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1.
Mol Cell ; 81(16): 3323-3338.e14, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34352207

RESUMO

The emerging "epitranscriptomics" field is providing insights into the biological and pathological roles of different RNA modifications. The RNA methyltransferase METTL1 catalyzes N7-methylguanosine (m7G) modification of tRNAs. Here we find METTL1 is frequently amplified and overexpressed in cancers and is associated with poor patient survival. METTL1 depletion causes decreased abundance of m7G-modified tRNAs and altered cell cycle and inhibits oncogenicity. Conversely, METTL1 overexpression induces oncogenic cell transformation and cancer. Mechanistically, we find increased abundance of m7G-modified tRNAs, in particular Arg-TCT-4-1, and increased translation of mRNAs, including cell cycle regulators that are enriched in the corresponding AGA codon. Accordingly, Arg-TCT expression is elevated in many tumor types and is associated with patient survival, and strikingly, overexpression of this individual tRNA induces oncogenic transformation. Thus, METTL1-mediated tRNA modification drives oncogenic transformation through a remodeling of the mRNA "translatome" to increase expression of growth-promoting proteins and represents a promising anti-cancer target.


Assuntos
Carcinogênese/genética , Metiltransferases/genética , Neoplasias/genética , tRNA Metiltransferases/genética , Guanosina/análogos & derivados , Guanosina/genética , Humanos , Metilação , Neoplasias/patologia , Oncogenes/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA de Transferência/genética
3.
Cell Cycle ; 18(15): 1798-1811, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31258013

RESUMO

Efforts to search for better treatment options for cancer have been a priority, and due to these efforts, new alternative therapies have emerged. For instance, clinically relevant tumor-suppressive microRNAs that target key oncogenic drivers have been identified as potential anti-cancer therapeutics. MicroRNAs are small non-coding RNAs that negatively regulate gene expression at the posttranscriptional level. Aberrant microRNA expression, through misexpression of microRNA target genes, can have profound cellular effects leading to a variety of diseases, including cancer. While altered microRNA expression contributes to a cancerous state, restoration of microRNA expression has therapeutic benefits. For example, ectopic expression of microRNA-34a (miR-34a), a tumor suppressor gene that is a direct transcriptional target of p53 and thus is reduced in p53 mutant tumors, has clear effects on cell proliferation and survival in murine models of cancer. MicroRNA replacement therapies have recently been tested in combination with other agents, including other microRNAs, to simultaneously target multiple pathways to improve the therapeutic response. Thus, we reasoned that other microRNA combinations could collaborate to further improve treatment. To test this hypothesis miR-34a was used in an unbiased cell-based approach to identify combinatorial microRNA pairs with enhanced efficacy over miR-34a alone. This approach identified a subset of microRNAs that was able to enhance the miR-34a antiproliferative activity. These microRNA combinatorial therapeutics could offer superior tumor-suppressive abilities to suppress oncogenic properties compared to a monotherapeutic approach. Collectively these studies aim to address an unmet need of identifying, characterizing, and therapeutically targeting microRNAs for the treatment of cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/terapia , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Biologia Computacional , Ensaios de Triagem em Larga Escala , Humanos , MicroRNAs/genética
4.
Mol Ther Nucleic Acids ; 16: 505-518, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31071527

RESUMO

The therapeutic promise of small-RNA therapeutics is limited, not only by the lack of delivery vehicles, but also by the inability of the small RNAs to reach intracellular compartments where they can be biologically active. We previously reported successful delivery of functionally active miRNAs via receptor-mediated endocytosis. This type of targeted therapy still faces a major challenge in the delivery field: endosomal sequestration. Here, a new method has been developed to promote endosomal escape of delivered miRNA. The strategy relies on the difference in solute contents between nascent endosomes and the cytoplasm; early endosomes are rich in sodium ions, whereas the intracellular fluid is rich is potassium ions. Exploiting this difference through favoring the influx of potassium into the endosomes without the exchange of osmotically active sodium, results in an osmotic differential leading to the endosomes swelling and bursting. One molecule that is able to exchange potassium for an osmotically inactive hydrogen ion is the ionophore nigericin. Through generating an intramolecular miRNA delivery vehicle, containing a ligand, in this case folate and nigericin, we enabled the escape of folate-RNA conjugates from their entrapping endosomes into the cytoplasm where they bound the RNA-induced silencing complex and activated the RNAi response.

5.
Bioconjug Chem ; 29(4): 1047-1059, 2018 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-29446616

RESUMO

Although peptides, antibodies/antibody fragments, siRNAs, antisense DNAs, enzymes, and aptamers are all under development as possible therapeutic agents, the breadth of their applications has been severely compromised by their inability to reach intracellular targets. Thus, while macromolecules can often enter cells by receptor-mediated endocytosis, their missions frequently fail due to an inability to escape their entrapping endosomes. In this paper, we describe a general method for promoting release of any biologic material from any entrapping endosome. The strategy relies on the fact that all nascent endosomes contain extracellular (Na+-enriched) medium, but are surrounded by intracellular (K+-enriched) fluid in the cytoplasm. Osmotic swelling and rupture of endosomes will therefore be facilitated if the flow of K+ down its concentration gradient from the cytosol into the endosome can be facilitated without allowing downhill flow of Na+ from the endosome into the cytosol. While any K+ selective ionophore can promote the K+ specific influx, the ideal K+ ionophore will also exchange influxed K+ for an osmotically inactive proton (H+) in order to prevent buildup of an electrical potential that would rapidly halt K+ influx. The only ionophore that catalyzes this exchange of K+ for H+ efficiently is nigericin. We demonstrate here that ligand-targeted delivery of nigericin into endosomes that contain an otherwise impermeable fluorescent dye can augment release of the dye into the cell cytosol via swelling/bursting of the entrapping endosomes. We further show that nigericin-facilitated escape of a folate-targeted luciferase siRNA conjugate from its entrapping endosomes promotes rapid suppression of the intended luciferase reporter gene. Taken together, we propose that ionophore-catalyzed entry of K+ into endosomal compartments can promote the release of otherwise impermeable contents from their encapsulating endosomes.


Assuntos
Endossomos/efeitos dos fármacos , Hidrogênio/metabolismo , Ionóforos/farmacologia , Nigericina/farmacologia , Potássio/metabolismo , Animais , Linhagem Celular Tumoral , Citosol/metabolismo , Endocitose , Endossomos/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Camundongos , Osmose , Células RAW 264.7 , RNA Interferente Pequeno/metabolismo , Sódio/metabolismo
6.
Oncotarget ; 8(57): 96470-96471, 2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-29228541
7.
Sci Transl Med ; 9(401)2017 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-28768807

RESUMO

MicroRNAs are small RNAs that negatively regulate gene expression posttranscriptionally. Because changes in microRNA expression can promote or maintain disease states, microRNA-based therapeutics are being evaluated extensively. Unfortunately, the therapeutic potential of microRNA replacement is limited by deficient delivery vehicles. In this work, microRNAs are delivered in the absence of a protective vehicle. The method relies on direct attachment of microRNAs to folate (FolamiR), which mediates delivery of the conjugated microRNA into cells that overexpress the folate receptor. We show that the tumor-suppressive FolamiR, FolamiR-34a, is quickly taken up both by triple-negative breast cancer cells in vitro and in vivo and by tumors in an autochthonous model of lung cancer and slows their progression. This method delivers microRNAs directly to tumors in vivo without the use of toxic vehicles, representing an advance in the development of nontoxic, cancer-targeted therapeutics.


Assuntos
Ácido Fólico/metabolismo , Técnicas de Transferência de Genes , MicroRNAs/administração & dosagem , Células A549 , Animais , Neoplasias da Mama/terapia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Feminino , Marcação de Genes , Humanos , Imunocompetência , Ligantes , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Bio Protoc ; 6(21)2016 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-28573164

RESUMO

The SRB assay has been used since its development in 1990 (Skehan et al., 1990) to inexpensively conduct various screening assays to investigate cytotoxicity in cell based studies (Vichai and Kirtikara, 2006). This method relies on the property of SRB, which binds stoichiometrically to proteins under mild acidic conditions and then can be extracted using basic conditions; thus, the amount of bound dye can be used as a proxy for cell mass, which can then be extrapolated to measure cell proliferation. The protocol can be divided into four main steps: preparation of treatment, incubation of cells with treatment of choice, cell fixation and SRB staining, and absorbance measurement. This assay is limited to manual or semiautomatic screening, and can be used in an efficient and sensitive manner to test chemotherapeutic drugs or small molecules in adherent cells. It also has applications in evaluating the effects of gene expression modulation (knockdown, gene expression upregulation), as well as to study the effects of miRNA replacement on cell proliferation (Kasinski et al., 2015).

9.
Cancers (Basel) ; 7(3): 1388-405, 2015 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-26226002

RESUMO

Recent progress in microRNA (miRNA) therapeutics has been strongly dependent on multiple seminal discoveries in the area of miRNA biology during the past two decades. In this review, we focus on the historical discoveries that collectively led to transitioning miRNAs into the clinic. We highlight the pivotal studies that identified the first miRNAs in Caenorhabditis elegans to the more recent reports that have fueled the quest to understand the use of miRNAs as markers for cancer diagnosis and prognosis. In addition, we provide insights as to how unraveling basic miRNA biology has provided a solid foundation for advancing miRNAs, such as miR-34a, therapeutically. We conclude with a brief examination of the current challenges that still need to be addressed to accelerate the path of miRNAs to the clinic: including delivery vehicles, miRNA- and delivery-associated toxicity, dosage, and off target effects.

10.
Appl Microbiol Biotechnol ; 99(8): 3715-28, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25672844

RESUMO

An endophytic fungus was isolated that produces a series of volatile natural products, including terpenes and odd chain polyenes. Phylogenetic analysis of the isolate using five loci suggests that it is closely related to Nigrograna mackinnonii CBS 674.75. The main component of the polyene series was purified and identified as (3E,5E,7E)-nona-1,3,5,7-tetraene (NTE), a novel natural product. Non-oxygenated hydrocarbons of this chain length are uncommon and desirable as gasoline-surrogate biofuels. The biosynthetic pathway for NTE production was explored using metabolic labeling and gas chromatography time of flight mass spectometer (GCMS). Two-carbon incorporation (13)C acetate suggests that it is derived from a polyketide synthase (PKS) followed by decarboxylation. There are several known mechanisms for such decarboxylation, though none have been discovered in fungi. Towards identifying the PKS responsible for the production of NTE, the genome of N. mackinnonii E5202H (ATCC SD-6839) was sequenced and assembled. Of the 32 PKSs present in the genome, 17 are predicted to contain sufficient domains for the production of NTE. These results exemplify the capacity of endophytic fungi to produce novel natural products that may have many uses, such as biologically derived fuels and commodity chemicals.


Assuntos
Ascomicetos/isolamento & purificação , Ascomicetos/metabolismo , Endófitos/isolamento & purificação , Endófitos/metabolismo , Redes e Vias Metabólicas/genética , Polienos/metabolismo , Ascomicetos/classificação , Ascomicetos/genética , DNA Fúngico/química , DNA Fúngico/genética , Endófitos/classificação , Endófitos/genética , Cromatografia Gasosa-Espectrometria de Massas , Genoma Fúngico , Marcação por Isótopo , Dados de Sequência Molecular , Análise de Sequência de DNA
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