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1.
Mol Cancer ; 15(1): 47, 2016 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-27296891

RESUMO

BACKGROUND: Fusion proteins have unique oncogenic properties and their identification can be useful either as diagnostic or therapeutic targets. Next generation sequencing data have previously shown a fusion gene formed between Rad51C and ATXN7 genes in the MCF7 breast cancer cell line. However, the existence of this fusion gene in colorectal patient tumor tissues is largely still unknown. METHODS: We evaluated for the presence of Rad51C-ATXN7 fusion gene in colorectal tumors and cells by RT-PCR, PCR, Topo TA cloning, Real time PCR, immunoprecipitation and immunoblotting techniques. RESULTS: We identified two forms of fusion mRNAs between Rad51C and ATXN7 in the colorectal tumors, including a Variant 1 (fusion transcript between Rad51C exons 1-7 and ATXN7 exons 6-13), and a Variant 2 (between Rad51C exons 1-6 and ATXN7 exons 6-13). In silico analysis showed that the Variant 1 produces a truncated protein, whereas the Variant 2 was predicted to produce a fusion protein with molecular weight of 110 KDa. Immunoprecipitation and Western blot analysis further showed a 110 KDa protein in colorectal tumors. 5-Azacytidine treatment of LS-174 T cells caused a 3.51-fold increase in expression of the fusion gene (Variant 2) as compared to no treatment controls evaluated by real time PCR. CONCLUSION: In conclusion we found a fusion gene between DNA repair gene Rad51C and neuro-cerebral ataxia Ataxin-7 gene in colorectal tumors. The in-frame fusion transcript of Variant 2 results in a fusion protein with molecular weight of 110 KDa. In addition, we found that expression of fusion gene is associated with functional impairment of Fanconi Anemia (FA) DNA repair pathway in colorectal tumors. The expression of Rad51C-ATXN7 in tumors warrants further investigation, as it suggests the potential of the fusion gene in treatment and predictive value in colorectal cancers.


Assuntos
Ataxina-7/genética , Clonagem Molecular/métodos , Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/genética , Proteínas de Fusão Oncogênica/genética , Ataxina-7/metabolismo , Azacitidina/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Simulação por Computador , Metilação de DNA/efeitos dos fármacos , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Variação Genética , Humanos , Peso Molecular , Proteínas de Fusão Oncogênica/efeitos dos fármacos , Proteínas de Fusão Oncogênica/metabolismo
2.
Anal Chem ; 85(23): 11265-74, 2013 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-24102152

RESUMO

Noninvasive early detection methods have the potential to reduce mortality rates of both cancer and infectious diseases. Here, we present a novel assay by which tethered cationic lipoplex nanoparticles containing molecular beacons (MBs) can capture cancer cell-derived exosomes or viruses and identify encapsulated RNAs in a single step. A series of ultracentrifugation and Exoquick isolation kit were first used to isolate exosomes from the cell culture medium and human serum, respectively. Cationic lipoplex nanoparticles linked onto the surface of a thin glass plate capture negatively charged viruses or cell-secreted exosomes by electrostatic interactions to form larger nanoscale complexes. Lipoplex/virus or lipoplex/exosome fusion leads to the mixing of viral/exosomal RNAs and MBs within the lipoplexes. After the target RNAs specially bind to the MBs, exosomes enriched in target RNAs are readily identified by the fluorescence signals of MBs. The in situ detection of target extracellular RNAs without diluting the samples leads to high detection sensitivity not achievable by existing methods, e.g., quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Here we demonstrate this concept using lentivirus and serum from lung cancer patients.


Assuntos
Doenças Transmissíveis/sangue , Doenças Transmissíveis/virologia , Líquido Extracelular/química , Neoplasias Pulmonares/sangue , Nanopartículas/química , RNA/sangue , Cátions/química , Linhagem Celular Tumoral , Exossomos/química , Humanos , Lipídeos/química
3.
Small ; 9(13): 2358-67, 2013 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-23471869

RESUMO

Many delivery methods have been developed to improve the therapeutic efficacy and facilitate the clinical translation of nucleic acid-based therapeutics. A facile surface-mediated nucleic acid delivery by lipoplexes is prepared in a microwell array, which combines the advantages of lipoplexes as an efficient carrier system, surface-mediated delivery, and the control of surface topography. Uniform disc-like lipoplexes containing nucleic acids are formed in the microwell array with a diameter of ∼818 nm and thickness of ∼195 nm. The microwell array-mediated delivery of lipoplexes containing FAM-oligodeoxynucleotides is ∼18.6 and ∼10.6 times more efficient than the conventional transfection method in an adherent cell line (A549 non-small cell lung cancer cells) and a suspension cell line (KG-1a acute myelogenous leukemia cells), respectively. MicroRNA-29b is then used as a model nucleic acid to investigate the therapeutic efficacy of lipoplexes delivered by the microwell array. Compared to conventional transfection methods, the effective therapeutic dosage of microRNA-29b is reduced from the microgram level to the nanogram level by lipoplexes prepared in the microwell array. The microwell array is also a very flexible platform. Both nucleic acid therapeutics and imaging reagents are incorporated in lipoplexes and successfully delivered to A549 cells, demonstrating its potential applications in theranostic medicine.


Assuntos
Técnicas de Transferência de Genes/instrumentação , Lipídeos/química , Ácidos Nucleicos/metabolismo , Carbocianinas/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/genética , Citometria de Fluxo , Humanos , Proteína 1 Inibidora de Diferenciação/genética , Proteína 1 Inibidora de Diferenciação/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Microscopia de Fluorescência , Oligodesoxirribonucleotídeos/metabolismo , Pontos Quânticos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Propriedades de Superfície
5.
Mol Pharm ; 8(4): 1381-9, 2011 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21648427

RESUMO

Lung cancer is the leading cause of cancer deaths in western countries and carries a poor overall five year survival rate. Several studies demonstrate that microRNAs (miRNAs or miRs) are actively involved in tumor development by serving as tumor suppressors, oncogenes or both. In lung cancer, miRNAs may serve as both diagnostic and prognostic biomarkers as well as regulate in vitro and in vivo tumor progression. However, miRNA-based therapy is faced with several challenges including lack of tissue specificity, lack of optimal delivery systems, poor cellular uptake and risk of systemic toxicity. Here, we report a cationic lipid based miRNA delivery system to address some of these challenges. Among many lung cancer related miRNAs, miR-133b, a tumor suppressor, was selected as a therapeutic target because it directly targets the prosurvival gene MCL-1 thus regulating cell survival and sensitivity of lung cancer cells to chemotherapeutic agents. The efficacy of pre-miR-133b containing lipoplexes was evaluated in A549 non-small cell lung cancer (NSCLC) cells. Compared with siPORT NeoFX transfection agent, lipoplexes delivered pre-miR-133b in a more efficient manner with ~2.3-fold increase in mature miR-133b expression and ~1.8-fold difference in MCL-1 protein downregulation in vitro. In the in vivo biodistribution study, lipoplexes achieved ~30% accumulation in lung tissue, which was ~50-fold higher than siPORT NeoFX transfection agent. Mice treated with pre-miR-133b containing lipoplexes had mature miR-133b expression in lung ~52-fold higher than untreated mice. Our results demonstrated that cationic lipoplexes are a promising carrier system for the development of miRNA-based therapeutics in lung cancer treatment.


Assuntos
Lipídeos/química , Neoplasias Pulmonares/terapia , MicroRNAs/química , MicroRNAs/metabolismo , Animais , Western Blotting , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos Endogâmicos ICR , MicroRNAs/genética , Tamanho da Partícula , Reação em Cadeia da Polimerase em Tempo Real
6.
Mol Pharmacol ; 78(5): 811-7, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20693279

RESUMO

Imatinib, a BCR-Abl inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). Despite the success of imatinib, multiple mechanisms of resistance remain a problem, including overexpression of Lyn kinase (Lyn) and Bcl-2 family antiapoptotic proteins. Profiling micro-RNA (miRNA) expression in a model of Lyn-mediated imatinib-resistant CML (MYL-R) identified approximately 30 miRNAs whose expression differed >2-fold compared with drug-sensitive MYL cells. In particular, the expression of the miR181 family (a-d) was significantly reduced (∼11- to 25-fold) in MYL-R cells. Incubation of MYL-R cells with a Lyn inhibitor (dasatinib) or nucleofection with Lyn-targeting short interfering RNA increased miR181b and miR181d expression. A similar Lyn-dependent regulation of miR181b and miR181d was observed in imatinib-resistant K562 CML cells. Sequence analysis of potential targets for miR181 regulation predicted myeloid cell leukemia-1 (Mcl-1), a Bcl-2 family member whose expression is increased in MYL-R cells and drug-resistant leukemias. Inhibition of Lyn or rescue of miR181b expression reduced Mcl-1 expression in the MYL-R cells. To further investigate the mechanism of Mcl-1 repression by miR181, a luciferase reporter construct incorporating the Mcl-1 3'-untranslated region was tested. Overexpression of miR181b reduced luciferase activity, whereas these effects were ablated by the mutation of the seed region of the miR181 target site. Finally, stimulation of Lyn expression by 1,25-dihydroxyvitamin D(3) treatment in HL-60 cells, a cell model of acute myelogenous leukemia, decreased miR181b expression and increased Mcl-1 expression. In summary, our results suggest that Lyn-dependent regulation of miR181 is a novel mechanism of regulating Mcl-1 expression and cell survival.


Assuntos
Antineoplásicos/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide/tratamento farmacológico , MicroRNAs/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Quinases da Família src/fisiologia , Benzamidas , Calcitriol/farmacologia , Linhagem Celular Tumoral , Humanos , Mesilato de Imatinib , Leucemia Mieloide/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Piperazinas/farmacologia , Pirimidinas/farmacologia
7.
Int J Cancer ; 127(2): 313-20, 2010 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19921694

RESUMO

Restoration of p53 function in tumor cells would be an attractive strategy for lung cancer therapy because p53 mutations are found in more than 50% of lung cancers. The small molecule PRIMA-1 has been shown to restore the tumor suppression function of p53 and to induce apoptosis in human tumor cells. The mechanism of apoptosis induced by PRIMA-1 remains unclear. We investigated the effects of PRIMA-1 in apoptosis with Western immunoblot analysis, TaqMan microRNA real-time PCR, cell viability analysis and flow cytometry using human lung cancer cell lines containing mutant (H211 and H1155), wild-type (A549) or null (H1299) p53. PRIMA-1 induced massive apoptosis in the H211 and H1155 cells, but was less toxic to the A549 and H1299 cells. Western immunoblot analysis showed cleavage of PARP in H211 and H1155 cells but not in A549 and H1299 cells following treatment with PRIMA-1. In addition, p53 protein was also phosphorylated in H211 and H1155 cells. TaqMan microRNA assay showed that the expression of microRNA-34a was increased in the H211 and H1155 cells posttreatment. Knockdown microRNA-34a decreased the rate of apoptosis caused by PRIMA-1. The above results suggest that microRNA-34a is one of the important components of PRIMA-1-induced apoptotic network in the cancer cells harboring mutant p53.


Assuntos
Apoptose , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Membrana/fisiologia , MicroRNAs/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Western Blotting , Proliferação de Células , Humanos , Neoplasias Pulmonares/metabolismo , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
8.
Am J Respir Crit Care Med ; 179(1): 4-10, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18787215

RESUMO

Over the last 15 years, investigators have identified small noncoding RNAs as regulators of gene expression. One type of noncoding RNAs are termed microRNAs (miRNAs). miRNAs are evolutionary conserved, approximately 22-nucleotide single-stranded RNAs that target genes by inducing mRNA degradation or by inhibiting translation. miRNAs are implicated in many critical cellular processes, including apoptosis, proliferation, and differentiation. Furthermore, it is estimated that miRNAs may be responsible for regulating the expression of nearly one-third of the human genome. Despite the identification of greater than 500 mature miRNAs, very little is known about their biological functions and functional targets. In the last 5 years, researchers have increasingly focused on the functional relevance and role that miRNAs play in the pathogenesis of human disease. miRNAs are known to be important in solid organ and hematological malignancies, heart disease, as potential modulators of the immune response, and organ development. It is anticipated that miRNA analysis will emerge as an important complement to proteomic and genomic studies to further our understanding of disease pathogenesis. Despite the application of genomics and proteomics to the study of human lung disease, few studies have examined miRNA expression. This perspective is not meant to be an exhaustive review of miRNA biology but will provide an overview of both miRNA biogenesis and our current understanding of the role of miRNAs in lung disease as well as a perspective on the importance of integrating this analysis as a tool for identifying and understanding the biological pathways in lung-disease pathogenesis.


Assuntos
Pneumopatias/fisiopatologia , MicroRNAs/fisiologia , Linfócitos B/fisiologia , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade Inata/fisiologia , Pneumopatias/metabolismo , MicroRNAs/efeitos dos fármacos , MicroRNAs/imunologia , MicroRNAs/metabolismo , Linfócitos T/fisiologia
9.
Biochem Biophys Res Commun ; 388(3): 483-9, 2009 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-19654003

RESUMO

Lung cancer is the most frequent cause of cancer-related death in this country for men and women. MicroRNAs (miRNAs) are a family of small non-coding RNAs (approximately 21-25nt long) capable of targeting genes for either degradation of mRNA or inhibition of translation. We identified aberrant expression of 41 miRNAs in lung tumor versus uninvolved tissue. MiR-133B had the lowest expression of miRNA in lung tumor tissue (28-fold reduction) compared to adjacent uninvolved tissue. We identified two members of the BCL-2 family of pro-survival molecules (MCL-1 and BCL2L2 (BCLw)) as predicted targets of miR-133B. Selective over-expression of miR-133B in adenocarcinoma (H2009) cell lines resulted in reduced expression of both MCL-1 and BCL2L2. We then confirmed that miR-133B directly targets the 3'UTRs of both MCL-1 and BCL2L2. Lastly, over-expression of miR-133B induced apoptosis following gemcitabine exposure in these tumor cells. To our knowledge, this represents the first observation of decreased expression of miR-133B in lung cancer and that it functionally targets members of the BCL-2 family.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Neoplasias Pulmonares/metabolismo , MicroRNAs/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Reguladoras de Apoptose/genética , Sequência de Bases , Linhagem Celular Tumoral , Feminino , Inativação Gênica , Humanos , Neoplasias Pulmonares/patologia , Masculino , MicroRNAs/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/genética
11.
Am J Respir Cell Mol Biol ; 39(6): 706-16, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18587055

RESUMO

Human herpesvirus-8 (HHV-8) is the causative agent of Kaposi's sarcoma and is associated with the angioproliferative disorders primary effusion lymphoma and multicentric Castleman's disease. Evidence of HHV-8 infection within the pulmonary vasculature of patients with idiopathic pulmonary arterial hypertension (IPAH) has been described. We hypothesize that HHV-8 infection of pulmonary microvascular endothelial cells results in an apoptotic-resistant phenotype characteristic of severe pulmonary arterial hypertension. Our objective was to investigate the ability of HHV-8 to infect human pulmonary microvascular endothelial cells in vitro and characterize the phenotypic effect of this infection. Human pulmonary microvascular endothelial cells were exposed to HHV-8 using two methods (direct virus and co-culture technique). The presence of lytic and latent infection was confirmed. Changes in endothelial cell gene and protein expression and effects on cellular apoptosis were measured. HHV-8 can both lytically and latently infect primary human pulmonary microvascular endothelial cells in vitro. HHV-8 infection results in significant changes in gene expression, including alterations of pathways important to cellular apoptosis. HHV-8 infection also alters expression of genes integral to the bone morphogenic protein pathway, including down-regulation of bone morphogenic protein-4. Other genes previously implicated in the development of PAH are affected by HHV-8 infection, and cells infected with HHV-8 are resistant to apoptosis.


Assuntos
Vasos Sanguíneos/citologia , Vasos Sanguíneos/virologia , Células Endoteliais/virologia , Infecções por Herpesviridae/metabolismo , Apoptose/efeitos dos fármacos , Vasos Sanguíneos/enzimologia , Proteína Morfogenética Óssea 4/farmacologia , Proteínas de Transporte/farmacologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 8 , Marcação In Situ das Extremidades Cortadas , Interleucina-6/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes
12.
Front Genet ; 9: 243, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30050561

RESUMO

Nearly all classes of coding and non-coding RNA undergo post-transcriptional modification, as more than 150 distinct modification types have been reported. Since RNA modifications were first described over 50 years ago, our understanding of their functional relevance in cellular control mechanisms and phenotypes has truly progressed only in the last 15 years due to advancements in detection and experimental techniques. Specifically, the phenomenon of RNA methylation in the context of ncRNA has emerged as a novel process in the arena of epitranscriptomics. Methylated ncRNA molecules may indeed contribute to a potentially vast functional panorama, from regulation of post-transcriptional gene expression to adaptive cellular responses. Recent discoveries have uncovered novel dynamic mechanisms and new layers of complexity, paving the way to a greater understanding of the role of such phenomena within the broader molecular cellular context of human disease.

13.
J Control Release ; 203: 140-9, 2015 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-25687308

RESUMO

Alveolar type II (ATII) respiratory epithelial cells are essential to normal lung function. They may be also central to the pathogenesis of diseases such as acute lung injury, pulmonary fibrosis, and pulmonary adenocarcinoma. Hence, ATII cells are important therapeutic targets. However, effective ATII cell-specific drug delivery in vivo requires carriers of an appropriate size, which can cross the hydrophobic alveolar surfactant film and polar aqueous layer overlying ATII cells, and be taken up without inducing ATII cell dysfunction, pulmonary inflammation, lung damage, or excessive systemic spread and side-effects. We have developed lipoplexes as a versatile nanoparticle carrier system for drug/RNA delivery. To optimize their pulmonary localization and ATII cell specificity, lipoplexes were conjugated to an antibody directed against the ATII cell-specific antigen surfactant protein-C (SP-C) then administered to C57BL/6 mice via the nares. Intranasally-administered, anti-SP-C-conjugated lipoplexes targeted mouse ATII cells with >70% specificity in vivo, were retained within ATII cells for at least 48h, and did not accumulate at significant levels in other lung cell types or viscera. 48h after treatment with anti-SP-C-conjugated lipoplexes containing the test microRNA miR-486, expression of mature miR-486 was approximately 4-fold higher in ATII cells than whole lung by qRT-PCR, and was undetectable in other viscera. Lipoplexes induced no weight loss, hypoxemia, lung dysfunction, pulmonary edema, or pulmonary inflammation over a 6-day period. These findings indicate that ATII cell-targeted lipoplexes exhibit all the desired characteristics of an effective drug delivery system for the treatment of pulmonary diseases that result primarily from ATII cell dysfunction.


Assuntos
Células Epiteliais Alveolares/imunologia , Técnicas de Transferência de Genes , Imunoconjugados/imunologia , Lipossomos/imunologia , MicroRNAs/administração & dosagem , Proteína C Associada a Surfactante Pulmonar/imunologia , Animais , Linhagem Celular , Células Cultivadas , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Lipossomos/química , Lipossomos/farmacocinética , Pulmão/imunologia , Camundongos Endogâmicos C57BL
14.
Mol Ther Nucleic Acids ; 2: e84, 2013 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-23591808

RESUMO

MicroRNA-29b (miR-29b) expression has been shown to be reduced in non-small-cell lung cancer (NSCLC) tissues. Here, we have identified the oncogene cyclin-dependent protein kinase 6 (CDK6) as a direct target of miR-29b in lung cancer. We hypothesized that in vivo restoration of miR-29b and thus targeting of genes important to tumor initiation and progression may represent an option for lung cancer treatment. We developed a cationic lipoplexes (LPs)-based carrier that efficiently delivered miR-29b both in vitro and in vivo. LPs containing miR-29b (LP-miR-29b) efficiently delivered miR-29b to NSCLC A549 cells, reduced the expression of key targets CDK6, DNMT3B, and myeloid cell leukemia sequence 1 (MCL1), as well as cell growth and clonogenicity of A549 cells. In addition, the IC50 for cisplatin in the miR-29b-treated cells was effectively reduced. In a xenograft murine model, LPs efficiently accumulated at tumor sites. Systemic delivery of LP-miR-29b increased the tumor miR-29b expression by approximately fivefold, downregulated the tumor mRNA expression of CDK6, DNMT3B, and MCL1 by ~57.4, ~40.5, and ~52.4%, respectively, and significantly inhibited tumor growth by ~60% compared with LP-miR-NC (negative control). Our results demonstrate that cationic LPs represent an efficient delivery system that holds great potential in the development of miRNA-based therapeutics for lung cancer treatment.Molecular Therapy-Nucleic Acids (2013) 2, e84; doi:10.1038/mtna.2013.14; published online 16 April 2013.

15.
PLoS One ; 7(11): e50837, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23226399

RESUMO

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that plays a critical role in the lung by maintaining fluid homeostasis. Absence or malfunction of CFTR leads to Cystic Fibrosis, a disease characterized by chronic infection and inflammation. We recently reported that air pollutants such as cigarette smoke and cadmium negatively regulate the expression of CFTR by affecting several steps in the biogenesis of CFTR protein. MicroRNAs (miRNAs) have recently received a great deal of attention as both biomarkers and therapeutics due to their ability to regulate multiple genes. Here, we show that cigarette smoke and cadmium up-regulate the expression of two miRNAs (miR-101 and miR-144) that are predicted to target CFTR in human bronchial epithelial cells. When premature miR-101 and miR-144 were transfected in human airway epithelial cells, they directly targeted the CFTR 3'UTR and suppressed the expression of the CFTR protein. Since miR-101 was highly up-regulated by cigarette smoke in vitro, we investigated whether such increase also occurred in vivo. Mice exposed to cigarette smoke for 4 weeks demonstrated an up-regulation of miR-101 and suppression of CFTR protein in their lungs. Finally, we show that miR-101 is highly expressed in lung samples from patients with severe chronic obstructive pulmonary disease (COPD) when compared to control patients. Taken together, these results suggest that chronic cigarette smoking up-regulates miR-101 and that this miRNA could contribute to suppression of CFTR in the lungs of COPD patients.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulação da Expressão Gênica , Pulmão/metabolismo , MicroRNAs/metabolismo , Regiões 3' não Traduzidas/genética , Poluentes Atmosféricos/toxicidade , Animais , Sequência de Bases , Brônquios/patologia , Cádmio/toxicidade , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Dados de Sequência Molecular , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/patologia , Fumar/efeitos adversos , Fumar/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
16.
J Thorac Oncol ; 4(8): 1028-34, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19474765

RESUMO

Lung cancer is the leading cause of cancer related deaths in the United States. It is estimated that in 2008 there were 215,000 new diagnoses of lung cancer and 163,000 deaths. Despite emerging technologies for potential early diagnosis and discovery of novel targeted therapies, the overall 5-year survival remains a disappointing 15%. Explanations for the poor survival include late presentation of disease, a lack of markers for early detection, and both phenotypic and genotypic heterogeneity within patients of similar histologic classification. To further understand this heterogeneity and thus complexity of lung cancer, investigators have applied various technologies including high throughput analysis of both the genome and proteome. Such approaches have been successful in identifying signatures that may clarify molecular differences in tumors, identify new targets, and improve prognostication. In the last decade, investigators have identified a new mode of gene regulation in the form of noncoding RNAs termed microRNAs (miRNAs or miRs). First determined to be of importance in larval development, microRNAs are approximately 19-22 nucleotide single stranded RNAs that regulate genes by either inducing mRNA degradation or inhibiting translation. MiRNAs have been implicated in several cellular processes including apoptosis, development, proliferation, and differentiation. By regulating hundreds of genes simultaneously, miRNAs have the capacity for regulation of biologic networks. Global alterations in miRNA expression in both solid organ and hematological malignancies suggest their importance in the pathogenesis of disease. To date, both in vivo and in vitro studies in lung cancer demonstrate a dysregulation of miRNA expression. Furthermore, investigators are beginning to identify individual targets and pathways of miRNAs relevant to lung tumorigenesis. Thus, miRNAs may identify critical targets and be important in the pathogenesis of lung cancer.


Assuntos
Neoplasias Pulmonares/etiologia , MicroRNAs/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia
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