Congenital cystic adenomatoid malformations of the lung: an epithelial transcriptomic approach

Guillaume Lezmi, Shamila Vibhushan, Claudia Bevilaqua, Nicolas Crapart, Nicolas Cagnard, Naziha Khen-Dunlop, Christine Boyle-Freyssaut, Alice Hadchouel, Christophe Delacourt, Guillaume Lezmi, Shamila Vibhushan, Claudia Bevilaqua, Nicolas Crapart, Nicolas Cagnard, Naziha Khen-Dunlop, Christine Boyle-Freyssaut, Alice Hadchouel, Christophe Delacourt

Abstract

Background: The pathophysiology of congenital cystic adenomatoid malformations (CCAM) of the lung remains poorly understood.

Aim: This study aimed to identify more precisely the molecular mechanisms limited to a compartment of lung tissue, through a transcriptomic analysis of the epithelium of macrocystic forms.

Methods: Tissue fragments displaying CCAM were obtained during planned surgical resections. Epithelial mRNA was obtained from cystic and normal areas after laser capture microdissection (LCM). Transcriptomic analyses were performed and the results were confirmed by RT-PCR and immunohistochemistry in independent samples.

Results: After controlling for RNA quality, we analysed the transcriptomes of six cystic areas and five control areas. In total, 393 transcripts were differentially expressed in the epithelium, between CCAM and control areas. The most highly redundant genes involved in biological functions and signalling pathways differentially expressed between CCAM and control epithelium included TGFB2, TGFBR1, and MAP 2 K1. These genes were considered particularly relevant as they have been implicated in branching morphogenesis. RT-qPCR analysis confirmed in independent samples that TGFBR1 was more strongly expressed in CCAM than in control tissues (p < 0.03). Immunohistochemistry analysis showed TGFBR1 (p = 0.0007) and TGFB2 (p < 0.02) levels to be significantly higher in the epithelium of CCAM than in that of control tissues.

Conclusions: This compartmentalised transcriptomic analysis of the epithelium of macrocystic lung malformations identified a dysregulation of TGFB signalling at the mRNA and protein levels, suggesting a possible role of this pathway in CCAM pathogenesis.

Trial registration: ClinicalTrials.gov Identifier: NCT01732185.

Keywords: Congenital thoracic malformations; Cystic lung; Laser capture microdissection; Transcriptome; Transforming growth factor.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Laser capture microdissection of epithelial samples
Fig. 2
Fig. 2
Unbiased clustering of genes based on their levels of expression in microarray analyses of epithelial samples from six cystic and five control areas. Samples CCAM_2_E, CCAM_6_E and CCAM_19_E are type I CCAM; samples CCAM_5_E, CCAM_14_E and CCAM_16_E are type II CCAM. The cluster correlation dendrogram showed a clear separation of CCAM samples from control samples. No clear clustering differentiated type I CCAM from type II CCAM
Fig. 3
Fig. 3
Gene ontology analysis of genes differentially expressed between CCAM epithelium and control epithelium. TGFB2 was identified in 17 of the top 20 biological processes, and TGFbR1 in 12 of the top 20 biological processes
Fig. 4
Fig. 4
Gene-set enrichment analysis of genes differentially expressed between cystic and control areas of epithelium. TGFB2 was identified in 10 of the top 20 signalling pathways, and TGFbR1 in 9 of the top 20 signalling pathways
Fig. 5
Fig. 5
RT-PCR (a) and immunostaining (b) results for TGFb2, TGFbR1 and MAP 2 K1 in control and CCAM samples. TGFB2: 5 controls vs. 17 CCAM samples and 9 controls vs. 9 CCAM samples for RT-PCR and immunostaining, respectively; TGFBR1: 4 controls vs. 11 CCAM samples and 13 controls vs. 13 CCAM samples for RT-PCR and immunostaining, respectively; MAP 2 K1: 4 controls vs. 17 CCAM samples and 13 controls vs. 11 CCAM samples for RT-PCR and immunostaining, respectively. * p < 0.05; ***p < 0.001
Fig. 6
Fig. 6
Immunostaining for TGFB2 (A, A', B, B') and TGFBR1 (C, C', D, D') in CCAM (B, B', D, D') and control samples (A, A', C, C') , at magnifications of × 20 (A, C, B, D) and × 40 (A', B', C', D'). Diffuse expression is observed in CCAM epithelium

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