RESUMEN
Immunotherapy has revolutionized cancer treatment, but inconsistent responses persist. Our study delves into the intriguing phenomenon of enhanced immunotherapy sensitivity in older individuals with cancers. Through a meta-analysis encompassing 25 small-to-mid-sized trials of immune checkpoint blockade (ICB), we demonstrate that older individuals exhibit heightened responsiveness to ICB therapy. To understand the underlying mechanism, we reanalyze single-cell RNA sequencing (scRNA-seq) data from multiple studies and unveil distinct upregulation of exhausted and cytotoxic T cell markers within the tumor microenvironment (TME) of older patients. Recognizing the potential role of gut microbiota in modulating the efficacy of immunotherapy, we identify an aging-enriched enterotype linked to improved immunotherapy outcomes in older patients. Fecal microbiota transplantation experiments in mice confirm the therapeutic potential of the aging-enriched enterotype, enhancing treatment sensitivity and reshaping the TME. Our discoveries confront the prevailing paradox and provide encouraging paths for tailoring cancer immunotherapy strategies according to an individual's gut microbiome profile.
Asunto(s)
Microbioma Gastrointestinal , Humanos , Animales , Ratones , Anciano , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia , Envejecimiento , Complejo CD3RESUMEN
BACKGROUND AND AIMS: Deregulation of RNA N6-methyladenosine (m6A) modification in intestinal epithelial cells (IECs) influences intestinal immune cells and leads to intestinal inflammation. We studied the function of fat mass-and obesity-associated protein (FTO), one of the m6A demethylases, in patients with ulcerative colitis (UC). METHODS: We analysed colon tissues of Ftoflox/flox; Villin-cre mice and their Ftoflox/flox littermates with dextran sulfate sodium (DSS) using real-time PCR and 16s rRNA sequencing. RNA and methylated RNA immunoprecipitation sequencing were used to analyse immunocytes and IECs. Macrophages were treated with conditioned medium of FTO-knockdown MODE-K cells or sphingosine-1-phosphate (S1P) and analysed for gene expression. Liquid chromatograph mass spectrometry identified C16-ceramide. RESULTS: FTO downregulation was identified in our in-house cohort and external cohorts of UC patients. Dysbiosis of gut microbiota, increased infiltration of proinflammatory macrophages, and enhanced differentiation of Th17 cells were observed in Ftoflox/flox;Villin-cre mice under DSS treatment. FTO deficiency resulted in an increase in m6A modification and a decrease in mRNA stability of CerS6, the gene encoding ceramide synthetase, leading to the downregulation of CerS6 and the accumulation of S1P in IECs. Subsequentially, the secretion of S1P by IECs triggered proinflammatory macrophages to secrete serum amyloid A protein 1/3, ultimately inducing Th17 cell differentiation. In addition, through bioinformatic analysis and experimental validation, we identified UC patients with lower FTO expression might respond better to vedolizumab treatment. CONCLUSIONS: FTO downregulation promoted UC by decreasing CerS6 expression, leading to increased S1P accumulation in IECs and aggravating colitis via m6A-dependent mechanisms. Lower FTO expression in UC patients may enhance their response to vedolizumab treatment.
Asunto(s)
Colitis Ulcerosa , Colitis , Humanos , Animales , Ratones , Colitis Ulcerosa/metabolismo , ARN Ribosómico 16S/metabolismo , Mucosa Intestinal/metabolismo , Colitis/inducido químicamente , Colitis/genética , Colon/metabolismo , Esfingolípidos/metabolismo , Sulfato de Dextran , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismoRESUMEN
The perturbations of the gut microbiota and metabolites are closely associated with the progression of inflammatory bowel disease (IBD). However, inconsistent findings across studies impede a comprehensive understanding of their roles in IBD and their potential as reliable diagnostic biomarkers. To address this challenge, here we comprehensively analyze 9 metagenomic and 4 metabolomics cohorts of IBD from different populations. Through cross-cohort integrative analysis (CCIA), we identify a consistent characteristic of commensal gut microbiota. Especially, three bacteria, namely Asaccharobacter celatus, Gemmiger formicilis, and Erysipelatoclostridium ramosum, which are rarely reported in IBD. Metagenomic functional analysis reveals that essential gene of Two-component system pathway, linked to fecal calprotectin, are implicated in IBD. Metabolomics analysis shows 36 identified metabolites with significant differences, while the roles of these metabolites in IBD are still unknown. To further elucidate the relationship between gut microbiota and metabolites, we construct multi-omics biological correlation (MOBC) maps, which highlights gut microbial biotransformation deficiencies and significant alterations in aminoacyl-tRNA synthetases. Finally, we identify multi-omics biomarkers for IBD diagnosis, validated across multiple global cohorts (AUROC values ranging from 0.92 to 0.98). Our results offer valuable insights and a significant resource for developing mechanistic hypotheses on host-microbiome interactions in IBD.
Asunto(s)
Enfermedades Inflamatorias del Intestino , Microbiota , Humanos , Multiómica , Enfermedades Inflamatorias del Intestino/metabolismo , Metaboloma , Biomarcadores/metabolismoRESUMEN
The effect of gut bacteria on the response to immune checkpoint inhibitors (ICIs) has been studied, but the relationship between fungi and ICI responses is not fully understood. Herein, 862 fecal metagenomes from 9 different cohorts were integrated for the identification of differentially abundant fungi and subsequent construction of random forest (RF) models to predict ICI responses. Fungal markers demonstrate excellent performance, with an average area under the curve (AUC) of 0.87. Their performance improves even further, reaching an average AUC of 0.89 when combined with bacterial markers. Higher enrichment of exhausted T cells is detected in responders, as predicted by fungal markers. Multi-kingdom network and functional analysis reveal that the fungus Schizosaccharomyces octosporus may ferment starch into short-chain fatty acids in responders. This study provides a fungal profile of the ICI response and the identification of multi-kingdom microbial markers with good performance that may improve the overall applicability of ICI therapy.
Asunto(s)
Microbioma Gastrointestinal , Neoplasias , Humanos , Metagenoma , Inmunoterapia , Bacterias/genética , Neoplasias/terapiaRESUMEN
Azathioprine (AZA) therapy failure, though not the primary cause, contributes to disease relapse and progression in inflammatory bowel disease (IBD). However, the role of gut microbiota in AZA therapy failure remains poorly understood. We found a high prevalence of Blautia wexlerae in patients with IBD with AZA therapy failure, associated with shorter disease flare survival time. Colonization of B. wexlerae increased inflammatory macrophages and compromised AZA's therapeutic efficacy in mice with intestinal colitis. B. wexlerae colonization reduced 6-mercaptopurine (6-MP) bioavailability by enhancing selenium-dependent xanthine dehydrogenase (sd-XDH) activity. The enzyme sd-XDH converts 6-MP into its inactive metabolite, 6-thioxanthine (6-TX), thereby impairing its ability to inhibit inflammation in mice. Supplementation with Bacillus (B.) subtilis enriched in hypoxanthine phosphoribosyltransferase (HPRT) effectively mitigated B. wexlerae-induced AZA treatment failure in mice with intestinal colitis. These findings emphasize the need for tailored management strategies based on B. wexlerae levels in patients with IBD.
Asunto(s)
Colitis , Enfermedades Inflamatorias del Intestino , Animales , Ratones , Mercaptopurina/uso terapéutico , Azatioprina/uso terapéutico , Inmunosupresores/uso terapéutico , Disponibilidad Biológica , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , BacteriasRESUMEN
Background: Up to 40 per cent of people with active inflammatory bowel disease (IBD) also suffer from mood disorders such as anxiety and depression. Notwithstanding, the fundamental biological pathways driving depression in IBD remain unknown. Methods: We identified 33 core genes that drive depression in IBD patients and performed consensus molecular subtyping with the NMF algorithm in IBD. The CIBERSORT were employed to quantify the immune cells. Metabolic signature was characterized using the "IOBR" R package. The scoring system (D. score) based on PCA. Pre-clinical models are constructed using DSS. Results: Using transcriptome data from the GEO database of 630 IBD patients, we performed a thorough analysis of the correlation between IBD and depression in this research. Firstly, the samples were separated into two different molecular subtypes (D. cluster1 and D. cluster2) based on their biological signatures. Moreover, the immunological and metabolic differences between them were evaluated, and we discovered that D. cluster2 most closely resembled IBD patients concomitant with depression. We also developed a scoring system to assess the IBD-related depression and predict clinical response to anti-TNF- therapy, with a higher D. score suggesting more inflammation and worse reaction to biological therapies. Ultimately, we also identified through animal experiments an antidepressant, paroxetine, has the added benefit of lowering intestinal inflammation by controlling microorganisms in the digestive tract. Conclusions: This study highlights that IBD patients with or without depression show significant variations and antidepressant paroxetine may help reduce intestinal inflammation.
Asunto(s)
Enfermedades Inflamatorias del Intestino , Paroxetina , Humanos , Paroxetina/uso terapéutico , Depresión/tratamiento farmacológico , Inhibidores del Factor de Necrosis Tumoral , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Antiinflamatorios/uso terapéutico , Antidepresivos , Inflamación/tratamiento farmacológicoRESUMEN
Long non-coding RNAs (lncRNAs) play important roles in carcinogenesis. However, the effect of lncRNA on chemoresistance and RNA alternative splicing remains largely unknown. In this study, we identified a novel lncRNA, CACClnc, which was upregulated and associated with chemoresistance and poor prognosis in colorectal cancer (CRC). CACClnc promoted CRC resistance to chemotherapy via promoting DNA repair and enhancing homologous recombination in vitro and in vivo. Mechanistically, CACClnc specifically bound to Y-box binding protein 1 (YB1, a splicing factor) and U2AF65 (a subunit of U2AF splicing factor), promoting the interaction between YB1 and U2AF65, and then modulated alternative splicing (AS) of RAD51 mRNA, and consequently altered CRC cell biology. In addition, expression of exosomal CACClnc in peripheral plasma of CRC patients can effectively predict the chemotherapy effect of patients before treatment. Thus, measuring and targeting CACClnc and its associated pathway could yield valuable insight into clinical management and might ameliorate CRC patients' outcomes.
Asunto(s)
Neoplasias Colorrectales , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Empalme Alternativo/genética , Resistencia a Antineoplásicos/genética , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Proliferación Celular/genética , Línea Celular Tumoral , Recombinasa Rad51/genéticaRESUMEN
Maladaptation of host-microbiota metabolic interplay plays a critical role in colorectal cancer initiation. Here, through a combination of single-cell transcriptomics, microbiome profiling, metabonomics, and clinical analysis on colorectal adenoma and carcinoma tissues, we demonstrate that host's urea cycle metabolism is significantly activated during colorectal tumorigenesis, accompanied by the absence of beneficial bacteria with ureolytic capacity, such as Bifidobacterium, and the overabundance of pathogenic bacteria lacking ureolytic function. Urea could enter into macrophages, inhibit the binding efficiency of p-STAT1 to SAT1 promotor region, and further skew macrophages toward a pro-tumoral phenotype characterized by the accumulation of polyamines. Treating a murine model using urea cycle inhibitors or Bifidobacterium-based supplements could mitigate urea-mediated tumorigenesis. Collectively, this study highlights the utility of urea cycle inhibitors or therapeutically manipulating microbial composition using probiotics to prevent colorectal cancer.
Asunto(s)
Neoplasias Colorrectales , Microbioma Gastrointestinal , Microbiota , Ratones , Animales , Microbioma Gastrointestinal/fisiología , Carcinogénesis , Neoplasias Colorrectales/patología , Transformación Celular NeoplásicaRESUMEN
BACKGROUND & AIMS: Enterotoxigenic Bacteroides fragilis (ETBF) is strongly associated with the occurrence of inflammatory bowel disease (IBD), colitis-associated colorectal cancer, and colorectal cancer (CRC). However, the mechanism of ETBF-induced intestinal inflammation and tumorigenesis remains unclear. METHODS: microRNA sequencing was used to detect the differentially expressed microRNAs in both ETBF-treated cells and exosomes derived from ETBF-inoculated cells. Cell Counting Kit 8 assays were used to evaluate the effect of ETBF and exosomes on CRC cell proliferation. The biological role and mechanism of ETBF-mediated miR-149-3p in colitis and colon carcinogenesis were determined both in vitro and in vivo. RESULTS: ETBF promoted CRC cell proliferation by down-regulating miR-149-3p both in vitro and in vivo. ETBF-down-regulated miR-149-3p depended on METTL14-mediated N6-methyladenosine methylation. As the target gene of miR-149-3p, PHF5A transactivated SOD2 through regulating KAT2A messenger RNA alternative splicing after ETBF treatment in CRC cells. miR-149-3p could be released in exosomes and mediated intercellular communication by modulating T-helper type 17 cell differentiation. The level of plasma exosomal miR-149-3p was gradually decreased from healthy control individuals to patients with IBD and CRC. miR-149-3p, existing in plasma exosomes, negatively correlated with the abundance of ETBF in patients with IBD and CRC. CONCLUSIONS: Exosomal miR-149-3p derived from ETBF-treated cells facilitated T-helper type 17 cell differentiation. ETBF-induced colorectal carcinogenesis depended on down-regulating miR-149-3p and further promoting PHF5A-mediated RNA alternative splicing of KAT2A in CRC cells. Targeting the ETBF/miR-149-3p pathway presents a promising approach to treat patients with intestinal inflammation and CRC with a high amount of ETBF.
Asunto(s)
Bacteroides fragilis/patogenicidad , Colitis Ulcerosa/microbiología , Colon/microbiología , Neoplasias Colorrectales/microbiología , Enfermedad de Crohn/microbiología , Exosomas/microbiología , MicroARNs/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Colitis Ulcerosa/genética , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Colon/metabolismo , Colon/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Enfermedad de Crohn/genética , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Modelos Animales de Enfermedad , Exosomas/genética , Exosomas/metabolismo , Células HCT116 , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , MicroARNs/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor that shows marked efficacy against many types of cancers and is approved to treat severe metastatic cutaneous T-cell lymphomas. In addition to its anticancer activity, SAHA has significant effects on the growth of many viruses. The effect of SAHA on replication of human cytomegalovirus (HCMV) has not, however, been investigated. Here, we showed that the replication of HCMV was significantly suppressed by treatment with SAHA at concentrations that did not show appreciable cytotoxicity. SAHA reduced transcription and protein levels of HCMV immediate early genes, showing that SAHA acts at an early stage in the viral life-cycle. RNA-sequencing data mining showed that numerous pathways and molecules were affected by SAHA. Interferon-mediated immunity was one of the most relevant pathways in the RNA-sequencing data, and we confirmed that SAHA inhibits HCMV-induced IFN-mediated immune responses using quantitative Real-time PCR (qRT-PCR). Fatty acid-binding protein 4 (FABP4), which plays a role in lipid metabolism, was identified by RNA-sequencing. We found that FABP4 expression was reduced by HCMV infection but increased by treatment with SAHA. We then showed that knockdown of FABP4 partially rescued the effect of SAHA on HCMV replication. Our data suggest that FABP4 contributes to the inhibitory effect of SAHA on HCMV replication.
Asunto(s)
Citomegalovirus , Inhibidores de Histona Desacetilasas , Replicación Viral/efectos de los fármacos , Vorinostat , Citomegalovirus/efectos de los fármacos , Citomegalovirus/fisiología , Proteínas de Unión a Ácidos Grasos , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Vorinostat/farmacologíaAsunto(s)
Betacoronavirus/genética , Colon/virología , Infecciones por Coronavirus/genética , Interacciones Huésped-Patógeno/genética , Neumonía Viral/genética , Receptores Virales/genética , Transcriptoma , Adenoma/etnología , Adenoma/genética , Adenoma/patología , Enzima Convertidora de Angiotensina 2 , Pueblo Asiatico , Basigina/genética , Basigina/metabolismo , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidad , COVID-19 , Catepsina B/genética , Catepsina B/metabolismo , Catepsina L/genética , Catepsina L/metabolismo , Colon/metabolismo , Colon/patología , Neoplasias Colorrectales/etnología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Infecciones por Coronavirus/etnología , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Células Epiteliales/virología , Furina/genética , Furina/metabolismo , Humanos , Interleucina-6/sangre , Interleucina-6/genética , Pandemias , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/etnología , Neumonía Viral/patología , Neumonía Viral/virología , Receptores Virales/metabolismo , SARS-CoV-2 , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Índice de Severidad de la Enfermedad , Análisis de la Célula Individual , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/genética , Internalización del VirusRESUMEN
BACKGROUND: Epithelial-Mesenchymal Transition (EMT) is a major process in the initiation of tumor metastasis, where cancer cells lose sessile epithelial potential and gain mesenchymal phenotype. Large-scale cell identity shifts are often orchestrated on an epigenetic level and the interplay between epigenetic factors and EMT progression was still largely unknown. In this study, we tried to identify candidate epigenetic factors that involved in EMT progression. METHODS: Colorectal cancer (CRC) cells were transfected with an arrayed shRNA library targeting 384 genes involved in epigenetic modification. Candidate genes were identified by real-time PCR. Western blot, RNA-seq and gene set enrichment analysis were conducted to confirm the suppressive role of ALKBH4 in EMT. The clinical relevance of ALKBH4 in CRC was investigated in two independent Renji Cohorts and a microarray dataset (GSE21510) from GEO database. In vitro transwell assay and in vivo metastatic tumor model were performed to explore the biological function of ALKBH4 in the metastasis of CRC. Co-IP (Co-Immunoprecipitation) and ChIP (Chromatin Immunoprecipitation) assays were employed to uncover the mechanism. RESULTS: We screened for candidate epigenetic factors that affected EMT process and identified ALKBH4 as a candidate EMT suppressor gene, which was significantly downregulated in CRC patients. Decreased level of ALKBH4 was associated with metastasis and predicted poor prognosis of CRC patients. Follow-up functional experiments illustrated overexpression of ALKBH4 inhibited the invasion ability of CRC cells in vitro, as well as their metastatic capability in vivo. Mechanistically, CO-IP and ChIP assays indicated that ALKBH4 competitively bound WDR5 (a key component of histone methyltransferase complex) and decreased H3K4me3 histone modification on the target genes including MIR21. CONCLUSIONS: This study illustrated that ALKBH4 may function as a novel metastasis suppressor of CRC, and inhibits H3K4me3 modification through binding WDR5 during EMT.
RESUMEN
BACKGROUND: Epigenetic alterations are involved in various aspects of colorectal carcinogenesis. N6-methyladenosine (m6A) modifications of RNAs are emerging as a new layer of epigenetic regulation. As the most abundant chemical modification of eukaryotic mRNA, m6A is essential for the regulation of mRNA stability, splicing, and translation. Alterations of m6A regulatory genes play important roles in the pathogenesis of a variety of human diseases. However, whether this mRNA modification participates in the glucose metabolism of colorectal cancer (CRC) remains uncharacterized. METHODS: Transcriptome-sequencing and liquid chromatography-tandem mass spectrometry (LC-MS) were performed to evaluate the correlation between m6A modifications and glucose metabolism in CRC. Mass spectrometric metabolomics analysis, in vitro and in vivo experiments were conducted to investigate the effects of METTL3 on CRC glycolysis and tumorigenesis. RNA MeRIP-sequencing, immunoprecipitation and RNA stability assay were used to explore the molecular mechanism of METTL3 in CRC. RESULTS: A strong correlation between METTL3 and 18F-FDG uptake was observed in CRC patients from Xuzhou Central Hospital. METTL3 induced-CRC tumorigenesis depends on cell glycolysis in multiple CRC models. Mechanistically, METTL3 directly interacted with the 5'/3'UTR regions of HK2, and the 3'UTR region of SLC2A1 (GLUT1), then further stabilized these two genes and activated the glycolysis pathway. M6A-mediated HK2 and SLC2A1 (GLUT1) stabilization relied on the m6A reader IGF2BP2 or IGF2BP2/3, respectively. CONCLUSIONS: METTL3 is a functional and clinical oncogene in CRC. METTL3 stabilizes HK2 and SLC2A1 (GLUT1) expression in CRC through an m6A-IGF2BP2/3- dependent mechanism. Targeting METTL3 and its pathway offer alternative rational therapeutic targets in CRC patients with high glucose metabolism.
Asunto(s)
Adenosina/análogos & derivados , Neoplasias Colorrectales/patología , Epigénesis Genética , Transportador de Glucosa de Tipo 1/metabolismo , Glucólisis , Hexoquinasa/metabolismo , Metiltransferasas/metabolismo , Adenosina/química , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Metilación de ADN , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Transportador de Glucosa de Tipo 1/genética , Hexoquinasa/genética , Humanos , Metiltransferasas/genética , Ratones , Ratones Desnudos , Pronóstico , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Late gene expression of betaherpesviruses and gammaherpesviruses is tightly controlled by virus-encoded transactivation factors (vTFs). We recently proved that the 6 vTFs of murine cytomegalovirus (MCMV) form a complex to regulate late gene transcription. pM49, one of the vTFs that has not been studied before, was identified to be a component of the complex that interacts with pM95. In this study, we began to investigate the potential role of pM49 in viral late gene expression. A recombinant MCMV expressing C-terminal FLAG-tagged pM49 was constructed to study the expression kinetics and localization of pM49. pM49 was expressed at the late time of virus infection. Inhibition of viral DNA synthesis by phosphonate sodium phosphonic acid (PAA) abolished pM49 expression, indicating that it is a late protein. pM49 colocalized with pM44 at the viral replication compartment, similarly to other viral vTFs that have been reported. Mutant virus lacking full-length pM49 expression failed to express viral late genes, leading to nonproductive infection. The expression of immediate early and early genes was not affected, and viral DNA synthesis was only minimally affected during pM49-deficient virus infection. All of these data support the role of pM49 in viral late gene expression. After a series of mutagenesis analyses, two key residues, K325 and C326, were identified as required for pM49-pM95 interaction. Cells expressing pM49 with either single mutation of these two residues failed to rescue the late gene expression and support the replication of pM49-deficient virus. Our results indicated that pM49-pM95 interaction is essential for viral late gene expression.IMPORTANCE Cytomegalovirus (CMV) infections result in morbidity and mortality in immunocompromised individuals, and the virus is also a major cause of birth defects in newborns. Currently, because of the unavailability of vaccines against this virus and restricted antiviral therapies with low toxicity, as well as the emergency of resistant strain of this virus, the understanding of viral late gene regulation may provide clues to study new antiviral drugs or vaccines. In this study, we report that MCMV protein pM49 is critical for viral late gene transcription, based on its interaction with pM95. This finding reveals the important role of pM49-pM95 interaction in the regulation of viral late gene expression and that it could be a future potential target for therapeutic intervention in CMV diseases.
Asunto(s)
ADN Viral/biosíntesis , Regulación Viral de la Expresión Génica , Infecciones por Herpesviridae/metabolismo , Muromegalovirus/metabolismo , Mutación , Proteínas Virales/metabolismo , Animales , Línea Celular , ADN Viral/genética , Infecciones por Herpesviridae/genética , Ratones , Muromegalovirus/genética , Proteínas Virales/genéticaRESUMEN
Antibodies are fundamental tools for basic science; however, high-quality antibodies suitable for multiple experimental applications are often inaccessible to research laboratories. To this end, a modular and low-cost pipeline for small-scale antibody customization is developed. First, soluble antigens are designed according to the secondary structure of a desired protein. Then, the antigens are efficiently displayed on a modular nanoplatform by intein-mediated trans-splicing (TS) that enables elicitation of high titers of protein-specific antibodies. After that, target antibodies are obtained by a modular HaloLink resin platform with antigens as the ligand that is devised by intein-mediated TS. Finally, purified antibodies show excellent properties in immunofluorescence, immunoprecipitation, and western blotting assays. Overall, these results suggest that the proposed pipeline is amenable to the generation of high-quality, research-grade antibodies and to aid in protein functional studies.
RESUMEN
OBJECTIVE: Colorectal cancer (CRC) is highly malignant and cancer metastasis remains the predominant cause of CRC death. The potential molecular mechanism of long non-coding RNA (lncRNAs) in CRC malignance is still poorly elucidated. METHODS: CCMAlnc expression was analyzed by using the Sequence ReadArchive (SRA) database. Target gene expression was examined by real-time PCR and Western blotting. The biological function of CCMAlnc and miR-5001-5p was detected by cell invasion, CCK8 proliferation, and colony formation assays in loss of function and gain of function experiments in vitro. A luciferase assay was performed to validate the target site of miR-5001-5p on the 3'-UTR of HES6 mRNA. RESULTS: CCMAlnc was identified as a novel functional lncRNA in CRC. Elevated CCMAlnc was detected in CRC cells as well as in clinical CRC tissue samples, and the expression of this lncRNA positively correlated with the poor prognosis of CRC patients. Functional validation assays revealed that downregulation of CCMAlnc impaired CRC cell proliferation and invasion in vitro, but upregulation of CCMAlnc reversed this effect. Moreover, CCMAlnc was validated to act as a competing endogenous RNA (ceRNA) that stabilizes the expression of HES6 by downregulating miR-5001-5p. CONCLUSION: CCMAlnc/miR-5001-5p/HES6 signaling is strongly activated to promote CRC malignance. CCMAlnc is defined as a potential candidate biomarker for metastasis prediction in CRC patients and as a potential therapeutic target for CRC treatment.
RESUMEN
DDX21 regulates the biogenesis of rRNA and transcription of ribonucleoprotein genes. Recently, it has been reported that DDX21 regulates the growth of some RNA viruses through various mechanisms, such as inhibiting viral genome replication, suppressing virion assembly and release, and modulating antiviral immune responses (Chen et al., Cell Host Microbe 15:484-493, 2014, https://doi.org/10.1016/j.chom.2014.03.002; Dong et al., Biophys Res Commun, 473:648-653, 2016, https://doi.org/10.1016/j.bbrc.2016.03.120; and Watanabe et al., PLoS Pathog 5:e1000654, 2009, https://doi.org/10.1371/journal.ppat.1000654). The relationship between DDX21 and DNA viruses has not yet been explored. In this study, we used human cytomegalovirus (HCMV), a large human DNA virus, to investigate the potential role of DDX21 in DNA virus replication. We found that HCMV infection prevented the repression of DDX21 at protein and mRNA levels. Knockdown of DDX21 inhibited HCMV growth in human fibroblast cells (MRC5). Immunofluorescence and quantitative PCR (qPCR) results showed that knockdown of DDX21 did not affect viral DNA replication or the formation of the viral replication compartment but did significantly inhibit viral late gene transcription. Some studies have reported that DDX21 knockdown promotes the accumulation of R-loops that could restrain RNA polymerase II elongation and inhibit the transcription of certain genes. Thus, we used the DNA-RNA hybrid-specific S9.6 antibody to stain R-loops and observed that more R-loops formed in DDX21-knockdown cells than in control cells. Moreover, an DNA-RNA immunoprecipitation assay showed that more R-loops accumulated on a viral late gene in DDX21-knockdown cells. Altogether, these results suggest that DDX21 knockdown promotes the accumulation of R-loops, which prevents viral late gene transcription and consequently results in the suppression of HCMV growth. This finding provides new insight into the relationship between DDX21 and DNA virus replication.IMPORTANCE Previous studies have confirmed that DDX21 is vital for the regulation of various aspects of RNA virus replication. Our research is the first report on the role of DDX21 in HCMV DNA virus replication. We identified that DDX21 knockdown affected HCMV growth and viral late gene transcription. In order to elucidate how DDX21 regulated this transcription, we applied DNA-RNA immunoprecipitation by using the DNA-RNA hybrid-specific S9.6 antibody to test whether more R-loops accumulated on the viral late gene. Consistent with our expectation, more R-loops were detected on the viral late gene at late HCMV infection time points, which demonstrated that the accumulation of R-loops caused by DDX21 knockdown prevented viral late gene transcription and consequently impaired HCMV replication. These results reveal that DDX21 plays an important role in regulating HCMV replication and also provide a basis for investigating the role of DDX21 in regulating other DNA viruses.
Asunto(s)
Citomegalovirus/fisiología , ARN Helicasas DEAD-box/fisiología , Replicación Viral/fisiología , Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN Viral/metabolismo , Fibroblastos/virología , Expresión Génica , Técnicas de Silenciamiento del Gen , Genes Virales , Células HEK293 , Humanos , Inmunoprecipitación , ARN Polimerasa II/metabolismo , Transcripción Genética , Ensamble de VirusRESUMEN
BACKGROUND: The long noncoding RNA (lncRNA) colon cancer-associated transcript-1 (CCAT1) has been reported to play a vital role in the development of cancer. Although the link between inflammation and cancer initiation is well established, whether CCAT1 is involved in inflammation and promotes inflammatory bowel disease (IBD) malignancy remains undetermined. We aimed to investigate the expression of CCAT1 in IBD and the effect of CCAT1 overexpression on intestinal epithelial barrier function. METHODS: The relationship between CCAT1 and the inflammation-related pathway was analyzed in both colorectal cancer (CRC) and IBD patients. Gene expression was detected by real-time polymerase chain reaction and Western blot. Transepithelial electrical resistance (TEER) and FD-4 flux measurement were used to test the effect of CCAT1 and miR-185-3p on intestinal epithelial barrier function. Luciferase assay was performed to validate the target site of miR-185-3p on 3'-UTR of MLCK mRNA. RESULTS: Gene set enrichment analysis revealed that several inflammation-related genes were enriched in the CCAT1 high-expressed group of CRC patients. The relationship between CCAT1 and inflammation activation in IBD patients was further confirmed. CCAT1 expression positively correlated with MLCK, which acts as a protein kinase to phosphorylate myosin light chain and induces tight junction protein distribution, whereas it was negatively correlated with miR-185-3p in IBD tissues. We also determined that CCAT1 overexpression increased Caco-2 monolayer permeability and upregulated MLCK. Furthermore, CCAT1-induced MLCK overexpression and IBD disease progression were significantly attenuated by miR-185-3p. CONCLUSIONS: The CCAT1/miR-185-3p/MLCK signaling pathway is strongly activated to destroy barrier function and promotes the pathogenesis of IBD.
Asunto(s)
Permeabilidad de la Membrana Celular , Neoplasias Colorrectales/patología , Enfermedades Inflamatorias del Intestino/patología , Intestinos/patología , MicroARNs/genética , ARN Largo no Codificante/genética , Uniones Estrechas/patología , Células CACO-2 , Estudios de Casos y Controles , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosforilación , Transducción de Señal , Uniones Estrechas/genética , Uniones Estrechas/metabolismoRESUMEN
Viral gene expression is tightly regulated during cytomegalovirus (CMV) lytic replication, but the detailed mechanism of late gene transcription remains to be fully understood. Previous studies reported that six viral proteins (named viral transactivation factors [vTFs]) supporting late gene expression were conserved in beta- and gammaherpesviruses but not in alphaherpesviruses. Here, we performed coimmunoprecipitation experiments to elucidate the organization of these six proteins in murine CMV. Our results showed that these proteins formed a complex by both direct and indirect interactions. Specifically, pM91 strongly bound to pM79 even in the absence of other vTFs. Similar to pM79, pM91 exhibited early-late expression kinetics and localized within nuclear viral replication compartments during infection. Functional analysis was also performed using the pM91-deficient virus. Real-time PCR results revealed that abrogation of M91 expression markedly reduced viral late gene expression and progeny virus production without affecting viral DNA synthesis. Using mutagenesis, we found that residues E61, D62, D89, and D96 in pM91 were required for the pM91-pM79 interaction. Disruption of the interaction via E61A/D62A or D89A/D96A double mutation in the context of virus infection inhibited progeny virus production. Our data indicate that pM91 is a component of the viral late gene transcription factor complex and that the pM91-pM79 interaction is essential for viral late gene expression.IMPORTANCE Cytomegalovirus (CMV) infection is the leading cause of birth defects and causes morbidity and mortality in immunocompromised patients. The regulation of viral late gene transcription is not well elucidated, and understanding of this process benefits the development of novel therapeutics against CMV infection. This study (i) identified that six viral transactivation factors encoded by murine CMV form a complex, (ii) demonstrated that pM91 interacts with pM79 and that pM91 and pM79 colocalize in the nuclear viral replication compartments, (iii) confirmed that pM91 is critical for viral late gene expression but dispensable for viral DNA replication, and (iv) revealed that the pM91-pM79 interaction is required for progeny virus production. These findings give an explanation of how CMV regulates late gene expression and have important implications for the design of antiviral strategies.
Asunto(s)
Muromegalovirus/fisiología , Proteínas Virales/química , Proteínas Virales/metabolismo , Sitios de Unión , Regulación Viral de la Expresión Génica , Células HEK293 , Humanos , Muromegalovirus/metabolismo , Unión Proteica , Multimerización de Proteína , Proteínas Virales/genética , Replicación ViralRESUMEN
Human cytomegalovirus (HCMV) protein pUL38 has been shown to prevent premature cell death by antagonizing cellular stress responses; however, the underlying mechanism remains unknown. In this study, we identified the host protein ubiquitin-specific protease 24 (USP24) as an interaction partner of pUL38. Mutagenesis analysis of pUL38 revealed that amino acids TFV at positions 227 to 230 were critical for its interaction with USP24. Mutant pUL38 TFV/AAA protein did not bind to USP24 and failed to prevent cell death induced by pUL38-deficient HCMV infection. Knockdown of USP24 suppressed the cell death during pUL38-deficient HCMV infection, suggesting that pUL38 achieved its function by antagonizing the function of USP24. We investigated the cellular pathways regulated by USP24 that might be involved in the cell death phenotype by testing several small-molecule compounds known to have a protective effect during stress-induced cell death. The iron chelators ciclopirox olamine and Tiron specifically protected cells from pUL38-deficient HCMV infection-induced cell death, thus identifying deregulated iron homeostasis as a potential mechanism. Protein levels of nuclear receptor coactivator 4 (NCOA4) and lysosomal ferritin degradation, a process called ferritinophagy, were also regulated by pUL38 and USP24 during HCMV infection. Knockdown of USP24 decreased NCOA4 protein stability and ferritin heavy chain degradation in lysosomes. Blockage of ferritinophagy by genetic inhibition of NCOA4 or Atg5/Atg7 prevented pUL38-deficient HCMV infection-induced cell death. Overall, these results support the hypothesis that pUL38 binds to USP24 to reduce ferritinophagy, which may then protect cells from lysosome dysfunction-induced cell death.IMPORTANCE Premature cell death is considered a first line of defense against various pathogens. Human cytomegalovirus (HCMV) is a slow-replicating virus that encodes several cell death inhibitors, such as pUL36 and pUL37x1, which allow it to overcome both extrinsic and intrinsic mitochondrion-mediated apoptosis. We previously identified HCMV protein pUL38 as another virus-encoded cell death inhibitor. In this study, we demonstrated that pUL38 achieved its activity by interacting with and antagonizing the function of the host protein ubiquitin-specific protease 24 (USP24). pUL38 blocked USP24-mediated ferritin degradation in lysosomes, which could otherwise be detrimental to the lysosome and initiate cell death. These novel findings suggest that iron metabolism is finely tuned during HCMV infection to avoid cellular toxicity. The results also provide a solid basis for further investigations of the role of USP24 in regulating iron metabolism during infection and other diseases.