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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system has emerged as a powerful tool for knock-in of DNA fragments via donor plasmid and homology-independent DNA repair mechanism; however, conventional integration includes unnecessary plasmid backbone and may result in the unfaithful expression of the modified endogenous genes. Here, we report an efficient and precise CRISPR/Cas9-mediated integration strategy using a donor plasmid that harbors 2 of the same cleavage sites that flank the cassette at both sides. After the delivery of donor plasmid, together with Cas9 mRNA and guide RNA, into cells or fertilized eggs, concurrent cleavages at both sides of the exogenous cassette and the desired chromosomal site result in precise targeted integration without plasmid backbone. We successfully used this approach to precisely integrate the EGFP reporter gene into the myh6 locus or the GAPDH locus in Xenopus tropicalis or human cells, respectively. Furthermore, we demonstrate that replacing conventional terminators with the endogenous 3UTR of target genes in the cassette greatly improves the expression of reporter gene after integration. Our efficient and precise method will be useful for a variety of targeted genome modifications, not only in X. tropicalis, but also in mammalian cells, and can be readily adapted to many other organisms.-Mao, C.-Z., Zheng, L., Zhou, Y.-M., Wu, H.-Y., Xia, J.-B., Liang, C.-Q., Guo, X.-F., Peng, W.-T., Zhao, H., Cai, W.-B., Kim, S.-K., Park, K.-S., Cai, D.-Q., Qi, X.-F. CRISPR/Cas9-mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis.
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CXCL10, the chemokine with potent chemotactic activity on immune cells and other non-immune cells expressing its receptor CXCR3, has been demonstrated to involve in myocardial infarction, which was resulted from hypoxia/ischemia. The cardiac microvascular endothelial cells (CMECs) are the first cell type which is implicated by hypoxia/ischemia. However, the potential molecular mechanism by which hypoxia/ischemia regulates the expression of CXCL10 in CMECs remains unclear. In the present study, the expression of CXCL10 was firstly examined by real-time PCR and ELISA analysis. Several potential binding sites (BS) for transcription factors including NF-kappaB (NFkB), HIF1 alpha (HIF1α) and FoxO3a were identified in the promoter region of CXCL10 gene from -2000 bp to -1 bp using bioinformatics software. Luciferase reporter gene vectors for CXCL10 promoter and for activation of above transcription factors were constructed. The activation of NFkB, hypoxia-inducible transcription factor-1 alpha (HIF-1α) and FoxO3a was also analyzed by Western blotting. It was shown that the production of CXCL10 in CMECs was significantly increased by hypoxia/ischemia treatment, in parallel with the activation of CXCL10 promoter examined by reporter gene vector system. Furthermore, transcription factors including NFkB, HIF1α and FoxO3a were activated by hypoxia/ischemia in CMECs. However, over-expression of NFkB, but not that of HIF1α or FoxO3a, significantly promoted the activation of CXCL10 promoter reporter gene. These findings indicated that CXCL10 production in CMECs was significantly increased by hypoxia/ischemia, at least in part, through activation of NFkB pathway and subsequently binding to CXCL10 promoter, finally promoted the transcription of CXCL10 gene.
Assuntos
Quimiocina CXCL10/metabolismo , Vasos Coronários/citologia , Células Endoteliais/metabolismo , NF-kappa B/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Western Blotting , Hipóxia Celular , Células Cultivadas , Quimiocina CXCL10/genética , Ensaio de Imunoadsorção Enzimática , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia , NF-kappa B/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
CXCL10 is a chemokine with potent chemotactic activity for immune and non-immune cells expressing its receptor CXCR3. Previous studies have demonstrated that CXCL10 is involved in myocardial infarction. However, the role of CXCL10 in cardiac microvascular endothelial cell (CMEC) regulation and related mechanisms remains unclear. In this study, we investigated the effects of CXCL10 on the CMEC migration and explored its potential molecular mechanism by wound healing, cell proliferation and viability analysis. Furthermore, migration-related signaling pathways, including FAK, Erk, p38 and Smad, were examined by Western blotting. We found that CXCL10 significantly promotes CMEC migration under normal conditions and during hypoxia/ischemia. However, no significant differences in CMEC proliferation and viability were observed with or without CXCL10 treatment. CXCL10-mediated CMEC migration was greatly blocked by treatment with an anti-CXCR3 antibody. Although CXCL10 treatment promoted phosphorylation and activation of the FAK, Erk, and p38 pathways during hypoxia/ischemia, CXCL10-mediated CMEC migration was significantly blocked by p38 and FAK inhibitors, but not by an Erk inhibitor. Furthermore, CXCL10-mediated FAK activation was suppressed by the p38 inhibitor. These findings indicated that the CXCL10/CXCR3 pathway promotes the migration of CMECs under normal conditions and during hypoxia/ischemia in a proliferation-independent manner, at least in part, through regulation of the p38/FAK pathways.
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Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL10/farmacologia , Células Endoteliais/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Receptores CXCR3/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Western Blotting , Hipóxia Celular , Células Cultivadas , Vasos Coronários/citologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Quinase 1 de Adesão Focal/antagonistas & inibidores , Expressão Gênica/efeitos dos fármacos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores CXCR3/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Triple-negative breast cancer (TNBC) is a unique breast cancer subtype characterized by a lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. Since TNBC lacks ER, PR, and HER2, there are currently no drugs that specifically target TNBC. Therefore, the development of new drugs or effective treatment strategies to target TNBC has become an urgent clinical need. Research has shown that the application of histone deacetylase (HDAC) inhibitors and DNA methyltransferase (DNMT) inhibitors leads to genomic and epigenomic instability. This, in turn, triggers the activation of pattern recognition receptors (PRRs) and subsequently activates downstream interferon (IFN) signalling pathways. In this study, the bifunctional HDAC and DNMT inhibitor J208 exhibited antitumour activity in TNBC cell lines. J208 effectively induced apoptosis and cell cycle arrest at the G0/G1 phase, inhibiting cell migration and invasion in TNBC. Moreover, this bifunctional inhibitor induced the expression of endogenous retroviruses (ERVs) and elicited a viral mimicry response, which increased the intracellular levels of double-stranded RNA (dsRNA) to activate the innate immune signalling pathway in TNBC. In summary, we demonstrated that the bifunctional inhibitor J208, which is designed to inhibit HDAC and DNMT, has potent anticancer effects, providing a new research basis for reactivating antitumour immunity by triggering innate immune signalling and offering a promising strategy for TNBC treatment.
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Inibidores de Histona Desacetilases , Imunidade Inata , Neoplasias de Mama Triplo Negativas , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/imunologia , Humanos , Linhagem Celular Tumoral , Imunidade Inata/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Feminino , Apoptose/efeitos dos fármacos , Antineoplásicos/farmacologia , Movimento Celular/efeitos dos fármacos , AnimaisRESUMO
PURPOSE: Leukaemia remains a major contributor to global mortality, representing a significant health risk for a substantial number of cancer patients. Despite notable advancements in the field, existing treatments frequently exhibit limited efficacy or recurrence. Here, we explored the potential of abolishing HVEM (herpes virus entry mediator, TNFRSF14) expression in tumours as an effective approach to treat acute lymphoblastic leukaemia (ALL) and prevent its recurrence. METHODS: The clinical correlations between HVEM and leukaemia were revealed by public data analysis. HVEM knockout (KO) murine T cell lymphoblastic leukaemia cell line EL4 were generated using CRISPR-Cas9 technology, and syngeneic subcutaneous tumour models were established to investigate the in vivo function of HVEM. Immunohistochemistry (IHC), RNA-seq and flow cytometry were used to analyse the tumour immune microenvironment (TIME) and tumour draining lymph nodes (dLNs). Immune functions were investigated by depletion of immune subsets in vivo and T cell functional assays in vitro. The HVEM mutant EL4 cell lines were constructed to investigate the functional domain responsible for immune escape. RESULTS: According to public databases, HVEM is highly expressed in patients with ALL and acute myeloid leukemia (AML) and is negatively correlated with patient prognosis. Genetic deletion of HVEM in EL4 cells markedly inhibited tumour progression and prolonged the survival of tumour-bearing mice. Our experiments proved that HVEM exerted its immunosuppressive effect by inhibiting antitumour function of CD8+ T cell through CRD1 domain both in vivo and in vitro. Additionally, we identified a combination therapy capable of completely eradicating ALL tumours, which induces immune memory toward tumour protection. CONCLUSIONS: Our study reveals the potential mechanisms by which HVEM facilitates ALL progression, and highlights HVEM as a promising target for clinical applications in relapsed ALL therapy.
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Immunosuppressive myeloid cells hinder immunotherapeutic efficacy in tumors, but the precise mechanisms remain undefined. Here, by performing single-cell RNA sequencing in colorectal cancer tissues, we found tumor-associated macrophages and granulocytic myeloid-derived suppressor cells increased most compared to their counterparts in normal tissue and displayed the highest immune-inhibitory signatures among all immunocytes. These cells exhibited significantly increased expression of immunoreceptor tyrosine-based inhibitory motif-bearing receptors, including SIRPA. Notably, Sirpa-/- mice were more resistant to tumor progression than wild-type mice. Moreover, Sirpα deficiency reprogramed the tumor microenvironment through expansion of TAM_Ccl8hi and gMDSC_H2-Q10hi subsets showing strong antitumor activity. Sirpa-/- macrophages presented strong phagocytosis and antigen presentation to enhance T cell activation and proliferation. Furthermore, Sirpa-/- macrophages facilitated T cell recruitment via Syk/Btk-dependent Ccl8 secretion. Therefore, Sirpα deficiency enhances innate and adaptive immune activation independent of expression of CD47 and Sirpα blockade could be a promising strategy to improve cancer immunotherapy efficacy.
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Antígeno CD47 , Neoplasias Colorretais , Camundongos , Animais , Antígeno CD47/genética , Antígeno CD47/metabolismo , Fagocitose , Macrófagos/metabolismo , Células Mieloides/metabolismo , Neoplasias Colorretais/patologia , Microambiente TumoralRESUMO
Polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts), a group of isoenzymes that initiate mucin-type O-glycosylation, have been shown to mediate tumor growth and metastasis in various cancer types. However, data on the clinical significance and features of GalNAc-Ts remain scant. Here, we used Oncomine and The Cancer Genome Atlas (TCGA) databases to analyze the transcription and survival effect of GALNTs (N-acetylgalactosaminyltransferase genes) in pan-cancer. The data showed that the GALNTs were aberrantly expressed in various human cancers and significantly associated with patients' clinical outcomes. The expression of 13 GALNTs were correlated with prognosis in brain low grade glioma (LGG) patients. In addition, based on the expression profiles of GALNT family genes in TCGA-LGG dataset, we identified 2 molecular subtypes (cluster1/2) by consensus clustering and analyzed tumor heterogeneity. Our results demonstrated that cluster 2 group was associated with poor prognosis, CD8+ T cells, macrophages and DCs infiltration, up-regulated expression of immune checkpoints, and higher tumor immune dysfunction and exclusion score, indicating that GalNAc-Ts might contribute to tumor immune escape. Furthermore, we employed LASSO regression and time-dependent ROC analysis to construct a GALNTs-related prognostic signature with the TCGA-LGG dataset, and then validated the signature using 2 external cohorts. Taken together, our study successfully developed a novel prognostic biomarker for LGG and provides a basis for personalized immunotherapy in brain cancer.
This study successfully developed a novel prognostic biomarker for LGG and provides a basis for personalized immunotherapy in brain cancer.
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Glioma , N-Acetilgalactosaminiltransferases , Biomarcadores , Linfócitos T CD8-Positivos/metabolismo , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Humanos , Isoenzimas , Mucinas , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Peptídeos , PrognósticoRESUMO
The forkhead-box transcription factors of O subfamily (FOXO) play important roles in regulation of various biological functions. We cloned foxo1, foxo3, foxo4, and foxo6 from Xenopus tropicalis (hereafter X. tropicalis), and examined their expression in embryos and adult tissues. Maternal transcripts of foxo1 and foxo3 genes are detected within the animal half of the early embryo, their zygotic transcripts show distinct patterns. At late tailbud stages, foxo1 expression is observed mainly in eye, brain, branchial arches, and pronephros. In addition to eye, brain, branchial arches and pronephros, foxo3 expression is also evident in heart and somites. Foxo4 expression was not detected in oocytes. At late tailbud stages, foxo4 is mainly expressed in eye, brain, branchial arches and otic vesicle. Foxo6 expression was not detectable until stage 36, with a specific expression in nasal pits. Obvious expression of foxo1, foxo3 and foxo4, but not foxo6, is detected by RT-PCR both in oocytes and in embryos at examined stages. The expression of foxo1, foxo3 and foxo4 is observed in all tested adult tissues including heart, muscle, liver, lung, stomach and small intestine, while foxo6 is only detectable in stomach and small intestine. The differential expression pattern of foxo genes suggests that they exert distinct functions during embryonic development and in various organs of X. tropicalis.
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Proteínas de Anfíbios/genética , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Anfíbios/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Brônquios/embriologia , Brônquios/metabolismo , Olho/embriologia , Olho/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Coração/embriologia , Rim/embriologia , Rim/metabolismo , Mesoderma/embriologia , Mesoderma/metabolismo , Miocárdio/metabolismo , XenopusRESUMO
AIMS: This study aims to investigate the pathophysiological role and mechanism of pigment epithelium-derived factor (PEDF) deletion in ovarian damage. METHODS: Female PEDF-knockout mice and their wild-type littermates were used in this study. Relevant tests were performed at 8-10â¯weeks or 32â¯weeks of age. KEY FINDINGS: Compared to the wild-type mice, the PEDF-knockout mice showed diminished ovarian reserve (DOR), worse ovum quality after injection to induce controlled ovarian stimulation, increased serum follicle stimulating hormone (FSH) level and an follicle stimulating hormone/luteinizing hormone (FSH/LH) ratio. Moreover, severe ovarian oxidative damage was found in ovaries of PEDF-knockout mice that mainly manifested as an accumulation of reactive oxygen species (ROS), NFE2-related factor 2 (Nrf2) pathway activation, significantly upregulated expression of ROS-generating genes. Correspondingly, the PEDF-knockout mice exhibited lipid metabolism disorder and insulin resistance, which mainly manifested as obesity, abdominal fat accumulation, adipocyte enlargement, severe ectopic fat deposition, dyslipidemia, changes in adipokine levels, hyperglycemia, hyperinsulinemia, impaired glucose tolerance, impaired insulin tolerance and significantly declined protein kinase B (Akt) phosphorylation levels. SIGNIFICANCE: Loss of PEDF leads to ovarian oxidative damage accompanied by DOR in mice, this is related to PEDF deficiency induced severe insulin resistance and lipid metabolism disorder. Therefore, PEDF may be a potential target for the treatment of diseases related to ovarian oxidative damage.
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Proteínas do Olho/genética , Fatores de Crescimento Neural/genética , Reserva Ovariana/fisiologia , Ovário/fisiopatologia , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Serpinas/genética , Gordura Abdominal/metabolismo , Animais , Proteínas do Olho/metabolismo , Feminino , Resistência à Insulina/genética , Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Crescimento Neural/metabolismo , Obesidade/patologia , Reserva Ovariana/genética , Estresse Oxidativo/genética , Serpinas/metabolismo , Regulação para CimaRESUMO
Recent studies suggest that ribosome-binding protein 1 (RRBP1) is involved in multiple diseases such as tumorigenesis and cardiomyopathies. However, its function during embryonic development remains largely unknown. We searched Xenopus laevis database with human RRBP1 protein sequence and identified two cDNA sequences encoding Xenopus orthologs of RRBP1 including rrbp1a (NM_001089623) and rrbp1b (NM_001092468). Both genes were firstly detected at blastula stage 8 with weak signals in animal hemisphere by whole mount in situ hybridization. Evident expression of rrbp1 was mainly detected in cement gland and notochord at neurula and tailbud stages. Heart expression of rrbp1 was detected at stage 36. RT-PCR results indicated that very weak expression of rrbp1a was firstly detected in oocytes, followed by increasing expression until stage 39. Differently, very weak expression of rrbp1b was firstly observed at stage 2, and then maintained at a lower level to stage 17 followed by an intense expression from stages 19-39. Moreover, both expression profiles were also different in adult tissues. This study reports Xenopus rrbp1 expression during early embryonic development and in adult tissues. Our study will facilitate the functional analysis of Rrbp1 family during embryonic development.
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Proteínas de Transporte/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Miocárdio/metabolismo , Transcriptoma , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis/genéticaRESUMO
Vascular endothelial growth factor (VEGF) plays important roles in improvement of cardiac function following myocardial infarction (MI). However, the lack of a steerable delivery system of VEGF targeting the infarcted myocardium reduces the therapeutic efficacy and safety. Here, we constructed a series of lentiviral vector systems which could express a fusion protein consisted of a collagen-binding domain (CBD) and hVEGF (CBDhVEGF), under the control of 5HRE-hCMVmp (5HRE), the hypoxia-inducible promoter consists of five copies of the hypoxia-responsive element (HRE) and a human cytomegalovirus minimal promoter (hCMVmp). We demonstrated that 5HRE has the comparable ability to strongly drive CBDhVEGF under hypoxic condition as the ubiquitous CMV promoter, but it can hardly drive target gene under normoxic condition. 5HRE-drived CBDhVEGF specifically bound to type I collagen and significantly promoted the viability of HUVEC cells. Moreover, after injection of lentivirus into heart of mouse with MI, CBDhVEGF was mainly retained in infarcted myocardium where containing rich collagen and significantly improved angiogenesis and cardiac function when compared with hVEGF. Moreover, CBDhVEGF mediated by lentivirus has little leakage from infarcted zone into blood than hVEGF. Taken together, our results indicate that 5HRE-CBDhVEGF lentiviral vector system could improve cardiac function in the collagen-targeting and hypoxia-inducible manners.