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1.
PLoS Biol ; 18(8): e3000792, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32745129

RESUMO

A ubiquitous feature of the circadian clock across life forms is its organization as a network of cellular oscillators, with individual cellular oscillators within the network often exhibiting considerable heterogeneity in their intrinsic periods. The interaction of coupling and heterogeneity in circadian clock networks is hypothesized to influence clock's entrainability, but our knowledge of mechanisms governing period heterogeneity within circadian clock networks remains largely elusive. In this study, we aimed to explore the principles that underlie intercellular period variation in circadian clock networks (clonal period heterogeneity). To this end, we employed a laboratory selection approach and derived a panel of 25 clonal cell populations exhibiting circadian periods ranging from 22 to 28 h. We report that a single parent clone can produce progeny clones with a wide distribution of circadian periods, and this heterogeneity, in addition to being stochastically driven, has a heritable component. By quantifying the expression of 20 circadian clock and clock-associated genes across our clone panel, we found that inheritance of expression patterns in at least three clock genes might govern clonal period heterogeneity in circadian clock networks. Furthermore, we provide evidence suggesting that heritable epigenetic variation in gene expression regulation might underlie period heterogeneity.


Assuntos
Proteínas CLOCK/genética , Relógios Circadianos/genética , Ritmo Circadiano/genética , Epigênese Genética , Redes Reguladoras de Genes , Animais , Proteínas CLOCK/metabolismo , Linhagem Celular Tumoral , Células Clonais , Perfilação da Expressão Gênica , Genes Reporter , Heterogeneidade Genética , Humanos , Padrões de Herança , Luciferases/genética , Luciferases/metabolismo , Camundongos , Células NIH 3T3 , Osteoblastos/citologia , Osteoblastos/metabolismo , Processos Estocásticos
2.
Life Sci ; 258: 118176, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32771556

RESUMO

AIMS: We investigated the anti-inflammatory activity of 3ß-hydroxycholest-5-en-7-one from Hippocampus trimaculatus leach and provided a theoretical basis for identifying its therapeutic targets. MAIN METHODS: Small-RNA libraries were constructed for untreated control RAW 264.7 cells and cells treated with lipopolysaccharide (LPS; 1.0 µg/mL) or 10 µM 3ß-hydroxycholest-5-en-7-one +1.0 µg/mL LPS. We constructed and tested a miR-98-5p-interfering lentivirus to evaluate the role of miR-98-5p in the 3ß-hydroxycholest-5-en-7-one-dependent regulation of inflammatory responses in LPS-induced macrophage and murine inflammation models. The small-RNA libraries were analyzed using high-throughput sequencing. KEY FINDINGS: Among the differentially expressed microRNAs, miR-98-5p showed the most significant difference. Bioinformatics tools were used to identify the potential regulatory targets of miR-98-5p, which were tested using dual-luciferase reporter assays. Our results demonstrated that 3ß-hydroxycholest-5-en-7-one exerted an anti-inflammatory effect via miR-98-5p, which negatively regulated the expression of its target gene TNFAIP3. The results indicate that miR-98-5p interference and 3ß-hydroxycholest-5-en-7-one treatment significantly upregulated the low TNFAIP3 expression induced by LPS stimulation, thereby inhibiting TRAF6, RIP, NF-κB, IL-1ß, and TNF-α secretion. SIGNIFICANCE: 3ß-Hydroxycholest-5-en-7-one alleviates inflammation by downregulating miR-98-5p and upregulating TNFAIP3, thereby blocking NF-κB pathway activation. These results reveal the specific anti-inflammatory mechanism of 3ß-hydroxycholest-5-en-7-one, providing a foundation for developing new drugs and identifying drug targets.


Assuntos
Colestenonas/farmacologia , Regulação para Baixo/genética , Inflamação/patologia , MicroRNAs/metabolismo , Smegmamorpha/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Colestenonas/química , Regulação para Baixo/efeitos dos fármacos , Genes Reporter , Inflamação/genética , Lentivirus/metabolismo , Lipopolissacarídeos , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , Células RAW 264.7 , Reprodutibilidade dos Testes
3.
Virology ; 548: 226-235, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32771769

RESUMO

Bovine leukemia virus (BLV) is a global problem that results in significant economic losses to the livestock industry. We developed three virus strains by inserting the HiBiT reporter tag from NanoLuc luciferase (NLuc) into limited sites within BLV molecular clones. Initial analysis for site selection of the tag insertion revealed a permissible site immediately downstream of the viral envelope gene. Therefore, NLuc activity could be used to measure virus copy numbers in the supernatant and the levels of cell infection. Productivity and growth kinetics of the reporter virus were similar to those of the wild-type strain; therefore, the reporter virus can be used to characterize the replication of chimeric viruses as well as responses to the antiviral drug, amprenavir. Collectively, our results suggest that the BLV reporter virus with a HiBiT tag insertion is a highly versatile system for various purposes such as evaluating virus replication and antiviral drugs.


Assuntos
Vírus da Leucemia Bovina/genética , Animais , Antivirais/farmacologia , Genes Reporter , Vírus da Leucemia Bovina/efeitos dos fármacos , Vírus da Leucemia Bovina/crescimento & desenvolvimento , Vírus da Leucemia Bovina/fisiologia , Luciferases/análise , Luciferases/genética , Luciferases/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Replicação Viral/efeitos dos fármacos
4.
Am J Chin Med ; 48(5): 1091-1102, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32668967

RESUMO

Black ginseng (BG), which is ginseng that has been steamed and dried nine times, and its main protopanaxatriol-type ginsenosides Rg4, Rg6, Rh4, and Rg2 have been reported to exhibit various forms of biological activity, including antiseptic, antidiabetic, wound-healing, immune-stimulatory, and anti-oxidant activity. The aim of the this study was to examine the effects of [Formula: see text] (a rare protopanaxatriol-type ginsenoside fraction; Rg2, Rg4, Rg6, Rh1, and Rh4) on heme oxygenase-1 (HO-1) induction and on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-)2 in lipopolysaccharide (LPS)-activated human pulmonary artery endothelial cells (HPAECs). [Formula: see text] was tested to determine its effect on iNOS protein expression and inflammatory markers (interleukin [IL]-1[Formula: see text] and tumor necrosis factor [TNF]-[Formula: see text] in the lung tissue of LPS-treated mice. The results showed that [Formula: see text] induced the expression of HO-1, reduced LPS-activated NF-[Formula: see text]B-luciferase activity, and inhibited iNOS/NO and COX-2/PGE2, which contributed to the inhibition of STAT-1 phosphorylation. In particular, [Formula: see text] induced the translocation of Nrf2 from the cytosol to the nucleus by increasing Nrf2-ARE activity and decreased IL-1[Formula: see text] production in LPS-activated HPAECs. This reduction in iNOS/NO expression due to [Formula: see text] was reversed by siHO-1 RNA transfection. In LPS-treated mice, [Formula: see text] significantly reduced lung tissue iNOS protein levels and TNF-[Formula: see text] levels in the bronchoalveolar lavage fluid. In conclusion, these findings indicate that [Formula: see text] has a critical anti-inflammatory effect due to its ability to regulate iNOS via the inhibition of p-STAT-1 and NF-[Formula: see text]B, and thus it may be suitable for the treatment of inflammatory disease.


Assuntos
Expressão Gênica/efeitos dos fármacos , Ginsenosídeos/farmacologia , Inflamação/genética , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Panax/química , Fator de Transcrição STAT1/metabolismo , Animais , Anti-Inflamatórios , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Ginsenosídeos/isolamento & purificação , Heme Oxigenase-1/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/tratamento farmacológico , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Luciferases/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Fitoterapia
5.
Virus Res ; 286: 198074, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32589897

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human coronavirus causing the pandemic of severe pneumonia (Coronavirus Disease 2019, COVID-19). SARS-CoV-2 is highly pathogenic in human, having posed immeasurable public health challenges to the world. Innate immune response is critical for the host defense against viral infection and the dysregulation of the host innate immune responses probably aggravates SARS-CoV-2 infection, contributing to the high morbidity and lethality of COVID-19. It has been reported that some coronavirus proteins play an important role in modulating innate immunity of the host, but few studies have been conducted on SARS-CoV-2. In this study, we screened the viral proteins of SARS-CoV-2 and found that the viral ORF6, ORF8 and nucleocapsid proteins were potential inhibitors of type I interferon signaling pathway, a key component for antiviral response of host innate immune. All the three proteins showed strong inhibition on type I interferon (IFN-ß) and NF-κB-responsive promoter, further examination revealed that these proteins were able to inhibit the interferon-stimulated response element (ISRE) after infection with Sendai virus, while only ORF6 and ORF8 proteins were able to inhibit the ISRE after treatment with interferon beta. These findings would be helpful for the further study of the detailed signaling pathway and unveil the key molecular player that may be targeted.


Assuntos
Betacoronavirus/genética , Interações Hospedeiro-Patógeno/genética , Interferon beta/genética , NF-kappa B/genética , Proteínas do Nucleocapsídeo/genética , Proteínas Virais/genética , Betacoronavirus/imunologia , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Interferon beta/imunologia , Luciferases/genética , Luciferases/metabolismo , NF-kappa B/imunologia , Proteínas do Nucleocapsídeo/imunologia , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Elementos de Resposta , Vírus Sendai/genética , Vírus Sendai/imunologia , Transdução de Sinais , Transfecção/métodos , Proteínas Virais/imunologia
6.
J Vis Exp ; (160)2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32597835

RESUMO

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising treatment option for heart disease, owing to its efficiency and exceptional therapeutic effects. In an effort to repair the damaged tissue post-MI, various studies have employed DNA-based or viral gene therapy but have faced considerable hurdles due to the poor and uncontrolled expression of the delivered genes, edema, arrhythmia, and cardiac hypertrophy. Synthetic modified mRNA (modRNA) presents a novel gene therapy approach that offers high, transient, safe, nonimmunogenic, and controlled mRNA delivery to the heart tissue without any risk of genomic integration. Due to these remarkable characteristics combined with its bell-shaped pharmacokinetics in the heart, modRNA has become an attractive approach for the treatment of heart disease. However, to increase its effectiveness in vivo, a consistent and reliable delivery method needs to be followed. Hence, to maximize modRNA delivery efficiency and yield consistency in modRNA use for in vivo applications, an optimized method of preparation and delivery of modRNA intracardiac injection in a mouse MI model is presented. This protocol will make modRNA delivery more accessible for basic and translational research.


Assuntos
Técnicas de Transferência de Genes , Infarto do Miocárdio/genética , Infarto do Miocárdio/terapia , RNA Mensageiro/administração & dosagem , RNA Mensageiro/uso terapêutico , Animais , Modelos Animais de Doenças , Terapia Genética/métodos , Injeções , Integrases/metabolismo , Ligadura , Luciferases/metabolismo , Camundongos , Infarto do Miocárdio/cirurgia , Miocárdio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
7.
J Vis Exp ; (158)2020 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-32391815

RESUMO

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA→RNA→protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.


Assuntos
Células Artificiais/metabolismo , Emulsões/química , Escherichia coli/metabolismo , Biossíntese de Proteínas , Biologia Sintética/métodos , Sistema Livre de Células/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Lipossomos/química , Luciferases/metabolismo
8.
J Vis Exp ; (158)2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32420988

RESUMO

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than Δcpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.


Assuntos
Bioensaio , Toxoplasma/crescimento & desenvolvimento , Animais , Antiparasitários/farmacologia , Luciferases/metabolismo , Proteínas de Protozoários/genética , Toxoplasma/efeitos dos fármacos , Toxoplasma/genética , Toxoplasmose
9.
Biol Res ; 53(1): 14, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32293550

RESUMO

BACKGROUND: Previous studies have shown that long noncoding RNA (lncRNA) LINC00483 was aberrantly expressed in human cancers, including gastric cancer. However, the regulatory mechanism of this lncRNA in gastric cancer remains largely unknown. The present study aimed to investigate the effect of LINC00483 on gastric cancer development and explore the potential regulatory network of LINC00483/microRNA (miR)-490-3p/mitogen-activated protein kinase 1 (MAPK1). METHODS: Thirty patients with gastric cancer were recruited for tissues collection. The expression levels of LINC00483, miR-490-3p and MAPK1 were detected by quantitative real-time polymerase chain reaction or western blot. Cell viability, apoptosis, migration and invasion were determined by MTT, flow cytometry, transwell assays and western blot, respectively. The target association between miR-490-3p and LINC00483 or MAPK1 was confirmed by luciferase reporter assay. Xenograft model was established to assess the function of LINC00483 in vivo. RESULTS: LINC00483 and MAPK1 levels were increased in gastric cancer tissues and cells. Knockdown of LINC00483 or MAPK1 inhibited cells viability, migration and invasion but promoted apoptosis in gastric cancer cells. Moreover, MAPK1 overexpression attenuated the effect of LINC00483 knockdown on gastric cancer development. LINC00483 could increase MAPK1 expression by competitively sponging miR-490-3p. miR-490-3p overexpression suppressed gastric cancer development, which was abated by introduction of LINC00483. Besides, inhibition of LINC00483 decreased xenograft tumor growth by regulating miR-490-3p/MAPK1 axis. CONCLUSION: Knockdown of LINC00483 inhibited gastric cancer development in vitro and in vivo by increasing miR-490-3p and decreasing MAPK1, elucidating a novel mechanism for understanding the development of gastric cancer.


Assuntos
MicroRNAs/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , RNA Longo não Codificante/metabolismo , Neoplasias Gástricas/metabolismo , Animais , Apoptose , Carcinogênese/metabolismo , Linhagem Celular Tumoral/metabolismo , Movimento Celular , Sobrevivência Celular , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Luciferases/metabolismo , Masculino , Camundongos Endogâmicos BALB C , MicroRNAs/genética , RNA Longo não Codificante/genética , Neoplasias Gástricas/genética , Ensaios Antitumorais Modelo de Xenoenxerto
10.
PLoS Negl Trop Dis ; 14(4): e0008182, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32243453

RESUMO

BACKGROUND: Studies of the human filarial parasite have been hampered by the fact that they are obligate parasites with long life cycles. In other pathogenic infections, in vivo imaging systems (IVIS) have proven extremely useful in studying pathogenesis, tissue tropism and in vivo drug efficacy. IVIS requires the use of transgenic parasites expressing a florescent reporter. Developing a method to produce transgenic filarial parasites expressing a florescent reporter would permit IVIS to be applied to the study of tissue tropism and provide a non-invasive way to screen for in vivo drug efficacy against these parasites. METHODOLOGY/PRINCIPAL FINDINGS: We report the development of a dual luciferase reporter construct in a piggyBac backbone that may be used to stably transfect Brugia malayi, a causative agent of human filariasis. Parasites transfected with this construct were visible in IVIS images obtained from infected gerbils. The signal in these infected animals increased dramatically when the transgenic parasites matured to the adult stage and began to produce transgenic progeny microfilaria. We demonstrate that the IVIS system can be used to develop an effective method for cryopreservation of transgenic parasites, to non-invasively monitor the effect of treatment with anti-filarial drugs, and to rapidly identify transgenic F1 microfilariae. CONCLUSIONS: To our knowledge, this represents the first application of IVIS to the study of a human filarial parasite. This method should prove useful in studies of tissue tropism and as an efficient in vivo assay for candidate anti-filarial drugs.


Assuntos
Brugia Malayi/genética , Elementos de DNA Transponíveis , Transfecção/métodos , Imagem Corporal Total , Animais , Animais Geneticamente Modificados , Brugia Malayi/crescimento & desenvolvimento , Criopreservação , Filariose/parasitologia , Gerbillinae , Humanos , Luciferases/genética , Luciferases/metabolismo , Masculino , Microfilárias/genética , Microfilárias/crescimento & desenvolvimento , Plasmídeos/genética , Plasmídeos/metabolismo
11.
PLoS One ; 15(4): e0232045, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32330156

RESUMO

The functional efficiency of the expression cassettes integrated into a plasmid and a PCR- amplified fragment was comparatively analyzed after transient transfection in vitro or introduction into the developing embryo of Danio rerio. The cassettes contained the reporter genes, luciferase of Photinus pyralis (luc) or enhanced green fluorescent protein, under the control of the promoter of human cytomegalovirus immediate-early genes. In the in vitro system, the efficiency of the circular plasmid was 2.5 times higher than that of the PCR- amplified fragment. The effect of mutations in the expression cassette on the efficiency of the transgene expression in the PCR- amplified fragment was quantitatively evaluated. The mutations generated after 25 amplification cycles with Taq DNA polymerase decreased luciferase activity in transfected cells by 65-85%. Thus, mutations are the key factor of decreased functional efficiency of the PCR- amplified fragment relative to the circular plasmid in this experimental model, while other factors apparently have a lesser impact. At the organism level, no significant difference in the expression efficiency of the plasmid and PCR- amplified fragment has been revealed. Comparison of the vector efficiencies in in vivo and in vitro systems demonstrates that the level of luciferase in the D. rerio cell lysate, normalized to the molar concentration of the vector, is by three orders of magnitude higher than that after the cell transfection in vitro, which indicates that the quantitative data obtained for in vitro systems should not be directly extrapolated to the organism level.


Assuntos
Genes Reporter/genética , Vetores Genéticos/genética , Reação em Cadeia da Polimerase/métodos , Animais , Linhagem Celular Tumoral , Eficiência/fisiologia , Vaga-Lumes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Luciferases/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Transfecção/métodos , Transgenes/genética , Peixe-Zebra/metabolismo
12.
Toxicology ; 439: 152476, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32335162

RESUMO

Two non-animal test methods, KeratinoSens™ and LuSens, have been approved by the Organization of Economic Cooperation and Development (OECD) test guidelines for evaluating the sensitization potential of chemicals, and been positioned as a method for appraising key event (KE)-2, namely, the keratinocyte response component of the Adverse Outcome Pathway (AOP) in sensitization process. However, these two methods require separate cytotoxicity tests to determine the concentrations to be tested in the main test. Therefore, we developed a simple and highly accurate KE-2 test method named α-Sens that uses the dual luciferase assay system and attempted a further application of luciferase-based determination of cell viability to calculate the normalized Antioxidant response element (ARE)-mediated transcriptional activity, named normalized ARE Activity (nAA), to evaluate the sensitizing potential of chemicals. A cell line carrying the ARE-inducible Firefly luciferase reporter gene and Thymidine kinase (TK) promoter-driven Renilla luciferase gene was established and used for the α-Sens. A total of 28 chemicals, consisting of 19 skin sensitizers and nine non-skin sensitizers were tested by this assay system. The α-Sens yielded an accuracy (%), sensitivity (%), and specificity (%) against corresponding values for local lymph node assay of 96.4 %, 95.0 %, and 100 %, respectively, and for human data of 100 % for all. The α-Sens gave clear positive results for phenyl benzoate and eugenol, chemicals for which KeratinoSens™ or LuSens yielded false-negative results, using a new parameter. Our results suggest that better prediction capacity could be achieved by using nAA as a classifier compared to other existing KE-2 test methods. In conclusion, the α-Sens is promising as a simple and highly accurate in vitro skin sensitization test method for evaluation of KE-2.


Assuntos
Elementos de Resposta Antioxidante/efeitos dos fármacos , Dermatite Alérgica de Contato/patologia , Avaliação Pré-Clínica de Medicamentos/métodos , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Alternativas aos Testes com Animais , Animais , Sobrevivência Celular/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Ensaio Local de Linfonodo , Luciferases/metabolismo , Renilla/enzimologia , Sensibilidade e Especificidade , Testes Cutâneos , Timidina Quinase/metabolismo
13.
J Vis Exp ; (157)2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32281984

RESUMO

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.


Assuntos
Luciferases/metabolismo , Fatores de Transcrição/metabolismo , Humanos
14.
Nat Commun ; 11(1): 1334, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170079

RESUMO

Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Éxons/genética , Vesículas Extracelulares/metabolismo , Nanopartículas/química , RNA Guia/metabolismo , Sequência de Bases , Sobrevivência Celular , Dimerização , Edição de Genes , Vetores Genéticos/metabolismo , Células HEK293 , Protease de HIV/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Ligantes , Luciferases/metabolismo , Processamento de RNA/genética , RNA Catalítico/metabolismo , Ribonucleoproteínas/metabolismo , Doadores de Tecidos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
15.
Nat Commun ; 11(1): 1211, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32139701

RESUMO

Tumor metastasis is a hallmark of cancer. Metastatic cancer cells often reside in distal tissues and organs in their dormant state. Mechanisms underlying the pre-metastatic niche formation are poorly understood. Here we show that in a colorectal cancer (CRC) model, primary tumors release integrin beta-like 1 (ITGBL1)-rich extracellular vesicles (EVs) to the circulation to activate resident fibroblasts in remote organs. The activated fibroblasts induce the pre-metastatic niche formation and promote metastatic cancer growth by secreting pro-inflammatory cytokine, such as IL-6 and IL-8. Mechanistically, the primary CRC-derived ITGBL1-enriched EVs stimulate the TNFAIP3-mediated NF-κB signaling pathway to activate fibroblasts. Consequently, the activated fibroblasts produce high levels of pro-inflammatory cytokines to promote metastatic cancer growth. These findings uncover a tumor-stromal interaction in the metastatic tumor microenvironment and an intimate signaling communication between primary tumors and metastases through the ITGBL1-loaded EVs. Targeting the EVs-ITGBL1-CAFs-TNFAIP3-NF-κB signaling axis provides an attractive approach for treating metastatic diseases.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Vesículas Extracelulares/metabolismo , Fibroblastos/patologia , Integrina beta1/metabolismo , Animais , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Luciferases/metabolismo , Camundongos , NF-kappa B/metabolismo , Metástase Neoplásica , Transdução de Sinais , Distribuição Tecidual , Transcrição Genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo
16.
Proc Natl Acad Sci U S A ; 117(14): 7745-7754, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32198205

RESUMO

Competence allows bacteria to internalize exogenous DNA fragments for the acquisition of new phenotypes such as antibiotic resistance or virulence traits. In most streptococci, competence is regulated by ComRS signaling, a system based on the mature ComS pheromone (XIP), which is internalized to activate the (R)RNPP-type ComR sensor by triggering dimerization and DNA binding. Cross-talk analyses demonstrated major differences of selectivity between ComRS systems and raised questions concerning the mechanism of pheromone-sensor recognition and coevolution. Here, we decipher the molecular determinants of selectivity of the closely related ComRS systems from Streptococcus thermophilus and Streptococcus vestibularis Despite high similarity, we show that the divergence in ComR-XIP interaction does not allow reciprocal activation. We perform the structural analysis of the ComRS system from S. vestibularis. Comparison with its ortholog from S. thermophilus reveals an activation mechanism based on a toggle switch involving the recruitment of a key loop by the XIP C terminus. Together with a broad mutational analysis, we identify essential residues directly involved in peptide binding. Notably, we generate a ComR mutant that displays a fully reversed selectivity toward the heterologous pheromone with only five point mutations, as well as other ComR variants featuring XIP bispecificity and/or neofunctionalization for hybrid XIP peptides. We also reveal that a single XIP mutation relaxes the strictness of ComR activation, suggesting fast adaptability of molecular communication phenotypes. Overall, this study is paving the way toward the rational design or directed evolution of artificial ComRS systems for a range of biotechnological and biomedical applications.


Assuntos
Feromônios/metabolismo , Transdução de Sinais , Streptococcus/metabolismo , Sequência de Aminoácidos , Luciferases/metabolismo , Modelos Moleculares , Mutação Puntual/genética , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína
17.
PLoS One ; 15(2): e0228005, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32027681

RESUMO

Targeted gene therapy using recombinant adeno-associated virus (rAAV) vectors is a potential therapeutic strategy for treating cancer, and tissue-specific promoters may help with tissue targeting. Medullary thyroid carcinoma (MTC) is a disease of the calcitonin secreting thyroid C cells, and calcitonin is highly expressed in MTC tumors compared to other cells. To target MTC cells, we evaluated an rAAV serotype 2 vector (rAAV2-pM+104-GFP) containing a modified calcitonin/calcitonin gene related peptide promoter (pM+104) and a green fluorescent protein (GFP) reporter gene. In vitro transduction experiments comparing the MTC TT cell line with non-MTC cell lines demonstrated that rAAV2-pM+104-GFP infection yielded significantly (p < 0.05) higher GFP expression in TT cells than in non-MTC cell lines (HEK293 and HeLa), and significantly higher expression than in TT cells infected with the positive control rAAV2-pCBA-GFP vector. The rAAV2-pCBA-GFP control vector included a well-characterized, ubiquitously expresses control promoter, the chicken beta actin promoter with a cytomegalovirus enhancer (pCBA). In vivo experiments using a TT cell xenograft tumor mouse model showed that tumors directly injected with 2 x 1010 vg of rAAV2-pM+104-GFP vector resulted in GFP expression detected in 21.7% of cells, 48 hours after the injection. Furthermore, GFP expression was significantly higher for rAAV-pM+104-GFP treatments with a longer vector treatment duration and higher vector dose, with up to 52.6% (q < 0.05) GFP cells detected 72 hours after injecting 1x 1011 vg/tumor. These data show that we have developed an rAAV vector with improved selectivity for MTC.


Assuntos
Calcitonina/genética , Carcinoma Neuroendócrino/terapia , Dependovirus/genética , Vetores Genéticos/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Neoplasias da Glândula Tireoide/terapia , Animais , Peptídeo Relacionado com Gene de Calcitonina/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Luciferases/metabolismo , Masculino , Camundongos SCID , Regiões Promotoras Genéticas , Transgenes , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Clin Exp Metastasis ; 37(2): 247-255, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32052231

RESUMO

Current laboratory models of lymphatic metastasis generally require either genetically modified animals or are technically challenging. Herein, we have developed a robust protocol for the induction of intralymphatic metastasis in wild-type mice with reproducible outcomes. To determine an optimal injection quantity and timeline for tumorigenesis, C57Bl/6 mice were injected directly into the mesenteric lymph duct (MLD) with varying numbers of syngeneic murine colon cancer cells (MC38) or gastric cancer cells (YTN16) expressing GFP/luciferase and monitored over 2-4 weeks. Tumor growth was tracked via whole-animal in vivo bioluminescence imaging (IVIS). Our data indicate that the injection of tumor cells into the MLD is a viable model for lymphatic metastasis as necropsies revealed large tumor burdens and metastasis in regional lymph nodes. This protocol enables a closer study of the role of lymphatics in cancer metastasis and opens a window for the development of novel approaches for treatment of metastatic diseases.


Assuntos
Neoplasias do Colo/patologia , Modelos Animais de Doenças , Metástase Linfática/diagnóstico por imagem , Neoplasias Gástricas/patologia , Animais , Linhagem Celular Tumoral/transplante , Feminino , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luciferases/química , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes , Linfonodos/diagnóstico por imagem , Linfonodos/patologia , Metástase Linfática/patologia , Vasos Linfáticos , Masculino , Mesentério , Camundongos , Camundongos Endogâmicos C57BL , Tomografia Óptica , Carga Tumoral
19.
Biochem Biophys Res Commun ; 524(4): 791-797, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32019676

RESUMO

Increased granulosa cell (GC) proliferation may contribute to abnormal folliculogenesis in patients with polycystic ovary syndrome (PCOS), which affects approximately 10% reproductive aged women. However, the mechanisms underlying increased GC proliferation in PCOS remain incompletely understood. In this study, we identified miR-3940-5p as a hub miRNA in GC from PCOS using weighted gene co-expression network analysis (WGCNA), and real-time polymerase chain reaction (RT-PCR) analysis confirmed that miR-3940-5p was significantly increased in GC from PCOS. Enrichment analysis of predicted target genes of miR-3940-5p indicated potential roles of miR-3940-5p in follicular development and cell proliferation regulation. Consistently, functional study confirmed that miR-3940-5p overexpression promoted granulosa cell proliferation. Integrated analysis of mRNA expression profiling data and predicted target genes of miR-3940-5p identified potassium voltage-gated channel subfamily A member 5 (KCNA5) as a potential target of miR-3940-5p, and was validated by luciferase reporter assay. Finally, functional analysis suggested that miR-3940-5p promoted GC proliferation in a KCNA5 dependent manner. In conclusion, miR-3940-5p was a hub miRNA upregulated in GC from PCOS, and promoted GC proliferation by targeting KCNA5.


Assuntos
Regulação Neoplásica da Expressão Gênica , Células da Granulosa/metabolismo , Canal de Potássio Kv1.5/genética , MicroRNAs/genética , Proteínas de Neoplasias/genética , Síndrome do Ovário Policístico/genética , Adulto , Antagomirs/genética , Antagomirs/metabolismo , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Genes Reporter , Células da Granulosa/patologia , Humanos , Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/metabolismo , Luciferases/genética , Luciferases/metabolismo , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Síndrome do Ovário Policístico/metabolismo , Síndrome do Ovário Policístico/patologia , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
20.
Biochem Biophys Res Commun ; 524(4): 798-802, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32037085

RESUMO

Inflammatory bowel disease (IBD) is a risk factor for the development of colorectal cancer (CRC) for which mutation to p53 is an early event leading to dysplasia. Interestingly, P2RY6 mRNA increases in both pathologies. In this study, we investigated if p53 and p53R273H mutant, commonly found in CRC and IBD, were involved in the transcriptional regulation of P2RY6. First, the P2RY6 promoter was defined as a region corresponding to -1600 to +273 nucleotides relative to the putative TATA-less transcriptional starting site found at position 73,264,505 of NCBI reference sequence NC_000010.11. We cloned this promoter region along with 5'-deletion constructs in the pGL4.10[luc2] vector for luciferase assays to delineate the minimal promoter region. We observed that p53 wt and p53R273H differentially regulated the transcription of the P2RY6 gene. In fact, increasing quantity of p53R273H enhanced the capacity of p53 wt to stimulate the transactivation of the P2RY6 promoter but this cooperative effect was lost when p53R273H was present in a ratio of 3:1. In accordance with the luciferase assays, ChIP analysis revealed that endogenous p53 wt was significantly associated with the P2RY6 proximal promoter, whereas the interaction of the p53R273H with the P2RY6 promoter was not significant. Although further studies are required to fully elucidate the molecular determinant controlling P2Y6 expression in diseases, we propose, for the first time, a molecular mechanism involving a collaboration between p53 wt and p53R273H to regulate the expression of this receptor.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Receptores Purinérgicos P2/genética , Transcrição Genética , Proteína Supressora de Tumor p53/genética , Células A549 , Substituição de Aminoácidos , Células CACO-2 , Proliferação de Células , Imunoprecipitação da Cromatina , Genes Reporter , Células HCT116 , Células HT29 , Humanos , Luciferases/genética , Luciferases/metabolismo , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
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