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1.
Cell Death Dis ; 11(1): 39, 2020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31959745

RESUMO

Ginsenosides exhibit a large variety of biological activities in maintaining physical health; however, the molecule underpinnings underlining these biological activities remain to be defined. Here, we took a cellular condition that compound K (CK) induces autophagic cell death in HeLa cells, and setup a high-throughput genetic screening using CRISPR technology. We have identified a number of CK-resistant and CK-sensitive genes, and further validated PMAIP1 as a CK-resistant gene and WASH1 as a CK-sensitive gene. Compound K treatment reduces the expression of WASH1, which further accelerates the autophagic cell death, highlighting WASH1 as an interesting downstream mediator of CK effects. Overall, our study offers an easy-to-adopt platform to study the functional mediators of ginsenosides, and provides a candidate list of genes that are potential targets of CK.

2.
Circulation ; 141(1): 67-79, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31779484

RESUMO

BACKGROUND: Mutations in low-density lipoprotein (LDL) receptor (LDLR) are one of the main causes of familial hypercholesterolemia, which induces atherosclerosis and has a high lifetime risk of cardiovascular disease. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is an effective tool for gene editing to correct gene mutations and thus to ameliorate disease. METHODS: The goal of this work was to determine whether in vivo somatic cell gene editing through the CRISPR/Cas9 system delivered by adeno-associated virus (AAV) could treat familial hypercholesterolemia caused by the Ldlr mutant in a mouse model. We generated a nonsense point mutation mouse line, LdlrE208X, based on a relevant familial hypercholesterolemia-related gene mutation. The AAV-CRISPR/Cas9 was designed to correct the point mutation in the Ldlr gene in hepatocytes and was delivered subcutaneously into LdlrE208X mice. RESULTS: We found that homogeneous LdlrE208X mice (n=6) exhibited severe atherosclerotic phenotypes after a high-fat diet regimen and that the Ldlr mutation was corrected in a subset of hepatocytes after AAV-CRISPR/Cas9 treatment, with LDLR protein expression partially restored (n=6). Compared with the control groups (n=6 each group), the AAV-CRISPR/Cas9 with targeted single guide RNA group (n=6) had significant reductions in total cholesterol, total triglycerides, and LDL cholesterol in the serum, whereas the aorta had smaller atherosclerotic plaques and a lower degree of macrophage infiltration. CONCLUSIONS: Our work shows that in vivo AAV-CRISPR/Cas9-mediated Ldlr gene correction can partially rescue LDLR expression and effectively ameliorate atherosclerosis phenotypes in Ldlr mutants, providing a potential therapeutic approach for the treatment of patients with familial hypercholesterolemia.

3.
Theranostics ; 9(12): 3501-3514, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31281493

RESUMO

Identification of proper agents to increase or activate UCP1+ cells in adipose tissues remains a potent therapeutic strategy to combat obesity. Screening systems for UCP1 activators have been previously established and allow for unbiased discovery of effective compound(s). Methods: A previously established Ucp1-2A-GFP reporter system was applied to a chemical library containing 33 phosphatase inhibitors. Compounds that can significantly activate UCP1 expression were further tested in vivo in mouse adipose tissues. Possible underlying mechanism was explored via RNA profiling, CMAP analysis, CRISPR targeting as well as inhibitor treatments. Results: We identified BML-260, a known potent inhibitor of the dual-specific phosphatase JSP-1, that significantly increased UCP1 expression in both brown and white adipocytes. BML-260 treatment also activated oxidative phosphorylation genes, increased mitochondrial activity as well as heat generation in vitro and in vivo. Mechanistic studies revealed that effect of BML-260 on adipocytes was partly through activated CREB, STAT3 and PPAR signaling pathways, and was unexpectedly JSP-1 independent. Conclusion: The rhodanine derivate BML-260 was previously identified to be a JSP-1 inhibitor, and thus was proposed to treat inflammatory and proliferative disorders associated with dysfunctional JNK signaling. This work provides evidences that BML-260 can also exert a JSP-1-independent effect in activating UCP1 and thermogenesis in adipocytes, and be potentially applied to treat obesity.

4.
Cell Rep ; 26(4): 884-892.e4, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30673611

RESUMO

DNA variants in the SLC16A11 coding region were identified to be strongly associated with type 2 diabetes (T2DM) in a Mexican population. Previous studies suggested that these variants disrupt SLC16A11 function and therefore proposed to revive SLC16A11 levels or activity to achieve therapeutic benefit. However, with knockout mouse models, here we show that Slc16a11 depletion has no significant metabolic defects. Further studies demonstrate that reconstitution of the mutant, but not the wild-type Slc16a11, in the liver of knockout mice causes more triglyceride accumulation and induction of insulin resistance via upregulation of lipin 1, suggesting gaining of aberrant functions of the mutant protein that affects lipid metabolism. Our findings offer a different explanation to the function of these diabetic variants, challenging the concept of enhancing SLC16A11 function to treat T2DM. The contradictory results by our and previous studies suggest that how the SLC16A11 locus contributes to human metabolism warrants further investigation.

5.
EBioMedicine ; 37: 344-355, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30348622

RESUMO

BACKGROUND: The pharmacological activation of thermogenesis in brown adipose tissue has long been considered promising strategies to treat obesity. However, identification of safe and effective agents remains a challenge. In this study, we addressed this challenge by developing a cellular system with a fluorescence readout, and applied in a high-throughput manner to screen for FDA-approved drugs that may activate endogenous UCP1 expression in adipocytes. METHODS: We have generated a Ucp1-2A-GFP reporter mouse, in which GFP intensity serves as a surrogate of the endogenous expression level of UCP1 protein; and immortalized brown adipocytes were derived from this mouse model and applied in drug screening. Candidate drugs were further tested in mouse models either fed with normal chow or high fat diet to induce obesity. FINDINGS: By using the cellular screening platform, we identified a group of FDA-approved drugs that can upregulate UCP1 expression in brown adipocyte, including previously known UCP1 activators and new candidate drugs. Further studies focusing on a previously unreported drug-sutent, revealed that sutent treatment could increase the energy expenditure and inhibit lipid synthesis in mouse adipose and liver tissues, resulting in improved metabolism and resistance to obesity. INTERPRETATION: This study offered an easy-to-use cellular screening system for UCP1 activators, and provided a candidate list of FDA-approved drugs that can potentially treat obesity. Further study of these candidates may shed new light on the drug discovery towards obesity. FUND: National Key Research and Development Program and the Strategic Priority Research Program of the Chinese Academy of Sciences, etc. (250 words).


Assuntos
Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Desacopladora 1/biossíntese , Adipócitos Marrons/patologia , Tecido Adiposo Marrom/patologia , Animais , Linhagem Celular Transformada , Aprovação de Drogas , Avaliação Pré-Clínica de Medicamentos , Camundongos , Camundongos Transgênicos , Proteína Desacopladora 1/genética , Estados Unidos , United States Food and Drug Administration
6.
Stem Cell Reports ; 11(1): 22-31, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29861165

RESUMO

Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) offer a promising cell resource for disease modeling and transplantation. However, differentiated HLCs exhibit an immature phenotype and comprise a heterogeneous population. Thus, a better understanding of HLC differentiation will improve the likelihood of future application. Here, by taking advantage of CRISPR-Cas9-based genome-wide screening technology and a high-throughput hPSC screening platform with a reporter readout, we identified several potential genetic regulators of HLC differentiation. By using a chemical screening approach within our platform, we also identified compounds that can further promote HLC differentiation and preserve the characteristics of in vitro cultured primary hepatocytes. Remarkably, both screenings identified histone deacetylase 3 (HDAC3) as a key regulator in hepatic differentiation. Mechanistically, HDAC3 formed a complex with liver transcriptional factors, e.g., HNF4, and co-regulated the transcriptional program during hepatic differentiation. This study highlights a broadly useful approach for studying and optimizing hPSC differentiation.


Assuntos
Diferenciação Celular , Hepatócitos/citologia , Hepatócitos/metabolismo , Histona Desacetilases/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Sistemas CRISPR-Cas , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Citometria de Fluxo , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Genes Reporter , Genes abl , Fator 4 Nuclear de Hepatócito/metabolismo , Histona Desacetilases/genética , Humanos , Modelos Biológicos , Fenilenodiaminas/farmacologia
7.
RNA ; 23(1): 1-5, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27742910

RESUMO

Multiplex genome engineering in vivo with CRISPR/Cas9 shows great promise as a potential therapeutic approach. The ability to incorporate multiple single guide RNA (sgRNA) cassettes together with Cas9 gene expression in one AAV vector could greatly enhance the efficiency. In a recent Method article, Mefferd and coworkers indicated that small tRNA promoters could be used to drive sgRNA expression to facilitate the construction of a more effective AAV vector. In contrast, we found that when targeting endogenous genomic loci, CRISPR/Cas9 with tRNA promoter-driven sgRNA expression showed much reduced genome editing activity, compared with significant cleavage with U6 promoter-driven sgRNA expression. Though the underlying mechanisms are still under investigation, our study suggests that the CRISPR/Cas9 system with tRNA promoter-driven sgRNA expression needs to be reevaluated before it can be used for therapeutic genome editing.


Assuntos
Exorribonucleases/genética , Edição de Genes/métodos , Regiões Promotoras Genéticas , RNA Guia/genética , RNA de Transferência/genética , Sistemas CRISPR-Cas , Expressão Gênica
9.
Yi Chuan ; 37(11): 1167-73, 2015 11.
Artigo em Chinês | MEDLINE | ID: mdl-26582531

RESUMO

The RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has offered a new platform for genome editing with high efficiency. Here, we report the use of CRISPR/Cas9 technology to target a specific genomic region in human pluripotent stem cells. We show that CRISPR/Cas9 can be used to disrupt a gene by introducing frameshift mutations to gene coding region; to knock in specific sequences (e.g. FLAG tag DNA sequence) to targeted genomic locus via homology directed repair; to induce large genomic deletion through dual-guide multiplex. Our results demonstrate the versatile application of CRISPR/Cas9 in stem cell genome editing, which can be widely utilized for functional studies of genes or genome loci in human pluripotent stem cells.


Assuntos
Sistemas CRISPR-Cas/genética , Genoma Humano/genética , Células-Tronco Pluripotentes/metabolismo , Edição de RNA , Mutação da Fase de Leitura , Humanos , RNA Guia/genética
10.
Yi Chuan ; 37(10): 983-91, 2015 10.
Artigo em Inglês | MEDLINE | ID: mdl-26496750

RESUMO

Precision medicine emerges as a new approach that takes into account individual variability. The successful conduct of precision medicine requires the use of precise disease models. Human pluripotent stem cells (hPSCs), as well as adult stem cells, can be differentiated into a variety of human somatic cell types that can be used for research and drug screening. The development of genome editing technology over the past few years, especially the CRISPR/Cas system, has made it feasible to precisely and efficiently edit the genetic background. Therefore, disease modeling by using a combination of human stem cells and genome editing technology has offered a new platform to generate " personalized " disease models, which allow the study of the contribution of individual genetic variabilities to disease progression and the development of precise treatments. In this review, recent advances in the use of genome editing in human stem cells and the generation of stem cell models for rare diseases and cancers are discussed.


Assuntos
Sistemas CRISPR-Cas , Doença/genética , Engenharia Genética/métodos , Genoma Humano/genética , Medicina de Precisão/métodos , Células-Tronco/metabolismo , Humanos , Modelos Genéticos , Neoplasias/genética , Neoplasias/patologia , Medicina de Precisão/tendências , Doenças Raras/genética , Doenças Raras/patologia
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