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Differential Expression Between Cufi cells and Nuli cells

Differential Expression Between Cufi cells and Nuli cells. Breathe Project Yves Berthiaume Grégory Voisin Chantal Massé September 2006. Methodology. Purpose: to note the differential expression between a CF cell and no- CF cell

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Differential Expression Between Cufi cells and Nuli cells

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  1. Differential Expression Between Cufi cells and Nuli cells Breathe Project Yves Berthiaume Grégory Voisin Chantal Massé September 2006

  2. Methodology • Purpose: to note the differential expression between a CF cell and no- CF cell to understand inflammation in CF disease. • Used cells: a new cellular model cell of epithelial pulmonary cells (immortalized lines) developped by J.Zabner. Nuli : non CF. Cufi : CF phenotype ( homozygote ∆F508) • Technologie: Microarray analysis with Affymetrix Array U133.2.0 ( 54 000 transcripts), 3 technicals replicats Nuli and 3 technicals replicats Cufi. • Statistic Analysis of microarray: bioconductor (Limma), based on a linear model (the expression probability ≥ to 50 % with a Pvalue minimum equals to 0,01). • Analysis of Gene Ontology with Onto-Express (Sorin Draghici). • Analysis of Pathway with Pathway-Express (Sorin Draghici).

  3. Results Gene Ontology analysis • 2335 modulated probesets = 1659 annoted genes (+202 NA annotation genes) 785 annotated Up-regulated genes + 32 not annotated Up-regulated genes 874 annotated Down-regulated genes + 170 not annotated Down-regulated genes • Inflammatory response excessive • Modification of metabolism: lipid, protein

  4. Conclusion of GO analysis…about inflammatory response • Up-regulation of GO = immune response, inflammatory response, chemotaxis, cell adhesion. • Inflammatory actors : IL6 , IL8, SPINK5, CXCL10, CXCL11, CXCL1, 2,3,5,6, IFIT1,3,IL1R2,TNFAIP6, S100A12.

  5. Results of Metabolic Pathway analysis • Two modulated pathways: Toll-like receptor Pathway (path: hsa04620): adjustedPvalue : 8.231x10e-3 Jak/Stat signaling pathway (path: hsa04630): adjustedPvalue : 3.285x10e-4

  6. Expression Ratio Q-PCR analysisin Toll-Like Receptor Signaling Pathway NULI CUFI

  7. In summary, ratio expression in Cufi versus Nuli (Q-PCR) • IL1beta: : expression X 5 • IL6 : : expression X 4 • IL8: : expression X 4 • CXCL10 : expression X 5 • CXCL11 : expression X 5

  8. GSTT1: x 0.1 (microarray ratio) CLCA4: : x 6 (microarray ratio) Other interesting observations

  9. Future experiments -1 • Principal conclusion: Activation of TLR signaling pathway in Cufi cells. • Experiments to be done to validate this conclusion: -NF-kB cascade. -activation (phosphorylation and translocation) of STAT 1,2. -activation of Myd 88. -measurement of protein expression (cytokines and signaling molecules).

  10. Future experiments -2 1. To complete analysis of this gene expression response following oxidative stress. 2. Validation of information in human primary cells.

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