RESUMEN
Exhausted CD8 T (Tex) cells in chronic viral infection and cancer have sustained co-expression of inhibitory receptors (IRs). Tex cells can be reinvigorated by blocking IRs, such as PD-1, but synergistic reinvigoration and enhanced disease control can be achieved by co-targeting multiple IRs including PD-1 and LAG-3. To dissect the molecular changes intrinsic when these IR pathways are disrupted, we investigated the impact of loss of PD-1 and/or LAG-3 on Tex cells during chronic infection. These analyses revealed distinct roles of PD-1 and LAG-3 in regulating Tex cell proliferation and effector functions, respectively. Moreover, these studies identified an essential role for LAG-3 in sustaining TOX and Tex cell durability as well as a LAG-3-dependent circuit that generated a CD94/NKG2+ subset of Tex cells with enhanced cytotoxicity mediated by recognition of the stress ligand Qa-1b, with similar observations in humans. These analyses disentangle the non-redundant mechanisms of PD-1 and LAG-3 and their synergy in regulating Tex cells.
Asunto(s)
Antígenos CD , Linfocitos T CD8-positivos , Antígenos de Histocompatibilidad Clase I , Proteína del Gen 3 de Activación de Linfocitos , Subfamília D de Receptores Similares a Lectina de las Células NK , Receptor de Muerte Celular Programada 1 , Animales , Antígenos CD/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Ratones , Receptor de Muerte Celular Programada 1/metabolismo , Subfamília D de Receptores Similares a Lectina de las Células NK/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismo , Ratones Endogámicos C57BL , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Citotoxicidad Inmunológica , Proliferación Celular , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/inmunologíaRESUMEN
Improving effector activity of antigen-specific T cells is a major goal in cancer immunotherapy. Despite the identification of several effector T cell (TEFF)-driving transcription factors (TFs), the transcriptional coordination of TEFF biology remains poorly understood. We developed an in vivo T cell CRISPR screening platform and identified a key mechanism restraining TEFF biology through the ETS family TF, Fli1. Genetic deletion of Fli1 enhanced TEFF responses without compromising memory or exhaustion precursors. Fli1 restrained TEFF lineage differentiation by binding to cis-regulatory elements of effector-associated genes. Loss of Fli1 increased chromatin accessibility at ETS:RUNX motifs, allowing more efficient Runx3-driven TEFF biology. CD8+ T cells lacking Fli1 provided substantially better protection against multiple infections and tumors. These data indicate that Fli1 safeguards the developing CD8+ T cell transcriptional landscape from excessive ETS:RUNX-driven TEFF cell differentiation. Moreover, genetic deletion of Fli1 improves TEFF differentiation and protective immunity in infections and cancer.
Asunto(s)
Linfocitos T CD8-positivos/citología , Proteína Proto-Oncogénica c-fli-1/metabolismo , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Sistemas CRISPR-Cas , Diferenciación Celular , Enfermedad Crónica , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Epigénesis Genética , Redes Reguladoras de Genes , Infecciones/inmunología , Ratones , Neoplasias/inmunologíaRESUMEN
Interferon-gamma (IFN-γ) has pleiotropic effects on cancer immune checkpoint blockade (ICB), including roles in ICB resistance. We analyzed gene expression in ICB-sensitive versus ICB-resistant tumor cells and identified a strong association between interferon-mediated resistance and expression of Ripk1, a regulator of tumor necrosis factor (TNF) superfamily receptors. Genetic interaction screening revealed that in cancer cells, RIPK1 diverted TNF signaling through NF-κB and away from its role in cell death. This promoted an immunosuppressive chemokine program by cancer cells, enhanced cancer cell survival, and decreased infiltration of T and NK cells expressing TNF superfamily ligands. Deletion of RIPK1 in cancer cells compromised chemokine secretion, decreased ARG1+ suppressive myeloid cells linked to ICB failure in mice and humans, and improved ICB response driven by CASP8-killing and dependent on T and NK cells. RIPK1-mediated resistance required its ubiquitin scaffolding but not kinase function. Thus, cancer cells co-opt RIPK1 to promote cell-intrinsic and cell-extrinsic resistance to immunotherapy.
Asunto(s)
Resistencia a Antineoplásicos , Inhibidores de Puntos de Control Inmunológico , Interferones , Neoplasias , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Animales , Inmunoterapia , Interferón gamma/metabolismo , Interferones/metabolismo , Ratones , FN-kappa B/metabolismo , Neoplasias/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
The transformed state in acute leukemia requires gene regulatory programs involving transcription factors and chromatin modulators. Here, we uncover an IRF8-MEF2D transcriptional circuit as an acute myeloid leukemia (AML)-biased dependency. We discover and characterize the mechanism by which the chromatin "reader" ZMYND8 directly activates IRF8 in parallel with the MYC proto-oncogene through their lineage-specific enhancers. ZMYND8 is essential for AML proliferation in vitro and in vivo and associates with MYC and IRF8 enhancer elements that we define in cell lines and in patient samples. ZMYND8 occupancy at IRF8 and MYC enhancers requires BRD4, a transcription coactivator also necessary for AML proliferation. We show that ZMYND8 binds to the ET domain of BRD4 via its chromatin reader cassette, which in turn is required for proper chromatin occupancy and maintenance of leukemic growth in vivo. Our results rationalize ZMYND8 as a potential therapeutic target for modulating essential transcriptional programs in AML.
Asunto(s)
Factores Reguladores del Interferón/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular/genética , Cromatina/genética , Elementos de Facilitación Genéticos/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Factores Reguladores del Interferón/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Proto-Oncogenes Mas , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Acute graft-versus-host disease (aGVHD) is primarily driven by allogeneic donor T cells associated with an altered composition of the host gut microbiome and its metabolites. The severity of aGVHD after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not solely determined by the host and donor characteristics; however, the underlying mechanisms remain unclear. Using single-cell RNA sequencing, we decoded the immune cell atlas of 12 patients who underwent allo-HSCT: six with aGVHD and six with non-aGVHD. We performed a fecal microbiota (16SrRNA sequencing) analysis to investigate the fecal bacterial composition of 82 patients: 30 with aGVHD and 52 with non-aGVHD. Fecal samples from these patients were analyzed for bile acid metabolism. Through multi-omic analysis, we identified a feedback loop involving "immune cell-gut microbes-bile acid metabolites" contributing to heightened immune responses in patients with aGVHD. The dysbiosis of the gut microbiota and disruption of bile acid metabolism contributed to an exaggerated interleukin-1 mediated immune response. Our findings suggest that resistin and defensins are crucial in mitigating against aGVHD. Therefore, a comprehensive multi-omic atlas incorporating immune cells, gut microbes, and bile acid metabolites was developed in this study and used to propose novel, non-immunosuppressive approaches to prevent aGVHD.
Asunto(s)
Ácidos y Sales Biliares , Heces , Microbioma Gastrointestinal , Enfermedad Injerto contra Huésped , Ácidos y Sales Biliares/metabolismo , Humanos , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/microbiología , Microbioma Gastrointestinal/inmunología , Femenino , Masculino , Heces/microbiología , Persona de Mediana Edad , Enfermedad Aguda , Adulto , Retroalimentación Fisiológica , Inmunidad , Metabolómica , Trasplante de Células Madre Hematopoyéticas , MultiómicaRESUMEN
Human infection of orthohantavirus can cause potentially fatal diseases, such as hemorrhagic fever with renal syndrome (HFRS) caused by Hantaan virus (HTNV) in Eurasia. Exosomes are new carriers for information exchange between cells. Cumulative findings suggest that exosomes released from parental infected cells can block or promote viral infection in recipient cells, but the role of exosomes in hantavirus infection is poorly understood. In our study, we identified the exosomes derived from HTNV-infected human vascular endothelial cells (HUVECs) (Exo-HV) and found the antiviral properties of Exo-HV in the uninfected recipient cells. High-throughput sequencing revealed the distinctly expressed miRNAs transcriptomes in Exo-HV. MiR-145-5p, one of the abundant miRNAs packaged into Exo-HV, was found to be able to transferred to recipient cells and functioned by directly targeting M RNA of HTNV 76-118 and inducing type I interferon (IFN-I) response, thus, blocking the viral replication. Concluding, this study indicated that exosomes released by HTNV-infected HUVECs were able to transfer active molecules, miR-145-5p as a proving sample, to mediate novel anti-HTNV activity in the neighboring uninfected cells, which will help us to explore new strategies for the treatment of infectious disease utilizing exosomes with miRNA.
Asunto(s)
Exosomas/genética , Virus Hantaan/fisiología , Células Endoteliales de la Vena Umbilical Humana/virología , MicroARNs/metabolismo , Orthohepadnavirus/patogenicidad , Replicación Viral , Exosomas/metabolismo , Virus Hantaan/patogenicidad , Interacciones Huésped-Patógeno , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Interferones/genética , Interferones/metabolismo , MicroARNs/genética , TranscriptomaRESUMEN
Hantaan virus (HTNV), member of the newly defined Hantaviridae family, within the order Bunyavirales, can cause a hemorrhagic fever with renal syndrome with high fatality rates in humans. However, no specific antiviral agents are currently available for HTNV infection approved by the US Food and Drug Administration. Although interferon lambdas (IFN-λs) have been shown to induce an antiviral state against HTNV, the molecular mechanisms remain to be determined. In this study, we found that IFN-λs exerted its anti-HTNV effect by activating Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway-mediated antiviral immunity in A549 cells. Simultaneously, IFN-λs downregulated suppressor of cytokine signaling proteins, which are the known negative feedback regulators of the JAK-STAT signaling pathway. Additionally, we demonstrated the role of IFN-λs-induced myxovirus resistance 2 (Mx2, also known as MxB) protein as a potential inhibitor for HTNV infection. These findings indicate that IFN-λs play an important role in cellular defenses against HTNV infection at an early stage and that human Mx2 may represent a potential therapeutic target for HTNV infection.
Asunto(s)
Antivirales/farmacología , Virus Hantaan/efectos de los fármacos , Fiebre Hemorrágica con Síndrome Renal/inmunología , Interferones/farmacología , Quinasas Janus/metabolismo , Proteínas de Resistencia a Mixovirus/metabolismo , Factores de Transcripción STAT/metabolismo , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Proteínas de Resistencia a Mixovirus/genética , Transducción de Señal/efectos de los fármacos , Células VeroRESUMEN
Increasing research has shown a link between viruses and miRNAs, such as miRNA-146a, in regulating virus infection and replication. In the current study, the association between miR-146a and hantaan virus (HTNV) infection in human umbilical vein endothelial cells (HUVECs) was investigated, with a focus on examining the expression of pro-inflammatory cytokines. The results showed that HTNV infection promoted the production of miR-146a in HUVECs and activated nuclear factor-κB (NF-κB) signaling, along with the upregulation of pro-inflammatory cytokines, including interleukin 8 (IL-8), C-C Motif Chemokine Ligand 5 (CCL5, also RANTES), interferon-inducible protein-10 (IP-10) and interferon beta (IFN-ß). Moreover, miR-146a exhibited a negative regulatory effect on the NF-κB pathway. Accordingly, a miR-146a inhibitor increased the expression of IL-8, CCL5, IP-10 and IFN-ß, whereas a miR-146a mimic reduced the levels of these cytokines. Consequently, exogenous transduction of miR-146a significantly enhanced HTNV replication in HUVEC cells. We also discovered that viral proteins (NP/GP) contributed to miR-146a expression via enhancement the activity of miR-146a promoter. In conclusion, these results imply the negative regulation of miR-146a on the production of HTNV-induced pro-inflammatory cytokines contributes to virus replication, which suggest that miR-146a may be regarded as a novel therapeutic target for HTNV infection.
Asunto(s)
Citocinas/inmunología , Células Endoteliales/inmunología , Células Endoteliales/virología , Virus Hantaan/inmunología , Fiebre Hemorrágica con Síndrome Renal/inmunología , MicroARNs/inmunología , Internalización del Virus , Células Cultivadas , Fiebre Hemorrágica con Síndrome Renal/patología , Fiebre Hemorrágica con Síndrome Renal/virología , Humanos , Mediadores de Inflamación/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
Schistosoma japonicum as a pathogeny requires dendritic cells to activate immune response. So, the research is to study the dynamic changes of CD3e-CD11c+ dendritic cells in mice infected with S. japonicum. Zero, 7, 28, 35, and 63 days were selected to study the variation of dendritic cells, and the proportions of CD3e-CD11c+ dendritic cells and CD86+ mature dendritic cells in spleens and bone marrow were tested by flow cytometry. As a result, the variation trends of dendritic cells in spleen and bone marrow are similar as follows: the proportions of CD3e-CD11c+ dendritic cells increased first and then decreased from day 35, but the percentages of CD86+ mature dendritic cells decreased from day 28 and increased in day 63. In vitro, cultured dendritic cells treated with SEA and SAWA were tested by flow cytometry, the variation trends of CD86 on dendritic cells are consistent with the results in days 28 and 63. Besides CD86, the expression of MHC-II also hints immune regulation. In conclusion, it is speculated that dendritic cells play a role of immune regulation through MHC-II and CD86 in S. japonicum infection. Immune regulation of dendritic cells is not only in favor of the survival of host and parasite but also can be used in the therapy for immune diseases.
Asunto(s)
Médula Ósea/inmunología , Células Dendríticas/inmunología , Schistosoma japonicum/inmunología , Esquistosomiasis Japónica/inmunología , Animales , Médula Ósea/parasitología , Antígeno CD11c/inmunología , Complejo CD3/inmunología , Células Dendríticas/parasitología , Citometría de Flujo , Humanos , Factores Inmunológicos , Ratones , Ratones Endogámicos C57BL , Schistosoma japonicum/parasitología , Schistosoma japonicum/fisiología , Esquistosomiasis Japónica/parasitología , Bazo/inmunologíaRESUMEN
Based on salting-out assisted liquid-liquid extraction (SALLE) and high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS), a simple, rapid pretreatment without complex clean-up for the determination of 22 veterinary drug residues in aquatic products was developed and validated. In order to improve the efficiency of the method, the key procedural parameters of SALLE were fabricated. Na2EDTA-Mcllvaine buffer/ACN was used as the extraction solvent, anhydrous MgSO4 and NaCl as the extraction salts. The relationship between extraction efficiency and logD was initially evaluated during the optimization process. This study was well validated in various aquatic samples such as bass, large yellow croaker, carp, and shrimp, the limits of detection (LOD) and accuracy for all compounds ranged from 0.5 to 1.0 µg/kg, 71.4% to 120%. This method has the advantages of rapidity, simplicity, low cost, and high efficiency, and has broad potential for risk monitoring and evaluation of veterinary antibiotics in aquatic products.
RESUMEN
The spindle assembly checkpoint (SAC) ensures chromosome segregation fidelity by manipulating unattached kinetochore-dependent assembly of the mitotic checkpoint complex (MCC). The MCC binds to and inhibits the anaphase promoting complex/cyclosome (APC/C) to postpone mitotic exit. However, the mechanism by which unattached kinetochores mediate MCC formation is not yet fully understood. Here, it is shown that CCDC68 is an outer kinetochore protein that preferentially localizes to unattached kinetochores. Furthermore, CCDC68 interacts with the SAC factor CDC20 to inhibit its autoubiquitination and MCC disassembly. Therefore, CCDC68 restrains APC/C activation to ensure a robust SAC and allow sufficient time for chromosome alignment, thus ensuring chromosomal stability. Hence, the study reveals that CCDC68 is required for CDC20-dependent MCC stabilization to maintain mitotic checkpoint activation.
RESUMEN
Transcriptional dysregulation is a prominent feature in leukemia. Here, we systematically surveyed transcription factor (TF) vulnerabilities in leukemia and uncovered TF clusters that exhibit context-specific vulnerabilities within and between different subtypes of leukemia. Among these TF clusters, we demonstrated that acute myeloid leukemia (AML) with high IRF8 expression was addicted to MEF2D. MEF2D and IRF8 form an autoregulatory loop via direct binding to mutual enhancer elements. One important function of this circuit in AML is to sustain PU.1/MEIS1 co-regulated transcriptional outputs via stabilizing PU.1's chromatin occupancy. We illustrated that AML could acquire dependency on this circuit through various oncogenic mechanisms that results in the activation of their enhancers. In addition to forming a circuit, MEF2D and IRF8 can also separately regulate gene expression, and dual perturbation of these two TFs leads to a more robust inhibition of AML proliferation. Collectively, our results revealed a TF circuit essential for AML survival.
RESUMEN
BACKGROUND: The role of the IL6 family members in organ fibrosis, including renal interstitial fibrosis (TIF), has been widely explored. However, few studies have ever simultaneously examined them in the same cohort of patients. Besides, the role of leukemia inhibitory factor (LIF) in TIF remains unclear. METHODS: RNA-seq data of kidney biopsies from chronic kidney disease (CKD) patients, in both public databases and our assays, were used to analyze transcript levels of IL6 family members. Two TIF mouse models, the unilateral ureteral obstruction (UUO) and the ischemia reperfusion injury (IRI), were employed to validate the finding. To assess the role of LIF in vivo, short hairpin RNA, lenti-GFP-LIF was used to knockdown LIF receptor (LIFR), overexpress LIF, respectively. LIF-neutralizing antibody was used in therapeutic studies. Whether urinary LIF could be used as a promising predictor for CKD progression was investigated in a prospective observation patient cohort. FINDINGS: Among IL6 family members, LIF is the most upregulated one in both human and mouse renal fibrotic lesions. The mRNA level of LIF negatively correlated with eGFR with the strongest correlation and the smallest P value. Baseline urinary concentrations of LIF in CKD patients predict the risk of CKD progression to end-stage kidney disease by Kaplan-Meier analysis. In mouse TIF models, knockdown of LIFR alleviated TIF; conversely, overexpressing LIF exacerbated TIF. Most encouragingly, visible efficacy against TIF was observed by administering LIF-neutralizing antibodies to mice. Mechanistically, LIF-LIFR-EGR1 axis and Sonic Hedgehog signaling formed a vicious cycle between fibroblasts and proximal tubular cells to augment LIF expression and promote the pro-fibrotic response via ERK and STAT3 activation. INTERPRETATION: This study discovered that LIF is a noninvasive biomarker for the progression of CKD and a potential therapeutic target of TIF. FUNDINGS: Stated in the Acknowledgements section of the manuscript.
Asunto(s)
Riñón , Insuficiencia Renal Crónica , Humanos , Ratones , Animales , Factor Inhibidor de Leucemia/genética , Riñón/metabolismo , Interleucina-6/genética , Estudios Prospectivos , Proteínas Hedgehog , Fibrosis , Insuficiencia Renal Crónica/patologíaRESUMEN
The ER tethers tightly to mitochondria and the mitochondrial protein FUNDC1 recruits Drp1 to ER-mitochondria contact sites, subsequently facilitating mitochondrial fission and preventing mitochondria from undergoing hypoxic stress. However, the mechanisms by which the ER modulates hypoxia-induced mitochondrial fission are poorly understood. Here, we show that USP19, an ER-resident deubiquitinase, accumulates at ER-mitochondria contact sites under hypoxia and promotes hypoxia-induced mitochondrial division. In response to hypoxia, USP19 binds to and deubiquitinates FUNDC1 at ER-mitochondria contact sites, which facilitates Drp1 oligomerization and Drp1 GTP-binding and hydrolysis activities, thereby promoting mitochondrial division. Our findings reveal a unique hypoxia response pathway mediated by an ER protein that regulates mitochondrial dynamics.
Asunto(s)
Dinaminas/genética , Endopeptidasas/genética , Proteínas de la Membrana/genética , Mitocondrias/genética , Proteínas Mitocondriales/genética , Retículo Endoplásmico/genética , Humanos , Hipoxia/genética , Dinámicas Mitocondriales/genética , Mitofagia/genética , Fosforilación/genética , Transducción de Señal/genéticaRESUMEN
Hantaviruses can cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and have led to public health threat in China. The pathogenesis of HFRS is complex and involves capillary leakage due to the infection of vascular endothelial cells. Accumulating evidence has demonstrated that hantavirus can induce apoptosis in many cells, but the mechanism remains unclear. Our studies showed that Hantaan virus (HTNV) infection could induce TNF-related apoptosis-inducing ligand (TRAIL) expression in primary human umbilical vein endothelial cells (HUVECs) and sensitize host cells toward TRAIL-mediated apoptosis. Furthermore, TRAIL interference could inhibit apoptosis and enhance the production of HTNV as well as reduce IFN-ß production, while exogenous TRAIL treatment showed reverse outcome: enhanced apoptosis and IFN-ß production as well as a lower level of viral replication. We also observed that nucleocapsid protein (NP) and glycoprotein (GP) of HTNV could promote the transcriptions of TRAIL and its receptors. Thus, TRAIL was upregulated by HTNV infection and then exhibited significant antiviral activities in vitro, and it was further confirmed in the HTNV-infected suckling mice model that TRAIL treatment significantly reduced viral load, alleviated virus-induced tissue lesions, increased apoptotic cells, and decreased the mortality. In conclusion, these results demonstrate that TRAIL-dependent apoptosis and IFN-ß production could suppress HTNV replication and TRAIL treatment might be a novel therapeutic target for HTNV infection.
Asunto(s)
Apoptosis/inmunología , Virus Hantaan/inmunología , Virus Hantaan/patogenicidad , Interacciones Microbiota-Huesped/inmunología , Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Animales , Animales Lactantes , Modelos Animales de Enfermedad , Femenino , Fiebre Hemorrágica con Síndrome Renal/inmunología , Fiebre Hemorrágica con Síndrome Renal/terapia , Fiebre Hemorrágica con Síndrome Renal/virología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Interferón beta/biosíntesis , Ratones , Ratones Endogámicos BALB C , Embarazo , ARN Interferente Pequeño/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/uso terapéutico , Carga Viral/inmunología , Replicación ViralRESUMEN
BACKGROUND: Gastric cancer is a severe disease with a high occurrence rate worldwide. And lncRNAs are demonstrated to be responsible for cancer growth and metastasis. So, it is of great importance to explore the lncRNAs involved mechanism of gastric cancer occurrence and development deeply. METHODS: Transfection was conducted to build over-expression and down-expression models. Moreover, RT-qPCR and western blot were used to detect the transcriptional and translational levels. The biological functions such as proliferation, migration and invasion of AGS cells were evaluated by MTT analysis, colony formation assay, scarification detection and transwell assay, respectively. The potential binding of miR-135b and its downstream and upstream molecules was validated by dual luciferase reporter gene assay or RIP. Also, the in-vivo mice model was further used to demonstrate the role of lncRNA PCAT18 in gastric cancer. RESULTS: PCAT18 down-expression promoted proliferation, migration and invasion of gastric cancer cells. Furtherly, over-expression of miR-135b also promoted these biological characteristics of AGS cells. Importantly, we found that PCAT18 could bind miR-135b which also was bound with CLDN11. We found that miR-135b is negatively correlated with CLDN11; PCAT18 and CLDN11 are positively correlated. Moreover, miR-135b mimics could down-regulate protein level of CLDN11, whereas CLDN11 could reverse this effect. In in-vivo experiment, PCAT18 over-expression restrained tumor growth and metastasis. CONCLUSIONS: Over-expressed lncRNA PCAT18 inhibits proliferation, migration and invasion of gastric cancer cells through regulation of miR-135b/CLDN11.
Asunto(s)
Proliferación Celular/genética , Claudinas/genética , MicroARNs/genética , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , ARN Largo no Codificante/genética , Neoplasias Gástricas/patología , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
CRISPR-based genetic screening has revolutionized cancer drug target discovery, yet reliable, multiplex gene editing to reveal synergies between gene targets remains a major challenge. Here, we present a simple and robust CRISPR-Cas12a-based approach for combinatorial genetic screening in cancer cells. By engineering the CRISPR-AsCas12a system with key modifications to the Cas protein and its CRISPR RNA (crRNA), we can achieve high efficiency combinatorial genetic screening. We demonstrate the performance of our optimized AsCas12a (opAsCas12a) through double knockout screening against epigenetic regulators. This screen reveals synthetic sick interactions between Brd9&Jmjd6, Kat6a&Jmjd6, and Brpf1&Jmjd6 in leukemia cells.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleasas/genética , Edición Génica , Regulación Leucémica de la Expresión Génica , Leucemia/genética , Animales , Proliferación Celular , Epigénesis Genética , Biblioteca de Genes , Ingeniería Genética , Genoma Humano , Células HEK293 , Humanos , Células K562 , Ratones , Células 3T3 NIH , Dominios Proteicos , ARN Guía de Kinetoplastida/genéticaRESUMEN
The endoplasmic reticulum (ER) is the largest membranous organelle, and its turnover ensures cellular homeostasis. The selective macroautophagy/autophagy of the ER (reticulophagy) guarantees the balance of ER turnover. However, the mechanism and physiological relevance of reticulophagy is largely unknown. Recently, we identified ATL3 (atlastin GTPase 3), generally considered to mediate ER fusion, as a receptor for reticulophagy. ATL3 specifically interacts with the GABARAP subfamily proteins of the Atg8-family, and this association is crucial for ATL3's role as a receptor for reticulophagy. Moreover, 2 hereditary sensory and autonomic neuropathies type 1 (HSANI)-associated mutations of ATL3 (Tyr192Cys and Pro338Arg) impair ATL3's binding to GABARAP and function in reticulophagy. Therefore, we illuminate a new function of ATL3 in reticulophagy and the potential physiological relevance of reticulophagy in neurodegenerative diseases.
Asunto(s)
Autofagia , GTP Fosfohidrolasas/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Humanos , Ratones Noqueados , Modelos BiológicosRESUMEN
The endoplasmic reticulum (ER) consists of the nuclear envelope and both peripheral ER sheets and a peripheral tubular network [1, 2]. In response to physiological or pathological conditions, receptor-mediated selective ER-phagy, engulfing specific ER subdomains or components, is essential for ER turnover and homeostasis [3-6]. Four mammalian receptors for ER-phagy have been reported: FAM134B [7], reticulon 3 (RTN3) [8], SEC62 [9], and CCPG1 [10]. However, these ER-phagy receptors function in subcellular- and tissue- or physiological- and pathological-condition-specific manners, so the diversity of ER-phagy receptors and underlying mechanisms remain largely unknown [3, 4]. Atlastins (ATL1, ATL2, and ATL3), in mammals, are a class of membrane-bound, dynamin-like GTPases that function in ER fusion [11, 12]. ATL1 is expressed mainly in the central nervous system, while ATL2 and ATL3 are more ubiquitously distributed [13]. Recent studies showed that ATL2 mainly affects ER morphology by promoting ER fusion, whereas alterations in ER morphology are hardly detectable after ATL3 depletion [14, 15]. Here, we show that ATL3 functions as a receptor for ER-phagy, promoting tubular ER degradation upon starvation. ATL3 specifically binds to GABARAP, but not LC3, subfamily proteins via 2 GABARAP interaction motifs (GIMs). ATL3-GABARAP interaction is essential for ATL3 to function in ER-phagy. Moreover, hereditary sensory and autonomic neuropathy type I (HSAN I)-associated ATL3 mutations (Y192C and P338R) disrupt ATL3's association with GABARAP and impair ATL3's function in ER-phagy, suggesting that defective ER-phagy is involved in HSAN I. Therefore, we reveal a new ATL3 function for GABARAP-mediated ER-phagy in the degradation of tubular ER.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Autofagia/genética , GTP Fosfohidrolasas/genética , Proteínas Asociadas a Microtúbulos/genética , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Células COS , Chlorocebus aethiops , Retículo Endoplásmico/fisiología , GTP Fosfohidrolasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
The ER is composed of distinct structures like tubules, matrices, and sheets, all of which are important for its various functions. However, how these distinct ER structures, especially the perinuclear ER sheets, are formed remains unclear. We report here that the ER membrane protein Climp63 and the ER luminal protein calumenin-1 (Calu1) collaboratively maintain ER sheet morphology. We show that the luminal length of Climp63 is positively correlated with the luminal width of ER sheets. Moreover, the lumen-only mutant of Climp63 dominant-negatively narrows the lumen of ER sheets, demonstrating that Climp63 acts as an ER luminal bridge. We also reveal that Calu1 specifically interacts with Climp63 and antagonizes Climp63 in terms of both ER sheet distribution and luminal width. Together, our data provide insight into how the structure of ER sheets is maintained and regulated.