differential expression between cufi cells and nuli cells breathe project yves berthiaume grégory...

Differential Expression Between Cufi cells and Nuli cells

Breathe Project

Yves Berthiaume Grégory VoisinChantal Massé

September 2006

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).

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

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.

Results of Metabolic Pathway analysis

• Two modulated pathways:

Toll-like receptor Pathway (path: hsa04620):

adjusted Pvalue : 8.231x10e-3

Jak/Stat signaling pathway (path: hsa04630):

adjusted Pvalue : 3.285x10e-4

Expression Ratio Q-PCR analysisin Toll-Like Receptor Signaling Pathway

expression de STAT2Pvalue = 0.03 , ratio 1.8

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.4000

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

Expression de IL1b par PCRp=0.0004 ratio = 3,6

0

0.5

1

1.5

2

2.5

3

3.5

4

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

Expression d'IL6p=0,01 , ratio= 5.4

0

1

2

3

4

5

6

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

Expression de IL8Pvalue= 0,002

0

0.5

1

1.5

2

2.5

3

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

EXPRESSION DE CXCL10p=0.028, ratio= 5,5

0.0000

1.0000

2.0000

3.0000

4.0000

5.0000

6.0000

7.0000

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

expression de CXCL11p=0,01 , ratio = 4,9

0

1

2

3

4

5

6

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

expression de STAT1Pvalue = 0.006 , ratio= 2.1

0.0000

0.5000

1.0000

1.5000

2.0000

2.5000

3.0000

Nuli_CTL1 Nuli_CTL2 Nuli_CTL3 Cufi_CTL1 Cufi_CTL2 Cufi_CTL6

expression de C-FOSpvalue = 0.63, ratio = 0.9

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

expression de MYD88pvalue= 0,88 , ratio =1,1

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6 expression de MAPK8pvalue = 0.6 , ratio = 0.9

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

CUFINULI

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

Other interesting observationsexpression de GSTT1

(Q PCR)

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

expression de CLCA4 (Q PCR)pvalue = 0.0001 , ratio = 4,7

0.0000

0.5000

1.0000

1.5000

2.0000

2.50003.0000

3.5000

4.0000

4.5000

5.0000

Nuli-CTL1 Nuli-CTL2 Nuli-CTL3 Cufi-CTL1 Cufi-CTL2 Cufi-CTL6

GSTT1: x 0.1 (microarray ratio)

CLCA4: : x 6 (microarray ratio)

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).

Future experiments -2

1. To complete analysis of this gene expression response following oxidative stress.

2. Validation of information in human primary cells.

Cufi - Physiological response - DMNQ 24h

Ctl

DMNQ 5

µM

DMNQ 1

0µM

DMNQ 1

5 µM

DMNQ 2

0 µM

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