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Z. Xiao



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    MINI 34 - RNA and miRNA (ID 162)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      MINI34.13 - Long Noncoding RNA BC070487 Represses ZNFX1 during Tobacco-Induced Lung Carcinogenesis (ID 2912)

      18:30 - 20:00  |  Author(s): Z. Xiao

      • Abstract
      • Presentation
      • Slides

      Background:
      Limited information exists regarding regulation of gene expression by long noncoding RNAs (lncRNAs) during initiation and progression of tobacco-associated lung cancers. In the present study, an in-vitro model system was used to examine the effects of cigarette smoke on lncRNA expression in human respiratory epithelia and lung cancer cells.

      Methods:
      Micro-array and qRT-PCR techniques were used to assess lncRNA and gene expression profiles in normal human small airway epithelial cells (SAEC) and immortalized human bronchial epithelial cells (HBEC) cultured in the presence or absence of cigarette smoke condensate (CSC). Quantitative chromatin immunoprecipitation (ChIP), methyl-DNA immunoprecipitation (MeDIP), and cross-link immunoprecipitation (CLIP) techniques were used to assess promoter occupancy, DNA methylation, and interaction of lncRNAs with target proteins. Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assays were used to identify a regulatory sequence for lncRNA BC070487.

      Results:
      Micro-array analysis demonstrated that under relevant exposure conditions, CSC consistently mediated a 2.5 fold increase in BC070487 expression, and 3.6 fold decrease in its immediate downstream target gene, ZNFX1, in SAEC. qRT-PCR experiments confirmed a 4-7 fold up-regulation of BC070487, with a 4-8 fold down-regulation of ZNFX1 in SAEC and HBEC, as well as Calu-6 and H841 lung cancer cells following CSC exposure. BC070487 expression correlated inversely with expression of ZNFX1 (BC070487: 1.38-10.31 fold up vs ZNFX1: 2.26-26.29 fold down) in primary lung cancers relative to adjacent normal lung parenchyma. Overexpression or depletion of BC070487 inhibited or enhanced expression of ZNFX1, respectively in normal respiratory epithelia and lung cancer cells. CSC as well as BC070487-mediated repression of ZNFX1 coincided with increased occupancy of EZH2, SUZ12, BMI1, and increased levels of H3K27Me3, with decreased H3K4Me3 within the ZNFX1 promoter region. CSC as well as BC070487 overexpression enhanced binding of BC070487 with EZH2, SUZ12 and BMI1 proteins in SAEC and Calu-6 cells. CSC exposure significantly increased DNA methylation in one of three CpG islands spanning the regulatory elements of ZNFX1. In addition, CSC enhanced binding of BC070487 with DNMT3A and DNMT3B with site-specific de novo DNA methylation of ZNFX1 in SAEC and Calu-6 cells. FAIRE assays identified a 800 bp regulatory sequence for BC070487; CSC-mediated activation of BC070487 coincided with increased levels of H3K4Me3, H3K27Ac, H3K36Me3, Sp1, and Ago proteins, with decreased levels of H3K27Me3 and H3K9Me3 within this regulatory region. miR-31, an oncomiR specifically induced by CSC, modulated the transcriptional activity of BC070487 via remodeling the distribution of histone modification marks (Up: H3K4Me3, H3K27Ac, H3K36Me3; Down: H3K27Me3 and H3K9Me3) in the regulatory region of BC070487. Overexpression or depletion of BC070487 increased or inhibited proliferation and invasion of lung cancer cells, and similarly modulated growth of SAEC and HBEC cells. In contrast, ZNFX1 overexpression or depletion decreased or enhanced growth of normal respiratory epithelial cells and lung cancer cells. BC070487 overexpression enhanced growth of lung cancer cells xenografts in athymic nude mice, while forced expression of ZNFX1 arrested growth of these xenografts.

      Conclusion:
      Collectively, these data suggest that CSC-mediated up-regulation of BC070487 suppresses ZNFX1 to promote pulmonary carcinogenesis.

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