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1.
Sci Rep ; 13(1): 43, 2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36593298

RESUMO

Notch1 plays important roles in T cell development and is highly expressed in activated CD4+ T cells. However, the underlying mechanism of Notch1 transcription in T cells has not been fully characterized. Therefore, we aimed to determine how Notch1 expression is regulated during the activation of CD4+ T cells. Both the surface expression and mRNA transcription of Notch1 were significantly higher in activated CD4+ T cells, but the inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 or deletion of the Pdk1 gene impaired this upregulation of Notch1. Interrogation of the Notch1 promoter region using serially deleted Notch1 promoter reporters revealed that the - 300 to - 270 region is crucial for its transcription in activated T cells. In addition, we found that nuclear factor (NF)-κB subunits containing RelA bind directly to this promoter region, thereby upregulating transcription. In addition, inhibition of NF-κB by SN50 impaired upregulation of Notch1 surface protein and mRNA in activated CD4+ T cells. Thus, we provide evidence that Notch1 transcription in activated CD4+ T cells is upregulated via the PI3K-PDK1-NF-κB signaling pathway.


Assuntos
NF-kappa B , Fosfatidilinositol 3-Quinases , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Regulação da Expressão Gênica , Fator de Transcrição RelA/metabolismo , Linfócitos T/metabolismo , Ativação Transcricional , Receptor Notch1/genética , Receptor Notch1/metabolismo , RNA Mensageiro/metabolismo
2.
J Chin Med Assoc ; 86(1): 39-46, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36599141

RESUMO

BACKGROUND: Hyperglycemia-induced advanced glycation end products (AGEs) and receptor for AGEs (RAGEs) play major roles in diabetic nephropathy progression. In previous study, both glucagon-like peptide-1 (GLP-1) and peroxisome proliferator-activated receptors delta (PPARδ) agonists were shown to have anti-inflammatory effect on AGE-treated rat mesangial cells (RMCs). The interaction among PPARδ agonists, GLP-1, and AGE-RAGE axis is, however, still unclear. METHODS: In this study, the individual and synergic effect of PPARδ agonist (L-165 041) and siRNA of GLP-1 receptor (GLP-1R) on the expression of GLP-1, GLP-1R, RAGE, and cell viability in AGE-treated RMCs were investigated. RESULTS: L-165 041 enhanced GLP-1R mRNA and protein expression only in the presence of AGE. The expression of RAGE mRNA and protein was enhanced by AGE, attenuated by L-165 041, and siRNA of GLP-1R reversed L-165 041-induced inhibition. Cell viability was also inhibited by AGE. L-165 041 attenuated AGE-induced inhibition and siRNA GLP-1R diminished L-165 041 effect. CONCLUSION: PPARδ agonists increase GLP-1R expression on RMC in the presence of AGE. PPARδ agonists also attenuate AGE-induced upregulated RAGE expression and downregulated cell viability. The effect of PPARδ agonists needs the cooperation of GLP-1R activation.


Assuntos
Células Mesangiais , PPAR delta , Ratos , Animais , Células Mesangiais/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Produtos Finais de Glicação Avançada/farmacologia , Produtos Finais de Glicação Avançada/metabolismo , PPAR delta/agonistas , PPAR delta/metabolismo , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glucose/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , RNA Mensageiro
3.
Mol Med ; 29(1): 1, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36604626

RESUMO

BACKGROUND: Long intergenic non-coding RNA 00963 (LINC00963) is an oncogenic lncRNA in human cancers. However, little is known on how it impacts the pathogenesis of lung adenocarcinoma (LUAD). METHODS: Biological effects on proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) were examined by CCK-8, colony formation, EdU incorporation, transwell, and immunofluorescence assays, respectively. Macrophage polarization was evaluated by flow cytometry. Ubiquitination of Zeb1 was examined by co-immunoprecipitation. The location of LINC00963 in LUAD tissues and cell lines was tested by FISH assay. The LINC00963/HNRNPA2B1/Siah1 mRNA complex interaction was verified using RNA pull-down and immunoprecipitation assays. The exact roles of LINC00963 were further validated in metastasis and xenograft models. RESULTS: Higher LINC00963 expression in LUAD patients positively correlated with shorter overall survival, higher stages, and metastasis. LINC00963 mainly localized in the cytoplasm and aggravated malignant phenotypes of LUAD cells in vitro and metastasis in vivo. Mechanistically, LINC00963 directly interacted HNRNPA2B1 protein to trigger the degradation of Siah1 mRNA, which inhibited the ubiquitination and degradation of Zeb1. Moreover, exosomal LINC00963 derived from LUAD cells induced M2 macrophage polarization and promoted LUAD growth and metastasis. CONCLUSION: By stabilizing Zeb1 in cancer cells and delivering exosomes to induce M2 macrophage polarization, LINC00963 promoted the malignancy and metastasis of LUAD. Targeting LINC00963 may become a valuable therapeutic target for LUAD.


Assuntos
Adenocarcinoma , Exossomos , Neoplasias Pulmonares , MicroRNAs , RNA Longo não Codificante , Humanos , MicroRNAs/genética , Neoplasias Pulmonares/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Exossomos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Pulmão/patologia , Adenocarcinoma/genética , RNA Mensageiro , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo
4.
Hum Exp Toxicol ; 42: 9603271221146780, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36607234

RESUMO

OBJECTIVES: Kaempferol (KMF), has beneficial effects against hepatic lipid accumulation. In this study, we aimed to investigate molecular mechanism underlying the protective effect of KMF on lipid accumulation. METHODS: HepG2 cells were treated with different concentrations of KMF and 0.5 mM palmitate (PA) for 24  h. The mRNA and protein levels of genes involved in lipid metabolism were evaluated using real-time PCR and western blot. The expression of Nrf2 was silenced using siRNA. RESULTS: Data indicated that KMF (20 µM) reversed PA-induced increased triglyceride (TG) levels and total lipid content. These effects were accompanied by down-regulation of the mRNA and protein levels of lipogenic genes (FAS, ACC and SREBP1), and up-regulation of genes related to fatty acid oxidation (CPT-1, HADHα and PPARα). Kaempferol significantly decreased the levels of the oxidative stress markers (ROS and MDA) and enhanced the activities of antioxidant enzymes SOD and GPx in PA-challenged cells. Luciferase analysis showed that KMF increased the transactivation of Nrf2 in hepatocytes. The results also revealed that KMF-mediated activation of Nrf2 target genes was suppressed by Nrf2 siRNA. Furthermore, Nrf2 siRNA abolished the KMF-induced reduction in ROS and MDA levels in PA treated cells. In addition, the inhibitory effect of KMF on TG levels and the mRNA and protein levels of FAS, ACC and SREPB-1 were significantly abolished by Nrf2 inhibition. Nrf2 inhibition also suppressed the KMF-induced activation of genes involved in ß oxidation (CPT-1 and PPAR-α). CONCLUSION: The results suggest that KMF protects HepG2 cells from PA-induced lipid accumulation via activation of the Nrf2 signaling pathway.


Assuntos
Fator 2 Relacionado a NF-E2 , Hepatopatia Gordurosa não Alcoólica , Humanos , Células Hep G2 , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Palmitatos/toxicidade , Quempferóis/farmacologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Metabolismo dos Lipídeos , Estresse Oxidativo , Transdução de Sinais , PPAR alfa/metabolismo , RNA Mensageiro/metabolismo
5.
PLoS One ; 18(1): e0279908, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36607980

RESUMO

BACKGROUND: Gasdermin (GSDM) B is a member of the GSDM family, which is a protein that may be involved in the cell pyroptosis process and is associated with inflammatory diseases. OBJECTIVE: To explore the correlation between GSDMB and psoriasis vulgaris. METHODS: Skin lesions from 33 patients with psoriasis vulgaris and 69 normal controls were collected. ELISA and Western blot were adopted to detect proteins. The HaCaT cell line was transfected with 3 sets of interfering sequence siRNA, and the mRNA and protein levels before and after the transfection were measured by qPCR and Western blot respectively, so as to establish a cell model with low GSDMB gene expression; the MTT method was used to detect cells viability, flow cytometry to detect cell apoptosis. RESULTS: The level of GSDMB protein in the skin lesions of patients with psoriasis vulgaris was lower than that in normal skin tissues (P < 0.05). The mRNA and protein expression levels of the target gene in the siRNA-GSDMB-3 group were lower than those in the control group (P < 0.05). The proliferation of HaCaT cells was decreased by MTT method and flow cytometry, and the apoptosis rate was increased (P < 0.05). CONCLUSION: The expression level of GSDMB in psoriasis vulgaris lesion tissue is lower than that of normal skin tissue. The down-regulation of GSDMB expression can inhibit cell proliferation and promote cell apoptosis. GSDMB may play a role in the pathogenesis of psoriasis by affecting the differentiation of keratinocytes and the function of T cells.


Assuntos
Psoríase , Humanos , Psoríase/patologia , Pele/metabolismo , Queratinócitos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proliferação de Células , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo
6.
Mol Cell ; 83(1): 9-11, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36608672

RESUMO

Wang et al. (2022)1 employ real-time single-molecule fluorescence spectroscopy to monitor eukaryotic translation initiation events, revealing that, while mRNA engagement by ribosomal 43S subunits is slow, the subsequent mRNA scanning process is rapid- ∼10 times faster than translation.


Assuntos
Biossíntese de Proteínas , Ribossomos , Códon de Iniciação/genética , Ribossomos/genética , Ribossomos/metabolismo , RNA Mensageiro/metabolismo , Iniciação Traducional da Cadeia Peptídica
7.
BMC Endocr Disord ; 23(1): 7, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36609306

RESUMO

BACKGROUND: Animal model studies suggest that change in the members of the suppressor of the cytokine signaling (SOCS) family (mainly SOCS1 and SOCS3) is linked to the pathogenesis of obesity-related metabolic disorders. Moreover, epigenetic modification is involved in the transcriptional regulation of the SOCS gene family. Here, we aimed to evaluate the mRNA expression as well as gene promoter methylation of SOCS1 and SOCS3 in subcutaneous adipose tissue (SAT) from obese women compared to normal-weight subjects. We also intend to identify the possible association of SOCS1 and SOCS3 transcript levels with metabolic parameters in the context of obesity. METHODS: This study was conducted on women with obesity (n = 24) [body mass index (BMI) ≥ 30 kg/m 2] and women with normal-weight (n = 22) (BMI < 25 kg/m 2). Transcript levels of SOCS1 and SOCS3 were evaluated by real-time PCR in SAT from all participants. After bisulfite treatment of DNA, methylation-specific PCR was used to assess the putative methylation of 10 CpG sites in the promoter of SOCS1 and 13 CpG sites in SOCS3 in SAT from women with obesity and normal weight. RESULTS: It was found that unlike SOCS3, which disclosed an elevating expression pattern, the expression level of SOCS1 was lower in the women with obesity as compared with their non-obese counterparts (P-value = 0.03 for SOCS1 transcript level and P-value = 0.011 for SOCS3 transcript level). As for the analysis of promoter methylation, it was found that SOCS1 and SOCS3 methylation were not significantly different between the individuals with obesity and normal weight (P-value = 0.45 and P-value = 0.89). Correlation analysis indicated that the transcript level of SOCS1 mRNA expression had an inverse correlation with BMI, hs-CRP levels, HOMA-IR, and insulin levels. However, the SOCS3 transcript level showed a positive correlation with BMI, waist-to-height ratio, waist circumference, hip circumference, hs-CRP, HOMA-IR, insulin, fasting blood glucose, and total cholesterol. Interestingly, HOMA-IR is the predictor of the transcript level of SOCS1 (ß = - 0.448, P-value = 0.003) and SOCS3 (ß = 0.465, P-value = 0.002) in SAT of all participants. CONCLUSIONS: Our findings point to alterations of SOCS1 and SOCS3 transcript levels, but not promoter methylation levels in subcutaneous adipose tissues from women with obesity. Moreover, mRNA expression of SOCS1 and SOCS3 in SAT was associated with known obesity indices, insulin resistance, and hs-CRP, suggesting the contribution of SOCS1 and SOCS3 in the pathogenesis of obesity-related metabolic abnormalities. However, further studies are required to establish this concept.


Assuntos
Proteína C-Reativa , Metilação de DNA , Feminino , Humanos , Proteína C-Reativa/metabolismo , Obesidade/genética , Obesidade/metabolismo , Gordura Subcutânea , Insulina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo
8.
J Exp Clin Cancer Res ; 42(1): 10, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36609396

RESUMO

BACKGROUND: Posttranscriptional modification of tumor-associated factors plays a pivotal role in breast cancer progression. However, the underlying mechanism remains unknown. M6A modifications in cancer cells are dynamic and reversible and have been found to impact tumor initiation and progression through various mechanisms. In this study, we explored the regulatory mechanism of breast cancer cell proliferation and metabolism through m6A methylation in the Hippo pathway.  METHODS: A combination of MeRIP-seq, RNA-seq and metabolomics-seq was utilized to reveal a map of m6A modifications in breast cancer tissues and cells. We conducted RNA pull-down assays, RIP-qPCR, MeRIP-qPCR, and RNA stability analysis to identify the relationship between m6A proteins and LATS1 in m6A regulation in breast cancer cells. The expression and biological functions of m6A proteins were confirmed in breast cancer cells in vitro and in vivo. Furthermore, we investigated the phosphorylation levels and localization of YAP/TAZ to reveal that the activity of the Hippo pathway was affected by m6A regulation of LATS1 in breast cancer cells.  RESULTS: We demonstrated that m6A regulation plays an important role in proliferation and glycolytic metabolism in breast cancer through the Hippo pathway factor, LATS1. METTL3 was identified as the m6A writer, with YTHDF2 as the reader protein of LATS1 mRNA, which plays a positive role in promoting both tumorigenesis and glycolysis in breast cancer. High levels of m6A modification were induced by METTL3 in LATS1 mRNA. YTHDF2 identified m6A sites in LATS1 mRNA and reduced its stability. Knockout of the protein expression of METTL3 or YTHDF2 increased the expression of LATS1 mRNA and suppressed breast cancer tumorigenesis by activating YAP/TAZ in the Hippo pathway. CONCLUSIONS: In summary, we discovered that the METTL3-LATS1-YTHDF2 pathway plays an important role in the progression of breast cancer by activating YAP/TAZ in the Hippo pathway.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Metilação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transformação Celular Neoplásica/genética , Carcinogênese/genética , Fatores de Transcrição/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo
9.
J Exp Clin Cancer Res ; 42(1): 9, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36609449

RESUMO

BACKGROUND: N4-acetylcytidine (ac4C), a widespread modification in human mRNAs that is catalyzed by the N-acetyltransferase 10 (NAT10) enzyme, plays an important role in promoting mRNA stability and translation. However, the biological functions and regulatory mechanisms of NAT10-mediated ac4C were poorly defined. METHODS: ac4C mRNA modification status and NAT10 expression levels were analyzed in gastric cancer (GC) samples and compared with the corresponding normal tissues. The biological role of NAT10-mediated ac4C and its upstream and downstream regulatory mechanisms were determined in vitro and in vivo. The therapeutic potential of targeting NAT10 in GC was further explored. RESULTS: Here, we demonstrated that both ac4C mRNA modification and its acetyltransferase NAT10 were increased in GC, and increased NAT10 expression was associated with disease progression and poor patient prognosis. Functionally, we found that NAT10 promoted cellular G2/M phase progression, proliferation and tumorigenicity of GC in an ac4C-depedent manner. Mechanistic analyses demonstrated that NAT10 mediated ac4C acetylation of MDM2 transcript and subsequently stabilized MDM2 mRNA, leading to its upregulation and p53 downregulation and thereby facilitating gastric carcinogenesis. In addition, Helicobacter pylori (Hp) infection contributed to NAT10 induction, causing MDM2 overexpression and subsequent p53 degradation. Further investigations revealed that targeting NAT10 with Remodelin showed anti-cancer activity in GC and augmented the anti-tumor activity of MDM2 inhibitors in p53 wild-type GC. CONCLUSIONS: These results suggest the critical role of NAT10-mediated ac4C modification in GC oncogenesis and reveal a previously unrecognized signaling cascade involving the Hp-NAT10-MDM2-p53 axis during GC development.


Assuntos
Helicobacter pylori , Neoplasias Gástricas , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias Gástricas/genética , Helicobacter pylori/genética , Acetiltransferases/metabolismo , Acetilação , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Acetiltransferases N-Terminal/metabolismo
10.
Sci Rep ; 13(1): 332, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36609461

RESUMO

microRNAs (miRNAs) are small non-coding ribonucleic acids that post-transcriptionally regulate gene expression through the targeting of messenger RNA (mRNAs). Most miRNA target predictors have focused on animal species and prediction performance drops substantially when applied to plant species. Several rule-based miRNA target predictors have been developed in plant species, but they often fail to discover new miRNA targets with non-canonical miRNA-mRNA binding. Here, the recently published TarDB database of plant miRNA-mRNA data is leveraged to retrain the TarPmiR miRNA target predictor for application on plant species. Rigorous experiment design across four plant test species demonstrates that animal-trained predictors fail to sustain performance on plant species, and that the use of plant-specific training data improves accuracy depending on the quantity of plant training data used. Surprisingly, our results indicate that the complete exclusion of animal training data leads to the most accurate plant-specific miRNA target predictor indicating that animal-based data may detract from miRNA target prediction in plants. Our final plant-specific miRNA prediction method, dubbed P-TarPmiR, is freely available for use at http://ptarpmir.cu-bic.ca . The final P-TarPmiR method is used to predict targets for all miRNA within the soybean genome. Those ranked predictions, together with GO term enrichment, are shared with the research community.


Assuntos
MicroRNAs , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Biologia Computacional/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plantas/genética , Plantas/metabolismo , RNA de Plantas/genética
11.
Arch Virol ; 168(2): 39, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36609933

RESUMO

The disease caused by Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the major causes of death of individuals with acquired immunodeficiency syndrome (AIDS). Development of anti-KSHV drugs is thus crucial. In this study, we investigated the effect of parthenolide (PTL) on the proliferation and NF-κB signaling pathway of KSHV-infected cells. iSLK.219 and KSHV-infected SH-SY5Y cells (SK-RG) were treated with PTL, TaqMan real-time quantitative PCR was used to determine the number of copies of the KSHV genome, and mRNA and protein expression of KSHV genes were analyzed by real-time PCR and immunocytochemistry. A cell viability test was used to measure cell proliferation, and flow cytometry was used to examine the effect of the drug on the cell cycle. Cyclin D1, CDK6, CDK4, and NF-κB-related proteins, including IKKß, P-p65, and P-IKB-α, were detected by Western blot. The results showed that PTL altered the morphology of the cells, reduced the KSHV copy number, and suppressed the production of ORF50, K8.1, and v-GPCR mRNA and the LANA, ORF50, and K8.1 proteins. It blocked the G1 phase in iSLK.219 cells and decreased the levels of cyclin D1, CDK6, and CDK4 as well as the levels of NF-κB signaling proteins, including IKKß, P-p65, and P-IKB-α. Together, these results suggest that PTL is a candidate drug that can decrease KSHV pathogenicity by suppressing cell proliferation and inhibiting the NF-κB signaling pathway in KSHV-infected cells.


Assuntos
Herpesvirus Humano 8 , Neuroblastoma , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Sarcoma de Kaposi/tratamento farmacológico , Sarcoma de Kaposi/genética , Ciclina D1/metabolismo , Quinase I-kappa B/metabolismo , Transdução de Sinais , Proliferação de Células , RNA Mensageiro/metabolismo
12.
Commun Biol ; 6(1): 18, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36611093

RESUMO

Aerobic exercise is well known to promote neuroplasticity and hippocampal memory. In the developing brain, early-life exercise (ELE) can lead to persistent improvements in hippocampal function, yet molecular mechanisms underlying this phenomenon have not been fully explored. In this study, transgenic mice harboring the "NuTRAP" (Nuclear tagging and Translating Ribosome Affinity Purification) cassette in Emx1 expressing neurons ("Emx1-NuTRAP" mice) undergo ELE during adolescence. We then simultaneously isolate and sequence translating mRNA and nuclear chromatin from single hippocampal homogenates containing Emx1-expressing neurons. This approach allowed us to couple translatomic with epigenomic sequencing data to evaluate the influence of histone modifications H4K8ac and H3K27me3 on translating mRNA after ELE. A subset of ELE mice underwent a hippocampal learning task to determine the gene expression and epigenetic underpinnings of ELE's contribution to improved hippocampal memory performance. From this experiment, we discover gene expression - histone modification relationships that may play a critical role in facilitated memory after ELE. Our data reveal candidate gene-histone modification interactions and implicate gene regulatory pathways involved in ELE's impact on hippocampal memory.


Assuntos
Histonas , Consolidação da Memória , Camundongos , Animais , Histonas/genética , Histonas/metabolismo , Epigenoma , Hipocampo/metabolismo , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Expressão Gênica
13.
J Biomed Sci ; 30(1): 2, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36611161

RESUMO

BACKGROUND: Heat shock protein 27 (HSP27) is overexpressed during pulmonary fibrosis (PF) and exacerbates PF; however, the upregulation of HSP27 during PF and the therapeutic strategy of HSP27 inhibition is not well elucidated. METHODS: We have developed a mouse model simulating clinical stereotactic body radiotherapy (SBRT) with focal irradiation and validated the induction of RIPF. HSP25 (murine form of HSP27) transgenic (TG) and LLC1-derived orthotropic lung tumor models were also used. Lung tissues of patients with RIPF and idiopathic pulmonary fibrosis, and lung tissues from various fibrotic mouse models, as well as appropriated cell line systems were used. Public available gene expression datasets were used for therapeutic response rate analysis. A synthetic small molecule HSP27 inhibitor, J2 was also used. RESULTS: HSP27 expression with its phosphorylated form (pHSP27) increased during PF. Decreased mRNA expression of SMAD-specific E3 ubiquitin-protein ligase 2 (Smurf2), which is involved in ubiquitin degradation of HSP27, was responsible for the increased expression of pHSP27. In addition, increased expression of miRNA15b was identified with decreased expression of Smurf2 mRNA in PF models. Inverse correlation between pHSP27 and Smurf2 was observed in the lung tissues of PF animals, an irradiated orthotropic lung cancer models, and PF tissues from patients. Moreover, a HSP27 inhibitor cross-linked with HSP27 protein to ameliorate PF, which was more effective when targeting the epithelial to mesenchymal transition (EMT) stage of PF. CONCLUSIONS: Our findings identify upregulation mechanisms of HSP27 during PF and provide a therapeutic strategy for HSP27 inhibition for overcoming PF.


Assuntos
MicroRNAs , Fibrose Pulmonar , Camundongos , Animais , Fibrose Pulmonar/genética , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP27/farmacologia , Transição Epitelial-Mesenquimal , Ubiquitina-Proteína Ligases/genética , MicroRNAs/metabolismo , RNA Mensageiro
14.
Eur J Med Res ; 28(1): 11, 2023 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-36611198

RESUMO

BACKGROUND: We used microarrays to analyse the changes in long non-coding RNAs (lncRNAs) and mRNAs in aorta tissue in model rats with lipopolysaccharide-induced sepsis and determined the lncRNA-mRNA and lncRNA-miRNA-mRNA functional networks. METHODS: Wistar rats were intraperitoneally injected with lipopolysaccharide, and the lncRNA and mRNA expression profiles in the aorta were evaluated using microarrays. The functions of the differentially expressed mRNAs were analysed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. We then constructed coding/non-coding co-expression and competing endogenous RNA networks to study the mechanisms related to sepsis in rats. RESULTS: We identified 503 differentially expressed lncRNAs and 2479 differentially expressed mRNAs in the model rats with lipopolysaccharide-induced sepsis. Mitochondrial fission process 1 (MTFP1) was the most significantly down-regulated mRNA. Bioinformatics analysis showed that the significantly down-regulated mRNAs in the sepsis models were in pathways related to mitochondrial structure, function, and energy metabolism. Coding/non-coding co-expression and competing endogenous RNA analyses were conducted using 12 validated lncRNAs in combination with all mRNAs. The coding/non-coding co-expression analysis showed that the 12 validated lncRNAs were mainly regulatory factors for abnormal energy metabolism, including mitochondrial structure damage and aberrant mitochondrial dynamics. The competing endogenous RNA analysis revealed that the potential functions of these 12 lncRNAs might be related to the inflammatory response. CONCLUSION: We determined the differentially expressed lncRNAs and mRNAs in the aorta of septic rats using microarrays. Further studies on these lncRNAs will help elucidate the mechanism of sepsis at the genetic level and may identify potential therapeutic targets.


Assuntos
MicroRNAs , RNA Longo não Codificante , Sepse , Ratos , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Lipopolissacarídeos , Ratos Wistar , Sepse/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Redes Reguladoras de Genes , Perfilação da Expressão Gênica
15.
Cells ; 12(1)2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36611995

RESUMO

Therapy resistance is still a major reason for treatment failure in colorectal cancer (CRC). Previously, we identified the E3 ubiquitin ligase TRIM25 as a novel suppressor of caspase-2 translation which contributes to the apoptosis resistance of CRC cells towards chemotherapeutic drugs. Here, we report the executioner caspase-7 as being a further target of TRIM25. The results from the gain- and loss-of-function approaches and the actinomycin D experiments indicate that TRIM25 attenuates caspase-7 expression mainly through a decrease in mRNA stability. The data from the RNA pulldown assays with immunoprecipitated TRIM25 truncations indicate a direct TRIM25 binding to caspase-7 mRNA, which is mediated by the PRY/SPRY domain, which is also known to be highly relevant for protein-protein interactions. By employing TRIM25 immunoprecipitation, we identified the heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) as a novel TRIM25 binding protein with a functional impact on caspase-7 mRNA stability. Notably, the interaction of both proteins was highly sensitive to RNase A treatment and again depended on the PRY/SPRY domain, thus indicating an indirect interaction of both proteins which is achieved through a common RNA binding. Ubiquitin affinity chromatography showed that both proteins are targets of ubiquitin modification. Functionally, the ectopic expression of caspase-7 in CRC cells caused an increase in poly ADP-ribose polymerase (PARP) cleavage concomitant with a significant increase in apoptosis. Collectively, the negative regulation of caspase-7 by TRIM25, which is possibly executed by hnRNPH1, implies a novel survival mechanism underlying the chemotherapeutic drug resistance of CRC cells. The targeting of TRIM25 could therefore offer a promising strategy for the reduction in therapy resistance in CRC patients.


Assuntos
Carcinoma , Neoplasias do Colo , Humanos , RNA Mensageiro/genética , Caspase 7 , Ubiquitina-Proteína Ligases/metabolismo , RNA , Neoplasias do Colo/genética , Linhagem Celular Tumoral , Ubiquitina , Apoptose/genética , Proteínas com Motivo Tripartido/genética , Fatores de Transcrição/genética
16.
J Exp Med ; 220(3)2023 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-36598533

RESUMO

Regulatory T cells (Tregs) suppress the activation and subsequent effector functions of CD4 effector T cells (Teffs). However, molecular mechanisms that enforce Treg-mediated suppression in CD4 Teff are unclear. We found that Tregs suppressed activation-induced global protein synthesis in CD4 Teffs prior to cell division. We analyzed genome-wide changes in the transcriptome and translatome of activated CD4 Teffs. We show that mRNAs encoding for the protein synthesis machinery are regulated at the level of translation in activated CD4 Teffs by Tregs. Tregs suppressed global protein synthesis of CD4 Teffs by specifically inhibiting mRNAs of the translation machinery at the level of mTORC1-mediated translation control through concerted action of immunosuppressive cytokines IL-10 and TGFß. Lastly, we found that the therapeutic targeting of protein synthesis with the RNA helicase eIF4A inhibitor rocaglamide A can alleviate inflammatory CD4 Teff activation caused by acute Treg depletion in vivo. These data show that peripheral tolerance is enforced by Tregs through mRNA translational control in CD4 Teffs.


Assuntos
Linfócitos T CD4-Positivos , Linfócitos T Reguladores , Ativação Linfocitária , Citocinas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
17.
Cell ; 186(1): 80-97.e26, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36608661

RESUMO

Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.


Assuntos
RNA Helicases DEAD-box , Glucose , Queratinócitos , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Glucose/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Humanos
18.
Allergol Immunopathol (Madr) ; 51(1): 54-62, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36617822

RESUMO

Acute lung injury causes severe inflammation and oxidative stress in lung tissues. In this study, we analyzed the potential regulatory role of nuclear factor erythroid-2-related factor 2 (Nrf2) on NADPH oxidase 1 (NOX1) in tumor necrosis factor-α (TNF-α)-induced inflammation and oxidative stress in human type II alveolar epithelial cells. In this study, A549 cells were transfected with Nrf2 siRNA and overexpression vectors for 6 h before being induced by TNF-α for 24 h. TNF-α upregulated the expression of NOX1 and Nrf2 in A549 cells. Furthermore, overexpression of Nrf2 could reduce TNF-α-induced NF-κB mRNA and protein expression after transfection with the Nrf2 siRNA vector, and the levels of IL-6, IL-8, ROS, and malondialdehyde (MDA) in TNF-α-induced A549 cells increased, while the level of total antioxidation capability (T-AOC) decreased. On the other hand, the overexpression of Nrf2 decreased the levels of IL-6, IL-8, ROS, and MDA, while increasing T-AOC. The mRNA and protein levels of NOX1 were dramatically increased by TNF-α, while those changes were notably suppressed by Nrf2 overexpression. Further studies demonstrated that Nrf2 suppressed NOX1 transcription by binding to the -1199 to -1189 bp (ATTACACAGCA) region of the NOX1 promoter in TNF-α-stimulated A549 cells. Our study suggests that Nrf2 may bind to and regulate NOX1 expression to antagonize TNF-α-induced inflammatory reaction and oxidative stress in A549 cells.


Assuntos
NADPH Oxidase 1 , Fator 2 Relacionado a NF-E2 , Fator de Necrose Tumoral alfa , Humanos , Células A549 , Inflamação/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , NADPH Oxidase 1/genética , NADPH Oxidase 1/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , RNA Mensageiro , RNA Interferente Pequeno/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
19.
JCI Insight ; 8(1)2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36625348

RESUMO

BACKGROUND: To minimize COVID-19 pandemic burden and spread, 3-dose vaccination campaigns commenced worldwide. Since patients who are pregnant are at increased risk for severe disease, they were recently included in that policy, despite the lack of available evidence regarding the impact of a third boosting dose during pregnancy, underscoring the urgent need for relevant data. We aimed to characterize the effect of the third boosting dose of mRNA Pfizer BNT162b2 vaccine in pregnancy. METHODS: We performed a prospective cohort study of anti-SARS-CoV-2 antibody titers (n = 213) upon delivery in maternal and cord blood of naive fully vaccinated parturients who received a third dose (n = 86) as compared with 2-dose recipients (n = 127). RESULTS: We found a robust surge in maternal and cord blood levels of anti-SARS-CoV-2 titers at the time of delivery, when comparing pregnancies in which the mother received a third boosting dose with 2-dose recipients. The effect of the third boosting dose remained significant when controlling for the trimester of last exposure, suggesting additive immunity extends beyond that obtained after the second dose. Milder side effects were reported following the third dose, as compared with the second vaccine dose, among the fully vaccinated group. CONCLUSION: The third boosting dose of mRNA Pfizer BNT162b2 vaccine augmented maternal and neonatal immunity with mild side effects. These data provide evidence to bolster clinical and public health guidance, reassure patients, and increase vaccine uptake among patients who are pregnant. FUNDING: Israel Science Foundation KillCorona grant 3777/19; Research grant from the "Ofek" Program of the Hadassah Medical Center.


Assuntos
COVID-19 , SARS-CoV-2 , Recém-Nascido , Feminino , Gravidez , Humanos , COVID-19/prevenção & controle , Vacina BNT162 , Imunidade Humoral , Pandemias , Estudos Prospectivos , Mães , RNA Mensageiro
20.
Hum Exp Toxicol ; 42: 9603271231151376, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36625353

RESUMO

The widespread use of acetaminophen (APAP) in children as an over-the-counter treatment can cause acute liver failure through accidental overdose or ingestion. Therefore, the current research sought to investigate the function of hemin in mitigating the acute hepatotoxic effect of APAP in rat offspring. Thirty-two rats were assigned into four groups: control, hemin, APAP, and hemin/APAP groups. Liver enzymes were measured in serum along with oxidative stress indicators, tumor necrosis factor-α (TNF-α), interleukin-1beta (IL-1ß), total nitrites (NOx), and caspase 3 in liver. Immunoblotting of heme oxygenase-1 (HO-1), interleukin-6 (IL-6), Janus kinase 2 (Jak2), and signal transducer and activator of transcription 3 (STAT3) was carried out. The Bax/Bcl2 mRNA expression ratio was determined. A histological study and an immunohistochemical study of phosphorylated STAT3 were also done. Hemin reduced liver enzymes, MDA, TNF-α, NOx, caspase 3, IL-1ß, p-STAT3 expression, p-Jak2 expression, IL-6 expression, and Bax/Bcl2 mRNA expression ratio. In contrast, hemin increased GSH, TAC, and the expression of HO-1, improving the histopathological picture of liver tissue. Thus, hemin could ameliorate APAP-induced hepatic toxicity in rat offspring through anti-oxidant, anti-apoptotic, and anti-inflammatory actions with a possible role for the IL-6/HO-1/Jak2/STAT3 pathway.


Assuntos
Acetaminofen , Doença Hepática Induzida por Substâncias e Drogas , Ratos , Animais , Acetaminofen/toxicidade , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Animais Recém-Nascidos , Caspase 3/genética , Caspase 3/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Hemina/farmacologia , Proteína X Associada a bcl-2/metabolismo , Fígado , Transdução de Sinais , RNA Mensageiro , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Doença Hepática Induzida por Substâncias e Drogas/patologia
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