Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling by array to investigate effect of repeated cigarette exposure on three-dimensional human bronchial epithelial cultures

ABSTRACT: Cigarette smoke (CS) is a major risk factor in the development of chronic inflammatory lung diseases such as chronic obstructive pulmonary disease. To evaluate the biological impact of CS on lung tissue, three-dimensional (3D) organotypic bronchial tissue cultures can be used to replicate in vivo conditions. We developed an original 3D human bronchial epithelial co-culture model to assess the biological impact of repeated CS exposure on cell differentiation and on the inflammatory response. We found that CS can disrupt homeostatic capacity in a dose-dependent manner, and that the activation of the EGFR pathway, which is involved in the early-stage pathogenesis of airway diseases, was predicted from transcriptomic data. We believe that our model of bronchial tissues, used for repeated CS exposure, can provide valuable information on tissue-specific alterations in biological systems.

ORGANISM(S): Homo sapiens

SUBMITTER: Kazushi Matsumura

PROVIDER: E-MTAB-6175 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In vitro toxicology approaches have evolved, from a focus on the molecular changes within a cell to understanding of toxicity-related mechanisms that simulate the in vivo environment. The recent development of three dimensional (3-D) organotypic nasal epithelial culture models offer a physiologically robust system for studying the effects of exposure through inhalation. Exposure to cigarette smoke (CS) is associated with nasal inflammation; thus the nasal epithelium is relevant for evaluating the pathophysiological impact of CS exposure. The present study investigated further the relevancy and application of in vitro human 3-D nasal epithelial culture models for toxicological assessment of inhalation exposure. The biological impact was assessed following exposure to aerosol generated from a candidate modified risk tobacco product (MRTP), the Tobacco Heating System (THS) 2.2, as compared with smoke generated from reference cigarette 3R4F using an in vitro human 3-D nasal epithelial cultures. A series of experimental repetitions where multiple doses of the aerosol and smoke were applied, were conducted to obtain reproducible measurements and reliable observations to understand the cellular/molecular changes that occur following exposure. Aligned with the 3Rs Strategy and the Vision-and-Strategy of the Toxicity Testing in the 21st Century, this study implemented a systems toxicology approach and found that for all tested concentrations, the impact of 3R4F smoke was considerably greater than that of THS2.2 aerosol in terms of cytotoxicity levels, alterations in the tissue morphology, secretion of pro-inflammatory mediators, impaired ciliary function, and increased perturbed transcriptomes and miRNA expression profiles. In addition, to evaluate further the possible adverse effects of THS2.2 aerosol, a dose range assessment was conducted. A broader range of THS2.2 concentrations were exposed to the nasal cultures. Various dilutions of THS2.2 were applied to the cultures using the VitrocellÂ® 24/48 exposure system, corresponding to the concentrations of nicotine between 0.15 mg/L and 1.79 mg/L

Project description:MicroRNAs are critical mediators of stem cell pluripotency, differentiation and malignancy. Limited information exists regarding microRNA alterations that facilitate initiation and progression of human lung cancers. In this study, array techniques were used to evaluate microRNA expression in normal human respiratory epithelia and lung cancer cells cultured in the presence or absence of cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly repressed miR-487b in these cells. Subsequent experiments demonstrated that miR-487b directly targets SUZ12, BMI1, Wnt5a, c-Myc and K-ras. Repression of miR-487b correlated with over-expression of these targets in primary lung cancers, and coincided with DNA methylation, de-novo nucleosome occupancy, and decreased H2AZ and TCF1 levels within the miR-487b genomic locus. Deoxyazacytidine de-repressed miR-487b, and attenuated CSC-mediated silencing of miR-487b. TGF-?1 recapitulated CSC-mediated repression of miR-487b. Constitutive expression of miR-487b abrogated Wnt signaling; inhibited in-vitro proliferation and invasion of lung cancer cells mediated by CSC or over-expression of miR-487b targets, and decreased growth and metastatic potential of lung cancer cells in-vivo. Collectively, these findings indicate that miR-487b is a novel tumor suppressor microRNA silenced by epigenetic mechanisms during tobacco-induced pulmonary carcinogenesis, and suggest that DNA demethylating agents may be useful for activating miR-487b for lung cancer therapy. MicroRNA PCR array analysis. Human RT2 miRNA PCR Arrays (MAH-3100E-12) were obtained from SA Biosciences. Two hundred ng of isolated miRNA was used for reverse transcription and the entire first strand cDNA was diluted and distributed amongst the 384 wells of the super-array plate. The reactions were performed with RT² SYBR Green / ROX PCR Master Mix (SABiosciences). Results were analyzed using software provided by the vendor (http://www.sabiosciences.com/pcr/arrayanalysis.php).

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Transcription profiling by array to investigate effect of repeated cigarette exposure on three-dimensional human bronchial epithelial cultures

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