest that LASr correlates with atrial COL1A1 gene expression associated with fibrosis-related gene expression. Patients with low LASr exhibit increased atrial fibrosis-related gene expression, even those with PAF, highlighting the utility of LAS as a marker for LA fibrosis in cardiovascular surgery patients.


Assuntos
Fibrilação Atrial , Remodelamento Atrial , Fibrose , Átrios do Coração , Humanos , Masculino , Feminino , Remodelamento Atrial/genética , Idoso , Pessoa de Meia-Idade , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Fibrilação Atrial/genética , Fibrilação Atrial/metabolismo , Fibrilação Atrial/cirurgia , Fibrilação Atrial/patologia , Fibrilação Atrial/fisiopatologia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Ecocardiografia , Cadeia alfa 1 do Colágeno Tipo I , Biomarcadores/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Função do Átrio Esquerdo
12.
Nat Commun ; 15(1): 5713, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38977661

RESUMO

Cellular senescence is characterized by a decrease in protein synthesis, although the underlying processes are mostly unclear. Chemical modifications to transfer RNAs (tRNAs) frequently influence tRNA activity, which is crucial for translation. We describe how tRNA N7-methylguanosine (m7G46) methylation, catalyzed by METTL1-WDR4, regulates translation and influences senescence phenotypes. Mettl1/Wdr4 and m7G gradually diminish with senescence and aging. A decrease in METTL1 causes a reduction in tRNAs, especially those with the m7G modification, via the rapid tRNA degradation (RTD) pathway. The decreases cause ribosomes to stall at certain codons, impeding the translation of mRNA that is essential in pathways such as Wnt signaling and ribosome biogenesis. Furthermore, chronic ribosome stalling stimulates the ribotoxic and integrative stress responses, which induce senescence-associated secretory phenotype. Moreover, restoring eEF1A protein mitigates senescence phenotypes caused by METTL1 deficiency by reducing RTD. Our findings demonstrate that tRNA m7G modification is essential for preventing premature senescence and aging by enabling efficient mRNA translation.


Assuntos
Senescência Celular , Guanosina , Metiltransferases , Biossíntese de Proteínas , RNA de Transferência , Senescência Celular/genética , RNA de Transferência/metabolismo , RNA de Transferência/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Guanosina/análogos & derivados , Guanosina/metabolismo , Metilação , Humanos , Ribossomos/metabolismo , Envelhecimento/metabolismo , Envelhecimento/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Animais , Fator 1 de Elongação de Peptídeos/metabolismo , Fator 1 de Elongação de Peptídeos/genética , Estabilidade de RNA
13.
Nat Commun ; 15(1): 5558, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38977672

RESUMO

Deletion of the maternal UBE3A allele causes Angelman syndrome (AS); because paternal UBE3A is epigenetically silenced by a long non-coding antisense (UBE3A-ATS) in neurons, this nearly eliminates UBE3A protein in the brain. Reactivating paternal UBE3A holds promise for treating AS. We previously showed topoisomerase inhibitors can reactivate paternal UBE3A, but their therapeutic challenges prompted our search for small molecule unsilencers with a different mechanism of action. Here, we found that (S)-PHA533533 acts through a novel mechanism to significantly increase paternal Ube3a mRNA and UBE3A protein levels while downregulating Ube3a-ATS in primary neurons derived from AS model mice. Furthermore, peripheral delivery of (S)-PHA533533 in AS model mice induces widespread neuronal UBE3A expression. Finally, we show that (S)-PHA533533 unsilences paternal UBE3A in AS patient-derived neurons, highlighting its translational potential. Our findings provide a lead for developing a small molecule treatment for AS that could be safe, non-invasively delivered, and capable of brain-wide unsilencing of paternal UBE3A.


Assuntos
Síndrome de Angelman , Modelos Animais de Doenças , Neurônios , Ubiquitina-Proteína Ligases , Síndrome de Angelman/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Camundongos , Neurônios/metabolismo , Humanos , Masculino , Feminino , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Encéfalo/metabolismo
14.
BMC Genom Data ; 25(Suppl 1): 67, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38978021

RESUMO

BACKGROUND: The competitive endogenous RNA (ceRNA) hypothesis suggests that microRNAs (miRNAs) mediate a regulatory relation between long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) which share similar miRNA response elements (MREs) to bind to the same miRNA. Since the ceRNA hypothesis was proposed, several studies have been conducted to construct a network of lncRNAs, miRNAs and mRNAs in cancer. However, most cancer-related ceRNA networks are intended for representing a general relation of RNAs in cancer rather than for a patient-specific relation. Due to the heterogeneous nature of cancer, lncRNA-miRNA-mRNA interactions can vary in different patients. RESULTS: We have developed a new method for constructing a ceRNA network of lncRNAs, miRNAs and mRNAs, which is specific to an individual cancer patient and for finding prognostic biomarkers consisting of lncRNA-miRNA-mRNA triplets. We tested our method on extensive data sets of three types of cancer (breast cancer, liver cancer, and lung cancer) and obtained potential prognostic lncRNA-miRNA-mRNA triplets for each type of cancer. CONCLUSIONS: Analysis of expression patterns of the RNAs involved in the triplets and survival rates of cancer patients revealed several interesting findings. First, even for the same cancer type, prognostic lncRNA-miRNA-mRNA triplets can be different depending on whether lncRNA and mRNA show opposite or similar expression patterns. Second, prognostic lncRNA-miRNA-mRNA triplets are often more predictive of survival rates than RNA pairs or individual RNAs. Our approach will be useful for constructing patient-specific lncRNA-miRNA-mRNA networks and for finding prognostic biomarkers from the networks.


Assuntos
Biomarcadores Tumorais , Redes Reguladoras de Genes , MicroRNAs , Neoplasias , RNA Longo não Codificante , RNA Mensageiro , Humanos , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , MicroRNAs/genética , Biomarcadores Tumorais/genética , Prognóstico , Neoplasias/genética , Neoplasias/mortalidade , Redes Reguladoras de Genes/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Regulação Neoplásica da Expressão Gênica/genética , Feminino
15.
Int J Biol Sci ; 20(9): 3675-3690, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38993567

RESUMO

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly tumors; however, its pathogenic mechanism remains largely elusive. In-depth researches are needed to reveal the expression regulatory mechanisms and functions of the RNA-binding protein RALY in HCC. Here, we identify RALY as a highly expressed oncogenic factor that affects HCC cells proliferation both in vitro and in vivo. O-GlcNAcylation of RALY at Ser176 enhances its stability by protecting RALY from TRIM27-mediated ubiquitination, thus maintaining hyper-expression of the RALY protein. Mechanistically, RALY interacts with USP22 messenger RNA, as revealed by RNA immunoprecipitation, to increase their cytoplasmic localization and protein expression, thereby promoting the proliferation of HCC cells. Furthermore, we develop a novel RALY protein degrader based on peptide proteolysis-targeting chimeras, named RALY-PROTAC, which we chemically synthesize by linking a RALY-targeting peptide with the E3 ubiquitin ligase recruitment ligand pomalidomide. In conclusion, our findings demonstrate a novel mechanism by which O-GlcNAcylation/RALY/USP22 mRNA axis aggravates HCC cells proliferation. RALY-PROTACs as degraders of the RALY protein exhibit potential as therapeutic drugs for RALY-overexpressing HCC.


Assuntos
Carcinoma Hepatocelular , Proliferação de Células , Neoplasias Hepáticas , Ubiquitina Tiolesterase , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Linhagem Celular Tumoral , Animais , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Camundongos , Camundongos Nus , Ubiquitinação , Transporte Ativo do Núcleo Celular
16.
Nat Commun ; 15(1): 5868, 2024 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-38997252

RESUMO

The 3' end of a gene, often called a terminator, modulates mRNA stability, localization, translation, and polyadenylation. Here, we adapted Plant STARR-seq, a massively parallel reporter assay, to measure the activity of over 50,000 terminators from the plants Arabidopsis thaliana and Zea mays. We characterize thousands of plant terminators, including many that outperform bacterial terminators commonly used in plants. Terminator activity is species-specific, differing in tobacco leaf and maize protoplast assays. While recapitulating known biology, our results reveal the relative contributions of polyadenylation motifs to terminator strength. We built a computational model to predict terminator strength and used it to conduct in silico evolution that generated optimized synthetic terminators. Additionally, we discover alternative polyadenylation sites across tens of thousands of terminators; however, the strongest terminators tend to have a dominant cleavage site. Our results establish features of plant terminator function and identify strong naturally occurring and synthetic terminators.


Assuntos
Arabidopsis , Poliadenilação , Zea mays , Zea mays/genética , Zea mays/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Regiões Terminadoras Genéticas/genética , Nicotiana/genética , Nicotiana/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
17.
Mol Biol Rep ; 51(1): 797, 2024 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-39001947

RESUMO

BACKGROUND: Parkinson's disease (PD) is a common neurodegenerative disorder characterized by a multifaceted genetic foundation. Genome-Wide Association Studies (GWAS) have played a crucial role in pinpointing genetic variants linked to PD susceptibility. Current study aims to delve into the mechanistic aspects through which the PD-associated Single Nucleotide Polymorphism (SNP) rs329648, identified in prior GWAS, influences the pathogenesis of PD. METHODS AND RESULTS: Employing the CRISPR/Cas9-mediated genome editing mechanism, we demonstrated the association of the disease-associated allele of rs329648 with increased expression of miR-4697-3p in differentiated SH-SY5Y cells. We revealed that miR-4697-3p contributes to the formation of high molecular weight complexes of α-Synuclein (α-Syn), indicative of α-Syn aggregate formation, as evidenced by Western blot analysis. Furthermore, our study unveiled that miR-4697-3p elevates SNCA112 mRNA levels. The resultant protein product, α-Syn 112, a variant of α-Syn with 112 amino acids, is recognized for augmenting α-Syn aggregation. Notably, this regulatory effect minimally impacts the levels of full-length SNCA140 mRNA, as evidenced by qRT-PCR. Additionally, we observed a correlation between the disease-associated allele and miR-4697-3p with increased cell death, substantiated by assessments including cell viability assays, alterations in cell morphology, and TUNEL assays. CONCLUSION: Our research reveals that the disease-associated allele of rs329648 is linked to higher levels of miR-4697-3p. This increase in miR-4697-3p leads to elevated SNCA112 mRNA levels, consequently promoting the formation of α-Syn aggregates. Furthermore, miR-4697-3p appears to play a role in increased cell death, potentially contributing to the pathogenesis of PD.


Assuntos
MicroRNAs , Doença de Parkinson , Polimorfismo de Nucleotídeo Único , RNA Mensageiro , alfa-Sinucleína , Humanos , Alelos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Linhagem Celular Tumoral , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , MicroRNAs/genética , MicroRNAs/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
18.
Neurobiol Aging ; 141: 160-170, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38964013

RESUMO

Women have a higher incidence of Alzheimer's disease (AD), even after adjusting for increased longevity. Thus, there is an urgent need to identify genes that underpin sex-associated risk of AD. PIN1 is a key regulator of the tau phosphorylation signaling pathway; however, potential differences in PIN1 expression, in males and females, are still unknown. We analyzed brain transcriptomic datasets focusing on sex differences in PIN1 mRNA levels in an aging and AD cohort, which revealed reduced PIN1 levels primarily within females. We validated this observation in an independent dataset (ROS/MAP), which also revealed that PIN1 is negatively correlated with multiregional neurofibrillary tangle density and global cognitive function in females only. Additional analysis revealed a decrease in PIN1 in subjects with mild cognitive impairment (MCI) compared with aged individuals, again driven predominantly by female subjects. Histochemical analysis of PIN1 in AD and control male and female neocortex revealed an overall decrease in axonal PIN1 protein levels in females. These findings emphasize the importance of considering sex differences in AD research.


Assuntos
Doença de Alzheimer , Cognição , Disfunção Cognitiva , Peptidilprolil Isomerase de Interação com NIMA , Neocórtex , Emaranhados Neurofibrilares , Caracteres Sexuais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/genética , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Humanos , Feminino , Neocórtex/patologia , Neocórtex/metabolismo , Masculino , Disfunção Cognitiva/genética , Disfunção Cognitiva/patologia , Disfunção Cognitiva/metabolismo , Idoso , Idoso de 80 Anos ou mais , Emaranhados Neurofibrilares/patologia , Emaranhados Neurofibrilares/metabolismo , Fenótipo , Sistema Límbico/patologia , Sistema Límbico/metabolismo , Expressão Gênica , Envelhecimento/patologia , Envelhecimento/genética , Envelhecimento/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Proteínas tau/metabolismo , Proteínas tau/genética , Fosforilação
19.
Respir Res ; 25(1): 276, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-39010105

RESUMO

BACKGROUND: The pathogenesis of acute lung injury (ALI) involves a severe inflammatory response, leading to significant morbidity and mortality. N6-methylation of adenosine (m6A), an abundant mRNA nucleotide modification, plays a crucial role in regulating mRNA metabolism and function. However, the precise impact of m6A modifications on the progression of ALI remains elusive. METHODS: ALI models were induced by either intraperitoneal injection of lipopolysaccharide (LPS) into C57BL/6 mice or the LPS-treated alveolar type II epithelial cells (AECII) in vitro. The viability and proliferation of AECII were assessed using CCK-8 and EdU assays. The whole-body plethysmography was used to record the general respiratory functions. M6A RNA methylation level of AECII after LPS insults was detected, and then the "writer" of m6A modifications was screened. Afterwards, we successfully identified the targets that underwent m6A methylation mediated by METTL3, a methyltransferase-like enzyme. Last, we evaluated the regulatory role of METTL3-medited m6A methylation at phosphatase and tensin homolog (Pten) in ALI, by assessing the proliferation, viability and inflammation of AECII. RESULTS: LPS induced marked damages in respiratory functions and cellular injuries of AECII. The m6A modification level in mRNA and the expression of METTL3, an m6A methyltransferase, exhibited a notable rise in both lung tissues of ALI mice and cultured AECII cells subjected to LPS treatment. METTL3 knockdown or inhibition improved the viability and proliferation of LPS-treated AECII, and also reduced the m6A modification level. In addition, the stability and translation of Pten mRNA were enhanced by METTL3-mediated m6A modification, and over-expression of PTEN reversed the protective effect of METTL3 knockdown in the LPS-treated AECII. CONCLUSIONS: The progression of ALI can be attributed to the elevated levels of METTL3 in AECII, as it promotes the stability and translation of Pten mRNA through m6A modification. This suggests that targeting METTL3 could offer a novel approach for treating ALI.


Assuntos
Lesão Pulmonar Aguda , Células Epiteliais Alveolares , Proliferação de Células , Metiltransferases , Camundongos Endogâmicos C57BL , PTEN Fosfo-Hidrolase , RNA Mensageiro , Animais , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Camundongos , Proliferação de Células/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/patologia , Masculino , RNA Mensageiro/metabolismo , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Metilação , Adenosina/análogos & derivados , Adenosina/metabolismo , Lipopolissacarídeos/toxicidade , Estabilidade de RNA , Células Cultivadas
20.
Artigo em Inglês | MEDLINE | ID: mdl-38965928

RESUMO

mRNA therapeutics have shown great potential for a broad spectrum of disease treatment. However, the challenges of mRNA's inherent instability and difficulty in cellular entry have hindered its progress in the biomedical field. To address the cellular barriers and deliver mRNA to cells of interest, various delivery systems are designed. Among these, lipid nanoparticles (LNPs) stand out as the most extensively used mRNA delivery systems, particularly following the clinical approvals of corona virus disease 2019 (COVID-19) mRNA vaccines. LNPs are comprised of ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol derived lipids (PEG-lipids). In this review, we primarily summarize the recent advancements of the LNP mRNA delivery technology, focusing on the structures of four lipid constituents and their biomedical applications. We delve into structure-activity relationships of the lipids, while also exploring the future prospects and challenges in developing more efficacious mRNA delivery systems. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology-Inspired Nanomaterials > Lipid-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.


Assuntos
Lipídeos , Nanopartículas , RNA Mensageiro , Humanos , Nanopartículas/química , RNA Mensageiro/metabolismo , Lipídeos/química , Animais , SARS-CoV-2 , COVID-19 , Sistemas de Liberação de Medicamentos , Vacinas contra COVID-19/química , Lipossomos
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