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1.
Cell ; 184(1): 92-105.e16, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33147445

RESUMO

To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.


Assuntos
/genética , Interações Hospedeiro-Patógeno , /fisiologia , Células A549 , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Vias Biossintéticas , Colesterol/biossíntese , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endossomos/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes/métodos , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Interferência de RNA , Análise de Célula Única , Carga Viral/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/genética
2.
Proc Natl Acad Sci U S A ; 117(37): 23182-23190, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32873645

RESUMO

Enzyme turnover numbers (k cats) are essential for a quantitative understanding of cells. Because k cats are traditionally measured in low-throughput assays, they can be inconsistent, labor-intensive to obtain, and can miss in vivo effects. We use a data-driven approach to estimate in vivo k cats using metabolic specialist Escherichia coli strains that resulted from gene knockouts in central metabolism followed by metabolic optimization via laboratory evolution. By combining absolute proteomics with fluxomics data, we find that in vivo k cats are robust against genetic perturbations, suggesting that metabolic adaptation to gene loss is mostly achieved through other mechanisms, like gene-regulatory changes. Combining machine learning and genome-scale metabolic models, we show that the obtained in vivo k cats predict unseen proteomics data with much higher precision than in vitro k cats. The results demonstrate that in vivo k cats can solve the problem of inconsistent and low-coverage parameterizations of genome-scale cellular models.


Assuntos
Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Técnicas de Inativação de Genes/métodos , Genoma/genética , Cinética , Aprendizado de Máquina , Modelos Biológicos , Proteômica/métodos
3.
Nat Protoc ; 15(10): 3478-3498, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32958931

RESUMO

Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR gene knockout methods in cell lines have relied on frameshifts caused by stochastic insertion/deletion in all alleles. However, this method is inefficient for genes with high copy number due to polyploidy or gene amplification because frameshifts in all alleles can be difficult to generate and detect. Here we describe a homology-directed insertion method to knockout genes in the polyploid Drosophila S2R+ cell line. This protocol allows generation of homozygous mutant cell lines using an insertion cassette which autocatalytically generates insertion mutations in all alleles. Knockout cells generated using this method can be directly identified by PCR without a need for DNA sequencing. This protocol takes 2-3 months and can be applied to other polyploid cell lines or high-copy-number genes.


Assuntos
Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Alelos , Animais , Sequência de Bases/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Drosophila/genética , Endonucleases/metabolismo , Homozigoto , Poliploidia , RNA Guia/genética
4.
Nucleic Acids Res ; 48(18): e108, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32941625

RESUMO

The auxin-inducible degron (AID) system enables rapid depletion of target proteins within the cell by applying the natural auxin IAA. The AID system is useful for investigating the physiological functions of essential proteins; however, this system generally requires high dose of auxin to achieve effective depletion in vertebrate cells. Here, we describe a super-sensitive AID system that incorporates the synthetic auxin derivative 5-Ad-IAA and its high-affinity-binding partner OsTIR1F74A. The super-sensitive AID system enabled more than a 1000-fold reduction of the AID inducer concentrations in chicken DT40 cells. To apply this system to various mammalian cell lines including cancer cells containing multiple sets of chromosomes, we utilized a single-step method where CRISPR/Cas9-based gene knockout is combined with insertion of a pAID plasmid. The single-step method coupled with the super-sensitive AID system enables us to easily and rapidly generate AID-based conditional knockout cells in a wide range of vertebrate cell lines. Our improved method that incorporates the super-sensitive AID system and the single-step method provides a powerful tool for elucidating the roles of essential genes.


Assuntos
Técnicas de Inativação de Genes/métodos , Ácidos Indolacéticos/química , Proteínas de Plantas/genética , Proteólise , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Galinhas , Humanos , Oryza/metabolismo
5.
Transplantation ; 104(8): 1566-1573, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32732833

RESUMO

BACKGROUND: Xenogeneic organ transplantation has been proposed as a potential approach to fundamentally solve organ shortage problem. Xenogeneic immune responses across species is one of the major obstacles for clinic application of xeno-organ transplantation. The generation of glycoprotein galactosyltransferase α 1, 3 (GGTA1) knockout pigs has greatly contributed to the reduction of hyperacute xenograft rejection. However, severe xenograft rejection can still be induced by xenoimmune responses to the porcine major histocompatibility complex antigens swine leukocyte antigen class I and class II. METHODS: We simultaneously depleted GGTA1, ß2-microglobulin (ß2M), and major histocompatibility complex class II transactivator (CIITA) genes using clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins technology in Bamma pig fibroblast cells, which were further used to generate GGTA1ß2MCIITA triple knockout (GBC-3KO) pigs by nuclear transfer. RESULTS: The genotype of GBC-3KO pigs was confirmed by polymerase chain reaction and Sanger sequencing, and the loss of expression of α-1,3-galactose, SLA-I, and SLA-II was demonstrated by flow cytometric analysis using fluorescent-conjugated lectin from bandeiraea simplicifolia, anti-ß2-microglobulin, and swine leukocyte antigen class II DR antibodies. Furthermore, mixed lymphocyte reaction assay revealed that peripheral blood mononuclear cells from GBC-3KO pigs were significantly less effective than (WT) pig peripheral blood mononuclear cells in inducing human CD3CD4 and CD3CD8 T-cell activation and proliferation. In addition, GBC-3KO pig skin grafts showed a significantly prolonged survival in immunocompetent C57BL/6 mice, when compared with wild-type pig skin grafts. CONCLUSIONS: Taken together, these results demonstrate that elimination of GGTA1, ß2M, and CIITA genes in pigs can effectively alleviate xenogeneic immune responses and prolong pig organ survival in xenogenesis. We believe that this work will facilitate future research in xenotransplantation.


Assuntos
Rejeição de Enxerto/prevenção & controle , Xenoenxertos/imunologia , Transplante de Órgãos/métodos , Transplante Heterólogo/métodos , Aloenxertos/provisão & distribução , Animais , Animais Geneticamente Modificados/imunologia , Sistemas CRISPR-Cas/genética , Modelos Animais de Doenças , Feminino , Galactosiltransferases/genética , Galactosiltransferases/imunologia , Técnicas de Inativação de Genes/métodos , Genes MHC da Classe II/genética , Genes MHC da Classe II/imunologia , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Xenoenxertos/transplante , Humanos , Masculino , Camundongos , Transplante de Órgãos/efeitos adversos , Suínos/genética , Suínos/imunologia , Transplante Heterólogo/efeitos adversos , Microglobulina beta-2/genética , Microglobulina beta-2/imunologia
6.
Sci Rep ; 10(1): 13332, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770110

RESUMO

Although electroporation has been widely accepted as the main gene transfer tool, there is still considerable scope to improve the electroporation efficiency of exogenous DNAs into primary cells. Here, we developed a square-wave pulsing protocol using OptiMEM-GlutaMAX for highly efficient transfection of murine embryonic fibroblasts (MEF) and induced pluripotency stem (iPS) cells using reporter genes as well as gRNA/Cas9-encoding plasmids. An electrotransfection efficiency of > 95% was achieved for both MEF and iPS cells using reporter-encoding plasmids. The protocol was efficient for plasmid sizes ranging from 6.2 to 13.5 kb. Inducing the error prone non-homologous end joining repair by gRNA/Cas9 plasmid transfection, a high rate of targeted gene knockouts of up to 98% was produced in transgenic cells carrying a single-copy of Venus reporter. Targeted deletions in the Venus transgene were efficiently (up to 67% deletion rate) performed by co-electroporation of two gRNA-encoding plasmids. We introduced a plasmid electrotransfection protocol which is straight-forward, cost-effective, and efficient for CRISPRing murine primary cells. This protocol is promising to make targeted genetic engineering using the CRISPR/Cas9 plasmid system.


Assuntos
Eletroporação/métodos , Fibroblastos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Transfecção/métodos , Animais , Animais Geneticamente Modificados/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Reparo do DNA por Junção de Extremidades/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Genes Reporter/genética , Camundongos , Plasmídeos/genética , RNA Guia/genética , Transgenes/genética
7.
Sci Rep ; 10(1): 10565, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601291

RESUMO

CRISPR/Cas9 has revolutionized the genome-editing field. So far, successful application in human adipose tissue has not been convincingly shown. We present a method for gene knockout using electroporation in preadipocytes from human adipose tissue that achieved at least 90% efficiency without any need for selection of edited cells or clonal isolation. We knocked out the FKBP5 and PPARG genes in preadipocytes and studied the resulting phenotypes. PPARG knockout prevented differentiation into adipocytes. Conversely, deletion of FKBP51, the protein coded by the FKBP5 gene, did not affect adipogenesis. Instead, it markedly modulated glucocorticoid effects on adipocyte glucose metabolism and, furthermore, we show some evidence of altered transcriptional activity of glucocorticoid receptors. This has potential implications for the development of insulin resistance and type 2 diabetes. The reported method is simple, easy to adapt, and enables the use of human primary preadipocytes instead of animal adipose cell models to assess the role of key genes and their products in adipose tissue development, metabolism and pathobiology.


Assuntos
Adipócitos/metabolismo , Diferenciação Celular/genética , Edição de Genes/métodos , Adipogenia/genética , Tecido Adiposo/metabolismo , Adulto , Idoso , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Feminino , Técnicas de Inativação de Genes/métodos , Humanos , Pessoa de Meia-Idade , PPAR gama/genética , Estudo de Prova de Conceito , Proteínas de Ligação a Tacrolimo/genética
8.
Mol Immunol ; 124: 100-108, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32554100

RESUMO

Programmed cell death protein 1 (PD1) is a cell-surface receptor that plays a vital regulatory role in suppressing inflammatory T cell activity; therefore, it is an ideal target for T cell-redirecting therapies. Here, we describe a cynomolgus macaque model for studying the transfer of PD1-modified T cells. We developed the first T cell engager targeting the disruption of PD1 by electroporation of plasmids encoding sgRNA and Cas9. There were no significant differences between mock T cells and PD1-knockout (PD1-KO) T cells in terms of cell viability, T cell signature marker expression, cell apoptosis, or cell cycling during prolonged in vitro culture. However, in a mixed lymphocyte reaction, PD1-KO T cells exhibited increased proliferation for both CD4+ and CD8+T cells and enhanced IFNγ release. We adoptively transferred autologous PD1-KO T cells into three cynomolgus monkeys. The PD1-KO T cells did not cause overt toxicity as measured by evaluating body weight, hematological parameters, and blood chemistry parameters. Histopathological analyses of tissues showed no lesions related to the infused PD1-KO T cells. Our findings demonstrate the utility of cynomolgus monkeys in expanding PD1-KO T cells and evaluating the safety of this immunotherapy and provide a new strategy for T cell-based adoptive cell therapies.


Assuntos
Receptor de Morte Celular Programada 1/deficiência , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Técnicas de Inativação de Genes/métodos , Macaca fascicularis , Linfócitos T/transplante
9.
Sci Rep ; 10(1): 9957, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561775

RESUMO

Severe immunodeficient mice are widely used to examine human and animal cells behaviour in vivo. However, mice are short-lived and small in size; while large animals require specific large-scale equipment. Rabbits are also commonly employed as experimental models and are larger than mice or rats, easy to handle, and suitable for long-term observational and pre-clinical studies. Herein, we sought to develop and maintain stable strains of rabbits with X-linked severe combined immunodeficiency (X-SCID) via the CRISPR/Cas9 system targeting Il2rg. Consequently, X-SCID rabbits presented immunodeficient phenotypes including the loss of T and B cells and hypoplasia of the thymus. Further, these rabbits exhibited a higher success rate with engraftments upon allogeneic transplantation of skin tissue than did wild type controls. X-SCID rabbits could be stably maintained for a minimum of four generations. These results indicate that X-SCID rabbits are effective animals for use in a non-rodent model of severe immunodeficiency.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Animais , Linfócitos B/imunologia , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , Feminino , Técnicas de Inativação de Genes/métodos , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/imunologia , Coelhos , Pele/imunologia , Linfócitos T/imunologia , Timo/imunologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia
10.
Biol Res ; 53(1): 18, 2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32349783

RESUMO

BACKGROUND: Cisplatin resistance (DDP-resistance) remains one of the major causes of poor prognosis in females with ovarian cancer. Long non-coding RNAs (lncRNAs) have been shown to participate in the regulation of cellular processes, including chemoresistance. The aim of this study was to explore the role of HOX transcript antisense RNA (HOTAIR) in DDP-resistant ovarian cancer cells. METHODS: DDP-resistant ovarian cancer cell lines (SKOV3/DDP and A2780/DDP) were established. Real-time PCR, western blot, dual-luciferase reporter assay, and flow cytometry were then used to evaluate the effect of HOTAIR/miR-138-5p axis on chemoresistance of DDP-resistant ovarian cancer cells to DDP. RESULTS: We found that HOTAIR was upregulated in DDP-resistant cells, while miR-138-5p was downregulated. Knockdown of HOTAIR increased the expression of miR-138-5p in DDP-resistant cells and miR-138-5p is directly bound to HOTAIR. Upregulation of miR-138-5p induced by HOTAIR siRNA or by its mimics enhanced the chemosensitivity of DDP-resistant cells and decreased the expression of EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) and SIRT1 (sirtuin 1). Furthermore, the HOTAIR silencing-induced chemosensitivity of DDP-resistant cells was weakened by miR-138-5p inhibitor. CONCLUSIONS: These data demonstrate that HOTAIR acts as a sponge of miR-138-5p to prevent its binding to EZH2 and SIRT1, thereby promoting DDP-resistance of ovarian cancer cells. Our work will shed light on the development of therapeutic strategies for ovarian cancer treatment.


Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Ovarianas/genética , RNA Longo não Codificante/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes/métodos , Humanos , MicroRNAs/antagonistas & inibidores , Reação em Cadeia da Polimerase em Tempo Real , Sirtuína 1/antagonistas & inibidores , Regulação para Cima
11.
PLoS One ; 15(5): e0232985, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32413049

RESUMO

Receptor tyrosine kinases, such as VEGFR, PDGFR and EGFR, play important roles in renal cancer. In this study, we investigated EGFR knockout as a therapeutic approach in renal cell carcinoma (RCC). We showed that a renal cell carcinoma cell line (RC21) has higher expression of EGFR as compared to other frequently used cell lines such as HEK293, A549, Hela and DLD1. Ablation of EGFR by CRISPR/Cas9 significantly restrained tumor cell growth and activated the MAPK (pERK1/2) pathway. The VEGFR and PDGFR inhibitor, sunitinib, attenuated the expression of MAPK (pERK1/2) and pAKT induced by EGFR loss and further inhibited EGFR-/- cell proliferation. We showed that loss of EGFR eventually leads to resistance to SAHA and cisplatin. Furthermore, EGFR loss induced G2/M phase arrest and resulted in an increased resistance to TNF-related apoptosis-inducing ligand (TRAIL) in renal cell carcinoma. Thus, ablation of overexpressed EGFR by CRISPR/Cas9 alone or in combination with sunitinib may be a new treatment option for renal cell carcinoma.


Assuntos
Sistemas CRISPR-Cas , Carcinoma de Células Renais/terapia , Técnicas de Inativação de Genes/métodos , Neoplasias Renais/terapia , Sunitinibe/uso terapêutico , Células A549 , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cisplatino/farmacologia , Terapia Combinada , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Genes erbB-1 , Células HEK293 , Células HeLa , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Modelos Biológicos , Inibidores de Proteínas Quinases/uso terapêutico
12.
PLoS One ; 15(3): e0229782, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32126126

RESUMO

African Oryza glaberrima and Oryza sativa landraces are considered valuable resources for breeding traits due to their adaptation to local environmental and soil conditions. They often possess superior resistance to endemic pests and tolerance to drought and nutrient deficiencies when compared to the "imported" high production Asian rice varieties. In contrast, "domestication traits" such as seed shattering, lodging, and seed yield are not well established in these African landraces. Therefore, the use of these African varieties for high production agriculture is limited by unpredictable yield and grain quality. We are addressing this shortcoming by developing protocols for genetically transforming African landraces to allow the use of CRISPR-Cas mediated breeding approaches. Here we use as proof of concept the cultivated African landrace Kabre to target selected known "domestication loci" and improve the agronomic potential of Kabre rice. Stable genetic transformation with CRISPR-Cas9-based vectors generated single and simultaneous multiple gene knockouts. Plants with reduced stature to diminish lodging were generated by disrupting the HTD1 gene. Furthermore, three loci shown to control seed size and/or yield (GS3, GW2 and GN1A) were targeted using a multiplex CRISPR-Cas9 construct. This resulted in mutants with significantly improved seed yield. Our study provides an example of how new breeding technologies can accelerate the development of highly productive African landrace rice varieties, an important advancement considering that Africa is a hotspot for worldwide population growth and therefore prone to food shortage.


Assuntos
Domesticação , Grão Comestível/genética , Edição de Genes/métodos , Oryza/genética , Melhoramento Vegetal/métodos , Aclimatação/genética , África , Sistemas CRISPR-Cas/genética , DNA de Plantas/genética , Técnicas de Inativação de Genes/métodos , Genes de Plantas/genética , Loci Gênicos/genética , Plantas Geneticamente Modificadas/genética , Estudo de Prova de Conceito , Fatores de Tempo
13.
Sci Rep ; 10(1): 5350, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32210344

RESUMO

The major barrier to a HIV-1 cure is the persistence of latent genomes despite treatment with antiretrovirals. To investigate host factors which promote HIV-1 latency, we conducted a genome-wide functional knockout screen using CRISPR-Cas9 in a HIV-1 latency cell line model. This screen identified IWS1, POLE3, POLR1B, PSMD1, and TGM2 as potential regulators of HIV-1 latency, of which PSMD1 and TMG2 could be confirmed pharmacologically. Further investigation of PSMD1 revealed that an interacting enzyme, the deubiquitinase UCH37, was also involved in HIV-1 latency. We therefore conducted a comprehensive evaluation of the deubiquitinase family by gene knockout, identifying several deubiquitinases, UCH37, USP14, OTULIN, and USP5 as possible HIV-1 latency regulators. A specific inhibitor of USP14, IU1, reversed HIV-1 latency and displayed synergistic effects with other latency reversal agents. IU1 caused degradation of TDP-43, a negative regulator of HIV-1 transcription. Collectively, this study is the first comprehensive evaluation of deubiquitinases in HIV-1 latency and establishes that they may hold a critical role.


Assuntos
Sistemas CRISPR-Cas , Enzimas Desubiquitinantes/genética , Técnicas de Inativação de Genes/métodos , HIV-1/fisiologia , Latência Viral , DNA Polimerase III/genética , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Enzima Desubiquitinante CYLD/genética , Enzimas Desubiquitinantes/antagonistas & inibidores , Endopeptidases/genética , Inibidores Enzimáticos/farmacologia , HIV-1/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Células Jurkat , Nucleoproteínas/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Ubiquitina Tiolesterase/genética , Latência Viral/efeitos dos fármacos
14.
Life Sci ; 253: 117360, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32001269

RESUMO

AIMS: Progesterone receptor membrane component 1 (PGRMC1) has been reported to mediate the neuroprotective effect of progesterone, but the exact mechanism has not been elucidated. Therefore, the purpose of this study was to investigate the signalling pathway downstream of PGRMC1 in progesterone-induced neuroprotection. Recognition of the mechanism of progesterone opens novel perspectives for the treatment of diseases of the nervous system. MAIN METHODS: The PGRMC1 protein level was knocked down in rat primary cortical neurons, and Aß25-35 was used to establish an Alzheimer's disease cell model. The neuroprotective effect of progesterone was assessed by Hoechst 33258 staining and a cell counting kit-8 (CCK-8) assay. Then, proteomic and bioinformatic methods were used to analyse the proteins altered in response to PGRMC1 silencing to identify target proteins and signalling pathways involved in PGRMC1-mediated progesterone-induced neuroprotection. These findings were further verified by using signalling pathway inhibitors and western blotting. KEY FINDINGS: The neuroprotective effect of progesterone was significantly attenuated with PGRMC1 silencing. The expression of many proteins in the Ras signalling pathway was significantly changed in response to PGRMC1 silencing. FTI-277 inhibited progesterone-induced neuroprotection. Progesterone increased the expression of total Ras and Grb2. SIGNIFICANCE: These findings provide new perspectives for understanding the mechanism of and role of PGRMC1 in progesterone-induced neuroprotection. The Ras signalling pathway is the signalling pathway downstream of PGRMC1 in the mediation of progesterone-induced neuroprotection.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/química , Proteínas de Membrana/metabolismo , Neuroproteção/efeitos dos fármacos , Progesterona/metabolismo , Receptores de Progesterona/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Apoptose , Sobrevivência Celular , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Proteína Adaptadora GRB2/metabolismo , Técnicas de Inativação de Genes/métodos , Inativação Gênica , Humanos , Metionina/análogos & derivados , Metionina/química , Metionina/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteômica , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Espectrometria de Massas em Tandem
15.
PLoS One ; 15(2): e0228910, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32053639

RESUMO

A fluorescence marker mOrange was inserted to the popular pLentiCrispr-V2 to create pLentiCrispr-V2-mOrange (V2mO) that contained both a puromycin selection and a fluorescent marker, making viral production and target transduction visible. Lentiviruses packaged with this plasmid and appropriate guide RNAs (gRNAs) successfully knocked out the genes RhoA, Gli1, and Gal3 in human gastric cancer cell lines. Cas9-gRNA editing efficiency could be estimated directly from Sanger electropherograms of short polymerase chain reaction products around the gRNA regions in Cas9-gRNA transduced cells. Single cloning of transduced target cell pools must be performed to establish stable knockout clones. Rescue of wildtype (RhoA and Gal3) and mutant (RhoA.Y42C) genes into knockout cells was successful only when cDNAs, where gRNAs bind, were modified by three nucleotides while the amino acid sequences remained unchanged. Stringent on-target CRISPR/Cas9 editing was observed in Gal3 gene, but not in RhoA gene since RhoA.Y42C already presented a nucleotide change in gRNA5 binding site. In summary, our improved strategy added these advantages: adding visual marker to the popular lentiviral system, monitoring lentiviral production and transduction efficiencies, cell-sorting Cas9+ cells in target cells by fluorescence-activated cell sorting, direct estimation of gene editing efficiency of target cell pools by short PCR electropherograms around gRNA binding sites, and successful rescue of wildtype and mutant genes in knockout cells, overcoming Cas9 editing by modifying cDNAs.


Assuntos
Técnicas de Inativação de Genes/métodos , Engenharia Genética/métodos , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Corantes Fluorescentes , Edição de Genes , Vetores Genéticos , Humanos , Lentivirus/genética , Plasmídeos , RNA Guia/genética
17.
J Insect Physiol ; 122: 104023, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32061647

RESUMO

Helicoverpa armigera is one of the most serious agricultural insect pests of global importance. It is highly polyphagous and depends on digestive serine proteases to degrade proteins to peptides and to amino acids. H. armigera has evolved adaptive ability to compensate for the inhibition of plant defensive protease inhibitors (PIs) in its diet by overproduction of digestive enzymes. As far as we know, compensation for deletion of serine protease genes has not yet been studied in any herbivorous insect. In this study, we used CRISPR/Cas9 to knock out a cluster of 18 trypsin-like genes in H. armigera. Compared with the wild type SCD strain, activities of the total proteases, trypsins and chymotrypsins were not significantly changed in the gene cluster knockout strain (Tryp-KO). RNA-seq data showed 1492 upregulated and 461 downregulated DEGs in Try-KO. GO function classification and KEGG pathway analyses revealed these differentially expressed genes were enriched for terms related to binding, catalytic activity, metabolic process and signal transduction. In regard to serine protease genes, 35 were upregulated and 12 downregulated in Tryp-KO strain. Our study indicated that H. armigera can compensate for the deleted protease genes by overexpression of other trypsin and chymotrypsin genes in order to maintain its genetic and metabolic robustness. It also suggests that genetic perturbations created by genome editing tools can induce global gene expression changes.


Assuntos
Sistemas CRISPR-Cas , Técnicas de Inativação de Genes/métodos , Mariposas/genética , Serina Proteases/genética , Animais , Proteína 9 Associada à CRISPR , Expressão Gênica , Inativação Gênica , Genes de Insetos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Mariposas/metabolismo , Família Multigênica , Serina Proteases/metabolismo , Transcriptoma , Tripsina/genética , Tripsina/metabolismo
18.
Int J Mol Sci ; 21(2)2020 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-31963842

RESUMO

Mammalian telomere lengths are primarily regulated by telomerase, a ribonucleoprotein consisting of a reverse transcriptase (TERT) and an RNA subunit (TERC). TERC is constitutively expressed in all cells, whereas TERT expression is temporally and spatially regulated, such that in most adult somatic cells, TERT is inactivated and telomerase activity is undetectable. Most tumor cells activate TERT as a mechanism for preventing progressive telomere attrition to achieve proliferative immortality. Therefore, inactivating TERT has been considered to be a promising means of cancer therapy. Here we applied the CRISPR/Cas9 gene editing system to target the TERT gene in cancer cells. We report that disruption of TERT severely compromises cancer cell survival in vitro and in vivo. Haploinsufficiency of TERT in tumor cells is sufficient to result in telomere attrition and growth retardation in vitro. In vivo, TERT haploinsufficient tumor cells failed to form xenograft after transplantation to nude mice. Our work demonstrates that gene editing-mediated TERT knockout is a potential therapeutic option for treating cancer.


Assuntos
Técnicas de Inativação de Genes/métodos , Telomerase/genética , Telomerase/metabolismo , Neoplasias do Colo do Útero/patologia , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Feminino , Haploinsuficiência , Células HeLa , Humanos , Mutação INDEL , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo
19.
Gut ; 69(2): 329-342, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31439637

RESUMO

OBJECTIVE: Facilitates Chromatin Transcription (FACT) complex is a histone chaperone participating in DNA repair-related and transcription-related chromatin dynamics. In this study, we investigated its oncogenic functions, underlying mechanisms and therapeutic implications in human hepatocellular carcinoma (HCC). DESIGN: We obtained HCC and its corresponding non-tumorous liver samples from 16 patients and identified FACT complex as the most upregulated histone chaperone by RNA-Seq. We further used CRISPR-based gene activation and knockout systems to demonstrate the functions of FACT complex in HCC growth and metastasis. Functional roles and mechanistic insights of FACT complex in oxidative stress response were investigated by ChIP assay, flow cytometry, gene expression assays and 4sU-DRB transcription elongation assay. Therapeutic effect of FACT complex inhibitor, Curaxin, was tested in both in vitro and in vivo models. RESULTS: We showed that FACT complex was remarkably upregulated in HCC and contributed to HCC progression. Importantly, we unprecedentedly revealed an indispensable role of FACT complex in NRF2-driven oxidative stress response. Oxidative stress prevented NRF2 and FACT complex from KEAP1-mediated protein ubiquitination and degradation. Stabilised NRF2 and FACT complex form a positive feedback loop; NRF2 transcriptionally activates the FACT complex, while FACT complex promotes the transcription elongation of NRF2 and its downstream antioxidant genes through facilitating rapid nucleosome disassembly for the passage of RNA polymerase. Therapeutically, Curaxin effectively suppressed HCC growth and sensitised HCC cell to sorafenib. CONCLUSION: In conclusion, our findings demonstrated that FACT complex is essential for the expeditious HCC oxidative stress response and is a potential therapeutic target for HCC treatment.


Assuntos
Carcinoma Hepatocelular/fisiopatologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Grupo de Alta Mobilidade/fisiologia , Chaperonas de Histonas/fisiologia , Neoplasias Hepáticas/fisiopatologia , Estresse Oxidativo/fisiologia , Fatores de Elongação da Transcrição/fisiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carbazóis/farmacologia , Carbazóis/uso terapêutico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/prevenção & controle , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Inativação de Genes/métodos , Proteínas de Grupo de Alta Mobilidade/antagonistas & inibidores , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/fisiopatologia , Neoplasias Hepáticas Experimentais/prevenção & controle , Camundongos Endogâmicos BALB C , Camundongos Nus , Estresse Oxidativo/genética , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Fatores de Elongação da Transcrição/antagonistas & inibidores , Fatores de Elongação da Transcrição/biossíntese , Fatores de Elongação da Transcrição/genética , Regulação para Cima/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Biol. Res ; 53: 18, 2020. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1124204

RESUMO

BACKGROUND: Cisplatin resistance (DDP-resistance) remains one of the major causes of poor prognosis in females with ovarian cancer. Long non-coding RNAs (lncRNAs) have been shown to participate in the regulation of cellular processes, including chemoresistance. The aim of this study was to explore the role of HOX transcript antisense RNA (HOTAIR) in DDP-resistant ovarian cancer cells. METHODS: DDP-resistant ovarian cancer cell lines (SKOV3/DDP and A2780/DDP) were established. Real-time PCR, western blot, dual-luciferase reporter assay, and flow cytometry were then used to evaluate the effect of HOTAIR/miR-138-5p axis on chemoresistance of DDP-resistant ovarian cancer cells to DDP. RESULTS: We found that HOTAIR was upregulated in DDP-resistant cells, while miR-138-5p was downregulated. Knockdown of HOTAIR increased the expression of miR-138-5p in DDP-resistant cells and miR-138-5p is directly bound to HOTAIR. Upregulation of miR-138-5p induced by HOTAIR siRNA or by its mimics enhanced the chemosensitivity of DDP-resistant cells and decreased the expression of EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) and SIRT1 (sirtuin 1). Furthermore, the HOTAIR silencing-induced chemosensitivity of DDP-resistant cells was weakened by miR-138-5p inhibitor. CONCLUSIONS: These data demonstrate that HOTAIR acts as a sponge of miR-138-5p to prevent its binding to EZH2 and SIRT1, thereby promoting DDP-resistance of ovarian cancer cells. Our work will shed light on the development of therapeutic strategies for ovarian cancer treatment.


Assuntos
Humanos , Feminino , Neoplasias Ovarianas/genética , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , RNA Longo não Codificante/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação para Cima , Apoptose/efeitos dos fármacos , MicroRNAs/antagonistas & inibidores , Linhagem Celular Tumoral , Técnicas de Inativação de Genes/métodos , Sirtuína 1/antagonistas & inibidores , Reação em Cadeia da Polimerase em Tempo Real , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores
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