ABSTRACT
Sex-based differences in obesity-related hepatic malignancies suggest the protective roles of estrogen. Using a preclinical model, we dissected estrogen receptor (ER) isoform-driven molecular responses in high-fat diet (HFD)-induced liver diseases of male and female mice treated with or without an estrogen agonist by integrating liver multi-omics data. We found that selective ER activation recovers HFD-induced molecular and physiological liver phenotypes. HFD and systemic ER activation altered core liver pathways, beyond lipid metabolism, that are consistent between mice and primates. By including patient cohort data, we uncovered that ER-regulated enhancers govern central regulatory and metabolic genes with clinical significance in metabolic dysfunction-associated steatotic liver disease (MASLD) patients, including the transcription factor TEAD1. TEAD1 expression increased in MASLD patients, and its downregulation by short interfering RNA reduced intracellular lipid content. Subsequent TEAD small molecule inhibition improved steatosis in primary human hepatocyte spheroids by suppressing lipogenic pathways. Thus, TEAD1 emerged as a new therapeutic candidate whose inhibition ameliorates hepatic steatosis.
Subject(s)
Fatty Liver , Non-alcoholic Fatty Liver Disease , Animals , Female , Humans , Male , Mice , Diet, High-Fat/adverse effects , Estrogens , Fatty Liver/genetics , Fatty Liver/metabolism , Gene Expression , Liver/metabolism , Mice, Inbred C57BL , Non-alcoholic Fatty Liver Disease/genetics , Non-alcoholic Fatty Liver Disease/metabolism , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Receptors, Estrogen/therapeutic use , TEA Domain Transcription FactorsABSTRACT
The CRISPR-Cas9 system is a powerful tool for studying gene functions and holds potential for disease treatment. However, precise genome editing requires thorough assessments to minimize unintended on- and off-target effects. Here, we report an unexpected 283-kb deletion on Chromosome 10 (10q23.31) in chronic myelogenous leukemia-derived HAP1 cells, which are frequently used in CRISPR screens. The deleted region encodes regulatory genes, including PAPSS2, ATAD1, KLLN, and PTEN We found that this deletion was not a direct consequence of CRISPR-Cas9 off-targeting but rather occurred frequently during the generation of CRISPR-Cas9-modified cells. The deletion was associated with global changes in histone acetylation and gene expression, affecting fundamental cellular processes such as cell cycle and DNA replication. We detected this deletion in cancer patient genomes. As in HAP1 cells, the deletion contributed to similar gene expression patterns among cancer patients despite interindividual differences. Our findings suggest that the unintended deletion of 10q23.31 can confound CRISPR-Cas9 studies and underscore the importance to assess unintended genomic changes in CRISPR-Cas9-modified cells, which could impact cancer research.
Subject(s)
CRISPR-Cas Systems , Neoplasms , Humans , CRISPR-Cas Systems/genetics , Gene Editing , Genome , Chromosome Structures , Phenotype , Neoplasms/genetics , PTEN Phosphohydrolase/geneticsABSTRACT
The next steps of deep space exploration are manned missions to Moon and Mars. For safe space missions for crew members, it is important to understand the impact of space flight on the immune system. We studied the effects of 21 days dry immersion (DI) exposure on the transcriptomes of T cells isolated from blood samples of eight healthy volunteers. Samples were collected 7 days before DI, at day 7, 14, and 21 during DI, and 7 days after DI. RNA sequencing of CD3+ T cells revealed transcriptional alterations across all time points, with most changes occurring 14 days after DI exposure. At day 21, T cells showed evidence of adaptation with a transcriptional profile resembling that of 7 days before DI. At 7 days after DI, T cells again changed their transcriptional profile. These data suggest that T cells adapt by rewiring their transcriptomes in response to simulated weightlessness and that remodeling cues persist when reexposed to normal gravity.
Subject(s)
Weightlessness , Humans , Weightlessness/adverse effects , Immersion , T-Lymphocytes , Volunteers , TranscriptomeABSTRACT
The liver holds central roles in detoxification, energy metabolism, and whole body homeostasis but can develop malignant phenotypes when being chronically overwhelmed with fatty acids and glucose. The global rise of metabolic dysfunction-associated fatty liver disease (MAFLD) is already affecting a quarter of the global population. Pharmaceutical treatment options against different stages of MAFLD do not yet exist, and several clinical trials against hepatic transcription factors and other proteins have failed. However, emerging roles of noncoding RNAs, including long (lncRNA) and short noncoding RNAs (sRNA), in various cellular processes pose exciting new avenues for treatment interventions. Actions of noncoding RNAs mostly rely on interactions with proteins, whereby the noncoding RNA fine-tunes protein function in a process termed riboregulation. The developmental stage-, disease stage-, and cell type-specific nature of noncoding RNAs harbors enormous potential to precisely target certain cellular pathways in a spatiotemporally defined manner. Proteins interacting with RNAs can be categorized into canonical or noncanonical RNA-binding proteins (RBPs) depending on the existence of classical RNA-binding domains. Both, RNA- and RBP-centric methods have generated new knowledge of the RNA-RBP interface and added an additional regulatory layer. In this review, we summarize recent advances in how RBP-lncRNA interactions and various sRNAs shape cellular physiology and the development of liver diseases such as MAFLD and hepatocellular carcinoma.
Subject(s)
Liver Neoplasms , Metabolic Diseases , RNA, Long Noncoding , RNA, Small Untranslated , Fatty Acids , Glucose , Humans , Pharmaceutical Preparations , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Transcription Factors/metabolismABSTRACT
OBJECTIVE: To better comprehend transcriptional phenotypes of cancer cells, we globally characterised RNA-binding proteins (RBPs) to identify altered RNAs, including long non-coding RNAs (lncRNAs). DESIGN: To unravel RBP-lncRNA interactions in cancer, we curated a list of ~2300 highly expressed RBPs in human cells, tested effects of RBPs and lncRNAs on patient survival in multiple cohorts, altered expression levels, integrated various sequencing, molecular and cell-based data. RESULTS: High expression of RBPs negatively affected patient survival in 21 cancer types, especially hepatocellular carcinoma (HCC). After knockdown of the top 10 upregulated RBPs and subsequent transcriptome analysis, we identified 88 differentially expressed lncRNAs, including 34 novel transcripts. CRISPRa-mediated overexpression of four lncRNAs had major effects on the HCC cell phenotype and transcriptome. Further investigation of four RBP-lncRNA pairs revealed involvement in distinct regulatory processes. The most noticeable RBP-lncRNA connection affected lipid metabolism, whereby the non-canonical RBP CCT3 regulated LINC00326 in a chaperonin-independent manner. Perturbation of the CCT3-LINC00326 regulatory network led to decreased lipid accumulation and increased lipid degradation in cellulo as well as diminished tumour growth in vivo. CONCLUSIONS: We revealed that RBP gene expression is perturbed in HCC and identified that RBPs exerted additional functions beyond their tasks under normal physiological conditions, which can be stimulated or intensified via lncRNAs and affected tumour growth.
ABSTRACT
Using AI, we identified baricitinib as having antiviral and anticytokine efficacy. We now show a 71% (95% CI 0.15 to 0.58) mortality benefit in 83 patients with moderate-severe SARS-CoV-2 pneumonia with few drug-induced adverse events, including a large elderly cohort (median age, 81 years). An additional 48 cases with mild-moderate pneumonia recovered uneventfully. Using organotypic 3D cultures of primary human liver cells, we demonstrate that interferon-α2 increases ACE2 expression and SARS-CoV-2 infectivity in parenchymal cells by greater than fivefold. RNA-seq reveals gene response signatures associated with platelet activation, fully inhibited by baricitinib. Using viral load quantifications and superresolution microscopy, we found that baricitinib exerts activity rapidly through the inhibition of host proteins (numb-associated kinases), uniquely among antivirals. This reveals mechanistic actions of a Janus kinase-1/2 inhibitor targeting viral entry, replication, and the cytokine storm and is associated with beneficial outcomes including in severely ill elderly patients, data that incentivize further randomized controlled trials.
Subject(s)
Antiviral Agents/pharmacology , Azetidines/pharmacology , COVID-19/mortality , Enzyme Inhibitors/pharmacology , Janus Kinases/antagonists & inhibitors , Liver/virology , Purines/pharmacology , Pyrazoles/pharmacology , SARS-CoV-2/pathogenicity , Sulfonamides/pharmacology , Adult , Aged , Aged, 80 and over , COVID-19/metabolism , COVID-19/virology , Cytokine Release Syndrome , Cytokines/metabolism , Drug Evaluation, Preclinical , Female , Gene Expression Profiling , Humans , Interferon alpha-2/metabolism , Italy , Janus Kinases/metabolism , Liver/drug effects , Male , Middle Aged , Patient Safety , Platelet Activation , Proportional Hazards Models , RNA-Seq , Spain , Virus Internalization/drug effects , COVID-19 Drug TreatmentABSTRACT
With the rise of new powerful genome engineering technologies, such as CRISPR/Cas9, cell models can be engineered effectively to accelerate basic and disease research. The most critical step in this procedure is the efficient delivery of foreign nucleic acids into cells by cellular transfection. Since the vectors encoding the components necessary for CRISPR/Cas genome engineering are always large (9-19 kb), they result in low transfection efficiency and cell viability, and thus subsequent selection or purification of positive cells is required. To overcome those obstacles, we here show a non-toxic and non-viral delivery method that increases transfection efficiency (up to 40-fold) and cell viability (up to 6-fold) in a number of hard-to-transfect human cancer cell lines and primary blood cells. At its core, the technique is based on adding exogenous small plasmids of a defined size to the transfection mixture.
Subject(s)
CRISPR-Cas Systems , Plasmids/genetics , Transfection/methods , Cells, Cultured , Genetic Vectors , Green Fluorescent Proteins/genetics , HumansABSTRACT
Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immunoprecipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both requires nuclear steps. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.
Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Uukuniemi virus/physiology , Animals , Antiviral Agents/pharmacology , Bunyaviridae Infections/virology , Cell Line, Tumor , Chlorocebus aethiops , Glycoproteins/metabolism , Host Microbial Interactions , Humans , Mass Spectrometry , Proteomics , Pyridines/pharmacology , Quinolines/pharmacology , RNA Interference , RNA Viruses/physiology , Uukuniemi virus/drug effects , Vero Cells , Viral Envelope Proteins/metabolism , Virus Release , Virus ReplicationABSTRACT
Amantadine is an established antiparkinsonian drug with a still unclear molecular site of action. In vivo studies on rodents, in vitro studies on tissue of rodents as well as binding studies on post mortem human tissue implicate monoamine transporters and NMDA receptors. In order to re-examine its action at human variants of these proteins on intact cells we established cells stably expressing the human NR1/2A NMDA-receptor, noradrenaline transporter (NAT) or dopamine transporter (DAT) and tested the activity of amantadine in patch-clamp, uptake, release, and cytotoxicity experiments. Amantadine was less potent in blockade of NMDA-induced inward currents than in blockade of noradrenaline uptake and in induction of inward currents in NAT expressing cells. It was 30 times more potent in blocking uptake in NAT- than in DAT cells. Amantadine induced NAT-mediated release at concentrations of 10-100 µM in superfusion experiments and blocked NAT-mediated cytotoxicity of the parkinsonism inducing neurotoxin 1-methyl-4-phenyl-pyridinium (MPP(+)) at concentrations of 30-300 µM, whereas 300-1000 µM amantadine was necessary to block NMDA-receptor mediated cytotoxicity. Similar to amphetamine, amantadine was inactive at α(2A)-adrenergic receptors and induced reverse noradrenaline transport by NAT albeit with smaller effect size. Thus, amantadine acted as "amphetamine-like releaser" with selectivity for the noradrenergic system. These findings and differences with memantine, which had been reported as less efficient antiparkinsonian drug than amantadine but in our hands was significantly more potent at the NMDA-receptor, suggest contributions from a noradrenergic mechanism in the antiparkinsonian action of amantadine.