Patients and specimens
According to the characteristics of CPFE addressed by Cottin and colleagues, six patients were selected for the current study. All six patients were originally diagnosed with lung cancer and high-resolution computed tomography showed not only the cancerous lesions but also emphysematous lesions in the upper zones and fibrotic lesions in the lower zones. The fibrotic and emphysematous lesions were located within the resected lobes. Further pulmonary function tests also indicated that impairment of carbon monoxide diffusing capacity and subnormal spirometry were present in these patients. CPFE was also diagnosed for these patients. Lung lobectomy was thought to be the optimal treatment for the lung cancer after consulting with chest surgeons. Specimens of fibrotic lesions and emphysematous lesions in lung tissues were sampled from the resected lobes of the six patients. Immediately after the lobectomy, the lung tissues with fibrosis and emphysema were separated from the resected lobes, and the lung tissue adjacent to each specimen was cut for histological confirmation of the absence of cancerous cells. The pathological types of fibrosis and emphysema were examined and confirmed after H & E staining by a pathologist who was blinded to the purpose of the study. This study was approved by the Ethics Committee of Shinshu University School of Medicine, and written informed consent was obtained from all patients.
Lung tissue specimens were immediately frozen in dry ice and stored at −80°C. RNA was extracted from the lung tissues using TRIzol Reagent (Life Technologies, Rockville, MD, USA) according to the manufacturer’s protocol. RNA for microarray analysis was purified using the RNeasy MinElute Cleanup Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. RNA quality was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).
GeneChip expression preprocessing
In accordance with the standard Affymetrix protocol from the GeneChip Expression Analysis Technical Manual Revision 5, 2 μg total RNA was processed, biotinylated, fragmented, and hybridized to the GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, USA). The Human Genome U133 Plus 2.0 chips consist of 54,765 probe sets and provide comprehensive coverage of the transcribed human genome on a single array, allowing the analysis of more than 47,000 transcripts and variants, including 38,500 well-characterized human genes plus approximately 6,500 new genes. Immediately following hybridization, the probe array underwent an automated washing and staining protocol on the Affymetrix Fluidics Station 450. The prepared GeneChips were then scanned using the Affymetrix GeneChip Scanner 3000. Each probe array was scanned twice and the software calculated an average of the two images, defined the probe cells, and computed the intensity for each cell. The double scan improved assay sensitivity and reduced background noise. Each complete probe array image was stored in a separate data file.
The scanned images were analysed with GeneChip Operating Software version 1.4 (Affymetrix 690036) and Microarray Suite version 5.0 (Affymetrix). The advantages of the Microarray Suite include the associated P values indicating statistical significance for detection and change calls, the confidence limits being associated with expression change values, and the negative expression values being eliminated. The profiling dataset from the emphysematous lesions was used as background when extracting the differentially expressed genes in the fibrotic lesions, and the data from the fibrotic lesions were used as background when extracting the differentially expressed genes in the emphysematous lesions. The trimmed mean target intensity of each array was set to 500. Differentially expressed genes were extracted using DNA Microarray Viewer (Kurabo, Osaka, Japan).
All microarray data of the individual lung tissue of the six specimens are freely available through the Gene Expression Omnibus repository [GEO:GSE38934].
The paired t test was performed for comparison of the detected signals between fibrotic and emphysematous lesions on the microarray data. The volcano plot was derived from the summary results of the paired t test to represent the point of intersection of fold-change (emphysema vs. fibrosis) relative to P value (emphysema vs. fibrosis). Clustering analysis was then carried out with log-transformed average signals of both fibrotic and emphysematous lesions on the microarray data and the heat map was built (Avadis 4.3; Strand Scientific Intelligence, San Francisco, CA, USA), according to the result of the hierarchical clustering analysis.
The SLR algorithm measures the magnitude and direction of the change between transcript levels of the experimental and background chips. A SLR of 1 represents a twofold increase in the abundance of an mRNA, and a value of −1 represents a twofold reduction in transcript expression. In the present analysis, we extracted genes with SLR >1 and SLR <1 for the fibrotic/emphysematous and emphysematous/fibrotic ratios, respectively. We then systematically annotated the large list of genes according to their biological functions using bioinformatics resources from the Database for Annotation, Visualization and Integrated Discovery, which provides the ability to explore and view functionally related genes together, as a unit, to concentrate on the large biological network rather than at the level of an individual gene. Condensing large gene lists into biologically meaningful modules greatly improves the ability to assimilate large amounts of information and thus switches functional annotation analysis from a gene-centric analysis to a biological module-centric analysis. Since an enrichment score of 1.3 is equivalent to 0.05 on the nonlog scale, we focused on gene clusters with scores ≥1.3 to address genes hypothetically involved in the phenotypes of the examined tissues.